simpósio comemorativo dos 50 anos do programa de pós ... · livro de resumos do simpÓsio...

Simpósio Comemorativo dos 50 anos do

Programa de Pós-Graduação em Ciências

(Bioquímica) da UFPR

LIVRO DE RESUMOS

2015

ii

Universidade Federal do Paraná

Programa de Pós-Graduação em Ciências (Bioquímica)

Simpósio Comemorativo dos 50 anos do Programa de Pós-

Graduação em Ciências (Bioquímica) da UFPR

Livro de Resumos

Curitiba-PR

2015

iii


REALIZAÇÃO:

Coordenação do Curso dePós-Graduação em Ciências (Bioquímica):

Glaucia Regina Martinez

Emanuel Maltempi de Souza

Chefia de Departamento:

Rose Adele Monteiro

Joana Léa Meira Silveira

Colegiado de Curso de Pós-Graduação:

David Alexander Mitchell

Sheila Maria Brochado Winnischofer

Miguel Daniel Noseda

Guilherme Lanzi Sassaki

Marcelo Muller Dos Santos

Leonardo Magalhães Cruz

Maria Eliane Merlim Rocha

Nadia Krieger

Alessandra Biz (Discente)

Alexsandro Vinícius Nogueira (Discente)

Rocio del Pilar Cuaspa Ropain (Discente)

Apoio discente na organização das atividades do evento:

Paloma Bonato

Sarah Sacks Timoteo

Maura Harumi Sugai

Ester Mazepa

Rafaela Perez

Shayla Fernanda Barbieri

Heloisa Bruna Soligo Sanchuki

Carlos Eduardo Sanchuki

Edileusa Cristina Marques Gerhardt

Vanessa Kessler Chicora

Manuel Jose Pinero Gavida

Fernanda Gravina

Montagem e organização do livro de resumos:

Otávio Martins Cruz (Discente)

Patrícia da Silva Peres (Discente)

iv

Programa de Pós-Graduação em Ciências – Bioquímica

APRESENTAÇÃO

Simpósio Comemorativo dos 50 anos do Programa de

Pós-Graduação em Ciências (Bioquímica) UFPR

O Simpósio Comemorativo dos 50 Anos visa fortalecer a interação dos

docentes e pós-graduandos, motivar os jovens pesquisadores, promover maior

integração entre pesquisadores de diferentes Instituições Nacionais e

Internacionais e enriquecer a formação dos pós-graduandos e docentes.

O Programa de Pós-Graduação em Ciências (Bioquímica) - PPGBq (conceito

6, triênio 2010-2012) agrega linhas de pesquisa bem estabelecidas e

diversificadas na área de bioquímica básica e aplicada com importante

inserção estadual e nacional.

APOIO:

PATROCÍNIO:


5 LIVRO DE RESUMOS DO SIMPÓSIO COMEMORATIVO DOS 50 ANOS DO PROGRAMA DE

PÓS-GRADUAÇÃO EM CIÊNCIAS (BIOQUÍMICA) UFPR _ setembro/2015

SUMÁRIO

APRESENTAÇÃO ................................................................................................................. iv

SUMÁRIO ............................................................................................................................... 5

RESUMOS .............................................................................................................................. 6

ÍNDICE DE AUTORES ........................................................................................................ 61

ANEXO .................................................................................................................................. 65

PROGRAMAÇÃO DO EVENTO ..................................................................................... 65




RESUMOS

ANTI-INFLAMMATORY EFFECTS OF THE POLYSACCHARIDES PRESENT

IN THE BLACKBERRY WINE

Caillot, A. R. C1; Bezerra, I. L.; Santana-Filho, A. P.; Sassaki, G. L.

Biochemistry and Molecular Biology Department, Federal University of Parana,

Curitiba, Brazil

Introduction: Blackberry wine is recognized as a natural source of essential

minerals and many bioactive phytochemicals that can play an important role in

health promotion. This work aims to evaluate receptor mediated anti-

inflammatory activity of the polysaccharides from blackberry wine. Methods:

The blackberry wine were precipitated by addition of EtOH-(3V) and

centrifuged. The precipitate was then dialyzed generating polysaccharide

fraction (PVA). Those were submitted to freeze-thawing and centrifugation,

resulting in soluble (PVAS) and insoluble (PVA-I) fractions. The fraction PVAS

was submitted the Fehling treatment, resulting in two new fractions: supernatant

(PVAFESB) and precipitate (PVAFEPPT). The fractions were hydrolyzed with

TFA at 100°C/14h. Thereafter the sample was dried and reduced with NaBH4

giving rise to alditols, which were acetylated. The resulting alditol-acetates were

analyzed by GC–MS. The fraction PVAS was evaluated regarding anti-

inflammatory activity. RAW-264.7 cells were treated with LPS alone (1µg/ml) or

in combination with PVAS (100µl/ml) for 24h. After 24h, anti-inflammatory

activity was evaluated on culture supernatants using Quanti-Blue and following

manufacture´s recommendations. Results: The monosaccharide composition of

the PVAS showed Man-(33.1%), Glc-(12.6%), Gal-(19.1%), Ara-(12.9%), Rha-

(5.5%), Xyl-(2.8%), Fuc-(1.3%) and GalpA-(12.9%). 1H/13C HSQC NMR

analysis of the fraction PVAFESB showed signals at chemical shift (C1/H1)

103.2/4.47, which are characteristic of (1→3)-linked b-D-Galp units present in

arabinogalactan-II. The fraction PVAFEPPT 1H/13C HSQC showed correlations

corresponding to a (1→6)-linked a-D-mannan, with chemical shift at 99.08/5.09

and 98.6/4.95. We also demonstrated that fraction PVAS inhibited receptor

mediated LPS triggered inflammation on RAW-264.7 cells using a reporter gene

assay based on NF-kb transcriptional activity.

Financial Support: CAPES




STRUCTURAL CHARACTERIZATION OF POLYSACCHARIDES

EXTRACTED BY INFUSION OF SEDUM DENDROIDEUM LEAVES

Oliveira A.F., Cordeiro L.M.C., Iacomini, M. and Cipriani, T.R.

Biochemistry and Molecular Biology Department, Federal University of Parana,

Curitiba, Brazil

Sedum dendroideum is a medicinal plant known in Brazil as bálsamo. Its leaves

are traditionally used to treat skin inflammations and gastric disorders. Infusion

of its lyophilized leaves (1.30 kg) was used to extract polysaccharides, which

were fractionated by freezing/thawing process, dialysis and anion exchange

chromatography. The cold water soluble fraction (6.66 g) was dialyzed with a

100 kDa cut-off membrane, yielding the fractions Rsbal (2.55 g), Esbal-I (3.35

g) and Esbal-II (0.55 g). Rsbal and Esbal-II showed homogeneous HPSEC

profiles. Monosaccharide analysis showed that Esbal-II contained GalA

(82.3%), Ara (4.5%) and Gal (4.2%), suggesting the presence of a

homogalacturonan, whereas Rsbal contained GalA (47.2%), Ara (23.2%), Gal

(24.8%) and Glc (4,9%), suggesting the presence of a homogalacturonan and

an arabinogalactan. Rsbal (100 mg) was subjected to anion exchange

chromatography yielding the fractions Rsbal-H2O (25 mg), Rsbal 0,5M (62.1

mg) and Rsbal-1M (1.2 mg). Rsbal-H2O showed GalA (60%), Gal (25.3%), Rha

(1.9%), Ara (3.9%) and Glu (6.8%). This acid polysaccharide was eluted with

H2O, probably because its GalA units are methyl-esterified. Rsbal-0,5M

presented GalA (62.1%), Gal (17.4%), Ara (17.1%) and Rha (1.0%), suggesting

the presence of a homogalacturonan and an arabinogalactan. Its NMR analysis

(HSQC) showed chemical shifts at 103.3/4.50 of C1/H1 of β-D-Galp;

106.5/5.80, 107.9/5.42 and 108.9/5.25 of C1/H1 of α-L-Araf; 100.2/4.82 and

99.0/5.15 of C1/H1 of 6-OMe-α-D-GalpA and α-D-GalpA, respectively;

52.9/3.81 of -COO-CH3; and 70.5/5.11 and 71.3/4.78 of C5/H5 of 6-OMe-α-D-

GalpA and α-D-GalpA, respectively. Methylation analysis will be performed to

obtain more structural informations.




MMP14 GENE REGULATION BY DNA METHYLATION IN THE INTRONIC

REGION

Chequin, A.1; Manica, G. C. M.1; Klassen, L. M. B.1; Ramos, E. A. S.1; Toledo,

M. B.1; Brandão, Y. O.; Souza, E. M.2; Klassen, G.1

1Dep. de Patologia Básica, Laboratório de Epigenética. Universidade Federal

do Paraná. UFPR, PR, Brasil.

2: Dep. de Bioquimica. Universidade Federal do Paraná. UFPR, PR, Brasil.

Metastases are responsible for 90% of deaths in breast cancer. The

metalloproteinase 14 (MMP-14) is an important protein related to metastatic

process, and present three CpG islands (CGIs), one on the promoter region,

which known regulates gene expression, and two located in intronic regions,

that haven´t been studied. Recent reports showed that methylation on intronic

regions may be important to regulate the human transcriptome. The aim of this

study was evaluate the effect of DNA methylation inhibitior (DAC) and inhibitor

of histone deacetylase (TSA) in the demethylation of intronic islands of MMP14

in breast tumor cells lines. The expression of the MMP14 in two breast tumor

cell lines was evaluated by qRT-PCR. The regions containing the CGIs were,

after sodium bisulfite treatment, cloned and sequenced. The cell line MMP14

negative was treated with DAC, TSA, or both, and also subjected to qRT-PCR

and sequencing. MCF7, which shows no expression of MMP14, showed high

levels of methylation in CGIs, as well as PMC-42, which expresses MMP14,

showed lower levels of methylation. After treatments, MCF7 passed to express:

2.6 (DAC), 3.2 (TSA) and 2.9 (DAC+TSA) more MMP14 than mock in its three

CGIs respectively, and the rates of methylation in first CGI had a reduction of

40,7%, 18.7%, 66%. On the intronics islands 2 and 3, these reductions were

more pronounced: 95%, 59%, 100% and; 81.3%, 64%, 73.3%, in the same

treatments. Apparently, MMP14 is being regulated by methylation on CGIs

located in first íntron in addition to promoter region.




SYD-1 INDUCE RESPIRATION DYSFUNCTION ON HEPG2 WITH

OXIDATIVE PHOSPHORYLATION PATHWAY ACTIVATED

Brandt, A.P.1; Pires, A. R. A. 1; Echevarria, A.2; Canuto, V.C.2; Cadena,

S.M.S.C.1

1Departamento de Bioquímica e Biologia Molecular, UFPR, PR;

2Departamento de Química,UFRRJ, RJ. Brazil.

An important antitumor effect of SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-

oxadiazolium-5-olate) has been shown. We previously reported that SYD-1

impairs oxidative phosphorylation in isolated mitochondria. The expressive

inhibition of electron transport throughout the respiratory chain was related with

its antitumor effect. Now, to better visualize the effects of SYD-1 under

metabolic parameters, with highlight to the mitochondrial function, we evaluated

the effects of this derivative on human liver carcinoma cells (HepG2) cultured

with free glucose DMEM medium supplemented with galactose and glutamine

(aiming to induce these cells to obtain energy through oxidative respiration).

SYD-

after 24h of treatment, as evaluated by MTT assay. Using crystal violet staining,

after 72h of treatment, SYD-1 decreased the viability of these cells by ~35% to

as MTT assay after 24h of treatment, reaching only ~43% to the high

inhibited by SYD-1. However, the compound strongly inhibited the leak state to

all concentrations used (15-

cultured with DMEM medium supplemented with high concentration of glucose

(previous data), these results were less pronounced indicating SYD-1 effects

may be related to impairment of oxidative respiration.

Financial Support: CNPq, CAPES, INCT-Redoxoma




ACTIVITY OF HEXAHYDROXYTRIPHENYLENE ON CELL VIABILITY AND

REACTIVE OXYGEN SPECIES LEVELS IN HUMAN GLIOMA T98G CELLS

Ribeiro, C.S.P¹; Cruz, O.M¹; Pino-Gomes, R.¹; Bark, J.M¹; Winnischofer, H.²;

Martinez, G.R¹; Winnischofer, S.M.B¹

¹ Departamento de Bioquímica e Biologia Molecular – Universidade Federal do

Paraná, Paraná, Brasil.

² Departamento de Química – Universidade Federal do Paraná, Paraná, Brasil.

Glioblastoma multiform is considered the most aggressive cancer of the central

nervous system, with a very high mortality rates. This scenario shows the

inefficiency of the current treatment, which is based on the use of temozolomide

chemotherapy followed by radiation therapy. Therefore, the aim of this study is

to evaluate the effects of hexahydroxytriphenylene (HHTP) on cell viability and

intracellular reactive oxygen species (ROS) levels in T98G cell line Cell viability

was assessed by MTT method, testing different concentrations of HHTP (10,

25and 50 μM) and drug exposure times (12, 24 and 48 hours). After 12 hours, it

was observed that the HHTP treatment did not alter cell viability at all times and

concentrations tested. After 24 hours exposure of 50μM HHTP, the viability of

the cells was reduced by 25%. At the time of 48h the reduction in the viability

was 44% and 46% with 25 and 50 μM HHTP, respectively. In addition, it was

also evaluated the levels of ROS by the oxidation of the fluorescent probe 2 ', 7'

- diclorodiidrofluorescina (DCFH-DA). Results showed that T98G cells treated

with HHTP at 50 μM for 12 hours displayed an increase in the ROS levels, by

40%. After 24 hours, it was observed a significant increase in ROS levels, at all

concentrations HTTP. In 48 hours, persistent high ROS levels were observed,

reaching 30% at 25 μM HHTP and 47% at 50 μM HHTP. Together, these data

suggest that treatment with HHTP in glioblastoma model, reduces the cell

viability by concentration-dependent, accompanied by increase in ROS levels.





DETERMINING THE BACTERIAL DIVERSITY IN THE MANGROVE SOIL IN

THE REGION OF PARANAGUÁ BAY, BRAZIL, AND THE NATURAL

FACTORS AFFECTING IT

Denny Marcel Seccon, Daniel Renato Lammel, Eduardo Balsanelli, Emanuel

Maltempi de Souza, Helisson Faoro, Michelle Zibetti Tadra Sfeir, Paulo da

Cunha Lana, Roseli Wassem, Fabio de Oliveira Pedrosa

Departamento de Bioquímica e Biologia Molecular – Universidade Federal do


Mangroves are unique habitats present in the interface of sea and land. The

peculiar characteristics of this environment are likely due to fluctuation in sea

salinity and to the anaerobicity of the soil. Mangroves provide shelter for many

terrestrial and sea animals, protect the shores from erosion and are well suited

for economic exploitation. As expected, the microorganisms inhabiting its

sediments play a pivotal role in maintaining its functionality and health by

interacting with the plants growing thereby. A better understanding of the

microbial communities of mangroves has become increasingly important since

mangroves have been strongly affected by human activities and pollution. This

work characterizes the biodiversity of the prokaryotic community living in the

mangrove soil of the region of Paranaguá Bay, Paraná, Brazil, depicting its

general profile and which environmental factors contribute to its constitution,

such as seasonal changes, the proximity to open sea, the rhizosphere of the

prevalent plants and the physicochemical parameters of the soil.

Financial Support: CAPES




ANTI-METASTATIC AND ANTIINFLAMATORY ACTIVITY OF A L-

GALACTAN TYPE POLYSACCHARIDE ISOLATED FROM A ASCIDIA

STYELA PLICATA

Diana C. Restrepo E.1, Felipe C.O.B. Teixeira2, Eliene O. Kozlowski 2, Jhonny

Colorado Ríos1, Alejandro Martinez1, Mauro S.G. Pavão2

1Universidad de Antioquia, Medellín, Colombia; Research group of Marine

Natural Products, Faculty of Pharmaceutical Chemistry.

2Universidade Federal do Rio de Janeiro, Brazil; Research group of

Biochemistry and Cellular Biology of Glycoconjugates, Biochemistry Institute.

Ascidians (Tunicata) are sessile marine invertebrates. Previous studies have

accessed novel biological activities for a sulfated polysaccharide isolated from

the tunic of the Brazilian ascidian Styela plicata. This carbohydrate is a high

molecular weight 3- -galactopyranosyl. The ability of a

cancer cell to metastasize successfully depends on its individual properties and

the properties of immune, bloodstream and lymphatic cells. It is known that

Heparin treatment attenuates metastasis and inflammation through inhibition of

P-selectin, present on platelets and neutrophil, binding to its ligands on tumor

cells. In this preliminary study, in vitro and in vivo assays were performed to

evaluate the polysaccharide cytotoxicity on LLC cell line, and its anti-

inflammatory and antimetastatic effect with the purpose of finding new therapies

to treat these diseases.

Financial Support: COLCIENCIAS (Administartive Department of Science,

Technology- Colombia)




Β-(1→4)-GALACTAN FROM SICANA ODORIFERA MODULATING TO

MACROPHAGES FOR IMMUNOSSUPRESSOR PROFILE

Abreu, E. C. A.; Noleto, G. R.

Departamento de Bioquímica e Biologia Molecular – Universidade Federal do


Polysaccharides from different plants have great potential of application. The

effects of β-(1→4)-galactan from fruit of S. odorifera on mice peritoneal

macrophages were investigated. The results show that in 24h of incubation

100µg/ml of the polymer reduced the cell viability of macrophages at 20%. In

contraste, an increase of 25% and ~32% with 50 µg/ml and 100 µg/ml,

respectively in 48h was observed. The treated groups with 50 µg/ml and 100

µg/ml of the β-(1→4)-galactan decreased at ~47% and 67%, respectively, the

macrophages number with activation morphologic profile. The phagocytic

activity was reduced in ~50% with 50µg/ml. When the macrophages were

incubated with LPS (100ng/ml) + β-(1→4)-galactan the phagocytosis was

reduced at ~40% and ~33% (50 µg/ml and 100 µg/ml, respectively) in

comparison with the cells treated with only LPS. In the presence of LPS

(100ng/ml) + polymer (50 and 100 µg/ml) during 1h, the superoxide anion

production by macrophages was reduced at 70%). The production of nitric

oxide in 48h by these cells in presence of LPS 100 ng/ml + 50 µg/ml of β-

(1→4)-galactan was reduced in ~20%. The pro- and anti-inflammatory

interleukins production was also evaluated. In 6h of incubation, macrophages

treated with β-(1→4)-galactan (50 µg/ml) increased at ~100% the TNF-α level.

But 100µg/ml of the polymer in 48h reduced at ~95% the IL-1β production. In

the same time of incubation, the β-galactan polymer (100µg/ml) increased at

~57% the IL-10 level. Taken together, the results of this study show that the β-

(1→4)-galactan of S. odorifera trigger an immunossupressor profile.





CHEMICAL MODIFICATION OF AGAROSE FOR THE DEVELOPMENT OF

NEW BIOMATERIALS

Estela M. Aranha, Miguel D. Noseda, Janaina G. Heuke, Diogo R. B. Ducatti,

Alan G. Gonçalves, Maria Eugênia D. Noseda

Laboratório de Glicobiologia Estrutural de Carboidratos - Algas Marinhas

(GLICAM).

Departamento de Bioquímica e Biologia Molecular, Setor de Ciências

Biológicas, Universidade Federal do Paraná, Paraná, Brasil.

The search for new materials from renewable and biodegradable sources has

increased, and red algae polysaccharides, for example agarose, have drawn

attention, due to its wide application and abundance. Inserting good leaving

groups in polysaccharides by semi-synthesis can transform them into potential

precursors in the development of new materials with technological and

pharmaceutical applications, such as biotechnology field. The objective of this

study was the inclusion of tosyl groups in agarose. The synthesis of tosyl

agarose was started by reducing the polysaccharide terminals with NABH4,

followed by tosylation in heterogeneous aqueous medium (yield of 93 and 59%,

respectively). The products were characterized by FT-IR and 1D and 2D NMR

spectroscopy, showing that two positions were substituted by tosyl groups: G-6

of the 3-linked β-D-galactosyl unit (G-2) and position C-2 of the 4-linked 3,6-

anyhdro-α-L-galactosyl unit (LA-2). NMR analysis showed a degree of tosylation

of approximately 2. Therefore the chemical tosylation of this important

commercial polysaccharide was successfully achieved, representing a new

agarose backbone for future applications.




EVALUATION OF THE ANTITUMOR ACTIVITY OF NATIVE AND MODIFIED

SULFATED HETERORHAMNANS OBTAINED FROM THE GREEN

SEAWEED GAYRALIA BRASILIENSIS

Ester Mazepa1; Miguel D. Noseda1; Diogo R.B. Ducatti1; Alan G. Gonçalves1;

Rafaela P. Gomes2; Sheila M.B. Winnischofer2; Maria Eugênia D. Noseda1

1Laboratório de Glicobiologia Estrutural de Carboidratos de Algas Marinhas

(GLICAM), Departamento de Bioquímica e Biologia Molecular, Setor de

Ciências Biológicas, Universidade Federal do Paraná, PR, Brasil

2Laboratório de Cultivo Celular e Oxidações Biológicas, Departamento de

Bioquímica e Biologia Molecular, Setor de Ciências Biológicas, Universidade

Federal do Paraná, PR, Brasil, Universidade Federal do Paraná, Paraná, Brasil.

Sulfated polysaccharides produced by seaweeds have great potential for

antitumoral therapy. Study of structure-activity relationship can provide better

understanding of mechanism actions, and enable chemical modifications

enhancing biological effect. The aim of the present study was to evaluate the

effect of the native and chemically modified sulfated polysaccharides isolated

from a seaweed on the viability of cancer cells. A sulfated heterorhamnan (Gb1,

33.6% NaSO3) was obtained by aqueous extraction (80°C) from the green

seaweed Gayralia brasiliensis. Besides rhamnose (63.3 mol%) it contains

xylose (8.8 mol%), galactose (6.4 mol%), glucose (10.1 mol%), and glucuronic

(8.6 mol%) and galacturonic (2.7 mol%) acids. Gb1 was submitted to chemical

modifications: partial depolimerization and oversulfation. The partially

depolymerized product Gb1-S (51.6% NaSO3) contains rhamnose, glucose,

xylose and galactose (91.5, 5.9, 1.1, 0.5 mol%, respectively). Gb1 was

submitted to chemical sulfation giving rise to an oversulfated polysaccharide

(Gb1-OS, 55.6% NaSO3). 1D and 2D NMR analyses of polysaccharides

showed different proportions of 3-linked, 3-linked-2-sulfated, 3-linked-4-sulfated,

3-linked-2,4-sulfated, 2-linked-4-sulfated, 2-linked-3,4-sulfated and 2,3-

disubstituted rhamnose units. The antitumor effect of Gb1, Gb1-S and Gb1-OS

(at 25, 100 and 500 μg/mL) was evaluated against cancer cells (MDA-MB-435)

by MTT assay. Gb1 at 100 and 500 μg/mL reduced cell viability in 23 and 32%,

Gb1-S at 25 μg/mL reduced 22%, and Gb1-OS at 25, 100 and 500 μg/mL

reduced 35, 47 and 48% of cell viability. The oversulfated polysaccharide

showed the highest effect against MDA-MB 435 cells.




EVALUATION OF SIX METHODS FOR TOTAL DNA EXTRACTION FROM

GUT EARTHWORMS FOR MICROBIAL COMMUNITY DIVERSITY AND

METAGENOMICS

Esther Dering Esteves1, George Gardner Brown2, Fábio de Oliveira Pedrosa 1,

Emanuel Maltempi de Souza1, Leda Satie Chubatsu1

1Universidade Federal do Paraná – Department of Biochemistry and Molecular

Biology.

2Embrapa Floresta Colombo.

DNA extraction is a crucial step for many molecular studies including microbial

community diversity in earthworms gut. A variety of methods have been used

for total DNA extraction from earthworms gut, including commercial kits and

manual methods. Extraction methods need to be evaluated for their efficiency,

as DNA degradation and fragmentation during extraction and other effects. In

this work genomic DNA was extracted from three different regions of the

earthworm gut, species Perionyx excavatus, using six different methods,

including commercial kits and manual procedures using SDS or CTAB . The

extracted DNA samples were compared for both yield and DNA quality.

Samples were also tested for efficient amplification of 16S rRNA by PCR.

Earthworm foregut samples had the lower DNA yield independent of the method

used for DNA extraction. Five of the methods had acceptable yields in DNA

extraction for samples from hindgut. All tested methods led to a fragmented

DNA, but one of the manual methods yield a high molecular weight DNA. Only

samples obtained from two of the manual methods and one of the commercial

kits were successful for DNA amplification to all earthworm gut regions. Results

indicate that three of methods could be used for the extraction of total DNA with

the purpose to analyze the microbial biodiversity of earthworm gut. However,

only one of these methods is suitable for metagenomic studies due to the high

molecular weight DNA obtained.

Financial Support: INCT, CAPES, CNPq, PNPD/CAPES




DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION OF

STAPHYLOCOCCUS AUREUS, STAPHYLOCOCCUS EPIDERMIDIS AND

ESCHERICHIA COLI TO DANOFLOXACINO INJECTABLE SOLUTION IN

THE PRESENCE OF THE DEGRADATION PRODUCT OBTAINED UNDER

PHOTOLYTIC CONDITIONS

Everson Willian Fialho Cordeiro¹,²; Renata Medeiros Hilgert¹; Luiz Alcides das

Chagas Batista¹; Cheila Denise Ottonelli Stopiglia², Clésio Sodatelli Paim¹

¹ Laboratório de Pesquisa em Desenvolvimento e Controle de Qualidade de

Medicamentos - Universidade Federal do Pampa, Uruguaiana-RS.

² Laboratório de Microbiologia - Universidade Federal do Pampa, Uruguaiana-

RS.

Danofloxacin is an antimicrobial chemotherapy for exclusive use in veterinary

medicine. The drug has a broad spectrum of action comprising Gram negative

bacteria, Gram positive and also mycoplasmas of clinical interest. The present

study aimed to determine the minimum inhibitory concentration (MIC) of the

drug and evaluating whether the degradation products, quantified by HPLC,

interfere in the activity of the quantitative in vitro assay. The microdilution assay

was performed according to the protocol M100-S25 CLSI (2015). Thus,

Advocin® injectable solution was diluted in Mueller Hinton culture medium and

performed in 96-well plates to give concentrations between 12.8 and 0.03

μg/mL. New tests were performed after the definition of MIC, to assess whether

the drug showed activity changes when it is exposed to forced degradation

conditions. The degradation study showed that the concentration of drug

reduced significantly with time, reaching 74.05% in the first 48 hours of

exposure to light. The results of the microdilution assay demonstrated that S.

aureus and S. epidermidis showed the same MIC (0.12 μg/mL) and E. coli was

more sensitive to the antimicrobial with a MIC of 0.06 μg/mL. However, assays

using the degradation products against S. epidermidis have shown that they

show antibacterial activity, since they reduced the MIC of 0.12 μg/mL to 0.06

μg/mL. Therefore the study demonstrates important contribution to evaluate the

antimicrobial activity and suggests that the degradation products present in vitro

antimicrobial activity.




ARAUCARIA ANGUSTIFOLIA CULTURES WITH DIFFERENT EMBRYOGENIC POTENTIAL PRESENT A DISTINCTIVE MITOCHONDRIAL

BIOENERGETICS

Fernando Diego Kaziuk, Ana Luiza D. M. Furlanetto, Andre L.W. Dos Santos,Eny I.S. Floh, Fabiane Fortes, Sílvia M. S. C. Cadena.

Departamento de Bioquímica e Biologia Molecular, Universidade Federal doParaná, Curitiba, Paraná, Brasil.

Departamento de Ciências Biológicas, Universidade Estadual do Paraná,Campus União da Vitória, União da Vitória, Paraná, Brasil.

Laboratório de Biologia Celular de Plantas (BIOCEL), Departamento deBotânica, Instituto de Biociências, Universidade de São Paulo, São Paulo,Brasil.

Araucaria angustifolia (Bert.) O. Kuntze is currently classified in the category CR (species critically endangered) by the IUCN red list of threatened species. Therefore, understanding this plant physiology is essential for its preservation. In this study, it was evaluated hot stress (30ºC) effects on A. angustifolia cell lines with different embryogenic abilities, one cell line being responsive to maturation conditions (SE1cell line) and one cell line that presented blocked development of mature somatic embryos (SE6 cell line). The cells were grown at 25 ± 1°C on semi-solid MSG culture medium (20-21 days of culture) in the dark and submitted to hot stress of 30 ± 1°C for 12h, 24h or 48h. The cells viability evaluated by MTT assay was not affect by all stress conditions. However, the viability of SE6 cells line was higher when compared to SE1 cells lines, submitted or not to stress conditions. As MTT method is based on the activity of mitochondrial dehydrogenases, was also evaluated the oxygen consumption (Oroboros 2k - Oxygraph) in mitochondria isolated from these cells not submitted to stress. Interestingly, the rate of oxygen consumption during states 3 and 4 of respiration and the Respiratory Control Coefficient (RCC) were higher in SE6 cells lines. These results suggest that mitochondrial functions linked to energy provision are stimulated in SE6 cells line and motivate further studies to clarify the involvement of this distinctive characteristic on propagation and maturation of embryogenic Araucaria angustifolia cells.





REDUCTIVE-AMINATION OF Κ-CARRAGEENAN HYDROLYSIS PRODUCTS

Franciely G. Colodi; Miguel D. Noseda; Estela M. Aranha; Mariana M. Carvalho;

Diogo R. B. Ducatti; Maria Eugênia D. Noseda.

Laboratório de Glicobiologia Estrutural de Carboidratos de Algas Marinhas

(GLICAM), Departamento de Bioquímica e Biologia Molecular, Universidade

Federal do Paraná, Curitiba, PR, Brazil.

Carrageenans are sulfated polysaccharides produced by red seaweeds that

present large industrial uses and biological interest. κ-Carrageenan consists of

alternating (1→3)-linked β-D-galactopyranose 4-sulfate and (1→4)-linked 3,6-

anhydro-α-D-galactopyranose units. This negative charged polysaccharide has

been used to assemble films for biomimetic surfaces with multiple functionalities

and to delineate protein delivery systems. The aim of this work was the

functionalization of κ-carrageenan hydrolysate products through reductive-

amination. κ-Carrageenan was obtained from red seaweed Kappaphycus

alvarezzi (aqueous extraction for 4 h at 80 °C), submitted to partial hydrolysis

using TFA (0.1 mol/L) at 65 °C for 15 and 35 min and at 80 °C for 180 min,

giving rise to partially depolymerized products K15, K35 and K180, respectively.

Reductive-amination was carried out in methanol (2.5% w/v) with propane-1,3-

diamine (2 eq., pH 5.5) and sodium cyanoborohydride (2.5 eq.). The mixtures

were stirred for 15 h at 55 °C, giving aminated fractions K15a, K35a and K180a.

κ-Carrageenan hydrolysis products K180, K35 and K15 are, respectively,

composed mainly by κ-carrabiose, κ-carratetraose and κ-oligosaccharides

larger than six units. HSQC NMR spectrum of K15a showed signals

corresponding to aminated 3,6-anhydro-α-D-galactopyranosyl units (C-1/H-1 at

57.1/3.50 ppm and C-2/H-2 at 69.1/3.98 ppm). Correlations at 50.0/3.32, 3.35;

32.8/2.78 and 45.0/3.24 ppm were attributed to -CH2- of N-linked propane-1,3-

diamine. Therefore, functionalized k-amino-oligosaccharides with different

degrees of polymerization can be obtained by reductive-amination, providing an

interesting approach to develop novel biomaterials.




SELECTIVE C-6 OXIDATION OF CARRAGEENANS AND EVALUATION OF

THE ANTICOAGULANT PROPERTIES

Gislaine C. dos Santos1, Diogo R. B. Ducatti1, Miguel D. Noseda1, Alan G.

Gonçalves2

1Departamento de Bioquímica, Universidade Federal do Paraná, Curitiba, Brasil

2Departamento de Farmácia, Universidade Federal do Paraná, Curitiba, Brasil

Carrageenans are polysaccharides extracted from extracellular matrix of red

seaweeds that belong to the family of sulfated galactans. They have a broad

spectrum of biological activities, including anticoagulant properties, which are

related to the sulfation pattern, molecular weight and chain conformations.

Chemical modifications of polysaccharides have been reported in order to

improve those properties. The selective C-6 oxidation of polysaccharides can

produce polyuronic acids derivatives with new biological and physical

properties, for example, the gelation, complexation, adhesion as well as a

variety of biological activities such as anticoagulant activity. Selective oxidative

methods using 2,2,6,6-tetramethylpiperidine-1-oxyl free radical (TEMPO) as

catalyst in water have been reported in the literature for several soluble and

insoluble polysaccharides such as starch, maltodextrin, cellulose,

galactomannan, glucan, hyaluronic acid, chitin, chitosan and xanthan in order to

generate polymers with potential biological and biotechnological applications.

Thus, the aim of this work was the selective C-6 oxidation of β-D-Galp units in

kappa-, iota-, iota/nu-, theta e lambda-carrageenan using TEMPO as catalyst

and trichloroisocyanuric acid (TCCA) in a carbonate buffer (pH 9.6). All the

samples were characterized by NMR (1D and 2D). The anticoagulant activity of

native and oxidized fractions was examined by aPTT in vitro.

Financial Support: CAPES, CNPq, Fundação Araucária




SCALE-UP OF BIODIESEL SYNTHESIS IN FIXED BED REACTORS USING

THE FERMENTED SOLID BY BURKHOLDERIA LATA

Dias, Glauco Silva1; Luz Jr., Luiz Fernando de Lima2; Krieger, Nadia3; Mitchell,

David Alexander1

1Laboratório de Tecnologia Enzimática e Biocatálise, Dep. de Bioquímica e

Biologia Molecular, UFPR, PR;

2Dep. de Engenharia Química, UFPR; (3) Dep. de Química, UFPR de

Farmácia, Universidade Federal do Paraná, Curitiba, Brasil

The biodiesel is produced industrially by homogeneous alkaline

transesterification of the triacylglycerols of vegetable oils with methanol. This

process requires steps for the removal from the biodiesel of the catalyst and of

the salt that is formed, for the treatment of the alkaline effluent that is generated

and for the recovery of the glycerol by product that is produced. One possible

strategy for avoiding problems associated with chemical catalysis is to use

lipases as catalysts. Recently, in our laboratory, a process was developed with

the intention of reducing the costs of the enzymatic route. The objective of the

current work was to increase the scale of this biodiesel production process. To

this end, a bioreactor consisting of three packed-beds in series was filled with a

total of 120 g of fermented solid, produced using Burkholderia lata and 1245 g

of a medium comprised of olein and ethanol was recirculated through the

reactor from a reservoir. The best results being a giving 88% conversion in 24

h. This system was operated for six consecutive 48-h cycles. In the first cycle, 1

kg of esters was produced, while the six cycles produced a cumulative total of

4.7 kg of esters, which is equivalent to 39 g of ester produced per g of

fermented solid that was used. These results are promising, since they

demonstrate that high yields can be maintained in the scale-up of enzymatic

biodiesel production processes involving fermented solids as the catalyst.




MONOCLONAL ANTIBODIES DEVELOPMENT FOR ADAM33 AND ITS

CLINICAL APPLICATION

Graciele C. M. Manica1, Marco A. S. de Oliveira2, Andressa Chequin1, Edneia

A. S. Ramos1, Liliane M. B. Klassen1, Mariana Busato Toledo1, Yara de Oliveira

Brandão1, Isis Venturi Biembengut1, Emanuel M. De Souza2, Giseli Klassen1

1 Dep. de Patologia Básica, Laboratório de Epigenética;

2 Dep. de Bioquímica e Biologia Molecular, UFPR, PR, Brasil

The biodiesel is produced industrially by homogeneous alkaline

transesterification of the triacylglycerols of vegetable oils with methanol. This

process requires steps for the removal from the biodiesel of the catalyst and of

the saltADAM33 gene is down-regulated in human breast cancer by promoter

hipermethylation. In order to make a clinical usefull tool, a recombinant

truncated ADAM33 protein containing the cystein rich domain was used to

immunize Balb/c mice and monoclonal antibody anti-ADAM33 was produced

(GMGK06). The specificity of the antibody was tested by western blot (WB) and

Immunocytochemical (IHQ). The WB results showed that GMGK06 has high

specificity for ADAM33 protein since no signal was detected in the MDA-MB-

231, a cell line ADAM33 negative. However, the ADAM33 expressing breast cell

lines MCF7 and PMC42, showed a single signal of approximately 37 kDa. The

antibody was used to test 44 cases of human breast cancer by

immunohistochemical assay. We found three different scores of ADAM33 in

breast cancer samples: 2 (weak), 3 (intermediate) and 4 (strong). Anti-ADAM33

antibodies may be useful in further studies to understand the biological

mechanisms of the breast cancer, furthermore, to evaluate the function of

ADAM33 in normal physiological states, different types of cancer and other

diseases.




ANTICOAGULANT AND ANTITHROMBOTIC ACTIVITY OF NATIVE AND

PARTIALLY DEGRADED GLYCOGLUCURONOMANNAN FROM VOCHYSIA

THYRSOIDEA AFTER CHEMICAL SULFATION

Helyn Priscila de Oliveira Barddal, Ana Helena Pereira Gracher, Fernanda

Fogagnoli Simas-Tosin, Marcello Iacomini, Thales Ricardo Cipriani

Dep. de Bioquímica e Biologia Molecular, UFPR, PR, Brasil

Heparin has great clinical importance as anticoagulant and antithrombotic

agent. However, because of its risks of causing bleeding and contamination by

animal pathogens, several studies aim to obtain alternatives to heparin. In the

search for anticoagulant and antithrombotic agents from a non-animal source, a

glycoglucuronomannan from the gum exudate of the plant Vochysia thyrsoidea

was partially hydrolyzed, and both native and partially degraded

polysaccharides were chemically sulfated, yielding VThS and Ph-VThS

respectively. Methylation analysis indicated that sulfation occurred preferentially

atthe O-5 position of arabinose units in the VThS and at the O-6 position of

mannose units in Ph-VThS. In vitro aPTT assay showed that VThS and Ph-

VThS have anticoagulant activity, which could be controlled by protamine, and

ex vivo aPTT assay demonstrated that Ph-VThS is absorbed by subcutaneous

route. Like heparin, they were able to inhibit α-thrombin and factor Xa by a

serpin-dependent mechanism. In vivo, VThS and Ph-VThS reduced thrombus

formation by approximately 50% at a dose of 40 IU/kg, similarly to heparin. The

results demonstrated that the chemically sulfated polysaccharides are

promising anticoagulant and antithrombotic agents.

Financial Support: CAPES, CNPq, UFPR, Fundação Araucária




THE EFFECTS OF XILOGLUCANA EXTRACTED FROM COPAIFERA

LANGSDORFFII (XGC) AND ITS COMPLEX WITH OXOVANADIUM IV/V

(XGC:VO) ON MITOCHONDRIAL BIOENERGETICS

Fernandes, K.L.M¹; Petkowicz, C. L. O¹; Noleto, G.R¹; Cadena, S.M.S.C¹

¹ Department of Biochemistry and Molecular Biology - Universidade Federal do

Paraná (UFPR), Paraná, Brasil.

It was shown that xiloglucana extracted from Copaifera langsdorffii (XGC) and

its complex with oxovanadium (XGC:VO) were cytotoxic to B16F10 murine

melanoma cells. This effect was related to the impairment of cells respiration. In

this study we evaluated the effects of these polymers on respiration parameters,

using glutamate plus malate as oxidizable substrates, in isolated rat liver

mitochondria. The activities of NADH and succinate oxidases in disrupted

organelle were also evaluated. XGC (0.5-25 μg.mL-1), did not affect the

respiration in intact organelle; however, the polysaccharide reduced the oxygen

consumption in uncoupled mitochondria (0.5 µM of FCCP) by ~30%.

Interestingly, XGC:VO reduced by 13% (0.5-25 μg.mL-1) the respiratory rate of

the state 3 but did not affect the state 4. The inhibition of state 3 resulted in a

decrease also by ~13% of Respiratory Control Coefficient (RCC) while ADP/O

ratio was unchanged. On the uncoupled state, the decrease of oxygen

consumption by XGC:VO was more pronounced (~32%). The activity of NADH

oxidase evaluated by oxygen consumption was not affected by both XGC and

XGC:VO. However, succinate oxidase activity was increased in the presence by

XGC in ~90% already at lower concentrations but, the enzyme activity was

unchanged by XGC:VO. These results suggest that the presence of vanadium

was essential for the inhibition of respiration in intact mitochondria oxidizing

substrates of complex I (glutamate plus malate). On the other hand, the

stimulation of succinate oxidase only for native XGC suggests that this effect is

abolished by the metal presence.




STRUCTURAL CHARACTERIZATION AND STUDY OF THE ANTI-

INFLAMMATORY POTENTIAL OF THE POLYSACCHARIDES OF

CABERNET FRANC, CABERNET SAUVIGNON AND SAUVIGNON BLANC

WINES

Iglesias de Lacerda Bezerra, Adriana Rute Cordeiro Caillot, Arquimedes Paixão

Santana Filho, Guilherme Lanzi Sassaki

Department of Biochemistry and Molecular Biology - Universidade Federal do

Paraná (UFPR), Paraná, Brasil.

Introduction: There are few works about characterization of polysaccharides of

wines. The structure and amounts of polysaccharides released depend on the

wine-making process and can influence the sensory properties and quality of

the wines. This work aimed structural characterization of the polysaccharides

found in three types of wines: Cabernet Franc (WCF), Cabernet Sauvignon

(WCS) and Sauvignon Blanc (WSB). Material and methods: The wines were

concentrated and the polysaccharides were obtained via ethanolic precipitation

followed centrifugation, dialysis and freeze dry. The polysaccharides were

analyzed by nuclear magnetic resonance. Monosaccharide composition was

determined after total hydrolysis with TFA 100°C/14h by quantitative-HSQC.

Homogeneity analyses were performed by HPSEC-MALLS. The anti-

inflammatory potential of the polysaccharides through inhibition of NF-Kβ in

Raw blue cells Results: Polysaccharide yields were: 1.5% (WCF), 0.5% (WCS)

and 0.2% (WSB). WCF showed Gal-(32.1%), Ara-(31.4%), Rha-(9.1%), GalA-

(9.2%), Glc-(10.6%) and Man-(6.3%); WCS showed Gal-(18.6%), Ara-(15.1%),

Rha-(9.2%), GalA-(12.0%), Glc-(18.7% ) and Man-(26.4%); and WSB showed

Gal-(19.1%), Ara-(19.2%), Rha-(4.7%), GalA-(4.1%), Glc-(12.7% ) and Man-

(40.2%). The total uronic acids content was determined for WCF, WCS and

WSB, giving rise to 15.2%, 16.4% and 8.6%, respectively. All the samples

showed a heterogeneous elution profile, suggesting the presence of

polysaccharide mixture. HSQC-NMR spectroscopy indicated the presence at

least four polysaccharides in all samples: An arabinogalactan type II, type I

rhamnogalacturanan, dextrin, and mannan. All the simples inhibited of NF-Kβ in

vitro. Conclusion: The results suggest that the HSQC NMR of the

polysaccharides can furnish a fingerprint for each wine, since the profile of the

mixtures had different yields and quantities, aiding for a non-volatile based

singular signature. As also, the simples showed anti-inflammatory potential, but

they will still be made other experiments to prove these results.




INTRAGENIC DNA METHYLATION REGULATES MMP9 GENE

EXPRESSION IN BREAST CANCER

Liliane M. B. Klassen1, Graciele C. M. Manica1, Isis V. Biembengut1, Edneia A.

S. Ramos1, Andressa Chequin1, Isis, Mariana B. Toledo1, Yara de O Brandão1,

Emanuel M. de Souza2, Giseli Klassen1

1Department of Basic Pathology, Federal University ofParana.

2Department of Biochemistry and Molecular Biology, Federal University of

Parana.

Breast cancer is the most frequently diagnosed cancer and the leading cause of

cancer death among women worldwide. Metastasis remains a major challenge

for the clinical management and prognosis of patients with cancer. The

metalloprotease MMP-9 play a critical role in the first step of metastasis through

extracellular matrix degradation. In cancer from breast, cervical, prostate and

many others MMP-9 have been correlated with poor prognosis. The MMP9

gene could be regulated by epigenetic mechanisms such as histone

modifications however DNA methylation remains to be further clarified. This

study starts with an in silico study of MMP9 gene showed two intragenic CpG

islands. Our goal was verify the effect of these DNA region in MMP9 gene

expression. The quantification in breast cancer cell lines with or without

decitabine (DAC) treatment showed that MCF7 and MDAMB436 expressed

MMP9 only after demethylating agent treatment. The sequencing data obtained

from the two DNA regions with high CpG content (CpG island) showed one

specific sequence with 273 pb between CpGs 11 and 29 in the second CpG

island differentially methylated. This specific region was studied in breast

cancer samples that reveal the similar results with demethylation in positive

MMP-9 samples. Taken together these results showed a new possible

mechanism of DNA methylation and gene expression regulation since no

description of intragenic DNA region was showed until now.




MICROPARTICLES OF CACAO POD HUSKS PECTIN AND 5-ASA: A

PRELIMINARY INVESTIGATION

Vriesmann, L.C.1; Franco, C.R.C.2; Lucinda-Silva, R.M.3 Petkowicz, C.L.O.1

1Departamento de Bioquímica e Biologia Molecular, UFPR, Curitiba-PR, Brazil

1Departamento de Biologia Celular, UFPR, Curitiba-PR, Brazil2

3Departamento de Ciências Farmacêuticas, UNIVALI, Itajaí-SC, Brazil

Pectins are polymers from plant cell wall widely used as gelling and stabilizing

agents in the food industry. Recently, they are suggested to the development of

colon-specific therapeutic systems. In this work, we employed a highly

acetylated HM pectin (OP) from cacao pod husks to prepare microparticles

containing 5-ASA, a drug employed in inflammatory bowel disease. The

microparticles were obtained by spray drying, using solutions of pectin and 5-

ASA in the proportions 1:1; 2:1; 3:1 and 4:1. Sample 1:1 has the higher yield

(68%) and 86% of drug incorporation efficiency. This efficiency was improved

when the proportion of pectin increased, with sample 4:1 reaching 98%

efficiency. From MEV, the microparticles presented <5 micrometers and an

irregular surface, more pronounced in sample 1:1. Liberation assays were

performed simulating the gastrointestinal transit. Although more investigation is

necessary to improve the drug protection in the gastric pH, the obtained results

indicate that this pectin showed better tendency of drug retention when its

content is increased, being a promissory polymer to be studied as an adjuvant

in systems containing drugs directed to colon liberation or action.




UNRAVELLING THE TRANSCRIPTIONAL REGULATORY NETWORK OF

THREE FNR PROTEINS FROM HERBASPIRILLUM SEROPEDICAE SMR1

USING CHIP-SEQ AND RNA-SEQ

Marcelo Bueno Batista1,2, Govind Chandra2, Emanuel Maltempi de Souza1,

Rose Adele Monteiro1, Ray Dixon2

1Department of Biochemistry and Molecular Biology, Universidade Federal do

Parana, P.O. Box 19046, Curitiba, PR 81531-990, Brazil

2Department of Molecular Microbiology, John Innes Centre, Colney Lane,

Norwich NR4 7UH, UK

H. seropedicae SmR1 is an endophytic aerobic bacterium capable of fixing

atmospheric nitrogen under conditions of nitrogen and oxygen limitation. To

efficiently adapt to low O2 levels, H. seropedicae, as in the case of other

bacteria, takes advantage of a branched respiratory chain comprising different

types of terminal oxidases, including oxidases predicted to have high affinity for

oxygen. Remarkably this organism has genes coding for three Fnr proteins,

designated as Fnr1, Fnr2 and Fnr3. Using genome-wide transcriptional

profiling in combination with physiological characterisation, we previously

observed that efficient reconfiguration of the respiratory chain in H. seropedicae

under low oxygen availability relies on transcriptional regulation of gene

expression by these Fnr proteins. However, we were not able to define the

specific regulons and functional roles of each of the three Fnr transcription

factors. In this study we have used a combination of RNA-Seq transcriptional

profiling of single fnr mutants together with a ChIP-Seq approach to address the

functions of three Fnr orthologs encoded by H. seropedicae genome. Although

Fnr1 and Fnr3 regulate discrete classes of genes, we have identified another

group of genes that are jointly regulated by both transcription factors.

Promoters in this class are bound by both Fnr1 and Fnr3, potentially indicative

of regulation by Fnr1-Fnr3 heterodimers. In contrast Fnr2 is apparently an

oxygen insensitive protein responsible for the transcriptional activation of the

bo3-type respiratory oxidase.




IDENTIFICATION OF AN EXOGLUCANASE OBTAINED FROM

METAGENOMIC LIBRARY OF BRAZILIAN ATLANTIC FOREST SOIL

Marcelo Scarduelli, Bruno Afonso Ramos Cassilha, Helisson Faoro, Fábio de

Oliveira Pedrosa, Luciano Fernandes Huergo e Emanuel Maltempi de Souza

Departamento de Bioquímica e Biologia Molecular- Universidade Federal do

Paraná

Cellulases have many industrials uses, from polishing fabrics to biofuel

production, especially second generation bioethanol. The group of cellulases

include endoglucanases, exoglucanases and beta-glucosidases. Searching for

new cellulases, prospection in metagenomic libraries could lead to discover

novel enzymes. This technique allows to capture all the genetic material in a

sample, covering nucleotidic sequences both cultivable and uncultivable

species in laboratory conditions. This study aimed to identify a

celulase/exoglucanase in metagenomic library of Brazilian Atlantic Forest soil.

This library was previously constructed. Sequence comparison was used for

identification of cellulases. Selected genes were cloned and overexpressed in

E. coli. Purification was performed by Ni2+ affinity chromatography (HiTrap

Chelating, GE Healthcare). The enzymatic activities were calculated with DNS

assay (3 5-dinitrosalicylic acid). After comparison analysis, one gene was

identified as exoglucanase, with 66 kDa and conserved domain for family 9

glycosyl hydrolases. This gene showed low identity with submitted genes: 70%

of identity with a hypothetical protein from Segetibacter koreensis or 66% with

glycosyl hydrolase from Chitinophaga pinensis. Preliminary results showed

enzymatic activity only in high level of NaCl (1 and 2 M), when using

regenerated amorphous cellulose (RAC) as substrate. Others enzymatic assays

will be made in order to quantify the cellulase activity and confirm the

halotolerance of this exoglucanase.




PERIODATE OXIDATION OF ULVANS FOR INTRODUCTION OF NEW

REACTIVE GROUPS

Mariana M. de Carvalho1, Miguel D. Noseda1, Franciely G. Colodi1, Rilton A. de

Freitas2, Maria Eugênia D. Noseda1

1Laboratório de Glicobiologia Estrutural de Carboidratos de Algas Marinhas

(GLICAM), Departamento de Bioquímica e Biologia Molecular, Universidade

Federal do Paraná, Paraná, Brasil.

2Biopol – Departamento de Química, Universidade Federal do Paraná, Paraná,

Brasil.

Green seaweeds from Ulva spp. biosynthesize sulphated polysaccharides

named ulvan. Considering they are still unexplored, chemical modifications may

increase their possible applications. The aim of this study was to introduce

aldehyde groups in ulvans by periodate oxidation, originating new reactive

groups for chemical modifications. Ulvans from Ulva fasciata were obtained by

aqueous extraction (80 ºC) resulting in F2 (20.9% SO3Na, 16.0% uronic acids),

which according to monosaccharide composition and NMR analyses (13C, 1H

and HSQC), was built up of the following diads: [→4)-β-D-glcpA-(1→4)-α-L-

rhap3S-(1→], [→4)-β-D-xylp-(1→4)-α-L-rhap3S-(1→], [→4)-β-D-xylp2S-(1→4)-

α-L-rhap3S-(1→] and ([→4)-α-L-idopA-(1→4)-α-L-rhap3S-(1→]). F2 was

submitted to periodate oxidation for 24, 48 and 72 h resulting in the partially

oxidized polysaccharide fractions F2-A, F2-A1 and F2-A2 respectively.

Oxidation was confirmed by FTIR (aldehyde bands at 1750 cm-1) and 13C

NMR (hemicetals at 90-91 ppm). Moreover in the 13C spectra, the decrease in

the C1 signals of [→4)-β-D-xylp-(1→] (104.8 ppm) and ([→4)-α-L-idopA-(1→]

(104.1 ppm) indicates that these units were, as expected, oxidized. Units with a

lack of vicinal hydroxyls such as ([→4)-β-D-xylp2S-(1→] and [→4)-α-L-rhap3S-

(1→) were not oxidized. Noteworthy, the units of [→4)-β-D-glcpA-(1→] were not

oxidized, although they contain vicinal hydroxyls. This may occur when the unit

adopts a conformation that prevents periodate oxidation or by the formation of

hydrogen bonds between COOH at C6 and OH at C3/C4. These results indicate

that the introduction of aldehydes groups was successful, giving rise to

aldheydic ulvans. Chemical modifications from periodate oxidized ulvans are in

progress. Physical-chemical properties and biological activities of these new

polysaccharides are under study.





NTRYX AND NTRBC SYSTEMS ARE REQUIRED TO INDUCE GENES

ENCODING THE ASSIMILATORY NITRATE REDUCTASE IN

HERBASPIRILLUM SEROPEDICAE SMR1

Bonato, P.1; Tadra-Sfeir, M. Z.1; Camilios-Neto, D.2; Wassem, R.1; Rigo, L.u.1;

Pedrosa, F. O.1; Souza, E.M.1; Chubatsu, L.S.1

1Núcleo de Fixação de Nitrogênio, Departamento de Bioquímica e Biologia

Molecular, Universidade Federal do Paraná;

2Departamento de Bioquímica e Biotecnologia, Universidade Estadual de

Londrina

Nitrate is the main inorganic source of nitrogen found in soil. The diazotrophic

and plant-associative Herbaspirillum seropedicae SmR1 has genes encoding

for two nitrate reductases: the assimilatory (NAS) and the respiratory nitrate

reductases (NAR). The ntrY and ntrC mutant strains do not grow on nitrate as

the only nitrogen source suggesting that the NtrYX and NtrBC systems are

required to induce genes important for nitrate assimilation. Given that, H.

seropedicae wild type, and ntrY and ntrC mutant strains were cultivated

aerobically in low nitrogen (1 mM NH4Cl) until cells reached OD600 0.4, then

cells were divided in three parts: one stored in RNAlater and the other two

incubated for more 30 minutes after addition of 10 mM KNO3 or 10 mM NH4Cl.

Samples from the three bacterial strains in these three different conditions were

submitted to RNA extraction, cDNA libraries construction and DNA sequencing

using an Ion Proton Sequencer. Reads were mapped against the H.

seropedicae genome using CLC Genomics Workbench 7.0 and differentially

expressed genes were analysed using DESeq 2.0. The

narKnirBDHSERO_RS14545nasA operon, which encodes the NAS system,

showed the highest induction rate after nitrate addition in the wild type strain,

when compared to ammonium or to low nitrogen conditions. In contrast these

genes were downregulated in both ntrY and ntrC mutant strains cultivated with

nitrate. These results were validated with lacZ reporter fusions. Together these

results indicate that NtrYX and NtrBC systems are required to activate the

narKnirBDHSERO_RS14545nasA operon.




MODIFICATION ON THE 2’-DEOXYGUANOSINE OXIDATION BY SINGLET

MOLECULAR OXYGEN PATHWAY BY GLUTATHIONE

Patrícia S. Peres1, Andressa Valerio1, Silvia M. S. C. Cadena1, Sheila M. B.

Winnischofer1, Alexsandra C. Scalfo2, Paolo Di Mascio2, Glaucia R. Martinez1

1Laboratório de Oxidações Biológicas, Departamento de Bioquímica e Biologia

Molecular, Setor de Ciências Biológicas, Universidade Federal do Paraná

(UFPR), Curitiba, PR, Brazil.

2Departamento de Bioquímica, Instituto de Química, Universidade de São

Paulo, São Paulo,SP,Brazil.

The oxidation of the free nucleoside 2’-deoxyguanosine (dGuo) by singlet

molecular oxygen (1O2) has been studied over the three last decades due to the

major role of DNA oxidation products in process such as ageing, mutation and

carcinogenesis. In the present work we investigated the dGuo oxidation by 1O2

in the presence of the important low molecular antioxidant, glutathione, in its

reduced (GSH) and oxidized (GSSG) forms. There were applied different

conditions of concentration, pH, time of incubation, and the use of a [18O]-

labeled thermolabile endoperoxide naphthalene derivative as a source of [18O]-

labeled 1O2. Data was obtained through high performance liquid

chromatography (HPLC) and HPLC coupled to micrOTOFQ-II analysis of the

main oxidation products: the diastereomers of spiroiminodihydantoin-2′-

deoxyribonucleosides (dSp) and 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-

oxodGuo). An intriguing result was that 8-oxodGuo levels increased by 100 fold

when dGuo was oxidized by 1O2 in the presence of GSH and by 2 fold in the

presence of GSSG, while dSp levels dropped to zero for both conditions. All

data from dGuo, 8-oxodGuo and dSp quantification together with the analysis of

residual GSH/GSSG content in each sample strongly suggest that glutathione

modifies the mechanism of dGuo oxidation by 1O2 by disfavoring the pathway of

dSp formation.




INFLUENCE OF MOLAR MASS AND CONCENTRATION ON THE

THERMOGELATION AND INTERFACIAL PROPERTIES OF

METHYLCELLULOSES

Pauline L. Nasatto 1,2, Joana L. M. Silveira1, Frédéric Pignon2, Marguerite

Rinaudo3, Miguel D. Noseda1, Maria Eugênia R. Duarte1.

1Laboratório de Glicobiologia Estrutural de Carboidratos de Algas Marinhas,

Departamento de Bioquímica e Biologia Molecular, Federal University of

Paraná, Curitiba, Paraná, Brazil.

2Laboratoire Réologie et Procedes, Université Grenoble Alpes, Grenoble,

France.

3Biomaterials Applications, Grenoble, France.

Four methylcelluloses having the same average degree of substitution and

distribution of methyl groups, but different molar masses were investigated, their

thermogelation correlation with the molar mass and concentration in aqueous

medium, as well their interfacial interactions, at room temperature and at very

low polymer concentrations were studied. Heating process was specially

studied to analyse the two steps of gelation using rheometry, a large hysteresis

between heating and cooling ramps was observed whatever the conditions. At

low temperature, in the sol state, viscosity depends on the concentration and

molar mass. Over 30 °C a gel like behaviours was observed including two steps

(the second step is a strong gel with phase separation) having storage moduli

which are nearly independent of polymer molar mass but directly related to

polymer concentration. The surface tension (σ) at the water/air interface was

determined for the progressive addition of methylcellulose up to 100 mg/L; σ

starts to decrease over 1 mg/L up to the critical aggregation concentration

(CAC) at 10 mg/L. Curves describing the influence of polymer concentration on

σ are independent of the molar mass at equilibrium. The adsorption of

methylcellulose on silica particles was estimated from ζ-potential

measurements. Those results were interpreted in terms of an increase of the

adsorbed layer thickness at the interface when the molar mass of

methylcellulose increases. It was demonstrated that methylcellulose is

adsorbed, forming trains and loops, at the interface based on the equilibrium

between surface free energy and solvent quality.




EVALUATION OF CYTOTOXICITY OF ZINC PHTHALOCYANINE LOADED

IN SUPERPARAMAGNETIC POLY(METHYL METHACRYLATE)

NANOPARTÍCLES VIA PHOTODYNAMIC THERAPY

Paulo Emilio Feuser1, Eduardo Ricci-Júnior2, Claudia Sayer1and Pedro

Henrique H. de Araújo1

1Department of Chemical Engineering and Food Engineering, Federal

University of Santa Catarina, Brazil

2Faculty of Pharmacy, Federal University of Rio Janeiro, Brazil

Superparamagnetic nanoparticles (MNPs) are promising materials for

hyperthermia treatment and magnetic targeting systems. PDT is a current

therapy that involves the administration of a non-toxic dye and the activation of

photosensitizers with visible light. The aim of this work was the simultaneous

encapsulation of magnetic nanoparticles (MNPs) and zinc (II) phthalocyanine

(ZnPc) in poly(methyl methacrylate) (PMMA) nanoparticles (NPs) by

miniemulsion polymerization and to evaluate the photobiological activity against

murine fibroblast (L929) and human lung adenocarcinoma epithelial cells

(A549). NPs presented an average diameter of 104 ±2.5 nm with a

polydispersity index (PdI) of 0.14 ±0.03 and negative surface charge - 47 ± 2.2

mV (pH 7.4 ±0.1). The release of ZnPc from PMMA NPs was slow and

sustained without the presence of burst effect, indicating a homogeneous

distribution of the drug in the polymeric matrix. NPs did not present any

cytotoxicity effect on L929 cells, after activation with visible light at 675 nm.

However, NPs showed considerable cytotoxic effect on A549 cells only after

activation with visible light at 675 nm photodynamic therapy (PDT). The

preparation of ZnPc loaded in superparamagnetic PMMA NPs with sustained

release can be a new alternative for cancer treatment leading to significant

tumor regression after minimum doses light with a targeted drug delivery.




EVALUATION OF B16-F10 CELL PROLIFERATION UNDER

MELANOGENESIS STIMULUS IN THE PRESENCE OF ANTIOXIDANT N-

ACETYLCYSTEINE

Paulo Szwarc, Tassiele A. Heinrich, and Glaucia R. Martinez

Departamento de Bioquímica e Biologia Molecular, Setor de Ciências

Biológicas, Universidade Federal do Paraná, Curitiba, PR, Brazil

Melanoma is a skin tumor with high pigmentation (melanin). Previous results by

our group have shown that B16-F10 cells (murine melanoma model) with

induced melanogenesis feature increased levels of reactive oxigen species

(ROS) and changes in metabolism, citoskeleton and celular resistance proteins,

cell cycle halt at G1 and fluroescent marking with Pyronin-Y, suggesting that

induced melanogenesis promotes entry into a quiescent state as a protection

mechanism against increased melanin production. This study's main objective

was to evaluate cell proliferation on B16-F10 cells in the presence of antioxidant

N-acetylcysteine (NAC), aiming to better understand the role that ROS plays in

relation to melanogenesis-induced decreased cell proliferation. The cells were

cultured, plated and treated with RPMI 1640 medium supplemented with L-

tyrosine and NH4Cl to induce melanogenesis, and also received NAC

treatment. Cell count was done using an hemocytometer between 12 h intervals

for 48 h, and the results were plotted in a cell growth graph. A decrease in

melanogenesis-induced cell proliferation was observed in comparison to non-

induced cells, and melanogenesis-induced cells that received NAC treatment

had higher numbers of cells than the control. This highlight a possible

involvement of ROS in melanogenesis-induced decreased cell proliferation.




STRUCTURAL CHARACTERIZATION OF A TYPE II ARABINOGALACTAN

FROM CHAMOMILLA RECUTITA [L.]RAUSCHERT INFUSION

Chaves, Pedro Felipe Pereira; Iacomini, Marcello; Cordeiro, Lucimara Mach

Côrtes

Biochemistry and Molecular Biology Department of Federal University of Paraná

Chamomile (Chamomilla recutita [L.] Rauschert) is one of most commonly used

species in phytotherapy and is included in the pharmacopoeia of almost all

countries. Some of the known pharmacological effects are attributed to the

presence of secondary metabolites, but it is not known whether other molecules

such as polysaccharides, are working together to these effects. The chemical

structure of polysaccharides have direct relationship with the biological activities

and elucidate this characteristics is of paramount importance. There are few

studies on the chemical structure of chamomile polysaccharides. Therefore, the

present study aimed to structurally characterize a type II arabinogactan

obtained from chamomile infusion. After a series of ultrafiltrations, a Fehling

precipitation, a enzyme treatment and a elution in chromatographic column a

fraction was obtained and analyzed by monosaccharide composition,

methylation and HSQC/DEPT. The mainly monosaccharides observed were

arabinose and galactose. The major methylated derivative observed was

corresponding of 3,6-O-substituted galactose units. Other methylated

derivatives observed, were corresponding to galactose (terminal 6-O, 3-O and

2,3,6-tri-O-substituted), to arabinose (terminal 5-O- and 3,5-di-O-substituted)

and terminal glucuronic acid units. The HSQC/DEPT spectrum showed signals

assigned to the C1/H1 of α-L-Araf-(1→, →5)-α-L-Araf-(1→, β-D-Galp-(1→, →3)-

β-D-Galp-(1→/→3,6)-β-D-Galp-(1→ e →6)-β-D-Galp-(1→ units, to C6/H6

substituted of the β-D-Galp units, to C5/H5 substituted of the α-L-Araf units and

to free C6/H6 and C5/H5 of the α-L-Araf-(1→, β-D-Galp-(1→ and →3)-β-D-

Galp-(1 units. In conclusion, a type II arabinogalactan that the main chain

consists of (1 → 3)-linked galactose units 6-O-substituted by side chains was

isolated from the chamomile infusion.




STEADY AND DYNAMIC SHEAR RHEOLOGICAL PROPERTIES

OFGABIROBA PULP

(CAMPOMANESIA XANTHOCARPA BERG)

Barbieri, Shayla F. 1; Petkowicz, Carmen L. O. 1; Ruthes, Andrea C.2; De

Godoy, Rossana C. B.3; Silveira, Joana L. M. 1

1Biochemistry and Molecular Biology Department, Federal University of Paraná,

CEP 81.531-980, Curitiba-PR, Brazil

2Division of Glycoscience, Royal Institute of Technology - KTH, Sweden

3Empresa Brasileira de Pesquisa Agropecuária - Embrapa Florestas, CEP

83.411-000, Colombo-PR, Brazil

Gabiroba (Campomanesia xanthocarpa Berg) is a Brazilian native fruit. Its pulp

presented 82 ± 0.8% of moisture content, while different polysaccharides:

pectin, hemicellulose and cellulose compose 17 ± 0.8% of dry weight.

Monosaccharide composition of pectin fractions showed mainly arabinose (Ara

40-60%) and galacturonic acid (GalA 20-42%). The rheological properties of

gabiroba pulp were evaluated by steady-state shear experiments where pulp

exhibited a non-Newtonian pseudoplastic behavior and also showed a yield

stress minimum to initiate the flow related to the material’s internal structure

which must be broken. The presence of a yield stress is a typical characteristic

of multiphase materials as fruit pulps and juices, which are formed by a

dispersion of insoluble components. In dynamic rheological analysis, the

gabiroba pulp presents gel behavior (frequencies 0.01-100 Hz, at 25°C).

Thermal stability as a gel behavior was observed for the gabiroba pulp at

temperatures from 5-95°C, 1 Hz. This stability is suitable for use of the pulp in

food formulations, such as the production of jelly.

Financial Support: Rede Nanoglicobiotecnologia MCT/CNPQ, Pronex

Carboidratos, CNPq




CONSTRUCTION AND CHARACTERIZATION OF A PILT MUTANT IN

HERBASPIRILLUM SEROPEDICAE SMR1

Vanessa Kessler Chicora, Vânia Carla Silva Pankievicz, Eduardo Balsanelli,

Rose Adele Monteiro, Emanuel Maltempi de Souza, Fábio de Oliveira Pedrosae

Leonardo Magalhães Cruz

Departamento de Bioquímica e Biologia Molecular, Universidade Federal do

Paraná

Herbaspirillum genus is mainly known for its capacity to colonize roots, stems

and leaves of grasses and fix nitrogen. The molecular mechanisms involved in

Herbaspirillum bacteria-plant association are still poorly understood, but

homologous genes to type III secretion system (T3SS), type IV pili (T4P),

adhesins/hemagglutinin and genes involved in lipopolysaccharide (LPS) and

exopolysaccharide (EPS) synthesis were identified in the genome of

Herbaspirillum seropedicae SmR1. Some of these mechanisms have been

elucidated in Herbaspirillum spp. and this work aims to unravel the role of type

IV pili of H. seropedicae motility and plant-bacteria interaction. Previous

transcriptomic analysis confirmed type IV pili genes induction in H. seropedicae

associated with roots of maize and wheat, including pilT gene. The pilT gene,

encoding an ATPase able to retract the pili and develop twitching motility, was

deleted in H. seropedicae. Twenty-nine genes homologous to the pil genes

were identified and are distributed on eight different regions of the genome. The

H. seropedicae PilT protein has four conserved motifs and sequences of the

common amino acids in this protein, however, the C-terminal domain did not

show high degree of conservation. Petri dishes tests with different agar

concentrations was performed to verify how the twitching motility would be

affected. Mutants deleted pilT gene shows growth similar to the wild strain in

1.5% agar, however, in 0.175% agar the mutants showed a greater spread than

wild strain, which can influence the association of bacteria with the plant.

Financial Support: CNPq, INCT




ESTROGEN RECEPTOR α GENE (ESR1) EXPRESSION EVALUATION IN

TWO HISTOLOGICAL TYPES OF CANINE MAMMARY TUMOR BY qPCR

Yara de Oliveira Brandão¹, Mariana Busato Toledo¹, Andressa Chequin¹,

Graciele Cristiane Moré Manica¹, Liliane Maria Bacaro Klassen¹, Thierry Grima

de Cristo², Renato Silva de Sousa², Ednéia Amancio de Souza Ramos

Cavalieri¹, Giseli Klassen¹

¹ Laboratório de Epigenética, Departamento de Patologia, Setor de Ciências

Biológicas- Universidade Federal do Paraná

²Laboratório de Patologia Veterinária- Hospital Veterinário- Universidade

Federal do Paraná

Mammary cancer is the mainly neoplasia found in non-spayed bitches Although

the estrogen receptor α (ERα) is one of the molecular marks for breast cancer

in women, it still has a few disagreements about its expression relevance in

canine mammary tumor. In this study the RNA of eight canine mammary

tumors, of which three tubulopapillary carcinoma and five solid carcinoma, was

extracted and used in cDNA synthesis. The transcript quantification was made

by qPCR technique, following the SYBR®Green protocol. The housekeeping

gene was RPS18. Even though solid adenocarcinoma is commonly related to

higher malignance neoplasia degree and many studies have found, by

immunohistochemical technique, lower ERα expression in more aggressive

tumors the qPCR results of this study didn’t show statistical significant

difference in ESR1 expression between both histological types tumors

evaluated. These finds agree with it was previously described in literature

regarding canine mammary adenoma and carcinoma. Once qPCR reaction was

standardized, it is proposed to increase the samples amount and include benign

tumors and non-neoplastic mamma.




CLATHRIN LIGHT CHAIN IN TRYPANOSOMA CRUZI: GENE

IDENTIFICATION AND SUBCELLULAR LOCALIZATION

Ligia Cristina Kalb, Yohana Camila Antunes Frederico, Cassiano Martin Batista,

Iriane Eger, Stênio Perdigão Fragoso and Maurilio José Soares

Laboratory of Cell Biology, Carlos Chagas Institute, Fiocruz-PR

Laboratory of Molecular Biology of Trypanosomatids, Carlos Chagas Institute,

Fiocruz-PR

Department of General Biology, State University of Ponta Grossa

Clathrin-mediated vesicular trafficking, the mechanism by which proteins and

lipids are transported between membrane-bound organelles, accounts for a

large proportion of import from the plasma membrane (endocytosis) and

transport from the trans-Golgi network towards the endosomal system. Clathrin-

mediated events are still poorly understood in the protozoan Trypanosoma

cruzi, the causative agent of Chagas disease in Latin America. In this study,

Clathrin light (TcCLC) chain gene expression and protein localization were

investigated in different developmental forms of T. cruzi (epimastigotes,

trypomastigotes and amastigotes), using polyclonal antibodies raised against T.

cruzi recombinant proteins.

Analysis by confocal microscopy revealed an accumulation of TcCLC at the cell

anterior, where the flagellar pocket and Golgi complex are located. TcCLC

partially colocalized with the Golgi marker TcRAB7-GFP and with ingested

albumin, but did not colocalize with transferrin, a protein mostly ingested via

uncoated vesicles at the cytostome/cytopharynx complex.

In conclusion, Clathrin light chain is expressed in T. cruzi. The protein typically

localize anterior to the kinetoplast, at the flagellar pocket and Golgi complex

region. Our data also indicate that in T. cruzi epimastigotes clathrin-mediated

endocytosis of albumin occurs at the flagellar pocket, while clathrin-independent

endocytosis of transferrin occurs at the cytostome/cytopharynx complex.

Financial Support: CAPES, CNPq, FUNDAÇÃO ARAUCÁRIA, FIOCRUZ




ANTICOAGULANT ACTIVITY OF FUCOGALACTAN SULFATED FROM

AGARICUS BISPORUS. OPTIMIZATION OF CHEMICAL SULFATION AND

STRUCTURAL CHARACTERIZATION

Yony Román, Thales R. Cipriani, Marcello Iacomini, Guillerme L. Sassaki

Department of Biochemistry and Molecular Biology, Federal University of

Paraná, Paraná, Brazil

Introduction: Mushrooms have been valued by human kind as an edible and

medical resource containing a number of bioactive molecules with therapeutic

properties, including polysaccharides. Several studies have shown the

anticoagulant activity of sulfated polysaccharides. The common mushroom

Agaricus bisporus is a good source of polysaccharides which can be chemically

sulfated to present anticoagulant activity. Objective: To optimize the chemical

sulfation of a fucogalactan from A. bisporus in order to obtain an anticoagulant

agent.Materials and methods: The fucogalactan was purified from an aqueous

extract of A. bisporus, and characterized by methylation, NMR and HPSEC-

MALLS analyses. It was sulfated in function of different factors [time, molar ratio

of ClSO3H to OH of the polysaccharide (ηClSO3H:ηOH), and weight ratio of

polysaccharide to total reaction volume (Wp/VT)]. The Degree of Sulfation (DS)

was measured and the anticoagulant activity was evaluated by Activated Partial

Thromboplastin Time (APTT) and Prothrombin Time (PT). Discussion and

results: The best anticoagulant activity was obtained with the sulfated

fucogalactan synthesized with a ηClSO3H:ηOH of 18:1 and a Wp/VT of 1:100 in

6 hours of reaction, named E100.. The results of anticoagulant activity of E100

showed a linear increment of APTT for concentrations of 15 to 45 µg.mL-1,

whereas PT was constant between 120 and 160 µg.mL-1. The NMR analyses

suggest that non-reducing end-units of α-L-Fucp and α-D-Galp were greatly

replaced by sulfate groups.Conclusions: E100 inhibited especially the intrinsic

pathway of blood coagulation. Different sulfation conditions generated different

sulfated polysaccharides, which varied at DS and anticoagulant activity.





DEEP EUTECTIC SOLVENTS AS NEW MEDIA FOR BIOCATALYSIS:

EXAMPLE OF LIPASE-CATALYZED SYNTHESIS OF PHENOLIPIDS

Erwann Durand

Cirad (La recherche agronomique pour le développement) - Montpellier, França

With the recent interest on green chemistry, the scientists have focused on

developing new and moreefficient solvents to carry out enzymatic-catalyzed

reactions with emphasis on reduced costs, risks and toxicity whileimproving

biodegradability. Among the new available solvents, the multimolecular-based

liquids (such as ionic liquidsand (natural) eutectic solvents) have been the

subject of most recent studies. Currently, and mainly due to its environmental

andeconomic features, (natural) eutectic solvents (NA)DES are arousing much

interest and curiosity. Regarding the biotransformations with lipases, theso-

called “lipophilization” reactions (grafting of lipid moiety onto a hydrophilic

molecule) are of major interest. Indeed, most of the bioactive molecules (e.g.

phenolic acids) express their functionalproperties in a hydrophilic environment,

resulting in a few efficientand advanced applications in formulated-lipid

dispersions.In that sense, lipophilization are particularly advantageousand

effective, and may be seen as a vectorizationkey unlocking the lipid barrier

encountered by the bioactivemolecule while maintaining its original functional

properties.In the case of phenolic acids, the methods commonly used consistin

attaching on the reactive carboxyl group, either a singleor double tail lipophilic

domain (usually an aliphatic witha different carbon backbone) resulting in new

molecule called “phenolipid” withemulsifying properties and often improved

antioxidantactivity.Although these reactions haveshowed tremendous potential,

theyare still complex to implement because of the difficulty in findinga suitable

reactionmedium.Hereby, we propose new perspectivesfor the enzymatic

modification of such polar substrates using the novel generation of green and

inexpensive easy-to-handle (NA)DES.




TARGETINGRESISTANT CANCER CELLS OVEREXPRESSING

MULTIDRUG ABC TRANSPORTERS WITH SELECTIVE DRUG-EFFLUX

INHIBITORS AND APOPTOSIS INDUCERS

Di Pietro, Attilio

BMSSI UMR5086 CNRS-University of Lyon,Institute of Protein Biology and

Chemistry, Passage du Vercors 7, 69367 Lyon, France

MultidrugABC (“ATP-binding cassette”) transporters overexpressed in

chemoresistanttumors are pumping anticancer drugsout of the cells. Totarget

the“breast cancer resistance protein”ABCG2, we have screened different series

of flavonoids and derivatives, such as chalcones,chromones,and

indenoindoles,as inhibitors of mitoxantrone efflux from transfected HEK293

human cells and chemosensitizers of cell proliferation. Two types of selective

and non-competitive inhibitorshave been characterized, either inhibiting or

stimulatingATPase activity.The most potent onewas efficient in vivo on SCID

mice, xenografted with human ABCG2-transfected cells, by chemosensitizing

tumor growth to thedrug-substrateirinotecan. These selective inhibitors

constitute good drug candidates, with low intrinsic toxicity, as sensitizers of cell

proliferation to conventional chemotherapeutics. The “Multidrug Resistance

Protein 1” ABCC1 is able tocatalyze the efflux of glutathione conjugates, or the

co-transport of free glutathione and hydrophobic substrate drugs.Modulators

such as verapamil mimick substratesandinduce a fast and massive efflux of

intracellular glutathione from ABCC1-overexpressing cells,leading to selective

cell death through apoptosis, due to“collateral sensitivity”, or

hypersensitivity.The overexpressed transporter then constitutes the

Achilles’heel of resistant cancer cells. Verapamil being known for cadiotoxic

effects, othermodulatorssuch as xanthones, flavones and flavonoid dimers were

investigated. Glutathione efflux appeared to be necessary, but not sufficient

alone, to trigger apoptosis, indicating the contribution of other partner(s) or

signaling pathway(s). Such apoptosis inducers may constitute a new type of

anticancer drugs operating through an original strategy aimed at selectively

targeting and eliminating multidrug-resistant tumors overexpressing the ABCC1

transporter.




IN SEARCH OF A MODEL SYSTEM TO STUDY ASSOCIATIVE,BIOLOGICAL

NITROGEN FIXATION

Vania C. S. Pankievicz1, Fernanda P. Amaral2, Beverly Agtuca1, Karina

Santos2, Abigail Ferrieri3, Richard Ferrieri4, Maria Berenice R. Steffens1, Ana

Carolina M. Arisi5,Emanuel de Souza1, Fabio Pedrosa1 and Gary Stacey2

1Federal University of Parana, Curitiba, Brazil; 2University of Missouri, Divisions

of Pant Sciences and Biochemistry, C.S. Bond Life Science Building, Columbia,

MO, USA;3Environmental and Molecular Science Laboratory, Pacific Northwest

National Laboratory, Richland, WA, USA;4Biosciences Department, Brookhaven

National Laboratory, Upton, NY 11973, USA; 5University of Santa Catarina -

UFSC, Dept. of Genetic Plant Resources, Florianopolis, Brazil

The world is facing a growing challenge with increasing population,

changing climate and a finite supply of arable land. Hence, perhaps no time in

our history has there been a greater need to invest in agricultural research to

develop sustainable cropping systems to meet the rising demand for food.

Nitrogen is an essential nutrient forplant growth. However, industrial production

of nitrogen fertilizerutilizes fossil fuel with the potential to negatively impact the

environment (e.g., through nitrogen runoff). Legumes and some grasses have

the ability to obtain nitrogen through biological nitrogen fixation (BNF) in

association with soil bacteria. For example, Azospirillumbrasilenseand

Herbaspirillumseropedicaeare examples of diazotrophicbacteria that associate

with several grasses and have been shown to release nitrogen to their host as a

result of BNF. However, it remains controversial whether biological nitrogen

fixation (BNF) contributes significantly to the ability of these bacteria to

promoteplant growth. We believe that this controversy can only be addressed

by more mechanistic studies that would be facilitated by adoption of a useful

plant model system. Our collaboration is focused on analyzing model grass

species for their suitability to support such mechanistic studies. For example,

we recently demonstrated that Setariaviridis, a model C4 grass,isstrongly

colonized by nitrogen fixing bacteria resulting in a significant increase of plant

growth, even under nitrogen limitation. Evidence of nitrogen incorporation by

plant was provided through13NN tracer studies. We were able to follow the

incorporation of 13NN by BNF into plant protein, demonstrating direct, plantuse

of the fixed nitrogen. The use of a genetically tractable, grass model system

should facilitate mechanistic studies of BNF in grass species;hopefully




contributing to the development of technologies to alleviate the need for

industrial nitrogen fertilizers in a moresustainable and environmentally friendly

cropping system.




UPDATE THE RESEARCH IN CARBOHYDRATE CHEMISTRY IN THE

DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY UFPR

Marcello Iacomini

Department of Biochemistry and Molecular Biology UFPR

Research in Carbohydrate Chemistry in Brazil has few groups. The group

of the Department of Biochemistry and Molecular Biology at the Federal

University of Paraná, probably is the most important group developing this type

of research in our country. Research on polysaccharides started in the 60s in

the last century. Gradually this group has improved the research, precisely

because of the arrival of new researchers have come very important to

strengthen the group and also this kind of research. Precisely because new

lines of research were introduced and with it numerous scientific papers were

published in international journals as well as numerous dissertations and theses

have been produced ever-increasing number and especially in research quality.

All research lines generated scientific production and also with high quality

science. Naturally the number of Master's and PhD students ever increasing

over the decades until the present day in this way, were responsible for the

scientific production of Carbohydrate Chemistry Group. In the last two decades

in addition to the Master's students and PhD to postgraduate absorbed

numerous post docs and today in significant numbers are absorbed by the

groups in the most diverse research areas, bringing a high level of research and

thus the results are observed by the originating publications with the

participation of these new researchers. In this way the results achieved by the

Carbohydrate Chemistry Graduate in Biochemistry and Molecular Biology a

promising future in relation to the quality of the science that has been

developed.




ENDOCRINE FUNCTION OF FIBROBLAST GROWTH FACTOR 21 (FGF21)

EXPRESSED IN THE LIVER IN HYPERCALORIC DIET-INDUCED OBESITY

Fabiana Rodrigues Silva Gasparin1, Juliana Moraes Mewes1, Eduardo Hideo

Gilglione1, Clairce Salgueiro Pagadigorria1, Karina Sayuri Utsunomya, Amanda

Tomie Ouchida2, Ingrid C. Gaemers3, Ronald P.J. Oude Elferink3, Jorgete

Constantin1 and Emy Luiza Ishii-Iwamoto1*

1Department of Biochemistry, Laboratory of Liver Metabolism, University of

Maringá, Maringá, Paraná, Brazil

2Laboratory of Bioenergetics, Faculty of Pharmaceutical Sciences, University of

São Paulo, Ribeirão Preto, São Paulo, Brazil.

3Tytgat Institute for Liver and Intestinal Research, Academic Medical Center,

University of Amsterdam, Amsterdam, The Netherlands.

Fibroblast growth factor 21 (FGF21) is an atypical member of the FGF21

superfamily that functions as an endocrine hormone. FGF21 is expressed in

liver, brown (BAT) and white (WAT) adipose tissue and pancreas. The

circulating FGF21 is derived mainly from the liver and coordinates a systemic

response to nutritional stress. In this report, we reported a sex-dependent

difference in hepatic FGF21 expression in response to cafeteria diet-induced

obesity. Swiss mice of both sexes were fed a standard diet or a cafeteria diet

for 14 weeks. Body weight gain, adiposity, liver lipid content, serum levels of

FGF21 and expression of several genes related to lipid and glucose metabolism

and antioxidant defense system were measured. In response to cafeteria diet,

males exhibited greater increases in FGF21 expression in the liver, WAT and

BAT, and a higher serum level of FGF21 compared with females. Metabolic

disturbances and lipotoxicity were found only in females, which exhibited

increased liver fat accumulation (steatosis) and increased cellular oxidative

stress. The analysis of the expression of transcript factors and key enzymes

indicated that the steatosis in females was consequence of metabolic changes

in peripheral tissues. An increased expression of receptor 1 for FGF21 in BAT

was found only in males along with increased expression of uncoupling protein-

1 (UCP1) and fat-specific protein 27 (FSFP). These findings indicated

that BAT is an important target for FGF21 action. These data suggested a

central role of hepatokine FGF21 in protecting the males, but not the females,

against steatosis and lipotoxicity-associated disorders in the cafeteria diet-

induced obesity.





REGULATION OF ALTERNATIVE NITROGENASE BIOSYNTHESIS IN

AZOTOBACTERVINELANDII

Ray Dixon

Department of Molecular Microbiology, John Innes Centre, Norwich Research

Park, Norwich, NR4 7UH UK

Azotobactervinelandiiis one of the few diazotrophs that can express three

different types of nitrogenase dependent upon metal availability, namely the

conventional molybdenum enzyme (encoded by nifgenes), the vanadium

nitrogenase (encoded by the vnfgene cluster) and the iron-only nitrogenase

(encoded by the anf gene cluster). In addition to the three specific activators,

NifA, VnfA and AnfA, required for transcriptional activation of the molybdenum,

vanadium and iron-only nitrogenase operons respectively, the A.vinelandii

genome encodes an additional homolog of NifA, designated NifA2 and two

homologs of VnfA, designated as VnfA2 and VnfA3. Each of these activator

homologs has a regulatory GAF domain, which in the case of the VnfA

homologs contains conserved cysteine residues thought to bind an iron-sulphur

cluster. A model to explain the complex hierarchy of metal-dependent gene

regulation mediated by the VnfA homologs will be discussed in this talk.Due to

the complexities of metal-dependent regulation and gene redundancy in A.

vinelandii, it has been difficult to determine the precise genetic requirements for

alternative nitrogen fixation. In this study we have utilized Escherichia coli as a

chassis to build an artificial iron-only (Anf) nitrogenase system comprised of

defined anf and nif genes. Using this system, we demonstrate that the pathway

for biosynthesis of the iron-only co-factor (FeFe-co) is likely to be more simple

than the Mo-dependent (FeMo-co) equivalent. This minimal Anf system has

potential implications for engineering diazotrophy in eukaryotes, particularly in

compartments (e.g. organelles) where molybdenum may be limiting.




THE LONG AND SUGARY ROAD: STRUCTURAL DIVERSITY AND

BIOLOGICAL SIGNIFICANCE OF GLYCOSPHINGOLIPIDS IN PATHOGENIC

AND OPPORTUNISTIC FUNGI

Helio K. Takahashi and Anita H. Straus

Division of Glycoconjugate ImmunochemistryDepartment of Biochemistry

Escola Paulista de MedicinaUniversidade Federal de Sao Paulo

Glycosphingo lipids (GSLs) are ubiquitous membrane components and

have key roles in biological systems, acting as second messengers or

modulators of signal transduction by affecting several cell events, ranging from

cell adhesion, cell growth, cell motility, regulation of apoptosis and cell cycle.

Over the last 20 years our laboratory and other research groups elucidated the

glycan and ceramide structures of more than 20 GSLs from several

pathogenic/opportunistic fungi and parasites, using a combination of nuclear

magnetic resonance, gas chromatography, mass spectrometry, as well as other

chemical, biochemical and immunochemical techniques. Fungal GSLs can be

divided in two major classes: neutral GSLs, e.g., galactosyl- and

glucosylceramide, and acidic GSLs, the glycosylinositol-phosphorylceramides

(GIPCs). Glycan structures in fungal GIPCs exhibited significant structural

diversity and distinct composition when compared to mammalian GSLs, e.g.,

the expression of inositol-mannose and inositol-glucosamine cores and the

terminal residue of β-D-galactofuranose which are absent in mammalian cells.

Studies performed by our group demonstrated that GIPC

(Galfβ6[Manα3]Manα2InsPCer) elicited in patients with paracoccidioidomycosis

an immune response with production of antibodies directed to the terminal

residue of β-D-galactofuranose. Further studies also showed that inhibition of

glucosylceramide (GlcCer) biosynthetic pathways affects fungal colony

formation, spore germination and hyphal growth, indicating that enzymes

involved in GlcCer biosynthesis may represent promising targets for the therapy

of fungal infections. Recently, it was shown that GlcCer and GIPCs are

preferentially localized in membrane microdomains and monoclonal antibodies

directed to these GSLs interfere in several fungal biological processes such as

growth and morphological transition. An in-depth knowledge of fungal

microdomain interactions in combination with the elucidation of the concerted

action of membrane microdomain, GSL and cell wall will certainly help to




elaborate a more refined concept of key events related to survival and

proliferation of parasite and pathogenic/opportunistic fungi in the human host.




ANTI-TUMOR COMPOUNDS: DIFFERENT APPROACHES TO INVESTIGATE

THE MECHANISMS OF ACTION

Maria Eliane Merlin Rocha, Silvia Maria Suter Correa Cadena, Glaucia Regina

Martinez, Guilhermina Rodrigues Noleto, andSheila Maria Brochado

Winnischofer

Department of Biochemistry and Molecular Molecular Biology, UFPR

Cancer treatments include surgery radiation, chemotherapy, hormone

therapy, immune therapy, and targeted therapy. Furthermore, the existing

pharmacological-based treatments are insufficiently effective and generate

many side effects. It is necessary new drugs to treatment of cancer. In the

Center of Studies in Bioenergetics and Biochemistry of Drugs and Xenobiotics

we study molecular mechanisms of action of flavonoids, mesoionic compounds,

statins and other xenobiotics in tumor various cell lines. New drugs studies of

structure-activity relationship and pro-oxidants on tumor cells effects are

investigated. Involvement of mitochondrial pathway on antitumor derivatives

1,3,4 thiadiazoles mesoionic; polysaccharides effects on modulation of immune

system are other focus of studies. Photodynamic action using dyes and UV

radiation to verify DNA damage promoved by singlet oxygen in melanoma cells

and analyses of role of the RECK tumor suppressor gene and their alternative

transcripts are verified too. Compounds studied can induce different cell

responses as cell death; increase of ROS levels; senescence; autophagy;

alteration of cell cycle progression; inhibition of cellular migration or are

imunomodulators. Furthermore, we have been studying the inhibition produced

by flavonoids on drug efflux by ABCG2.

Financial Support: CNPq, CAPES, INCT-REDOXOMA and Fundação Araucária




FORMATION OF PROTEIN PARTICLES AND THEIR INFLUENCE ON THE

STRUCTURE AND RHEOLOGY OF POLYSACCHARIDE SOLUTIONS AND

GELS

Bach T. Nguyen, Christophe Chassenieux, Lazhar Benyahia, Taco Nicolai

LUNAM, Université du Maine, IMMM UMR CNRS 6283, PCI, 72085 Le Mans

cedex 9, France

β-lactoglobulin (β-lg) is a globular protein and is the major protein

component of whey. β-lg aggregates irreversibly when heated and gels above a

critical concentration. In a narrow range of conditions heated β-lg forms

spherical microgels the size of which can be controlled by the pH and the

concentrations of added CaCl2. Here I will discuss how the presence of protein

microgels influences the behaviour of the polysaccharide -carrageenan (κ-car).

κ-car gels reversibly below a critical temperature that can be reduced by adding

small amounts of specific ions such as K+. κ-car is sometimes added to milk

based products in order to improve the texture. The aggregation and gelation of

both κ-car and β-lg are very sensitive to the presence of calcium ions that are

most often present in the food products. Therefore it is important to understand

the effect of Ca2+ on gelation of mixtures. The effect of adding CaCl2 to mixtures

of κ-car and β-lg microgels at neutral pH on the morphology and the elasticity is

presented. The β-lg microgels were formed by heating either before or after

mixing with κ-car. It will be shown that both β-lg and κ-car specifically bind Ca2+

and that in mixtures they compete for Ca2+. This influences aggregation and

gelling of each biopolymer. In addition, thermodynamic incompatibility drives

micro phase separation above a critical κ-car concentration with β-lg microgels.

An attempt is made to disentangle the effects of competition for Ca2+ and

thermodynamic incompatibility on the structure and the elasticity of the

mixtures.




GLYCOCONJUGATES FROM PATHOGENIC FUNGI

Mariana I. D. S. Xisto1, Vera C. B. Bittencourt2, Livia C. Liporagi-Lopes3, Rosa

M. T. Haido2, MorenaS. A. Mendonça 4, Guilherme Sassaki5, Rodrigo T.

Figueiredo6 , Maria Teresa V. Romanos 7 and Eliana Barreto-Bergter1

1Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de

Góes – UFRJ, Rio de Janeiro, Rio de Janeiro, Brazil

2Departamento de Microbiologia e Parasitologia, Instituto Biomédico, UNIRIO

3Departamento de Análises Clínicas e Toxicológicas, Faculdade de Farmácia -

UFRJ, Rio de Janeiro, Rio de Janeiro, Brazil

4Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, RJ

5Departamento de Bioquímica e Biologia Molecular, Universidade Federal do

Paraná, Curitiba, Paraná.

6Campus de Xerém, Universidade Federal do Rio de Janeiro, Instituto de

Ciências Biomédicas, Universidade Federal do Rio de Janeiro

7Departamento de Virologia, Instituto de Microbiologia Paulo de Góes – UFRJ,

Rio de Janeiro, Rio de Janeiro, Brazil

A peptidorhamnomannan (PRM) that consists of a peptide chain

substituted both by O- and N-linked glycans was isolated from the cell wall of S.

prolificans, an emerging opportunistic fungus, with remarkable resistance to

antifungal agents and able to cause localized infection in immune competent

patients and disseminated infection with high rate of mortality among immune

compromised patients. Its chemical structure was elucidated using methylation

analysis and 13C - nuclear magnetic resonance. The importanceofO-

linkedoligosaccharidespresent in peptidorhamnomannan (PRM)

fromthecellwallof the fungusScedosporiumprolificans for

recognitionandphagocytosisofconidiabymacrophageswasanalyzed.Adding PRM

ledto a dose-dependentinhibitionofconidiaphagocytosis, whereas de-O-

glycosylated PRM didnot show anyeffect. PRM inducedthe release

ofmacrophage-derivedantimicrobialcompounds. However, O-

linkedoligosaccharides do notappeartoberequired for such induction. The

effectof PRM onconidia-

inducedmacrophagekillingwasexaminedusinglatexbeadscoatedwith PRM or de-




O-glycosylated PRM. A decrease in macrophageviability similar

tothatcausedbyconidiawasdetected. However,

macrophagekillingwasunaffectedwhenbeadscoatedwith de-O-glycosylated PRM

wereused, indicatingthetoxiceffectofO-linkedoligosaccharidesonmacrophages.

In addition, PRM triggered TNF release bymacrophages. ChemicalremovalofO-

linkedoligosaccharidesfrom PRM abolishedcytokineinduction,

suggestingthattheO-linkedoligosaccharidicchains are importantmoietiesinvolved

in inflammatory responses throughtheinductionof TNF-α secretion. In summary,

we show thatO-glycosylation plays a role in therecognitionanduptakeofS.

prolificansbymacrophages, killingofmacrophagesandproductionofpro-

inflammatorycytokines.

Financial Support: CNPq, CAPES, FAPERJ, UFRJ




SEARCHING FOR TARGET MOLECULES FOR DIAGNOSTICS AND

THERAPEUTICS OF DEGENERATIVE DISEASES (CANCER, DIABETES)

AND REGENERATIVE MEDICINE

Marina Trombetta-Lima, Fernando Henrique Lojudice, Aline Maia Lobba, Ana

Claudia Oliveira Carreira, Sheila Maria Brochado Winnischofer, Renato Astorino

Filho, Túlio Felipe Pereira, Patricia Mayumi Kossugue, Fernando Janczur

Velloso, Raquel Arminda Carvalho Machado, Marluce da Cunha Mantovani,

Suely Kazue Marie, Maria Lucia Correa Giannella, Mari Cleide Sogayar

NUCEL/NETCEM Cell and Molecular Therapy Center, Internal Medicine Dept.,

Medical School, University of São Paulo, São Paulo, 055360-130 SP, Brazil

The incidence of degenerative diseases, such as cancer and diabetes

mellitus, has been increasing at alarming rates due, mainly, to greater life

expectancy, population ageing and obesity. Therefore, tools for early diagnosis

and efficient therapeutics are essential.We focused on the deadliest tumorof the

central nervous system, namely: Glioblastoma (GBM), using both a rat glioma

cellular model and human tumor samples to search for genes involved in the

action of chemotherapeutics (glucocorticoids-GCs) and in chemoresistance to

these drugs, and, also, on the role played by the RECK tumor and metastasis

suppressor gene alternatively spliced variants, finding important correlations

with patients survival rates and prognosis.Straighforward, uncontroversial,

molecular markers for mammary carcinoma are still lacking, but the recent

tumor stem cell theory provided some insights that led us to find two different

stem cell markers with high diagnostic and therapeutic potential for these

tumors.Insulin-dependent or type 1 diabetes mellitus arises from the auto-

immune destruction of insulin-producing pancreatic beta-cells. Exogenous

insulin replacement does not perfectly mimick the physiological blood glycemic

control, leading to renal failure, cardio-vascular diseases, blindness and other

problems. Whole pancreas and pancreatic islet transplantation are possible

therapeutic options, however, both require immunossupression of the patients.

Islet encapsulation, stem cell differentiation into insulin producing cells and

Tissue Bioengineering are promising new alternatives arising from

Regenerative Medicine, which is based on: a) isolated and well-characterized

stem cells; b) recombinant peptide growth factors; c) extracellular matrix

scaffolds. Personalized therapeutics, tissue repair/reconstruction and organs




replacement, employing different cell types, growth factors and 3D printers are

concrete perspectives for the near future.

Financial Support: BNDES, CNPq, CAPES, FAPESP, MCTI, MS-DECIT




BIOLOGICAL NITROGEN FIXATION- A FRUITFUL IDEA

José Ivo Baldani

Embrapa Agrobiologia, Km 07, 23891-000 – Seropédica, RJ

Biological nitrogen fixation (BNF) is a natural process realized by a small

group of Prokaryotes that lives in terrestrial and aquatic environment. Their

contribution to the global nitrogen cycle vary from 100 to 300 million tons N/year

with about 1/3 derived from terrestrial microorganisms and the rest from the

oceans. The main sources of terrestrial contribution are from the association of

rhizobia with legumes and of associative/endophytic bacteria with

graminaceous plants. Soybean is the best example of the symbiosis mainly in

Brazil where all the nitrogen required for high yield comes from the BNF and

provides an economy of about U$ 10 billion with N fertilizer. Other legumes

such as common beans and cowpea are also benefited from the symbiosis with

rhizobia including the new species such as Microvirga vignae described

recently. In addition, the recovery of mine areas using the tripartite symbiosis

(legume tree, rhizobia and micorrhizae) reinforce the importance of the

biological nitrogen fixation to the environment. On the other hand, the BNF

contribution of the associative/endophytic association is lower; nevertheless, the

economy with N fertilizer application is quite substantial such it was

demonstrated for sugarcane. In contrast, the BNF contribution to cereals (rice,

sorghum, wheat and corn) does not meet the N requirement despite the

diversity of nitrogen–fixing bacterial species (Gluconactobacter diazotrophicus,

Azospirillum lipofereum, A. brasilense, Nitrospirillum amazonense,

Herbaspirillum seropedicae and Burkholderia tropica ) that have been isolated

and tested in association with these cereals. Therefore, strains more efficient

and competitive should be selected including other additional characteristics (N-

use efficiency, phytohormones and siderophere production, Pi and Zn

solubilization, etc). About 3,000 strains, phenotypically characterized as rhizobia

and associative/endophytic bacteria, are deposited at the Embrapa Agrobiology

Culture Collection and now are molecularly and physiologically characterized

envisaging the creation of the Genetic Resource Center-CRB Johanna

Döbereiner at Embrapa and their bioprospection for agriculture application.

Financial Support: CNPq, CAPES, FAPERJ and Embrapa.




FROM STRUCTURE TO CATALYSIS: RECENT ADVANCES IN THE

BIOTECHNOLOGICAL APPLICATIONS OF LIPASES

Erika C. G. Aguieiras and Denise M. G. Freire

Lipases are highly appreciated as biocatalysts due to their peculiar

characteristics such as the ability to utilize a wide range of substrates, high

activity and stability in organic solvents, and regio- and/or enantioselectivity.

These biocatalysts account for 5% of the global market for industrial enzymes

and can be applied in a variety of biotechnological processes, including food

modification, detergent formulation, cosmetics, pharmaceutical, leather, textile,

and paper industries, biodiesel and biopolymer synthesis, or pretreatment of

lipid-rich wastewaters. However, in certain segments of industry such as

biodiesel production and treatment of wastewaters, the use of lipases is still

limited by their high cost. Thus, there is a great interest in obtaining low- cost,

highly active, and stable lipases that can be applied in several different

industrial branches. The use of low-cost enzyme preparations obtained by solid

state fermentation (SSF) and application of the solid enzymatic preparation

(SEP) is an alternative for reduction of costs with extraction, purification and

enzyme immobilization. In addition, by SSF is possible to remove toxic and/or

anti-nutritional compounds and to enhance the nutritional value of the fermented

solids through protein enrichment of the residues, that can be potential

substitutes for animal feed components. Moreover, currently the design of

specific enzymes for each type of process has been used as an important tool

to address the limitations of natural enzymes. Nowadays, with the progress in

protein engineering and structure-based rational design it is possible to “order”

a “customized” lipase that has ideal properties for the development of the

desired bioprocess.




AN OMICS PERSPECTIVE OF BENEFICIAL CEREAL-DIAZOTROPHIC

BACTERIA INTERACTIONS

Emanuel Maltempi de Souza

Núcleo de Fixação de Nitrogênio

Department of Biochemistry and Molecular Biology, UFPR.

Nitrogen is main factor limiting plant growth and productivity. However,

increasing amounts of nitrogen fertilizers cannot be indefinitely used to meet the

demandfor world food production due to high costs and environmental impacts.

Two major groups of diazotrophic bacteria associate closely with plants and can

transfer fixed nitrogen. The symbiotic organisms that form differentiated

structures on plant roots and the associative bacteria that colonize roots both

epiphytically and endophytically. Both groups can promote plant growth and

improve productivity in a sustainable way. The mechanism through which

symbiotic association is formed and the exchange of nitrogen and carbon

between plant and bacteria is well studied, and this group of bacteria is largely

used in agriculture. On the other hand, the molecular mechanisms of plant

recognition, colonization and nutrient exchange between diazotrophic

epiphytes/endophytes and plants are hardly known. Azospirillumbrasilenseand

Herbaspirillumseropedicae are promising plant growth promoting bacteria

capable of colonizing roots of important cereals. We used RNA-Seq

transcriptional profiling and proteomics to further the understanding of the

association of wheat, maize and rice with these bacteria. Genes

codingplantdefence proteins, nutrient uptake andsecondary metabolites were

differentially expressed in response to the bacteriam while the bacteria express

nitrogenfixation,cellmotilityandcellwallbiogenesis genes. These studies

represent an advancement in the understanding of the diazotrophic bacteria-

cereals interaction.

Financial Support: CNPQ/INCT-FBN, CAPES and Fundação Araucária




NEW LIPASES FROM METAGENOMIC LIBRARIES: FROM

CHARACTERIZATION TO THE DEVELOPMENT OF APPLICATIONS

Nadia Krieger

Department of Biochemistry and Molecular Biology, UFPR

In recent years, the application of traditional methods for the isolation and cultivation of microorganisms in samples collected from the environment has resulted in a high rate or rediscovery of known enzymes that have already been well characterized. These enzymes do not necessarily have characteristics suitable for use in reaction media containing organic solvents that are often used in biocatalysis. On the other hand, since metagenomic prospection allows access to bacteria that are not cultivatable using traditional techniques, it has a good chance of identifying new enzymes. The metagenomic technique consists of the extraction of DNA fragments from samples collected from the environment, with the construction of a library of clones, called a “metagenomic library”. The cloned genes are sequenced and, in the case of enzymes, screened for activity and the ability to maintain stability under the proposed reaction conditions. This talk will show how we have used the metagenomic technique to isolate new lipases with interesting characteristics that may allow their use in the organic reaction media and, consequently, in biocatalytic processes.




ÍNDICE DE AUTORES

Abreu, E.C.A. 13

Agtuca, B. 44

Aguieras, E.C.G. 58

Amaral, F.P. 44

Aranha, E.M. 14; 19

Araújo, P.H.H. 34

Arisi, A.C.M. 44

Baldani, J.I. 57

Balsanelli, E. 11; 38

Barbieri, S F. 37

Barddal, H.P.O. 23

Bark, J.M. 10

Barreto-Bergter, E. 53

Batista, C.M.40

Batista, L.A.C. 17

Batista, M.B. 28

Benyhmia, L. 52

Bezerra, I.L. 6; 25

Biembengut, I.V 22; 26

Bittencourt, V.C.B. 53

Bonato, P. 31

Brandão, Y. O. 8; 22; 26; 39

Brandt, A.P. 9

Brown, G.G. 16

Cadena, S.M.S.C. 9; 18; 24; 32; 51

Caillot, A. R. C.25

Camilios-Neto, D. 31

Canuto, V.C.9

Carreira, A.C.O.

Carvalho, M.M. 19; 30

Cassilha, B.A.R. 29

Cavalieri, E.A.R.39

Chandra, G. 28

Chassenieux, C. 52

Chaves, P.F.P.36

Chequin, A. 22; 26; 39

Chicora, V.K. 38;

Chubatsu, L.S. 16; 31

Cipriani, T.R. 7; 23; 41

Colodi, F.G. 19; 30

Constantin, A. 47

Cordeiro L.M.C. 7; 36

Cordeiro, E.W.F. 17

Cristo, T.G.C.39

Cruz, L.M. 38

Cruz, O.M. 10




De Godoy, R. C. B. 37

Di Mascio, P. 32

Di Pietro, A. 43

Dias, G.S. 21

Dixon, R. 28; 48

Ducatti, D.R.B. 14; 15; 19; 20

Durand, E. 42

Echevarria, A.9

Eger, I. 40

Elferink, R.P.J.O. 47

Esteves, E.D.16

Faoro, H.29

Fernandes, K.L.M. 24

Ferrieri, A. 44

Ferrieri, R. 44

Feuser, P.E. 34

Figueiredo, R.T. 53

Filho, R.A.

Floh, E.I.S. 18

Fortes, F. 18

Fragoso, E.P. 40

Franco, C.R.C. 27

Frederico, Y.C.A. 40

Freire, D.M. 58

Freitas, R.A. 30

Furlanetto, A.L.D.M. 18

Gaemers, I.C. 47

Gasparin, F.R.S. 47

Gianella, M.L.C. 55

Gilglione, E.H. 47

Gonçalves, A.G. 14; 15; 20

Gracher, A.H.P. 23

Haido, R.M.T. 53

Heinrich,T.A. 35

Heuke, J.G. 14

Hilgert, R.M. 17

Huergo, L.F. 29

Iacomini, M.7; 23; 36; 41; 46

Ishii-Iwamoto, E.L 47

Kalb, L.C. 40

Kaziuk, F.D.18

Klassen, G. 8; 22; 26; 39

Klassen, L. M. B. 8; 22; 26; 39

Kossugue, P.M. 55

Kozlowski, E.O. 12

Krieger, N. 21; 60

Lammel, D.R. 11

Lana, P.C. 11

Liporagi-Lopes, L.C. 53

Lobba, A.M. 55




Lojudice, F.H. 55

Lucinda-Silva, R.M. 27

Luz Jr., L.F.L. 21

Machado, A.C. 55

Manica, G. C. M. 8; 22; 26; 39

Mantovani, M.C. 55

Marie, S.K. 55

Marinez, A. 12

Martinez, G.R. 10; 32; 35; 51

Mazepa, E. 15

Mendonça, M.S.A. 53

Mewes, J.M. 47

Mitchell, D.A. 21

Monteiro R.A. 28; 38

Nassato, P.L. 33

Nguyen, B.T 52

Nicolai, T. 52

Noleto, G.R. 13; 24; 51

Noseda, M.D. 14; 15; 19; 20; 30; 33

Noseda, M.E.R.D. 14; 15; 19, 30; 33

Oliveira A.F. 7

Oliveira, M.A.S. 22

Ouchida, A.T. 47

Pagadigorria, C.S. 47

Paim, C.S. 17

Pankievicz, V. 38; 44

Pavao, M.S.G. 12

Pedrosa, F.O. 11; 16; 29; 31; 38; 44

Pereira, T.F. 55

Peres, P.S. 32

Petkowicz, C.L.O. 24; 27; 37

Pignon, F. 33

Pino-Gomes, R. 10; 15

Pires, A. R. A. 9

Ramos, E. A. S. 8; 22; 26; 39

Restrepo, D.C. 12

Ribeiro, C.S.P. 10

Ricci-Júnior, E. 34

Rigo, L. 31

Rinaudo, M. 33

Rios, J.C. 12

Rocha, M.E.M. 51

Román, Y. 41;

Romanos, M.T.V. 53

Ruthes, A.C. 37

Santana-Filho, A. P. 6; 25

Santos, A.L.W. 18

Santos, G.C.S. 20

Santos, K. 44

Sassaki, G. L. 6; 25; 41; 53




Sayer, C. 34

Scalfo, A.C. 32

Scarduelli, M. 29

Seccon, D.M. 11

Silveira, J.L.M. 33; 37

Simas-Tosin, F.F. 23

Soares, M.J. 40

Sogayar, M.C. 55

Sousa, R.S.39

Souza, E. M. 8; 11; 16; 22; 26; 28; 29;

31; 38; 44; 59

Stacey, G. 44

Stefens, M.B.R. 44

Stopiglia, C.D.O. 17

Straus, A.M. 49

Szwarc, P. 35

Tadra-Sfeir, M. Z. 11; 31

Tagahashi, M.K 49

Teixeira, F.C.O.B. 12

Toledo, M. B. 8; 22; 26; 39

Trombetta-Lima, M.

Utsunomya, K.S. 47

Valerio, A. 32

Velloso, F.J. 55

Vriesmann, L.C. 27

Wassem, R. 11; 31

Winnischofer, H. 10

Winnischofer, S.M.B. 10; 15; 32; 51; 55

Xisto, M. 53




ANEXO

PROGRAMAÇÃO DO EVENTO

Quarta-feira – 23 de setembro de 2015

18:00 Abertura e Coquetel

Local: Teatro da Reitoria - UFPR

Rua XV de Novembro, 1299 – Centro - Curitiba – PR

***

LOCAL dos Simpósios e cerimônia de encerramento: Auditório do Setor

de Ciências Sociais Aplicadas – Campus do Jardim Botânico

Av. Pref Lothario Meissner, 3400 – Jardim Botânico

***

Quinta-feira - 24 de setembro 2015

9:00 – 12:00 Simpósio Biotecnologia

Coordenador: David Mitchell

Departamento de Bioquímica e Biologia Molecular – UFPR




“Deep eutectic solvents as new media for biocatalysis: Example of lipase-

catalyzed synthesis of phenolipids”

Palestrante : Erwann Durand

Cirad (La recherche agronomique pour le développement) - Montpellier, França

“From structure to catalysis: recent advances on the biotechnological

applications of lipases”

Palestrante :Erika Cristina G. Aguieiras

UFRJ (Instituto de Química) - Rio de Janeiro

“New lipases isolated from metagenomic libraries: from characterization to the

development of applications”

Palestrante :Nadia Krieger

Departamento de Química – UFPR

Quinta-feira - 24 de setembro 2015

12:00 – 14:00 Sessão de Pôsteres

14:00 – 17:00 Simpósio Fixação Biológica de Nitrogênio

Coordenador: Fábio Oliveira Pedrosa


“Biological Nitrogen Fixation, from the discovery of nif gene to nitrogen fixation

in planta”

Palestrante :Ray Dixon

John Innes Centre – Inglaterra




“Nitrogen fixation in non-leguminous plants”

Palestrante :Gary Stacey

Missouri University – Estados Unidos

“Fixação biológica de nitrogênio: uma ideia fértil”

Palestrante :Ivo Baldani

Embrapa –Rio de Janeiro Agrobiologia

“Fixação de nitrogênio: uma perspectiva ômica”

Palestrante :Emanuel Maltempi de Souza


Sexta-feira - 25 de setembro 2015

9:00 – 12:00 Simpósio Bioquímica Farmacológica

Coordenador: Maria Eliane Merlin Rocha


“Targeting resistant cancer cells overexpressing multidrug ABC transporters

with selective drug-efflux inhibitors and apoptosis inducers”

Palestrante :Dr. Attilio Di Pietro

Institut de Biologie et Chimie des Protéines (IBCP)- Université Lyon 1 (França)




“Busca de moléculas-alvo para doenças degenerativas (cancer, diabetes) e

Medicina Regenerativa”

Palestrante : Mari Cleide Sogayar

Universidade de São Paulo

“Função endócrina do fator de crescimento de fibroblastos 21 (FGF21)

expresso no fígado na obesidade induzida por dieta hipercalórica”

Palestrante : Emy Luiza Ishii Iwamoto

Universidade Estadual de Maringá

“Anti-tumorais: diferentes abordagens na investigação dos mecanismos de

ação”

Palestrante : Maria Eliane Merlin Rocha



12:00 – 14:00 Sessão de Pôsteres

14:00 – 17:00 Simpósio Estrutura e Propriedades de Carboidratos

Coordenador: Marcello Iacomini


“Formation of protein particles and their influence on the structure and rheology

of polysaccharide solutions and gels”

Palestrante :Taeke Nicolai

Institute des Molecules et des Materiaux – CNRS Maine – France




“Glicoconjugados de fungos patogênicos”

Palestrante : Eliana Barreto Bergter

Instituto de Microbiologia Paulo de Góes, UFRJ

“The long and sugary road: Structural diversity and biological significance of

glycosphingolipids in pathogenic and opportunistic fungi”

Palestrante:Helio K. Takahashi

Escola Paulista de Medicina - Universidade Federal de Sao Paulo

“Atualização da Pesquisa em Química de Carboidratos no Departamento de

Bioquímica e Biologia Molecular”

Palestrante: Marcello Iacomini



17:30 Cerimônia de encerramento

simpósio comemorativo dos 50 anos do programa de pós ... · livro de resumos do simpÓsio...

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