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IV Encontro Annual do INBEB – www.inbeb.org.br Página 1

Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem - INBEB

O Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem (INBEB) é uma iniciativa pioneira com a missão de criar e consolidar uma infraestrutura técnico-científica que permita o estudo das estruturas de sistemas biológicos, desde o nível macromolecular até o organismo inteiro, lançando mão das técnicas mais avançadas e de mais alta resolução possível.

O INBEB promove a interdisciplinaridade, fazendo com que áreas convencionais como biofísica, parasitologia, microbiologia, imunologia, bioquímica, farmacologia, química e computação tenham suas fronteiras estendidas. Isto permite a maior interação entre grupos para solucionar diversos problemas biológicos. Outro objetivo fundamental do Instituto é contribuir para o treinamento de pesquisadores em Biologia Estrutural e Bioimagem em vários níveis.

É neste âmbito que apresentamos no Programa do IV Encontro Anual do INBEB a programação do evento, assim como os resumos dos 163 pôsteres inscritos no encontro. Esperamos que todos possam aproveitar a oportunidade para interagir, aperfeiçoar seus trabalhos e estabelecer novas parcerias.

Prof. Jerson Lima da Silva Prof. Wanderley de Souza

Coordenador do INBEB Vice-coordenador do INBEB

Apoio:

IV Encontro Anual do INBEB – www.inbeb.org.br Página 2

Índice Programação .......................................................................................................................................................................... 3

Quarta-feira - 08/05/2013 .................................................................................................................................................. 3

Quinta-feira - 09/05/2013 .................................................................................................................................................. 5

Sexta-feira - 10/05/2013 ..................................................................................................................................................... 7

Sessões de pôsteres ............................................................................................................................................................... 9

Data das apresentações ...................................................................................................................................................... 9

Certificados ......................................................................................................................................................................... 9

Listas de apresentadores .................................................................................................................................................. 10

Graduandos ................................................................................................................................................................... 10

Mestrandos ................................................................................................................................................................... 12

Doutorandos ................................................................................................................................................................. 13

Pós-doutorandos .......................................................................................................................................................... 14

Pesquisadores ............................................................................................................................................................... 15

Resumos ............................................................................................................................................................................... 16

Graduandos....................................................................................................................................................................... 16

Mestrandos ....................................................................................................................................................................... 30

Doutorandos ..................................................................................................................................................................... 39

Pós-doutorandos .............................................................................................................................................................. 58

Pesquisadores ................................................................................................................................................................... 66


Programação

Quarta-feira - 08/05/2013 Auditório Hélio Fraga, CCS/UFRJ 9:00 - 9:30h Cadastramento e entrega dos materiais. 9:30 - 10:00h Apresentação - Prof. Jerson Lima da Silva, IBqM / UFRJ / Coordenador do INBEB. 10:00 – 10:30h LA 14: “Potencial imunomodulador de compostos naturais com propriedades leishmanicida”. - Profa. Edilene Oliveira da Silva, UFPA. 10:30 – 11:00h: Coffee-Break. 11:00 – 11: 30h LA 2: “O papel das microglias nas amiloidoses”. - Profa. Débora Foguel, IBqM/UFRJ. 11:00 – 11:30h LA 3: “BEX3 (Brain expressed x-linked), an apoptotic protein with intriguing structural behaviour”. - Prof. Fabio Almeida, IBqM/UFRJ. 11:30 – 12: 00h LA18: “Trypanosoma cruzi, a flagellated protozoan surfing on a proteolytic tzunami: an adaptive strategy leading to persistent infection in the edematous heart?”. - Prof. Julio Scharfstein, IBCCF/UFRJ


12:00 – 13:30h: Almoço. 13:30 – 15:00h Mesa redonda: “PESQUISA TRANSLACIONAL: COLABORAÇÃO IDOR – INBEB” Chair: DR

A. LÉA MIRIAN/FACULDADE DE MEDICINA/UFRJ 1.Dra. Fernanda Tovar Moll, Profa. Adjunta/ICB/UFRJ, Responsável pela RMN do CENABIO/UFRJ e Diretora Científica do Instituto D'Or de Pesquisa e Ensino: “POSSIBILIDADES DE APLICAÇÃO DA RESSONÂNCIA MAGNÉTICA DE 7T E

3T” 2.Dr. Jorge Moll, Diretor de Pesquisa do Instituto D'Or de Pesquisa e Ensino: “RESSONÂNCIA MAGNÉTICA

ESTRUTURAL E FUNCIONAL NA INVESTIGAÇÃO DE DOENÇAS PSIQUIÁTRICAS” 3.Dr. Fernando Bozza, Pesquisador da Fundação Oswaldo Cruz e Chefe do Centro de Terapia Intensiva do Instituto de Pesquisa Clínica Evandro Chagas/FIOCRUZ: “ESTUDO DA INFLAMAÇÃO PULMONAR, AVALIADA ATRAVÉS DE

MODELOS CLÍNICOS E EXPERIMENTAIS” 15:00 – 15:30h LA 6: “Protein phosphatases: model systems for biomimetics”. - Prof. Hernán Terenzi, UFSC. 15:30 - 16:00h LA 15: “Caracterização de novos fármacos anti-inflamatórios”. - Profa. Christina Peixoto, Fiocruz/INT-CETENE. 16:00 – 16:30h LA 8: “Planejamento in silico de candidatos à fármacos contra doença tropicais negligenciadas”. - Prof. Marcelo Castilho, FF/UFBA. 16:30 – 17:0h LA 10: “Novos microscópios de força atômica do inbeb” - Prof. Gilberto Weissmuller, IBCCF/UFRJ. 17:00 – 19:00h: Sessão de Pôsteres / Coquetel.


Quinta-feira - 09/05/2013

Auditório Hélio Fraga, CCS/UFRJ 9:30 – 10:00h LA 9: “Em busca de compostos mais eficientes e menos tóxicos para quimioterapia das leishmanioses". - Profa. Juliany Cola Fernandes Rodrigues, IBCCF/UFRJ. 10:00 – 10:30h LA 1: “Structural biology and bioimaging studies on cancer, neurodegenerative and viral diseases”. – Prof. Jerson Lima da Silva, IBqM/UFRJ. 10:30 – 11:00h: Coffee-Break 11:00 – 11:30h LA 4: “A venômica e antivenômica revelando novas moléculas com potencial biotecnológico para o desenho de novos fármacos e controle de qualidade de soros antiofídicos”. - Profa. Lina Zingali, IBqM/UFRJ e Dr. Carlos Correa Netto, Pós-doc IBqM/UFRJ. 11:30 – 12:00h LA 16: “Abordagens experimentais no estudo das células-tronco”. - Profa. Regina Goldenberg, IBCCF/UFRJ. 12:00 – 13:00h Conferência Magna: - Dr. Kurt Wuthrich (Prêmio Nobel de Química em 2002), Prof. Visitante do IBqM/UFRJ/INBEB: “Historical Development and Current Trends of NMR in Structural Biology and Biotechnology“. 13:00 – 14:30h: Almoço


14:30 – 16:00h Mesa Redonda

“DIVULGAÇÃO CIENTÍFICA NO INBEB: INTERAÇÃO UNIVERSIDADE E ESCOLA” Chair: PROF

A. MARTHA SORENSON/IBQM/UFRJ 1.Dr. Emiliano Medei/Prof. Adjunto/IBCCF/UFRJ: “CONTEXTUALIZAÇÃO CIENTÍFICA PARA ALUNOS DA EDUCAÇÃO BÁSICA DE

UMA REGIÃO RURAL DO ESTADO DO RJ” 2.MsC. Josineide Pantoja/Profa. da Rede Municipal/Igarapé Miri/PA: “ATIVIDADES CIENTÍFICAS NA CIDADE DE

IGARAPÉ-MIRI/PA” 3.Dra. Patricia S. dos Santos, Coordenadora do Núcleo de Educação e Divulgação Científica do INBEB: “ATIVIDADES DO NEDICI/INBEB” 16:00 – 16:30h LA13: “Biogênese do flagelo de leishmania amazonensis durante a diferenciação celular”. – Dra. Ana Paula, INMETRO. 16:30 – 17:30h: Reunião do Comitê Gestor do INBEB 17:00 – 19:00h: Sessão de pôsteres/Coffee-break.


Sexta-feira - 10/05/2013 Auditório Hélio Fraga, CCS/UFRJ 10:00h: Coffee-break 10:00 – 10:30h LA 12: “Identificação de uma nova estrutura que participa na formação da parede cística em Giardia intestinalis”. - Profa. Marlene Benchimol, USU. 10:30 – 11:00h LA 19: “Terapia celular em modelos animais de doenças neurológicas”. - Prof. Marcelo Santiago, IBCCF/UFRJ. 11:00 – 11:30h

LA 5:“Novos métodos em cálculos estruturais e dinâmicos de biomoléculas: aplicações em biomembranas e enzimas”. - Prof. Pedro Pascutti, IBCCF/UFRJ. 11:30 – 12:00h “Caracterização do padrão de captação de 18-FDG na lesão pulmonar aguda”. - Prof. Alysson Roncally, IBCCF COPPE/UFRJ. 12:00 – 13:30h: Almoço 13:30 – 14:00h LA 7: “Eukariotic molecular chaperones: sturctural and cell biology”. - Prof. Carlos Ramos, IQ/Unicamp. 14:00 – 14:30h LA 11: "Trypanosoma abeli: um novo Trypanosoma de peixes". – Dra. Moara Lemos, Pós-doc/IMPPG/UFRJ.


14:30 – 15:00h LA 17: “Uma visão do realizado no biênio 2011-2012 no laboratório 17 do INBEB”. - Prof. Adalberto Vieyra, IBCCF/UFRJ. 15:00 – 15:30h Conferência de encerramento: -Premiação dos melhores pôsteres -Resultado do concurso do logo do INBEB -CENABIO: O novo Órgão Suplementar da UFRJ – Apresentação do Diretor. 15:30 – 17:00h: Confraternização.


Sessões de pôsteres

Data das apresentações Trabalhos de número par: dia 08/05

Trabalhos de número ímpar: dia 09/05

Certificados Após a arguição dos avaliadores, serão entregues certificados de apresentação apenas em nome dos apresentadores inscritos que forem avaliados. Os demais autores terão este caderno de resumos como certificado de inscrição dos trabalhos no evento.


Listas de apresentadores Os resumos estão separados por titulação e ordenados alfabeticamente pelo primeiro nome do apresentador.

Graduandos

G 1 - Ana Beatriz Padilha de Figueiredo

G 2 - Arthur Daniel Rocha Alves

G 3 - Brunno Renato Farias Verçoza

G 4 - Camila Cristina da Silva

G 5 - Carolina Andrade

G 6 - Caroline Lauritzen da Costa

G 7 - Caroline Mendes Ferreira

G 8 - Cássia Netto de Araújo

G 9 - Clarice de Souza Cerqueira Machado

G 10 - Claudia Monteiro da Rocha

G 11 - Daniel Antônio Shimizu Kitagawa

G 12 - Daniel Meira dos Anjos

G 13 - Eliã Barbosa Marins

G 14 - Elias Ataide Mendonça

G 15 - Felipe Henrique Souza daSilva

G 16 - Gabriel Nascimento dos Santos

G 17 - Geovana Vargas Silva

G 18 - Gerson Duarte Guercio

G 19 - Giselle Souza Moreira

G 20 - Gusthavo figueira Barbosa

G 21 - Hortencia Almeida da Silva Monteiro

G 22 - Jéssica Monteiro de Oliveira

G 23 - João Paulo Bortot Soares

G 24 - José Eduardo Teixeira Pinto Junior

G 25 - Juliana Santos Santana

G 26 - Leandro Braga Rodrigues

G 27 - Lilian Goulart Schultz

G 28 - Lilianne Gomes de Magalhães LAmeira

G 29 - Marcella Valentim Monteiro Ferreira

G 30 - Marina Chao Campello

G 31 - Matheus Esteves Ferreira

G 32 - Mayara Gil de Castro Santos


G 33 - Mayra de Amorim Marques

G 34 - Michelle Guimarães Furtado

G 35 - Nathali Pereira da Costa Campos

G 36 - Neilton Cesar Araujo da Cruz

G 37 - Nicoli Cardoso Mortari

G 38 - Roger Borges dos Santos

G 39 - Tarcísio Nascimento Corrêa

G 40 - Thais Cordovil

G 41 - Thaís de Oliveira Silva

G 42 - Veronica da Silva Ferreira

G 43 - Wesley Júnio Alves da Conceição

G 44 - William Yutaka Ohashi

G 45 - Yasmin Lemos Rollemberg Cruz Machado


Mestrandos

M 1 - Aline Miyoko Sakaguchi Yamashita

M 2 - Almir Jordão da Silva Junior

M 3 - Alvaro Carrier Ruiz

M 4 - Ana Clara Vicente dos Santos

M 5 - Ana Paula Miranda Mendonça

M 6 - André Roberto de Oliveira Fredi

M 7 - Beatriz Bastos Fonseca

M 8 - Bruno José Martins da Silva

M 9 - Camila Carane Bitencourt Brito

M 10 - Camila Silva Gonçalves

M 11 - Carolina De Lima Alcantara

M 12 - Caroline Martins Almeida

M 13 - Cinthia Lima

M 14 - Clara Simões

M 15 - Diego de Souza Gonçalves

M 16 - Gabriel Barros Rodrigues

M 17 - Gabriela Veras de Moraes

M 18 - Jorge Augusto Leão Pereira

M 19 - Júlia Araújo de Freitas

M 20 - Laise Aline Martins dos Santos

M 21 - Luiz Carlos dos Santos Ribeiro

M 22 - Luiz Felipe Garcia e Souza

M 23 - Luiza Fernandes

M 24 - Maria Eduarda Rocha de França

M 25 - Paula Cristina Rodrigues Frade

M 26 - Pedro Ernesto Lopes Leão

M 27 - Rachel de Pinho Rachid

M 28 - Tatiana Kelly da Silva Fidalgo

M 29 - Wilma Helena de Oliveira


Doutorandos

D 1 - Adolfo Henrique de Moraes Silva

D 2 - Alane Bernardo Ramos

D 3 - Aline Lidiane Batista de Amorim

D 4 - Amanda Karolina Soares e Silva

D 5 - Ana Karolina de Santanna Nunes

D 6 - Andréa Marins Damasceno Bomfim

D 7 - Anne Cristine Silva Fernandes

D 8 - Aracelys López Castilla

D 9 - Bruna Santos da Silva

D 10 - Bruno Macedo

D 11 - Camila Salata

D 12 - Carlos Alberto Marques de Carvalho

D 13 - Carlos Henrique Dumard

D 14 - Carolina Catta Preta

D 15 - Caroline Rezende Guerra

D 16 - Cherley Borba Vieira de Andrade

D 17 - Cícero Figueiredo Freitas

D 18 - Daniel Sanches

D 19 - Denise Cristian Ferreira Neto

D 20 - Edlene Lima Ribeiro

D 21 - Erivan Schnaider Ramos Junior

D 22 - Estefania Pereira Cardoso Azevedo

D 23 - Éverton Dias D'Andréa

D 24 - Fabian Gilberto Villalta Romero

D 25 - Fernando Antonio de Oliveira Adnet

D 26 - Franco Henrique Andrade Leite

D 27 - Gilson Costa dos Santos Junior

D 28 - Glaucia Melina Squizato Pinheiro

D 29 - Gloria Maria Castañeda Valencia

D 30 - Gustavo Monnerat Cahli

D 31 - Ivone Rosa de Andrade

D 32 - Joseane Lima Prado Godinho

D 33 - Josineide Pantoja da Costa



D 36 - Juliana Cunha Vidal

D 37 - Juliana Pandini Castelpoggi

D 38 - Leandro Teixeira de Oliveira


D 39 - Letícia Maria Zanphorlin Murakami

D 40 - Luana Borba

D 41 - Marcelo de Lima Sant Anna

D 42 - Marco Antonio Vidal Jimenez

D 43 - Mariana Araya de Godoy

D 44 - Matheus Pinto de Oliveira

D 45 - Natália do Carmo Ferreira de Araújo

D 46 - Nathalia dos Santos Alves

D 47 - Rayana Leal de Almeida Luna

D 48 - Renata Travassos de Lima

D 49 - Ricardo Santanna Oliveira

D 50 - Roberta Ferreira Cura das Neves

D 51 - Samir pereira da costa campos

D 52 - Sara Teixeira de Macedo Silva

D 53 - Sirlene Oliveira Francisco de Azeredo

D 54 - Sura Wanessa Santos Rocha

D 55 - Tatiana Christina Paredes Santos

D 56 - Tiago Bortolotto

D 57 - Tiago Veltri Ormastroni da Trindade

D 58 - Vanessa Lopes de Azevedo Braga

D 59 - Victor do Valle Pereira Midlej

D 60 - Viviane Cristina Heinzen da Silva

Pós-doutorandos

PD 1 - Bruno Kaufmann Robbs

PD 2 - Camila Marques Adade

PD 3 - Camila Zaverucha do Valle

PD 4 - Carlos Correa Netto

PD 5 - Carolina Galvão Sarzedas

PD 6 - Caroline Madeira Moreira

PD 7 - Catarina Raposo

PD 8 - Clarissa Rodrigues Nascimento

PD 9 - Danielly Cristiny Ferraz da Costa

PD 10 - Fabiana Pestana Albernaz

PD 11 - Fabio Mendonça Gomes

PD 12 - Fernanda de Avila Abreu


PD 13 - Fernanda Gubert

PD 14 - Joana da Costa Pinto d'Avila

PD 15 - Juliana M. F Dutra Santiago

PD 16 - Leandro Lara de Carvalho

PD 17 - Mariana Pierre de Barros Gomes

PD 18 - Moara Lemos

PD 19 - Nathalia Varejão

PD 20 - Patricia Alves Reis

PD 21 - Priscila Ferreira

PD 22 - Shana Priscila Coutinho Barroso

PD 23 - Tuane Cristine Ramos Goncalves Vieira

PD 24 - Viviane Silva de Paula

Pesquisadores

P 1 - Ana Carolina Oliveira

P 2 - Gisele Cardoso de Amorim

P 3 - Karla Patricia de Sousa Barbosa

P 4 - Marcela Cristina de Moraes

P 5 - Mariana Aragão Matos Donato


Resumos Graduandos

G1. ANÁLISE POR RESSONÂNCIA MAGNÉTICA DA PERMANÊNCIA DE CÉLULAS DE MEDULA ÓSSEA NO VÍTREO APÓS LESÃO DO NERVO ÓPTICO

1,2- FIGUEIRÊDO, A.B.P.; 1,2- MESENTIER-LOURO, L.; 1,2- ZAVERUCHA-DO-VALLE, C.; 1,2- NASCIMENTO-DOS-SANTOS, G.; 2- TEIXEIRA, C.; 2- TOVAR-MOLL, F.; 1,2- MENDEZ-

OTERO, R.; 1,2- SANTIAGO, M.F. 1- Instituto de Biofísica Carlos Chagas Filho; 2- Instituto Nacional de Ciência e Tecnologia

de Biologia Estrutural e Bioimagem – INBEB "Em mamíferos adultos, lesões no nervo óptico provocam degeneração retrógrada das células ganglionares da retina e morte progressiva, especialmente por apoptose. Várias abordagens terapêuticas vem sendo estudadas para aumentar a sobrevida neuronal e regeneração axonal daqueles neurônios em diferentes modelos de lesão, incluindo a terapia celular. Nesse sentido, em trabalhos anteriores demonstramos que tanto as células mononucleares quanto as células mesenquimais derivadas da medula óssea geram neuroproteção e aumentam a extensão axonal após lesão no nervo óptico. No presente trabalho, procuramos, então, analisar o destino das células transplantadas in vivo por imagem através de ressonância magnética e ex vivo através de reações histoquímicas. Para isso, utilizamos ratos adultos da variedade Lister-hooded e as células derivadas da medula óssea foram marcadas com nanopartículas superparamagnéticas (SPIO - Feratrack) e injetadas no vítreo dos animais imediatamente após lesão no nervo óptico. Os animais foram então analisados por ressonância magnética 1 dia e 2, 14 e 18 semanas após o transplante. Demonstramos, então, que por ressonância magnética foi possível observar um sinal hipointenso derivado das células marcadas em todos os períodos analisados e não houve redução evidente de sinal ao longo do experimento. Além disso, foi possível identificar células marcadas com SPIO ex vivo 2 e 18 semanas após a injeção através da reação de azul da Prússia. Dezoito semanas após a injeção, observamos uma concentração de células marcadas no vítreo próximas à região do disco óptico. Isso sugere a permanência a longo prazo das células injetadas nos olhos dos animais hospedeiros, demonstrando a eficiência do transplante e levantando novas questões sobre o possível efeito terapêutico dessas células." CNPq, FAPERJ, CAPES, INBEB

G2. PREHISTORICAL PEDICULUS HUMANUS CAPITIS INFESTATION: QUANTITATIVE DATA AND LOW VACUUM SCANNING MICROSCOPY 1 - ALVES, A. D.; 1 - DUTRA, J. M. F.; 1 - PESSANHA, T.; 2 - RACHID, R.; 2 - SOUZA, W.; 3 -

LINARDI, P. M.; 1 - FERREIRA, L. F.; 1 - SOUZA, S. M.; 1 - ARAUJO, A. 1 - Laboratório de Paleoparasitologia - Escola Nacional de Saúde Pública Sérgio Arouca,

Fundação Oswaldo Cruz, Rio de Janeiro; 2 - Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de

Janeiro; 3 - Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais.

Há décadas um escalpo peruano, datado do período pré)colombiano, foi examinado por um pesquisador da Fundação Oswaldo Cruz. O Professor Olympio da Fonseca Filho descreveu lêndeas e adultos fixos a fios de cabelos e fez comentários sobre a origem da infecção por piolhos na espécie humana. Este mesmo escalpo foi enviado ao nosso laboratório e é objeto deste artigo. Sua análise mostrou maciça infestação, com 9 lêndeas/ cm2 em impressionante número de adultos muito bem preservados. O tempo de infestação foi estimado em cerca de 9 meses antes da morte, baseado na maior distância entre lêndeas e o couro cabeludo, levando em consideração uma taxa média de crescimento capilar de 1cm por mês. Um pequeno pedaço de tecido tradicional peruano foi encontrado associado ao escalpo, provavelmente pertencente ao conjunto de peças usado no ritual funerário. Aqui, apresentamos alguns aspectos morfológicos de adultos e lêndeas visualizados por microscopia eletrônica de varredura de baixo vácuo. FAPERJ

G3. EVALUATION OF A NOVEL HISTONE DEACETYLASES INHIBITOR AGAINST LEISHMANIA AMAZONENSIS

1,2,3 – VERÇOZA, B. R. F.; 4 - HUBER, K.; 4 - BRACHER, F., 1,2,3,5 - DE SOUZA, W.; 1,2,3,5 - RODRIGUES, J. C. F.

1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2- Polo Avançado de Xerém, Universidade Federal do Rio de Janeiro; 3- Instituto Nacional

de Ciência e Tecnologia de Biologia Estrutural e Bioimagem; 4- Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universitt Mnchen, Germany;

5- Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro. Leishmaniasis is one of the most important neglected tropical diseases caused by protozoan parasites of the Leishmania genus. The treatment is based on the use of antimonials, miltefosine, amphotericin B and pentamidine. However, all these drugs cause several side effects for the patients. In this context, there is an urgent necessity to develop new compounds safer, more efficacious and accessible. Histone desacetilases inhibitors are new compounds that inducing alterations on the gene expression that promoting apoptosis in the treated-cells. Thus, the aim of this work is study the effects of a novel histone desacetilases inhibitors, against Leishmania amazonensis promastigotes and intracellular amastigotes. The effects induced by TFMDI were evaluated using different techniques such as: growth curve, immunofluorescence microscopy, scanning and transmission electron microscopy, western blotting, and fluorimetry. For promastigotes, the IC50 value was 2μM. Against intracellular amastigotes, the IC50 value was around 3μM. Immunofluorescence, DIC and scanning electron microscopy revealed an alteration on the promastigote' shape, that presented elongated and thinner, and increased expression of acetylated tubulin. Furthermore,


transmission electron microscopy revealed several ultrastuctural alterations. These results together indicated that this compound is a promising molecule against leishmaniasis, however new studies are necessary to understand better this mechanism of action. CNPq, CAPES, FAPERJ

G4. CAMPTOTECINA, UM INIBIDOR DA TOPOISOMERASE I, QUE PODE SER USADO COMO FERRAMENTA PARA O ESTUDO DA BIOLOGIA CELULAR DE TRIPANOSOMATÍDEOS 1,2 - ZUMA, A.A.; 1,2- DA SILVA, C.C.; 3- ELIAS, M.C.; 1,2,4- DE SOUZA, W.; 1,2- MOTTA,

M.C.M. 1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2 - Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Imagem; 3 - Instituto

Butantan; 4 - Instituto Nacional de Metrologia, Qualidade e Tecnologia - Inmetro Os protozoários Trypanosoma cruzi e Strigomonas culicis pertencem à família Trypanosomatidae, ordem Kinetoplastida. O primeiro é um parasita heteroxênico e o agente etiológico da Doença de Chagas, enquanto que o segundo é um monoxênico não-patogênico que abriga uma bactéria simbiótica em seu citoplasma. A organização do DNA do cinetoplasto, assim como a do DNA nuclear, conta com a participação das topoisomerases, enzimas que atuam nos processos de replicação, transcrição e reparo. Inibidores de topoisomerases vêm sendo amplamente usados em estudos quimioterápicos, porém também podem ser empregados para estudar a biologia celular destes protozoários. Neste trabalho nós avaliamos os efeitos da camptotecina, um inibidor da topoisomerase do tipo I, na proliferação, no ciclo celular e na ultraestrutura de T. cruzi e S. culicis. Para isto, as células foram cultivadas na presença de diferentes concentrações do inibidor e a cada 24h, uma alíquota foi retirada para contagem em câmara de Neubauer e para análises por microscopia óptica de fluorescência e por microscopia eletrônica de transmissão. A proliferação de ambas as espécies diminuiu na presença da camptotecina, sendo S. culicis mais sensível ao inibidor. A camptotecina também afetou o ciclo celular de ambos tripanosomatídeos, promovendo em T. cruzi a parada na fase S/G2 e induzindo em S. culicis a formação de simbiontes filamentosos. Do ponto de vista ultraestrutural, observamos que células tratadas apresentam a heterocromatina nuclear fortemente descompactada e inchaço mitocondrial. Além disso, houve um aumento do número de corpos lipídicos, especialmente no tripanosomatídeo com simbionte, como verificado em análises por microscopia de fluorescência e fluorimetria. Juntos, nossos resultados mostram que estas duas espécies podem ser usadas como modelos comparativos para a melhor compreensão da biologia celular da família Trypanosomatidae. CNPq e FAPERJ

G5. SERUM TRANSTHYRETIN LEVELS IN BRAZILIAN PATIENTS WITH FAMILIAL AMYLOID POLYNEUROPATHY.

1-LIMA, C.; 1-FERREIRA, P. S.;1- ANDRADE, C.; 2-CRUZ, MÁRCIA WADDINGTON;1-FOGUEL, D.

1- Instituto de Bioquímica Médica-UFRJ Brazil. 2- Hospital Universitário Clementino Fraga Filho, Rio de Janeiro, Brazil

Familial amyloid polyneuropathy (FAP) is an autosomal dominant polyneuropathy of adult onset which used to lead to death within 10 years on average after the first symptoms. Serum transthyretin (TTR) levels are reduced in familial amyloidotic

polyneuropathy (FAP) in others populations. A single study of patients with senile systemic amyloidosis (SSA) in Sweden found that those individuals also had a significantly lower mean serum TTR concentration. It is noteworthy that there are no reports of levels of TTR in the serum of patients with PAF in Brazil. The objective of this work is to assess the TTR serum levels of patients with FAP and compare these levels to those of healthy patients and to correlate these levels with the clinical development of the disease. Initial patient data collection includes information on family and medical history, physical and laboratory exam results, and patient reported outcomes. Results are shown for Brazilian patients enrolled in THAOS. We compared the serum TTR levels, as determined by ELISA method. Some of the patients tested had low levels of TTR in serum compared to controls. Our data shows that patients that presents a late onset of disease has lower levels of serum TTR in comparison with the patients with early onset of symptoms. In addition we also verified that the levels of serum TTR decreases with clinical disease progression. These data lead us to believe that serum levels of TTR can be used as markers for the clinical development of the disease. INBEB, FAPERJ, CNPq, CAPES

G6. A NEW MECHANISM OF ACTION FOR PRIMA-1 ON P53 REACTIVATION 1,2- LAURITZEN, C.; 1,2- RANGEL, L.P.; 1,2- SILVA, J.L.

1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade

Federal do Rio de Janeiro. Introduction: p53, among other functions, acts as a transcription factor and is involved in cell cycle control, leading to apoptosis or to the arrest of the cell cycle for DNA damage repair. It is mutated in around 50% of all tumors. In general, mutations occur on the DNA-binding domain, leading to its loss of function as a transcription factor. The mutation in a single allele leads to the complete loss of function due to a phenomenon called negative dominance. Prima-1 is a classical drug described to reestablish the structure and function of p53, leading to apoptosis. Our group has previously described that p53 is able to form amyloid aggregates, in which the mutant form of the protein is responsible for the conversion of the wild type form into oligomers and amorphous aggregates [1, 2]. Material & Methods: Recombinant p53 central core domain (p53C) was submitted to aggregation at 37oC for 1 h in the presence of increasing concentrations of Prima-1 and 2-methylene-3-quinuclidinone hydrate (MQ) and the fluorescence of ThT was measured at 440 nm (excitation) and 480 nm (emission). For immunostaining, cells were incubated with anti-p53 antibody DO-1 and anti-oligomer antibody A11 for posterior confocal microscopy visualization. Results and discussion: In the present work, we used Prima-1 and its active metabolite, MQ, and both of them demonstrated the ability to inhibit mutant p53C aggregation. The WT form has been protected in a lower degree. Also, we have observed the ability of Prima-1 to revert p53 aggregation in MDA-MB-231 cells, which express mutant p53. Conclusion: These results might lead to a different understanding on the controversial mechanism of action of Prima-1. We suggest that this compound is able to recover p53 from aggregates accumulated into cancer cells. CNPq, FAPERJ , MS-DECIT, INBEB.

G7. THE ENEMY WITHIN: IMPAIRMENT OF HEME CRYSTALLIZATION BY A QUINOLINE DRUG DRIVES BIOCHEMICAL, CELLULAR AND PHYSIOLOGICAL SHIFTS ON AN INSECT VECTOR


Ferreira, C.M.¹, Stiebler, R¹, Gandara, A.C.P.¹, Nuno,A.B.W.¹, Paiva-Silva, G.O.¹, Mena-Barreto, R.B.², Oliveira, M.F.1

1 - Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro 2 - Fundação Oswaldo Cruz, FIOCRUZ

Introduction: Hematophagous organisms digest hemoglobin and release large amounts of heme in their digestive tracts. When free, heme is toxic and promotes redox imbalance and membrane destabilization. Thus, blood-feeding organisms require protective mechanisms to deal with excessive heme. Hemozoin (Hz) is a heme crystal produced by Plasmodium sp, Schistosoma sp and triatomine insects as the main heme detoxification mechanism. Previous evidence demonstrated that quinoline drugs are antimalarial and antischistosomal agents by inhibiting Hz formation. Here, we investigated the effect of quinidine, an antimalarial quinoline, in R. prolixus. Material and methods: Hz was extracted from posterior midgut of R. prolixus by differential solubilization. Urate and hemoglobin levels were determined by colorimetric assays. Lipid peroxidation was determined by the TBARS assay in hemolymph. Total heme levels were measured by the alkaline-pyridine method. Reactive species in posterior midgut were analyzed by fluorescence microscopy using the dihydroethidium (DHE) probe. The hemolymph levels of Rhodnius heme binding protein (RHBP) were determined spectrophotometricaly and transcript levels assessed by real time PCR in fat bodies. Posterior midguts were fixed with glutaraldeyde and submitted to transmission electron microscopy routine. Results and discussion: Quinidine did not affect blood ingestion or digestion, but severely affected Hz formation. Heme levels in the hemolymph were increased by quinidine, which promoted lipid peroxidation, reactive species generation and reductions in urate levels. As a compensatory mechanism, protein and transcript levels of RHBP increased by quinidine. Ultrastructural analyses of posterior midgut indicate general loss of cellular organization with reduced densities of mitochondria, together with the presence of myelin figures as well as numerous electron dense structures (haemoxysomes), indicating autophagic pathways. Although there were no changes on insect viability after quidinine treatment, the most striking physiological observation was a decrease in eggs laying. Conclusion: Impairment of heme crystallization within the Rhodnius midgut results in severe physiological changes involving redox imbalance and autophagy. INBEB, FAPERJ, CNPq, CAPES G8. ANTIPROLIFERATIVE AND ULTRASTRUCTURAL EFFECTS OF BFDI, A HISTONE DESACETILASES INHIBITOR, IN LEISHMANIA AMAZONENSIS

ARAÚJO, C. N.1,2,3, VERÇOSA, B. R. F1,2,3., BRACHER, F4.,DE SOUZA, W1,3,5., RODRIGUES, J. C. F. R.1,2,3,5

1-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brasil. 2- Polo Avançado de Xerém, Universidade Federal do Rio de Janeiro 3- Instituto

Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Brasil 4- Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universitt

Mnchen, Germany 5- Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro, Brasil.

Leishmaniasis is one of the most important neglected tropical disease distributed around the world, and is caused by protozoan parasite of the Leishmania genus. In Brazil, endemic areas are mainly concentrated in the North and Northeast regions, however there is a significant increase in other regions more developed regions. In Brazil, Leishmania amazonensis is one important species responsible for cutaneous and diffuse cutaneous leishmaniasis, which is so difficulty to treat. The treatment is based on the use of pentavalent antimonials, miltefosine, amphotericin B and pentamidine. Therefore, there is a urgent necessity to develop new drugs more efficient and less

toxic. Recently, histone deacetylases inhibitors have been studied as potential target for the treatment of cancer. These enzymes act directly on the packaging of the DNA, thus regulating the expression of various genes related to apoptotic cell death. Thus, the aim of our work is study the effects of a novel histone deacetylases inhibitor on Leishmania amazonensis. The present study focuses on the effect of BTFDI in promastigote forms. The IC50 value found was 2 μM, with total inhibition of the growth after treatment with 4μM. DIC and Immunofluorescence microscopy with anti-α-tubulin antibody showed that the promastigotes became swollen mainly in the nuclear region of the cell body after treatment with concentrations up 1 μM. Scanning electron microscopy confirmed this alteration on the cell body. Furthermore, transmission electron microscopy was carried out to identify the organelles and structures affected by the treatment. Several alterations were observed, such as: mitochondrial swelling, presence of many lipid bodies, and changes in the chromatin condensation. These initial results obtained suggest that BTFDI is a potential new compound against Leishmania, which needs to be tested in intracellular amastigotes and in murine model of leishmaniasis, thus demonstrating its real efficacy. New experiments also have to be carry out to understand better this mechanism of action. CNPq, CAPES and FAPERJ. G9. ANÁLISE DAS INTERAÇÕES ENTRE A PROTEÍNA PRION E DIFERENTES LIGANTES ATRAVÉS DE DOCKING MOLECULAR

1-MACHADO, C. S. C.;1- ARAUJO, N. C. F.;1- ALVES, W.;2- MASCARELLO, A. 2- CHIARADIA, L. D.;2- NUNES; 2- R. J. YUNES, R.3- CAUGHEY, B.; 1-CORDEIRO, Y.

1- Faculdade de Farmácia, Universidade Federal do Rio de Janeiro; 2- Departamento de Química, Universidade Federal de Santa Catarina; 3- National Institutes of Health,

Hamilton, Montana As Encefalopatias Espongiformes Transmissíveis (EETs), compreendem um grupo de doenças neurodegenerativas que acometem seres humanos e animais. A proteína príon, agente causador das EETs, na sua forma celular (PrPC) está majoritariamente presente na membrana de células nervosas, organizada em três alfa-hélices, 1 fita beta antiparalela e uma região desestruturada e, até o presente momento, sua função biológica não é elucidada. As EETs desenvolvem-se após a conversão da PrPC em uma isoforma patológica, scrapie (PrPSc) que pode formar agregados que se depositam no sistema nervoso central. Quando comparadas por espectroscopia óptica as estruturas secundárias da PrPC e da PrPSc se mostram bastante distintas, a primeira é rica em alfas-hélices, enquanto a segunda apresenta um maior conteúdo de folhas-beta. Em nosso laboratório foram identificados 46 compostos eficazes em diminuir os níveis de PrPSc em células de neuroblatoma ScN2a. Neste trabalho nos propomos a analisar os tipos de interações entre a PrP e esses compostos. Utilizamos a técnica de “docking” molecular, que permite pesquisar potenciais de interação entre proteína e ligante, buscando os complexos moleculares menos energéticos, levando em consideração os aspectos geométricos e eletrostáticos das moléculas envolvidas. Através do SwissDock, um servidor online, realizamos o “docking” da PrPC com os compostos, o qual forneceu informações sobre as forças, sítios e estequeometria dessas interações. Observamos diferentes padrões de afinidade para os complexos, mas sempre com energias favoráveis. Para todos os “dockings”, verificou-se que uma cavidade presente entre a hélice 2 e as fitas betas da PrPC, sítio preferencial da interação, com maior número de clusters conformacionais para todos os ligantes. Uma vez que esses compostos são possíveis candidatos a fármacos, objetivamos também realizar testes in silico com o intuito de predizer diferentes propriedades desses compostos, como sua capacidade de atravessar a barreira hematoencefálica e seus possíveis efeitos tóxicos. CAPES, FAPERJ, INBEB, CNPQ


G10. INVESTIGATION OF YELLOW FEVER VIRUS-INDUCED ENDOPLASMIC RETICULUM STRESS

SANCHES, D.1; ROCHA, C.M. 1; CAMPOS, S.P.C.1 ; GASPAR, L.P.2; FREIRE, M.S.2; GONÇALVES, B.S. 3; CHIARINI, L.B.3; SILVA, J.L.1; GOMES, A.M.O.1 & OLIVEIRA, A.C.1 1Instituto de Bioquímica Médica/UFRJ 2ITI/FIOCRUZ/RJ 3Instituto de Biofísica Carlos

Chagas Filho/UFRJ INTRODUCTION:The yellow fever is a hemorrhagic disease of great relevance in Africa and South America, where it occurs as small outbreaks. It is caused by the yellow fever virus (YFV), a flavivirus such as the Dengue Virus, both being transmitted by mosquitos. During its life cycle, the YFV uses the endoplasmic reticulum for translation of viral proteins and assembly of new viral particles. The accumulation of misfolded proteins in this organelle triggers the endoplasmic reticulum stress (ERS), which leads to the dissociation of the binding immunoglobulin protein (BiP) from ATF6, PERK e IRE1. Once released, these factors become active and start to mediate the ERS. ATF6 is transported to Golgi, where is cleaved. PERK and IRE1 homodimerize, are phosphorylated and become active. PERK phosphorylates and inactivates eIF2a. IRE1 is a RNAse that alternatively splices XBP1 mRNA, leading to the production of a response for ERS. One of these responses is the overexpression of the nuclear transcription factor CHOP, which regulates the expression of pro and anti-apoptotic genes. MATERIAL AND METHODS: In this study, we investigate the ERS induced by the infection of VERO cells by YFV through western-blotting and fluorescence microscopy. We infected VERO cells with YFV, using a multiplicity of infection of 1. RESULTS AND DISCUSSION: We analyzed cell viability by the LIVE/DEAD assay and we observed that by 72 hours post infection, cells undergo cell death. The ERS induction was noticed by CHOP overexpression. Moreover, we observed phosphorylation of eIF2a, ATF6 cleavage and spliced XBP1 18 hours post infection. BiP expression levels remained unaltered. Apoptosis was analyzed by the TUNEL assay and it was observed 72 hours post infection. CONCLUSION: Our results suggest that the YFV induces ERS in VERO cells through PERK, ATF6 cleavage, XBP1 splicing and CHOP overexpression. Capes, CNPq, FAPERJ, Pronex, INBEB G11. SYNTHESIS AND EVALUATION OF AROMATIC HYDRAZONES, GUANYL HYDRAZONE AND OXIMES WITH ELECTRON-WITHDRAWING SUBSTITUENTS AS POTENTIAL AGENTS AGAINST CHEMICAL WARFARE

1- PETRONILHO, E. C.; 1- KITAGAWA, D. A. S.; 1- FIGUEROA-VILLAR, J. D. 1- Group of Medicinal Chemistry, Department of Chemistry, Military Institute of

Engineering, Square Gen. Tiburcio, 80, Red Beach, 22290-070, Rio de Janeiro, RJ, Brazil. The neurotoxic chemical warfare agents are a major threat to humanity, those deserve special attention by the high lethality and danger. Most neurotoxic agents are organophosphorus compounds (OP) that act primarily by inhibiting acetylcholinesterase enzyme which is essential to control the transmission of nervous impulses. When inhibited by OPs, AChE may be reactivated by cationic oximes such as pralidoxime (2-PAM) and other analogs, however, none are effective against all known neurotoxic agents. To deal with OPS intoxication efficiently it is necessary to find out the OP used, a long process that generally leads to death of the victim. This highlights the need for development of potential new drugs that are potent and effective against all OPs and low toxicity. In previous research it was found that the more acidic oximes have greater efficiency in the reactivation of AChE. With this objective in this study were synthesized and characterized new aromatic hydrazones, guanyl hydrazones and oximes that have electron-withdrawing groups to assist the reactivation process. These compounds were

tested as reactivators of AChE Electrophorus electricus inhibited by paraoxon-ethyl, using as positive reference pralidoxime (2-PAM), through the Ellman method. INBEB, CAPES, FAPERJ, CNPQ, MINISTÉRIO DA DEFESA G12. PRION PROTEIN AGGREGATION TRIGGERED BY NEUTROPHIL EXTRACELLULAR TRAPS (NETS)

1- DOS ANJOS, D.M.; 1- AZEVEDO, E.P.; 1- VIEIRA T.C.R.G.; 1- FOGUEL, D.; 2- SARAIVA, E.M.; 1- SILVA, J.L.

1- Inst. de Bioquímica Médica, IBqM-UFRJ, RJ, Brazil; 2- Inst. de Microbiologia, IMPPG-UFRJ, RJ, Brazil

"INTRODUCTION: Prion diseases are fatal neurodegenerative protein-misfolding diseases related to a protein known as prion protein (PrP). The constitutive cellular isoform of PrP (PrPC), present in cell surface, can be converted into its abnormal, β-sheet-rich isoform (PrPSc) that undergoes aggregation. PrPSc can be transmissible and infectious. The evidences so far suggest that adjuvant factors, such as glycosaminoglycans and nucleic acids, may play a role in the conversion process. Our group previously reported that DNA can convert PrPC into the β-sheet conformation leading to aggregation. Neutrophil extracellular traps (NETs) are large DNA webs decorated with histones and granule proteins released by neutrophils that trap and kill pathogens. Recently, NETs were found in association with amyloid fibrils in tissues of patients with other protein-misfolding diseases. Furthermore, amyloid fibrils triggered the release of NETs [1]. Following peripheral exposure, the replication of prions within lymphoid tissue has been shown to be important for the efficient spread of the disease to the brain. Moreover, NETs were reported of being released in lymphoid tissues. Regarding these observations and considering the large quantity of DNA provided by NETs we asked if NETs could induce PrPC aggregation in vitro. MATERIALS AND METHODS: Murine recombinant PrP (23-231) was added into the supernatant from activated human neutrophils containing NETs. Aggregation was measured by turbidimetry (330 nm) and electron microscopy. RESULTS AND DISCUSSION: Aggregation was detected and reached a maximum after the first 10 minutes of incubation and then gradually declined remaining detectable after 1 hour. The aggregates showed amorphous morphology. This results report that NETs can trigger a stable aggregation of PrPC. However, fibrillar structures were not detected. CONCLUSION: Our data suggest that NETs are an intriguing factor that must be appraised in the studies concerning prion propagation mechanisms. [1] Azevedo EP, Guimarães-Costa AB, Torezani GS, Braga CA, Palhano FL, Kelly JW, Saraiva EM, Foguel D. (2012) J. Biol. Chem. 287, 37206-37218." FAPERJ, CNPq, PRONEX, CAPES, IMBEBB G13. SELEÇÃO DE INIBIDORES DA ACETILCOLINESTERASE EM EXTRATOS BRUTOS DA FLORA FLUMINENSE

1- MARINS, E. B. ; 1- TINOCO, L. W. ; 2- HAMERSKI, L.; 2- PINTO, A.C. 1- Laboratório de Análise e Desenvolvimento de Inibidores Enzimáticos, NPPN, CCS,

UFRJ; 2 - NPPN, UFRJ "O sistema mais comprometido no portador do Mal de Alzheimer é o colinérgico1. Uma das soluções é prolongar o tempo de permanência do neurotransmissor acetilcolina na fenda sináptica, através da inibição da enzima acetilcolinesterase(AChE). Este projeto tem como objetivo identificar novas moléculas que inibam a acetilcolinesterase a partir da prospecção direta de extratos de plantas da Mata Atlântica do Rio de Janeiro. A atividade da acetilcolinesterase (Eletric eel -TipoVI-S-SIGMA) foi medida pelo método de Ellman.2 A enzima hidrolisa o substrato iodeto de acetiltiocolina(ATCI), produzindo tiocolina e acetato. A tiocolina e o reagente de Ellman(DTNB) reagem, produzindo dois


ácidos detectados a 405 nm. Os extratos etanólicos(folhas, galhos, frutos e/ou flores) das espécies de plantas coletadas foram submetidos ao teste de inibição enzimática. O ensaio foi realizado com 100 µL do substrato ATCI 15mM, 500 µL de DTNB 3mM, 200 µL de tampão Tris HCl 50mM pH8 com BSA 0,1%, 100 µL de amostra em metanol 1% na concentração final de 0,1mg/uL. Antes da adição da enzima, mediu-se absorbância 5 vezes a cada 13 segundos. Em seguida adicionou-se 100 µL da enzima AChE 0,22U/mL, e a absorbância foi medida 8 vezes a cada 13 segundos. Os inibidores Eserina e Carbacol foram os controles positivos da inibição. Dos 20 extratos testados até o momento, 3 mostraram atividade inibitória. Após os testes de inibição, foram feitos ensaios de interação ligante-proteína com estes 3 extratos através da diferença da transferência de saturação (STD-SaturationTransferDifference) em um espectrômetro AgilentVNMRS500MHz a 25°C. As soluções estoque dos extratos de foram preparadas em DMSO_d6 e diluídas em D2O para uma concentração final de 0,02mg/uL da amostra e 10% DMSO_d6. Após a análise dos extratos, adicionou-se AChE para uma concentração final de 1:100(proteína:ligante). Através dos espectros STD foi possível observar os picos dos componentes do extrato que interagem com a acetilcolinesterase. 1 - MINETT, T.S.C. & BERTOLUCCI, P.H.F. Neurociências 8(1): 11-14, 2000. 2 - RHEE, MEENT, INGKANINAN, VERPOORTE, Journal of Chromatography A, 915 (2001) 217–223" INCT-INBEB, FAPERJ, CNPq G14. EMBRYONIC STEM CELLS LABELED WITH SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES ARE DETECTED BY MAGNETIC RESONANCE IMAGING IN THE MURINE HEART 1- ATAIDE-MENDONCA, E.; 1- BRASIL, G. V.; 1- SILVA DOS SANTOS, D.; 2- VASCONCELOS-

DOS-SANTOS, A; 3- AZEVEDO, C. F.; 3,4- TOVAR-MOLL, F.; 2- MENDEZ-OTERO, R.; 1- GOLDENBERG, RC.; 1,5- DE CARVALHO, ANTONIO C.

1- Laboratory of Cellular and Molecular Cardiology - Carlos Chagas Filho Institute of Biophysics (IBCCF) - Federal University of Rio de Janeiro (UFRJ); 2- Laboratory of Cellular

and Molecular Neurobiology - Carlos Chagas Filho Institute of Biophysics (IBCCF) - Federal University of Rio de Janeiro (UFRJ); 3- D’Or Institute for Research and Education

(IDOR); 4- National Institute of Science and Technology for Structural Biology and Bioimaging (INBEB - CENABIO); 5- National Institute of Cardiology (INC)

"Background: Embryonic stem cells (ESC) differentiate into several cell types and have been considered a promising source for cellular therapy. However, the lack of non-invasive tracking of cells has represented a serious obstacle to understand the mechanisms responsible for functional improvement of host tissue. Cellular magnetic resonance imaging (MRI) is a rapidly growing field that aims to visualize and track cells in living organisms. Objectives: To label beating cells derived from an ESC with superparamagnetic iron oxide nanoparticles (SPIONs) and track these cells in the murine heart by MRI after intramyocardial injection. Methods: Mouse ESC line E14TG2A was cultured and differentiated in embryoid bodies (EB) by the hanging drop method. On the fifth day, the EB were plated on adherent dishes and incubated with SPIONs (FeraTrack™). After 2-days, labeled beating cells were trypsinized and 3x105cells were injected into mice hearts using echocardiography to guide the closed chest intramyocardial injection. Then, heart images were obtained by MRI. Results: ESC expressed undifferentiated markers (Oct-3/4 and SSEA-1) in immunofluorescence assays. Beating cells were observed after 7-days of EB cultivation. Labeling efficiency was evaluated by dextran immunofluorescence and prussian blue reaction in vitro. The labeled cells were detected by MRI. Conclusion: The ESC derived beating cells were efficiently labeled with FeraTrack™ and detected in the murine heart by MRI showing

that this approach is suitable for in vivo cell tracking of transplanted cells in the host organism. Approved by the Committee for the Use of Experimental Animals (UFRJ)" CNPq, Faperj, CAPES and Ministério da Saúde G15. ESTUDOS ESTRUTURAIS DO INIBIDOR DA COAGULAÇÃO SANGUÍNEA IXOLARIS

1-SILVA, F. H. S.; 2-FRANCISCHETTI, I. M. B.; 1- MONTEIRO, R. Q.; 1- VALENTE, A. P.; 1- DE PAULA, V.S.

1- Intituto de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil; 2- Section of Vector Biology, Laboratory of Malaria

and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, USA

"A cascata de coagulação sanguínea é um mecanismo fundamental para a manutenção da homeostasia do corpo humano. Diversos estudos demonstram que pacientes com câncer apresentam alterações no sistema de coagulação sanguínea que levam a um quadro de hipercoagulabilidade, conduzindo ao acontecimento de eventos trombóticos que são os principais responsáveis pelo óbito de pacientes com câncer. Em virtude disso, tem-se feito o uso de anti-coagulantes a fim de diminuir o avanço do tumor e melhorar o prognóstico do paciente. O Ixolaris é uma proteína de 140 aminoácidos presente na saliva do carrapato Ixodes scapularis. O Ixolaris se liga ao fator Xa e posteriormente forma um complexo quaternário com o fator tecidual e fator VIIa, resultando na inativação do complexo tenase extrínseco. Alguns estudos demonstram que o Ixolaris possui propriedades anti-coagulantes e anti-tumorais, caracterizando-o como um possível alvo terapêutico no tratamento do câncer. O modelo proposto para o Ixolaris mostra uma proteína com dois domínios bastante homólogos chamados domínio kunitz 1 e kunitz 2 (K1 e K2). O objetivo deste trabalho é mapear os sítios de interação no Ixolaris com o complexo binário FVIIa-Fator Tecidual através de estudos estruturais por RMN e calcular sua estrutura tridimensional. Até o momento, foi possível definir um protocolo eficiente para expressão e purificação das proteínas recombinantes na forma solúvel. Estes dados foram confirmados através de análise por espectrometria de massas e western blotting. Os espectros de 1H de RMN mostram linhas finas e boa dispersão de deslocamento químico, indicando que as proteínas estão enoveladas. Ensaios de coagulação sanguínea estão sendo realizados para verificar a atividade das proteínas purificadas, como também a obtenção das proteínas isotopicamente marcadas com para iniciarmos o assinalamento de 1H, 13C e 15N, utilizando experimentos de tripla ressonância para o cálculo da estrutura." CNPq G16. MESENCHYMAL STEM CELLS PROMOTE NEUROPROTECTION AND AXONAL REGENERATION IN A MODEL OF OPTIC NERVE CRUSH

1,2- MESENTIER-LOURO, L.; 1,2- ZAVERUCHA-DO-VALLE, C.; 1,2- FIGUEIRÊDO, A.B.P.; 1,2- NASCIMENTO-DOS-SANTOS, G.; 1,2- SILVA JÚNIOR, A.J.; 1,2- TORRES, A.L.; 1,2-

PAREDES, B.D.; 1,2- MENDEZ-OTERO, R.; 1,2- SANTIAGO, M.F. 1- Instituto de Biofísica Carlos Chagas Filho; 2- Instituto Nacional de Ciência e Tecnologia

de Biologia Estrutural e Bioimagem – INBEB Bone marrow derived cells have been used in different animal models of neurologic diseases. In this study we have investigated the therapeutic potential of mesenchymal stem cells (MSC) injected into the vitreous body in a model of optic nerve injury. Adult (3-5 months old) Lister rats underwent unilateral optic nerve crush followed by MSC or vehicle injection into the vitreous body. Sixteen and 28 days after injury, RGC survival was evaluated assessing the number of Tuj1 or Brn3a-positive cells in flat-mounted retinas, and optic nerve regeneration was investigated after anterograde labeling of the


optic axons with cholera toxin B conjugated to Alexa 488. Cell therapy with MSC increase the number of Tuj1 and Brn3a positive cells in the retina and the number of axons distal to the crush site both at 16 and 28 days after optic nerve crush. In summary, MSC protects RGC and stimulates axon regeneration after optic nerve crush. The long permanence of the transplanted cells in the eye may account for the sustained effect observed. Further studies are necessary to elucidate mechanisms of action of MSC in the visual system. INBEB, FAPERJ, CNPq, CAPES. G17. STRUCTURAL AND DYNAMIC PROPERTIES OF THE GLYCOPROTEIN E – DOMAIN III OF DENGUE VIRUS FREE AND IN COMPLEX WITH SPECIFIC SINGLE CHAIN FRAGMENTS (SCFV) BY NMR SPECTROSCOPY

1- SILVA, G. V.; 1- MORAES, A.H.; 1- ALMEIDA, F.C.L.; 2- VARANI, L. ; 1- VALENTE, A.P. 1- Centro Nacional de Ressonância Magnética, IBQM-UFRJ,RJ, Brazil; 2- Medical Institute

for Research in Biomedicine, Bellinzona, Switzerland The dengue virus belongs to the family of flavivirus and is responsible for more than 100 million cases annually. The viral envelope glycoprotein E is the major constituent of dengue virus surface and is the main target for antibody response against DENV. The crystal structures of the glycoprotein E demonstrate this protein as dimer, where each monomer is composed of three domains: DI, DII and DIII. The development of a dengue vaccine has been hampered by the fact that there are four distinct serotypes of dengue (DENV 1 - 4), and due to a poorly understood process: antibody depended enhancement (ADE). In an ADE, antibodies generated against a dengue serotype, facilitate the infection by a different one later, even leading to dengue hemorrhagic fever, a lethal form of the disease. In this work, we characterized the dynamics and the structural properties of the domain III of serotypes 1 and 4 of protein E using NMR spectroscopy. We are mapping the residues that participate of the interaction with scFv using chemical shift perturbation. The molecular dynamics on ps-ns time scale, which is related to thermal flexibility, is being accessed using 15N relaxation experiments. Also, movements on us-ms time scale, which are associated to conformation exchange events, are being studied using relaxation dispersion experiments based on CPMG pulse sequences. This work will present the characterization of glycoprotein E-domain III dynamics free and with scFv, along with a structural description of DIII-scFv interaction. This information set will be crucial for a better understanding of domain III-antibody interaction and might help the development of a dengue virus vaccine. FAPERJ, CNPq, CAPES and INBEB G18. D-SERINA REVERTE DÉFICITS COGNITIVOS E NA INIBIÇÃO POR PRÉ-PULSO CAUSADOS PELO ESTRESSE AGUDO: RELEVÂNCIA PARA A ESQUIZOFRENIA 1- GUERCIO, G.D.; 1- BEVICTORI, L.; 1- MADEIRA, C.;1- VARGAS-LOPES, C.;1- MELLO, I.;1-

PANIZZUTTI, R. 1- Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro

"A esquizofrenia é um grave transtorno que acomete cerca de 0,7 % da população. Apesar de vários genes terem sidos relacionados com o transtorno, estudos com gêmeos homozigotos mostram que fatores ambientais podem estar envolvidos com sua etiologia. Dentre eles, destaca-se o estresse. Nosso objetivo é investigar se o estresse em camundongos provoca alterações bioquímicas e comportamentais observadas na esquizofrenia e desenvolver estratégias para revertê-las. Após 90 minutos de estresse, sacrificamos camundongos adultos e dissecamos o cérebro. Animais controles foram sacrificados após serem retirados de suas caixas. Os animais estressados possuíam menos D-serina no córtex pré-frontal (média ± erro padrão, 5,78 µM ± 0,18 controle, 5,04 µM ± 0,13 estresse, t test p < 0,005). Testamos os animais na tarefa de

reconhecimento de objetos, na qual eles são expostos a dois objetos iguais (treino) e após 24h são reexpostos a um objeto familiar e outro novo (teste). Animais controle exploram mais o objeto novo independentemente se recebem D-serina (1g/kg, I.P.) ou salina (razão de exploração do objeto novo/ exploração total, 0,64 ± 0,03 e 0,66 ± 0,03, respectivamente), mas animais estressados que recebem D-serina após o treino exploram mais o objeto novo quando comparados a animais que receberam salina (0,65 ± 0,3 e 0,54 ± 0,3, p < 0,05 ANOVA de duas vias seguida de Bonferroni post test). No teste de inibição por pré-pulso, animais estressados possuem inibição menor do que animais controle (-0,68% ± 8,5 e 31,77% ± 5,54, p < 0,005), o que é prevenido pela D-serina (-0,68% ± 8,5 e 34,36% ± 4,19, p < 0,005). Em suma, o estresse é capaz de diminuir o nível de D-serina no córtex pré-frontal, e a administração de D-serina recupera déficits comportamentais causados pelo estresse. Este trabalho pode ajudar a entender como o estresse pode ser relevante para a esquizofrenia." FAPERJ, CNPq G19. ESTUDOS DE INTERAÇÃO ENTRE OS RECEPTORES DE QUIMIOCINA CXCR4 e CCR6 E β-DEFENSINA 6

1 - MOREIRA, G.S., 2 - ALMEIDA, F.L.C., 3 - VALENTE, A.P., 4- DE PAULA, V.S.1 Centro Nacional De Ressonância Magnética Nuclear, Instituto de BioquímicaMédica,

Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil Os receptores de quimiocina pertencem a família de receptores acoplados a proteína G e possuem importante papel na resposta inflamatória e estão associados em câncer metastático e infecção por HIV-1.O domínioN-terminal destesreceptores, localizados na região extracelular, tem sido empregado em estudos de interação quimiocina-receptor, entretanto atualmente nenhum dado de interação foi mostrado com -defensinas.Estes receptores normalmente apresentam uma sulfatação nos resíduos de tirosina no N-terminal, aumentando ainteração com quimiocinas.As -defensinas humanas são proteínas antimicrobianas importantes no sistema imunológico, produzidas principalmente por leucócitos e células epiteliais e apresentam atividade quimiotática para diferentes tipos de células nos sítios de inflamação, através dos receptores CCR2 e CCR6.Aβ-defensina 6 (hBD6) possui uma estrutura tridimensional constituída de uma alfa-hélice e três fitas beta, estabilizadas por pontes dissulfeto entre as cisteínas conservadas, e uma região C-terminal flexível. O objetivo deste trabalho é mapear os sítios de interação entre os peptídeos N-terminal do CCR6 e CXCR4 e a hBD6 através da Ressonância Magnética Nuclear (RMN). Nós estamos produzindo peptídeos correspondentes ao N-terminal do CXCR4 (resíduos 1-30) e CCR6 (resíduos 1-28) através de síntese em fase-sólida. O resíduo C28 do CXCR4 foi substituído por uma alanina para prevenir a formação de dímero e o resíduo tirosina sulfatadafoi introduzido na posição 21.A defensina hBD6foi expressa marcada isotopicamente com 15N e purificada por cromatografia de afinidade a níquel e fase reversa.Atualmente, estamos otimizando o protocolo de purificação dos peptídeos para posteriormente mapearmos através de RMN as regiões de interação na 15N-hBD6como também medirmos a dinâmica do complexo. INBEB, FAPERJ, CNPq, CAPES G20. INJEÇÃO INTRAESTRIATAL DE ALFA-SINUCLEÍNA CAUSA DÉFICITS MOTORES EM CAMUNDONGOS

1- Barbosa, G.F.; 2 - Melo, I.; 2 - Foguel, D.; 2 - Braga, C.A.; 3- Romão, L. 1 - Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro; 2- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 3 - Universidade Federal

do Rio de Janeiro - Campus Macaé


"A doença de Parkinson é a segunda desordem neurodegenerativa mais comum no mundo, sendo superada apenas pelo Mal de Alzheimer e acometendo de 1 a 2% da população mundial com mais de 65 anos. No alicerce da patologia está a proteína alfa-sinucleína, que pode sofrer mutações pontuais do tipo missense, gerando a forma mutante A30P que está propensa a transitar de alfa-hélice para folhas-beta, agregar e formar fibras amiloides. Eventualmente, na cinética de agregação, diferentes espécies oligoméricas são extremamente deletérias acarretando em morte de neurônios dopaminérgicos. O objeto deste estudo foi determinar se a injeção intraestriatal de oligômeros de alfa-sinucleína selvagem e a forma mutante A30P em camundongos é capaz de desenvolver a morfofisiologia da doença de Parkinson em humanos - tendo como controles animais injetados com veículo e 6-OHDA. Utilizamos o teste de campo aberto para analisar a atividade locomotora e o pole teste para mensurar bradicinesia. Para estudar assimetria lateral foi feito o teste do cilindro, assim como a discriminação olfatória para análise de perda de olfato. A morte de neurônios dopaminérgicos, rearranjo sináptico e migração astrocitária serão visualizados por imunohistoquímica após diferentes intervalos de tempo da cirurgia. Nossos resultados sugerem que na primeira semana pós-cirugia, tanto os animais injetados com a alfa-sinucleína selvagem, quanto os injetados com a forma mutante A30P apresentam redução de 40% da atividade locomotora. Quanto a redução do controle motor fino, embora ela aconteça em ambas as condições experimentais, ela é mais acentuada nos animais injetados com o mutante A30P. Além disso, ao longo das 8 semanas de experimento, nenhum dos grupos apresentou assimetria lateral, porém existe uma substancial perda de olfato, levando crer que este possa ser um bom modelo para pesquisas e testes de fármacos em estágios precoces da patologia." FAPERJ, CNPq, PIBIC-UFRJ G21. IDENTIFICAÇÃO E QUANTIFICAÇÃO DE CAPSAICINA POR RMN EM EXTRATOS DE HÍBRIDOS DE Capsicum chinense E C. annum

1-MONTEIRO H. S. M.; 2-ANDRADE V. C. J.; 1-TINOCO L. W. 1-Núcleo de Pesquisa de Produtos Naturais da UFRJ; 2-Departamento de Agronomia –

UFVJM-Diamantina-MG "A capsaicina (8-metil-N-vanilil-trans-6-nonenamida) é um metabólito secundário encontrado nas pimentas, sendo facilmente reconhecida pela sua pungência e de grande importância econômica e medicinal. Sendo uma substância de alto valor econômico, há um grande interesse pelo aumento da sua produção por hectare. Para isto, vem sendo desenvolvido o cultivo de plantas híbridas que produzam capsaicina em maiores quantidades. Este trabalho teve como objetivo quantificar o teor de capsaicina produzida pelos diferentes híbridos de Capsicum chinense e C. annum fornecidos pelo grupo do Prof. Valter da UFVJM. A partir das análises dos espectros de RMN 1D e 2D da amostra padrão (15 mg de Nonivamide - SIGMA em 600uL de MeOD) foi feita a atribuição completa dos deslocamentos químicos dos hidrogênios da capsaicina, para que fosse possível identificar os sinais correspondentes à capsaicina diretamente nos extratos. Foram usados extratos dos frutos com e sem semente e das sementes dos 32 híbridos previamente secos e pulverizados. Os extratos foram preparados com 0,2 g de cada amostra em 1mL de MeOD, sonicados e centrifugados. O sobrenadante (600uL) foi usado para as análises de quantificação feita através da integração do sinal em 2,20 ppm em relação ao do padrão interno (3 L de dioxano - sinal em 3,67 ppm). Para a quantificação, os espectros de RMN de 1H (499,79 MHz –VNMRS-500 Agilent) foram adquiridos com 48 scans, d1 de 20 s e com pulsos de 90o. O processamento dos espectros foi feito com o programa MestRenova. Foi possível observar que o teor de capsaicina nos frutos inteiros, nos frutos sem semente e na semente varia de acordo com o tipo de híbrido. Em 16 híbridos o teor de capsaicina foi

maior no fruto sem semente, em 7 no fruto inteiro e em apenas dois híbridos o maior teor foi na semente." FAPERJ, CNPq, FINEP G22. ESTRUTURA E FUNÇÃO DE β-DEFENSINAS HUMANAS E INTERAÇÃO COM GLICOSAMINOGLICANO

de Oliveira, J.M., Almeida, F.L.C., Pomin, V., Valente, A.P., De Paula, V.S. ¹ Centro Nacional De Ressonância Magnética Nuclear, Instituto de Bioquímica Médica

UFRJ β-defensinas humanas são proteínas catiônicas, com estruturas em folha β estabilizada por três pontes dissulfeto intramolecular. Embora 28 genes de β-defensinas humanas tenham sido identificados, apenas seis foram caracterizadas até o momento. Estas proteínas apresentam atividade antimicrobiana frente a bactérias Gram positiva e negativa, e também apresentam efeito anti-HIV. Além disso, apresentam atividade quimiotática para diferentes tipos de células nos sítios de inflamação, através dos receptores de quimiocina CCR2 e CCR6. A estrutura tridimensional de três β-defensinas (hBD1, hBD2 e hBD3) já foram resolvidas em solução por RMN. O nosso objetivo inicial é expressar e purificar β-defensinas, e posteriormente determinar a estrutura tridimensional de uma delas por meio da técnica de Ressonância Magnética Nuclear (RMN). Neste trabalho, nós realizamos um screening de seis β-defensinas testando solubilidade e enovelamento, afim de selecionarmos as melhores candidatas para o cálculo de sua estrutura tridimensional. A partir do testes realizados, três defensinas foram selecionadas (hBD4, hBD5 e hBD18) e foram expressas marcadas isotopicamente com 15N. O espectro de 1H,15N HSQC para hBD4 mostra linhas finas e boa dispersão de deslocamento químico, indicando que a proteína está enovelada. Nós também utilizamos RMN para mapear os sítios de ligação na 15N hBD6 na presença de pentassacarídeo sintético fondaparinux (Arixtra®), um mimético da heparina altamente sulfatado. Neste estudo nós identificamos um sítio de ligação para o fondaparinux e analisamos o complexo utilizando experimentos de relaxação. Iniciaremos o assinalamento de 1H, 13C e 15N da hBD4, utilizando experimentos de tripla ressonância para o cálculo da estrutura tridimensional. A partir do cálculo da estrutura, poderemos realizar experimentos de interação com seus alvos biológicos e assim poderemos mapear na sua estrutura os sítios de interação e comparar com os dados da hBD6 a fim de compreender melhor o seu papel fisiológico e a sua relação com certos tipos de patologias. PIBIC-UFRJ G23. THE “SWEET” SIDE OF VIRUSES: HOW GLYCOSYLATION AFFECTS THE BIOLOGICAL ACTIVITY OF AN EMERGING VIRUS?

Carvalho, C.A.M.; Bortot, J.P.S.; Piazza, T.; Silva, J.L.; Gomes, A.M.O. Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro

Mayaro virus (MAYV) is an alphavirus widespread in South America in an endemic manner and represents an interesting case to consider with regards to potential for urban emergence. The viral envelope proteins mediate cell recognition and entry and present both an N-glycosylation motif along their primary structures. These motifs are conserved amongst other alphaviruses, what suggests that they play an important role for the virus particle. The aim of this work was to address the role of glycans N-linked to MAYV envelope proteins on its infectivity, via specific cleavage by N-glycosidase F. Our results showed that digestion with N-glycosidase F promotes a shift in the electrophoretic mobility of MAYV envelope proteins E1 and E2, but not in the capsid protein C. Cleavage of N-linked oligosaccharides also interfered with MAYV infectivity. Analysis of MAYV morphology by negative-staining electron microscopy revealed that


removal of N-linked glycans from MAYV envelope proteins led to an unusual viral structure. Further experiments are under course in order to evaluate possible effects of N-deglycosylation on MAYV entry into host cells. Our preliminary results points to virus glycosylation as an important issue in virus biology. CAPES, CNPq, FAPERJ, FINEP, INBEB, PRONEX G24. RABBITS AND THEIR RESISTANCE TO PRION INFECTION: A MATTER OF ALTERED PRP- NUCLEIC ACID INTERACTIONS?

Casali, S.1 , Teixeira, J.E1. , Macedo, B.1 , Silva, J.L. , Gomes2, M. P. B.1 , Cordeiro, Y.1 1Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

2Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

"Prion protein (PrP) is known for its vulnerability to suffer structural changes. The conversion of its native conformation (PrPC) into a misfolded form (prion scrapie, PrPSc) is associated to neurodegenerative diseases, such as bovine spongiform encephalopathy (BSE), Creutzfeldt-Jakob disease and Gerstmann-Sträussler- Scheinker Syndrome (GSS). PrP is highly conserved among mammals, but some aminoacid variations can lead to differences in protein structure and stability. Previous studies have indicated that rabbit prion protein (raPrP) is more stable and resistant to structural conversion than other mammalian PrPs, due to differences in its primary sequence. It has been shown that murine PrP binds to RNA and DNA molecules and that this interaction may trigger the misfolding process. However, nucleic acid (NA) interaction with rabbit PrP has not been investigated yet. In this work, we evaluated the effects of small nucleic acid molecules (DNA and RNA) in recombinant raPrP tertiary and secondary structures. These NA sequences were previously shown to induce murine recombinant PrP (mPrP) conformational changes leading to aggregation and cytotoxicity. Static light scattering (LS) data indicate that both selected DNA and RNA sequences (21bp DNA sequence and a 21mer RNA sequence) change the tertiary structure of raPrP similarly to mPrP. Moreover, intrinsic fluorescence emission of recombinant PrPs was equally suppressed by NA interaction. In general, our preliminary results indicate that, although raPrP seems to be resistant to conversion into PrPSc, its interaction with nucleic acids parallels previously reported mPrP:NA binding. It remains to be established if raPrP interaction with nucleic acids results in aggregated species also toxic to cells in culture and/or with distinct biophysical properties in comparison with mPrP:NA complexes. Keywords: prion, protein, nucleic acids, spectroscopy, cytotoxicity" CAPES, CNPq and FAPERJ G25. STRUCTURAL CHARACTERIZATION OF M-TTR AMYLOID FIBRILS BY SOLID-STATE NMR APPROACHES

ANDRADE, F. G.1, SANT’ANNA, R.O.1, SANTANA, J.S. 1, CORRÊA, D.H A., GAVA, L.M., DIEHL, A.2, BRAGA, C.A.1, RAMOS, C.H., OSCHKINAT, H.2, FOGUEL, D.1 AND FREITAS

M.S.1 1.University Federal of Rio de Janeiro, Medical Biochemistry Institute, The National

Institute of Science and Technology in Structure and Bio-image. Rio de Janeiro, Brazil. 2. Leibniz-Institute für Molekulare Pharmakologie, Structural Biology, Berlin, Germany.

Amyloidosis is a clinical disorder caused by extracellular deposition of proteins that are normally soluble in their native conformation, but suffer conformational modifications resulting in insoluble, abnormal fibrils that impair organ function. Parkinson’s disease (PD) and Alzheimer’s disease (AD) are the two major common neurodegenerative diseases. In this work we are using transthyretin (TTR) fibrils formed from the monomeric construction of TTR protein (MTTR). The solid state NMR data has shown narrow peaks as an indication of well ordered fibrils. However, only few peaks were

observed in comparison to the length of the protein that suggest us different degrees of mobility on fibril structure. In this way, a combination of solution and solid-state NMR has been applied in this work. Amyloid fibrils are interesting polymers whose the structure use to adopt a cross-b-sheet organization, indicating a common core structure shared by them. On the other hand, the protein-protein association seems not to be working in a promiscuous fashion, but it requires amino acids specificity. This behavior can be illustrated in a cross-seeding approach, in which an inter-species barrier can be observed, as in the case of prion protein. Applying atomic resolution techniques is possible to observe differences in fibrils structure at the quaternary level. For instance, X-ray diffraction map can provide slights differences in the diffraction pattern among different b-sheet structure conformations. In general, there exist a signal rising up at 4.7A and another at 10A that are regarding to the distances inter-strands and inter-sheets, respectively. The X-ray information taken together Solid-state NMR results had innovated the field of protein structure. The dipolar-dipolar interaction has added precise distance measurements between two spin-systems. This parameter has been reintroduced by application of some pulse sequences such as Transferred Echo Double Resonance (TEDOR). Indeed, 1H back exchanged samples with different levels of deuterium in combination to Radiofrequency Driven dipolar Recoupling (RFDR) has showed up as a nice tool to observe mobile parts of fibrils structure. Our work has collecting several structural information concerning fibrils formation and organization, as well, structural mobility that could help a future drug design to stop or force non toxic species of oligomers. Alexander von Humboldt, FAPERJ, CNPq, UFRJ, DAAD G26. TEMPO DE FORMAÇÃO, ESTABILIDADE ESTRUTURAL E INCORPORAÇÃO DE PROTEÍNA SOLÚVEL EM AGREGADOS FORMADOS A PARTIR DOS PRODUTOS DE DISSOCIAÇÃO DAS FIBRILAS DE TRANSTIRRETINA.

RODRIGUES, L.B.; SUAREZ, M. C. AND FOGUEL, D. Instituto de Bioquímica Médica, UFRJ

"Muitas doenças amilodoigênicas são causadas por agregação de proteínas celulares solúveis. Entretanto, as amiloidoses não são doenças de perda de função. Em nenhuma das doenças amiloidogênicas descritas, há evidências que uma quantidade suficiente de proteína é removida de fluidos fisiológicos a ponto de comprometer sua função. A hipótese amilóide considera que o processo de formação de fibrilas é a principal causa das doenças amiloidogênicas. Apesar das inúmeras evidências a favor desta hipótese, falta um entendimento detalhado de como a auto-associação de uma proteína malformada leva a disfunções de órgãos e às características neurodegenerativas. Diversos estudos apontam que intermediários precocemente associados são muito mais tóxicos que intermediários de alto peso molecular ou fibrilas. A proteína tetramérica transtirretina (TTR), encontrada no plasma humano e fluido cérebro-espinhal é responsável pelo transporte de retinol e tiroxina. Esta proteína está envolvida em duas doenças amiloidogênicas: a polineuropatia amilóide familiar, que afeta aproximadamente 1 em 100.000 pessoas e a amiloidose senil sistêmica, que afeta 25% das pessoas com mais de 80 anos. A primeira delas é causada por variantes da TTR ao passo que a proteína tipo selvagem tem sido encontrada nas fibrilas de pessoas acometidas pela forma senil da doença. Em trabalhos anteriores verificamos que fibrilas da TTR selvagem e do variante L55P podem ser dissociadas pelo emprego de alta pressão hidrostática. Neste trabalho, utilizando estas mesmas proteínas constatamos: 1) que a estabilidade estrutural das fibrilas diminui à medida em que elas se tornam mais “velhas”; 2) as espécies geradas a partir da dissociação das fibrilas podem sofrer reassociação após descompressão e “sequestrar” proteína solúvel marcada com


acrilodan adicionada ao meio antes da pressurização. Tais resultados parecem redimensionar o papel das fibrilas amilóides no desenvolvimento das patologias." FAPERJ, CNPq G27. STRUCTURAL INSIGHTS ON ONE FLAGELLAR CHAPERONE FROM XANTHOMONAS AXONOPODIS PV. CITRI 1-SCHULTZ,L.G.; 2-FATTORI,J.; 3-PRANDO, A.; 2-ASSIS, L.H.P.; 4-APARICIO, R.; 1-TASIC, L.

1- Laboratório de Química Biológica, Universidade Estadual de Campinas; 2- LNBio,LNLS,CNPEM,Campinas; 3- Syngenta Braul, São Paulo; 4- Laboratório de Biologia

Estrutural e Cristalografia, Universidade Estadual de Campinas. "The Gram-negative bacterial pathogens use several secretion systems to deliver effectors to eukaryotic cells. The type III secretion system (T3SSs) is the most complex of all, due to a kind of a needle that is formed in on the pathogenic cell capable to deliver virulence proteins directly into eukaryotic cells. These processes require customized chaperones whit high specificity for binding partners, thus providing the secretion to occur. The flagellar system, where our protein of interest acts, is considered a precursor of the type III secretion system (T3SSs). Due to the low similarity of sequence and annotation of secretion chaperones their identification is a very difficult task. However they present some common characteristics as being small (<20 kDa), have low pI, amphipathic helix and have a tendency to dimerize, which make the work less arduous. Concerning about this, herein, we present structural features on one flagellar secretion chaperone belonging to plant pathogen Xanthomonas axonopodis pv. citri and suggest how low resolution models based on Small Angle X-ray Scattering (SAXS) patterns can provide new structural insights that could be very helpful in their analysis and posterior classification." INBEB G28. ACTIVITY OF SQ-109, A SQUALENE SYNTHASE INHIBITOR, AND ANALOGUES AGAINST TRYPANOSOMA CRUZI 1 - LAMEIRA, L.; 1 - VEIGA-SANTOS P; 2 - LI, K.; 2 - OLDFIELD, E.; 3 - URBINA, J.A.; 1 - DE

SOUZA, W.; 1 - CARVALHO, T.M.U. 1Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos

Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil, 2Departments of Chemistry and Biophysics, University of Illinois at Urbana-Champaign, USA, 3Emeritus Investigator, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones

Cientificas, Caracas, Venezuela. Chagas’ disease is caused by the protozoan Trypanosoma cruzi. Currently available treatments use compounds such as nifurtimox and benznidazole, which have a wide range of side effects and an unsatisfactory response during the chronic phase. Thereby, it is important to find new safer and more active drugs. Previous studies have validated the ergosterol biosynthesis pathway as a chemotherapeutic target in T. cruzi and Leishmania spp. In the present work we investigated for the first time the anti-T. cruzi activity of SQ-109 (N-[(2E)-3,7-dimethyl-2,6-octadienyl]-N’-tricyclo[3.3.1.13,7]dec-2-yl-1,2-ethanediamine), a compound in clinical development for the treatment of drug-resistant tuberculosis, which has also been shown to inhibit T. cruzi ’s squalene synthase. We also evaluated the effects of SQ-109 and three analogs (1281, 1283 and 1304) against both proliferative stages of T. cruzi, cultured in vitro. All compounds inhibited the proliferation of epimastigote forms with IC50 values (144 h of exposure), of 4.2 µM for SQ-109 and 6.9 µM, 9.3 µM, and 6.1µM, respectively, for the analogs. The compounds also inhibited the proliferation of intracellular amastigote in cultured murine peritoneal macrophages, with IC50 values (96 h of exposure) of 1.4 µM for SQ-109, and 1.6 µM, 1.9 µM and 1.7 µM, respectively, for the analogs. The selectivity index

of SQ-109 activity was 12. Analyses by scanning electron microscopy revealed that SQ-109 caused depressions of the plasma membrane and rounding of the cell body. Thin sections of treated epimastigotes observed by transmission electron microscopy showed drastic changes in the Golgi complex, mitochondrial swelling, formation of myelin-like figures and cytoplasmic vesiculation. Taken together, our observations show that SQ-109 and analogs are promising selective inhibitors of T. cruzi proliferation. Further studies will evaluate their potential antiproliferative synergism with posaconazole. CNPq, CAPES, FINEP, FAPERJ, USPHS G29. A LACTOFERRINA INIBE A ENTRADA DO VÍRUS DA FEBRE AMARELA VIA LIGAÇÃO À SUPERFÍCIE CELULAR

1- Ferreira, M.V.M.; 1- Mendes, Y.S.; 1- Alves, N.S.; 1- Carvalho, C.A.M.; 1- Campos, S.P.C.; 2- Schwarcz, W.D.; 3- Silva, J.L.; 1,4- Gonçalves, R.B.; 1- Gomes, A.M.O.; 1-

Oliveira, A.C. 1- LABEV - Instituto de Bioquímica Médica/UFRJ; 2- LATEV - Instituto de Tecnologia em

Imunobiológicos/FIOCRUZ; 3- LTPV - Instituto de Bioquímica Médica/UFRJ; 4- LAAB - Departamento de Bioquímica/UNIRIO.

O vírus da Febre Amarela (YFV) é um Flavivírus endêmico em regiões tropicais, principalmente África e América do Sul, provocando uma doença febril aguda de grande impacto na saúde pública. Ainda não existem tratamentos específicos para esta infecção, tornando a busca por antivirais um alvo de grande importância médica. A lactoferrina bovina (bLf), uma glicoproteína presente em diversas secreções, como leite, lágrima e saliva, apresenta diversas funções biológicas, incluindo modulação da resposta imune e defesa contra diversos patógenos, como diferentes vírus de importância médica e socioeconômica. O objetivo deste estudo é avaliar a atividade antiviral da bLf contra a infecção pelo YFV. Nossos resultados mostram que a bLf apresenta uma atividade de inibição viral de aproximadamente 70%, sem provocar efeitos citotóxicos em nosso modelo celular. Buscando investigar quais etapas e que mecanismos estão envolvidos nesta inibição, nossos dados indicam que, ao pré-tratarmos a célula com bLf ou adicionarmos somente na etapa de ligação ao receptor celular (adsorção viral), a infecção é inibida em torno de 60%. Em contrapartida, a presença da bLf apenas após os processos iniciais de infecção (pós adsorção e internalização viral) leva a uma inibição inferior a 10%. Além disso, ao avaliarmos a capacidade da bLf em se ligar às partículas virais, notamos que não houve alteração significativa no título viral. Juntos, nossos resultados fortemente sugerem que a bLf apresenta atividade antiviral, atuando majoritariamente sobre os eventos iniciais do ciclo de infecção do YFV por se ligar à superfície celular e possivelmente dificultar a interação vírus-célula. O presente estudo pode ajudar na melhor compreensão do ciclo, além de auxiliar importantes estratégias para o desenvolvimento de antivirais eficazes contra a infecção por diferentes flavivírus. CNPq, CAPES, FAPERJ, PRONEX, INBEB G30. POLARIDADE MAGNÉTICA EM COCOS MAGNETOTÁTICOS CULTIVADOS DA LAGOA DE ITAIPU, RJ

1 - CAMPELLO, M.; 1- KARINA, V.; 1- LINS, U. 1 - Instituto de Microbiologia Paulo de Goes, Universidade Federal do Rio de Janeiro.

Bactérias magnetotáticas formam um grupo diverso de procariontes aquáticos que produzem organelas compostas de cristais magnéticos de magnetita (Fe3O4) ou greigita (Fe3S4), envoltos por uma bicamada lipídica, conhecidos como magnetossomos. Os magnetossomos conferem às bactérias a capacidade de orientação ao longo de linhas de campos magnéticos através da movimentação por flagelos. As bactérias no


hemisfério Norte nadam preferencialmente para o norte geográfico (tipo N), enquanto as do hemisfério Sul nadam para o Sul (tipo S). Como a componente vertical do campo geomagnético aponta para baixo no Hemisfério Norte e para cima no Hemisfério Sul, as bactérias magnetotáticas nadam para baixo nos dois hemisférios devido á polaridade invertida de seus flagelos. Contudo, há relatos de bactérias magnetotáticas com comportamento semelhante ao tipo S no hemisfério Norte. Neste trabalho, será testada a influência da inversão do campo magnético sobre a polaridade de um grupo de cocos magnetotáticos isolado em cultura axênica da lagoa de Itaipu, RJ. As bactérias foram crescidas em tubos de ensaio com meio de cultura semi-sólido heterotrófico com gradiente de oxigênio, usando quinato férrico como fonte de ferro e succinato de sódio, acetato de sódio e bicarbonato de sódio como fontes de carbono. As culturas serão crescidas em duas condições: a) em um campo magnético Norte, gerado por uma bobina magnética e (b) sob a influência do campo geomagnético Sul. A cada 24 horas, o padrão de formação das bandas no meio de cultura será observado. Por microscopia óptica, conferiremos a polaridade das bactérias magnetotáticas crescidas sob o campo magnético Norte e Sul e, por Microscopia Eletrônica de Transmissão, a produção de magnetossomos e outras características morfológicas na célula. Os resultados obtidos permitirão uma melhor compreensão do papel dos magnetossomos e da magnetotaxia para a sobrevivência dos micro-organismos magnetotáticos no ambiente. Financiamento: CNPq, CAPES, FAPERJ G31. ESTUDOS DE INTERMEDIÁRIOS DE AGREGAÇÃO DA PROTEÍNA SUPRESSORA DE TUMOR P53

1,2- FERREIRA, M. E.; 1,2- Rangel, L.P.; 1,2- Silva, J.L. 1-Instituto de Bioquímica Médica UFRJ 2-Instituto Nacional de Ciência e Tecnologia de

Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.

"Introdução A proteína supressora de tumor p53 é uma das principais proteínas regulatórias do ciclo celular, responsável por levar a célula à apoptose caso ocorra um dano irrevesível no material genético. Aproximadamente 50% dos tumores humanos estão relacionados a mutações dessa proteína. Estudos com a p53 selvagem e sua forma mutante R248Q levam a acreditar que a p53 quando sofre mutação pode formar agregados e flibrilas oligoméricas, de forma parecida com o que ocorre no prion, o que levariam a uma mudança conformacional e a perda de função. O objetivo desse trabalho é estudar a agregação da p53 selvagem e tentar caracterizar possíveis intermediários relacionados com esse processo. Métodos Como metodologia de estudo, foram utilizadas técnicas de espectroscopia para analisar a fluorescêcia e o espalhamento de luz da p53 quando submetida a desnaturação por alta pressão. Foi analisado também o efeito de diferentes concentrações de guanidina (0 a 1M) na reversibilidade da desnaturação da p53. A desnaturação por pressão é resultado da força hidrostática que leva a entrada de moléculas de água nas cavidades hidrofóbicas de uma proteína quando essa é submetida a pressões na ordem de Kpsi. O espectro de fluorescência de resíduos de triptofano, normalmente não expostos, foi usado como forma de quantificar o grau de desnaturação da p53. Estudos de espalhamento de luz foram usados para analisar a agregação proteica. Resultados e discussão Estudos iniciais mostram que há uma reversibilidade da desnaturação da p53 por pressão quando exposta a diferentes concentrações de guanidina. Apesar de reversível, é possível perceber uma diferença na cinética de desnaturação quando a p53 volta para seu estado nativo, podendo então mostrar a presença da formação de intermediários após o desenovelamento." INBEB

G32. PRODUÇÃO DE PHA E MAGNETOSSOMOS EMPREGANDO BACTÉRIAS MAGNETOTÁTICAS

1-SANTOS, M.G.C.; 1- LEÃO, P..E.L;1- CORREA,T.N.;2-GUTARRA, M.L.E; 3- LINS, U.G.C. 1- Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro; 2- Escola de Química, Polo Avançado de Xerém, Universidade Federal do Rio de Janeiro; 3-

Instituto de Química, Universidade Federal do Rio de Janeiro "As bactérias magnetotáticas são microrganismos encontrados em ambientes de água doce e salgada, são caracterizadas por produzirem nanocristais magnéticos compostos de magnetita ou greigita envoltos por uma membrana, denominados magnetossomos. Além dos magnetossomos, algumas bactérias magnetotáticas têm a capacidade de produzir grânulos lipídicos intracelulares geralmente em condições de desequilíbrio da razão carbono/nitrogênio no meio. Esses grânulos são utilizados como reserva energética por esses microrganismos, nesse trabalho analisamos a produção de grânulos lipídicos e magnetossomos pela bactéria Magnetovibrio blakemorei (cepa MV-1). A bactéria foi cultivada em meio específico contendo: solução de minerais, succinato de sódio, cloreto de amônio, hidrolisado de caseína, tampão fosfato, cisteína, solução de vitamina e sulfato ferroso, à 24°C, 100 rpm e em pH 7,5 por 168h. As amostras foram retiradas em intervalos de 24h para análise de crescimento celular em espectrofotometria à 600nm. A analise e quantificação da produção de grânulos lipídicos e magnetossomos foi realizada por microscopia eletrônica de transmissão. Com o sobrenadante foi analisado o consumo da fonte de nitrogênio pela determinação de FAN (Free Amino Nitrogen) e o consumo de succinato de sódio por HPLC. Como resultado observamos que mesmo em condições de desequilibrio das concentrações de carbono e nitrogênio o MV-1 apresentou cinética de crescimento semelhante à observada nas condições do meio controle (1,69x109 células/ml em 96h). Em 96h observamos 4,23x109 magnetossomos/ml. Após 168h de cultivo ocorreu o consumo de 61% das fontes de nitrogênio. Estes resultados demonstram que esta condição empregada foi eficiente para induzir o acumulo de grânulos lipídicos no interior das células. Novas análises de consumo de carbono estão sendo realizadas para caracterizar a produção e a natureza do lipídeo produzido." CNPq, CAPES, FAPERJ G33. ESTUDOS DE ENOVELAMENTO PROTEICO NOS DOMÍNIOS –N E –C DA TROPONINA C MEDIANTE LIGAÇÃO A IONS DE CÁLCIO

1-MAYRA A. MARQUES, 1-GUILHERME A. P. DE OLIVEIRA, 2-CRISTIANE B. ROCHA, 3-YRAIMA CORDEIRO, 1-MARTHA M. SORENSON, 1-DÉBORA FOGUEL, 1-JERSON L. SILVA E

1-MARISA C. SUAREZ 1-Instituto de Bioquímica Médica, UFRJ, Rio de Janeiro, RJ, Brasil 2-Centro de Ciências Biológicas e da Saúde, Instituto Biomédico – IB, Departamento de Bioquímica, Rio de

Janeiro, Brasil. 3-Faculdade de Farmácia, UFRJ, Rio de Janeiro, RJ, Brasil. A Troponina C (TnC), é uma subunidade do complexo Troponina encontrado no músculo vertebrado esquelético e cardíaco; possui dois domínios estruturalmente homólogos: o domínio N e C terminais conectados entre si por uma alfa hélice central e é capaz de se ligar a íons cálcio (Ca+2) através de uma estrutura denominada “EF-hand”. Ensaios de mutagênese sítio dirigida tem possibilitado a produção de mutantes, da construção inteira da TnC e dos seus domínios isolados, com resíduos de fenilalanina (Phe) substituídos por Triptofano (Trp). Estudos anteriores, utilizando esses mutantes submetidos à alta pressão hidrostática, propõem que o domínio C-terminal, na ausência de cálcio é menos estável que o domínio N-terminal. Este não parece exercer efeito na estabilidade do C-terminal. Analisamos a estabilidade da construção completa da F29W TnC, utilizando abordagens estruturais, sob condições desnaturantes de ureia e pressão; F29W TnC é um mutante fluorescente, na qual a Phe 29, localizada no domínio N foi


substituída por Trp. Foram calculados parâmetros termodinâmicos ( V e G atm) que regem o enovelamento da TnC F29W intacta, na presença ou ausência de Ca+2. Os dados sugerem que o domínio C tem um pequeno efeito sobre a estrutura do domínio N, na ausência de Ca+2. No entanto, usando espectroscopia de fluorescência, foi demonstrada uma queda significativa na estabilidade do domínio N ligado a Ca+2 quando, os sítios III e IV do domínio C estavam ligados ao Ca+2. Observou-se um decréscimo na estabilidade termodinâmica do domínio N promovendo uma redução de

G atm, em termos absolutos, além da afinidade do domínio N pelo Ca+2 mostrar-se alterada. Dados de espalhamento de raios-X a baixos ângulos revelam que a interação entre os domínios C e N pode ser mediada pela hélice central, que possui menor volume e aparenta maior rigidez e estabilidade, após a ligação do Ca+2 aos sítios EF-hands. INBEB, FAPERJ, CNPq G34. ANÁLISE DA EXPRESSÃO DO GANGLIOSÍDEO 9-O-ACETIL GD3 NA ZONA SUBVENTRICULAR 1-FURTADO, M; 1-MORTARI, N; 1-GUBERT, F;1- ZAVERUCHA-DO-VALLE, C;1- SANTIAGO,

MF;1- MENDEZ-OTERO, R 1-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro

"As células-tronco neurais são uma esperança para o tratamento de doenças neurodegenerativas. Desta forma, a descoberta dos mecanismos envolvidos com a proliferação e o recrutamento dessas células para áreas de lesão é essencial para que se possam desenvolver terapias celulares baseadas no estímulo as células-tronco endógenas. Em cultura, as células-tronco neurais são caracterizadas pela capacidade de formar neuroesferas em placas não-aderentes na presença dos fatores EGF e FGF-2, mas in vivo ainda não existem marcadores eficazes para que se possa distinguir claramente uma célula-tronco neural de um progenitor já comprometido. Nesse trabalho estamos propondo a análise do gangliosídeo 9-O-acetil GD3 como um possível marcador de células-tronco neurais. O gangliosídeo 9-O-acetil GD3 está presente no sistema nervoso central e periférico de roedores durante o desenvolvimento e foi descrito como uma molécula associada a eventos de migração celular e extensão axonal nesse período. Em ratos adultos, o gangliosídeo 9-O-acetil GD3 deixa de ser expresso na maior parte do sistema nervoso central, sendo observado apenas na zona subventricular, na via migratória rostral, na retina e no cerebelo. Como a zona subventricular é a região onde encontram-se as células-tronco neurais em mamíferos adultos, o objetivo desse trabalho é analisar uma possível associação do gangliosídeo 9-O-acetil GD3 com as células-tronco neurais. Para isso, analisamos a expressão do gangliosídeo 9-O-acetil GD3 na zona subventricular de ratos adultos e de ratos em idade pós-natal 21 (P21) e embrionários (E16), por imunohistoquímicas combinado o anticorpo contra o gangliosídeo 9-O-acetil GD3 com anticorpos específicos para os diferentes tipos celulares da região. Comparamos também a população de células da zona subventricular positiva para o gangliosídeo 9-O-acetil GD3 com a população negativa em ensaio de formação de neuroesferas utilizando as mesmas idades." INBEB, FAPERJ, CNPq, CAPES G35. CYTOTOXIC AND PROAPOPTOTIC EFFECTS OF PTEROSTILBENE ON BREAST CANCER CELLS

Campos, N.P.C.1; Costa, D.C.F.1; Fialho, E.2, Silva, J.L1 1Instituto de Bioquímica Médica, IBqM-UFRJ, 2Instituto de Nutrição Josué de Castro,

INJC-UFRJ. "Pterostilbene, a dimethyl ester derivative of resveratrol, is a bioactive food compound that mediates many cellular targets involved in cancer signaling pathways. The p53

tumor suppressor protein plays an essential role in preventing cancer development by inducing cell cycle arrest or apoptosis in response to cellular stress. This protein has been suggested to have a role in the anticancer properties of resveratrol and its structural analogues. Thus, the present study was aimed to check the cytotoxic and pro-apoptotic effects of pterostilbene on MCF-7 cells, a p53-positive human breast cancer cell line. MTT reduction cell viability assay showed that pterostilbene (10–200 µM) promoted a cytotoxic effect on MCF-7 cells in a dose- and time-dependent manner. In a concentration of 50 µM, this compound was able to impair approximately 30% and 75% of cell viability after 24 and 48h of treatment, respectively. These effects were more pronounced than those induced by resveratrol in MCF-7, at the same experimental conditions. Furthermore, cell treatment with 100 µM of pterostilbene for 24h increased the exposition of phosphatidylserine on cell surface, which is suggestive of apoptosis. This effect was partially prevented when cells were pretreated with pifithrin-α, a specific p53 inhibitor. Taken together, our results indicate that pterostilbene can be suggested as a promising chemopreventive agent and the cytotoxicity promoted by this compound in tumor cells possibly requires p53 function. Word Keys: resveratrol derivatives, pterostilbene, MCF-7, p53, apoptosis." Supported by: INBEB, FAPERJ and CNPq. G36. EFFECTS OF THE COMBINATION THERAPY BETWEEN ALQUILPHOSPHOLIPIDS AND DINITROANILINES AGAINST LEISHMANIA AMAZONENSIS

NEILTON CESAR ARAUJO DA CRUZ- 1; JOSEANE LIMA PRADO GODINHO - 2; CHISTINA MAEDA TAKIYA - 3; WANDERLEY DE SOUZA - 4; JULIANY COLA FERNANDES RODRIGUES -

5. 1.POLO AVANÇADO DE XERÉM, UNIVERSIDADE FEDERAL DO RIO DE JANEIRO;-

2,3.INSTITUTO DE BIOFÍSICA CARLOS CHAGAS FILHO, UNIVERSIDADE FEDERAL DO RIO DE JANEIRO; -4.UFRJ,IBCCF,IMETRO,INBEB; -5.POLO AVANÇADO DE

XERÉM,UFRJ,IBCCF,IMETRO,INBEB. Protozoan parasites of the Leishmania genus are able to cause leishmaniasis around the world. In Brazil, Leishmania amazonensis is responsible for cutaneous and diffuse cutaneous leishmaniasis. The current chemotherapy is based on pentavalent antimonials, amphotericin B, miltefosine and pentamidine. Nowadays, miltefosine has been implemented as oral treatment for visceral and cutaneous leishmaniasis in several countries. Trifluralin, is a dinitroaniline that displays important effects in some protozoan parasites, and its main action is specific against parasite tubulin. In this work, we evaluated the effect of miltefosine and trifluralin alone or in combination against L. amazonensis in vitro and in vivo. For promastigotes in vitro, the IC50 values found were around 25 µM and higher than 20 µM for miltefosine and trifluralin, respectively. Both compounds, when tested alone against intracellular amastigote, showed IC50 values higher than 50 µM. Microscopy techniques showed alterations on the shape of promastigotes and the presence of lipid bodies. The first combination assay was done in Balb/C-infected mice with L. amazonensis. After the development of the lesions at the basis of the tail, the mice were divided in different groups: Glucantime group (50 mg/kg/ day), Trifluralin groups (30, 50 mg/kg/day), Miltefosine-treated groups (20; 30; 40; 50 mg/kg/day), and combination of Miltefosine/Trifluralin [(20 x 30 mg/kg/day;(20 x 50 mg/kg/day). Miltefosine and trifluralin were administrated by oral gavage, while Glucantime by intraperitoneal route, during 21 days of treatment. The efficacy was evaluated by measuring the size of the lesions, presence of parasite in lesions stained with Giemsa and histopathological analysis. The results obtained with miltefosine showed a significant dose-dependent decrease in the lesion size, which was not observed after treatment with trifluraline alone. Combination of trifluralin with miltefosine resulted in an effect similar to those obtained for miltefosine alone.


Histophatological analysis showed an important immune response in the groups treated with miltefosine alone or in combination. Reduction of the number of foam macrophages and resident inflammatory cells was observed. Thus, this study suggests that miltefosine alone or in combination with trifluralin could be an alternative treatment for cutaneous leishmaniasis caused by L. amazonensis. CNPq, CAPES, and FAPERJ. G37. ANÁLISE DA NEUROGÊNESE NA ZONA SUBVENTRICULAR DE CAMUNDONGOS NOCAUTE PARA A ENZIMA GD3 SINTASE 2- NICOLI MORTARI ; 2- MICHELLE GUIMARÃES 2; 1,2- CAMILA ZAVERUCHA-DO-VALLE;

1-2- FERNANDA GUBERT; 1,2- MICHELLE BARGAS REGA, 1,2- MARCELO F. SANTIAGO; 1,2- ROSALIA MENDEZ-OTERO.

1- Programa de Terapias Celulares; 2- Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro.

"Gangliosídeos são glicoesfingolipídeos que contêm ácidos siálicos e estão presentes na membrana plasmática celular, com altas concentrações no sistema nervoso central. Eles desempenham importantes papéis no desenvolvimento e diferenciação do sistema nervoso de vertebrados. O gangliosídeo 9-O-acetil GD3 é formado a partir da acetilação do gangliosídeo GD3 e está presente em eventos de migração celular e extensão axonal durante o desenvolvimento. Em roedores adultos, esse gangliosídeo deixa de ser expresso na maior parte do sistema nervoso central, mantendo-se expresso, entretanto, na zona subventricular, principalmente. Como a zona subventricular é o principal nicho neurogênico em mamíferos adultos, procuramos avaliar uma possível relação entre o gangliosídeo 9-O-acetil GD3 e a neurogênese em animais adultos. Para isso, utilizamos camundongos nocaute para a enzima GD3 sintase, responsável pela síntese do gangliosídeo GD3 – precursor do gangliosídeo 9-O-acetil GD3 – e crítica para a formação de todos os gangliosídeos da série b. Desta forma, ao analisar a neurogênese nos animais nocaute para a enzima, estamos analisando não só a perda do gangliosídeo 9-O-acetil GD3, mas de todos os gangliosídeos da série b. Dissecamos a zona subventricular de camundongos adultos nocaute para a enzima GD3 sintase (GD3KO) e de camundongos selvagens e comparamos as duas populações em ensaios de formação de neuroesferas. Para isso, as células foram mantidas em condições não-aderentes por 7 dias. Conseguimos estabelecer um protocolo para o ensaio de formação de neuroesferas e observamos que as células dos dois animais são capazes de formar neuroesferas primárias e secundárias. Observamos nos dois casos a diferenciação das esferas em astrócitos e neurônios. Desta forma, demonstramos que as células da zona subventricular dos camundongos GD3KO são capazes de formar neuroesferas, mas temos evidências de que a neurogênese nesse animais possa estar de alguma forma alterada." INBEB, FAPERJ, CAPES, CNPq, DECIT, PROTECEL G38. INHIBITOR OF APOPTOSIS PROTEIN XIAP: A STRUCTURAL AND THERMODYNAMIC ANALYSES OF ITS INHIBITION FOR COMPOUNDS SMAC MIMETICS.

1-SANTOS, R.B. , 2- OLIVEIRA, A.C. & 1- SOUZA, T.L.F. 1-Faculdade de Farmácia, UFRJ, Rio de Janeiro,Brazil. 2-Programa de Biologia Estrutural,

IBqM, UFRJ, Rio de Janeiro,Brazil. Apoptosis (programmed cell death) resistance can be generated by high expression of inhibitor of apoptosis proteins (IAPs) family, which are responsible by the inhibition of effector proteases. Smac/DIABLO, is an endogenous inhibitor of XIAP due to its interaction on the XIAP-BIR3 domain. In cancer, the high expression of XIAP leads to apoptosis resistance and Smac/DIABLO becomes inefficient. Smac mimetics have been considered potential drug candidates, owing to its capacity to bind on XIAP-BIR3 and to

sensitize apoptosis in cancer cell. The goal of this work is better understand of the mechanisms of the XIAP-BIR3 inhibition. We used circular dichroism (CD), fluorescence spectroscopy and isothermal titration calorimetric (ITC) to perform structural and thermodynamic analyses of the effects of the binding of Smac mimetics on XIAP-BIR3. Compounds with similarities in structure, but with different activities, were selected. Our data showed that this domain has a high stability and that the chemical denaturation occurs with two transitions. In the presence of the different compounds, a differential stabilization of the domain was observed and the chemical denaturation, differently, occurred with a single transition. Additionally, the Smac mimetics promote to a significant change in the CD spectra of free XIAP-BIR3 domain, indicating changes in the secondary structure content. Results obtained by ITC revealed that, although present different affinities, the interaction is entropically and enthalpically favored to all compounds. In conclusion, we found that the XIAP-BIR3 domain undergoes significant changes in the structure, stability and thermodynamic parameters of binding when associated to different ligands, what may be a relevant factor to be considered in affinity optimization of anti-IAP drug candidates. CNPq, CAPES, FAPERJ, INBEB G39. CONSUMO DE ÓXIDO NITROSO RELACIONADO AO CRESCIMENTO DE MAGNETOVIBRIO BLAKEMOREI EM BIORREATOR DE BANCADA 1- CORREA, T.N.; 1- LEÃO, P.E.L.; 1- SANTOS, M.G.C.; 2- GUTARRA, M.L.E.; 3- LÓPEZ, J.A.

1- LINS, U.G.C. 1- Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro; 2- Escola de Química, Polo Avançado de Xerém, Universidade Federal do Rio de Janeiro; 3-

Instituto de Química, Universidade Federal do Rio de Janeiro Bactérias magnetotáticas apresentam a capacidade de produzir nanopartículas magnéticas denominadas magnetossomos. Formados por cristal de magnetita ou greigita envolto por uma membrana biológica, os magnetossomos possuem inúmeras aplicações no campo biomédico, tais como no carreamento de drogas. A bactéria alvo de nossos estudos é o vibrião marinho Magnetovibrio blakemorei, que produzem cristais prismáticos de magnetita, exibindo uma grande versatilidade metabólica, podendo utilizar diferentes compostos como aceptores finais de elétrons. A maior produção de magnetossomos por célula, porém, é atingida no crescimento com óxido nitroso (N2O) como aceptor final de elétrons. Pouco se conhece, entretanto, sobre a fisiologia desta bactéria e a relação entre o óxido nitroso e a produção de magnetossomos. Neste trabalho, o consumo de óxido nitroso pelo Magnetovibrio blakemorei foi medido durante o cultivo em biorreator de bancada e correlacionado com o crescimento celular e o consumo dos demais nutrientes presentes no meio. O meio de cultivo empregado continha succinato de sódio (11,3 g/L) e hidrolisado de caseína (3,3 g/L) como fonte de carbono e nitrogênio respectivamente, sulfato ferroso (130µmol/L) como fonte de ferro para o crescimento e formação dos magnetossomos, além de encontrar-se saturado com N2O (10mmol/L). Em 48h de crescimento, aproximadamente 50% do óxido nitroso presente no meio havia sido consumido, sendo que ao fim de 120h sua concentração havia se esgotado, assim como o ferro (II). A exaustão destes dois componentes no meio causou uma redução na produção de cristais por célula, observada através da contagem por microscopia eletrônica de transmissão. UFRJ G40. INTRACARDIAC INJECTION OF DM28C TRYPANOSOMA CRUZI PROVIDES A MODEL OF INFECTION-ASSOCIATED MYOCARDITIS AND HEART FIBROSIS THAT DEPENDS


CRITICALLY ON COOPERATIVE ACTIVATION OF THE KALLIKREIN/KININ SYSTEM AND THE ENDOTHELIN PATHWAY

ANDRADE D1., SERRA RR1., CORDOVIL, T1., BRASIL, G.V1., RAMOS-JUNIOR E.S1., CARVALHO-PINTO, C.E2., VAIRO L1., FORTES F3., GOLDENBERG, R1., CARVALHO A.C.C1.,

SVENSJÖ, E1, SCHARFSTEIN J1. 1Instituto de Biofísica Carlos Chagas Filho, UFRJ, CCS, Bl. D sala 7, Cidade Universitária, CEP 21944-900, Rio de Janeiro; 2 Instituto de Biologia - Departamento de Imunologia –

UFF, Niterói, RJ; 3CentroUniversitário Estadual da Zona Oeste -UEZO In this study, we tested the hypothesis (Andrade et al., 2012; Scharfstein and Andrade, 2011) that kinins released intramyocardiacally by Dm28c trypomastigotes, acting cooperatively with cardiac endothelins, infect heart tissues through the activation of bradykinin (BKRs) and endothelin receptors (ETRs). Guided by high-resolution echocardiography, we injected tissue-culture derived trypomastigotes (TCTs) in the left ventricle of C57BL/6 BK2R+/+(wt), BK2R-/- or Balb/c (naïve). One hour before parasite inoculation, wt mice were treated with a single dose (systemic) of (i) HOE-140 (BK2R antagonist) (ii) BK1R antagonist (iii) Bosentan (ETAR/ETBR antagonist). The functional parameters analyzed p.i. were intracardiac oedema (2 h p.i.); parasite load and mRNA levels of chemokines/cytokine in heart tissues (qPCR); myocarditis and fibrosis at 30 d p.i. As predicted, Dm28c TCTs evoked an early-phase intracardiac oedema in wt mice. In contrast, plasma extravasation was virtually abolished in the heart of BK2R-/- mice, or in infected wt mice that were pretreated with a single dose of BKR antagonists or with bosentan. Parasite load in the heart tissues (3 d p.i.) was markedly reduced by these GPCR antagonists. Combined, these results suggest that Dm28c TCTs might exploit the transient availability of pro-edematogenic peptides in the interstitial spaces to efficiently invade cardiovascular cells through the signaling of GPCRs. We then checked whether the benefits brought about by the pharmacological interventions, all of which made at the onset of infection, mitigated subsequent development of heart pathology. Our results (30 d p.i.) showed that myocarditis and fibrosis were virtually absent in infected mice that were pretreated with BKR or ETR antagonists whereas control mice exhibited conspicuous pathology. Additional studies are required to evaluate whether these anti-inflammatory drugs might ameliorate heart pathology during the chronic stage of Dm28c infection. CNPq, FAPERJ, INBEB G41. INIBIÇÃO DO SISTEMA TIOREDOXINA POR POLIFENÓIS NO TRATAMENTO DO CÂNCER

1- SILVA, T.; 2- ALMEIDA, F.; 3- AMORIM, G.; 1- Universidade Federal do Rio de Janeiro, Instituto de Bioquímica Médica, Centro

Nacional de Ressonância Magnética Nuclear 2- Universidade Federal do Rio de Janeiro, Instituto de Bioquímica Médica, Centro Nacional de Ressonância Magnética Nuclear 3-

Universidade Federal do Rio de Janeiro, Instituto de Bioquímica Médica, Centro Nacional de Ressonância Magnética Nuclear

"As tioredoxinas (Trxs) são enzimas antioxidantes ubíquas de baixo peso molecular (12kDa) que estão ligadas ao controle da homeostasia oxido-redutora das células e desempenham um importante papel em diversos processos celulares relacionados ao equilíbrio saúde-doença. Em diversas patologias, incluindo o câncer, o balanço oxido-redutor das células é afetado. Células tumorais possuem alta taxa metabólica e, consequentemente, nível elevado de espécies reativas de oxigênio (ROS). Dessa forma, interferir na homeostasia redox destas células representa uma abordagem promissora na terapia do câncer. Devido a seu papel essencial na regulação redox, as tioredoxinas representam um alvo importante na busca por novos tratamentos quimioterápicos. Muito já foi feito no sentido de mostrar a eficácia da inibição do sistema tioredoxina no

tratamento do câncer. Na busca por novos inibidores, os flavonóides foram identificados como potenciais agentes quimioterápicos, e seu mecanismo de ação é provavelmente mediado pelo sistema tioredoxina. O principal objetivo deste trabalho de pesquisa é selecionar compostos naturais, especialmente flavonóides, com potencial ação inibitória do sistema tioredoxina, e estudar as interações do complexo enzima-inibidor com enfoque bioquímico e estrutural. Alguns dados já foram obtidos, dentre os quais foi possível identificar uma amostra de flavonóide que mostrou-se capaz de interagir com a tioredoxina. Os possíveis resíduos envolvidos na interação já foram identificados. Estes resultados serão utilizados no desenho racional de drogas, visando a obtenção de moléculas mais eficazes e menos tóxicos na terapia contra o câncer, que estarão na base do desenvolvimento de futuras abordagens terapêuticas." FAPERJ, CNPq G42. SELEÇÃO DE MICROALGAS OLEAGINOSAS PARA A PRODUÇÃO DE BIODIESEL

1 - FERREIRA, V.S.; 1,2 -PINTO, R.F; 1- SANT'ANNA, C.B 1 -Inmetro (Instituto Nacional de Metrologia Normalização e Qualidade Industrial) - 2-

Instituto de biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro O uso de combustíveis fósseis será futuramente insustentável devido à depleção de suas fontes e ao acúmulo de gases do efeito estufa gerado por seu consumo. O biodiesel é um combustível derivado de biomassa renovável e seu uso traz substancial redução na emissão de gases estufa. Atualmente, este é produzido a partir de óleos vegetais ou gorduras animais, entretanto o uso de micro-organismos oleaginosos têm se mostrado interessante para produção de biodiesel devido ao rápido crescimento e menor gasto de energia e espaço para produção. As microalgas, organismos fotossintéticos, são promissoras fontes de lipídeos para a produção de biodiesel devido às altas taxas de produção de biomassa e à síntese de óleos em altas quantidades, podendo alcançar 60-70% de lipídeos em relação ao seu peso seco total. O objetivo deste trabalho é fazer a bioprospecção de microalgas capazes de produzir e armazenar grande quantidade de lipídeos. As microalgas foram coletadas de ambientes aquáticos diversos, e isoladas por estriamento em placa de ágar. A morfologia das espécies isoladas foi observada por microscopia óptica convencional, microscopia eletrônica de transmissão e varredura. Para a detecção de lipídeos neutros nestes microorganismos foi utilizado um corante lipofílico fluorescente, o Nile Red, e a observação foi realizada em um microscópio confocal de varredura a laser. Foram feitas as curvas de crescimento das células na presença e ausência de nitrogênio, e em seguida a presença e quantia de lipídeos foi comparada por citometria de fluxo. Pudemos observar que todas as cepas isoladas são capazes de armazenar lipídeos, e que há redução no crescimento de células que foram cultivadas na ausência de nitrogênio, no entanto, estas cepas tiveram uma maior produção de lipídeos. CNPq, Inmetro G43. CONFORMATIONAL ENSEMBLES OF GLYCOSYLATED PRION PROTEIN: A STRATEGY FOR MAPPING INTERACTIONS WITH PHYSIOLOGICAL AND THERAPEUTIC LIGANDS 1,2- ALVES, W. J. C.; 2- MACEDO, B.; 2- GONÇALVES, K. M.; 1- OLIVEIRA JUNIOR, R. S.; 1-

TORRES, P. M.; 3- POL-FACHIN, L.; 3- VERLI, H.; 1- LINDEN, R.; 2- CORDEIRO, Y.; 1- PASCUTTI, P.

1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2- Faculdade de Farmácia, Universidade Federal do Rio de Janeiro; 3- Centro de

Biotecnologia, Universidade Federal do Rio Grande do Sul Introduction: The prion protein (PrPC) is ubiquitously expressed in mammals, and anchored by GPI on the outer leaflet of the plasma membrane. Three alpha-helices, one


short anti-parallel beta-sheet, a large highly flexible domain, and two glycans compose the structure of PrPC. The functional properties of PrPC are controversial, although a number of its natural ligands have been described. Laminin receptor precursor(LRP), stress inducible protein-1(STI1) and neural cell adhesion molecule(NCAM), are three such proteins ligands for which cognate interaction domains have been mapped onto each pair of ligands. Our goal is to test whether the main function of PrPC may be to scaffold signaling modules at the cell surface, through allosteric interactions with its protein ligands, thus leading to effects on cell proliferation, differentiation and death. Materials/Methods: We use techniques of molecular modelling and dynamics, generalized simulated annealing and docking, to generate conformational ensembles including PrPC with its flexible N-terminus, and to stabilize structural models of interaction of PrPC together with its ligands. In addition, we use circular dichroism(CD), X-ray small angle scattering, and calorimetry for in vitro assays of binding and conformational effects among recombinant mouse PrPC and synthetic cognate peptides from each ligands. Results/Discussion: An initial CD experiment showed apparent conformational changes undergone by PrPC in the presence of cognate peptides added in a particular order, consistent with results from docking, and suggesting that the affinity of the globular domain of PrPC for a given peptide is distinct, depending on the previous binding of another ligand peptide. A novel protocol of molecular dynamics was produced, specific for the generation of conformational ensembles based on PrPC, including parameters for glycans. Conclusions: Promissory data are consistent with the hypothesis that PrPC scaffolds cell surface protein ensembles, and additional spectroscopy and simulation will provide further testing of allosteric regulation of PrPC structure. FAPERJ, CNPq, INBEB G44. 1H NMR METABOLIC INTERACTION EVALUATION BETWEEN Citrus cinensis AND Cadidatus Liberibacter asiaticus

1- OHASHI, W.; 1- ESPINDOLA, A.; 2- FERREIRA, M.; 3- COLETTA, H.; 1- TASIC, L. Universidade Estadual de Campinas - Instituto de Química – 1- Laboratório de Química

Biológica, 2- Laboratório de Quimiometria Teória e Aplicada. 3- Centro APTA Citros Sylvio Moreira IAC – Cordeirópolis.

"Candidatus liberibacter spp. is the pathogen associated with the Huanglongbing (HLB), a high economic impact disease present all over the world’s mayor citrus producing areas. The infection diagnosis can be confirmed by PCR after visual inspection. Our objective was to evaluate pathogen induced changes in the metabolic profile shown in 1H NMR spectra obtained from different polarity orange leaf extracts. By comparing these spectra of heathy, symptomatic and asymptomatic Citrus cinensis leafs using chemometric tools, such as, Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA), we attempted to visualize clustering on the chemometric models indicating the possibility of creating a HLB diagnostic method. The leaf samples were processed by liquid nitrogen grinding, separated in four fractions for extraction with phosphate buffer (A), methanol (B), chloroform/methanol (1:1, v v-1, C) and chloroform (D), evaporated and diluted in DMSO d-6 for 1H NMR analysis on a Bruker Avance III 600

MHz spectrometer. Resultant spectra were standardized, normalized and processed with Infometrix Pirouette® software. Initial results show that we were able to classify healthy and sympthomatic samples with a variance of PC1: 60.85%; PC2: 28.75% and PC3: 3.70%. Further analysis are currently being made for a wider statistical significance. Keywords: Citrus sinensis, Liberibacter asiaticus, Huanglongbing, plant-pathogen interaction, metabolomics, 1H NMR." INBEB G45. ANÁLISE E DETECÇÃO DE MUTAÇÕES NO GENE TP53 EM CASOS DE CÂNCER DE MAMA EM MULHERES DO ESTADO DO RIO DE JANEIRO E ESTUDO DE SUA PROTEÍNA MUTANTE P53 EM LINHAGENS CELULARES DE CARCINOMAS MAMÁRIOS 1- MACHADO, Y.L.R.C.; 1- ALMEIDA DA SILVA, A. P.; 2- PORTARI, E.A.; 3- SILVA JL; 1- DE

MOURA GALLO, C.V. 1- Departamento de Genética, IBRAG, Universidade do Estado do Rio de Janeiro; 2-

Instituto Fernandes Figueira, Fundação Oswaldo Cruz; 3-Instituto de Bioquimica Medica, Universidade Federal do Rio de Janeiro

O câncer de mama é o tipo de neoplasia maligna mais freqüente entre mulheres, com incidência de mais de um milhão de novos casos/ano no mundo. O gene TP53 apresenta-se mutado em 50% dos carcinomas humanos e em torno de 25% no câncer de mama, cujas alterações localizam-se majoritariamente nos éxons 5-8, domínio de ligação da proteína com o DNA. Além disso, verificou-se que a proteína p53 quando mutada apresenta uma tendência à agregação, exibindo um comportamento dominante negativo. O objetivo deste trabalho foi detectar mutações neste gene, associando-as com a agressividade do tumor, obtido dos prontuários médicos, e observar o comportamento da proteína p53 em linhagens celulares de carcinomas mamários. Foi realizada a análise da presença de mutações nos éxons 4-10 do TP53, em amostras de câncer de mama de 98 pacientes do IFF-FIOCRUZ e de 68 pacientes do INCA. Para avaliar a existência de co-localização da proteína p53 com agregados protéicos, utilizou-se linhagens celulares de carcinomas mamários e anticorpos A11 e DO-1 para reconhecimento de agregados protéicos e da p53, respectivamente. Após extração de DNA genômico, realizaram-se as técnicas de PCR e seqüenciamento automático para verificação da presença de mutações. As linhagens MDA-MB231 (p53 mutante p.R280K), T47-D (p53 mutante p.L194F), MCF-7 (p53 selvagem) e 21-NT (p53 nula) foram cultivadas em meio de cultura contendo 10% FBS, a 37oC e atmosfera de 5% de CO2, tratadas com os anticorpos conjugados a fluoróforos. Dos casos analisados, mutações no gene TP53 foram detectadas em 18 (20,45 %) dos 98 casos do IFF, sendo 12 (66,66 %) do tipo missense; já para os casos do INCA, foram detectadas apenas 3 mutações (4,41%), sendo 2 do tipo missense. Nas linhagens celulares, foi observada por análise em microscopia confocal co-localização entre a p53 mutante e os agregados protéicos, onde observaram-se diferentes perfis de agregação.

FAPERJ


Mestrandos

M1. O AUMENTO DA FUSÃO E PROLIFERAÇÃO DE MIÓCITOS MODULADO PELA GSNO-R

1- Yamashita, A.M.S., 1- Figueiredo-freitas, C., 2- Possidonio, A.C., 2- Soares, C.P., 2- Mermelstein, C.S., 1- Sorenson, M.M.

1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil, 2- Departamento de histologia e embriologia, universidade Federal do

rio de Janeiro, rio de janeiro, Brasil. "O óxido nítrico (NO) é produzido fisiologicamente no músculo durante a contração. Uma das vias de sinalização por NO encontra-se a oxidação dos grupos tiol em proteínas, formando SNO-proteína. Para controlar o estado redox intracelular, todas as células sintetizam antioxidantes como a glutationa (GSH). Excesso de NO pode levar a um aumento na produção de S-nitrosoglutationa (GSNO). Uma enzima importante para controlar os níveis de GSNO é a S-nitrosoglutationa redutase (GSNO-R), que reduz GSNO para GSH promovendo a denitrosilação de proteínas. Diversos tecidos expressam GSNO-R, porém muito pouco se sabe sobre essa enzima no músculo esquelético. O objetivo é caracterizar a expressão e a atividade da GSNO-R no músculo esquelético (peitoral) de embrião de galinha (11 dias) e analisar o papel durante a diferenciação celular, usando um inibidor específico para GSNO-R (Sanghani, 2009). A cultura primária de galinha foi crescida por 24h, seguido por 48h de tratamento com DMSO (veículo) ou inibidor (10 ou 30µM) e o controle foi crescido por 72h. Foi medida a atividade enzimática da GSNO-R no homogenato de miócitos (Liu, 2001). Foi feito Western blot para GSNO-R das amostras de fibroblasto e miócito (72h e 96h). Imunofluorescência para núcleos DAPI, anti-GSNO-R e anti-desmina, permitiram quantificar o número e a espessura dos miotubos, número de células mononucleadas e multinucleadas (Image J Fiji) acompanhando a diferenciação. A atividade, expressão e imuno-marcação da GSNO-R na cultura primária de miócito foi detectada. Não foi encontrada diferença no número de miotubos entre os grupos. Um aumento da espessura dos miotubos tratados com inibidor sugerindo um aumento na fusão. O número de núcleos associado aos miotubos não aumentou, porém o número de células mononucleadas do grupo com inibidor aumentou 40%, sugerindo um aumento da proliferação. Os dados sugerem que a atividade da GSNO-R pode modular a proliferação e fusão celular." INBEB, FAPERJ, CNPq, CAPES M2. EFEITOS DA ADMINISTRAÇÃO SISTÊMICA DE GUANOSINA EM MODELO DE LESÃO NO NERVO ÓPTICO

1- SILVA-JUNIOR, AJ; 1- MESENTIER-LOURO, LA; 1- ZAVERUCHA-DO-VALLE, C; 1- MENDEZ-OTERO, R e 1- SANTIAGO, MF.

1- Institudo de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro As lesões no nervo óptico podem levar desde a perda parcial da visão à cegueira total. Estas lesões afetam especialmente as células ganglionares da retina (CGRs). Nosso grupo obteve resultados positivos com a terapia celular utilizando tanto a população de células mononucleares da medula óssea, quanto as células-tronco mesenquimais da medula óssea., após lesão no nervo óptico. Outra terapia potencial é a administração sistêmica de guanosina. O tratamento com guanosina tem demonstrado efeito neuroprotetor em diferentes modelos. Neste trabalho nós investigamos se a administração sistêmica de guanosina tem um efeito neuroprotetor e regenerativo sobre as CGRs após lesão no nervo óptico. Ratos da variedade Lister-Hooded passaram

por um modelo de lesão no nervo óptico seguida de administração intraperitonial de guanosina (7,5 mg/kg) ou veículo, separados em grupos que receberam uma única injeção, ou uma injeção diária por 7 dias. Quatorze dias após este procedimento foram analisadas a sobrevivência das CGRs por imunomarcação para Tuj-1 (β-III-tubulina) ou Brn3a em montagens planas de retinas, e sua extensão axonal por imunomarcação para GAP-43 no nervo óptico. Nossas análises não demonstram diferenças significativas na sobrevivência das CGRs nos grupos de animais tratados em comparação aos grupos não-tratados, em todos os protocolos utilizados. Foi observada diferença significativa no número de axônios se estendendo além de 1,5mm do sítio de lesão no grupo que recebeu uma única injeção i.p. de guanosina em comparação ao grupo não-tratado. A avaliação da extensão axonal nos grupos que receberam 7 injeções ainda está em curso. Até o presente momento, pode-se concluir que o tratamento utilizando estas doses de guanosina não foi eficaz para aumentar o número de CGRs sobreviventes após a lesão no nervo óptico.Entretanto, as células sobreviventes são capazes de emitir um maior número de axônios através do nervo óptico em regeneração. INBEB, FAPERJ, CNPq, CAPES M3. BIOLOGICAL BEHAVIOR OF MESENCHYMAL STEM CELLS ON POLI-Ε-CAPROLACTONE FILAMENTS AND A POTENTIAL STRATEGY FOR TISSUE ENGINEERING OF PERIPHERAL NERVES

1- CARRIER-RUIZ, A.; 1- VASCONCELOS-DOS-SANTOS, A.; 1- CARVALHO, L.R.P.; 1- MENDEZ-OTERO, R.; 1- RIBEIRO-RESENDE, V.T.

1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro "Peripheral human nerves fail to regenerate across long tube implants, probably because implants lack the microarchitecture of native nerves. Bone marrow mesenchymal stem cells (MSC) contribute to the regeneration of the peripheral nervous system (PNS) in many aspects under cell therapies approaches. To optimize tubular implants by integrating artificial bands of Bungner and a biological component capable of enhancing that regenerative capacity, we study the interaction between poly-caprolactone (PCL) filaments and MSC. Rat’s MSC were placed on PCL filaments treated with plasma O2, poli-D-lysine and laminin. Cells were plated in three different concentrations, 2x10^5, 2x10^6 and 8x10^6 cells/ml, and, after 48h of incubation, the adhesion profile, viability and proliferation capacity were analyzed. We also plated rat’s Schwann cells at the concentration of 5x10^5 cells/mL on PCL filaments covered with MSC, 24h after the MSC’s plating, and let the co-culture for another 24h to analyze the feasibility of the system. Embryonic rat’s dorsal root ganglia (DRG) were plated in contact with PCL filaments, with or without MSC. The samples were incubated for 4 days then we analyzed the neurite extension of DRG’s neurons. MSC showed an alignment along the longitudinal microstructure, however, as we increased the concentration of cells, the rate of alignment gradually decreased. MSC showed high viability and their proliferation capability was not completely inhibited by the filaments. Schwann cells were able to adhere to the filaments plated with MSC maintaining also high viability. Neurites were able to grow and extend over the surface of the PCL filaments when they were previously covered with MSC. Data were analyzed using ANOVA with Neuman-Keuls post-test for multiple comparisons. We provide evidence for the interaction between MSC, Schwann cells and PCL filaments, as they can constitute a stable system permissive for neurite extension and possibly events for the regeneration of the PNS." CNPq, CAPES, FAPERJ, INBEB M4. STRUCTURAL ANALYSIS OF A VACCINE PLATFORM BASED ON MS2 VIRUS-LIKE PARTICLES


1- VICENTE, A.C.; 1- BARROSO, S.P.C.; 2- PEABODY, D.S.; 3- FERREIRA, D.F.; 4- DE MESQUITA, J.F.; 1- SILVA, J.L.; 1- 0LIVEIRA, A.C.

1- Inst. de Bioquímica Médica, IBqM-UFRJ, RJ, Brazil; 2- Department of Molecular Genetics and Microbiology, University of New Mexico; 3- Inst. de Microbiologia, IMPPG-

UFRJ, RJ, Brazil; 4- Universidade Federal do Estado do Rio de Janeiro. Virus-like particles (VLPs) can be considered as dense arrays of one or more repetitive subunits of a protein and this characteristic confers highly advantageous properties for their use as vaccines platforms. The platforms used here are VLPs of the bacteriophage MS2, an E. coli phage. We evaluated the structural stability of VLPs containing highly immunogenic peptides related to the infectious cycle of HIV-1 by submitting them to high hydrostatic pressure (HHP) and other chemical and physical denaturant agents and evaluating the changes by means of light scattering, intrinsic and extrinsic fluorescence and circular dichroism (CD). We analyzed the morphology of VLPs by transmission electron microscopy. In addition, the structure prediction of the coat protein with peptide insertions was done by homology modeling approach. The results obtained so far were performed with VLPs formed by a single chain dimer of the coat protein, the native coat protein, two constructions with the Flag epitope and with VLPs containing peptides of the extracellular loop of CCR5 cell co-receptor and the V3 loop of gp120 of HIV-1. The spectral center of mass and light scattering data indicate that there are differences in the structure and stability of VLPs with insertion of the epitopes, except for results using HHP, in which the construction with inserts showed the largest shift of the center of mass. CD measurements indicate no changes in secondary structure between dimer and native protein, but single chain constructs with Flag epitope had a different behavior. Coat proteins with peptide insertions show small RMSD when aligned with the native protein. Our results demonstrate that the VLPs assembled from coat protein containing peptides insertions behave differently from the ones assembled from native coat protein, however they showed structural stability under most of the conditions used, suggesting that these particles are very promising for application as a vaccine platform. CAPES-FAPERJ-CNPq-PRONEX-INBEB M5. METABOLIC CHANGES IN A GLIOBLASTOMA CELL LINE INDUCED BY IRON

Mendonça,A.P.M; Amoedo, N.D.;Oliveira, M.F. Instituto de Bioquímica Médica, IBqM-UFRJ, RJ; Brasil

"Introduction: The intracerebral hemorrhage (ICH) represents one of most severe events in the CNS with poor prognosis and outcome. ICH is caused by the extravasation of blood into the brain parenchyma as a result of blood brain barrier disruption. Blood derived products (BDPs) such as hemoglobin, heme and iron, are powerful toxic components which are released and metabolized in cerebral parenchyma after the ICH onset. Here, we investigated the effects of iron on energy and redox metabolism in U-87 glioblastoma cells. Material and methods: U-87 cells were cultured for 24 hours in the presence of iron (up to 50 mM) and several cellular and biochemical parameters were evaluated. Cell viability was assessed by the MTT reduction assay. Redox imbalance was determined by measuring the TBARS levels, whereas mitochondrial physiology assessment was carried out by measuring the oxygen fluxes on intact cells in glucose containing medium by high-resolution respirometry (O2k). Citrate synthase activity was determined spectrophotometrically. Results and discussion: Iron promoted lipid peroxidation without affecting cell viability. Mitochondrial content, determined by the activity of citrate synthase, was also not affected by iron up to 50 μM. High-resolution respirometry analyses showed that iron caused an overall reduction of the oxygen consumption rates, regardless the mitochondrial metabolic states. Conclusion: Iron triggered a signaling cascade that promote functional mitochondrial remodeling in a

glioblastoma cell line, which may represent a cellular survival mechanism in response to the BDPs during the ICH events. Keywords: mitochondria, oxidative stress, stroke" Faperj, CNPq M6. ANÁLISE DO PROCESSO DE AGREGAÇÃO DA ESFINGOSINA POR MEDIDAS DE RELAXAÇÃO E EFEITO DE TROCA H/D.

1- ANDRÉ ROBERTO DE OLIVEIRA FREDI; 1- LUZINEIDE W. TINOCO 1- Núcleo de Pesquisa de Produtos Naturais, Universidade Federal do Rio de Janeiro

A esfingosina (2S,3R,4E)-2amino-4-octadeceno-1,3-diol) é um derivado da cadeia de formação de esfingolipídios e atua como um mensageiro estando envolvida nos processos de inibição do crescimento celular e na estimulação da apoptose. A natureza anfifílica destes compostos leva a sua agregação, com a formação de micelas. A compreensão deste processo, em diferentes condições, pode contribuir para o entendimento da desordem no armazenamento lisossomal dos glicoesfingolipídios. Este trabalho teve como objetivo analisar o processo de agregação da esfingosina em função do pH através da análise dos espectros de RMN de 1H e de medidas do tempo de relaxação longitudinal. As amostras da D-eritro-esfingosina 1 mM (Sigma Aldrich), foram preparadas em solução tampão fosfato pH 5,0 e 7,2, em 10% de D2O e 100% D2O e analisadas por RMN [VNMRS-500 (Agilent) 499,8 MHz para 1H] a 25°C. A intensidade dos sinais e os valores de T1 sofrem uma significativa variação em função do pH e do percentual de D2O usado. Interessantemente, os sinais que, provavelmente, correspondem aos hidrogênios da amina (~8 ppm) somente são observados nos espectros adquiridos em 100% de D2O. Além disso, os hidrogênios da cabeça polar da esfingosina apresentam valores baixos de T1 que os da cadeia carbônica, principalmente na região da dupla ligação. Assim como o efeito de troca H-D, as medidas de T1 fornecem resultados opostos ao esperado. Uma vez que, os hidrogênios da cabeça polar estando de em uma região maior mobilidade deveriam ter valores de T1 mais altos. Isto sugere que o processo de troca H-D e os tempos de relaxação dos hidrogênios da cabeça polar devam ser mais bem investigados. Estes dados poderão fornecer informações importantes acerca do processo de agregação através da formação de ligações de hidrogênio intra e intermoleculares. CNPq, INBEB, FAPERJ, FINEP, CAPES M7. COMPARATIVE ANALYSIS OF DIFFERENT CHEMICAL FIXATIVES FOR SCANNING ELECTRON MICROSCOPY TO STUDY FUNGAL BIOFILM STRUCTURE FORMED BY CANDIDA ALBICANS.

1- FONSECA, B. B., 1- VILA, T.V.M., 2- ISHIDA, K., 1- ROZENTAL, S. 1- Laboratório de Biologia Celular de Fungos, Instituto de Biofísica Carlos Chagas Filho,

Universidade Federal do Rio de Janeiro. 2- Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo.

"Biofilms are frequently related to invasive fungal infections and are reported to be more resistant to antifungal drugs than planktonic cells. Biofilm cells are known to synthesize a polymeric extracellular matrix (ECM). In this work, we make a comparative analysis of different chemical fixatives for scanning electron microscopy (SEM) to study fungal biofilm. Candida albicans biofilms were formed on 5 mm sections of central venous catheters (CVC) and fixed by 3 different chemical fixatives: (a) Routine fixative method: glutaraldehyde (2.5%) formaldehyde (4%) in cacodylate buffer; (b) Sucrose fixative method: glutaraldehyde (2.5%) in cacodylate buffer, then a post-fixative with 0.2 M sucrose and 2 mM MgCl2; (c) Ruthenium red fixative method: glutaraldehyde (2.5%) and ruthenium red (0.5 mg/mL) in cacodylate buffer; all the samples are processed as routine and observed under a scanning electron microscope (FEI Quanta


250, Japan). Utilization of both routine and sucrose fixative methods allowed a good preservation of cell distribution inside the biofilm. However, the ECM was extracted from the biofilm. Although the sucrose fixative method seemed to preserve some ECM filaments interconnecting cells, the absence of a post-fixation step with OsO4 may be responsible for the reduced quality of images obtained from these samples. Ruthenium red fixative method leaded to the formation of a large amount of precipitate upon the biofilm structure. This result was confirmed by incubating uncultivated CVC and isolated C. albicans yeasts with ruthenium red as controls. Ruthenium red protocol was also adapted for lower concentrations (0,1mg/mL) but even so precipitates was formated. Routine and sucrose fixative methods were helpful to analyze cell distribution inside the biofilm but did not preserve the ECM. Unhappily, addition of ruthenium red to chemical fixative solutions did not improve biofilm ECM preservation. Other methods as cryothecniques should to be used to evaluate these structures." FAPERJ, CNPq, CAPES M8. IMMUNOMODULATORY EFFECTS OF AQUEOUS EXTRACT OF ROOT PLANT Physalis angulata ON CELL OBTAINED OF BONE MARROW SILVA, B. J. M 1,2; RODRIGUES, A. P. D. 1,2,3; FARIAS, L.H.S. 1,2; HAGE, A, A, P1,2.; SILVA,

E. O. 1,2 ¹ Laboratório de Biologia Estrutural/Laboratório de Parasitologia, Instituto de Ciências

Biológicas, Universidade Federal do Pará, Brazil. 2Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Brazil 3Laboratório de Microscopia, Instituto Evandro Chagas, secretaria

de vigilância em saúde, ministério da saúde, Belém, Pará, Brazil. "The bone marrow is a hematopoietic tissue that in the presence of cytokines and growth factors generates all circulating blood cells. These cells are important to protect the organism against pathogens and for establishing an effective immune response. Some studies in literature have been showing action of immunomodulatory effects of different products obtained of plant extracts. Thus, this study aim to evaluate the immunomodulatory properties of the root extract from Physalis angulata (REPa), a plant widely used in popular medicine. Bone marrow cells were obtained by flushing femurs, and maintained in cultures treated with REPa at a concentration of 100 µg/mL. It was observed by optical microscopy that REPa stimulates the process of cell adhesion, increase of cytoplasm, spreading ability, cytoskeleton alterations and high number of cytoplasmatic projections. Furthermore, REPa did not promote the proliferation of lymphocytes and polymorphonuclear leukocytes, however promotes increased the number of macrophages in the culture. Immunophenotyping was performed by immunofluorescence and flow cytometry. Labeling CD11b and F4/80, a marker specific for mononuclear phagocytes revealed that REPa seems to stimulate differentiation of bone marrow cells in macrophages. Labeling using a specific dendritic cells CD11c marker, showed that REPa did not stimulate differentiation of the treated cells into dendritic cells. No cytotoxic effect was observed in cells treated with REPa when compared to the untreated control. Thereby, these results demonstrate that REPa can promote the differentiation of bone marrow cells into macrophages in just 96 hours culture. These results demonstrate that REPa can be used as an immunomodulator agent. Keywords: Cell proliferation and differentiation, Bone marrow, Physalis angulata." Supported by CAPES, CNPq/UFPa, CNPq/MCT/CT-INFRA/CT-PETRO (Processo nº 620179/2008), MCT/CNPq/FNDCT/PROCAD-NF CAPES/FAPERJ. M9. TRIAGEM IN VITRO E PLANEJAMENTO IN SILICO DE INIBIDORES DE SUPERÓXIDO DISMUTASE DE TRYPANOSOMA BRUCEI (TbSOD)

1- BRITO,C.C.B.; 1- CASTILHO, M.S. 1- Faculdade de Fármacia, Universidade Federal da Bahia

A Doença de chagas, causada pelo Trypanosoma cruzi, ainda é um problema de saúde publica e uma das maiores causas de morbidade e mortalidade da América Latina. O sistema de defesa antioxidante é uma importante rota na sobrevivência deste parasita no hospedeiro e as enzimas chaves envolvidas nesse processo são vitais para o crescimento e desenvolvimento do parasita. Por essa razão, a enzima superoxido dismutase (SOD), que dismuta radicais superóxido em oxigênio (O2) e peróxido de hidrogênio (H2O2), é um alvo interessante para o desenvolvimento de fármacos antichagásicos. Devido a identidade sequencial elevada, a enzima do parasita T. brucei pode ser utilizada como modelo para identificação de moléculas que inibam a enzima de T. cruzi. A fim de alcançar esse objetivo, realizou-se a avaliação biológica de 33 derivados de tiossemicarbazonas, sendo a fenantrenoquinona -4 fenil tiossemicarbazona identificada como um inibidor de Tbsod em ensaio de dose única. O IC50 calculado para essa molécula foi em torno de 200µM. Para verificar o modo de interação proteina-ligante utilizou-se a técnica de acoplamento molecular, através dos programas SurflexDock e o AutoDock Vina. Os resultados do acoplamento sugerem que a Fenantrenoquinona 4-fenil tiossemicarbazona interage num bolsão que não faz parte do sítio ativo de Tbsod, sugerindo um modo de inibição não competitiva. As informações provenientes dos experimentos in silico guiarão o planejamento de inibidores mais potentes que possam ser considerados como compostos protótipos para o desenvolvimento de fármacos contra a doença de Chagas. INBEB M10. TÉCNICAS DE MICROSCOPIA COMO FERRAMENTA PARA ESTUDO DO DNA MITOCONDRIAL DE TRIPANOSOMATÍDEOS

1-CAMILA S. GONÇALVES, 1-MARCELO ZOGOVICH, 1-GABRIEL FELIPE B. D. da SILVA, 1-LUANA PORTELLA, 1-DANIELA L. GONÇALVES, 1,2-WANDERLEY de SOUZA e 1-

DANIELLE P. CAVACALTI 1-Instituto Nacional de Metrologia, Qualidade e Tecnologia, INMETRO; 2-Instituto de

Biofísica Carlos Chagas Filho, UFRJ Os tripanosomatídeos possuem estruturas peculiares, como o cinetoplasto, que está localizado dentro da matriz mitocondrial e contêm parte do genoma do protozoário. O DNA mitocondrial ou kDNA é a mais complexa e incomum forma de DNA extranuclear encontrada na natureza, sendo composto de milhares de moléculas circulares, os minicírculos e os maxicírculos, que se encontram topologicamente relaxadas e interligadas formando uma única rede. O objetivo do nosso trabalho é caracterizar ultraestruturalmente o cinetoplasto e a rede de kDNA de diferentes membros da família. Análises por Microscopia Eletrônica de Transmissão (MET) demonstram que, na maioria dos tripanosomatídeos, as fibrilas de kDNA apresentam-se bastante compactadas e contidas em um cinetoplasto em forma de disco. Já na forma tripomastigota de T. cruzi e nos tripanosomatídeos que contêm endossimbionte, as fibrilas de DNA estão organizadas em um arranjo mais largo e frouxo, que preenche toda a matriz do cinetoplasto. Além disso, nosso grupo desenvolveu uma metodologia para analisar a rede de kDNA isolada utilizando a Microscopia de Força Atômica (AFM). Os resultados obtidos mostram que as redes de kDNA de Crithidia fasciculata, Angomonas deanei, Strigomonas culicis, Leishmania amazonensis e Trypanosoma cruzi apresentam um padrão de organização muito similar, sendo possível identificar minicírculos interligados e um agrupamento de moléculas de DNA na periferia da rede, formando rosetas. Recentemente, utilizamos o AFM para analisar a ação de drogas intercalantes de DNA sobre a rede e entender melhor o mecanismo de ação destes


compostos. O próximo passo do trabalho é obter modelos tridimensionais do cinetoplasto dos diferentes tripanosomatídeos utilizando a tomografia de elétrons. INBEB, FAPERJ, CNPq. M11. EXPLORING THE ULTRASTRUCTURE AND FUNCTION OF THE CYTOSTOME-CYTOPHARINX COMPLEX OF TRYPANOSOMA CRUZI EPIMASTIGOTES

1,2- ALCANTARA, C.L.; 1,2- VIDAL.J.C; 1,2,3- DE SOUZA, W.; 1,2- CUNHA-E-SILVA, N. 1- Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373,

bloco G subsolo, Cidade Universitária, Ilha do Fundão, Rio de Janeiro 21941-902, Brazil. 2- Instituto Nacional de Biologia Estrutural e Bioimagem (INBEB), 3- Diretoria de

Programas, INMETRO" The cytostome is an aperture at the plasma membrane close to the flagellar pocket (FP) followed by a profound invagination called cytopharinx. This complex is the major portal for endocytosis in T. cruzi epimastigotes. Data from literature has shown that this region presents a prominent glycocalix continuous to the preoral ridge (POR), the membrane domain between the cytostome and the FP, and an uncharacterized set of microtubules (MTs) and vesicles along the cytopharinx. Little is known about its detailed structure and the correlation with the endocytic function. In this work we focused on the 3D structure of the cytostome-cytopharinx complex to describe how endocytic cargo passes through and leaves it. Epimastigotes were submitted to endocytosis of transferrin-gold particles with short incubations times. The material was processed to electron microscopy and serial semi-thin sections were observed by electron tomography. The structures of interest were segmented using appropriated software. We observed seven MTs supporting the opening of cytostome and the cytopharinx. These MTs could be separated in two sets: a quartet coming from the FP that also sustains the POR and a triplet coming from the subpellicular microtubules. The MTs arrangement around the cytopharinx leaves a free side, where aligned vesicles were seen. As the invagination becomes deeper and thinner, two MTs finish, one from the quartet and one from the triplet. Moreover, the MTs continue even beyond the end of the cytopharinx and accompany vesicles and tubules that seem to be originated from the cytopharinx. We also used horseradish peroxidase as a tracer to follow the cytopharinx in FIB-SEM preparations. We measured the cytopharinx length in 25 whole cells and found out that the length is independent of cell cycle or differentiation stage. Together these data provides a new vision about the structure and dynamics of endocytic pathway of epimastigotes. INBEB, CNPQ, CAPES M12. KOJIC ACID , A SECONDARY METABOLITE OBTAINED FROM ASPERGILLUS FUNGI, INDUCES THE DIFFERENTIATION OF MONONUCLEAR CELLS OBTAINED FROM MOUSE BONE MARROW IN VITRO ALMEIDA, C. M. 1,4; SILVA, B. J. M 1,4; RODRIGUES, A. P. D. 1,3,4; FARIAS, L.. H. S. 1,3;

HAJE, A. A. P. 1,4; SILVA, E. O. 1,4 Laboratório de Biologia Estrutural/Laboratório de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal do Pará, Brasil. ² Laboratório de Neuroinflamação,

Instituto de Ciências Biológicas, Universidade Federal do Pará, Brasil. 3 Laboratório de Microscopia, Instituto Evandro chagas, Pará, Brasil. 4 Instituto Nacional de Ciência e

Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Brasil

"Bone marrow is a tissue responsible for hematopoiesis. Cell proliferation and differentiation depend of a complex interaction between the stromal cells, cytokines, growth factors and extracellular matrix that compose the bone marrow

microenvironment. The most of the cells generated in the bone marrow give rise to circulating blood cells that play a important role in the immune response against pathogens. In this context, the research for drugs that enhance the innate immune response is needed to restore the homeostasis and the immune response in immunocompromised patients. Thus, in this study we evaluated the immunomodulatory effects of Kojic Acid (KA), a secondary metabolite obtained from Aspergillus fungi, on the bone marrow cells of mice. These cells were obtained by flushing femurs, and maintained in cultures treated with KA at the concentration of 50 and 100 µg/mL at four different times of culture. Analysis by optical microscopy revealed that KA promoted increased cell adhesion, spreading ability and high number of cytoplasmatic projections and vacuoles in cytoplasm of bone marrow mononuclear cells. These cells seem represent mature macrophages as indicated by the presence of mature-cell markers like CD11b (specific marker of mononuclear phagocytes). In addition, the absence of CD11c (a dendritic cell marker) showed that KA did not stimulate the differentiation of the treated bone marrow cells into dendritic cells. Also, no cytotoxic effects were observed in cells treated with KA when compared to the untreated bone marrow cells. Thus, these results suggested that KA can promote the differentiation of mononuclear cells obtained from mouse bone marrow in vitro. Keywords: Cell proliferation and differentiation, Kojic Acid, Bone marrow." Supported by CAPES, PRONEX/FAPESPA/CNPq, CNPq/MCT/CT-INFRA/CT-PETRO (Processo nº 620179/2008), MCT/CNPq/FNDCT/PROCAD-NF CAPES/FAPERJ. M13. DESCRIPTION OF A NOVEL TRANSTHYRETIN VARIANT WITH CARDIAC INVOLVEMENT: A STUDY OF STRUCTURAL PREDICTION 1-FERREIRA, P. S.; 1-VAREJÃO, N.; 1-LIMA, C;1-SANT'ANNA, R. O.;2-CALDEIRA, C. M.;1-FRANKLIN, D. W.;3-CRUZ, MARCIA WADDINGTON; 1-FOGUEL, D.

1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro.2-LABORATÓRIO SONDA-UFRJ; 3- HOSPITAL UNIVERSITÁRIO CLEMENTINO FRAGA FILHO-

UFRJ Mutations in the TTR gene are known to destabilize the structure of protein and facilitate the aggregation process causing an amyloidosis which can be characterized by the involvement of peripheral nerves, cardiac function and other disorders. Here we report a patient from Santa Catarina, state in the south region of Brazil, and his family is from Swedish/German origin, with a rare mutation in exon 2 of TTR gene where we have a substitution of a Ala for a Asp at the codon 19, causing a severe compromise of cardiac function characterizing the Familial Amyloidotic Cardiomyopathy (FAC). To predict the stability of this mutant we use bioinformatics tools how FoldX and PDBSum to analyze the thermodynamic stability of this mutant. We predicted that the tetramers of A19D presented a decreased thermodynamic stability, when compared to the WT-TTR, and is more amyloidogenic than V30M tetramers. The PDBSum analyses demonstrated that the strongest interface of TTR (AB) loses two hydrogen bonds and drastic changes in the orientation of some amino acids in C2 axis can facilitate the dissociation process in this interface. Taken together, all the structural changes imposed by the mutation A19D to the tetramers resulted in a destabilized protein that presents alterations in the monomer-monomer and dimer-dimer interface. Indeed the patient that presents this mutation has a severe cardiomyopathy which developed very fast in a short period of time. We describe for the first time in Brazil this mutation. INBEB, FAPERJ, CAPES, CNPq M14. CONFORMATIONAL ANALYSIS OF 9,10-PHENANTHRENEQUINONE GUANYLHYDRAZONE BY NUCLEAR MAGNETIC RESSONANCE AND MOLECULAR MODELING.


1 -SIMOES, C.; 2 -AZEREDO, S.O.F.,3- VILLAR, J.D.F 1,2,3 - Instituto Militar de Engenharia, Seção de Engenharia Química – Praça General

Tibúrcio, 80, 22290-270, Praia Vermelha, Rio de Janeiro, RJ, Brasil. The novel compound 9,10-phenantrenequinone guanylhydrazone is a promising molecule with potential biological activity such as antibiotic and anticancer (through interactions with DNA). This is possible because yours structural features (fused aromatic rings, Intracellular hydrogen bond with an oxygen atom and amines terminals). The structure has a rotation angle that allows changes in amine terminal nevertheless was observed there is a minimum energy to obtain this rotation angle and this occurs when the compound is submitted to a specific temperature. The objective of this work is study the 9,10-phenantrenequinone guanylhydrazone conformational changes versus temperature via NMR and molecular modeling to calculate the minimum energy value to achieve the rotation angle. The experimental conformational changes were studied via Nuclear Magnetic Resonance (Spectrometer Varian 600 MHz) through 1H experiments to elucidate the structure after submitted it to a different temperatures. The theoretical energy values were obtained through molecular modeling studies (Spartan 06). The NMR study showed that the rotation angle is achieved when the compound is submitted to a temperature above 34 °C, and this data was confirmed by quantum mechanics. This information is important to understand the form of interaction of this compound with DNA and with enzymes of microorganisms." INBEB M15. SERINA PROTEASES EM CANDIDA ALBICANS: IDENTIFICAÇÃO E PROCURA POR FUNÇÃO BIOLÓGICA

Gonçalves D.S1, Braga-Silva L.A1, Gandra R.M1, Seabra S.H2, Sodré C.L1, Santos A.L.S1 1 Laboratório de Estudos Integrados em Bioquímica Microbiana (LEIBM), UFRJ -

Universidade Federal do Rio de Janeiro, Instituto de Microbiologia Paulo de Góes, Centro de Ciências da Saúde - Bloco E-subsolo 2 Universidade Estadual do Estado do Rio

de Janeiro- UEZO "Introdução: O aumento expressivo das infecções fúngicas nas últimas décadas pode ser atribuído ao grande número de casos de pacientes imunocomprometidos / imunossuprimidos. Candida albicans é responsável por 43% das infecções causadas por leveduras, fato atribuído a seus fatores de virulência e sua capacidade de se adaptar a diversos nichos ecológicos. Nosso grupo descreveu a secreção de serina peptidase em C. albicans, capaz de degradar componentes proteicos essenciais do hospedeiro. Objetivos: Avaliar a presença de serina protease secretada em diferentes isolados clínicos e estudar os efeitos de diferentes inibidores de serina protease sobre múltiplos aspectos da biologia celular e virulência de C. albicans. Resultados: Sobrenadantes de cultivo de seis diferentes cepas de C. albicans, cultivadas em meio BHI por 48h a 37°C, foram concentradas 40 em sistema AMICON e analisados em gelatina-SDS-PAGE. Dois perfis de protease foram detectados: (1) 90kDa e 50kDa (cepas ATCC10231, ATCC38688, 2A e 72) e (2) 50kDa (ATCC24433 e 11). A protease de 50kDa foi inibida por PMSF, um inibidor de serina proteases. A cepa ATCC10231 foi selecionada para os testes com inibidores de serina protease. Dos inibidores de serina proteases (n=7) testados a 100 M, somente TPCK e TLCK foram capazes de inibir significativamente a proliferação celular em 100% e 62% respectivamente. O tratamento de C. albicans (103 leveduras) com TPCK gerou um IC50 de 11,5µM. A inibição da proliferação também foi dependente da densidade celular. A análise ultraestrutural evidenciou alterações na superfície das leveduras bem como células rompidas. TPCK apresentou uma redução significativa na diferenciação (transformação de levedura a tubo germinativo) e na formação do biofilme. Conclusão: Os resultados sugerem a importância da serina protease na

biologia celular de C. albicans, uma vez que funções como crescimento e diferenciação foram sensivelmente alterados quando expostas a inibidores de serina proteases. Suporte Financeiro: ." CNPq, CAPES & FAPERJ M16. EVALUATION OF ANTI-INFLAMMATORY POTENTIAL OF DIETHYLCARBAMAZINE COMPARED TO CELECOXIB IN MODEL OF LIVER INFLAMMATION INDUCED BY ALCOHOL 1-RODRIGUES, G.B., 1-ROCHA, S.W., 1-SILVA, B.S., 1-GOMES, F.O.S., 1-BARBOSA,K.P.S.,

1-RIBEIRO, E.L., 1-SANTOS, L.A.L.M., 1-FRANCA,M.E.R., 1,2-PEIXOTO,C.A. 1-Centro de Pesquisas Aggeu Magalhães – FIOCRUZ, Recife; 2-Centro de Tecnologias

Estratégicas do Nordeste - MCT, Recife. "Iintroduction: The Diethylcarbamazine (DEC) presents several different biochemical effects on enzyme systems and potential anti-inflammatory properties. The Celecoxib is part of the class of nonsteroidal anti-inflammatory drugs (NSAIDs). This study aims to evaluate the potential anti-inflammatory of DEC compared with celecoxib in liver inflammation induced by alcoholism in C57BL/6J mice wild. Materials and Methods: Solutions of DEC and celecoxib (concentration 50 mg / kg both) were administered by gavage for 12 days. Forty strain C57BL/6J male mice were divided into four groups: (I) control group that received only water, (II) alcoholic group, (III) alcohol + DEC (50 mg / kg) and (IV) alcohol + celecoxib group (50 mg / kg). Ethanol was supplied in the drinking water at crescent concentrations of 10%, 15% and 20%. The liver fragments were processed according to routine protocol and stained with hematoxylin-eosin (HE) and picro-sirius red for histopathological analysis and evaluation of collagen fibers. Immunohistochemistry was performed for markers: IL-1β (1:100, Genway), IL-17 (1: 100, Abcam) and malondialdehyde (1:50, Abcam). Results/Discussion: In alcoholic group several histological changes were observed, which were reduced in the groups III and IV. By picro-sirius staining was noted increased production of collagen in alcoholic group (II) compared to control (I), and this production was decreased only in group + DEC alcohol (III). By immunohistochemistry analysis intense staining of all inflammatory markers in group II was observed. There was a significant decrease in labeling of these markers in groups treated with DEC (III) and celecoxib (IV). Conclusion: Due to lack of efficacies therapies for the treatment of advanced stages of alcoholic liver disease, DEC shows up as a possible therapeutic alternative for the treatment of the condition compared to other drugs used already. Key-words: Diethylcarbamazine, inflammation, celecoxib." INBEB, CAPPES M17. ASPECTOS ULTRAESTRUTURAIS DA INFECÇÃO POR TOXOPLASMA GONDII EM INTESTINO DE FELINOS

VERAS, G. M¹. DUBEY, JP, DE SOUZA, W¹, ATTIAS, M.¹, ¹ Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,

Brasil. A toxoplasmose é uma doença de distribuição mundial causada pelo protozoário Toxoplasma gondii, um parasito intracelular obrigatório capaz de invadir e infectar qualquer célula nucleada de todos os animais homeotermos. A transmissão da doença pode ocorrer por via placentária, pela ingestão de carne crua ou mal cozida contendo cistos teciduais e ainda por alimentos e água contaminada com oocistos esporulados. O ciclo sexuado deste parasito acontece apenas nos felinos com a formação de oocistos, que são eliminados junto com as fezes e liberados no ambiente, onde se tornam infecciosos. Neste trabalho objetivamos visualizar por microscopia eletrônica de varredura as formas enteroepiteliais no intestino dos felinos, recorrendo à criofratura destes tecidos. Para isto, fixamos nossas amostras com glutaraldeído, pós-fixamos com


tetróxido de ósmio e ferrocianeto de potássio e desidratamos em uma bateria crescente de etanol. A seguir, congelamos em nitrogênio líquido e criofraturamos longitudinalmente o epitélio intestinal e finalmente, secamos no ponto crítico. As primeiras observações mostraram a presença do parasito em diversos estágios exclusivos da porção mais superficial das vilosidades intestinais, local em que ocorre a descamação do epitélio. Merozoítas, esquizontes, gamontes e oocistos puderam ser observados. INBEB, FAPERJ, CNPq M18. EFFECTS OF 5-HYDROXY-2-HYDROXYMETHYL-GAMA-PYRONE (HMP) IN FILAMENTOUS FUNGAL Curvularia pallescens 1- PEREIRA, J.A.L.; 1,2,4 - RODRIGUES, A.P.D.; 1,4 - FARIAS, L.H.S.; 1- OLIVEIRA, D.M.S.; 3-

SANTOS, A.S.; 1,4- SILVA, E.O. 1 Laboratório de Parasitologia e Biologia Estrutural, Instituto de Ciências Biológicas,

Universidade Federal do Pará, Brasil; 2 Instituto Evandro Chagas, Laboratório de Microscopia Eletrônica; 3- Universidade Federal do Pará, Laboratório de

Desenvolvimento e Planejamento de Fármacos; 4- Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro,

Ilha do Fundão, Brasil. "The species Curvularia pallescens is a entophytic fungi and producer of melanin that occasionally causes a variety of human infections. Nowadays, there are some studies about antifungi effects of bioproducts which have low toxicity. The hydroxy-2-hydroxymethyl-γ-pyrone (HMP) is a secondary metabolite synthesized by some species of fungi from Aspergillus genera. The HMP has several applications mainly as tyrosinase inhibitor, enzyme that works in biosynthetic pathway for melanin formation in various mammalian and fungi cells. This study evaluated the HMP effect in C. pallescens morphology cultivated and treated with 25, 50, 100 e 200 µg/ml of HMP. The morphological analysis by optical microscopy, scanning electron microscopy and transmission electron microscopy of treated cells showed accumulation of many cytoplasmic vesicles like lipid droplets as well external vesicles. In addition, it was showed conidia became wilted after HMP treatment. These results suggested HMP caused many morphological alterations in C. pallescens that could be involved in fungi death. Further studies are needed to identify the mechanisms that induce these alterations. Keywords: HMP; Fungal; Morphological alterations; Curvularia pallescens." CAPES, CNPq/UFPA, CNPq/MCT/CT-INFRA/CT-PETRO (Processo nº 620179/2008), MCT/CNPq/FNDCT/PROCAD-NF CAPES/FAPERJ. M19. ENVOLVIMENTO DO FATOR DE TRANSCRIÇÃO NFAT NA DOENÇA DE PARKINSON: CONTRIBUIÇÃO PARA A INFLAMAÇÃO E APOPTOSE.

1- DE FREITAS, J.A; 1- ROBSS, B.K.; 1- FOGUEL, D. 1- Instituto de Biquímica Médica

"A Doença de Parkinson (DP) é uma doença neurodegenerativa, progressiva e irreversível associada a um déficit da função motora. Em termos patológicos, a DP é descrita através da perda de neurônios dopaminérgicos. O mecanismo que leva a perda de função dos neurônios na DP consiste de um acúmulo anormal de uma proteína denominada α-sinucleína e posterior formação de agregados proteicos intracelulares chamados de corpúsculos de Lewy. Estudos demonstraram que agregados da α-sinucleína são capazes de induzir influxo de Ca+2 e levar a ativação da fosfatase calcineurina A (CnA). Aparentemente, tanto o influxo de Ca+2 quanto a ativação da CnA estão fortemente ligados com os processos de neurodegeneração. Apesar do fator de transcrição nuclear de célula T ativada (NFAT) ser um dos alvos da ação da via de

sinalização de Ca2+/CnA, pouco sabe-se sobre sua contribuição para a DP. As proteínas NFAT regulam diretamente a expressão de genes envolvidos no controle da morte celular por apoptose como Fas-L, TNF-α, c- Nur-77, FLIP, TRAIL, assim como genes envolvidos no processo inflamatório como IL1β, Cox-2, TNF-α, MCP1, iNOS. Tendo em vista que a desregulação dos processos de inflamação e de morte celular programada das células do sistema nervoso possui um papel central na neurodegeneração e que um dos eventos importantes para o processo é o aumento do Ca2, o objetivo principal do projeto é avaliar o envolvimento do NFAT no processo de neurodegereração induzido pelos agregados de α-sinucleina. Para isso serão avaliados a capacidade do agregado da α-sinucleína de ativar o fator de transcrição NFAT, através de ensaios que avaliam os processos de desfosforilação, transativação e translocação do NFAT, além de determinar a relevância da ativação da proteína NFAT para os processos de morte celular e desintegração de neuritos e por fim caracterizar quais genes são ativados pelo NFAT em modelo de DP." INBEB, CNPq, FAPERJ M20. DIETILCARBAMAZINA (DEC) CONFERE PROTEÇÃO ANTI-INFLAMATÓRIA EM MODELO MURINO DE PLEURISIA INDUZIDA POR CARRAGENINA SANTOS, L.A.M.; 1- RIBEIRO, E. L.; 1- FRAGOSO, I. T.; 1- BARBOSA, K.P.S.; 1- DONATO, M.

A.; 1- ROCHA, S.W.S.; 1- OLIVEIRA, W.B.; 1- FRANÇA, M.E.R.; 1- SILVA, A.K.S.; 1- RODRIGUES, G.B.; OLIVEIRA, F.G.; SILVA, B. S.; 1,2 - PEIXOTO, C.A;

1- Centro de Pesquisas Aggeu Magalhães 2- Centro de Tecnologias Estratégicas do Nordeste

A Dietilcarbamazina (DEC) é um fármaco utilizado no tratamento da filariose linfática. Apesar de seu longo período de utilização, desde 1947, seu mecanismo de ação permanece obscuro. Alguns estudos mostram que a DEC detém provável ação anti-inflamatória por interferir sobre o metabolismo do ácido araquidônico e atuar no sistema imune do hospedeiro. Nesta perspectiva, a DEC pode ser útil no tratamento da inflamação aguda pulmonar, grave problema de saúde mundial, segundo a OMS. O objetivo deste trabalho foi avaliar o efeito protetor da DEC sobre o dano tecidual, a produção de mediadores pro-inflamatórios e infiltração leucocitária em modelo murino de pleurisia induzida por carragenina. Metodologia: Os camundongos foram distribuídos em três grupos experimentais; Grupo 1- Controle SHAM (n=10); Grupo 2- Carragenina CAR (n=10); Grupo 3- Dietilcarbamazina, DEC (50 mg/kg dissolvida em água, n=10). Os animais do grupo 3 receberam o tratamento acima por três dias consecutivos antes da administração de carragenina intrapleural (CAR 1%, i.pl.); O grupo SHAM, recebeu uma injeção de salina 0,9% i.pl.; 4hs após a indução, os animais foram eutanaziados em câmara de CO2. Em seguida, recolheu-se o exsudato pleural para quantificação dos níveis de óxido nítrico (NO) e contagem celular total. Os fragmentos do pulmão foram processados para análise histológica, imunohistoquímica (iNOS, IL-1β). O resultado sobre a análise histológica demonstra o efeito protetor da DEC sobre as alterações teciduais, com redução de áreas de dano alveolar difuso, acompanhada de significativa redução da expressão de iNOS e IL-1β (p < 0,05) quando comparados ao grupo CAR. Paralelamente, houve significativa redução dos níveis de NO (p <0,05) e recrutamento de leucócitos no grupo DEC (p < 0,001) confirmando o efeito anti-inflamatório da DEC neste modelo experimental. FACEPE, INBEB, CNPQ M21. INVESTIGATION ON THE BEHAVIOR OF TRICHOMONAS TENAX

1,2- Ribeiro, L. C. S. and 1- Benchimol, M. 1- Laboratório de Ultraestrutura Celular da Universidade Santa Úrsula; 2- Instituto de

Biofísica Carlos Chagas Filho, UFRJ


T. tenax is a commensal of the human mouth found under conditions of poor oral hygiene or associated with periodontal diseases. There are some controversies concerning the pathogenicity of this protozoan. T. tenax is very similar to Trichomonas vaginalis, a parasite that inhabits the human genitourinary tract. This parasite is known to induce the disruption of various mammalian cells. Currently, there is a discussion whether T. tenax would be a genetic variant of T. vaginalis. The aim of this study was to investigate the capable of T. tenax to cause damage to mammalian cells and to compare their cytotoxicity with T.vaginalis. For this, the detection and distribution of the proteins membrane of T. vaginalis were verified in T. tenax by immunofluorescence using the antibodies anti-P270 and anti-AP65. In addition, interaction assays of this protozoan with MDCK and HeLa cell lines were performed with the ratio of 5:1. The samples were examined by scanning electron microscope (SEM) and the cytotoxic effects of the protozoa were analyzed by MTT assay. Our results show several similarities between the species. T. tenax and T. vaginalis displays four anterior flagella and one recurrent flagellum and the same proteins membrane distribution. Moreover, the interaction assay showed that both trichomonad species were able to adhere on mammalian cells. Interestingly, T. tenax provoked injury to mammalian cells in the same way of T. vaginalis. Take together, these results indicated that T. Tenax could be a parasite, not a commensal, but further experiments are needed to confirm its pathogenicity. AUSU, FAPERJ, CNPq, PRONEX M22. THROMBIN TRIGGER MITOCHONDRIAL FUNCTIONAL REMODELING IN HUMAN PLATELETS INVOLVING TWO DISTINCT MECHANISMS

1-Garcia-Souza L.F., 2-Hottz E.D., 3-Morton K.A., 1-Santiago A.P.S.A., 2-Bozza F.A., 1-Oliveira M.F.

1-Instituto de Bioquímica Médica, UFRJ, Rio de Janeiro, Brasil. 2-Fiocruz, Rio de Janeiro, Brasil. 3-University of Utah, Salt Lake City, USA.

Introduction Evidence has indicated that pro-coagulant factors modulate platelet energy and redox metabolism pathways. However, the involvement of mitochondria during platelet activation remain poorly understood and was investigated in the present work. Material and Methods Human platelets were collected from healthy volunteers, isolated in M199 medium, and subsequently challenged with different thrombin concentrations. Several parameters were analyzed in activated platelets such as P-selectin externalization, respiration, reactive species generation, mitochondrial membrane potential, lactate and NO production. These were assessed by specific probes fluorescence detection by flow cytometry or by high-resolution respirometry. Results and Discussion When platelets were exposed to low doses of thrombin (0-0.3 IU/mL) increased oxygen consumption parallel to a reduction of mitochondrial membrane potential (Δψm) was observed. This effect was strongly inhibited by cyclosporin A, implicating the opening of mitochondrial permeability transition pore (MPTP) in this process. At higher thrombin doses (0.4-1.0 IU/mL) changes in both parameters were less pronounced, being undistinguishable from quiescent platelets at 1.0 IU/mL thrombin. In this condition, nitric oxide (NO) is implicated in the partial impairment of electron transport system (ETS) resulting in a decrease in maximum oxygen consumption. Maintenance of Δψm seems to involve the reversion of F1Fo ATP synthase activity, since the presence its inhibitor oligomycin collapsed the Δψm when the electron transport system is impaired by antimycin a. Conclusions Our data indicate that platelet mitochondrial function is affected by thrombin activation and seems to involve two distinct mechanisms, depending on the stimuli intensity. The dominant mechanism during low activation seems to be the mitochondrial permeability transition, whereas at high activation, reversibility of ATP synthase prevails. FAPERJ, CNPq and CAPES

M23. ENTENDENDO A TOXICIDADE DA PROTEÍNA Α-SINUCLEÍNA E O EFEITO DE POSSÍVEIS COMPOSTOS ANTI-PARKINSONIANOS

1- FERNANDES, L ; 1- MIRANDA, M.C.; 1 - FOGUEL, D.; 1- BRAGA, C. 1 - Instituto de Bioquimica Médica, Universidade Federal do Rio de Janeiro

Doença de Parkinson (DP) é a segunda desordem neurodegenerativa mais comum em humanos, caracterizando-se, principalmente, por perda de neurônios dopaminérgicos na substância nigra (SN) e presença de inclusões proteicas intracelulares, denominadas Corpos de Lewy (CL). Os CL são majoritariamente compostos por agregados amilóides de alfa-sinucleína (α-sin), uma proteína abundantemente expressa no cérebro e localizada nos terminais pré-sinápticos. A função fisiológica da α-sin ainda é desconhecida e seu papel na degeneração, observada na DP, foi ressaltado após serem identificados mutantes dessa proteína (A53T, A30P e E46K) envolvidos em uma forma precoce e hereditária da doença. Acredita-se que a agregação de α-sin desencadeie estresse oxidativo e processos inflamatórios, causando dano aos neurônios dopaminérgicos. No entanto, os mecanismos moleculares pelo qual a agregação contribui para a neurodegeneração e as vias de sinalização afetadas não foram elucidados. A selegilina (Sel) é um composto inibidor de Monoamina Oxidase B (MAO-B) utilizado na terapia da DP e a Edaravona (Ed) um composto antioxidante utilizado em isquemias cerebrais, ambos tem demonstrado efeito neuroprotetor em modelos da DP induzida por drogas como a 6-hidroxidapamina (6-OHDA) ou 1-metil-4-fenil-1,2,3,6-tetraidropiridina (MPTP). Além disso, estudos do nosso grupo demonstram que tanto Sel quanto ED modulam a agregação da α-sin in vitro e levam à formação agregados não tóxicos aos neurônios em cultura. No presente estudo, pretendemos elucidar o mecanismo de neuroproteção destes compostos contra a toxicidade de agregados de α-sin. Utilizaremos culturas celulares de neuroblastoma submetidas à pré-tratamento com esses compostos e, posteriormente, à presença de diferentes espécies oligoméricas da α-sin. Será, então, avaliado o papel do pre-tratamento com os compostos frente à toxicidade dessas da proteína através de testes de viabilidade celular como MTT, Tunel e Live/Dead. INBEB, FAPERJ, CNPq M24. EVALUATION OF ANTI-INFLAMMATORY ACTIVITIES OF DIETHYLCARBAMAZINE ON ACUTE HEPATIC INFLAMMATION IN MICE 1- FRANÇA, M.E.R. ; 1- ROCHA, S.W.S. ; 2- ARAUJO, S. M. R. ; 2- SILVA, J.C. ;1- BARBOSA,

K.P.S. ; 1- RODRIGUES, G.B. ;1- RIBEIRO, E.L. ; 1- SPINOLA, M.G.D. ; 1- OLIVEIRA A.C. ; 1,2- PEIXOTO, C.A.

1. Centro de Pesquisas Aggeu Magalhães- CpqAM/FIOCRUZ. 2. Centro de Tecnologias Estratégicas do Nordeste- CETENE/MCT.

Pharmacological studies showed that Diethylcarbamazine (DEC) interferes with the arachidonic acid metabolism, acting as an anti-inflammatory drug. This study investigated the anti-inflammatory effects of DEC on the CCl4-induced hepatotoxicity in Swiss mice. 20 male S. Webster mice were separated in groups: control group (C) (n=6), CCl4 group (CCl4) (n=6) and CCl4 + DEC 50mg/kg group (CCl4DEC) (n=8). DEC solutions were administered in dose 50mg/kg in drink bottle for 3 days before CCl4 administration. After 24h of intraperitoneal injection (i.p.) of CCl4 (0,1µl/kg, in olive oil), the animals were euthanized. Liver fragments were processed for light microscopy and immunohistochemical assays. Liver sections from control group presented normal liver architecture, showed well preserved tissue, composed of radially arranged cords of hepatocytes distributed in hepatic lobules. Histological analyses of the CCl4 group showed large areas of extensive necrose, mainly pericentral, loss of hepatic architecture, lipid accumulation, perivenular hemorrhage and inflammatory cell


infiltration. The treatment with DEC before CCL4 administration completely prevented liver necrosis reduced the inflammatory infiltrates and hemorrhagic foci. In fact, the DEC+CCL4 group showed minimal hepatic damage. Immunohistochemical analyses of the control group did not show substantial TNF- immunopositivity. On the other hand, strong TNF-expression was found in the CCl4 group. TNF- immunoreactivity was observed mainly in mononuclear infiltrates and necrotic areas, predominantly in pericentral areas. No apparent TNF-expression was detected in nonparenchymal cells. DEC+CCl4 group substantially reduced TNF-expression. Quantification staining analyses were performed using the image program Gimp 2.6 software. According to present results DEC can be used as a potential anti-inflammatory drug for acute hepatic inflammation. Further assays are in development in our laboratory in order to clarify the role of DEC on the molecular mechanisms involved in the inflammation signaling network. INBEB, FACEPE, CNPq. M25. EFFECTS OF 5-HYDROXY-2-HYDROXYMETHYL-GAMMA-PYRONE (HMP), A BIOPRODUCT OBTAINED FROM ASPERGILLUS FUNGI, ON HUMAN NEUTROPHILS IN VITRO 1,3 - FRADE, P. C. R.; 1,3 - COSTA, J. P.; 1,3,4 - RODRIGUES, A. P. D.; 1,3 - FARIAS, L.H.S.; 2

- SANTOS, A.S. ; 1,3 - SILVA, E.O. 1 - Laboratório de Parasitologia e Laboratório de Biologia Estrutural, Instituto de

Ciências Biológicas da Universidade Federal do Pará; 2 - Laboratório de Desenvolvimento e Planejamento de Fármacos, Instituto de Ciências Exatas e Naturais

da Universidade Federal do Pará; 3 - Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem da Universidade Federal do Rio de Janeiro; 4 - Laboratório de Microscopia Eletrônica, Instituto Evandro Chagas, Secretaria de

Vigilância em Saúde do Ministério da Saúde. Neutrophils are cells that participate in innate immune system as professional phagocytes to eliminate pathogens. The mainly mechanisms that cells use are mediated by reactive oxygen species and proteolytic enzymes. The 5-hydroxy-2-hydroxymethyl-gamma-pyrone (HMP) is a secondary metabolite synthesized by some species of fungi from Aspergillus, Penicillium and Acetobacter genera. The HMP has several applications, being used as a food additive, tyrosinase inhibitor, antitumor agent and macrophage activator. Thus, this study evaluated the in vitro effect of HMP in functional properties related to human neutrophils activation, such as morphological changes, production of ROS (reactive oxygen species) and phagocytosis. Human peripheral leucocytes were obtained from blood bag donated from Fundation Hemocenter of Para State. Cells isolation was performed using HISTOPAQUE® 1077-density-gradient. Neutrophils were treated for 1 hour with 50 and 100 μg/mL of HMP. Cytometric analysis was performed for measurement of neutrophils viability by Mitochondrial Membrane Potential Detection Kit (JC-1) and Propidium Iodide (PI) exclusion test. These results showed that HMP has no citotoxicity effect on neutrophils. The morphological analysis by optical microscopy and electron microscopy of treated neutrophils showed extensive lamellipodia formation, pseudopodia extention, high spreading ability and increase of cell volume, features that are often observed in activating cells. The increased cytoplasmic area was confirmed by morphometric analysis. Regarding the microbicidal activity, HMP increased the production of ROS and phagocytosis activity, but not NO (nitric oxide) production. In conclusion, this study demonstrates a role for HMP as a human neutrophil activator. INBEB, CAPES, CNPq/UFPa.

M26. PERFIL DE PRODUÇÃO DE MAGNETOSSOMOS PELA BACTÉRIA MAGNETOVIBRIO BLAKEMOREI AO LONGO DE UM CULTIVO EM BATELADA SIMPLES. 1-PEDRO E. LEÃO, 1-TARCÍSIO CORREA, 2-MAYARA G.C. SANTOS, 2-MELISSA GUTARRA E

1-ULYSSES LINS 1-Instituto de Microbiologia Paulo de Goes, Universidade Federal do Rio de Janeiro; 2-

Escola de Química, Universidade Federal do Rio de Janeiro, Xerém Nanopartículas magnéticas são estruturas de grande interesse biotecnológico. O principal desafio para uma maior aplicação dessas partículas é a sua obtenção em larga escala. Os processos físico-químicos de síntese destas estruturas apresentam um baixo rendimento e um elevado custo. Uma alternativa é o uso de nanopartículas magnéticas de origem biológica. A bactéria Magnetovibrio blakemorei (cepa MV-1) é capaz de sintetizar em seu interior nanocristais magnéticos pseudo-hexagonais prismáticos de magnetita, com 50-70nm de diâmetro, envoltos por uma bicamada lipídica. Tais estruturas são chamadas magnetossomos. Neste trabalho foi analisado o perfil da produção de magnetossomos pela cepa MV-1, quando cultivado em um biorreator de tanque agitado com uma estratégia de batelada simples. O meio de cultura utilizado disponibiliza acetato de sódio e succinato de sódio como fontes de carbono, hidrolisado de caseína com fonte de nitrogênio e óxido nitroso como aceptor final de elétrons da cadeia respiratória. Amostras foram retiradas a cada 24h de cultivo para acompanharmos a cinética de consumo dos nutrientes e para a observação do tamanho e da forma dos magnetossomos produzidos. Os resultados mostram que após 3 dias de cultivo o meio de cultura está esgotado, em relação a disponibilidade de ferro. Nas 24 horas seguintes o número de magnetossomos por célula se mantêm inalterado (14 magnetossomos/célula). Após mais 24 horas, no quinto dia de cultivo, ocorre uma queda (8 magnetossomos/célula). Eletromicrografias dos magnetossomos mostram que alem de estarem em menor número nas células, os magnetossomos produzidos após 96h de cultivo são menores do que aqueles observados no inicio da cinética. Estes cristais menores e em menor quantidade podem comprometer tanto a produtividade quanto a qualidade destas nanopartículas. Uma nova cinética, envolvendo uma estratégia de suplementação de ferro esta em andamento. Com esta estratégia de batelada alimentada, pretendemos obter cristais maduros durante toda a cinética de crescimento celular. CNPq, FAPERJ, CAPES, INBEB M27. ESTABELECIMENTO DE METODOLOGIAS DE CRIOFIXAÇÃO E SUBSTITUIÇÃO A FRIO PARA ANÁLISE POR MICROSCOPIA ELETRÔNICA DE PROTOZOÁRIOS PARASITAS

RACHID,R.P.; MIRANDA, K. R Instituto de Biofísica Carlos Chagas Fillho, Universidade Federal do Rio de Janeiro

A malária persiste ainda como uma doença responsável por altos níveis de mortalidade no mundo. Esta é causada por protozoários parasitos do gênero Plasmodium, entre os quais o P. falciparum e o P. vivax são os de maior importância médica. Parasitos deste gênero apresentam estruturas peculiares, não encontradas em células de mamíferos e constituindo, portanto, interessantes modelos de estudo tanto do ponto de vista da biologia celular quanto para o desenvolvimento de fármacos. A maior parte do conhecimento sobre a estrutura e organização morfoestrutural de protozoários se deu com a utilização de técnicas de microscopia eletrônica. Estas requerem uma serie de etapas no preparo de amostras, que notadamente provocam significativas alterações na sua estrutura. O desenvolvimento de métodos menos evasivos, como técnicas de fixação física e metodologias complementares, surgiu então como alternativa de preparo para manter a integridade estrutural o mais próximo do nativo. Neste trabalho, utilizamos técnicas de criofixação de protozoários parasitas como método de preparo para microscopia eletrônica. Células de Plasmodium chabaudi foram criofixadas por


congelamento ultrarrápido por alta pressão e em seguida submetidas à substituição a frio. Os resultados mostraram maior preservação estrutural em células submetidas às criotécnicas, quando comparadas às submetidas a técnicas de fixação química. Em geral, estruturas com organização menos rugosa foram observadas em diferentes domínios da célula hospedeira e dos parasitas, como a membrana do vacúolo parasitóforo e o conjunto de membranas do parasito, indicando sua acentuada preservação estrutural, e estrutura íntegra dos cristais de hemozoína. Em conjunto, os resultados mostram um papel potencial de métodos de preparo menos invasivos para a compreensão detalhada sobre a organização estrutural destes parasitas, contribuindo assim para diversos estudos que envolvem a biologia da malária. CNPq, FAPERJ, FINEP, CAPES, INBEB. M28. SALIVARY METABOLITE SIGNATURES OF CHILDREN WITH AND WITHOUT DENTAL CARIES LESIONS 1-FIDALGO, T. K. S. ; 1-FREITAS-FERNANDES, L. B. ; 2-MUNIZ, A. M. S. ; 3-ANGELI, R. ; 4-

GONÇALVES, E. ; 1-PINHEIRO, R. ; 5-NADAL, J. ; 3-ALMEIDA, F. ; 3-VALENTE, A. P. ; 1-SOUZA, I. P. R.

1-Faculdade de Odontologia, Universidade Federal do Rio de Janeiro; 2-Escola de Educação Física do Exército; 3- Centro Nacional de Ressonância Magnética Nuclear,

Universidade Federal do Rio de Janeiro; 4-Escola de Física, Universidade Federal do Rio de Janeiro; 5-COPPE, Universidade Federal do Rio de Janeiro

A metabolomic approach was used to analyze endogenous metabolites and to correlate with a specific biological state. The analysis of salivary metabolites is a growing area of investigation with potential for basic and clinical applications. Analyses of children’s saliva in different dentitions and with or without caries could potentially reveal a specific profile related to oral disease risk. Nuclear Magnetic Resonance (NMR) is well suited for mixture analysis followed by Principal Component Analysis combined with Linear Regression (PCA-LR) statistics and was used to identify differences in the salivary metabolites. The classificatory analysis was performed using PCA-LR based on 1,000 cross-validation bootstrap runs from both classifiers in order to increase the data information from a small sample size. The PCA-LR presented a statistically good classificatory performance for children with and without caries with an accuracy of 90.11 % (P < 0.001), 89.61 % sensitivity (P < 0.001), and 90.82 % specificity (P < 0.001). Children with caries lesions presented higher levels of several metabolites, including lactate, fatty acid, acetate and n-butyrate. Saliva from subjects with different dentition stages was also analyzed. Although the salivary samples were poorly classified, permanent dentition presented increased levels of acetate, saccharides and propionate. The NMR data and PCA-LR were able to classify saliva from children with or without caries, with performance indexes comparable to the partial least-squares regression

discriminant analysis (PLS-DA) results also performed. Our data also showed similar salivary metabolite profiles for healthy subjects despite the differences in their oral hygiene habits, socioeconomic status and food intake. FAPERJ, CNPq. M29. THE AMPK-eNOS PATHWAY IS INVOLVED IN THE NEUROPROTECTIVE ROLE OF SILDENAFIL (VIAGRA®) IN CUPRIZONE-DEMYELINATION MODEL

4- OLIVEIRA, W.H., 4- NUNES, A.K.S.; 3- RAPÔSO, C.; 1- ARAÚJO, S.M.R.; 2- OLIVEIRA, A.G.V.; 4- ROCHA, S.W.; 1- BARBOSA, K.P.S.; 4- LUNA, R.L.A.; 3- HÖFLING, M.A.; 1, 2-

PEIXOTO, C. 1Laboratório de Ultraestrutura - Centro de Pesquisas Aggeu Magalhães - FIOCRUZ; 2Departamento de Microscopia - Centro de Tecnologias Estratégicas do Nordeste

(CETENE ) – MCT; 3Departamento de Histologia e Embriologia, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP); 4 Centro de Ciências Biológicas-

Universidade Federal de Pernambuco It was recently demonstrated that sildenafil (Viagra®) has neuroprotective role, inhibiting inflammation and demyelination in the cerebellum, in cuprizone-demyelination model. However, the mechanisms of sildenafil neuroprotection are unclear. AMPK is a regulatory protein of lipid and glucose metabolism and it activation modulates inflammatory processes, possibly through eNOS activation. The present work investigated if sildenafil acts through AMPK-eNOS pathway, modulating neuroinflammation. Five male mice (C57BL/6), 6 weeks-old, were used per group. The groups received for four weeks: 0.2% Cuprizone (CPZ) mixed into the chow; CPZ into the chow plus sildenafil (Viagra®, Pfizer, 25 mg/kg) in the drinking water, starting concomitantly (sild-T0) or fifteen days (sild-T15) after initiation of CPZ; Controls received pure chow/water. Cerebella were processed for western blotting, immunofluorescence, transmission electron microscopy and Luxol Fast Blue staining (LFB); serum was used for nitrite dosage by ELISA. Results showed demyelination after CPZ treatment, with decreased MBP (myelin basic protein) expression, light labeling for LFB and damaged myelin sheath ultrastructure, comparing to control group. The inactive-AMPK (unphosphorylated) expression increased and eNOS levels decreased after CPZ administration. Sildenafil treatment (T0 and T15) showed significant increase of MBP expression and more intense LFB staining, comparing to CPZ group. Sildenafil-T0, but not -T15, improved ultrastructural aspect of myelin sheath, significantly increased the eNOS expression and decreased inactive-AMPK levels. These data suggest that sildenafil activates the AMPK-eNOS signaling pathway. The sildenafil treatment beginning concomitantly to CPZ was more effective then beginning 15 days after CPZ. This work clarifies the mechanism of sildenafil neuroprotection in a demyelinating condition. INBEB, FACEPE, CNPq


Doutorandos

D1. STRUCTURE AND DYNAMICS OF ALLERGEN GAD M 1 FREE AND IN COMPLEX WITH SINGLE CHAIN VARIABLE FRAGMENT BY NMR SPECTROSCOPY

1- MORAES, A.H.; 2- ACKERBAUER, D.; 2- KOSTADINOVA, M.; 2- BUBLIN, M.; 3- FERREIRA, F.; 1- ALMEIDA, F.C.L.; 2- BREITENEDER, H.; 1- VALENTE, A.P.

1- Centro Nacional de Ressonância Magnética, IBQM-UFRJ,RJ, Brazil; 2- Medical University of Vienna, Vienna, Austria; 3- University of Salzburg, Salzburg, Austria.

Allergenic proteins are able to stimulate an inappropriate IgE production in atopic individuals, which results in manifestations of clinical symptoms such as asthma, rhinitis and atopic dermatitis. Gad m 1 is the major allergen from Atlantic cod and belongs to b-parvalbumin protein family, which are the most important fish allergens and their high cross-reactivity is the cause of the observed polysensitization in allergic patients. Despite extensive efforts, the complete elucidation of b-parvalbumin-IgE complexes has not been achieved yet. In this work, we solved the solution structure of Gad m 1 using NMR spectroscopy. Concomitantly, scFv interaction studies were performed, using chemical shift perturbation assays. We compared our results with other sites mapped in homologous parvalbumins: Cyp c 1, Sco j 1 and Gad c 1. Residues around positions 30 to 40 matched epitopes mapped for the three allergens. Residues around position 50 to 60 were only observed for Gad m 1 and Gad c 1, while residue around 80 was mapped on Cyp c 1. On the other hand, the N-terminal was only mapped in Gad m 1 and our results did not showed the participation of residues around position 90. The dynamic properties of Gad m 1 in the free state and in the complex with scFv were obtained from relaxation experiments. Residues 31-33 and 75-78 exhibit increased R2/R1 ratios. These residues are located in two regions mapped by CSP. The big ratios observed may be due to chemical exchange between bound and free forms. This work will present for the first time the solution structure of a b-parvalbumin as well as the characterization of Gad m 1-scFv interaction as a first and crucial step in the development of hypoallergenic vaccine and to an understanding of the molecular interactions between allergens and IgE’s. FAPERJ, CNPq, CAPES and INBEB

D2. EFFECT OF BONE MARROW MONONUCLEAR CELLS IN NEURODEGENERATION, NEURONAL SURVIVAL AND REACTIVE MICROGLIOSIS IN HIPPOCAMPAL CA1 LAYER AFTER GLOBAL CEREBRAL ISCHEMIA 1- RAMOS, A.B.; 1- CHAN, J.M.; 2- CINTRA, W.M.; 1- MENDEZ-OTERO, R.

1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2- Instituto de Ciências Biomédicas , Universidade Federal do Rio de Janeiro

"Introduction: The pyramidal neurons of the hippocampal CA1 layer are essential for cognitive functions and selectively destroyed after global cerebral ischemia. Bone marrow mononuclear cell (BMMC) therapy has shown positive results in preclinical models of focal cerebral ischemia, but has not been studied in transient global ischemia. Aim: The aim of our study was to investigate whether BMMC treatment could reduce the neurodegeneration and the inflammation observed in CA1 layer of ischemic animals. Methods: Transient forebrain ischemia was performed by four vessels occlusion method. To establish the time course of CA1 cell death, the rats were transcardially perfused 3, 7, 14, 21 and 28 days after ischemia (DAI). To analyze the

effect of BMMC therapy in neurodegeneration, neuronal survival and reactive microgliosis in CA1 layer, ischemic animals received 3x107 BMMC 3 DAI, in the left carotid, and were sacrificed different days after ischemia. For quantification of neuronal degeneration, survival and reactive microgliosis, we counted Fluoro-Jade C, NeuN and ED-1 positive cells in CA1 layer, respectively. Results: We observed a greater number of FJC positive cells in CA1 layer of ischemic animals 7 DAI when compared with the others days. The time course of neuronal survival shows a reduction of pyramidal neurons 7 DAI and in the days after. In the analysis of the effect of BMMC in neurodegeneration and neuronal survival, we observed a significant reduction of FJC cells and increase of NeuN cells in CA1 of animals injected with BMMC when compared to the saline group. Furthermore, we observed a lower number of ED-1 positive cells in ischemic animals treated with BMMC compared with ischemic animals that received only saline. Conclusion: We suggest that the BMMC therapy in transient global ischemia has a neuroprotective effect in CA1 layer that was followed by reduction of microgliosis in this region." CNPq; FAPERJ; CAPES;

D3. STRUCTURAL DYNAMICS AFFECT THE ALLERGENIC POTENTIAL OF BET V1 1- A.L. BATISTA, 1- C. ASAM, 1- A. MORAES, 1- F.C.L. ALMEIDA, 2- F. FERREIRA, 2- M.

WALLNER AND 1- A.P. VALENTE 1- CNRMN, Dep de Bioquímica Médica, IBqM-UFRJ, Brasil; 2- Christian Doppler

Laboratory for Allergy Diagnosis and Therapy, University of Salzburg, Salzburg, Austria Bet v 1 is the major allergen of birch pollen with molecular mass of 17.5 kDa. It is estimated that 100 million people are sensitized to Bet v 1, in Northern American and Western Europe. Bet v 1 secondary structure arrangement is β-α2-β6-α where seven anti-parallel β-sheet surround a long C-terminal α-helix. Also, a Y-shaped hydrophobic pocket is present inside the protein. Its biological function is associated with the transport of hydrophobic molecules such as brassinosteroid (plant hormone). In this work we used Na-deoxycholate (Doc) that is structurally similar to plant steroids, in order to analyze the influence of ligand binding on dynamics, conformation and IgE-binding. Recombinant 15N isotope labeled Bet v 1 was produced in E. coli and purified. We used NMR spectroscopy to probe dynamics aspects using R1, R2 and 1H - 15N-NOE for free and bound state. To further understand the free state dynamics we performed 15N CPMG relaxation dispersion experiments at 800 and 600 MHz. Moreover, we performed experiments to map IgE epitopes in the free and Doc-bound state of Bet v 1 using purified human IgE antibodies from birch pollen allergic patients. Bet v 1 is a dynamic protein with several residues in conformational exchange. The epitopes for three patients were slightly different but several residues are in common. Doc interaction changed slightly the protein conformation and profoundly the dynamics. These changes did not affected the IgE epitope but there was an increase in antibody afinity. Understanding the structural dynamics of Bet v 1 and its interaction with IgE with or without ligands may help to develop hypoallergic derivatives for immunotherapy and as well as understand the correlation between dynamics of allergens and the forces that drive the allergenicity. INBEB, FAPERJ, CNPq, CAPES.

D4. EFFECTS OF LPSF/GQ-02 ON NONALCOHOLIC FATTY LIVER DISEASE 1– SILVA, A.K.S., 1– SPINOLA, M.G.D., 1- GOMES, F.O.S., 1- OLIVEIRA, A.C., 1- OLIVEIRA,W. B., 1– SANTOS, L.A.M., 1- DONATO, M.A.M., 1,2- PEIXOTO, C.A.


1 - Centro de Pesquisas Aggeu Magalhães- CPqAM/FIOCRUZ, Brasil. 2 - Centro de Tecnologias Estratégicas do Nordeste- CETENE/MCT, Brasil.

Nonalcoholic fatty liver disease (NAFLD) defines a wide spectrum of liver diseases that extend from simple steatosis to nonalcoholic steatohepatitis. Although the pathogenesis of NAFLD remains undefined, it is recognized that insulin resistance is present in almost all patients who develop this disease. The Thiazolidinediones act as an insulin sensitizer and have been used in the treatment of patients with type 2 diabetes and other insulin-resistant conditions, including NAFLD. Hence, therapy of NAFLD with insulin-sensitizing drugs should ideally improve the key hepatic histological changes, but should also reduce cardiometabolic and cancer risk. The aim of our study was to evaluate the therapeutic effects of LPSF/GQ-02 on the liver the LDLR-/- mice after a diet rich in fat. 40 male mice were divided into four groups: 1 - received a standard diet of laboratory, 2- fed with high-fat diet (HFD), 3–HFD+pioglitazone, 4–HFD+LPSF/GQ-02. The experiments were conducted for 10 weeks and in the last two weeks the drugs were administered daily by gavage. After experimental protocols, the liver was processed for optical microscopy, Oil Red O and immunohistochemistry for interleukin-6 (IL-6). The control group showed well-preserved tissue with low lipid content and a low reactivity for IL-6. The HFD group presented steatosis, inflammatory infiltrates and a high reactivity for IL-6. Treatment with pioglitazone was not effective in reversing the changes caused by high-fat diet, showing disorganization in liver tissue, with accumulation of lipid and a strong reactivity for IL-6. However, treatment with LPSF/GQ-02 improved organization of liver tissue with a significant decrease of lipid inclusions and cytokine IL-6 levels. This study showed that pioglitazone, a drug used to treat type 2 diabetes mellitus was unable to reverse the condition caused by the diet rich in fat. However LPSF/GQ-02, showed to be effective in reducing the accumulation of lipid and also reduced significantly IL-6 expression in liver of LDLR-/- mice. INBEB, CNPq, FACEPE

D5. SILDENAFIL PREVENT MICRO AND ASTROGLIOSIS POSSIBLY VIA NFKB IN MODEL OF DEMYELINATION 1,2- NUNES,AKS.;1,3- RAPÔSO C.;1,2- LUNA RLA.; 2- ARAÚJO SMR.;1- OLIVEIRA WH.; 2-

OLIVEIRA AGV.;1- BARBOSA KP.; 1- ROCHA SWS.;3- Cruz-Hofling MA.;1,2- PEIXOTO C. 1- Laboratório de Ultraestrutura - Instituto de Pesquisas Aggeu Magalhães - FIOCRUZ; 2- Departamento de Microscopia - Centro de Tecnologias do Nordeste (CETENE ) – MCT; 3-

Departamento de Histologia e Embriologia,Universidade Estadual de Campinas - UNICAMP

Sildenafil (Viagra ®) induces cGMP accumulation by PDE5 inhibition. Recent studies have demonstrated that sildenafil inhibits demyelination process, decreases micro- and astrogliosis and proinflammatory cytokines expression in cuprizone EM-model. The NF-Кβ plays an important role in the regulation of expression of inflammatory mediators. This study investigated whether treatment of chronic and late Sildenafil inhibit the activation of microglia and astrocytes via NFkB pathway in a model of demyelination. Five C57BL/6 mice, 6-weeks-old, were used/group. The groups received: 1) Cuprizone (CPZ) 0,2% mixed into a chow/4 weeks, 2) CPZ in chow while Sildenafil (Viagra®) 25mg/kg in the drinking water (T0) , or 3) CPZ in chow while Sildenafil (Viagra®) 25mg/kg in the drinking water after 15 days administration the CPZ (T15), 4) Controls received pure chow and water. The cerrebellums were processed for western blotting (WB), immunohistochemistry (IH) and immunofluorescence (IF) in frozen sections. Results showed treatment with CPZ increased GFAP levels and induced morphological changes indicating activation of astrocytes compared to the control group. Sildenafil (T0) decreased levels of expression of GFAP compared with the group CPZ. However

treatment with Sildenafil (T15) was not able to significantly reduce levels of GFAP. Double staining with Iba-1 and NFkB in the control group showed cytoplasmic labeling in the resting state indicating microglia cells and inactivation of NFkB. CPZ treatment showed labeling of NFkB nearest the core of microglial cells indicating activation. Corroborant these results, WB data showed that treatment CPZ inhibit NFkB inhibitory protein (IKβα) and increased expression indicating NFkB translocation from the cytoplasm to the nucleus and activate transcription of genes for pro-inflammatory. Sildenafil (T0) was effective in decreasing expression and increased NFkB levels IKβα. Consequently the marking to Iba-1 was decreased, and prolongations with thinner and branched. However late treatment with Sildenafil was not effective in reducing the marking and Iba-1 expression levels of NFkB. The increase in cGMP levels by inhibiting PDE5, probably acts by inhibiting NFkB via gene transcription and pro-inflammatory activation of microglial cells and prevent astrocytes in a model of demyelination. Keywords: Microglia, inflammation,sildenafill CNPq, CAPES, FAPESP, FACEPE, INBEB

D6. EFFECT OF BONE MARROW MONONUCLEAR CELLS THERAPY IN A MODEL OF SPINAL CORD IN HEMITRANSECTION: CORRELATION MORPHOLOGICAL AND FUNCTIONAL

1- BOMFIM, A. M. D.; 1- RAMOS, A. B.; 2- CASTRO, N. G. D.; 1- MENDEZ-OTERO, R. 1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2-

Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro "Aim: The aim of our study was to evaluate the potential of bone marrow mononuclear cells (BMMC) therapy in a rat model of unilateral T10 spinal cord injury (SCI). Methods: Wistar rats were divided into 3 groups: false-operated group, underwent laminectomy, SCI + saline group and SCI+ BMMC group. The locomotor recovery was assessed by Basso Beattie Bresnahan scale (BBB) from 3 days after surgery. In histochemical analysis, immunostaining was performed using markers as ED-1 (CD68), GFAP and NF-200 marking macrophages and microglia, astrocytes and axons respectively. Results: Functional analysis showed that the treated group (SCI + BMMC) showed better locomotor recovery through the BBB scale compared with SCI + saline group. In the histochemical analysis of bone marrow mononuclear cells were found 3, 7 and 14 days after injury (DAI). The SCI + BMMC group showed more axons in all time windows observed and less activated microglia and macrophages at 7 and 14 DAI, compared with SCI + saline group. Morphological differences associated with reactive astrogliosis was also observed in the treated group. Conclusion: Our data suggest that injection of bone marrow mononuclear cells after spinal cord injury provided better locomotor recovery associated with a higher presence of axons and reduction of reactive microglia in different time windows." CNPq D7. ENVOLVIMENTO DA IPLA2 NA ADERÊNCIA, INTERNALIZAÇÃO E INFECTIVIDADE DE LEISHMANIA AMAZONENSIS EM MACRÓFAGOS PERITONEAIS

Fernandes ACS, Soares DC, Saraiva EM, Souto-Padrón T* 1Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro,

Rio de Janeiro, Brasil *[email protected] "O processo de interação parasito hospedeiro depende do reconhecimento mútuo de moléculas e receptores. A exposição de moléculas na superfície de ambas as células ou sua secreção para o meio extracelular dependem das vias endo / exocíticas, onde a fusão entre seus diferentes compartimentos é mediada por múltiplos fatores, entre eles a ação das fosfolipases. Nosso grupo vem investigando a participação da fosfolipase A2


independente de cálcio (iPLA2) na fusão de compartimrntos das vias endo / exociticas de Leishmania amazonensis e no processo de interação com macrófagos peritoneais. Promastigotas de L. amazonensis foram mantidos em meio de Schneider durante 144h e tratados durante 1 h com 2,5 M de bromenolactone (BEL), um inibidor irreversível da iPLA2. Após o tratamento, parasitas controle e tratados foram incubados com macrófagos. Observamos os processos de adesão, internalização e sobrevivência do parasito por até 48 horas no interior da célula hospedeira. BEL reduziu aproximadamente em 80% a adesão de promastigotas à superfície de macrófagos. Ao contrário do controle, parasitos tratados com BEL aderiram-se aos macrófagos exclusivamente pela região do flagelo. BEL também reduziu a percentagem de macrófagos infectados e o número de parasitas no interior do vacúolo parasitóforo (PV) em 80 e 90%, respectivamente, em um período de até 48 h após a infecção. O PV em macrófagos infectados com parasitos previamente tratados com BEL apresentaram diferenças significativas em relação a parasitos controle. Observamos um atraso na diferenciação de promastigota em formas amastigotas, redução na acidez do PV e alteração no processo de fusão com de lisossomos ao PV. Nossos resultados indicam que a inibição da iPLA2 em promastigotas de L. amazonensis reduz a adesão e a infecção na célula hospedeira com inibição da diferenciação do parasito, inibição na multiplicação de formas amastigota e alteração na formação e morfologia do PV." Apoiado pelo CNPq, CAPES, FAPERJ, Pronex, CONCEA (11.794/08). D8. NMR STRUCTURAL CHARACTERIZATION OF Q4D059 AND Q4DRX8, CONSERVED HYPOTHETICAL PROTEINS FROM TRYPANOSSOMA CRUZI

1-LOPEZ-CASTILLA A.; 1-DE MENEZES R.; 1-DOS SANTOS T.; 1-PIRES J.R 1-Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro

Q4D059 and Q4DRX8 (Uniprot ID) are hypothetical proteins of 84 and 104 amino acids respectively from T.cruzi. A PSI-BLAST search using Q4D059 and Q4DRX8 sequences as query only identified hypothetical proteins from trypanosomatids (E-value above the threshold), so failed to provide insight into the protein function. Both proteins are conserved in the kinetoplastid genomes and show low-sequence homology with mammal proteins; therefore are considered potential targets for drug development against trypanosomatids parasites. Structural studies of these proteins could give information about their function and facilitate drug screening. In this work, the genes codifying for Q4D059 and Q4DRX8 were cloned into a pGEX expression vector and 13C- and 15N- NMR protein samples were overexpressed in Escherichia coli as a GST-fusion protein. Protein purification was performed by GST-affinity chromatography. GST was removed by thrombine protease digestion and as last step a size-exclusion chromatography was carried out. 1D 1H and 15N-1H HSQC spectra of Q4D059 showed a well-behaved and structured protein in solution. The NMR spectra required for protein structure determination were recorded and the assignment process of all atoms in Q4D059 is in progress. CNPq D9. DIETHYLCARBAMAZINE REDUCED THE NF- B ACTIVATION IN MODEL OF CHRONIC ETHANOL CONSUMPTION

1- SILVA, B.S.; 1- RODRIGUES, G.B.; 1- ROCHA, S.W.S.; 1- GOMES, F.O.S.; 1- RIBEIRO, E.L.;1- SILVA, A.K.S.; 1- SPINOLA, M.G.D.; 1- OLIVEIRA, A.C.;1- OLIVEIRA, W.B.; 1,2-

PEIXOTO, C.A. 1- Centro de Ciências Biológicas, Universidade Federal de Pernambuco; 2- Laboratório

de Microscopia do Centro de Tecnologias Estratégicas do Nordeste (INT-CETENE - Pernambuco)

Alcoholic liver disease (ALD) is considered as a complex and multifaceted pathological process, involving oxidative stress, inflammation and excessive fatty acid synthesis. Progression of disease involves various proinflammatory molecules such as interleukins, cytokines, adhesion molecules and nuclear factor-kB (NF-kB). NF-kB is a transcription factor that is implicated in inflammation and immune response and is activated by oxidants and cytokines that play important roles in the inflammation and development of ALD. Therapy for the treatment of ALD is limited and ineffective. Diethylcarbamazine (DEC) has anti-inflammatory properties as a result of its interference with the arachidonic acid metabolism and few studies focus their role in the pathophysiology of inflammation. The objective of this work was to investigate the role of DEC on NF-kB pathways in hepatic inflammation induced by alcoholism. Forty male C57BL were separated in four groups (n=10): control group (C) that received just distilled water, DEC-treated group (D50) that received 50 mg/kg DEC for twelve days by gavage, alcoholic group (EtOH) that received ethanol and alcoholic plus 50 mg/kg DEC group (EtOH50). After 5 weeks the alcoholism induction fragments of liver were processed for immunohistochemistry and western blot for an antibody against the activated p65 subunit of NF-κB and their regulatory enzyme, IkBa. High level of NF-κB expression was seen in hepatocytes exposed to alcohol, which was significantly decreased in the hepatocytes of the EtOH50 group. Activation of NF-kB occurs secondarily to the degradation of IkBa. Ours findings support a role for IkBa in alcoholic liver injury because the activation of NF-kB was accompanied by a loss of IkBa in alcoholic group. The inhibition of NF-kB activation in EtOH50 was accompanied by preservation of IkBa expression. These results suggest that agents that prevent the activation of the transcription factor NF-kB, will thereby prevent ALD. INBEB, FACEPE, CNPq, UFPE D10. CROSS-TALK BETWEEN PRION PROTEIN AND NUCLEIC ACIDS: PROTEIN MISFOLDING LEADING TO CYTOTOXICITY 1- MACEDO, B.; 2- MILLEN, T. A.; 1- FERREIRA, N. C.; 1- GOMES, M. P. B.; 2- BRAGA, C. A.

C. A.; 2- FERREIRA, P. S.; 2- SILVA, J. L.; 1- CORDEIRO, Y. 1- Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, ²Instituto de

Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro; 2- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro

"Prion proteins (PrPs) are involved in lethal neurodegenerative diseases, that affect humans and other animals. So far, many issues remain unclear about PrP physiopathological role. The misfolding of its normal cellular form (PrPC) into an abnormal form (PrPSc) is thought to be the central event of these pathogenesis. Some studies have suggested nuclear localization for PrPC and functions derived from its interaction with nuclear components. Having a common local to cross-talk, nucleic acids (NAs) are really intriguing PrP molecular partners, and have been proposed to work as catalysts in the protein misfolding process. To clarify the biological relevance of PrP-NA interactions, we worked with several oligonucleotides (RNA and DNA) and different cell lineages (neuroblastoma - N2a - and kidney cells – HK2), using diverse cellular assays to evaluate cytotoxicity and cell viability. We have found that interactions of PrP with NAs can lead to different aggregates and neurotoxic species, depending on the nucleic acid molecule (1). Interestingly, the number of N2a cells was reduced and their morphology also changed in the presence of the cytotoxic PrP-NA complexes, which indicate that they are indeed affecting cell signaling. We are currently investigating the mechanism through which such complexes mediate cell death. Understanding the cellular and structural effects observed for PrP and nucleic acid cross-talks may enlighthen the still obscure prion physiopathology.


Reference: (1) Macedo, B., Millen, T. A., Braga, C. A. C. A., Gomes, M. P. B., Ferreira, P. S., Kraineva, J., Winter, R., Silva, J. L. and Cordeiro, Y. (2012) Biochemistry, 51: 5402−5413" FAPERJ, CNPq, CAPES, INBEB D11. MOLECULAR EVALUATION OF THE RENIN ANGIOTENSIN SYSTEM COMPONENTS BY RT-QPCR AFTER BREAST CANCER TREATMENT

1,2SALATA C; 1,3FERREIRA-MACHADO SC; 4MENCALHA AL; 1CAMPOS VMA; 1,5ANDRADE CBV; 2MANDARIM-DE-LACERDA CA; 1DEALMEIDA CE.

1. Laboratório de Ciências Radiológicas/UERJ/RJ, Brasil; 2. Laboratório de Morfometria e Morfologia Cardiovascular/UERJ/RJ, Brasil; 3. Departamento de Biologia Geral/UFF/RJ,

Brasil; 4. Departamento de Biologia e Biometria/UERJ/RJ, Brasil; 5. Laboratório de Ultraestrutura e Biologia Tecidual/UERJ/RJ, Brasil

"Introduction: New treatment protocols and technologies for Breast Cancer (BC) have allowed that more women with early stage BC survive their disease. This increases their chances of developing late cardiovascular effects, as a result of BC treatments, chemotherapy and radiotherapy. It is known the relevance of the local cardiac Renin Angiotensin System (RAS) in the pathofisiology of cardiac damage. The angiotensin II (Ang II) in the heart can induce hypertrophy, inflammation and fibrosis, by activation of AT1 receptors. The present study aims to evaluate the mRNA expression of different components of RAS associate it with cardiac damage induced by breast cancer treatment. Methods: Female Wistar rats, were divided into groups (5 animals per group): control, chemotherapy (cyclophosphamide + docetaxel) + radiotherapy (TC+IR) and radiotherapy only (IR). The animals were euthanized 5 months after the treatment. RT-qPCR of angiotensinogen, renin, ECA, AT1R, TGF-ß1 and procollagen type I of left ventricle tissue was performed. Values are expressed as (mean±SEM). This study was submitted and approved by the UERJ ethical committee (n° CEUA/010/2012). Results/Discussion: Results showed no mRNA expression of angiotensinogen in heart tissue, as expected, because the angiotensinogen used in the local heart RAS is produced in the liver. Renin was only expressed in treated groups, and not in control. AT1R expression was different (p<0.001) between IR+TC and control, and between IR and control, with p<0.05. Procollagen type I expression was different (p<0.001) between TC + IR group and control. Results of TGF-ß1 showed significant difference (p<0.05) between TC + IR group and control group. Parameters as TGF-ß1 and procollagen type I were up-regulated, probably because of AT1R activation. These preliminary results indicate that the association of chemotherapy and radiotherapy cause more injuries in the heart tissue than radiotherapy alone. We will confirm these results using another techniques such stereology." CAPES, FAPERJ D12. THE “INS” AND “OUTS” OF VIRAL INFECTION: DISSECTING THE DYNAMICS OF ENTRY AND EXIT OF AN EMERGING VIRUS

CARVALHO, C.A.M.; VIGNOLI, K.; SILVA, J.L.; GOMES, A.M.O. Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro

Mayaro virus (MAYV) is an alphavirus widespread in South America in an endemic manner and represents an interesting case to consider regarding the potential for urban emergence. Alphavirus entry into target cells is supposed to occur by receptor-mediated endocytosis followed by fusion between the viral envelope and the endosomal membrane, although non-endocytic penetration of the viral genetic material into the cytoplasm without membrane fusion has also been suggested. On the opposite way, alphavirus exit from host cells is supposed to occur by budding from the plasma membrane, although egress by exocytosis may also be observed depending on cell

lineage. The aim of this work is to analyze the behavior of MAYV particles and their structural components during the entry into and exit from host cells. To study viral entry, MAYV was labeled with DiD and the fluorescent signals were tracked in Vero cells by laser-scanning confocal fluorescence microscopy (LSCFM) in real time. Our results show that MAYV entry into cells occurs by a fast endocytic mechanism: following DiD fluorescence dequenching at the single particle level, fusion between the viral envelope and the endosomal membrane was shown to occur at around 3 min post-binding. To study viral exit, Vero cells previously transfected with expression vectors for MAYV structural proteins fused to fluorescent proteins were infected and the interaction between these viral components was evaluated by LSCFM over viral infection. Our results suggest that plasma membrane is the viral budding site in these cells and that it seems to occur a rearrangement of the intracellular distribution of the fluorescent viral structural proteins during infection. This work provides unique dynamic insights into the entry and exit of MAYV particles in living cells. Understanding the dynamics of virus infection may provide important insights to the development of antiviral strategies. CAPES, CNPq, FAPERJ, FINEP, INBEB, PRONEX D13. HYDROSTATIC PRESSURE FULL INACTIVATED INFLUENZA VIRUS PRESERVES BINDING AND MEMBRANE FUSION WITH GENERATION OF IMMUNE RESPONSE AND PROTECTION OF MICES AGAINST INFECTION

1-DUMARD, C.H.; 1-SOUZA-SANTOS, P.; 1-BARROSO, S.P.C.; 1- OLIVEIRA, G.A.P.; 1-CARVALHO, C.A.M.; 2- COUCEIRO, J.N.S.S.; 2-FERREIRA, D.F.; 1- SILVA, J.L.

1-Laboratório de Termodinâmica de Proteínas e Estruturas Virais Gregorio Weber, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2- Instituto de

Microbiologia Professor Paulo de Goes, Universidade Federal do Rio de Janeiro Whole inactivated vaccines (WIV) present higher immunogenicity then split and subunit vaccines. Recent studies have demonstrated that WIV with preserved fusogenic activity are more protective then non-fusogenic WIV. In this work, we show the inactivation of human influenza virus X-31 by hydrostatic pressure (HP) and analyze the effects on the structure by spectroscopic measurements, light scattering, and electron microscopy. We also investigated the HP effects in the glycoproteins activity. Fusogenic activity was evaluated by confocal microscopy. Immune response and disease were tested by Elisa and weight loss respectively. Electron microscopy data show pore formation on viral envelope but general morphology was preserved, and small variations were seen in the particle structure. The activity of hemagglutinin in the process of binding and fusion was affected in time dependent-manner but neuraminidase activity was not affected. Infectious activities were abolished after three hours of pressurization as observed by RT-PCR. Vaccinated mices presented high levels of IgG2a, IgG1 and IgA after immunization and were protected from weight loss after infection. Our results show fully viral inactivation with preservation of viral structure and maintenance of fusogenic activity, with strong immune response and protection of mices against infection. Our data strongly support the idea of applying the hydrostatic pressure to develop new vaccines for influenza A and other viruses as well. INBEB, FAPERJ, CAPES, CNPq D14. ENDOSSIMBIOSE EM TRIPANOSSOMATÍDEOS, A ASSOCIAÇÃO DA BACTÉRIA COM OUTRAS ESTRUTURAS DO PROTOZOÁRIO HOSPEDEIRO

CATTA-PRETA, C.M.C. 1,2; MACHADO, A.C.L. 1,2; DE SOUZA, W. 1,2,3; MOTTA, M.C.M. 1,2

1 Laboratório de Ultraestrutura Celular Hertha Meyer, IBCCF, UFRJ 2 Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, UFRJ 3 Instituto Nacional de

Metrologia, Qualidade e Tecnologia (INMETRO)


O estudo da co-evolução entre dois seres primitivos fornece informações relevantes sobre a origem de organelas na célula eucariota. Alguns protozoários monoxênicos da família Trypanosomatidae mantém uma relação mutualística, e portanto obrigatória, com uma bactéria simbiótica. A divisão do simbionte é coordenada com outras estruturas do hospedeiro, de modo que cada célula filha herda uma única bactéria simbiótica. Outro aspecto interessante desta relação são as intensas trocas metabólicas que ocorrem entre os dois seres associados, como a cooperação na produção de aminoácidos, vitaminas e grupamentos heme. O objetivo deste trabalho é verificar do ponto de vista ultraestrutural, usando a tomografia eletrônica, a associação do simbionte com organelas do hospedeiro, como o núcleo, o retículo endoplasmático e organelas energéticas. Utilizando técnicas de preservação como congelamento em alta pressão e crio-substituição é possível observar com precisão se as estruturas que interagem somente se tocam ou se fundem. Nossos resultados mostram que o envoltório do simbionte encontra-se justaposto à membrana nuclear ao longo do ciclo celular do hospedeiro, o que pode estar relacionado com a simetria do eixo de divisão do protozoário e com o controle do número de bactérias por célula. Também foram flagrados toques entre as membranas do simbionte e dos glicossomos, organela envolvida na produção de ATP, reforçando a ideia que a bactéria simbiótica se beneficia da produção de energia do hospedeiro. Observamos também que o retículo endoplasmático apresenta íntima associação com o simbionte, e que em alguns momentos, estas estruturas parecem estar fundidas. Esta associação pode estar relacionada com a importação pelo simbionte de lipídeos e glicoproteinas produzidos pelo hospedeiro, como sugerem os nossos dados genômicos e bioquímicos. INBEB, CNPq e FAPERJ D15. IMAGING CRYPTOCOCCUS NEOFORMANS-MACROPHAGE INTERACTION

1-GUERRA, C.R.; 2-SEABRA, S.H.; 1-ROZENTAL, S. 1-Laboratório de Biologia Celular de Fungos, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2-Laboratório de Tecnologia em Bioquímica e

Microscopia, Centro Universitário Estadual da Zona Oeste. "Cryptococcus neoformans is an encapsulated yeast that causes disease in immunocompromised patients. Cryptococcosis is considered an AIDS-defining condition and it is the third most frequent neurological complication in AIDS patients. Macrophages represent first line of defense of cryptococcosis. During the last decade, studies have focused on how this yeast can survive and proliferate within macrophages. However, studies dealing with exact mechanisms used by the yeast to enter host cells are still incipient. Therefore, we focus this study using cytoskeleton inhibitors to observe C. neoformans interaction with peritoneal macrophages by confocal microscopy and scanning electron microscopy. Four different C. neoformans strains differing in serotypes and capsule size (ATCC28957, H99 and acapsular mutants CAP59 and CAP67) were allowed to interact with peritoneal macrophages, previously adhered on glass cover slips in a ratio of 50 yeast per macrophage, for 4 hours. Interactions occurred in absence or presence of cytoskeleton inhibitors: Cytochalasin D or Nocodazole and then cells were processed for flow cytometry (FACS), scanning electron microscopy (SEM) or confocal microscopy (actin filaments were labeled with AlexaFluor 488 phalloidin and α-tubulin was labeled with anti-α-tubulin AlexaFluor 546 conjugate). FACS experiments revealed that actin and tubulin inhibitors diminished yeast internalization process by macrophages. Confocal microscopy revealed the presence of actin surrounding endosomes vacuoles containing yeasts, whereas α-tubulin apparently was less recruited after yeast internalization. Macrophage membranes were extracted after interaction and visualized by SEM demonstrating cytoskeleton attachment to yeast cells and its important role upon host cell entry. Taken together, these results indicate a more

prominent role of actin recruitment than α-tubulin and helps us to better understand how the initial process of C. neoformans-macrophage interaction may occur. Further studies are ongoing." CNPq, FAPERJ, Capes D16. ASSOCIAÇÃO ENTRE OSTEOPOROSE E QUIMIOTERAPIA PARA O TRATAMENTO DO CÂNCER DE MAMA

ANDRADE, CBV1,2,3*; SALATA, C1,4; SILVA, CM1; FERREIRA-MACHADO, SC1,5; C. L. MOTA7; A. PICKLER7; A. MANTUANO7; ALMEIDA, AP6; BRAZ D6; NOGUEIRA, LP7;

BARROSO, RCR7; DEALMEIDA, CE1 1. Laboratório de Ciências Radiológicas/UERJ/RJ 2. Universidade Severino

Sombra/USS/RJ 3. Ultraestrutura e Biologia Tecidual/UERJ/RJ 4. Laboratório de Morfometria e Morfologia Cardiovascular/UERJ/RJ 5. Departamento de Biologia

Geral/UFF/ 6. Laboratório de Instrumentação Nuclear/COPPE/UFRJ 7. Instituto de Fisica/UERJ

"Introdução: Uma das mais utilizadas estratégias de tratamento para o câncer de mama (CM) é a quimioterapia. O regime TC (docetaxel e ciclofosfamida). TC é mais recente, portanto existem poucos estudos relacionados a ele em relação a efeitos tardios, como a osteoporose precoce. Mulheres na pré-menopausa submetidas à quimioterapia para o tratamento do CM apresentam significante perda óssea a partir do primeiro ano após o início do tratamento. A mais provável causa desta perda deve-se ao fato destes quimioterápicos levarem a menopausa precoce. A diminuição da densidade mineral óssea pode levar ao aumento do risco de fraturas na pós-menopausa. Sabendo-se que alguns quimioterápicos para o tratamento do CM, aumentam o risco de menopausa precoce, e subsequente osteoporose, espera-se neste estudo, determinar se o recente regime quimioterápico TC pode induzir aos mesmos danos. Metodologia: Ratas Wistar, com 3 meses, foram divididas em 2 grupos, quimioterapia (TC) e controle. Os animais foram eutanaziados 5 meses após o término do tratamento. Foi realizada a coleta de sangue, dos fêmures e dos úteros para posterior análise. As técnicas utilizadas foram: aferição da massa uterina; concentração de estradiol sorológico por radioimunoensaio; distribuição espacial dos elementos cálcio e zinco na matriz óssea através da técnica de Microfluorescência de raios X (µXRF) Resultados/Discussão: Os animais do grupo TC demonstraram redução significativa (p<0.01) da massa do útero em relação ao controle, sugerindo atrofia uterina gerada pela baixa de estrogênio (p<0.05), devido à ação dos quimioterápicos. Foram obtidas as distribuições bidimensionais de Ca e Zn dos grupos controle e TC, pois ambos são elementos importantes na composição da matriz óssea. Embora a concentração de Ca não tenha variado entre os grupos, a concentração de Zn no grupo TC é significativamente menor comparado ao controle (p<0.001). Futuramente serão realizadas análises por imunohistoquímica do número de osteoclastos e osteoblastos." CAPES, FAPERJ D17. S-NITROSYLATION DECREASES CA2+ SENSITIVITY AND ACTOMYOSIN ATPASE ACTIVITY OF CONTRACTILE PROTEINS IN CARDIAC MYOFIBRILS

1,2,3Figueiredo-Freitas C.,4,5Foster M.W., 6Nogueira L., 3Liang J.S., 2Yamashita A.M.S.,7Dulce R., 4,5Thompson J.W., 7Hare J.M., 4,5Moseley A., 2Sorenson M.M.,

1,3Pinto J.R. 1- Department of Biomedical Sciences, Florida State University, College of Medicine,

Tallahassee, FL, USA; 2- Instituto de BioquímicaMédica, UFRJ, Brazil; 3- Department of Molecular and Cellular Pharmacology, University of Miami, Miller School of Medicine, Miami, FL; 4- Pulmonary, Allergy and Critical Care Medicine, Duke University Medical

Center, Durham, NC; 5- Institute for Genome Sciences and Policy, Duke University


Medical Center, Durham, NC;6-Division of Physiology, Department of Medicine, University of California, San Diego, CA, USA, 7- Interdisciplinary Stem Cell Institute,

University of Miami, Miami, FL, USA. Increases in muscle nitric-oxide (NO) production can be buffered by reaction with intracellular glutathione, forming S-nitrosoglutathione (GSNO). GSNO has been shown to S-nitrosylate Cys thiols of cardiac contractile proteins in vivo and in vitro, but effects on maximal force, thin-filament Ca2+ sensitivity and actomyosin ATPase activity are unknown. Here, we analyzed the targets of S-nitrosylation in mouse cardiac contractile proteins, and examined the effects of these modifications on function in myocytes and skinned cardiac myofibrils. S-Nitrosylation and denitrosylation were detected using resin-assisted capture (SNO-RAC) and targets for S-nitrosylation were identified by quantitative LC-MS/MS. Isolated cardiomyocytes treated with S-nitrosocysteine (CysNO, 500µM, 10min) showed an increase in total protein-SNO, followed by progressive denitrosylation (30-60min with CysNO). At 10min, CysNO dose-dependently increased S-nitrosylation of specific Cys thiols in myosin heavy chain, actin, TnC, TnI, myosin-binding protein C and other muscle proteins. Myofibril thin-filament Ca2+ sensitivity decreased (P<0.05) after in-vitro treatment with pharmacological GSNO concentrations (1, 10, 100 µM), but maximum force did not change (P>0.05). Loss of Ca2+ sensitivity was partially reversed by the denitrosation agent, ascorbate. Relaxation kinetics of skinned fibers, as measured by flash photolysis, were also significantly reduced by100µM GSNO (k1,15.33 to 11.68/s; k2 2.33 to 0.87/s; fit to double exponential).. Maximal myofibrillar ATPase activity (pCa 5.0) was also dose-dependently inhibited (8, 15, 30%) by 50, 100 and 500µM GSNO, an effect that was reversed by ascorbate. The findings suggest that S-nitrosylation of regulatory Cys thiol(s) can reversibly modulate cardiac muscle contraction and may be able to protect the heart by reducing myosin ATPase demand without affecting the maximal force of contraction. CAPES, CNPq, FAPERJ D18. YELLOW FEVER VIRUS-INDUCED MITOCHONDRIAL DYSFUNCTION: CHANGES IN MITOCHONDRIAL ENERGETIC METABOLISM AND APOPTOSIS INDUCTION

Sanches, D.1; Campos, S.P.C.1; Rocha, C.M. 1; Gaspar, L.P.2; Freire, M.S.2; Silva, J.L.1; Gomes, A.M.O.1 & Oliveira, A.C.1

1Instituto de Bioquímica Médica – CCS – Universidade Federal do Rio de Janeiro, RJ, Brasil.

Flaviviruses cause diseases like Dengue and Yellow fever. These viruses are transmitted by mosquitoes mainly in South America, Central America and Asiatic southeast, where they have a particular importance for public health. Virus-induced apoptosis is related to a cytopathological consequence of an infection in vivo or in vitro. During apoptosis, mitochondrial pathway has been described as a crucial step during viruses-induced apoptosis. Once the mitochondrial pathway is activated, loss of mitochondrial membrane potential (Δ m) occurs and caspases can be activated, culminating in apoptotic process. Here, we investigate the role of mitochondrial cell death pathway during Yellow Fever Virus (YFV) infection and its consequence to mitochondrial energetic metabolism. We infected Vero cells with YFV using a MOI=1. We analyzed the cell viability using Live/Dead and LDH assay. Apoptosis was analyzed by PhosphatidylSerine (PS) exposure and TUNEL, while the role of mitochondrial pathway was followed by Δ m through fluorescence microscopy. The importance of mitochondrial pathway was investigated by Bongkrekic acid, an adenine nucleotide translocator (ANT) inhibitor. The mitochondrial energetic metabolism was studied by oxygraphy. Apoptosis was observed after 72 hours post infection (h.p.i.) through TUNEL and PS exposure. The dependence of caspases activation during the apoptosis process was also observed, using z-Vad-fmk, a pancaspase inhibitor. We also observed loss of

Δ m 72 h.p.i. demonstrating that the apoptotic mitochondrial pathway is being activated and apoptosis is dependent of ANT activity. Oxygraphy results show a slighter increase of routine respiration, but a significant increase of oligomycin-sensitive oxygen consumption at 48 h.p.i., that indicate an increase in oxygen consumption rate coupled to ATP synthesis. Our results suggest that the mitochondrial pathway is activated, contributing partially for the caspase-dependent cell death process induced by YFV. Our data also suggest changes on mitochondrial energetic metabolism associated to virus infection. CNPq, CAPES, FAPERJ, INBEB, PRONEX D19. INTERACTION STUDY OF BOVINE SERUM ALBUMIN (BSA) WITH GUANYLHYDRAZONES BY NUCLEAR MAGNETIC RESONANCE FERREIRA NETO, D.C.; AZEREDO, S.O.F.; PETRONILHO, E. C. AND FIGUEROA VILLAR, J. D.

Grupo de Química Medicinal, Instituto Militar de Engenharia, Seção de Engenharia Química – Praça General Tibúrcio, 80, 22290-270, Praia Vermelha, Rio de Janeiro, RJ.

The interaction between biomolecules and ligands has aroused interest in recent years and new researches have been developed aiming to study how these interactions occurs. Albumin is a important protein, and its main function is to transport and bind to substances in the blood. Substances that interact strongly with albumin present difficulty in their pharmacological function. However, molecules that do not interact with the albumin are likely to be destroyed by other proteins present in blood. Thus, to take appropriate action, the drug must provide a medium interaction with albumin. The objective of this study was to examine the possibility of interaction of bovine serum albumin (BSA) with guanylhydrazones synthesized by our research group, via Nuclear Magnetic Resonance (NMR). We used the experimental measurements of T1 and T2 (relaxation time), differences transferring saturation (Saturation Transfer Difference, STD), Nuclear Overhauser Effect (NOE), and Diffusion Ordered Spectroscopy (DOSY). Preliminary results by NMR using the techniques described, showed average changes in the values of T1 and T2 of some guanylhydrazones, which interact with albumin. These changes in relaxation times, which are confirmed by diffusion coefficient variations indicate the interaction ligand-protein. These preliminary results indicate that the tested guanylhydrazones are potentially stable drugs for cancer and bacterial infections. CAPES/Pró-defesa, INBEB e ao Exército Brasileiro. D20. DIETHYLCARBAMAZINE ATTENUATES THE DEVELOPMENT OF MONOCROTALINE-INDUCED LUNG INJURY IN MICE

1-Ribeiro E. L.; 1-Fragoso I. T.; 1- Silva A. K. S.; 1- Donato M.A.M.; 1-Olibeira A. C.; 1- Oliveira W. B. 1, 2- Peixoto C. A.

1- Universidade Federal de Pernambuco; 2- Centro de Tecnologia Estrategicas do Nordeste

The alkaloid monocrotaline (MCT) is a toxin from plants, mainly employed to establish a model of pulmonary dysfunction. Clinical reports have described favorable results with the use of diethylcarbamazine (DEC) in bronchial asthma. This drug has an important anti-inflammatory role since it interferes with arachidonic acid metabolism. In the present study, we investigated the efficacy of oral DEC treatment in mice model of injury pulmonary. Forty C57/BL6 male mice, weighting 20-25g, were used in all experiments. Four groups were studied: control; MCT7; MCT14; MCT14/DEC14 (50mg/Kg per day of DEC from 1 to day 14). MCT solution intraperitoneal injection (600mg/kg once per week). The animals were anesthetized and collection of bronchoalveolar lavage fluid. Lung tissues were collected and processed for light microscopy and immunohistochemistry (COX-2 and F4/80). The histological analysis revealed that the animals of group MCT7 and MCT14 exhibited discrete alveolar


thickening due to increased cellularity, mild hemorrhage and congestion, inflammatory cells, pulmonary edema and emphysema. No group MCT14/DEC14 there was a decrease of injury and the infiltration of PMNs. The lungs in the group control exhibited preserved morphological characteristics. The lung tissue sections obtained from mice in the group MCT7 and MCT14 revealed considerable COX-2 expression, whereas COX-2 expression was significantly reduced in lung sections obtained from mice treated with DEC. Lung sections from the mice in the group control expressed baseline levels of COX-2. Lung sections obtained from mice in the group MCT7 and MCT14 revealed positive staining for F4/80 in alveolar macrophages, whereas DEC treatment significantly attenuated F4/80 expression. Little staining for F4/80 was observed in the lung tissue obtained from the group control. In conclusion, the MCT induced pulmonary dysfunction was attenuated by DEC treatment, probably via inhibition of a number of steps in arachidonic acid pathway, thereby preventing the production of cyclooxygenase or lipoxygenase metabolites, important inflammatory intermediates. INBEB, FACEPE, CNPq, CPqAM D21. CROSS-TALK BETWEEN FIBROBLASTS AND DENDRITIC CELLS UPREGULATES IL-23 PRODUCTION DURING PORPHYROMONAS GINGIVALIS INFECTION IN GINGIPAIN-DEPENDENT MANNER

1-RAMOS-JUNIOR, E.S., 1-NASCIMENTO, C.R.,1-MORANDINI, A.C., 1-SCHARFSTEIN, J. Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro

Using a murine model of mucosal P. gingivalis (P.g) infection, a bacterium that causes periodontal disease, we have demonstrated that bradykinin, a proinflammatory peptide released by gingipain, upregulates generation of INF-γ and IL-17-producing T cells in submandibular LN. IL-23 produced by dendritic cells (DCs) is essential for Th17 expansion. Fibroblasts contribute to the gingival microenvironment, and it is known that DC/fibroblast interaction is important to cytokine modulation. Here we investigated the interplay between DCs/gingival fibroblasts (g-FB) in innate responses elicited by P.g. Mouse BMDCs were co-cultured with g-FBs and stimulated either with P.g (strain W83) or a triple gingipain knockout P.g (KRAB), in the presence of IFN-γ. We found that g- FBs/DCs exposed to W83 upregulated IL-23 in the supernatant as compared to DC alone or g- FBs/DCs exposed to KRAB. IL-10 was exclusively produced by W83-stimulated DCs (alone), and the levels were decreased upon g-FB co-culturing. IL-23 production is dependent on IL-6, TNF-α and IL-1β. IL-6 was produced by g-FB exposed to W83, but not by KRAB. TNF-α and IL-1β, were not observed in g-FB. DCs exposed to W83 or KRAB upregulated same levels of TNF-α and IL-1β, and the presence of g-FB decreased the levels of both cytokines. Furthermore, these differences were not related to DC activation, since CD40 and CD86 surface expression were upregulated independently of co-culturing with g-FB or bacteria strains. g- FBs and DCs are involved in a cellular “cross-talk” leading to the upregulation of IL-23. Ongoing studies should clarify whether bradykinin receptors and/or alternative molecular targets may account for the gingipain-dependent induction of the IL-23/Th17 pathway. FAPERJ, CNPq and INCT-CNPq/INBEB D22. THE ROLE OF MICROGLIA ACTIVATION IN SYNAPSE LOSS AND SHORT-TERM MEMORY DEFICITS IN OCULOLEPTOMENINGEAL AMYLOIDOSIS RELATED TO TRANSTHYRETIN

1-ESTEFANIA P.C.AZEVEDO, 1-JOSÉ HENRIQUE LEDO, 2-GUSTHAVO BARBOSA, 2-MORGANA SOBRINHO, 2-LUAN DINIZ, 2-ANNA C. C. FONSECA, 2-FLÁVIA GOMES, 2,3-

LUCIANA ROMÃO, 1-FERNANDO L. PALHANO, 2-FLÁVIA R. S. LIMA, 1-SÉRGIO T. FERREIRA AND 1-DEBORA FOGUEL

1-Instituto de Bioquímica Médica, 2-Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro , 3-Pólo de Macaé, Universidade Federal do Rio de Janeiro,

Macaé Oculoleptomeningeal amyloidosis (OA) is a fatal and untreatable hereditary disease characterized by the accumulation of transthyretin (TTR) amyloid within the central nervous system. The mechanistic basis of OA pathogenesis is still unknown, thus, we aimed to understand how amyloid triggers neuronal damage in this context. Herein we showed that amyloid formed by a TTR mutant, A25T, activates microglia leading to the secretion of TNF-α, IL-6 and nitric oxide. Also, we observed that A25T amyloid induces Akt activation culminating in NFκB translocation to the nucleus of microglia cells. A25T fibrils are not directly toxic to neurons, however when exposed to the conditioned media of fibrils-activated microglia, neuronal death via apoptosis was extensive. Prior, the conditioned media of fibrils-activated microglia led to synapse loss in neuron in culture. We tested A25T fibrils toxicity in vivo, using an i.c.v. procedure, and observed microgliosis and cognitive dysfunction in mice that received fibrils as treatment. These data indicates that A25T amyloid fibrils act mostly as inflammatory agents, activating microglia and leading to bystander neuronal damage. INBEB, FAPERJ, CNPq D23. DETERMINAÇÃO ESTRUTURAL DA PROTEÍNA HIPOTÉTICA Q4DY78 CONSERVADA EM CINETOPLASTÍDEOS POR RMN

1 - D'ANDRÉA, E. D.; 1 - HEREDIA, G.P.; 1 - PIRES, J.R. 1 - Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro

Os tripanossomatídeos Trypanosoma cruzi, Trypanosoma brucei e Leishmania major são causadores das doenças negligenciadas como a doença de Chagas, a doença do sono e leishmaniose, respectivamente. A partir do sequenciamento genômico destes cinetoplastídeos foi possível identificar genes que codificam proteínas hipotéticas específicas destes parasitas. A determinação estrutural de proteínas auxilia o processo de caracterização funcional e validação dessas macromoléculas como alvo terapêutico. O presente trabalho tem como objetivo primeiramente a seleção de genes codificadores de proteínas hipotéticas específicas de tripanossomatídeos e posteriormente a sua determinação estrutural através de ressonância magnética nuclear (RMN). A partir da análise das estruturas espera-se obter informações sobre suas funções. Para a seleção foi utilizado o banco de dados TritrypDB disponível na rede. O gene Q4DY78, que é predito codificar uma proteína alfa/beta e apresenta homologia com outras proteínas sem estrutura conhecida, foi obtido comercialmente clonado no plasmídeo pUC57. Ele foi sub-clonado no plasmídeo de expressão pGEX-4T2, seguido da expressão e purificação da proteína. A proteína se expressou solúvel e o espectro de RMN de hidrogênio mostrou que ela é enovelada. A partir daí, os seguintes experimentos de RMN foram obtidos: com uma amostra não marcada - 2D TOCSY, 2D NOESY (em H2O e em D2O); com uma amostra uniformemente marcada com 15N - 15N HSQC, 15N HSQC TOCSY, 15N HSQC NOESY, série de 15N HSQC com diversos tempos de relaxação para medida de T1 e T2 de 15N e heteronuclear NOE; com uma amostra duplamente marcada com 15N e 13C – 13C HMQC, 13C HSQC, CBCANH, CBCACONH, HCCH-COSY, HNCO, HBHACONH, 13C HSQC NOESY, além de espectros utilizando metodologia de Fast NMR, bCBCANH, bCBCACONH, bHNCO e bHNCOi. 15N HSQCs em diferentes temperaturas foram feitos para verificar ligações de hidrogênio e se o aminoácido está em estrutura secundária. O assinalamento do backbone da proteína está em andamento. CNPq


D24. IDENTIFICATION OF ZINC-BINDING GROUPS AS SPECIFIC SNAKE VENOM METALLOPROTEINASE INHIBITORS

1- Villalta-Romero, F.; 1- Espíndola A. P.; Tasic. L. Instituto de Química, UNICAMP

"Snakebite envenomation represents a highly relevant public health problem, mostly affecting poor people living in rural settings of Asia, Africa and Latin America. The majority of snakebite envenomations in Latin America are caused by the viperid species, whose venom contains a high proportion of zinc-dependent metalloproteinases that play a relevant role in the pathogenesis of hemorrhage characteristic of these envenomations. Antivenoms have proved to be highly effective in the neutralization of systemic effects induced by snake venoms; however, they are only partially effective in abrogating the local pathological alterations induced by viperid snake venoms. The use of broad MP inhibitor, such as Batimastat®, has shown to be effective in decreasing hemorrhagic, dermonecrotic and edema-forming effects of various snake-venom metalloproteinases. However, the difficulty of open public access to Batimastat® legitimates the design of new inhibitors for SVMP. More recently, reports of matrix metalloprotease inhibitors (MMPi) with novel zinc-binding groups for these inhibitors (ZBGs) have been found to influence inhibitor potency and selectivity. These ZBG effects have been attributed to hydrogen-bonding interactions with the protein and van der Waals contacts. ZBGs figure as potent leads, which can be further optimized to improve solubility and pharmacokinetics via traditional medicinal chemistry. Protein (MP) and ligand interactions were monitored using Saturation Transfer Difference (STD) NMR experiments. Screening different zing binding molecules in terms of their affinity to target protein (BaP1) enabled us to discriminate investigated compounds and to propose new and potent drugs. The desing of small molecules with affinity for the metalloproteinase active site, coupled to zinc-binding groups offers promising possibilities." CAPES, INBEB, FAPESP D25. SEMI-CORRELATIVE MICROSCOPY OF THE CUTICLE OF THE PARASITIC NEMATODE HASSALSTRONGYLUS EPISLON

Adnet, F. A. O.; de Souza, W.; Attias, M. Instituto de Biofísica Carlos Chagas Filho-UFRJ

Hassalstrongylus epsilon (Travassos 1937), is a nematode parasite of the duodenum of the water rat Nectomys squamipes, Brants, 1827 (Rodentia: Muridae). Hassalstrongylus epsilon is a member of Trichostrongyloidea superfamily that includes gastrointestinal parasites of ruminants such as Haemonchus and Trichostrongylus, which cause important diseases and economic loss in ruminants. The members this superfamily have longitudinal cuticular ridges that fix the parasite to the intestine or stomach of the host and that are sustained by the so called struts. These cuticular structures have been used as basis for taxonomic classification of trichostongylides. By observing in light microscopy semithin transversal sections of H. epsilon fixed and embedded in epoxy resin and comparing it with ultrathin sections subsequently taken at the same region and observed by Transmission electron microscopy. These observations revealed that each cuticular ridge can have a different appearance in cross section, indicating that this is not a good parameter for taxonomical classification. Electron tomography of the cuticle revealed a complex structure of the cuticle, with striated fibers associated with the struts and several layers of varied morphology. This complex structure needs to be further explored in order to establish its association with the muscular layer and its role in the parasite motility. INBEB, FAPERJ, CNPQ e CNPQ-PROTRAX

D26. EFEITO DO ESTADO DE PROTONAÇÃO DO ÁCIDO ASPÁRTICO NA ESTABILIDADE DA PTERIDINA REDUTASE DE LEISHMANIA MAJOR

1,2- LEITE, F. H. A.; 2- LACERDA, P. S.; 2- PITA, S. S. R.; 2- CASTILHO, M. S. 1- Programa de Pós-Graduação em Biotecnologia, Universidade Estadual de Feira de

Santana; 2- Faculdade de Farmácia, Universidade Federal da Bahia. Segundo Organização Mundial de Saúde (OMS) a Leishmaniose é a segunda protozoose mais importante em termos de prevalência e mortalidade. Entretanto, o repertório de fármacos é limitado e apresenta, na maioria dos casos, baixos índices de eficácia e segurança. Embora os protozoários do gênero Leishmania sejam auxotróficos para folatos, inibidores da enzima Diidrofolato redutase (DHFR) são pouco eficazes, pois a Pteridina redutase (PTR1) atua como via bioquímica alternativa. Diante desse cenário, inibidores de PTR1 e DHFR poderão ser compostos promissores para o desenvolvimento de novos fármacos. O conhecimento do mecanismo catalítico em nível molecular da PTR1 pode auxiliar no planejamento de fármacos leishmanicidas. Portanto, simulações de Dinâmica Molecular (DM) foram empregadas para investigar a influência do estado de protonação do resíduo catalítico ASP181 sobre a estabilidade da PTR1 (APO), bem como sobre a afinidade da enzima pelo cofator (NADPH). Os resultados mostram que após 50 ns de simulação (programa GROMACS 4.5.5, campo de força GROMOS 53a6, T= 303 K, pH= 4,7 e p=1atm), o valor de desvio em relação a estrutura inicial da DM para o sistema APO protonado foi 0,59±0,12 nm, enquanto que o sistema APO não protonado, 0,41±0,08 nm. Por outro lado, o valor de desvio para o sistema PTR1-NADPH protonado foi 0,41±0,06 nm e o sistema PTR1-NADPH não protonado, 0,46±0,08 nm. Deve-se ressaltar também que o sistema PTR1-NADPH protonado apresentou 2,8±0,8 ligações de hidrogênio com uma vida média de 705ps, enquanto que o sistema PTR1-NADPH não protonado apresentou 3,0±1,1 ligações de hidrogênio com uma vida média de 1305 ps. As informações estruturais obtidas através da DM revelam que o estado de protonação do resíduo ASP181 é essencial para estabilidade de PTR1 e modulação de sua afinidade pelo cofator. Esses dados serão cruciais para guiar estudos de triagem virtual baseados na estrutura tridimensional de PTR1 de L. major. FAPESB D27. ESTUDO EPIGENÉTICO NA PROGRESSÃO TUMORAL MAMÁRIA 1- GILSON C. SANTOS JR; 1- ANA P. SILVA; 1- LUCAS FELDMAN; 1- JULIANA CONSTANTE;

1- CLAUDIA V. DE MOURA-GALLO 1- Departamento de Genética, Universidade do Estado do Rio de Janeiro (UERJ), Instituto de Biologia Roberto Alcântara Gomes, Rio de Janeiro, 20550-013, Brasil.

A progressão tumoral mamária depende de uma série de alterações epigenéticas, dentre as quais se destacam a metilação e a acetilação. A metilação pode ocorrer tanto no genoma quanto em determinados tipos de histonas, a acetilação, por sua vez, ocorre praticamente apenas nas histonas. No intuito de analisar as principais alterações epigenéticas na progressão do carcinoma mamário, utilizamos as linhagens celulares da série de progressão tumoral mamária 21T(H16N2, 21PT, 21NT, 21MT1 e 21MT2), provenientes da mesma paciente, apresentando portanto o mesmo “background” genético. Com os resultados obtidos, através da digestão pelas enzimas de restrição MspI e HpaII, foi possível verificar um aumento significativo na metilação global genômica nas linhagens metastáticas, assim como um aumento da trimetilação na lisina 9 da histona H3 (H3K9me3), através de microscopia confocal e western blot, marcador de heterocromatina constitutiva. Também foi detectada uma diminuição da acetilação na histona H4, marcadora de eucromatina, através dos mesmos ensaios descritos anteriormente. Uma diferença significativa na distribuição da eucromatina e da heterocromatina ao longo da progressão do carcinoma mamário foi observada. Estes resultados ressaltam a importância das alterações epigenéticas na progressão da câncer


e apontam para a extensa mudança espacial que ocorre na cromatina no núcleo das células ao longo da progressão para malignidade. FAPERJ, CAPES, CNPq D28. BIOPHYSICAL STUDIES OF SUGARCANE MITOCHONDRIAL SHSP22

PINHEIRO,G.M.S; TIROLI-CEPEDA,A.O.; RAMOS, C.H.I. Instituto de Química, UNICAMP, Campinas, SP-Brasil.

"Heat shock proteins (HSPs) can be classified into six structurally conserved classes according to their molecular weight, namely, HSP100, HSP90, HSP70, HSP60, small heat shock proteins (sHSPs) and ubiquitin (8.5 kDa). SHSPs form large polydisperse oligomers that are exceptionally dynamic and important for the functional ability of protecting substrate proteins from aggregation. The diversity of sHSP in plants is intriguing and characterization of their chaperone activity is important to understand plant tolerance to heat stress. In the present work we describe the structural characterization of a recombinant SsHSP22 using circular dichroism, intrinsic/extrinsic fluorescence and size exclusion chromatography. The SsHSP22 was obtained 95% pure, and was folded as deemed by CD and fluorescence emission spectra. The chaperone was purified as a single species constituted of an oligomer with approximately 26 monomers. CD analyses showed that SsHSP22 had a secondary structure consisting primarily of β-sheets (~45%) due to the presence of a minimum at approximately 217 nm. Biophysical and biochemical characterization of a recombinant mitochondrial SsHSP22 from sugarcane are in progress and the results may be significant to the study of the ethanol production as a renewable energy source. Keywords: small heat-shock proteins, protein folding, chaperone activity, sugarcane, SsHSP22." FAPESP and CNPq. D29. ANÁLISE POR RMN DAS INTERAÇÕES INTERMOLECULARES DAS CATEQUINAS NO CHÁ VERDE (CAMELLIA SINENSIS)

1- CASTAÑEDA-VALENCIA, G; 2- TINOCO, L 1,2-Nucleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro

As catequinas são componentes fenólicos extraídos de plantas, possuem atividades anticancerígenas, antioxidantes, etc. e são encontradas em alimentos e bebidas, como no chá verde. Embora a agregação de compostos fenólicos já tenha sido descrita na literatura, ainda há pouca informação sobre os processos de agregação das catequinas diretamente no chá verde. As interações intermoleculares podem ser identificadas como variações nos deslocamentos químicos, nos tempos de relaxação e nos coeficientes de difusão translacional. A infusão do chá verde foi liofilizada e preparada nas concentrações de 0,5; 1; 2 e 5 mg/mL em D2O. Foram feitos espectros de RMN de 1H a 499,79 MHz ( VNMRS-500 –Agilent) e medidos os tempos de relaxação T1 e T2 a 25 o C. Nos espectros de RMN de 1H foram identificados os sinais representativos dos componentes catequinicos: Epigalocatequina galato (EGCG), Epicatequina (EC), Epigalocatequina (EGC) e Catequina (CATE). Foi observado que há uma diminuição dos deslocamentos químicos com o aumento da concentração, sendo esta variação mais evidente para os sinais de H-3, H-2 e H-2´´ da EGCG, para H-2, H-6 e H-2´ da EGC, para H-2´ da EC e para H-8, H-4α e H-2´ da CATE. Na amostra mais concentrada (5 mg/mL) houve uma maior sobreposição e alargamento dos sinais, não sendo possível identificar alguns dos hidrogênios, claramente identificados nas amostras de 0,1 e 1 mg/mL. Os tempos de relaxação T1 e T2 para a maioria dos hidrogênios representativos da EC, EGC e EGCG diminuem com o aumento da concentração, mas os hidrogênios da CATE apresentaram uma variação irregular dependo do hidrogênio analisado. Estes resultados demonstram que as catequinas, mesmo na presença dos demais componentes presentes no chá verde, sofrem um processo de agregação, o que deve

ser levado em consideração na avaliação dos ensaios de atividade biológica feitos com o chá verde. INBEB, FAPERJ, CNPq D30. BONE MARROW MESENCHYMAL STROMAL CELLS RESCUE CARDIAC FUNCTION IN STREPTOZOTOCIN-INDUCED DIABETIC RATS

1-MONNERAT-CAHLI, G; 1-GUERRA, B; 1-MANSO, G; 1-FERREIRA, A; 1-GRAN DA SILVA, D; 2-COUTINHO, D.C; 3-CARNEIRO-RAMOS, M.S.; 3-TRENTIN-SONODA, M; 1-

RODRIGUES, D; 1-CABRAL-DA-SILVA, M.C; 1-GOLDENBERG, R; 1-NASCIMENTO, J.H.M; 1,4-CAMPOS-DE-CARVALHO, A.C; 1-MEDEI, E

1 Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil. 2 Department of Morphology, Institute of Biological Sciences, Federal

University of Minas Gerais, Belo Horizonte, Brazil; 3 Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo Andre, Brazil; 4 Instituto Nacional de

Cardiologia. Rio de Janeiro. Brasil. Diabetes Mellitus can lead to cardiac dysfunction. Several studies have shown the anti-diabetic effects of bone marrow mesenchymal stromal cells (MSC). In the present study, we investigate whether MSC transplantation can revert cardiac dysfunction in streptozotocin-induced diabetic rats. The rats were divided in three groups: Non-diabetic, Diabetic (DM) and Diabetic-Treated (DM+MSC) that received 5x106 MSC 4 weeks after establishment of diabetes. Four weeks after MSC-therapy, systemic metabolic parameters and cardiac function were assessed. MSC transplantation was able to revert the hyperglycemia and increase the body weight of the animals, while decreasing sera corticosterone levels without restoring insulin secretion and oral glucose tolerance. Also, MSC improved electrical remodeling, shortening the QT and QTc on the ECG and the action potential duration of left ventricle myocytes. MSC rescued the cardiac beta-adrenergic sensitivity by increasing beta-1 adrenergic receptor expression. Both alpha and beta cardiac AMPK and P-AMPK returned to baseline values after MSC-therapy. Toll-like receptor signaling pathways are not involved in the mechanism of MSC-improved cardiac function. The results indicate MSC-therapy was able to rescue cardiac impairment induced by diabetes, normalize cardiac AMPK subunit expression and activity and decrease corticosterone and glycemia. CNPq, FAPERJ, INBEB D31. VISUALIZATION OF CYTOSKELETON OF TRITRICHOMONAS FOETUS BY XHR-SEM (EXTREME HIGH RESOLUTION SCANNING ELECTRON MICROSCOPY)

1,2- De Andrade Rosa, I; 3,4-De Souza, W.; 2,5 Benchimol, M. 1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 2-

Universidade Santa Úrsula; 3- Laboratório de Ultraestrutura Celular Hertha Meyer – Universidade Federal do Rio de Janeiro; 4- Instituto Nacional de Metrologia, Qualidade e Tecnologia-Inmetro, Duque de Caxias, Rio de Janeiro; 5- Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro Tritrichomonas foetus is the causative agent of bovine trichomoniasis, one of the most widespread sexually transmitted diseases in cattle. T. foetus is also studied because of its particular cell structure such as the mastigont system. This system is formed mainly for microtubular structures, the pelta and axostyle; the costa, a periodic rootlet; the parabasal fibers and several other filaments. The goal of this study was obtain a better understanding of T. foetus cytoskeleton using the extreme high resolution scanning electron microscopy (HXR-SEM). For this, the plasma membrane of T. foetus was extracted with detergent. Afterwards, the cytoskeleton was fixed with 2.5% glutaraldehyde, post-fixed with 1% osmium tetroxide, dehydrated in graded ethanol, critical point-dried in CO2, coated with carbon layer and observed on Magellan (FEI


Company). The Magellan microscopy offer subnanometer resolution over the full 1 kV to 30 kV electron energy range, effectively establishing a new performance category known as Extreme High Resolution Scanning Electron Microscopy (XHR-SEM). The results showed details of structures such as the costa and associated filaments, microtubules of axostyle and their connections, the comb and infrakinetosomal body. The costa was seen as a large striated root fibril that emerges from the anterior region and its bands could be observed. In addition, a new filament associated to the costa and connections between this structure and recurrent flagellum were also observed. A higher magnification revealed that these connections are formed to filaments and globular structures. The microtubule ribbons that form the pelta and axostyle were seen as two distinct orientations of microtubules. The posterior part of the axostyle turns upon itself forming a tube. Higher magnification of the axostyle allowed the observation of the oriented microtubules that form this structure. Thus, the results obtained show that XHR-SEM contributes to adding new data on the cytoskeleton of this protist. AUSU, INMETRO, INBEB, CNPq, FAPERJ and PRONEX D32. A NOVEL ALKYL PHOSPHOCHOLINE-DINITROANILINE HYBRID MOLECULE EXHIBITS BIOLOGICAL ACTIVITY IN VITRO AGAINST LEISHMANIA AMAZONENSIS

1 - GODINHO, J.L.P; 5 - GEORGIKOPOULOU, K.; 5 - CALOGEROPOULOU, T. ;1, 2, 3 - DE SOUZA, W. ; 1 ,2, 3, 4 - RODRIGUES, J.C.F.

1Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brasil. 2 Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro, Brasil. 3

Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Brasil 4 Polo Avançado de Xerém, Universidade Federal do Rio de Janeiro. 5National Hellenic

Research Foundation, Institute of Organic and Pharmaceutical Chemistry, Athens, Greece.

Leishmaniasis is one of the most important neglected tropical diseases caused by parasites of the Leishmania genus. The current chemotherapy is based on pentavalent antimonials, miltefosine, amphotericin B and pentamidine. However, there is an urgent need for safer and more efficacious drugs. An interesting approach in drug development is the combination therapy using drugs with known activity against the parasites. Both, trifluralin, a dinitroaniline herbicide, and miltefosine have activity against protozoan parasites, but with high IC50 and cytotoxicity for host cells. In this work, we investigated the effects of TC95 on Leishmania amazonensis. TC95 is a hybrid compound that combines trifluralin and miltefosine in only one molecular scaffold. The antiproliferative effects of TC95 against promastigotes were dose-dependent, with a total inhibition at concentrations of 4 and 5 μM. Against intracellular amastigotes, the effect was also dose-dependent after 24 h of treatment, however the effect was more pronounced after 48 h of treatment for all concentrations tested [1, 5 and 10 μM]. The IC50 values obtained after 48 h of treatment was 2.6 μM and 1.2 μM for promastigotes and intracellular amastigotes, respectively. Cytotoxicity assays against murine macrophages revealed that the CC50 was 60 μM, thus presenting a selective index of 24x. From 12 h until 72 h of treatment, promastigotes displayed profound alteration in the shape appearing rounded and swollen, with alterations in the cell cycle and in the cytoskeleton constituted mainly by microtubules. Transmission electron microscopy revealed that the mitochondrion is the main organelle affected, presenting an intense swelling with loss of the matrix content and disorganization of the mitochondrial membranes. Sometimes, lysis of the mitochondrion was also observed. Furthermore, alterations in the flagellar membrane, Golgi complex, increase in the number of lipid bodies and appearance of structures typically found in autophagic processes were also observed. Against intracellular amastigotes, TC95 also induced significant alterations such as: mitochondrial swelling with vesiculation of the mitochondrial membranes and

alterations in the kinetoplast; significant dilation of the trans-Golgi network; presence of lipid bodies in the amastigotes and inside the macrophages; and changes in the flagellar structure. Taken together, these results indicate that TC95 is a promising compound against Leishmania sp. CNPq, CAPES, and FAPERJ D33. IMMUNOMODULATORY EFFECT ON HUMAN MONOCYTES PROMOTES BY 5-HYDROXY-2-HYDROXYMETHYL-GAMMA-PYRONE (HMP), A SECONDARY METABOLITE OBTAINED FROM ASPERGILLUS FUNGI

Costa, J.P. 1,2*; Frade, P.C. R 1,2; Rodrigues, A. P. D.1,2,4; Farias, L.H.S. 1,2; Hage, A.A.P1,2; Silva, B. J. M 1,2; Santos, A.S. 3; Silva, E.O. 1,2

1Laboratório de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, Pará, Brazil 2 Laboratório de Biologia Estrutural, Instituto de Ciências

Biológicas, Universidade Federal do Pará, Belém, Pará, Brazil 3Laboratório de Planejamento e desenvolvimento de Fármacos, Instituto de Ciências Exatas e Naturais,

Universidade Federal do Pará, Belém, Pará, Brazil 4Laboratório de Microscopia Eletrônica, Instituto Evandro Chagas, secretaria de vigilância em saúde, ministério da

saúde, Belém, Pará, Brazil; "The 5-hydroxy-2-hydroxymethyl-gamma-pyrone (HMP) is a secondary metabolite synthesized by some species of fungi from Aspergillus, Penicillium and Acetobacter genera. The HMP has several applications, being used as antioxidant, tyrosinase inhibitor, protective agent against radiation and antitumor. Recently, it was also shown that this metabolite acts as a macrophage activator. However, the effect of HMP in human monocytes is unknown. Thus, the aim of this study was to evaluate the effects of HMP on the cell viability and differentiation of human blood monocytes in vitro. Human peripheral leucocytes were obtained from blood bag donated from Foundation Hemocenter of Para State. Cell isolation was performed using HISTOPAQUE® 1077-density-gradient. Monocytes were treated for 24, 48 and 72 hours with 50 and 100 μg/mL of HMP. The ultrastructural analysis of treated monocytes showed spreading ability, high number of cytoplasmatic projections and vacuoles, features that are often observed in activating cells. Immunofluorescence analysis of the expression of surface protein specific for the macrophage (F4/80), demonstrated that human monocytes treated with 50 and 100μg/mL for 48 and 72h showed the similar pattern of expression of proteins to that of human monocytes differentiated by macrophage colony-stimulating factor (M-CFS). The viability test used showed that HMP has no citotoxicity effect on human monocytes when treated with 50 and 100 μg/mL of HMP. These results demonstrated a new role for HMP as an immunomodulator agent, in inducing the differentiation of monocytes into macrophages. Keywords: HMP; Macrophages differentiation; Human monocytes." Supported by CAPES, CNPq/UFPa, CNPq/MCT/CT-INFRA/CT-PETRO (Processo nº 620179/2008), MCT/CNPq/FNDCT/CAPES/FAPERJ. D34. PRODUÇÃO DE CREME NATURAL A PARTIR DE PLANTAS DA AMAZÔNIA COM ATIVIDADE MICROBICIDA

*SANTOS, J.P2., COSTA, M.C2., PIMENTEL, B.W.M.2, COSTA1,2, J.P., JUNIOR, H.N.P2., SILVA, E.O2.

1Laboratório de Parasitologia e Laboratório de Biologia Estrutural, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, Pará, Brasil 2Colégio Estadual de

Ensino Médio Manoel Antônio de Castro, Secretaria Executiva Estadual de Educação (SEDUC) Belém, Pará, Brasil.

As mãos constituem a principal via de transmissão de microrganismos, pois a pele é um grande reservatório de diversos microrganismos que são transferidos de uma superfície


para outra, por meio de contato direto ou indireto com a pele. Na camada mais superficial da pele pode-se encontrar bactérias Gram-negativas, como a enterobactéria Escherichia coli e bactérias não fermentadoras como a Pseudomonas aeruginosa, além de fungos e vírus diversos. Assim, as mãos constituem uma importante maneira de veicular doenças que variam desde uma simples gripe até uma infecção mais severa, podendo levar a morte. Logo, o simples fato de higienizar as mãos, é um procedimento de grande importância e muito eficaz para evitar a propagação de muitas doenças contagiosas, sobretudo em ambientes frequentados por um grande numero de pessoas como nas Escolas Públicas. De acordo com o Centro de Controle e Prevenção de Doenças, dos EUA, a correta higiene das mãos é um dos mais importantes passos na prevenção de doenças e no controle da proliferação de germes. Para a ANVISA, o álcool em gel é o produto mais eficaz para higienização das mãos, pois é capaz de reduzir de forma significativa o número de bactérias e fungos que parasitam a pele. Entretanto, estudos mencionam que muitos microoganismos estão adquirindo resistência a vários produtos sintéticos, havendo portanto uma grande tendência a utilização de produtos de origem natural. Assim, este projeto tem como objetivo utilizar a rica biodiversidade da Amazônia para produção de um cosmético que tenha ação microbicida similar a verificada no álcool em gel. Para tanto, bactérias Staphylococcus aureus foram tratadas com extratos aquosos, infusões e extratos alcoólicos de 13 diferentes espécies de plantas na concentração de 0,1g/mL. Os ensaios preliminares mostraram que a o extrato aquoso da folha do jambo apresenta considerável ação microbicida. INBEB D35. DOENÇAS HELMÍNTICAS NO MUNICÍPIO DE IGARAPÉ-MIRI

2GOMES, D.A.O., 1,2 COSTA, J.P., 2JUNIOR, H.N.P.,1SILVA, E.O. 1Laboratório de Parasitologia e Laboratório de Biologia Estrutural, Instituto de Ciências

Biológicas, Universidade Federal do Pará, Belém, Pará, Brasil 2Colégio Estadual de Ensino Médio Manoel Antônio de Castro, Secretaria Executiva Estadual de Educação

(SEDUC) Igarapé-Miri, Pará, Brasil. "As enteroparasitoses, em especial as causadas por helmintos, constituem um grave problema de saúde pública no Brasil, estando diretamente relacionadas com as condições de saneamento básico, nível socioeconômico, grau de escolaridade, hábitos de higiene, entre outras variáveis. O município de Igarapé-Miri apresenta características favoráveis à ocorrência dessas doenças, pois apresenta saneamento básico precário, além de outros fatores que também favorecem a ocorrência dessas parasitoses. Assim, este estudo investiga quais helmintos encontram-se com maior frequência em amostras fecais de moradores do município de Igarapé-Miri. O estudo foi dividido em duas etapas: pesquisa de campo e pesquisa laboratorial, tendo como público alvo moradores da comunidade escolar do Colégio Manoel Antônio de Castro. Para a pesquisa de campo, foi realizada aplicação de questionários na comunidade escolar, a fim de fazer o levantamento do grau de conhecimento que esses indivíduos detêm em relação às parasitoses intestinais causadas por helmintos. Os resultados da pesquisa de campo mostraram que 86% dos entrevistados não sabiam informar as formas de transmissão de ao menos uma helmintíase 54% utilizam água sem nenhuma forma de tratamento. Para pesquisas laboratoriais, utilizou-se 50 amostras fecais que foram avaliadas através dos métodos coproscópicos Hoffman e Direto. Os testes laboratoriais mostraram que 42% das amostras fecais apresentavam ovos de T.trichiuris, 18% ovos de A. lumbricoides e 2% ovos de ancilostomídeo. Somente 38% das amostras, não apresentaram nenhuma forma de ovos helmínticos. Assim, este projeto é de grande relevância para a realização de um trabalho de educação preventiva no municpio de Igarapé-Miri.

Palavras-chave: helmintíases, Educação preventiva, Municipio de Igarapé-Miri, Métodos de Hoffman e Direto." INBEB, UFPA D36. LOSS OF ENDOCYTIC ACTIVITY DURING TRYPANOSOMA CRUZI METACYCLOGENESIS

1-NARCISA LEAL DA CUNHA-E-SILVA;1,2-WANDERLEY DE SOUZA,1-CAROLINA DE LIMA ALCANTARA; 1- JULIANA CUNHA VIDAL

1-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro Trypanosomatids require essential exogenous nutrients and growth factors in order to survive and divide. Trypanosoma cruzi epimastigotes acquire them by endocytosis via the cytostome and the flagellar pocket, both localized at the parasite’s anterior region. The cytostome is an opening at the plasma membrane surface that deepens in a membrane invagination called cytopharinx. This structure is responsible for 85% of endocytic activity in epimastigotes, delivering cargo to reservosomes. T. cruzi loses its endocytic ability somewhere during the transformation into trypomastigotes (metacyclogenesis), as these infective forms are not able to uptake exogenous cargo and do not present the cytostome-cytopharinx complex. Between epimastigotes and trypomastigotes, three subsequent types of T.cruzi intermediate forms were described: Ia, where the kinetoplast is close to the elongated nucleus, Ib where the kinetoplast is located side by side with nucleus, and Ic that presents the kinetoplast posterior to the nucleus. The latter is the form that just precedes the complete morphological transformation into trypomastigotes (Ferreira et al., 2008). We have analyzed changes in the ultrastructure of endocytic apparatus and the endocytic activity along in vitro metacyclogenesis. After four hours in TAU3AAG, we have obtained 30% of intermediate forms; using transmission electron microscopy we observed that Ic intermediate forms still present a cytostome. Interestingly, this invagination maintained its typical morphology, even after the migration backwards, accompanying the kinetoplast. Moreover, the endocytic apparatus proved to be functional in Ic intermediate forms, as they were able to uptake transferrin-gold offered after metacyclogenesis and store in typical reservosomes. Our data indicate that the disassembly and loss of function of the endocytic machinery is a very late event in metacyclogenesis. INBEB, CAPES, FAPERJ, CNPq D37. INTRA-ABDOMINAL ABSCESSES IN MICE CHALLENGED BY THE COMMENSAL BACTERIA BACTEROIDES FRAGILIS: EVIDENCE FOR INVOLVEMENT OF INFLAMMASOMES/IL-1BETA PATHWAY

CASTELPOGGI, J.1, TORRES, M.E.W.1, PEREIRA, J.S.1, LOBO, L.2, FERREIRA, E.O.2, DOMINGUES, R.M.C.P.2, ZAMBONI, D.3, BELLIO, M.2, SCHARFSTEIN, J.1 AND OLIVEIRA,

A.C.1 1- Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro; 2- Instituto de Microbiologia Paulo de Góes, UFRJ, Rio de Janeiro; 3 - Faculdade de Medicina de

Ribeirão Preto, USP, São Paulo. Bacteroides fragilis is a strictly anaerobic Gram-negative bacterium frequently isolated from patients with clinical infections, such as intra-abdominal abscesses and bacteremia. Its major source is the normal colonic microbiota where Bacteroides spp. outnumbers aerobic and facultative anaerobic bacteria. When intestinal integrity is disrupted, B. fragilis and colonic contents escape into the peritoneum. Although proinflammatory cytokines (e.g. IL-1 TNF ) may enhance host inflammatory response during sepsis and intra-abdominal abscess formation, these innate responses are also require to ensure the containment and elimination of intestinal microorganisms. Here we have evaluated the participation of IL-1 and inflammasome


pathway in the formation of intra-abdominal abscesses in mice injected intraperitoneally with B. fragilis plus sterile cecal contents (SCC). Although infected C57BL/6 (WT) mice present an intense cellular infiltration into the peritoneal cavity, the pathology was dramatically reduced in caspase-1-, ASC- and IL-1R-deficient mice. Consistently, caspase-1, ASC and IL-1R knockout mice were protected from early decrease on body weight and exhibit reduced inflammatory abscesses as compared to WT and IL-18R-/- mice subjected to the same challenge. Of further interest, we found that SCC alone induces in vitro IL-1 production by dendritic cells and macrophages in a MyD88-dependent manner, whereas no effect was observed with B. fragilis alone. The same results were obtained in vivo when we injected bacteria or SCC alone into peritoneal cavity and the IL-1 production was analyzed. Taken together, our results strongly suggest that the development of intra-abdominal abscesses containing this commensal microbe is critically dependent on adverse activation of the inflammasome/IL- pathway by colonic contents. Based on our mice studies, we suggest that the development of such abscesses in susceptible patients might be controlled by procedures that restore the epithelium barrier, preventing the transposition of colonic contents to the peritoneal cavity, and/or by inhibitors of the inflammasome/IL-1 pathway. INBEB, CNPq D38. IMPACT OF SOLUBLE Β-AMYLOID PEPTIDE ACCUMULATION ON INTRACELLULAR TRAFFICKING IN CULTURED NEURONS. OLIVEIRA, L.T.1,4; LEON, G.V.O.4; PROVANCE, D.W.2; MELLO, F.G.3; SORENSON, M.M.1;

SALERNO V.P.1,4 1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2- Centro

Desenvolvimento Tecnológico de Saúde, Fiocruz; 3- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro; 4- Departamento de Biociências da

Atividade Física - EEFD, Universidade Federal do Rio de Janeiro. It is widely recognized that β-amyloid (Aβ) also accumulates within neurons causing disruption of synapses and cognitive function. Here, we report the internalization and transport of a fluorescently labeled Aβ peptide and suggest its impact on intracellular trafficking. Using cultured chick neurons, we have shown previously that exogenous Aβ42 is internalized and leads to the redistribution of Myosin Vb (MVb), a molecular motor protein involved in intracellular traffic. The internalization process is specific for the 1-42 isoform, exhibits characteristics of a constitutive process without a specific point of saturation and depends on Aβ40/Aβ42 (Cytoskeleton. 69(3):166, 2012). Co-labeling for caveolin, EEA1 and Lamp1 after 10 min with Aβ42 suggested that a portion of the Aβ42 oligomers was internalized by caveolin-coated vesicles and localized with primary endosomes, but not lysosomes. Internalized Aβ42 evolved from a diffuse to a punctate distribution that shifted from the processes to the perinuclear region. These changes in pattern of Aβ coincided with an increased coincidence with MVb. A similar profile was observed for Rabs 8 and 10, which co-localized with internalized Aβ42 and suffered a shift in distribution. The localization of MVb with Rabs 8 and 10 is consistent with the early endosomes distribution suggested by labeling by with EEA1. It also suggested that some of the Aβ42 may be transported to the Golgi network. These observations are consistent with the hypothesis that AD proceeds as a result of an imbalance between Aβ production and Aβ clearance. Furthermore, it suggests a role for MVb and its binding partners in this process with the changes in MVb and Rab proteins distribution as possible consequences of Aβ42 internalization that could impact intracellular transport pathway(s) involving myosin Vb, thereby sequestering myosin Vb away from its normal sites of action and compromising overall neuronal function. INBEB, FAPERJ, CNPq, CNPq/INCT

D39. INSIGHTS INTO INTERACTION BETWEEN THE C-TERMINUS OF HUMAN 90 KDA HEAT SHOCK PROTEIN HSP90 AND THE MITOCHONDRIAL TRANSLOCASE OF OUTER MEMBRANE TOM70 ZANPHORLIN LM1, SQUILACE ALA1, FIGUEIREDO A1, GOZZO FC1, BARBOSA L2, RAMOS

CHI1. 1Instituto de Química, Unicamp, Campinas, SP, Brasil; 2Instituto de Física, USP, SP,

Brasil. “The mitochondrial import receptor Tom70 (translocase of the mitochondrial outer membrane 70) is a TPR-like protein, which interacts with chaperone–preprotein complexes and Hsp90 is a crucial molecular chaperone required for folding. Although it is known that the pentapeptide MEEVD at the C-terminus of Hsp90 is the motif involved in the recognition of TPR proteins, the molecular mechanism by which Tom70 co-ordinates interactions with preproteins and chaperones is still elusive. The interaction was investigated by cross-linking coupled with LC-MS/MS analysis, small-angle X-ray scattering (SAXS), isothermal tritration calorimetry (ITC) and in silico methods. Mass spectrometry (MS) analysis of the tryptic products of the complex revealed four interpeptide cross-link sites in the complex, from which two were sites between Tom70 and C-Hsp90. The interpeptide cross-link sites were confirmed by ITC experiments in which a model peptide of the region acted as a competitor for the complex interaction. Structural modeling based on cross-linking MS analysis was employed to unveil the molecular interfaces involved in the interaction and the complex model, which was supported by SAXS analysis. This study constitutes the first direct mapping of interaction sites in the C-Hsp90/Tom70 complex and is a significant contribution to the mechanism of interaction between Hsp90 and co-chaperones. Key Words: Hsp90, Tom70, interface mapping, crosslinking, ITC, SAXS.” Financial Support from Fapesp, Cnpq and INBEB D40. DIFERENÇAS ULTRAESTRUTURAIS ENTRE LEVEDURAS DE SPOROTHRIX SCHENCKII E SPOROTHRIX BRASILIENSIS

1-BORBA-SANTOS, L.P.;1- FONSECA, B., B.;2- ISHIDA, K.2; 3-LOPES-BEZERRA, L.M.;1- ROZENTAL, S.

1-Laboratório de Biologia Celular de Fungos, IBCCF, UFRJ. 2-Departamento de Microbiologia, ICB, USP. 3-Laboratório de Micologia Celular e Proteômica, UERJ.

"Espécies dimórficas do complexo Sporothrix schenckii são causadoras da esporotricose, micose subcutânea mais frequente no Brasil e de grande incidência no Estado do Rio de Janeiro. O objetivo deste estudo foi caracterizar aspectos ultraestruturais das leveduras de Sporothrix schenkii e Sporothrix brasiliensis, que são as espécies mais virulentas do complexo. Leveduras de dois isolados (ATCC MYA-4821 de S. schenckii e ATCC MYA-4823 de S. brasiliensis) crescidas em meio RPMI 1640 por 24 horas à 35°C, foram coletadas por centrifugação, lavadas em PBS e fixadas em solução de 2,5% glutaraldeído, 4% paraformaldeído em 0,1M tampão cacodilato de sódio por 24 horas à 4°C. As células foram pós fixadas em 1% OsO4 e 1,25 M K4[Fe(CN)6].3H2O por 2 horas à 4°C, lavadas em tampão cacodilato e desidratadas em etanol. Amostras para microscopia eletrônica de transmissão foram incluídas em resina Spurr e os cortes ultrafinos foram contrastados em acetato de uranila e citrato de chumbo. As imagens foram obtidas em um microscópio eletrônico de transmissão JEOL 1200 EX. Amostras para microscopia eletrônica de varredura foram aderidas em lamínulas recobertas com poli-L-lisina antes da pós fixação, desidratadas, secas em ponto crítico de CO2 e metalizadas com ouro. As imagens foram obtidas em um microscópio eletrônico de varredura FEI Quanta 250. Leveduras de S. schenckii exibiram morfologia alongada enquanto leveduras de S. brasiliensis exibiram morfologia ovóide. Leveduras de S.


schenckii apresentaram uma segunda camada da parede celular que não foi observada em leveduras de S. brasiliensis. Por outro lado, leveduras de S. brasiliensis apresentaram a parede celular e a camada microfibrilar mais espessa. Além disso, a parede celular de leveduras de S. brasiliensis apresentou acúmulo de material elétron denso. Estes estudos ainda são preliminares, entretanto, alguns aspectos ultraestruturais podem ser considerados na distinção entre S. schenckii e S. brasiliensis." Capes, FAPERJ e CNPq D41. CYSTATIN S/SA/SN IS SECRETED IN WORKLOAD-DEPENDENT MANNER

1,3-Sant’Anna, M.L.; 1,2-Almeida, A.C.P.A.; 1-Sorenson, M.M.; 2-Salerno, V.P 1-Instituto de Bioquímica Médica/UFRJ; 2-Escola de Educação Física e Desportos/UFRJ;

3-Centro de Instrução Almirante Sylvio de Camargo/RJ. "Ain: Physical exercise generates multiple physical and biochemical changes in the human body that can be monitored in various manners. The measurement of these changes can be good indicators of workload, redox state and the state of the immune system. To evaluate a group of cysteine protease inhibitors involved in several diseases, we following the salivary cystatin secretion at rest and after four incremental bouts on a cycle ergometer. Methods and results: The procedures in this study were approved by the Ethics Committee of the Hospital Universitario Clementino Fraga Filho. The subjects were 6 career enlisted men from the Brazilian Navy (age 25.8 ± 9.4 years). Exercise on the cycle ergometer consisted of four bouts at different intensities (65%, 75%, 85% and 95% of heart rate reserve). Saliva was collected before and after each bout (5, 10 and 15 min), centrifuged, and the supernatant stored at – 20° C. Accutrend indicator strips (Roche Diagnostic) were used to determine blood lactate concentration at rest and immediately after each test, with significant increase from 3 to 13 mM. Cystatin S/SA/SN was identified by Western blot and quantified by densitometry using a two-channel infrared scanner (Odyssey®). Cystatin increased significantly, 5 min after the highest exercise efforts 54% and 81% compared to resting values and returned toward normal values after 10 and 15 min post-exercise. Conclusion: Salivary cystatin secretion is correlated with exercise workload but recovers within 15 min." CAPES, CNPq, FAPERJ, INBEB/INCT, Centro de Instrução Almirante Sylvio de Camargo/MB D42. CONTRIBUIÇÃO DOS SOROS DE PACIENTES CHAGÁSICOS CRÔNICOS NA GÊNESE DE ARRITMIAS QUANDO PRESENTES EM UM AMBIENTE COM AUMENTADA DISPERSÃO TRANSMURAL DA REPOLARIZAÇÃO

1- VIDAL, M.A.; 1- MACIEL, L.; 2- PEDROSA R.C.; 1- NASCIMENTO, J. H. M.; 1- CAMPOS DE CARVALHO A. C; 1- CABRAL DA SILVA M. C.; 1- MEDEI, E.

1- Laboratório de Eletrofisiologia cardíaca Antonio Paes de Carvalho / Instituto de Biofísica Carlos Chagas Filho / UFRJ; 2- Hospital Universitário Clementino Fraga Filho /

UFRJ. "Introdução: A presença de anticorpos funcionais capazes de ativar receptores β1-adrenergicos e muscarínicos acoplados a proteínas G em pacientes chagásicos crônicos (PChC) foi descrita. Objetivo: Observar o efeito dos soros dos PChC na eletrogênese cardíaca, quando colocados no modelo farmacológico (E-4031) da Síndrome do QT longo do tipo 2 (SQTL2). Comparar a incidência de arritmias destes soros tanto em corações sadios como no modelo da SQTL2. Métodos: Foram utilizados corações de coelhos Nova Zelândia, machos. A SQTL2 foi induzida mediante perfusão do bloqueador da corrente IKr, E-4031 (5 µM) ao longo do experimento. Foram utilizados os soros de 52 PChC, previamente, funcionalmente caracterizados como tendo efeito: muscarínico (Soros-M; n=10), adrenérgico (Soros-β1; n=15) e ambos, muscarínico e adrenérgico (Soros-Mβ; n=17). Os soros sem atividade na bioeletrogenese foram denominados de

soros não-reativos (Soros-NR; n=10). Foi analisado o efeito de cada um dos soros no modelo farmacológico da SQTL2 visando estudar a incidências de arritmias. Resultados: A incidência de arritmias pelos soros dos PChC no coração sadio e na SQTL2 foi respectivamente: soros-β1: 0% vs. 75%, soros-M: 50% vs. 78% e os soros-Mβ: 67 vs. 64%. Já os soros-NR não induziram arritmias. Conclusão: Os soros-β1 em um coração sadio não apresentam eventos arrítmicos, entretanto, quando presentes em um modelo da SQTL-2 induziram arritmias em 75% dos casos. Um ambiente de maior dispersão transmural da repolarização, também, favoreceu uma maior incidência de arritmias na presencia dos soros-M. Todavia, os soros-Mβ apresentaram incidência semelhante de arritmias em ambos os casos. Estes achados preliminares sugerem que, para os soros dos PChC induzir arritmias ou aumentar a incidência destas, é necessário da presença de um microambiente conspícuo." FAPERJ, CNPq e CAPES D43. NEUROPROTECTIVE POTENTIAL OF BONE MARROW MESENCHYMAL STEM CELLS IN A MODEL OF ALZHEIMER´S DISEASE

1-GODOY, M.A.; 2-SARAIVA, L.; 1-VASCONCELOS-DOS-SANTOS, A.; 1-BEIRAL, H.J.V.; 1-SILVA, L.R.P.; 1-BRAGA, C.V.; 1-LEAL, R.B.; 1-SILVA, C.A.A.; 1-LIMA, A.P.C.A.; 1-VIEYRA,

A.; 2-DE FELICE, F.G.; 2-FERREIRA, S.T.; 1-MENDEZ-OTERO, R. 1-Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.2-Institute of Medical Biochemistry, Federal University of Rio de Janeiro,

Rio de Janeiro, Brazil. "Alzheimer's disease is a neurodegenerative disease whose incidence is expected to increase with the ageing of the world population. So far, there are no effective therapeutic alternatives. Thus, cellular therapy is a perspective for treatment of this neurodegenerative disorder. In bone marrow there are hematopoietic stem cells (HSC), which give rise to hematopoietic lineage and mesenchymal stem cells (MSCs), which form the connective tissue cells of bone marrow. The main hypothesis on the mechanism of action of MSCs is their paracrine action, releasing trophic factors that could act in the stimulation of neuronal survival, neurogenesis and modulation of inflammation. In this work, we aim to evaluate the effects of treatment with bone marrow mesenchymal cells in hippocampal cultures exposed to Aβ-derived diffusible ligands (ADDLs), the main neurotoxins responsible for the adverse effects observed in the beginning of disease. We found that ADDLs were able to bind to MSCs, without change the proliferation after 24h, cell respiration until 48h and viability during the whole period analyzed (maximum of 72h of exposure). In addition, MSCs were resistant to the oxidative stress generated by ADDLs after 24h of exposure, unlike observed in neurons. Furthermore, initial analysis suggests that the co-culture with mesenchymal stem cells prevents oxidative stress in hippocampal cultures exposed to Aβ oligomers (ADDLs). Thus, our data suggest that MSCs can represent a good therapeutic approach for the treatment of initial signs of Alzheimer’s disease. Key words: Alzheimer’s disease, mesenchymal stem cells, in vitro model" INBEB, FAPERJ, CNPq, PROTECEL, CAPES D44. STUDIES ON THE ENERGY AND REDOX METABOLISM OF THE BLOOD FLUKE SCHISTOSOMA MANSONI

1 - Oliveira, MP.; 1- -Oliveira, M.F 1 - Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro

The blood fluke Schistosoma mansoni is an intravascular parasite which causes human schistosomiasis. Within the human host, the adult forms of S. mansoni depend on the ingestion of large amounts of blood to supply their energy demands. Furthermore, evidence indicates that important metabolic changes occur in worms in several stages


of their life cycle, so that the cercariae present essentially an oxidative metabolism, while the adult forms living within the vertebrate blood vessels have a more fermentative metabolism. Furthermore, the activity and expression of antioxidant enzymes increases during development of the worm in the vertebrate host which is parallel with the ingestion of blood. Thus, we aim to investigate the redox and energy metabolism in adult forms of S. mansoni. We observed that the oxygen consumption associated with the Electron Transport System (ETS) is significantly lower in females than in males. This phenomenon occurs independently of the type of substrate used by worms. Glucose uptake was higher in females than in males suggesting increased dependence on glucose metabolism in females. Evaluation of ETS complexes activities showed that females have higher complex I-III and lower complex IV compared to males. Hydrogen peroxide production was significantly higher in whole females than in males. Pro-oxidant challenge with menadione showed that females are significant more resistant than males. We therefore conclude that there is a difference in energy metabolism and redox between adult female and adult male worms, which may be relevant to allow these organisms to the blood feeding habit. INBEB, FAPERJ, CNPq D45. AVALIAÇÃO DA ATIVIDADE DE MODULADORES DA AGREGAÇÃO DA PROTEÍNA PRION: ABORDAGENS TERAPÊUTICAS

1- ARAUJO, N. C. F.;2- MASCARELLO, A.;2- CHIARADIA, L. D.;2- NUNES, R. J.;2- YUNES, R.;1- MACEDO, B.;1- ALVES, W. J.;1- MACHADO, C. S. C.; 3- CAUGHEY, B.; 1-CORDEIRO, Y. 1- Faculdade de Farmácia, Universidade Federal do Rio de Janeiro; 2- Departamento de

Química, Universidade Federal de Santa Catarina; 3- National Institutes of Health, Hamilton, Montana

"As Encefalopatias Espongiformes Transmissíveis (EETs) são doenças neurodegenerativas fatais que afetam humanos e animais. Normalmente os indivíduos infectados apresentam sintomas clínicos de disfunção tanto cognitiva quanto motora e, em geral, os pacientes morrem cerca de doze meses depois do surgimento dos primeiros sintomas. Essas doenças ocorrem quando a proteína príon celular (PrPC) é convertida na sua isoforma infecciosa, a PrP scrapie (PrPSc). Essa conversão é caracterizada por uma alteração na estrutura secundária dessa proteína, que passa de uma proteína rica em alfa-hélices (PrPC) para uma proteína rica em folhas-beta (PrPSc), que forma agregados amiloides que se depositam no sistema nervoso central. Apesar do grande número de estudos na área, não há até o momento terapia para estas doenças, reforçando a necessidade de se buscar compostos eficazes em inibir essa conversão. Neste trabalho utilizamos como modelo de estudo o peptídeo da PrP de hamster sírio (Sha 109-149), que compreende um domínio hidrofóbico envolvido nessa conversão e que agrega prontamente em solução aquosa. Através da técnica de espalhamento de luz estático avaliamos a capacidade de uma biblioteca de compostos em inibir a agregação deste peptídeo. A redução da formação de agregados estruturalmente organizados foi acompanhada através da técnica de fluorescência da tioflavina T. A toxicidade desses compostos foi analisada através do ensaio de disfunção celular por redução de MTT e, através da técnica de dot blot verificamos a capacidade dos compostos em inibir o acúmulo da PrPSc em células de neuroblastoma infectadas (ScN2a). Através do “docking” molecular foram gerados modelos de interação entre a PrP e os compostos. Nossos resultados preliminares mostraram que alguns dos compostos avaliados apresentaram atividade moduladora da agregação do peptídeo da PrP e não mostraram toxicidade frente ao ensaio de viabilidade celular. Além disso, 47 compostos foram eficazes em reduzir o acúmulo da PrPSc em células ScN2a." CAPES, FAPERJ, INBEB, CNPQ

D46. THE PUTATIVE HEPATITIS C VIRUS FUSION PEPTIDE INTERACTS WITH MODEL MEMBRANES AND GAINS HELIX CONTENT 1- ALVES, N.S.; 1- MENDES, Y.S.; 2- SOUZA, T.L.F.; 1- BIANCONI, M.L.; 3- ANOBOM,C.D.;

1- VALENTE,A.P.; 1- SILVA, J.L.; 1- GOMES, A.M.O. & 1- OLIVEIRA, A.C. 1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro; 2- Faculdade

de Farmácia, Universidade Federal do Rio de Janeiro; 3- Instituto de Química, Universidade Federal do Rio de Janeiro

Introduction: The Hepatitis C virus (HCV) is an enveloped RNA virus, and the most common cause of chronic liver disease. Although much information has been gathered in recent years, the exact mechanisms that lead to membrane fusion are poorly understood. The envelope glycoprotein, E1 or E2, directly interacts with biological membranes, throughout the fusion peptide (FP). As the localization of FP remains unknown, we aim to characterize the interaction of a putative HCV FP, corresponding to residues 421-445 (HCV421-445), present in E2, with different membrane models. Methods: We used spectrophotometry, fluorescence spectroscopy, circular dichroism (CD), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), dynamic light scattering (DLS) and nuclear magnetic resonance (NMR) to evaluate peptide-membrane interaction and conformational changes due to this interaction. Results: ITC and fluorescence polarization data showed that HCV421-445 can interact with large unillamelar vesicles (LUV). However, DSC revealed that the peptide cannot change the phase transition temperature of multilamellar vesicles. Photometry analyses revealed that the peptide can induce LUV aggregation, but DLS data revealed that the peptide can either disrupt or aggregate the same vesicles. Since the interaction of fusion peptides with membranes can lead to an important structural gain, we also aim to solve HCV421-445 structure. NMR data of the peptide free in solution indicated low structure content but addition of SDS micelles leaded to a structural gain, confirmed by CD data. The preliminary structure calculation showed that the C-terminal of the peptide in the presence of micelles presents a tendency to form an alpha-helix, which could be stabilized by a helix stabilization motif, GXXXG. Conclusions: Altogether our results indicate that HCV421-445 peptide is able to perturb lipid membranes, and becomes structured. These studies are important to better understand the fusion mechanisms of HCV, which could help in the development of new antiviral therapies. Capes, CNPq, FAPERJ, PRONEX, INBEB D47. ANTI-INFLAMMATORY ACTION OF SILDENAFIL (VIAGRA®) TO PROTECT THE PLACENTAL INTEGRITY IN ABORTION MODEL INDUCED BY LIPOPOLYSACCHARIDES RAYANA LEAL DE ALMEIDA LUNA1, ANA KAROLINA SANTANA NUNES1, ANNE GABRIELLE

VASCONCELOS DE OLIVEIRA2, SURA WANESSA SANTOS ROCHA1, KARLA PATRÍCIA DE SOUSA BARBOSA1, WILMA HELENA DE OLIVEIRA2, SHYRLENE MERY DA ROCHA, MARIA

DA CONCEIÇÃO CARVALHO2, CHRISTINA ALVES PEIXOTO1,2* 1Laboratório de Ultraestrutura, Centro de Pesquisas Aggeu Magalhães, Fundação

Oswaldo Cruz, Recife-PE, Brasil; 2Laboratório de Microscopia, Centro de Tecnologias Estratégicas do Nordeste (MCT/CETENE

Infertility is an important problem of worldwide public health, but the mechanisms related to gestational complications are not completely understood yet. Abortion is an inflammatory and thrombotic process. Sildenafil plays an important role as vasodilator and anti-inflammatory properties. This study investigated the action of Sildenafil on placental integrity in a model of loss pregnancy induced by LPS, on treatment alone or in coadjutant treatment with Heparin. Eight females Albino Swiss aged 60, and weighing 30g were used per group. Control-group: females did not receive any treatment neither LPS administration; LPS-group: females received only the administration of LPS; Sildenafil-group: treated with 50mg/Kg of Sildenafil (Viagra®) administrated through the


drinking and received LPS administration; Heparin-group: treated with low weight molecular Heparin 500UI/Kg (Fragmin®) per day and received LPS administration; the Sild+Hep-group received both treatments and the LPS administration. Treatments were realized since first day of pregnancy. The model was induced by LPS injection on the 15th day and after 2h the females were euthanized. Placentas were processed for Histopathology, Electron Transmission Microscopy, immunohistochemistry and western blotting to TNF-α. In placental labyrinth region, the LPS caused edema, congestion and degeneration; all treatments resulted in the improvement of such conditions. Ultrastructural analysis of the labyrinth showed that exposure to LPS induced drastic alterations such as intense degenerations in the labyrinth cells. The treatments protected in different levels the tissue. The LPS group increased immunostaining for TNF-α, mainly in the labyrinth area. In contrast, the Sildenafil group reduced TNF-α labeling, the treatment was effective in reducing inflammation. The Sil + Hep group had the best results, showing significant immunostainnig reduction. These results were confirmed by western blotting analysis. Further studies are necessary to establish if Sildenafil can become a future treatment to female infertility especially those with inflammatory component. INBEB, FACEPE, Capes, CNPq D48. MODELAGEM TRIDIMENSIONAL DO COMPLEXO APICAL DE T. GONDII

1,2- TRAVASSOS, R.; 1,2- PAREDES-SANTOS, T.C.; 1,2- DE SOUZA, W.; 1,2- ATTIAS, M. 1-Laboratório de Ultraestrutura Celular Herta Meyer, Instituto de Biofísica Carlos Chagas

Filho, Universidade Federal do Rio de Janeiro; 2-Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro;

Situado na região apical do Toxoplasma gondii, o complexo apical é constituído pelo conóide e estruturas do citoesqueleto a ele associadas e pelas organelas secretórias, róptrias e micronemas. Essas organelas liberam proteínas diretamente envolvidas com os processos de reconhecimento, adesão e formação do vacúolo parasitóforo. As róptrias e os micronemas possuem forma e distribuição peculiar dentro da célula. As róptrias são alongadas e costumam estender-se desde a região próxima ao núcleo até o interior do conóide. Os micronemas são menores em tamanho, mas estão em maior quantidade e em geral se localizam na região abaixo do conóide. Pouco se sabe sobre a forma e o local de secreção dessas organelas. Utilizando cortes ultrafinos e espessos de amostras submetidas a congelamento por alta-pressão e crio-substituição e a elétron-tomografia, buscamos demonstrar com maior precisão a localização dos micronemas dentro do corpo do parasita e com isso demonstrar seu possível local de secreção. Através das imagens por microscopia eletrônica de transmissão conseguimos observar a distribuição dos micronemas, em forma radial, logo abaixo do anel polar apical. Essa distribuição ocorre de forma constante em vários materiais observados. Nos relatos da literatura, assume-se que a secreção dos micronemas e das róptrias se dá pelo interior do conóide, entretanto com o uso da elétron-tomografia reconstruímos essa região do parasita e não observamos a presença de micronemas dentro do conóide, todos sempre se localizavam abaixo e em torno do mesmo. A tomografia eletrônica de taquizoítas extraídos e contrastados negativamente, também acrescentou maior detalhamento da estrutura do citoesqueleto apical. CAPES, FAPERJ, CNPq D49. THE ROLE OF LYSINE 35 AS AN IMPORTANT TRANSTHYRETIN ANTI-AMYLOIDOGENIC GATEKEEPER RESIDUE

1-SANT'ANNA,R.O.; 2-VENTURA, S.; 1-BRAGA, C.A.; 1-FOGUEL D. 1-INSTITUTO DE BIOQUÍMICA MÉDICA, UFRJ; 2-UNIVERSIDADE AUTONOMA DE

BARCELONA

The aggregation of proteins into amyloid fibrils maybe is a generic feature of most polypeptides sequences, if not all, under appropriated conditions. Besides the ones those find their biological function on the amyloid state, the great majority have to remain on their soluble state. It seems that nature has raised mechanisms to counteract the tendency for aggregation. An example of such strategies is the placement of charged residues, known as gatekeepers, in key positions of the protein primary sequence. This idea has been supported by numerous recent in silico studies using whole proteomes as object of research. Here, we aim to explore if this evolutionary constrain is acting on the amyloidogenic protein transthyretin (TTR).We choose as model a stretch of TTR sequence that has been pointed out as decisive for the formation of the TTR amyloidogenic intermediate as well as concentrates many known natural amyloidogenic mutants. This segment comprises the residues 26 to 57. In fact, using a variety of bioinformatic tolls we found that this stretch has a very high tendency for aggregation in comparison to the entire protein sequence. Moreover, the presence of a lys (gatekeeper) at position 35, flanking this prone to aggregation segment, encouraged us to evaluate the importance of this residue in playing with the aggregation propensity of the peptide and confirm its rule as a TTR gatekeeper. As this peptide has other lys (position 48) we performed two rational changes by a highly aggregation favorable residue leu (K35L and K48L). Our experimental data support this hypothesis and the predictions of the algorithms. We found that secondary structure propensity vary dramatically between the three sequences, being the K35L by far the most prone for beta sheet formation. Analyzing amyloid formation by a lot of techniques we found that the substitution at position 35 converts the WT peptide into a sequence that display all classic characteristics of amyloid proteins, like seeding dependent aggregation, presence of spherical nucleus on the pathway for fibril formation and highly ordered fibrils that bind thioflavin-T. The K48L substitution did not trigger such behavior, confirming the importance of the right position for a gatekeeper. The WT sequence is by far the less prone to aggregation and only forms fibrils when induced by drastic conditions like incubation with SDS and HCl for long periods. Its aggregation pathway goes through a different intermediate, that displays alpha helical secondary content. More interestingly, is the recovery of the amyloidogenic potency of the WT peptide when it is in the presence of the physiologically relevant co-factor heparin, like has been demonstrated for the entire protein. To further confirm the rule of residue 35 on converting the sequence into aggregation prone, we performed limited proteolysis with proteinase K to map the fibrils architecture and confirmed the presence of this residue on fibrils core by mass spectrometry sequencing. These data together help to understand how proteins have escaped from aggregation and shed light on the field of rational design of proteins and formation of fibrils for nanotechnology. INBEB, FAPER e CNPQ D50. INCORPORAÇÃO DAS VESÍCULAS LIBERADAS PELAS FORMAS TRIPOMASTIGOTAS PELA CÉLULA LLC-MK2 E ULTRAESTRUTURA DAS VESÍCULAS DE AMASTIGOTAS DO CLONE CL-BRENER DO Trypanosoma cruzi 1-

NEVES, R.F.C., 1- SOUTO-PADRÓN, T. 1-Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio

de Janeiro, Brasil Patógenos liberam para o meio extracelular fatores de virulência associados a vesículas, oriundas de brotamentos (microvesículas) ou fusão de corpos multivesiculares com pequenas vesículas (exossomos) com a membrana plasmática. Vesículas podem ser incorporadas pelas células através de eventos de fusão entre as membranas vesícula-célula-alvo ou endocitose pela célula recipiente, interferindo ou modulando a resposta imune ou ação de células hospedeiras. Formas tripomastigotas do Trypanosoma cruzi,


liberam constitutivamente vesículas de “shedding”. Sabendo da importância das vesículas na comunicação celular, analisamos a interação das vesículas liberadas pelos tripomastigotas de cultura do clone CL Brener do T. cruzi com a célula LLC-MK2 e a ultraestrutura das vesículas de formas amastigotas desse parasito, que são importantes para o estabelecimento da infecção no vertebrado. Formas tripomastigotas foram incubadas em Hank’s sem soro por 3h-37°C e as vesículas, obtidas da ultracentrifugação do sobrenadante, foram marcadas com análogo lipídico fluorescente (DiI). Células LLC-MK2 internalizaram as vesículas, sendo a entrada não dependente de “lipid rafts”. Por imunofluorescência, após 2h-37°C de interação, observamos vesículas direcionadas para compartimentos endocíticos acídicos, lisossomos e endossomos tardios da LLC-MK2. Assim como tripomastigotas, observamos por microscopia de transmissão, que amastigotas liberam vesículas de 40-170nm para espaço extracelular apresentando glicocálice semelhante ao observado na sua superfície. Nas amastigotas, observamos estruturas semelhantes a corpos multivesiculares contendo múltiplas vesículas com aproximadamente 100nm de diâmetro que também puderam ser encontradas no interior da bolsa flagelar do parasito, sugerindo uma possível liberação das vesículas oriundas de compartimentos intracelulares do parasito para o meio extracelular. CNPq, CAPES, FAPERJ, Pronex D51. INTERACTION OF DENGUE AND YELLOW FEVER VIRUS WITH MEGAKARYOBLASTS: ROLE IN HEMOSTATIC ALTERATIONS 1- CAMPOS, S.P.C.;1- CASTRO, M. G.;1- SANCHES; D.;1- ROCHA, C.M.;2- RODRIGUES, M.

F.;3- PAREDES, B. D.; 1- SILVA, J.L.; 1- GOMES, A.M.O. & 1- OLIVEIRA, A.C. 1- Laboratório de Termodinâmica de Proteínas e Estruturas Virais Gregorio Weber -

IBqM/UFRJ, 2- Laboratório de Biologia Molecular do Câncer - IBqM/UFRJ; 3- Laboratório de Cardiologia Celular e Molecular – IBCCF/UFRJ

"Introduction: Dengue Virus (DENV) and Yellow Fever Virus (YFV) have great importance in economy and public health in Africa, South America and Asia. They are the etiologic agents of acute hemorrhagic fevers that are related to hemostasis dysfunction, with coagulation factors consumption and thrombocytopenia. The low platelet count is related to the evolution of the disease severity. Platelets play a crucial role in hemostasis and are cytoplasmic fragments of megakaryocytes. Each megakaryocyte produces from 5.000 to 10.000 platelets. The processes that lead to disease severity evolution have not yet been elucidated. Aim: To better clarify the processes in which viral infection leads to thrombocytopenia, we aim to study the interaction between DENV and YFV with megakaryocyte precursors. Material and Methods: We infected MEG-01 cells (Human Megakaryoblastic cell line) with DENV-2 and YFV 17 DD in a multiplicity of infection of 1. Results and Discussion: We confirmed YFV infection by detecting intracellular YFV proteins since 24h post infection (p.i.) by confocal microscopy. We analyzed the production of infectious particles by plaque assay and observed increasing production until 96h p.i. and followed by decrease. We analyzed cell viability by extracellular activity of LDH and trypan blue exclusion. We observed higher LDH activity from 96h p.i. with YFV but not with DENV-2. A decrease of cell number was evident after 72h p.i. but we only observed an increase of cell mortality from 120h p.i. for both viruses. We observed mitochondrial physiology changes during DENV and YFV infection by measuring oxygen consumption. We also did not observe cell differentiation profile changes from the control. Conclusion: Our data suggest that YFV can infect and replicate in MEG-01 cells. Our data also suggest that DENV-2 and YFV infections inhibit cell growth until 72h and induce cell death from 120h p.i., with mitochondrial alterations without changing cell differentiation kinetics." CAPES, CNPQ, FAPERJ, FINEP, INBEB, PRONEX

D52. EVALUATION OF THE SYNERGIC EFFECTS OF ERGOSTEROL BIOSYNTHESIS INHIBITORS IN LEISHMANIA AMAZONENSIS

1- MACEDO-SILVA, S. T; 2- URBINA, J. A.; 1- DE SOUZA, W.; 1- RODRIGUES, J. C. F. 1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,

Brazil; 2- Instituto Venezolano de Investigaciones Científicas, Venezuela. Leishmaniases are among the most prevalent neglected tropical diseases and are endemic in 98 countries. The treatments include the pentavalent antimonials, miltefosine, amphotericin B, and pentamidine. However, such drugs are unsatisfactory due to toxicity, limited efficacy, cost and administration. Therefore, there is an urgent need to develop new drugs that are efficacious, safe, and more accessible. Trypanosomatids have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. The inhibition of ergosterol biosynthesis (EB) has been recognized as a promising target for the development of new chemotherapeutic agents. Thus, the aim of this work was to investigate the effects of E5700, a squalene synthase inhibitor, in combination with itraconazole (ITZ) or posaconazole (POSA), two known inhibitors of the sterol C14α-demethylase (CYP51), against Leishmania amazonensis in vitro. E5700, ITZ and POSA alone produced a marked reduction in the viability of L. amazonensis promastigotes, with MIC (minimum inhibitory concentration) values of 30 nM, 1 µM, and 1 µM, respectively. Several combinations were tested and the most efficient was the combination of 1.25 nM E5700 with 40 nM ITZ or 0.625 nM E5700 with 5 nM POSA, which resulted in FIC (fractional inhibitory concentration) values of 0.082 for the combination of E5700 with ITZ and 0.026 for E5700 with POSA, indicating a potent synergistic effects. Against intracellular amastigotes, the MICs for E5700, ITZ and POSA alone were 30 nM, 1 µM, and 1 µM, respectively. The results indicated again strong synergism, with MICs of 2.5 nM E5700 plus 20 nM ITZ (FIC=0.103) and 2.5 nM E5700 plus 2.5 nM POSA (FIC=0.085). Differential interference contrast microcopy (DIC) revealed a significant alteration on the shape of promastigotes after treatment with E5700 in combination with POSA, more than that observed with ITZ. Transmission electron microscopy of treated-parasites showed several alterations such as: 1) Presence of lipid bodies; 2) Intense mitochondrial swelling followed by the loss of matrix content; and 3) Presence of autophagosome-like structures. In summary, our results indicate that combinations of EB inhibitors acting at different steps of the pathway have synergistic activity against L. amazonensis and open up the possibility of a novel combination therapies for the treatment of leishmaniasis. CNPq, CAPES, FAPERJ. D53. SYNTHESIS AND CHARACTERIZATION OF DERIVATIVES OF CHLORINATED COMPOUNDS PHENANTHRENEQUINONE USING TRICHLOROISOCYANURIC ACID

1-AZEREDO, S,O,F; 1-BARRETO, R,B,L; 1-FIGUEROA-VILLAR, J.D. 1-Military Institute of Engineering

Halogenated aromatic compounds are used in the synthesis of natural products and with important biological activity. Many of the methods of direct halogenation of aromatic systems involve the use of elemental halogen or expensive transition metal based catalysts. A reagent that has been used for chlorination of aromatic compounds enabled or disabled is trichloroisocyanuric acid (TCCA), which is a reagent that provides "Cl +". TCCA is easy to access and manipulate, being also stable and inexpensive. Reactions of aromatic substitution electrophilic using trichloroisocyanuric acid can be performed on solutions polar aqueous solution of sulfuric acid 50% (v / v) or using a Lewis acid as catalyst . The objective of this work is to use acid trichloroisocyanuric for chlorination phenantrenequinone and use chlorinated compounds obtained as intermediates for the synthesis of compounds with potential biological activity. CAPES, CNPq, FAPERJ, INBEB, MINISTÉRIO DA DEFESA


D54. DIETHYLCARBAMAZINE REDUCES THE INFLAMMATORY PROCESS IN CHRONIC HEPATIC C57BL/6J MICE

1-ROCHA, S.W.S.; 1-FRANÇA, M.E.R; 1-BARBOSA, K.P.S.; 1-NUNES, A.K.S; 2-OLIVEIRA, A.G.V. ; 2-OLIVEIRA, W.H.; 1-RODRIGUES, G.B.; 1-ARAÚJO, S.N.R.; 1,2-PEIXOTO, C.A

1- Centro de Pesquisas Aggeu Magalhães- CpqAM/FIOCRUZ, Brazil 2- Centro de Tecnologias Estratégicas do Nordeste- CETENE/MCT, Brazil

Pharmacological studies show that DEC interferes in the arachidonic acid metabolism, acting as an anti-inflammatory drug [1]. Rocha et al (2012) demonstrated that DEC can be a potential drug for the treatment of chronic inflammation induced by chronic alcoholism and as a hepatoprotective drug in reducing lesions in mice malnourished [2,3]. In the present study we investigated the effect of DEC on chronic liver inflammation induced by carbon tetrachloride (CCl4). Forty C57BL/6J male mice were separated in groups (n=10): control group, DEC 50mg kg group, CCl4 group and CCl4 + DEC group. DEC (50mg/kg) was administered in bottle for 12 days. CCl4 (0.5 ml/g) was administered for 6 weeks (2 injections per week). Liver fragments were processed for histological (HE and Sirius red), immunofluorescence and molecular analysis. Histopathological findings in the control group and DEC 50mg/kg group showed no change in their morphology pattern. In the group of animals exposed to CCl4 was observed striking cytoplasmic and nuclear degeneration, with the presence of fibrosis, inflammatory infiltrates and hemorrhagic foci. The animals treated with CCl4 + DEC showed a decrease of all lesions observed in CCl4 group. In staining specific for collagen CCl4 group intense staining was observed in fibrotic areas. However, CCl4 + DEC group showed reduced collagen labeling. Results of immunofluorescence revealed strong increase of inflammatory markers such as IL-1β and α-SMA in fibrotic areas and mononuclear infiltrates, especially in perivenulares areas in CCl4 group. However, a reduction of immunoreactivity of these markers was observed after treatment with DEC 50 mg/kg. Western blot analyzes showed increased expression of COX-2, IL-1β and NF-kB in the group subjected to chronic liver injury and there was a significant decrease in expression of these proteins after treatment with DEC 50mg/kg. According to the present results, DEC is a possible alternative treatment for chronic liver inflammation induced by CCl4. INBEB, FACEPE, CNPq. D55. A TOXOPLASMA GONDII STRAIN THAT SPONTANEOUSLY FORM CYSTS IN DIFFERENT CELL TYPES

PAREDES-SANTOTS T.C.1, TIBAU R. 1, DE SOUZA W. 1, ATTIAS M. 1, VOMMARO R.C. 1 Instituto de Biofísica Carlos Chagas Filho

Conversion of Toxoplasma gondii tachyzoites to the bradyzoite stage and tissue cyst formation have crucial roles in the establishment of chronic toxoplasmosis. In this work we investigated the in vitro cystogenesis and behavior of the EGS strain, isolated from human amniotic fluid. We observed that tachyzoites of the EGS strain converted to intracellular cysts spontaneously in LLC-MK2 epithelial cells, HSFS fibroblasts and C6 glial cell lineage. The peak of conversion occurred in the LLC-MK2 cells after 4 days of infection, when 72.3±15.9 of the infected cells contained DBA positive cysts. Using specific markers against bradyzoite, tachyzoite and cyst wall components, we confirmed stage conversion and distinguished immature from mature cysts. It was also observed that the deposition of cyst wall components occurred before the total conversion of parasites. Transmission electron microscopy confirmed the full conversion of parasites presenting the typical characteristics of bradyzoites such as the posterior position of the nucleus and the presence of amylopectin granules. A thick cyst wall was also present. Furthermore, scanning electron microscopy revealed that the intracystic matrix had

tubules that were shorter than those from the intravacuolar network of the parasitophorous vacuole and were immersed in a granular electron dense material. The EGS strain spontaneously forms high burden of cysts in cell culture without artificial stress conditions, and constitutes a useful tool to study this stage of the T. gondii life cycle. INBEB, FAPERJ, CNPq, CAPES D56. DNA BINDING AND CLEAVAGE ANALYSIS OF TWO NEW FEIIIZNII COMPLEXES WITH PYRENE MOTIFS

1-BORTOLOTTO, T.; 2-CAMARGO, T.P.; 2-NEVES, A.; 1-TERENZI, H. 1- Centro de Biologia Molecular Estrutural, Departamento de Bioquímica, Universidade

Federal de Santa Catarina, Santa Catarina, Brazil; 2- Departmento de Química, Universidade Federal de Santa Catarina, Santa Catarina, Brazil.

Purple acid phosphatases (PAPs) are a group of metallohydrolases that contain a heterovalentdinuclear FeIIIMII center (MII = Fe, Mn, Zn) in the active site, and which are able to catalyze the hydrolysis of a variety of phosphoric acid esters and anhydrides. Recently, we have demonstrated the DNA cleavage ability of FeIIIZnII complexes using the unsymmetrical donor ligand 2-[N-bis-(2 pyridylmethyl)-aminomethyl]-4-methyl-6-*N’-(2-pyridylmethyl)(2-hydroxybenzyl) aminomethyl]phenol (H2L-H) that mimics the active site of red kidney bean PAP. To improve this ability, the complex ligand was specifically altered by the addition ofone or twopyrene motifs that tightly binds to the DNA structure, facilitating the nucleophilic attack of phosphodiester bonds. Herein, we report the initial DNA binding and cleavage studies of the complex [FeIII-(μ-OH)ZnIIL-Pyrene] (1) compared to its parent complex with two pyrene motifs [FeIII-(μ-OH)ZnIIL-Pyrene2] (2).Thermal denaturation of DNA was used to evaluate the binding of the complexes to the DNA structure and their ability to cleave DNA was examined following the conversion of supercoiled form of pBSK-II plasmid into its cleaved forms, using agarose gel electrophoresis. Both complexes showed to interact to dsDNA with similar patterns on short DNA fragments (10-bp, 60% GC) or long fragments (CT-DNA, ~1-10kb, 42% GC). In terms of DNA cleavage, both complexes can cleave the DNA molecule introducing single- and double-strand breaks in a time and concentration-dependent manner. The pH-activity profile showed a peak of cleavage around 7.0 due to the formation of nucleophilic hydroxyl group at the metal center, in agreement with the hydrolytic pathway of strand scission. In conclusion, new pyrene derived metal complexes seem to be interesting models to develop improved synthetic nucleases. CNPq, CAPES, MCT, FINEP, FAPESC, INCT - Biologia Estrutural e Bioimagem. D57. INSTABILITY OF A CARDIOMYOPATHY MUTANT OF CARDIAC TROPONIN C AMPLIFIES ITS CA2+-SENSITIZING EFFECT AT PHYSIOLOGICAL TEMPERATURES 1,2- VELTRI, T.; 1- Bienkiewicz, E.A.; 2- PALHANO F.L.; 2- SORENON, M.M.; 1- PINTO, J.R. 1- Department of Biomedical Sciences, Florida State University, College of Medicine; 2-

Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro Although it has been linked to hypertrophic cardiomyopathy in humans, the cardiac troponin C mutant D145E has never been investigated for its impact on the thin filament at physiological temperatures. Here, we demonstrate that this C-domain mutation causes generalized instability of the isolated protein and that this leads to defective Ca2+ regulation in the N-domain. Circular dichroism of the isolated HcTnC isoforms revealed that D145E was more sensitive to thermal unfolding, with Tmelt at 52-56oC for apo, Mg2+ and Ca2+ states compared to 67-78oC for the WT protein. Even at 10-20oC in the presence of Ca2+ or Mg2+, D145E was almost completely unfolded by 3 M urea, while the WT protein remained intact up to 50o/60oC. Remarkably, physiologically relevant temperatures were associated with progressive increases in


Ca2+ affinity for D145E but not for the WT protein, as measured using a fluorescent probe attached to Cys residues: pCa50 increased by 0.98 log units from 21o to 45oC, compared to an increase of 0.26 units for WT . As expected, both proteins became more sensitive to Ca2+ when incorporated into cardiac skinned fibers, but this increase was greater near physiological temperatures. Maximal isometric force in HcTnC-reconstituted fibers was essentially identical with both proteins at 21oC, but at 30oC the force was 40% lower with the mutant, reflecting its instability. Thus, D145E destabilizes regions of HcTnC that lie closer to the N terminus, especially near physiological temperatures, where a higher Ca2+ affinity is correlated with loss of secondary structure and becomes an important hallmark of its impaired regulatory function. Future studies of cardiomyopathy mutants can benefit from including experiments at or near physiological temperatures, where the full range of their functional defects is exposed. INCT-INBEB, CNPq, FAPERJ, CAPES, NIH D58. STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE HEPATITIS C VIRUS CORE PROTEIN

BRAGA, V. L. A.1; MENDES-SILVA, A.1 ; CARVALHO, C. A. M.1; BIANCONI, M. L.1; PEABODY, D.2; SILVA, J. L.1; GOMES, A. M. O.1; SOUZA, T. L. F.2& OLIVEIRA, A. C.1 1Programa de Biologia Estrutural/IBqM/UFRJ, Brasil. 2Department of Molecular

Genetics and Microbiology and Cancer Research and Treatment Center/UNMSM/USA. 3Faculdade de Farmácia/UFRJ/Brazil.

Approximately 170 million people are infected worldwide with the Hepatitis C Virus (HCV), which clearly represents a public health problem. The HCV core protein (HCVCP) is responsible for the interaction with the viral RNA and capsid assembly. Three regions of the HCVCP are specifically important during nucleocapsid assembly: the sequences containing the amino acids 22-39 (VKFPGGGQIVGGVYLLPR), 50-67 (RKTSERSQPRGRRQPIPK) and 85-102 (PWPLYGNEGMGWAGWLLSPRG). To better understand the structural and physicochemical aspects of their interactions with the RNA and the viral envelope during HCV assembly we used membrane models (micelles), such as sodium-dodecyl-sulfate, n-octyl-b-D-glucopyranoside, Hexadecyltrimethylammonium-bromide and n-dodecylphosphocholine, and non specific nucleic acids. In the presence of different micelles, only the peptide 85-102 adopted an alpha-helix structure as verified by circular dichroism. Analyses of tryptophan intrinsic fluorescence and acrylamide quenching reveal that the interaction between peptide 85-102 and micelles probably involves a partial internalization of the tryptophan residue. Calorimetric measurements reveal different heat profiles for the interactions of micelles and the different peptides and showed similar thermodynamics of the interaction of peptide 50-67 with different DNAs. Fluorescence polarization data showed that, in the range of concentrations used, the peptides do not prevent the formation of NLPs by the interaction between core protein and nucleic acids. We also investigate the cellular localization and the assembly process in HepG2 and Huh7. Confocal microscopy data showed that 24 hours post transfection the HCVCP fused with the Green Fluorescent Protein (GFP) (HCVCPGFP) is located in the nucleus. In Huh7 cells, HCVCPGFP seems to be located on lipid droplets surface. Our data reveal a new approach to understand the HCV assembly, which is a great target for the development of drugs anti-HCV. CNPQ, CAPES, FAPERJ, FUJB, INBEB, PRONEX, FINEP D59. DIFFERENTIATION OF GIARDIA LAMBLIA: THE CYST WALL UNDER CONSTRUCTION

1,2- Midlej, V.; 3, 4, 5- de Souza, W.; 1, 4, 5- Benchimol, M.

1- Universidade Santa Úrsula. Rio de Janeiro, Brazil; 2- Programa de Pós-graduação em Ciências Morfológicas, Instituto de Ciências Biológicas, Universidade Federal do Rio de

Janeiro – Rio de Janeiro, Brazil; 3- Instituto de Biofísica Carlos Chagas Filho- Universidade Federal do Rio de janeiro, Brazil;4- Instituto Nacional de Metrologia e

Qualidade Industrial – Inmetro, Rio de Janeiro, Brazil; 5- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem.

Differentiation from one life cycle stage into another is an elegant adaptation by which many parasites ensure their transmission and survival. The protozoan Giardia lamblia is a major cause of water-borne diarrheal disease. Nonetheless, the basic biology of this protist is incompletely understood. This parasite exhibits two forms in its life cycle that includes trophozoite and cyst. The formation of the resistant extracellular cyst wall (CW), which is composed by proteins and carbohydrates, begins with intestinal signals, where the cyst wall proteins (CWPs) are synthesized. All of the CWPs are transported via encystation-specific secretory vesicles (ESVs) and are released at the site of CW assembly. Although the presence of carbohydrates in cyst wall has been reported, the detection and origin of the cyst wall carbohydrate-rich moieties is completely unknown. Moreover, the changes that happen in the cyst wall (CW) during the excystation process are poorly studied. Encystation and excystation are crucial processes for establishment and maintenance of Giardia infection. Thus, the aim of this work is to better understand the CW: verifying the origin of the carbohydrate components and observe the ultrastructural modification during the differentiation process. Here we tested the differentiation process in vitro and analyzed the formation and changes in the CW of the parasite by complementary techniques, such as scanning and transmission electron microscopy, cytochemistry for carbohydrates and immunocytochemistry. Immunofluorescence microscopy using anti-cyst wall protein 1 (CWP1) and gold- and fluorescent-conjugated DBA lectin were also used. A field emission scanning electron microscopy (FESEM) was also used to compare the mature cysts and the beginning of excystation process. Interestingly, encystation carbohydrate-containing vesicles (ECVs), positive for N-acetyl-galactosamine, were found in the encysting cells. The ECVs are distinct from the encystation specific vesicles (ESVs) because: (1) they are electron lucent whereas ESVs are electron dense; (2) they do not react with antibodies against cyst wall proteins; (3) their contents are positive for carbohydrates, whereas ESVs display a negative reaction; and (4) this cell compartment exhibits a positive labeling for DBA lectin indicating the presence of N-acetyl-galactosamine, whereas ESVs are negative. During the excystation changes in the cyst form and the presence of electron-dense vesicles near the CW were observed. The CW was seen in detailed using FESEM. In mature cyst, the cyst wall microfibrils were seen in a tighter arrangement. However, when excystation process begins this tight arrangement of the wall microfibrils is lost. In conclusion, our results suggest that the ECVs may represent a new structure involved in the cyst wall formation and alteration of CW fibrils is needed for the development of excystation. INBEB, AUSU, CNPq, FAPERJ, PRONEX D60. BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF A CYTOSOLIC HSP90 FROM SUGARCANE 1,2- SILVA,V.C.H.;1,2- CAGLIARI, T.C.; 1- LIMA, T.B.;1- GOZZO, F.C.; 1,2,3- RAMOS, C.H.I.

1-Institute of Chemistry;2-Institute of Biology;3-Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem

Hsp90s are involved in several cellular processes, such as signaling, proteostasis, epigenetics, differentiation and stress defense. Although Hsp90s from different organisms are highly similar, they usually have small variations in conformation and function. Thus, the characterization of different Hsp90s is important to gain insight into


the structure-function relationship that makes these chaperones key regulators in protein homeostasis. This work describes the characterization of a cytosolic Hsp90 from sugarcane and its comparison with Hsp90s from other plants. Previous expressed sequence tag (EST) studies in Saccharum spp. (sugarcane) predicted the presence of an mRNA coding for a cytosolic Hsp90. The corresponding cDNA was cloned, and the recombinant protein was purified and its conformation and function characterized. The structural conformation of Hsp90 was assessed by chemical cross-linking and

hydrogen/deuterium exchange using mass spectrometry and hydrodynamic assays, which revealed regions accessible to solvent and that Hsp90 is an elongated dimer in solution. The in vivo expression of Hsp90 in sugarcane leaves was confirmed by western blot, and in vitro functional characterization indicated that sugarcane Hsp90 has strong chaperone activity. INBEB, CAPES, CNPq, FAPESP


Pós-doutorandos

PD1. ENVOLVIMENTO DO FATOR DE TRANSCRIÇÃO NFAT NAS DOENÇAS DE ALZHEIMER E PARKINSON: CONTRIBUIÇÕES PARA A INFLAMAÇÃO E APOPTOSE

1- ROBBS, B.K.; 1- SPÍNDOLA, T; 1- DE FREITAS, J.A.; 1- LEDO, J.H.; 1- FERREIRA, S.T.; 1- FOGUEL, D.

1- Instituto de Bioquímica Médica Juntas, a Doença de Alzheimer (DA) e de Parkinson (DP) afetam mais de 7 milhões de pessoas nos Estados Unidos e, são a primeira e segunda causas de demência na população acima dos 65 anos de idade. Apesar do grande investimento para o estudo dessas patologias, pouco sabe-se sobre os mecanismos de controle da expressão gênica que levam à neurodegeneração. Aparentemente o processo de influxo de Ca2+ e ativação da fosfatase calcineurina A (CnA) podem estar envolvidos no controle da morte celular programada e do processo de inflamação crônica, que são mecanismos centrais para a progressiva perda cognitiva e motora em pacientes com essas patologias. Um dos principais fatores de transcrição ativado pela via de Ca2+/CnA é o fator nuclear de célula T ativada (NFAT). As funções das proteínas da família NFAT foram inicialmente descritas e caracterizadas no sistema imunológico. Contudo, o NFAT desempenha um importante papel na regulação de genes envolvidos no controle da morte celular como TNF-α; c-Flip; Fas-L; de mediadores inflamatórios como IL1β; Cox-2; TNF-α, além de estar envolvido no desenvolvimento e manutenção do sistema nervoso central. Portanto, postulamos que a regulação gênica mediada pelo NFAT poderia desempenhar um papel fundamental na DA e DP. Nesta proposta, temos interesse em investigar os mecanismos moleculares através dos quais as proteínas NFAT regulam a apoptose bem como a inflamação, e a sua potencial relação com a patogênese dessas doenças neurodegenerativas. Para tanto, avaliaremos a função das proteínas NFAT na indução do processo de morte celular programada e inflamação; na regulação de genes envolvidos nesses processos, e a relevância do NFAT na perda cognitiva e motora de modelos animais. Os estudos propostos auxiliarão na caracterização dos mecanismos moleculares que regulam a DA e DP e sua relação com a neurodegeneração, podendo trazer inovações para definir novos biomarcadores e/ou podendo contribuir para o desenvolvimento de novas terapias. INBEB, FAPERJ, CNPq PD2. DIFERENTES VIAS DE MORTE CELULAR SÃO INDUZIDAS NO TRYPANOSOMA CRUZI PELO PEPTIDEO MELITINA

1- ADADE, C.M.; 1- OLIVEIRA, I.R.; 1- PAIS, J.; 1- SOUTO-PADRÓN, T. 1- Instituo de Microbiologia Paulo de Góes

A doença de Chagas, causada pelo protozoário Trypanosoma cruzi, afeta cerca de 16-18 milhões de pessoas nas Américas Central e Sul, e o tratamento é baseado no uso do Benznidazol. Este, no entanto, possui eficácia variável, limitada à fase aguda da doença e acarreta em diversos efeitos colaterais. Desta forma, novos agentes quimioterápicos obtidos de fontes naturais são uma fonte a ser explorada. O veneno da abelha Apis mellifera é uma complexa mistura onde a sua fração majoritária é representada pela melitina, objeto deste estudo. O presente trabalho avaliou a atividade da melitina sobre o T. cruzi e sua toxicidade sobre células hospedeiras. Formas epimastigotas e tripomastigotas (clone CL-Brener) foram tratadas com melitina, e o seu efeito sobre o crescimento dos epimastigotas e lise dos tripomastigotas foi avaliado por contagem em câmara de Neubauer. O IC50/ 24h de inibição do crescimento dos epimastigotas foi 2,88

µg/ml e o DL50/ 24h para lise dos tripomastigotas foi 0,14 µg/ml. A viabilidade dos parasitos foi avaliada através da incubação com iodeto de propídio e analisados por citometria de fluxo onde os epimastigotas apresentaram marcação positiva de 70 a 99%, e os tripomastigotas exibiram de 62 a 69%. Os efeitos sobre a morfologia foram avaliados por microscopia eletrônica, onde a ultraestrutura sugeriu fenótipos de morte distintos, onde epimastigotas estariam morrendo por autofagia e tripomastigotas, por apoptose. A metodologia do MTS foi empregada para análise de citotoxicidade sobre culturas de macrófagos peritoneais, tratados ou não com o peptideo por 48h. Somente o tratamento com 5 µg/ml gerou citotoxicidade com relação às culturas controle. Estes dados demonstram que a melitina é eficaz sobre as formas epimastigotas e tripomastigotas do T. cruzi em concentrações não tóxicas às células hospedeiras. Estudos estão em andamento para analisar os possíveis alvos intracelulares do parasito e sobre o seu ciclo intracelular. FAPERJ, CNPq, INBEB, CAPES, Pronex PD3. SUSTAINED EFFECT OF BONE-MARROW MONONUCLEAR CELL THERAPY IN AXONAL REGENERATION AFTER OPTIC NERVE CRUSH

1,2- ZAVERUCHA-DO-VALLE,C.; 1,2- MESENTIER-LOURO, L.; 1,2- SANTIAGO, M.F.; 1,2- MENDEZ-OTERO, R.

1- Instituto de Biofísica Carlos Chagas Filho; 2- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem – INBEB

"PURPOSE: In adult mammals, the regeneration of the optic nerve is very limited and at the moment there are several groups trying different approaches to increase retinal ganglion cell (RGC) survival and axonal outgrowth. One promising approach is cell therapy using bone marrow cells. In previous work, we performed intravitreal transplantation of syngeneic bone-marrow mononuclear cells (BMMCs) after optic nerve crush in adult rats and we demonstrated an increase in RGC survival and axon outgrowth 14 days after injury. In the present work, we investigated if these results could be sustained for a longer period of time. METHODS: Optic nerve crush was performed in Lister-hooded adult rats and BMMC or saline injections were performed intravitreally shortly after injury. Neuronal survival and regeneration were evaluated in rats' retina and optic nerve after 28 days. RESULTS: We demonstrated that there is an increase of 5.2 fold in the axon outgrowth 28 days after lesion, but the BMMCs effect on RGC survival was not sustained. In an attempt to prolong RGC survival, we established a new protocol with two BMMC injections, the first one soon after the lesion and the second one 7 days after the injury. Untreated animals received two injections of saline. We observed that although the axonal outgrowth was still increased after the second BMMC injection, the RGC survival was not prolonged. CONCLUSIONS: These results demonstrate that BMMCs transplantation promotes neuroregeneration 28 days after injury. However, the effects on RGC survival previously observed by us at 14 days were not sustained at 28 days and could not be prolonged with a second dose of BMMC." CNPq, FAPERJ, CAPES, INBEB PD4. THE ANTIVENOMIC CHARACTERIZATION OF ANTIBODIES AGAINST ELAPID THREE FINGER TOXIN AND PLA2 MOLECULES FROM MICRURUS VENOMS.

Correa-Netto, C1,2., Araújo, R. T1,2., Lima L.M.T.R3., Perez-Payá. E4,5., Calvete, J. J4., Foguel, D & Zingali, R. B2.

1Instituto Vital Brazil; 2IBQM-UFRJ; 3Faculdade de Farmácia-UFRJ; 4Instituto de Biomedicina de Valencia, CSIC, Espanha; 5CIPF, Valencia, Espanha.

Polyclonal antibodies have been used for over a century for the treatment of snake bite envenoming. New strategies and approaches to understand how antibodies recognize and neutralize snake toxins represent a challenge to improve the antivenoms.


Previously we characterized the venom proteome of M. altirostris and M. corallinus venom and Elapid 3FTx and PLA2 molecules are the major toxic compounds. These molecules were purified from venoms and used as antigen to produce polyclonal and monoclonal antibodies against them. Moreover some regions of 3FTx were used as model to produce synthetic peptides. Three antisera against synthetic peptides were generated following different procedures. The serum produced by peptides encapsulated in liposome vesicles were the most reactive to 3FTxs molecules. However, the antisera were unable to neutralize the neurotoxicity of these proteins in vivo. Antivenomic analyses showed a poor recognition for many toxic compounds. We also tested the generation of monoclonal antibodies against the most toxic components. M. altirostris venom was fractionated, its major toxic proteins identified, and a fraction containing 3FTx and PLA2 were used to generate a panel of nine monoclonal antibodies. Six mAbs showed high affinity towards 3FTx and two against PLA2. We characterized the specificity of all mAb by antivenomic approach and identified a pair of antibodies able to recognize all PLA2 molecules of these venoms. These results showed that PLA2 of M. altirostris venom share a pair of conserved antigenic regions and draw attention to use these epitopes as antigen to generate antibodies against this class of protein. Support: CNPq, CAPES, FAPERJ, INBEB, Ministerio de Ciencia e Innovación, Madrid, Spain. PD5. INTERACTION STUDIES OF YERSINIA PESTIS PLASMIN (BUBONIC PLAGUE AGENT) AND HUMAN PLASMINOGEN BY CIRCULAR DICHROISM AND NMR

1-Sarzedas, C.G.; 1-Vidal, T.J.; 1-Barreto, J.R., 1-Tinoco, L.W. 1-Núcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro

Yersinia pestis is the agent of bubonic plague classified in category A of bioterrorism agents according with Center for Disease Control and Prevention, USA. Plasmin (Pla) of Y. pestis is a membrane protein that cleaves/activates the human plasminogen in the cell invasion process, destabilizing the fibrinolitic cascade causing bleeding. Our goal was to characterize the pH effect in the Pla folding and their interaction with human plasminogen peptide (PK2). The structural characterization of PK2-Pla interaction could be useful to plan mimetic peptides to inhibit such interaction. Gel filtration chromatography showed that Pla is dimeric at pH 6.5 and monomeric at pH 8.0. CD and NMR analyses were performed to evaluate the pH effect in the Pla folding and in the PK2-Pla interaction. CD studies showed structural changes of PK2 in the presence of Pla at pH 8.0, indicating Pla-PK2 interaction in this condition. NMR spectra of PK2 in the presence of Pla were acquired for the sample at pH 5.0. It was observed an increase in the peaks intensities compared with the PK2 free spectra, suggesting less conformational variability of PK2, probably due PK2-Pla interaction. However, a slight chemical shift change was observed only for H of VV residues of PK2 in the presence of Pla, suggesting that Pla-PK2 interaction is affecting specifically the RVV region. In the plasminogen activation process, Pla cleaves the R561-V562 bond in the sequence PGRVVGG. The absence of significant alteration on the NMR spectra suggests that PK2-Pla interaction at pH 5.0 could be weak, probably because Pla in this condition is in the dimeric form. These data show that Pla folding and aggregation state is sensitive to pH changes and interfere in the PK2-Pla interaction. INBEB, CNPq PD6. ALTERAÇÃO DOS NÍVEIS CEREBRAIS DE GLUTAMATO E GLUTAMINA NA SEPSE EXPERIMENTAL

1- MADEIRA, C.; 1- VARGAS-LOPES, C.; 2- D'ÁVILLA, J.; 3- KURTZ, P.; 3- AZEVEDO, L.; 2- BOZZA, F. e 1- PANIZZUTTI, R.

1- Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro; 2- Fundação Oswaldo Cruz, FIOCRUZ, Rio de Janeiro; 3- Instituto Sírio-Libanês de Ensino e Pesquisa,

São Paulo. A encefalopatia associada à sepse (EAS) é uma manifestação comum de quadros infecciosos graves, acometendo até 70% dos pacientes. Alguns pacientes apresentam disfunção cognitiva meses após o episódio de EAS. Apesar de sua enorme importância clínica, a fisiopatologia da EAS ainda não é completamente esclarecida. Glutamato é o principal neurotransmissor do cérebro e participa de diversos processos cognitivos. Nós investigamos os níveis de glutamato e seu precursor glutamina durante a evolução da sepse. Induzimos sepse em porcos através da injeção de fezes na cavidade abdominal e monitoramos os níveis extracelulares dos aminoácidos utilizando um cateter de microdiálise no hemisfério cerebral esquerdo. Nós observamos maiores níveis de glutamina no fluido extracelular dos porcos sépticos nos tempos de 12 horas (p< 0,05) e 18 horas (p< 0,01) quando comparados aos porcos controles. Entretanto, não encontramos diferença nos níveis de glutamato no fluido extracelular entre os porcos sépticos e os controles. Após o experimento sacrificamos os animais e observamos que os níveis de glutamato e glutamina totais no tecido estavam significativamente maiores no córtex frontal dos porcos sépticos do que nos porcos controles (p= 0,017, t= 2,87, para glutamato; p= 0,013, t= 3,01, para glutamina). Nossos resultados sugerem que durante a evolução da sepse experimental pode ocorrer uma lesão neuronal ativa, promovendo alteração nos níveis de glutamato e glutamina. INBEB, FAPERJ, CNPq PD7. THE ROLE OF INOS ON THE GLIAL CELLS ACTIVATION AND NEUROINFLAMMATION IN DEMYELINATING MODEL

1,2- RAPÔSO, C.; 2,3- NUNES, A.K.S.; 2,3- LUNA, R.L.A.; 3- ARAÚJO, S.M.R.; 1- CRUZ-HÖFLING, M.A.; 2,3- PEIXOTO, C.A.

1Departamento de Histologia e Embriologia, Instituto de Biologia, Universidade Estadual de Campinas/UNICAMP, Campinas-SP; 2Laboratório de Ultraestrutura -

CPqAM/FIOCRUZ, 3Centro de Tecnologias Estratégicas do Nordeste /CETENE, Recife-PE, Brazil

The protective role of iNOS/NO system in neurodegenerative diseases has been matter of debate. Recently, we have demonstrated that sildenafil protects the neural tissue in a cuprizone(CPZ)-induced demyelination model in C57BL/6 wild-type (WT) mice. Sildenafil accumulates cGMP by inhibiting PDE5, but the mechanism of neuroprotection is unknown. Here, we used male iNOS-/- mice (6 week-old) treated with sildenafil to investigate the nitric oxide/cyclic guanosine 3',5'-monophosphate/PDE5 (NO/cGMP/PDE5) signaling pathway to examine sildenafil protection. iNOS-/- mice received for four weeks (n = 5/group): Pure chow and water (control group); 0.2% CPZ into the chow; CPZ into the chow plus sildenafil (Viagra®, Pfizer, 25 mg/kg) in the drinking water. Cerebella were processed for immunofluorescence or western blotting. CPZ significantly increased GFAP, Iba-1, TNF-α, COX-2, IL-1β and IFN-γ expressions in iNOS-/- mice. Comparison of eNOS level in WT and iNOS-/- animals showed that the enzyme expression was higher in iNOS-/- than in WT mice in all treatments. In mice lacking iNOS, sildenafil did not decrease GFAP, Iba-1, TNF-α and COX-2; however, IFN-γ and IL-1β were diminished by sildenafil. All proteins were decreased by sildenafil in previous study using WT mice. The present findings reveal that the absence of iNOS limited the protective sildenafil effects. iNOS have an important feedback mechanism in inflammatory conditions, when chronic increases of this enzyme is self-regulated and induces decrease of some pro-inflammatory proteins. This feedback mechanism is co-regulated by high concentration of cGMP. The results suggest that sildenafil may exert its anti-inflammatory effect through iNOS inhibition by cGMP-iNOS feedback; in iNOS-/-


mice the inflammatory regulation through iNOS-feedback would be absent. On the other hand, the constant eNOS overexpression can generate high NO concentrations, damaging the tissue. Further studies are required to explain the the NO/cGMP/PDE5 mechanism underlying sildenafil effect. CNPq,CAPES,FACEPE,FAPESP, INBEB. PD8. A BI-PHASIC MODEL OF MICROVASCULAR LEAKAGE EXPLAINS HOW ACTIVATION OF THE MAST CELL/CONTACT SYSTEM PROPAGATES INFLAMMATION NASCIMENTO CR1, SERRA R1, JULIANO MA2, JULIANO L2, SVENSJÖ E1, SCHARFSTEIN J1. 1 Laboratório de Imunologia Molecular, Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro; 2 Departamento de Biofísica, Escola Paulista de Medicina, UNIFESP, São

Paulo, Brasil The contact system/Kallikrein-Kinin System (KKS) is activated by negatively charged molecules released from mast cell (MC) granules. We investigated the functional interplay between MCs and the KKS in a hamster model. Male hamsters (5 groups of n = 4 - 8) were anesthetized and their cheek pouches (HCPs) were prepared for intravital microscopy, (Svensjö et al. 2012). HCPs were subjected to topical applications of the contact system activator dextran sulfate 500 kDa (DXS) alone or in the presence of the ACE-inhibitor captopril, BK2R-antagonist HOE-140, cromoglycate (a stabilizer of MC granules) or PKSI-527 (a selective inhibitor of plasma kallikrein). Application of DXS alone during 30 min caused no plasma leakage (indicated with FITC-dextran) but it started to appear at 30 min. The dramatic increase in plasma leakage was blunted by HOE-140 and cromoglycate evidencing bradykinin (BK) and MC involvement. The leakage started in a few postcapillary venules suggesting that a discrete increase in plasma leakage was required for contact system (DXS)-dependent intensification of plasma leakage. We then found that leakage induced by histamine (4 μM) was not inhibited by HOE-140, cromoglycate or PKSI-527. However, subtle increases in plasma leakage (950 ± 490 RFU) provoked by histamine were greatly enhanced in the presence of DXS, (3907 ± 533 RFU) and were further enhanced by DXS+captopril (11340 ± 733 RFU). Conversely, the plasma leakage induced by histamine combined with DXS+captopril was almost completely inhibited by HOE-140, cromoglycate and PKSI-527 (924 ± 552, 1235 ± 497 and 2042± 879 RFU, respectively). Conclusions: plasma leakage induced by histamine may intensify inflammatory edema through contact-system dependent activation of the KKS and release of BK. Studies are underway to investigate the interplay between MS/KKS and BKRs in infection by T. cruzi, Andrade et al. 2012, L. chagasi, Svensjö, unpublished and bacteria (Porphyromonas gingivalis, Monteiro et al. 2009). FAPERJ, CNPq e INCT-CNPq/INBEB PD9. RESVERATROL PREVENTS P53 CORE DOMAIN AGGREGATION

1,2- COSTA, D.C.F.; 1,2- CAMPOS, N.P.C.; 1,2- PEDROTE, M. M.; 2,3- ZANPHORLIN, L.; 2,3- RAMOS, C.H.I.; 1,2- SILVA, J.L.

1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; 2- Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de Janeiro, Brazil; 3- Instituto de Química, Universidade Estadual de

Campinas, São Paulo, Brazil. INTRODUCTION: p53 has an essential role in preventing cancer development by inducing cell cycle arrest and/or apoptosis in response to cellular stress. Mutations in the p53 gene are described in over 50% of all human cancers. Besides the mutations, cellular aggregation of p53 can also inactivate the protein, leading to malignancy. Resveratrol, a natural polyphenol found in grapes and red wine, is able to induce p53-dependent cell death in a variety of cell lines. Although several indirect mechanisms of

p53 activation by resveratrol have been proposed, there is no evidence that this bioactive compound can interact with p53. Thus, we investigated a possible interaction between resveratrol and the p53 core domain (p53C). In addition, we tested the potential of resveratrol in preventing wild-type and mutated p53 aggregation in vitro. MATERIAL AND METHODS: Experiments were performed by using fluorescence spectroscopy techniques. RESULTS AND DISCUSSION: Our data suggest that an interaction between resveratrol and the wild-type p53C does occur. Additionally, we found that resveratrol has the ability to inhibit the wild-type p53 core domain as well as the R248Q p53 mutant to undergo aggregation. CONCLUSIONS: Our study provides evidence that resveratrol can directly modulate p53 and may pave the way for a better understanding of the mechanisms involved in p53 protein aggregation as a therapeutic strategy for cancer treatment. Key words: resveratrol, cancer, p53, aggregation. INBEB, FAPERJ, CNPq and Fundação do Câncer PD10. STRUCTURAL AND THERMODYNAMIC CHARACTERIZATION OF THE CAPSID PROTEIN OF HEPATITIS C VIRUS AND ITS INTERACTION WITH THE TUMOR SUPPRESSOR PROTEIN P53 1- Albernaz, F. P.;1- Braga, V. L. A.; 2- Souza, T. L. F.; 1- Rodrigues-Pinto, T.; 1- Rangel, L. P.; 3- Peabody, D.;1- Silva, J. L. ; 1- Gomes, A. M. O.;1- Almeida, F. C. L.; 1- Oliveira, A. C.

1- Programa de Biologia Estrutural and Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas -Instituto de Bioquímica Médica - UFRJ, RJ, Brazil; 2- Faculdade de Farmácia - UFRJ, RJ, Brazil; 3-Department of Molecular Genetics and

Microbiology and Cancer Research and Treatment Center, University of New Mexico School of Medicine, USA

Hepatitis C virus (HCV) acts as a major agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The HCV core protein (HCVcp) is involved with the nucleocapsid assembly and with different cellular processes. The interaction of HCVcp with the tumor suppressor protein p53 has been described in hepatocellular carcinoma but the mechanism of this interaction remains unknown. In this work we use thermodynamic and spectroscopic techniques to achieve the structural characterization of HCVcp and its interaction with p53. Although HCVcp is an intrinsically unstructured protein, our fluorescence spectroscopy results demonstrated a significant decrease in the spectral center of mass of the protein in function of increasing concentrations of chemical denaturants, indicating the presence of structural elements. These data are corroborated by Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR). HCVcp backbone assignments strategy based on projection spectroscopy, a way of reducing the experiment time when recording high-dimensional NMR experiments and Triple resonance experiments are in course. To evaluate the interaction between HCVcp and p53, 1H/15N HSQC NMR spectra of labeled HCVcp were acquired in the absence and in the presence of unlabeled p53. Changes in the chemical shift of some resonances of the HCVcp were observed, showing changes in the chemical environment of these amino acid residues and suggesting interaction with p53. Fluorescence polarization assays of FITC-labeled p53 in the presence of HCVcp corroborate the interaction in vitro between both proteins. Additionally to these studies, we are also investigating the interaction of the HCVcp with the p53 in HepG2 and H1299 cells. We constructed vectors to express both HCVcp and p53 fused to the proteins GFP and DsRed-Monomer, respectively, in order to perform Fluorescence Resonance Energy Transfer analyses and these studies are in progress. Our data reveal a new approach to understand the HCVcp-p53 interaction. FAPERJ, CAPES, CNPq and INBEB


PD11. IMAGEAMENTO POR ESPECTROMETRIA DE MASSA COMO FERRAMENTO DE ESTUDO DA FISIOLOGIA DE INVERTEBRADOS 1,2 GOMES, F.M., 3 ZECCHI, R., 4 BARACCHI, D., 3 PIERACCINI, G., 2, 5 MIRANDA, K., 1,5

MACHADO, E.A., 3 MONETI, G., 4 TURILLAZZI, S. 1. Laboratório de Entomologia Médica, Universidade Federal do Rio de Janeiro, Rio de

Janeiro, Brazil 2. Laboratório de Ultraestrutura Celular Hertha Meyer, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil 3. Centro di Servizi di Spettrometria di Massa dell’Università degli Studi di Firenze, viale G.Piaraccini, 50139, Firenze, Italy 4.

Università degli Studi di Firenze, Dipartimento di Biologia Evoluzionistica ‘Leo Pardi’, Via Romana 17, 50125, Firenze, Italy 5. Divisão de Biologia Estrutural, Diretoria de

Metrologia Aplicada a Ciências da Vida, Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMETRO), Xerém, RJ, Brazil

O imageamento por espectrometria de massa acoplado a ionização e dessorção a laser assistida por matriz (MALDI-IMS) é uma metodologia que vem se popularizado como ferramenta de estudo da fisiologia de vertebrados. Sua utilização permite que, por meio da análise por espectrometria de massa em modo de varredura de cortes de material biológico, sejam geradas imagens da distribuição de moléculas. Assim, presente trabalho pretendeu avaliar o potencial do MALDI-IMS como ferramenta de estudo em invertebrados, utilizando cortes de larvas não-fixada da abelha europeia Apis mellifera. Inicialmente, visou-se a otimização da composição de matriz a ser utilizado a fim de maximizar a variedade de analitos detectados e a sua relação sinal-ruído. Posteriormente, os padrões de segmentação de peptídeos no intestino e na hemolinfa – o líquido circulante dos insetos - foram analisados. Nossos resultados sugerem um protocolo inicial baseado na utilização de uma mistura contendo ácido alfa-ciano-4-hidroxicinamínico (CHCA) e anilina, na presença de ácido trifluoroacético e acetonitrila. Posteriormente, identificamos padrões que evidenciaram a segmentação do intestino médio da abelha em quatro regiões não-sobrepostas. A análise da distribuição de peptídeos na hemolinfa revelou a presença de gradientes previamente desconhecidos, cuja categorização era mais facilmente realizada quanto a proximidade destes com o intestino - sugerindo que este órgão atue na organização e, possivelmente na secreção, desses componentes para a hemolinfa. Finalmente, pelo acompanhamento da variação dos níveis de complexidade dos espectros ao longo do lúmen do intestino, corroboramos a hipótese de que a rota digestiva de proteínas ocorra em um modelo de contra-corrente organizado pela segmentação do lúmen em espaço ecto- e endoperitrófico. Em conjunto, nossos resultados corroboram que a análise por MALDI-IMS fornece uma valiosa ferramenta para o estudo da fisiologia de insetos e apontam para a presença de níveis de complexidade previamente desconhecidos no intestino e hemolinfa desses animais. CAPES, CNPq, FAPERJ PD12. DESVENDANDO A FISIOLOGIA DA BACTÉRIA MULTICELULAR MAGNETOTÁTICA CANDIDATUS MAGNETOGLOBUS MULTICELLULARIS ATRAVÉS DA ASSOCIAÇÃO DA ANÁLISE POR MICROSCOPIA E METAGENÔMICA 1- ABREU, F.; 1- MORILLO, V.; 1- NASCIMENTO, F.F.; 1- WERNECK, C.; 2- CANTÃO, M.E.;

2- CIAPINA, L.P.; 2- ALMEIDA, L.G.P.; 3- LEFÈVRE, C.T.; 4- BAZYLINSKI, D.A.; 2- VASCONCELOS, A.T.R.; 1- LINS, U.

1- Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro; 2- Departamento de Matemática Aplicada e Computacional, Laboratório Nacional de

Computação Científica; 3- UMR7265 Service de Biologie Végétale et de Microbiologie Environnementale, CEA Cadarache/CNRS/Aix-Marseille Université; 4- School of Life

Sciences, University of Nevada at Las Vegas Candidatus

Magnetoglobus multicellularis é um procarioto multicelular magnetotático não cultivável formado por células Gram-negativas dispostas lado a lado, formando uma esfera. Cada célula possui inúmeras inclusões ricas em ferro chamadas magnetossomos, organelas responsáveis pela orientação ao longo do campo magnético. Devido à sua capacidade de responder a campos magnéticos, é possível seu enriquecimento a partir de amostra ambiental. A caracterização morfológica por microscopia eletrônica de transmissão e varredura e a análise do comportamento do micro-organismo por microscopia de luz é feita desde sua descoberta em 1983. Nesse trabalho, a análise das sequencias geradas pelo pirosequenciamento de amostra enriquecida em Ca. M. multicellularis foi feita tendo como base os dados observados por microscopia, de forma que as características do micro-organismo descritas por microscopia garantissem que genes detectados realmente pertencessem ao micro-organismo. Dessa forma, características definidas por proteínas envolvidas na adesão e morfogênese multicelular, divisão celular, produção de grânulos e cápsula, quimio-, foto- e magnetotaxia, entre outras, foram identificadas, permitindo o entendimento da fisiologia do micro-organismo. As conclusões obtidas a partir da integração dos dados obtidos pelo emprego das técnicas de microscopia e dos dados genômicos levaram ao desenvolvimento de um meio de enriquecimento para o micro-organismo, condição em que alguns dados ultraestruturais foram confirmados, e ao entendimento das taxias observadas, indicando que o deslocamento do micro-organismo no ambiente é dependente não só da magnetotaxia como também da fototaxia e quimiotaxia, como observado por experimentos de quantificação dos micro-organismos em diferentes camadas de sedimento e da análise por vídeo microscopia do comportamento do micro-organismo quando expostos a diferentes substratos em ambiente anaeróbico. Assim, esse trabalho exemplifica como dados morfológicos, ultraestruturais e de análise de comportamento obtidos por técnicas de microscopia podem ser utilizados para refinar, ou mesmo, transformar dados brutos provenientes de sequenciamento em estudos de caracterização detalhada de micro-organismos específicos. INBEB, CNPq, CAPES e FAPERJ PD13. BONE MARROW CELL THERAPY DURING PRE AND POST SYMPTOMATIC PHASE IN A MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS 1,2- GUBERT, F.; 1,2-PEREIRA, I.B.; 1,2-DECOTELLI-SILVA, A.; 1,2-ZAVERUCHA-DO-VALLE, C.; 1,2-FIGUEIREDO, F.R; 2-TOVAR-MOLL, F.; 1,2-SANTIAGO, MF; 1,2-MENDEZ-OTERO, R. 1- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 2-

Insituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disease that affects selectively the motor neurons. The detail mechanisms of selective motor neuron death remain unknown. and no effective therapy has been developed. The aim of this work is to investigate the therapy with bone marrow cells (BMC) in a mouse model of ALS (SOD1-G93A mice). We injected 106 BMC (mononuclear fraction) in the lumbar portion of the spinal cord of the SOD1-G93A mice at: 9 weeks (pre-symptomatic) and 14weeks (post-symptomatic). In each condition, we analyzed the progression of disease and the lifespan of animals. We did not observed increase in the lifespan of the animals injected with BMC at 9weeks of life, although there is slightly delay in the disease onset in the treated animals. When we injected the BMC at 14weeks, we observed only an increase in the animal’s performance at the rotarod test when compared with the animals that receive saline injection one week after the treatment. In this protocol, we also did not observe difference in the animal’s lifespan. Using different strategies to track the BMC, we notice that these cells do not remain in the spinal cord after injected. One week after the transplant, approximately 3% of BMC still could be observed, however we did


not observe significantly BMC after a longer period. These results indicate that, although the treatment with BMC in the spinal cord of a mouse model of ALS, delayed the progression of the symptoms, it did not increase the lifespan of the animals, probably because the BMC did not remain in the injected site. INBEB, Protecel, FAPERJ, CNPq, CAPES PD14. BIOENERGETIC IMBALANCE AND OXIDATIVE STRESS IN THE PATHOPHYSIOLOGY OF SEPTIC ENCEPHALOPATHY 1- D'AVILA, JC; 1- REIS, PA; 2- RODRIGUES, RS; 1- CASTRO-FARIA-NETO, H; 3- OLIVEIRA,

MF; 4- BOZZA, F. 1- Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz - FIOCRUZ; 2- Radiologia

do Hospital Universitário Clementino Fraga Filho, UFRJ; 3- Instituto de Bioquímica Médica - UFRJ; 4- Instituto de Pesquisa Clínica Evandro Chagas - FIOCRUZ e Instituto

D'Or de Pesquisa e Ensino. "Septic encephalopathy is a frequent complication in severe sepsis but its pathogenesis and mechanisms are not fully understood. Oxygen supply and utilization are critical for organ function, especially for the brain, a tissue extremely dependent on oxygen and glucose. Disturbances in oxygen utilization are common in sepsis and a number of mitochondrial dysfunctions have been described in different tissues in septic animals as well as in septic patients. Our group described brain mitochondrial dysfunctions in experimental sepsis. But mitochondria isolated from septic brains generated less ROS in vitro. This led us to investigate the role of NADPH oxidase as a source of reactive oxygen species in the brain during sepsis. Experimental sepsis was induced by endotoxemia (LPS 10mg/kg i.p.) in Sprague Dawley rats and by polymicrobial fecal peritonits in Swiss mice. Brain glucose uptake was observed in vivo in endotoxemic rats using Positron Emission Tomography (PET) with [18F]Fluorodeoxyglucose (FDG) and autoradiography with 2- deoxy-14C-glucose. Mice with polymicrobial sepsis present hypoglycemia, hyperlactatemia and long-term cognitive impairment. We observed a rapid increase in the uptake of fluorescent glucose analog 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4- yl) amino]-D-glucose (NBDG) in brain slices from septic mice in vitro. A similar increase in brain glucose uptake was observed in vivo in endotoxemic rats. The brains of mice with experimental sepsis presented neuroinflammation, mitochondrial dysfunctions and oxidative stress, NADPH oxidase inhibitor apocynin prevented brain oxidative stress and long term cognitive impairment in survivors from experimental sepsis. Our data indicate that a bioenergetic imbalance and oxidative stress are associated to the pathophysiology of septic encephalopathy. We are observing a new metabolic phenotype in the brain during sepsis, characterized by a rapid increase in glucose uptake and mitochondrial dysfunctions that may be secondary to inflammation and hypoxia." FAPERJ, CNPq PD15. PALEOPARASITOLOGY REVISITED BY SCANNING ELECTRON MICROSCOPY

1-DUTRA JMF; 1-CAMACHO M;2- RACHID R; 2- DE SOUZA W; 1- ARAÚJO A. 1-Laboratório de Paleoparasitologia - Escola Nacional de Saúde Pública Sérgio Arouca,

Fundação Oswaldo Cruz; 2-Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro.

Microscopy techniques are used since 16th century to make visible what our eyes are not able to distinguish. For paleoparasitology studies, coprolites – desiccated or mineralized feces - are rehydrated to recover consistence of fresh feces before observation. After applying recommended techniques to concentrate cysts and eggs of parasites, samples are prepared for light microscopy analysis. Only in 1960 the first study using electron microscopy described a method to identify intestinal parasites,

bones or injuries in mummified soft tissues. Until today, scanning electron microscopy (SEM) in ancient samples is made following a routine protocol that passes through rehydration, fixation, dehydration and critical point drying steps. We describe here some alternative methodologies to prepare archeological samples for scanning electron microscopy to improve paleoparasitological findings. Natural desiccated, rehydrated samples and even parasites eggs trapped between glass slides and coverslip are submitted to different techniques for SEM analysis. CAPES, FAPERJ, CNPq. PD16. NEW 2-AMINO-5-BENZYL-4,6-DIHYDROXYPYRIMIDINES: SYNTHESIS, STRUCTURAL ELUCIDATION AND BIOLOGICAL ACTIVITY EVALUATION

1- CARVALHO, L.L.; 1- FIGUEROA-VILLAR, J.D. 1- Grupo de Química Medicinal, Departamento de Química, Instituto Militar de

Engenharia In Brazil, the diseases such as dengue, malaria, leishmaniasis, among others, are considered a serious public health problem. According to the National Institute of Science and Technology for Innovation in Neglected Diseases (INCT-IDN), these diseases prevail in poverty conditions and contribute to the maintenance of social inequality in the country. Following this judging, today there is a priority to research new prototype of drugs capable for more effective treatment of these diseases. Our group has developed research on novel substances able to act as antifolates inhibiting the enzyme dihydrofolate reductase (DHFR) present in malaria protozoa. Pyrimidine frameworks such as pyrimethamine (PYR) and trimethoprim (TMP) are a good example of potent and selective drugs capable to inhibit the dihydrofolate reductase present in the Plasmodium falsiparum (pfDHFR) responsible to the most dangerous type of malaria. Herein we wish to report our initial investigations on the synthesis and structural elucidation of new 5-benzylpyrimidines derivatives. The synthetic route was initiated by Knoevenagel condensation reaction between substituted aromatic aldehydes and a malonic ester in 40-74% yield. The benzylidene malonates generated in the first step were reduced easily from NaBH4 in 70-80% yield and, sequentially, the respective benzyl malonates can react with guanidine to furnish the desired 2-amino-5-benzyl-4,6-dihydroxypyrimidines, which are in process of the respective evaluation of their biological activity as bactericidal, fungicide and antimalarial. INBEB PD17. ISOLATION AND CHARACTERIZATION OF RNA MOLECULES BOUND TO RECOMBINANT MOUSE PRION PROTEIN

Mariana P. B. Gomes1, Luciana P. Rangel2, Murilo S. Amaral3, Sergio Verjovski-Almeida3, Yraima Cordeiro1 and Jerson L. Silva2

1Departamento de Fármacos, Faculdade de Farmácia, UFRJ; 2Instituto de Bioquímica Médica, UFRJ; 3Departamento de Bioquímica, Instituto de Química, USP

It is accepted that the prion protein (PrP) interacts with nucleic acids (NA) and that these interactions might be related to the early events in prion conformational changes and subsequent appearance of prion diseases. Our group has been studying PrP:NA interactions and our findings show that mouse recombinant PrP (rPrP) binds with high affinity to DNA in vitro. Recently, we demonstrated that rPrP:DNA interactions lead to different aggregated species and induce cell dysfunction depending on the DNA sequence tested, which appears to be correlated with the biophysical properties of the complex. Our studies with RNA showed that these molecules can also modulate rPrP aggregation and form toxic species, depending on RNA source. In the present study we use total RNA extracted from neuroblastoma cells (N2aRNA) to search for sequences that would bind rPrP with high affinity, being responsible for the effects observed


previously. N2aRNA was incubated with rPrP and after that, RNA was recovered using different isolation protocols. The samples were analyzed to determine quality, integrity and composition. In all conditions tested, large amounts of rRNA were recovered. When rPrP:N2aRNA complex was treated with RNase, the obtained RNA was partially degraded, confirming that rPrP-bound RNA is protected from digestion. The set of molecules recovered is still heterogeneous, suggesting that more than one RNA sequence contained in this extract can be responsible for inducing aggregation and toxicity. This finding is in agreement with recent studies that show that PrP is part of a ribonucleoprotein complex, as well as studies that identify large amounts of rRNA in tissues infected with PrPSc. Despite these findings, we cannot rule out the possibility of other RNA types being involved in this interaction. We believe that RNA encoded by the host itself or derived of foreign sources, may be directly involved in the PrPC/PrPSc conversion. CNPq, CAPES, INCT-INBEB, FAPERJ PD18. CARACTERIZAÇÃO ULTRAESTRUTURAL DE TRIPANOSSOMAS ENCONTRADOS EM PEIXES CASCUDOS, HYPOSTOMUS AFFINIS E HYPOSOTMUS LUETKENI

1-LEMOS, M. & 1-SOUTO-PADRÓN, T. 1-Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio

de Janeiro, Brasil. Os tripanossomas que parasitam peixes são transmitidos por sanguessugas durante a hematofagia. Formas metacíclicas presentes na probóscide de sanguessugas entram em contato com a lesão provocada no peixe e alcançam a circulação sanguínea. Neste estudo procedeu-se a caracterização ultraestrutural dos tripanossomas que infectam peixes cascudos identificados como Hypostomus affinis e Hypostomus luetkeni, procedentes do Rio Pomba (21º21'07“S, 43º02'49” O) Guarani, MG. Foi verificada a presença de sanguessugas do gênero Haementeria, aderidas à superfície corporal dos peixes, infectadas por tripanossomas. Os tripomastigotas foram isolados em 9 tipos distintos de meios de cultura a partir do sangue de 9 peixes da espécie H. affinis e de 6 da espécie H. luetkeni. Os tripomastigotas sanguíneos e os epimastigotas, esferomastigotas e tripomastigotas observados no estômago das sanguessugas e in vitro foram analisados por microscopia eletrônica de varredura (MEV) e de transmissão (MET). A caracterização ultraestrutural das formas sanguíneas por MEV revelou variação morfológica sendo encontrados tripomastigotas de corpo alongado e largo e de corpo curto e delgado. Nos cecos estomacais das sanguessugas foram observados tripomastigotas de corpo alongado e delgado e epimastigotas piriformes. Por MET os tripomastigotas sanguíneos apresentaram estruturas esféricas semelhantes a acidocalcisomas e o cinetoplasto alongado em forma de barra com arranjo compacto das fibrilas de kDNA. A ultraestrutura das formas de cultura revelou cinetoplasto semelhante ao observado para os tripomastigotas sanguíneos e a presença de complexo citóstoma-citofaringe em epimastigotas e tripomastigotas além de inclusões lipídicas, estruturas semelhantes à acidocalcisomas, organelas relacionadas a lisossomas, corpos multivesiculares. CNPq, INBEB PD19. CRATYLIA MOLLIS SEED LECTIN (CRAMOLL 1) CAN CAMOUFLAGE LEISHMANIA PROMASTIGOTES IMPAIRING THE RELEASE OF NEUTROPHIL EXTRACELLULAR TRAPS (NETS): IMPACT IN CUTANEOUS LEISHMANIASIS

NATHALIA VAREJÃO1, PEDRO TOJAL1, THIAGO VIEIRA2, TEREZA CORREIA3, ELVIRA SARAIVA2, HERBERT GUEDES4, DEBORA FOGUEL1

1Instituto de Bioquímica Médica, Federal University of Rio de Janeiro, Rio de Janeiro, BR, 2Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro,

Rio de Janeiro, BR, 3Departamento de Bioquímica, Federal University of Pernambuco, Recife, BR, 4Institutode Biofísica Carlos Chagas Filho, Federal University of Rio de

Janeiro, Rio de Janeiro, BR. rCRAMOLL 1 is the bacterial recombinant form of lectin found in seeds of Cratylia mollis (Leguminosae family, Diocleinae subtribe). We have demonstrated that this tetrameric lectin (236 amino acids per monomer) retained sugar-binding activities of the plant lectin counterpart that could result in agglutination of Trypanosome epimastigotes and Leishmania promastigotes, and, possibly induction of Th1 cell response. So, we hypothesized if rCRAMOLL 1 could promote a protective effect in a murine model of American cutaneous leishmaniasis acting as a healing chemical in initial stages of footpad lesions by intradermal or ointment applications or as an adjuvant molecule in vaccination designs (oral, nasal). Surprisingly, our results pointed to an aggravation of the lesions in the animals treated with rCRAMOLL 1 associated with enhanced parasite burden. In 2009, Dr. Saraiva and cols. showed that neutrophils play an important role in control of leishmanial infection by a peculiar cellular death named NETosis which is trigger by interaction of neutrophils with lipophosphoglycan (LPG) present in Leishmania surface that results in fibrilar DNA release that traps and kills the parasites. Now, we are investigating how the interaction of rCRAMOLL 1 with L. amazonensis promastigotes could affect NETs release and parasite survival. Interestingly, our data show that neutrophils lost the ability to recognize and release the extracellular traps in the presence of promastigotes that were previously incubated with subagglutinating concentration of rCRAMOLL 1. On another hand, when the neutrophils were pre-incubated with our lectin they engulfed it and even so they can perfectly respond to the presence of Leishmania, which indicates that rCRAMOLL 1 does not affect any mechanism of NETs formation and release, instead it acts as a parasite camouflage. Taken together our findings could help to explain the negative effects observed in the model disease treatments and point an alert in the use of plant lectins in this field. CAPES, FAPERJ, INBEB PD20. STATINS PREVENT BRAIN MICROVASCULAR ALTERATIONS AND COGNITIVE IMPAIRMENT DUE TO SEPSIS

1-REIS, P.A.; 1-ALEXANDRE, P.C.B.; 2-ESTATO, V.; 2-TIBIRIÇÁ, E.; 1-BOZZA, F.A.; 1-CASTRO-FARIA-NETO, H.C..

Lab. de Imunofarmacologia, Lab Invest. Cardiovascular -IOC-FIOCRUZ-Rio de Janeiro, Brazil

Sepsis is a major cause of mortality in intensive care units, and brain dysfunction is frequently observed as a consequence of changes in cerebral structure and metabolism. Statins have been prescribed extensively for their cholesterol-lowering properties and efficacy in cardiovascular disease and also present antiinflammatory and antioxidant effects due to pleiotropic mechanisms. The objective of the present study was investigate the effect of statins on the brain microcirculatory changes and on the cognitive impairment due to sepsis. Feces were extracted (5 mg/g b.w.) from large intestine of SW mice and diluted in saline, centrifuged and the supernatant was collected and injected in the animals (n=5-8/group). Control animals received 0.5 ml of saline. Animals were treated at 6, 24 and 48 hours after that with imipenem (10 mg/kg b.w., 0.2 ml s.c.) and 1.0 ml of saline (s.c.). The statins healthy or septic groups were treated 1 h before to 48 hours after the infection (20 mg/kg b.w., p.o.). Mortality was observed for 96 h and the severity score was evaluated. After 24 hours, groups of animals were anesthetized and intravital fluorescence videomicroscopy was performed. Cognitive damage was evaluated by inhibitory avoidance task 15 days post-sepsis. Mice developed a moderate sepsis 6 and 24 h post feces injection which is reduced in 48 h. Treatment with atorvastatin or simvastatin did not reduced the clinical score during


sepsis development or the mortality. Statins therapy reduced the leukocyte rolling and adhesion induced by sepsis, and reversed brain functional capillary rarefaction. Animals that received statins were able to keep the avoidance memory missed in sepsis-untreated mice. The present findings indicate that the treatment with statins was able to protect the brain microvascular alterations and reverse the cognitive impairment during sepsis. FIOCRUZ, FAPERJ, CNPq, CAPES PD21. DISSECTING THE MECHANISM OF CITOTOXICITY TRIGGERED BY AGGREGATES OF TRANSTHYRETIN

1-FERREIRA, P. S.;1- OLIVEIRA, L. T.;1- FOGUEL, D.. 1- Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro.

Familial amyloidotic polyneuropathy (FAP) is a neurodegenerative autosomal dominant disorder characterized by the extracellular deposition of transthyretin (TTR) fibrils in several tissues. There is a debate in the literature concerning the identification of the toxic species present in the pathway to fibril formation (native àprotofibrilà fibril) trying to elucidate which species would be responsible for cellular damage caused in PAF. In the present study we evaluated which are the most toxic species in the aggregation pathway of TTR, and the mechanism by which they promote their citotoxic effects in different cell lineages and in primary culture of retinal neurons. The viability assays show that the oligomers formed during the aggregation process are the most toxic species and that have a tissue specific toxicity. Our data also shows that these oligomers trigger cell death by activating the caspase-dependent apoptotic pathway. Furthermore, we verify the internalization of these oligomers only in cells which were toxic, suggesting that perhaps this internalization has some correlation with toxicity. All together these data show that oligomers of TTR were the most toxic species formed during the aggregation process of TTR and account for much of the cell damage caused by the TTR amyloidosis. INBEB, FAPERJ, CAPES, CNPq PD22. VACCINATION WITH A PRESSURE INACTIVATED AVIAN INFLUENZA VIRUS PROTECTS MICE AGAINST INFECTION WITH INDUCTION OF GOOD IMMUNE RESPONSE AND PRESERVATION OF HA AND NA ACTIVITY BARROSO, S.P.C.1, NICO, D.2, VICENTE, A.C.S.1, COUCEIRO, J.N.S.S.2, NASCIMENTO, D.3, BOZZA, F.3, FERREIRA, DF2, PALATNIK-DE-SOUSA, C.B.2, SILVA, J.L.1 & OLIVEIRA, A.C.1

1Institute of Medical Biochemistry - Federal University of Rio de Janeiro (UFRJ); 2Institute of Microbiology Paulo de Goes - UFRJ, Rio de Janeiro; 3Oswaldo Cruz Institute

- FIOCRUZ H3N8 is an avian influenza virus that was originally isolated from birds, and later found in horses and dogs. Here, we used 12 h of incubation under hydrostatic pressure (HP) to achieve this virus inactivation without damage to its hemagglutinin and neuraminidase activities and study its protective capability in vaccination against H3N8 infection. Balb/c mice were treated by the intranasal route, with 3 doses of the pressure-inactivated virus. Mice were challenged with native H3N8 on fourth week, and monitored for: virus-specific antibodies in serum, nasal lavage and faeces, CD4+ and CD8+ virus-specific T cells, cytokine ELISA, clinical symptoms and inflammatory parameters. After immunization and challenge we found an increase of IgG1, IgG2a, and IgA antibodies. These antibodies found in serum are neutralizing. The analysis of cytokine production by CD4+ and CD8+ T cells showed a mixed Th1/Th2 pattern after vaccination. Two weeks after the challenge, we observed an increase in the production of antibodies and IL-6, IFN gamma and TNF alpha. The control group (saline) showed more clinical signs of disease (lethargy, weight loss and huddling) than vaccinated animals and more Evans

blue leakage than the vaccinated group. In the same way differential cell counts in bronchoalveolar lavage showed more cells in control group. HP-inactivated H3N8 virus vaccine induced significant protection against the infection by H3N8 influenza virus. Our work reaffirms the use of HP as an interesting tool in the development of viral vaccines at low cost and good immune response. Support:CAPES,PRONEX,INBEB,CNPq,FAPERJ. PD23. HEPARIN MODULATION OF PRION SEEDING ACTIVITY

1- VIEIRA, T.C.R.G.; 2- CAUGHEY, B.;1- SILVA, J.L. 1-Inst de Bioquímica Médica, IBqM-UFRJ, RJ, Brazil; 2-LPVD, RML, NIAID, NIH, MT, USA.

"INTRODUCTION. The conversion of PrP into scrapie PrP is the central event of prion diseases (TSEs). Such conversion and propagation is seeded or templated by a polymerization mechanism. Some authors have suggested that glycosaminoglycans (GAGs) directly convert PrP into a protease resistant form, while others have proposed that these molecules have a protective activity. Our group recently reported that low molecular weight heparin (LMWHep) does not induce recombinant mouse prion protein (rPrP23-231) conversion, protecting rPrP23-231 from RNA-induced aggregation (1). MATERIAL AND METHODS: Real-time quaking-induced conversion (RT-QuIC) is an assay in which disease-associated PrP initiates a rapid conformational transition in recombinant PrP, resulting in the formation of amyloid fibrils that can be monitored in real time using the dye thioflavin T. We used rPrP from mouse and hamster as substrate (23-231 and 90-231), and TSE-associated forms were from mouse and hamster brain homogenates (RML and 263K strain respectively). LMWHep was used in order to determine the effect of this GAG on PrP fibrillization. RESULTS AND DISCUSSION: In the present work, we show that LMWHep delays and decrease fibril formation. It also inhibits fibrilization depending on the seed used. There is no effect when rPrP 90-231 is used, or with high salt concentration. Moreover, it is effective when added at the lag phase of the polymerization process. When a soluble LMWHep-rPrP complex is added to the reaction, no fibrils are detected. On the contrary, the addition of a LMWHep-rPrP aggregated complex results in conversion. CONCLUSIONS: Through electrostatic interactions, LMWHep interaction with PrP N-terminal domain, modulates PrP fibrilization. It affects the nucleation processes and the formation of oligomers, the first step of fibrilization. Our findings may explain the protective effect of these molecules in different models. References: Vieira TCRG, et AL. J Amer Chem Soc 2011; 133:334-344. Keywords: Prion, Gycosaminoglycan, Aggregation, Neurodegeneration" NIAID (NIH), CNPq, INBEB and FAPERJ. PD24. STRUCTURAL BASIS FOR THE INTERACTION OF HUMAN Β-DEFENSINS 1 AND 6 AND ITS PUTATIVE CHEMOKINE RECEPTOR CCR2 AND BREAST CANCER MICROVESICLES De Paula, V.S.1, Gomes, N.S.F.1, Lima, L.G.1, Monteiro, R.Q.1, Almeida, F.C.L. 1, Valente,

A.P. 1-Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de

Janeiro, RJ 21941-902, Brasil Human β-defensins (hBD) are believed to function as alarm molecules that stimulate the adaptive immune system when a threat is present. In addition to its antimicrobial activity, defensins present other activities such as chemoattraction of a range of different cell types to the sites of inflammation. We have solved the structure of the human β-defensins 6 (hBD6) by NMR spectroscopy that contains a conserved β-defensin domain followed by an extended C-terminus. We also investigated the interaction of β-defensin 1 and 6 with microvesicles shed by breast cancer cell lines using NMR.


Chemical shift mapping of the interaction showed that both defensins interact with microvesicles but in slightly different way, suggesting an inverse correlation with the aggressiveness potential of the cell. Furthermore, molecular docking using restraints derived from the NMR chemical shift data produced a model of the complex between hBD6 and a peptide derived from the extracellular domain of CC chemokine receptor 2 (Nt-CCR2) that reveals a contiguous binding surface on hBD6, which comprises amino

acid residues of the α-helix, loop between β2-β3 and C-terminal. The microvesicles binding surface partially overlaps with the chemokine receptor interface. These data offer new insights into the structure–function relation of the hBD6–CCR2 interaction and may be helpful for the design of novel anti-cancer agents. INBEB, CAPES, FAPERJ,CNPq


Pesquisadores

P1. MICE DEFICIENT IN BRADYKININ B1 RECEPTOR, A GPCR UPREGULATED IN INFLAMED TISSUES, ARE PROTECTED FROM CHRONIC MYOCARDITIS AND ADVERSE CARDIAC REMODELLING

OLIVEIRA AC1, ANDRADE, D1, CORDOVIL, T1, RAMOS-JUNIOR E.S1, ALMEIDA, LN1; CARVALHO-PINTO, C.E2, MONTEIRO, RQ3, SVENSJO, E1, SIROIS, P.4, SCHARFSTEIN, J1 1Instituto de Biofísica Carlos Chagas Filho, UFRJ; 2Instituto de Biologia, Departamento

de Imunologia, UFF; 3 Departamento de Bioquímica Médica, UFRJ; 4CHUL Research Center, Laval University, Quebec, Canada

Chronic chagasic myocarditis (CCM) depends on Trypanosoma cruzi persistence in the myocardium. In a recent article (Scharfstein et al., Frontiers in Immunology, 2013) we have suggested that extracellular trypomastigotes released from heavily parasitized heart cells evoke intermittent “flares” of plasma-leakage through the activation of the kallikrein-kinin cascade. Based on multiple evidences, we hypothesized that T. cruzi may exploit this window of opportunity (formation of a transient intramyocardial edema) to proteolytically generate infection-promoting signals, such as bradykinin (BK) and Des-Arg-BK, respectively the agonists of BK2R (constitutive) and BK1R, a NFk-B-inducible GPCR upregulated in inflamed tissues. Consistent with this hypothesis, here we show evidence that R954 (BK1R antagonist) reduced the parasite tissue load (skeletal muscle) in LPS-treated infected (3 d pi) mice, but not PBS-treated infected. Extending this analysis to the classical intraperitoneal model of infection, we found that BK1R-/- mice displayed reduced heart parasitism as compared to WT B6 mice (14 d pi). Immunological studies revealed that adaptive responses were fairly similar in both strains. Interestingly, analysis of innate response profiles revealed a significant reduction (p<0.05) of intracardiac frequencies of Ly6C low monocytes, a subset implicated in tissue repair and collagen deposition. Reminiscent of the cardioprotective phenotype of BK1R-/- mice in models of diabetic cardiomyopathy, phenotypic analysis of chronic chagasic mice (90 d) revealed that myocarditis and heart fibrosis were profoundly reduced in BK1R-/- mice. Combined with the in vivo pharmacological data, these studies support the hypothesis that BK1R, GPCR upregulated in inflamed tissues, might serve a preferential gateway for T. cruzi infection. INBEB, CNPq, FAPERJ P2. CARACTERIZAÇÃO ESTRUTURAL DA TXNIP, UMA PROTEINA CHAVE NO METABOLISMO CELULAR

1-AGUIAR, R.P.; 1-AMORIM, G.C.; 1- VALENTE, A.P.; ALMEIDA, F.C.L. 1- Centro Nacional de Ressonância Magnética Nuclear, Instituto de Bioquimica Médica,

UFRJ "A Txnip (Thioredoxin interacting protein) é um importante regulador multifuncional do metabolismo celular, o que vem atraindo muita atenção para esta proteína atualmente. Entre suas funções está a regulação do crescimento, diferenciação, sinalização e morte celular. Ela atua ainda no metabolismo da glicose e de lipidios. A Txnip faz parte da família das α-arrestinas, que inclui outras cinco proteínas, Arrdc1-5. No entanto, a Txnip é a única proteína desta família capaz de interagir com a Tioredoxina (Trx), uma enzima antioxidante ubíqua que regula a homeostasia oxido-redutora das células. A Trx desempenha papel importante em diversos processos celulares relacionados ao

equilíbrio saúde-doença. Pouca informação estrutural e funcional está disponível para a família das α-arrestinas e, especialmente, para a Txnip e sua interação com a Trx. Com o objetivo de compreender estes mecanismos, estamos fazendo a caracterização estrutural e funcional da Txnip e sua interação com a Trx utilizando RMN, SAXS e fluorescência. Por se tratar de uma proteína grande (390 resíduos), estudamos seus domínios estruturais isolados. Além disso, também utilizamos peptídeos correspondentes a prováveis regiões de interação. Esta abordagem nos permitiu caracterizar a participação de cada domínio na interação com a Trx. Os primeiros experimentos de RMN e SAXS para o cálculo da estrutura tridimensional já foram realizados e os peptídeos sintetizados. Estes dados fornecerão ferramentas para a melhor compreensão dos mecanismos de ação e das funções desempenhadas por essa classe de proteínas, pouco conhecidas, mas de grande importância para a regulação das funções celulares." FAPERJ, CNPq P3. NEW THIAZOLIDINEDIONE DERIVATIVE RA-4 DECREASES THE EXPRESSION OF INFLAMMATORY MARKERS IN MURINE MODEL OF CARRAGEENAN-INDUCED PLEURISY

1- BARBOSA,K.P.S.; 1- ROCHA, S.W.S.; 1- SANTOS, L.A.M.; 1- SANTANA, A.K.; 1- FRAGOSO, I.T.; 1- FRANÇA, M.E.R.; 1- GOMES, F.O.S.; 1- RIBEIRO, E.L; 1- SILVA, B.S.; 1-

SILVA, A.K.S.; 1- DONATO, M.M.A.; 3- SILVA, T.G; 3- LIMA, M.C.A.; 3- PITTA, I.R.; GALDINO, S.L.; 1,2- PEIXOTO, C.A.

1- Centro de Pesquisas Aggeu Magalhães - CPqAM/FIOCRUZ; 2- Centro de Tecnologias Estratégicas do Nordeste - CETENE/MCT, Universidade Federal de Pernambuco; 3-

Departamento de Antibióticos, Universidade Federal de Pernambuco Background: Many studies have demonstrated several biological activities of peroxisome proliferator-activated receptor (PPAR), however, few studies have been conducted on the effects of agonist these receptors on lung diseases, which pose a serious public health problem. Objective: The present study evaluated the anti-inflammatory action of a new synthetic thiazolidinedione derivative RA-4 on acute lung inflammation (pleurisy) induced by carrageenan. Methodology: Forty mice were randomly allocated into the following groups: Control group (sham) (n=10); carrageenan group (CAR) (n=10); CAR + LPSF/RA-4 group (n=10); which was treated with LPSF/RA-4 (60 µMol/kg); CAR + INDO group (5 mg/kg) (n=10). The mice were anaesthetized and submitted to injection into the pleural cavity with Saline (0.1ml) or saline containing 1% carrageenan (0.1ml) injected into the pleural cavity. After 4h, the animals were sacrificed under CO2 vapors. The levels of nitric oxide (NO) quantified on pleural exudates and lung fragments were processed for light microscopy, transmission electron microscopy, immunohistochemistry and Western blotting assays. Results: The influx of leukocytes and nitric oxide levels were significantly reduced after treatment with RA-4 and INDO, compared with the CAR group. Histopathological and ultrastructural analysis showed that tissue injury was significantly reduced in the groups treated with RA-4 or INDO. Immunohistochemistry showed an increase of inflammatory markers such as COX-2, iNOS, TNF-α, IL-1β in lung tissue CAR group, whereas the groups treated with RA-4 and INDO presented significant reduction of these immunomarkers. Analysis by Western blotting revealed increased expression of COX-2 and IL-1 in the CAR group, which were significantly reduced by treatment with RA-4. Conclusions: These results demonstrate the potent anti-inflammatory action of the new derivative thiazolidinedione RA-4 (60µMol/kg) in acute lung injury induced by carrageenan. Therefore, assays are in development in our laboratory in order to clarify the role of RA-4 on the molecular mechanisms involved in the inflammation. INBEB, FACEPE


P4. ESTUDO DA IMOBILIZAÇÃO DA PROTEÍNA PRÍON EM NANOPARTÍCULAS MAGNÉTICAS

1 - de MORAES, M.C.; 1- dos ANJOS, D.M.; 1- BOSCO, J.; 1- da SILVA, J.L. Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro

Encefalopatias espongiformes transmissíveis são desencadeadas quando a proteína príon nativa é convertida em sua isoforma infecciosa, o que provavelmente requer um fator celular. Assim, diversos compostos têm sido avaliados como inibidores desta conversão. Considerando a importância das interações ligante-proteína nos sistemas biológicos, estudos de afinidade podem contribuir para a compreensão do mecanismo de conversão e da avaliação de possíveis inibidores. Neste trabalho, foi selecionado como modelo o estudo de afinidade por "fishing" de ligantes. Para o desenvolvimento desta metodologia, a proteína príon deve ser imobilizada covalentemente em nanopartículas magnéticas, sem afetar significativamente a estrutura da proteína. Assim, foram preparadas nanopartículas magnéticas (NPMs) de Fe3O4 pelo método da co-precipitação de íons Fe3+ e Fe2+. Posteriormente, as MNPs foram derivadas com tetraetoxisilano, aminopropriltrietoxisilano e glutaraldeído, com a posterior ligação covalente da proteína através da formação de bases de Schiff. Cada etapa de derivação foi confirmada pela análise do potencial zeta de amostras coletadas após cada reação. As NPMs foram caracterizadas também por infravermelho, morfologicamente por microscopia eletrônica de transmissão, e a distribuição do tamanho das NPMs foi investigada por DLS (tamanho médio de 51,12nm). A ligação da proteína príon foi estimada medindo-se a absorbância a 280nm da solução protéica antes e após a reação com as NPMs. Com o estudo de otimização, observou-se que aproximadamente 0,6mg de proteína príon se liga a 2,5mg de NPMs. A próxima etapa do trabalho consiste em imergir as NPMs com o príon ligado em soluções contendo compostos com conhecida afinidade pela proteína e, após incubação, realizar magneticamente uma extração líquido-sólido. Após algumas lavagens, pode-se identificar, por espectrometria de massas, os compostos com maior afinidade pela proteína, objetivo dos ensaios de “fishing” de ligantes, obtendo-se assim um método de triagem rápida para ligantes do príon.

FAPERJ P5. SILDENAFIL ESTIMULA A PROLIFERAÇÃO ENDOMETRIAL DE CAMUNDONGOS C57BL/6

1- DONATO, M. A. M.; 1- RIBEIRO, E. L.; 1- GOMES, F. O. S.; 1- OLIVEIRA, W. B.; 1- FRAGOSO, I. T.; 1- SANTOS, L. A. M.; 1,2- PEIXOTO, C. A.

1 - Centro de Pesquisas Aggeu Magalhães, FIOCRUZ, Recife-PE; 2 - CETENE, Recife-PE Recentemente, o sildenafil tem sido utilizado como estratégia terapêutica no tratamento de mulheres com problemas de fertilidade, sugerindo uma possível ação no trato reprodutor feminino. Vários autores sugerem que o tratamento com o Sildenafil estimula o crescimento endometrial ou aumenta a possibilidade de implantação. No presente trabalho, camundongos C57BL/6 selvagens e knocauteados para o gene da iNOS (iNOS-/-) foram tratados com Sildenafil na concentração de 25 mg/kg/dia por 60 dias. Após esse período, os animais foram eutanasiados, os úteros dissecados e submetidos a análises de microscopia óptica e eletrônica, assim como a análise imunohistoquímica para avaliar se houve alteração na expressão de proteínas essenciais para a receptividade endometrial. Nossos resultados confirmam que o Sildenafil induziu a proliferação celular no endométrio e miométrio no útero em camundongos C57Bl/6 wild type assim como aumentou a expressão de enzimas angiogênicas VEGF e eNOS no tecido estromal, além de reduzir a expressão de TGF-β. O grupo controle knockout para a iNOS apresentou miométrio e endométrio com maior espessura em relação ao grupo controle selvagem, apresentando glândulas endometriais com lúmen reduzido e células cilíndricas, indicando que o gene da iNOS apresenta um possível papel na preparação endometrial pré-implantacional e na implantação embrionária. Após o tratamento dos camundongos knockout para a iNOS com sildenafil, foram obtidos resultados similares em relação à expressão de VEGF, eNOS e TGF-β, indicando que a ação do sildenafil no endométrio independe da presença do gene iNOS. INBEB

instituto nacional de ciência e tecnologia de biologia ... iv encontro anual do... · 3.dr....

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