universidade federal do rio grande do norte · obrigada por me abençoar muito mais do que mereço....

MINISTÉRIO DA EDUCAÇÃO

UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE CIÊNCIAS DA SAÚDE

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE

INTERAÇÃO DE POLISSACARÍDEOS SULFATADOS DA MACROALGA MARINHA Caulerpa cupressoides var. flabellata COM CRISTAIS DE

OXALATO DE CÁLCIO

DAYANNE LOPES GOMES

NATAL/RN 2017

DAYANNE LOPES GOMES


OXALATO DE CÁLCIO

Tese apresentada ao Programa de Pós-

Graduação em Ciências da Saúde da

Universidade Federal do Rio Grande do Norte

como requisito para a obtenção do título de

Doutor em Ciências da Saúde.

Orientador: Prof. Dr. Hugo Alexandre de O. Rocha

NATAL/RN 2017

Universidade Federal do Rio Grande do Norte - UFRN

Sistema de Bibliotecas - SISBI

Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial do Centro Ciências da Saúde - CCS

Gomes, Dayanne Lopes.

Interações de polissacarídeos sulfatados da macroalga marinha Caulerpa cupressoides var. flabellata com cristais de oxalato de

cálcio / Dayanne Lopes Gomes. - Natal, 2017. 98f.: il.

Tese (Doutorado) - Programa de Pós-graduação em Ciências da

Saúde. Centro de Ciências da Saúde. Universidade Federal do Rio Grande do Norte.

Orientador: Hugo Alexandre de Oliveira Rocha.

1. Urolitíase - Tese. 2. Alga verde - Tese. 3. Estabilização em COD - Tese. 4. COD haleteres - tese. I. Rocha, Hugo Alexandre

de Oliveira. II. Título.

RN/UF/BS-CCS CDU 616.62-003.7

i

MINISTÉRIO DA EDUCAÃO UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE

CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE

Coordenador do Programa de Pós-Graduação em Ciências da Saúde:

Prof. Dr. Eryvaldo Sócrates Tabosa do Egito

ii

DAYANNE LOPES GOMES


OXALATO DE CÁLCIO

Aprovada em: 20 / 07 / 2017

Banca Examinadora:

Presidente da Banca:

Prof. Dr. Hugo Alexandre de Oliveira Rocha

Membros da Banca

Profa. Dra. Julliane Tamara Araújo de Melo

Profa. Dra. Vanessa de Paula Soares Rachetti

Profa. Dra. Heryka Myrna Maia Ramalho

Profa. Dra. Luciana Nunes Menoli Lanza

iii

Dedico esta obra

A Deus.

Obrigada por me abençoar muito mais do que mereço. Quando deixei de olhar tão

ansiosamente para o que me faltava e passei a olhar com gentileza para o que eu

tinha, descobri que, de verdade, há muito mais a agradecer do que a pedir.

Aos meus pais.

Os maiores incentivadores! Papi Soberano (Zeca) e Mami Poderosa (Célia), os

apelidos retratam a figura grandiosa que vocês representam na minha vida e agradeço

à Deus por tê-los em meu convívio me vendo e me ajudando a trilhar meus caminhos.

Quando falamos de herança, legado, futuro, percebo e reconheço isso porque vocês

me fizeram enxergar que nenhuma herança é capaz de superar o valor da educação.

iv

Dedico esta obra

Aos meus filhos.

Minha força, meu estímulo, minha superação, minha vida. Helena, Heitor e Heloisa:

com vocês aprendo a todo instante, e desses aprendizados o maior deles é saber que

eu sempre posso mais.

A Hugo Rocha (Profi),

Minha enorme gratidão por todos esses anos de orientação. Agradeço pela dedicação,

paciência e, principalmente, pela amizade ao longo desses 11 anos de convivência.

Construímos uma relação de muita confiaça e carinho que sei que jamais se apagará.

“Vivemos com o que recebemos, mas marcamos a vida com o que damos.”

Winston Churchill

v

Agradecimentos especiais

“Eu reconheço que para ti nada é impossível e que nenhum dos teus planos pode ser impedido”. Jó 42:2. Obrigada meu Deus!

Aos meus pais, pelo amor incondicional.

Aos meus filhos, por cada interrupção ao tentar elaborar essa tese. Vocês me fizeram valorizar muito mais essa conquista e também foram o meu refúgio onde eu renovava

minhas forças e retomava a caminhada.

Aos meus irmãos, Dastaev, Dmitryev, Drielle e Dmetryus. Unidos pelo sangue e inseparáveis pelo coração! Sei que posso contar com vocês sempre;

Aos meus compadres e comadres, em especial França por oferecer “amor de graça” a

nós e principalmente a meus filhos, se sacrificando para que eu mantenha minha jornada diária de trabalho. A minha amigan (Jailma) que tanto me ajuda na vida, você é luz, é calmaria, é inspiração. Espero ser para vocês um pouco do que são para mim;

Ao meu esposo Leonardo, pelo companheirismo e paciência. Estando lado a lado, ou

a quilômetros de distância, mas sempre concetados a nossa família pelo amor;

A toda família Rocha de Medeiros, em especial minha sogra Ceiça, que sempre exaltou a importância das conquistas acadêmicas;

Obrigada a todos àqueles do laboratório que fazem ou que já fizeram parte desta

história...

Jailma (Amigan), Mariana, Sara, Karol, Cinthia, Leandro, Diego (Popó), Leonardo Nobre, Ruth, Rafael, Gabriel, Moacir, Talita, Jefferson, Maíra, Polyana, Sr. Paulo, Raniere, Joanna, Letícia, Kaline, Arthur, Vinicius, Max, Rony, Marília, Monique,

Larisse, Mônica, Pablo, Danielle, Almino, Jéssica, Cinthya, Raynara, Adriana, Lucas, Carla, Fernanda (Pôia), Profa. Fabiana, Ana Karina, Ana Karinne (Donana), Fernando,

Ivan, Nednaldo, Duda, Edjane, Valquíria.

Agradeço de forma especial aos amigos do grupo dos cristais: Kerol, Momoxi, Luquete, Pablito, Rafildo. Vocês são top de line!

À UFRN, à Pós-Graduação em Ciências da Saúde e ao Departamento de Bioquímica pela oportunidade de concluir esse curso de Pós-Graduação, assim como as agências

Financiadoras CAPES e CNPq.

A todos os professores do CCS (UFRN), aos coordenadores e equipe do Programa de Pós-Graduação (PPGCSa).

A todos os professores do DBQ (UFRN), em especial, a Profa. Edda Lisboa Leite por ser grande incentivadora e referência pessoal e profissional. Também aos técnicos do

DBQ (UFRN), em especial a Ana Katarina e Francisco Freire pelo apoio direto.

As professoras da banca de qualificação: Profa. Joana Cristina Tavares e Profa. Ariane Lacerda.

Agradeço novamente ao meu orientador Prof. Dr. Hugo Rocha por toda disponibilidade

e tempo dispendido nesse trabalho.

Obrigada a todos!

vi

“Não é sobre chegar no topo do mundo

E saber que venceu

É sobre escalar e sentir

Que o caminho te fortaleceu”.

(Ana Vilela)

vii

RESUMO

A urolitíase afeta aproximadamente 10% da população mundial e está associada

fortemente a formação de cristais de oxalato de cálcio (CaOx). Atualmente não

existe nenhum composto eficiente que possa ser utilizado para prevenir esta

doença. No entanto, os polissacarídeos sulfatados (PS) de algas possuem a

capacidade de alterar a carga superficial dos cristais de CaOx e assim modificar

a dinâmica da cristalização, devido à interação das cargas negativas desse

polímero com a superfície do cristal durante sua síntese. Nestre trabalho foi

verificado o efeito de quatro populações de PS extraídos da alga verde Caulerpa

cupressoides var. flabellata na formação de cristais de CaOx in vitro. Os PS

extraídos foram nomeados de CCB-F0.3, CCB-F0.5, CCB-F1.0 e CCB-F2.0.

Análises de microscopia eletrônica de varredura e de potencial zeta mostraram

que os polissacarídeos extraídos modificam a morfologia, o tamanho e a carga

superficial dos cristais de CaOx formados na presença dos PS. Todos os PS de

C. cupressoides induziram o aumento da quantidade de cristais CaOx formados.

Contudo, com exceção de CCB-F2.0, a presença dos demais PS levou à

formação de cristais de CaOx dihidratados (COD), que são comuns em pessoas

não formadoras de cálculos. Além disso, esses cristais COD apresentaram

morfologia arredondada ou em formato de halteres. As análises de

infravermelho, miscroscopia de fluorescência, citometria de fluxo e a análise de

composição atômica (EDS) permitiram a proposição de um modelo de interação

entre os PS de Caulerpa e os cristais COD. Neste modelo, a distribuição de PS

na estrutura do cristal de CaOx ideal para que haja maior estabilização dos

cristais COD é de 2:1:1 entre a base: ápice: face. Acredita-se que nessa

distribuição os PS consigam evitar a desidratação dos cristais COD, tornando-

os mais estáveis. Este estudo é o primeiro passo para entender as interações

entre PS de C. cupressoides e os cristais de CaOx, que são a principal causa de

cálculos renais.

Palavras-chave. Urolitíase; alga verde; COD halteres; Estabilização em COD.

viii

LISTA DE ABREVIATURAS E SIGLAS

ANOVA Análise de Variância

BIOPOL Laboratório de biotecnologia de polímeros naturais

ºC Graus Celsius

Ca2+ Cálcio

CaOx Oxalato de cálcio

COD Cristal de CaOx Dihidratado

COM Cristal de CaOx Monohidratado

COT Cristal de CaOx Trihidratado

CCB-F0.3 Polissacarídeo precipitado com 0,3 volumes de acetona




ERPS Extrato Rico em Polissacarídeos Sulfatados

EDS Espectroscopia de energia dispersiva

FITC Isotiocianato de fluoresceína

g Grama

GAGs Glicosaminoglicanos

h hora

Hz Hertz de frequência

kV quilovolts

L Litros

M Molar

MEV Microscópio eletrônico de varredura

mg Miligrama

Min. Minutos

mL Mililitros

mM Milimolar

pH Potencial de hidrogênio

IV Infravermelho

MEV Microscopia Eletrônica de Varredura

PBS Tampão salino-fosfato

PS Polissacarídeo (s) Sulfatado (s)

rpm Rotações por minuto

ix

LISTA DE FIGURAS Figura 01. Prevalência Mundial da Urolitíase.............................................................. 13

Figura 02. Alga verde Caulerpa cupressoides var. flabellata em exsicata (Foto:

Mariana Santana).......................................................................................

19

x

SUMÁRIO

RESUMO................................................................................................................................... viii LISTA DE ABREVIATURAS E SIGLAS................................................................................... ix LISTA DE FIGURAS.................................................................................................................. x 1. INTRODUÇÃO....................................................................................................................... 12 2. JUSTIFICATIVA.................................................................................................................... 17 3. OBJETIVOS........................................................................................................................... 18

3.1. GERAL................................................................................................................... 18 3.2. ESPECÍFICOS....................................................................................................... 18

4. MÉTODOS............................................................................................................................. 19 4.1. MATERIAL BIOLÓGICO........................................................................................ 19

4.1.1. Algas....................................................................................................... 19 4.2. EXTRAÇÃO DOS POLISSACARÍDEOS SULFATADOS DA C. cupressoides...... 19

4.2.1. Obtenção de Extratos Ricos em Polissacarídeos Sulfatados (ERPS)... 19 4.2.2. Fracionamento do ERPS com concentrações crescentes de acetona... 20

4.3. SÍNTESE E CARACTERIZAÇÃO DOS CRISTAIS................................................ 21 4.3.1. Ensaio de cristalização de CaOx............................................................ 21 4.3.2. Microscopia Eletrônica de Varredura (MEV) e Espectroscopia de

Energia Dispersiva (EDS).....................................................................

21 4.3.3. Medida do Potencial Zeta (ζ) dos cristais de CaOx............................... 22 4.3.4. Espectroscopia de infravermelho........................................................... 22 4.3.5. Microscopia de fluorescência para análise da morfologia dos cristais

de CaOx................................................................................................

22 4.4. ANÁLISE ESTATÍSTICA........................................................................................ 23

5. ARTIGOS PRODUZIDOS...................................................................................................... 24 5.1. ARTIGO 1 (SUBMETIDO)...................................................................................... 25 5.2. ARTIGO 2............................................................................................................... 57

6. COMENTÁRIOS, CRÍTICAS E SUGESTÕES...................................................................... 76 7. REFERÊNCIAS..................................................................................................................... 78 8. APÊNDICES

8.1. ARTIGO 3............................................................................................................... 82 8.2. ARTIGO 4............................................................................................................... 84 8.3. ARTIGO 5............................................................................................................... 86 8.4. ARTIGO 6............................................................................................................... 88

9. ANEXOS

9.1. NORMAS PARA FORMATAÇÃO DA TESE (CCS)............................................... 91 9.2. NORMAS DA REVISTA PARA SUBMISSÃO (OXIDATIVE MEDICINE AND

CELLULAR LONGEVITY)....................................................................................

94

12

Gomes, D.L. PPGCSA/CCS

1. INTRODUÇÃO

Os rins sãos os órgãos responsáveis pela filtração do plasma, esse é um

processo que ocorre de forma permanente e em grande escala (180 L/dia).

Nesse processo, muitos íons e outras moléculas pequenas vão passar pelos

túbulos renais. Porém, além da alta taxa de filtração, também ocorre uma intensa

reabsorção de moléculas, levando à excreção de apenas algo entre 1 e 1,5 L [1].

Nesse contexto, os íons presentes na urina ficam supersaturados. Alguns

desses íons tendem a interagir com outros e formar sais insolúveis, um processo

comum e natural a todos os indivíduos [2,3]. Esses sais insolúveis vão se

associando de forma lenta, e o produto desta associação vai absorvendo cada

vez mais íons da solução, o que culmina com o surgimento de estruturas

cristalinas em escala nanométrica [4]. A interação entre as cargas dessas

nanoestruturas faz com que elas se atraiam e se agreguem, sendo essa

agregação uma segunda forma de crescimento de cristais, caracterizada pela

rápida deposição de íons e associação a matrizes orgânicas presentes no trato

urinário, fazendo com que a estrutura orgânica/inorgânica formada passe a ser

chamada de cálculo renal ou urolitíase [5].

Do ponto de vista epidemiológico, o cálculo renal atinge cerca de 10% da

população mundial, com maior prevalência nos Estados Unidos [6]. A maior

incidência de cálculo renal ocorre em homens, entre 20 e 60 anos de idade e

tem uma altíssima taxa de recorrência após a primeira crise renal do paciente

[7]. Por acometer uma grande parte da população, a urolitíase gera um grande

impacto na economia e, somente no ano de 2010, os custos associados ao

tratamento hospitalar da urolitíase no sistema público de saúde brasileiro

chegaram a 29,2 milhões no Brasil. Além do alto custo relativo às complicações

que a urolitíase pode gerar, existe o impacto econômico direto por acometer uma

faixa de idade economicamente ativa (20 aos 60 anos) [8].

Do ponto de vista clínico, estes cálculos podem causar dor suprapubica, na

glande do pênis, disuria, hematuria, jato de urina fraco e entrecortado, dentre

outros. Atualmente, o recurso mais utilizado diante de uma crise renal causada

pelo cálculo é a analgesia. Já o tratamento tradicional se baseia na eliminação

de forma espontânea do cálculo, ou ainda procedimentos mais invasivos e com

grau de risco mais crítico, como o cirúrgico tradicional ou bombardeamento a

13


laser [9]. Nota-se que mesmo com todos os avanços recentes na medicina ainda

não existe um tratamento realmente eficaz para impedir o surgimento dos

cálculos renais [10].

Figura 01. Prevalência Mundial da Urolitíase. Em vermelho, destacam-se as prevalências de cálculos renais. Em amarelo, destacam-se o crescimento no número de pessoas com cálculos, principalmente de Oxalato de Cálcio [6].

O estágio de formação dos cálculos que consiste na interação inicial dos íons

e na formação das nanoestruturas é chamado de nucleação, pois forma o que

virá a ser um núcleo de crescimento do cristal. Esse núcleo formado serve como

arcabouço para agregação de íons e moléculas e/ou macromoléculas, que vão

levar ao crescimento dos cristais. Existem dois destinos possíveis para esse

núcleo formado: ele pode ser eliminado naturalmente ou pode crescer e agregar

mais cristais, íons e moléculas orgânicas, formando os cálculos renais [11]. Se

ele seguir o segundo caminho, o próximo passo para a formação de cristais é

denominado de agregação, que consiste na união de dois ou mais núcleos entre

si. Essa agregação resulta em cristais grandes e pesados que podem se

precipitar e crescer cada vez mais [12].

Apesar da existência de um mecanismo de formação quase comum a todos

os cálculos, estes podem ser formados a partir de diversos componentes como:

oxalato de cálcio, fosfato de cálcio, ácido úrico, cistina [10] e cristais derivados

de infecção, como estruvita e carbonato de hapatita, além dos cristais mistos

[13]. Apesar dessa grande variedade, mais de 70% dos casos de urolitíase são

formados principalmente por cristais de oxalato de cálcio (CaOx) [14].

14


Três tipos de cristais de CaOx podem ser formados no tecido renal, os

cristais de CaOx monohidratados (COM), dihidratados (COD) e trihidratados

(COT) [15]. Devido à supersaturação urinária, naturalmente os três tipos de

cristais de CaOx se formam em todas pessoas. COD e COT são instáveis e tem

uma tendência a se converter em COM, que é o cristal mais estável e o principal

cristal detectado na urina de pacientes formadores de cálculos. Porém, em

pacientes assintomáticos, o cristal mais encontrado na urina é o cristal do tipo

COD [4].

Uma das hipóteses que mostra o papel dos cristais de CaOx na formação

dos cálculos renais relata que o COM possui uma carga positiva, que lhes

permite interagir com moléculas carregadas negativamente encontradas na

superfície celular do epitélio renal, o que leva a sua adesão à superfície da célula

e, por conseguinte, endocitose. A presença desses cristais dentro do citoplasma

celular provoca estresse oxidativo e lesão celular. Um dos resultados desse

processo é a peroxidação lipídica da superfície celular, o que,

consequentemente, provoca a perda de várias moléculas do glicocálice. Este

acontecimento é fundamental para a formação de cálculos renais, pois partes

dessas moléculas do glicocálice que são perdidas tem como função impedir a

interação/adesão de cristais de CaOx com as células. Ou seja, esta perda de

glicocálice permitirá que mais cristais COM se depositem na superfície, não

sejam endocitados e cresçam. Alguns desses cristais vão se combinando entre

si e com outras moléculas para formar cálculos renais, que levam a dano físico

no epitélio e lesão [16, 17].

Uma alternativa interessante para a prevenção da recorrência dos casos de

urolitíase seria o uso de alimentos ou suplementos que pudessem inibir

profilaticamente a formação de cristais [18]. Nessa perspectiva, inserem-se os

glicosaminoglicanos (GAGs), que são polissacarídeos sulfatados (PS)

encontrados nos tecidos, em sua maioria ligados covalentemente a proteínas,

formando os proteoglicanos [19]. No trato urinário, os proteoglicanos da

superfície celular são constituídos de moléculas grandes de GAGs que, por

serem negativamente carregadas, interagem com os cristais de CaOx. Isto

impede a endocitose desses cristais, como também leva à estabilização do

cristal na forma COD. Acredita-se que estes cristais associados aos GAGs

15


cresçam até um ponto em que o complexo cristal-fragmento de GAG se solte do

proteoglicano e, consequentemente, da superfície celular. Uma vez solto, o

fragmento do GAG ainda age como estabilizador do cristal, o que justificaria a

presença de cristais COD na urina [20]. Apesar desse papel benéfico, o uso

dessas moléculas como tratamento apresenta uma série de contraindicações,

tais como: i) a sua obtenção a partir de tecido animal, o que implica em alto custo,

baixo rendimento e risco de contaminação do paciente; ii) inúmeras atividades

são exercidas pelos GAGs dentro do organismo, implicando numa grande

chance de ocorrerem efeitos indesejados [19].

Na medicina oriental, algumas algas já são usadas no combate aos cálculos

de CaOx [21]. Essas informações sugerem que as algas marinhas são uma

potencial fonte de moléculas para o tratamento e prevenção de cálculos renais.

Vale salientar que as macroalgas marinhas são fonte de PS e, quando

comparadas a animais, as algas produzem grandes quantidades de PS.

Semelhante aos GAGs, já foi mostrado que PS de algas também tendem a se

associar, por atração eletrostática, às porções positivamente carregadas dos

cristais de CaOx em formação, alterando seu padrão de crescimento [22]. Ao

longo da última década, foi crescente o número de estudos que avaliou atividade

inibidora da formação de cristais de CaOx por PS, especialmente os de algas

marinhas [23].

Tanto Zhang e colaboradores (2012) [24] quanto Melo e colaboradores

(2013) [25], trabalharam com PS de diferentes algas marrons e obtiveram

resultados semelhantes. Zhang e colaboradores trabalharam com um extrato

bruto da alga Sargassum graminifolium e foi capaz de inibir nucleação e

agregação dos cristais de CaOx in vitro. Ainda, os cristais obtidos tinham

morfologia mais arredondada e eram mais estáveis. Já Melo e colaboradores

(2013) trabalharam com 4 polissacarídeos purificados da alga Dictyopteris justii.

Todos eles foram capazes de inibir o crescimento e a agregação de cristais de

CaOx, com destaque para uma glucana sulfatada que também foi capaz de

estabilizar cristais do tipo COD.

Nos dois trabalhos, o mecanismo de ação dos PS foi semelhante ao

observado nos GAGs: interação com o cristal, alteração da carga superficial e

estabilização de formas mais estáveis e menos propensas a causar dano.

16


Outro grupo de pesquisadores busca correlacionar esse efeito inibidor da

formação de cristais de CaOx [26, 27] ou de proteção do epitélio renal contra os

danos causados por esses cristais [28] com a quantidade de grupamentos

sulfatos existentes nos PS, já que na maioria dos casos são esses grupamentos

que conferem a carga negativa do polímero. Por exemplo, PS com baixa massa

molecular e diferentes conteúdos de grupamentos sulfato, extraídos de algas

marrons (Undaria pinnatifida, Laminaria japonica, Sargassum fusiforme) e

vermelhas (Porphyra yezoensis, Gracilaria lemaneiformis, Eucheuma) foram

avaliados quando à capacidade de reparar células epiteliais tubulares proximais

de rim humano (HK-2) danificadas com oxalato in vitro. Esse estudo mostrou

uma correlação positiva entre o conteúdo de sulfato e atividade reparadora do

epitélio renal [28]. Essa correlação corrobora com aqueles descritas para PS de

algas que apresentam outras atividades biológicas. Porém, nestes estudos

também é apontado que o fator mais importante para o PS ser bioativo não é só

o fato dele ser sulfatado, mas sim, como estes grupos sulfatos estão distribuídos

ao longo da cadeia molécula de PS [29, 30]. Além disso, os tipos de resíduos

de monossacarídeos que constituem os polímeros parecem também ter

influência sobre as atividades [31].

Ensaios in vivo também mostraram a importância do PS no combate à

urolitíase. Veena e colaboradores trabalhando com PS extraídos da alga marrom

Fucus vesiculosus mostraram que camundongos hiperoxalúricos suplementados

com esses PS tiveram redução do estresse oxidativo em seus organismos,

devido ao aumento da atividade das enzimas antioxidantes e consequente

diminuição da peroxidação lipídica. Este efeito dos polissacarídeos por

conseguinte diminuiu o acúmulo de cristais nos rins dos animais [32]

Ainda não foi proposto um mecanismo completo que explique a ação dos PS

de algas no processo de inibição dos cristais de CaOx [23]. Todavia, essas

moléculas se mostram como uma interessante alternativa na prevenção da

formação de cálculos renais e o entendimento das interações dos PS de algas

com os cristais de CaOx é imprescindível para que eles possam ser utilizados

na terapêutica.

17


2. JUSTIFICATIVA

O impacto causado pela urolitíase na sociedade é enorme e cada vez

mais é necessário encontrar fármacos e mecanismos alternativos para que esse

impacto seja reduzido.

Os glicosaminoglicanos (GAGs) são polissacarídeos sulfatados (PS) que

são encontrados na urina e que são tidos como agente inibidores da formação

de cristais de oxalato de cálcio (CaOx). Na década de 1990, vários estudos

avaliaram o efeito de GAGs exógenos na formação de cristais de CaOx [33].

Apesar dos resultados terem sido animadores, um fator importante que levou à

estagnação das pesquisas: a dificuldade de se obter os GAGs em grande

quantidade.

Posteriormente, outros PS passaram a ser estudados com a mesma

finalidade. Dentre eles, PS de algas marinhas (marrons e vermelhas) que,

diferente dos GAGs, são obtidos em grande quantidade. Além disso, muitos

desses PS de algas apresentam atividade antioxidante, que é uma atividade

procurada em agentes utilizados no tratamento de urolitíase [24, 25, 32, 34, 35].

A alga verde Caulerpa cupressoides é encontrada em grande quantidade

no litoral nordestino. Ela sintetiza quatro populações de polissacarídeos, que já

foram caracterizadas pela equipe do Laboratório de Biotecnologia de Polímeros

Naturais-UFRN, sob a coordenação do Prof. Dr. Hugo Rocha, e verificou-se que

estes têm excelente atividade antioxidante em comparação aos PS encontrados

em outras algas do litoral potiguar [30, 36]. Isso associado ao fato de que

nenhuma alga verde teve seus PS avaliados como agentes inibidores da

formação de cristais de CaOx, faz dessa alga uma excelente escolha para a

prospecção de PS com potencial uso no tratamento da urolitiase.

18


3. OBJETIVOS

3.1. GERAL

Verificar o efeito dos polissacarídeos sulfatados (PS) da alga verde

Caulerpa cupressoides var. flabellata na formação de cristais de oxalato

de cálcio (CaOx) in vitro.

3.2. ESPECÍFICOS

Extrair PS da alga marinha C. cupressoides utilizando uma metodologia

já desenvolvida pelo nosso grupo de pesquisa;

Analisar o efeito dessas populações de PS sobre a formação de cristais

in vitro;

Caracterizar os cristais de CaOx formados na presença dos PS de C.

cupressoides.

19


4. MÉTODOS

4.1. MATERIAL BIOLÓGICO

4.1.1. Algas

A alga marinha verde Caulerpa cupressoides var. flabellata (Figura 02) foi

coletada na Praia de Búzios, município de Nísia Floresta (litoral sul do Rio

Grande do Norte), no mês de março do ano de 2015, em marés baixas entre 0,0

a 0,2 metros a uma temperatura situada entre 28-30°C. As algas foram

recolhidas quando já desprendidas do substrato, mas permanecendo flutuando

nas águas de maré-baixa.

As algas foram acondicionadas em sacos de polietileno e trazidas ao

laboratório no mesmo dia da coleta e, lavadas em água corrente, examinadas

cuidadosamente para remoção de epífitas, inclusões calcárias e sais, sendo

postas para secar em estufa aerada a 45°C. Em seguida, foram trituradas em

liquidificador, pesadas e guardadas em frascos de vidro hermeticamente

fechados.

Figura 02. Alga verde Caulerpa cupressoides var. flabellata em exsicata (Foto: Mariana Santana).

4.2. EXTRAÇÃO DOS POLISSACARÍDEOS SULFATADOS DA C.

cupressoides.

4.2.1. Obtenção de Extratos Ricos em Polissacarídeos Sulfatados (ERPS)

20


A obtenção dos ERPS da C. cupressoides seguiu o método aplicado pelo

grupo pesquisa que em está inserido esta tese e, mais recentemente, utilizado

por Costa e colaboradores (2010) [30] durante a obtenção de ERPS de algas do

litoral potiguar. Para tanto, as algas secas e pulverizadas foram tratadas com

dois volumes de etanol para despigmentação e delipidação do material. A cada

saturação do álcool, que foi observada visualmente, realizava-se a troca deste

reagente. Posteriormente, o resíduo foi separado do álcool, e colocado para

secar à temperatura ambiente. Após seco, este material despigmentado foi

utilizado para a obtenção dos ERPS. Para a realização dessa etapa, foram

adicionados dois volumes de NaCl a 0,25 M ao pó delipidado (100 g) e o pH

ajustado para 8,0 com NaOH. A esse material foi adicionado a enzima

proteolítica prozima (15 mg/g de pó). Essa suspensão permaneceu em banho-

maria a 60 °C durante um período de 18 h. Depois, foi filtrado e o sobrenadante

submetido a uma centrifugação 10.000 x g por 15 minutos à temperatura de 4

°C. Após a centrifugação, o sobrenadante, que contém os polissacarídeos

solúveis, foi denominado de ERPS, sendo seco à pressão reduzida, triturado,

pesado e guardado para posteriores análises.

4.2.2. Fracionamento do ERPS com concentrações crescentes de acetona

O sobrenadante obtido após proteólise foi fracionado com volumes

crescentes de acetona. Este fracionamento foi realizado de acordo com método

descrito por Costa (2012) [36]. Os valores de acetona adicionados foram

determinados pela turvação da solução, que caracteriza a precipitação de

polissacarídeos devido à adição desse solvente. Inicialmente, foi adicionado 0,3

volumes de acetona, sob agitação leve, volume necessário para que se visualize

uma turvação da solução. Esta foi mantida em repouso a 4 ºC durante 18 h. O

precipitado foi coletado por centrifugação a 8.000 x g por 15 min. a 4 ºC e seco

a pressão reduzida. Em seguida, esse procedimento foi repetido utilizando-se

0.3, 0.5, 1.0 e 2.0 volumes de acetona, obtendo-se as quatro populações

polissacarídicas, denominadas de CCB-F0.3, CCB-F0.5, CCB-F1.0 e CCB-F2.0,

de acordo com o volume de acetona acrescentado.

21


4.3. SÍNTESE E CARACTERIZAÇÃO DOS CRISTAIS 4.3.1. Ensaio de cristalização do CaOx

Os cristais de CaOx podem ser formados in vitro a partir de Ca2+ e oxalato.

Para tal, se mistura de cloreto de cálcio (8 mmol/L), oxalato de sódio (1 mmol/L),

cloreto de sódio (200 mmol/L) e acetato de sódio (10 mmol/L), sendo as

concentrações finais desta solução semelhantes às das concentrações

fisiológicas da urina, quando consideradas separadamente. A formação dos

cristais de CaOx foi avaliada a partir da junção dessas soluções formando um

meio superconcentrado favorável à formação de cristais de CaOx. Os cristais

foram formados na presença dos polissacarídeos (250 µg/mL) ou na ausência

(controle) dos polissacarídeos [24]. Após um período de 30 minutos, as soluções

contendo estes cristais foram centrifugadas a 5000 x g e o sobrenatante foi

descartado. O precipitado, composto principalmente por cristais de CaOx, foi

ressuspendido em 0,5 mL de água e uma alíquota de 0,1 mL foi colocada em

lâmina histológica e observada em microscópio ótico (600x) imediatamente após

a ressuspensão. Foram obtidas imagens de 10 campos diferentes aleatórios

para cada lâmina. Em seguida, os diâmetros e tamanhos dos cristais foram

analisados utilizando o software NIS Elements AR Ver4.30.01 DU1 64 bit, ano

de 2014 (Melville, NY, EUA). As conclusões acerca das aferições dos cristais de

CaOx foram obtidas após a realização de experimentos distintos, repetidos

quatro vezes.

4.3.2. Microscopia Eletrônica de Varredura (MEV) e Espectroscopia de

Energia Dispersiva (EDS)

Para se caracterizar a morfologia e composição dos cristais de CaOx na

presença dos polissacarídeos de C. cupressoides, foi realizada a microscopia

eletrônica de varredura MEV (modelo Hitachi Tabletop Microscope TM-3000,

com aceleração de voltagem de 5 kV, frequência de 50/60Hz, magnificação da

imagem de 15 à 30000 vezes), e a Espectroscopia de Energia Dispersiva EDS

(o equipamento utilizado foi Swift ED TM-3000, fabricante Oxford Instruments

detector). As imagens foram geradas com a resolução 1280x960 pixels. Essas

22


análises foram realizadas no Departamento de Engenharia de Materiais da

Universidade Federal do Rio grande do Norte.

4.3.3. Medida do Potencial Zeta (ζ) dos cristais de CaOx

Após 30 minutos do início da formação dos cristais na presença e na

ausência dos polissacarídeos, as soluções foram centrifugadas (5000 x g). O

sobrenadante foi descartado e o precipitado rico em cristais de CaOx foi

ressuspendido em 1,5 mL de água e avaliado no Zeta Plus® (controle de

temperatura ativo entre -5°C a 110°C, ±0.2°C, 1 a 4 s/ciclo, faixa de pH: 2 a 12,

condutividade: 0 a 7,5 mS/cm, intensidade do campo elétrico: 0 a 3.2 kV/m,) para

obtenção do Potencial Zeta.

4.3.4. Espectroscopia de infravermelho

A espectroscopia de infravermelho foi realizada em espectrômetro Perkin-

Elmer de 4400 a 400 cm-1 no Departamento de Química da Universidade Federal

do Rio grande do Norte. Os cristais de CaOx controle e os cristais de CaOx

formados na presença de cada polissacarídeo de C. cupressoides (~5 mg) foram

analisados após secagem em aparelho de Abdenhalden sob a forma de pastilha

de KBr contendo P2O5 a 60ºC.

4.3.5. Microscopia de fluorescência para análise da morfologia dos cristais

de CaOx

Para melhor compreensão acerca da relação entre a morfologia dos

cristais e disposição dos polissacarídeos sulfatados nos cristais de CaOx, as

populações polissacarídicas foram marcadas com isotiocianato de fluoresceína

(FITC) (Sigma). Resumidamente, 5 mg de cada população foram solubilizadas

numa solução 0,1 M de tampão fosfato (PBS) em pH 7,0 contendo 0,1 mg de

FITC. A solução foi mantida em ambiente com redução de luminosidade, sob

leve agitação, à temperatura ambiente, por 1 hora. Em seguida, o material foi

dialisado contra água deionizada em membranas com poros de 6 kDa de

diâmetro e, posteriormente, liofilizado. As amostras sem polissacarídeos

sulfatados, assim como as amostras de populações marcadas com FITC foram

submetidas a nova produção cristais de CaOx e as lâminas montadas conforme

23


descrito no item 4.3.1. Para a visualização das imagens (10 campos), foi utilizado

o Microscópio TE-Eclipse 300, Nikon, Melville, NY, USA (Objetiva 60x) e as

imagens foram analisadas através do software NIS Elements AR Ver4.30.01

DU1 64 bit, ano de 2014 (Melville, NY, EUA).

4.4. ANÁLISE ESTATÍSTICA

Todos os dados dos experimentos realizados no item foram expressos

como média ± desvio padrão. Foi utilizado o teste de análise paramétrica de

análise de variância (ANOVA) seguido do teste de Tukey (Nível de significância

de p<0,05) como GraphPad InStat® Software versão 3.05 para Windows 95

(GraphPad Software Incorporation, San Diego, CA, EUA).

24


5. ARTIGOS PRODUZIDOS 5.1. Artigo 1 (SUBMETIDO)

Interaction of antioxidant sulfated polysaccharides from green seaweed

Caulerpa cupressoides var. flabellata with calcium oxalate crystals

Periódico: Oxidative Medicine and Cellular Longevity

The reference number for the article is 1284653

Fator de impacto: 4.593

ISSN: 1942-0900 (Online version)

Qualis: Medicina II – A1

5.2. Artigo 2

Methanolic Extracts from Brown Seaweeds Dictyota cilliolata and Dictyota

menstrualis Induce Apoptosis in Human Cervical Adenocarcinoma HeLa

Cells

Periódico: Molecules



Qualis: Medicina II – B1

25


5.1 ARTIGO 1

Interaction of antioxidant sulfated polysaccharides from green

seaweed Caulerpa cupressoides var. flabellata with calcium oxalate

crystals

Dayanne L. Gomes1,2, Karoline R. T. Melo1, Moacir Queiroz Fernandes1, Lucas A. N.

C. Batista1, Pablo C. Santos3, Mariana S. S. P. Costa4, Jailma Almeida-Lima1, Rafael

B. G. Câmara1, Hugo A. O. Rocha1*

1 Laboratório de Biotecnologia de Polímeros Naturais (BIOPOL), Departamento de

Bioquímica, Centro de Biociências, Universidade Federal do Rio Grande do Norte

(UFRN), Natal, Rio Grande do Norte- RN 59078-970, Brazil.

2 Programa de Pós-graduação em Ciências da Saúde, Universidade Federal do Rio

Grande do Norte (UFRN), Natal, Rio Grande do Norte – RN 59078-970, Brazil.

3 Universidade Estadual do Rio Grande do Norte (UERN), Mossoró, Rio Grande do

Norte – RN, 59.610-210, Brazil.

4 Instituto Federal de Educação, Ciência e Tecnologia do Rio Grande do Norte (IFRN),

Macau, Rio Grande do Norte – RN, 59.500-000, Brazil.

Corresponding author: H. A. O. Rocha

* Email: [email protected]; Tel.: +55-84-32153416 (Branch line: 207).

26


Abstract

Urolithiasis affects approximately 10% of the world population and is

strongly associated with calcium oxalate (CaOx) crystals. Currently, there is no

efficient compound that can be used to prevent this disease. However, seaweeds’

sulfated polysaccharides (SPs) have the ability to change the CaOx crystals surface’s

charge and thus modify the crystallization dynamics, due to the interaction of the

negative charges of these polymers with the crystal surface during their synthesis.

We observed that the SPs of C. cupressoides modify the morphology, size and

surface charge of CaOx crystals. Thus, these crystals are similar to those found in

healthy persons. In the presence of SPs, dihydrate CaOx crystals showed rounded

or dumbbell morphology. Infrared analyzes, fluorescence microscopy, flow

cytometry (FITC-conjugated SPs) and atomic composition analysis (EDS) let us

propose the mode of action between the Caulerpa’s SPs and the CaOx crystals. This

study is the first step in understanding the interactions between SPs, which are

promising molecules for the treatment of urolithiasis, and CaOx crystals, which are

the main cause of kidney stones.

Key words. Urolithiasis; Green algae; COD dumbbell; COD stabilization;

27


1. Introduction

Urinary lithiasis or urolithiasis is a pathophysiological condition resulting from

the formation of kidney stones. The incidence of urinary lithiasis in the world is increasing

rapidly. In the last decade, prevalence doubled from approximately 6 to 11% among men

and 4 to 7% among women, respectively [1]. One of the major problems with urolithiasis

is the risk of developing chronic kidney disease (CKD) and end-stage renal disease

(ESRD), a serious form of CKD [2]. This disease has a high social and economic cost,

since in the USA alone, CKD treatment exceeds $50 billion a year [3]. In England,

according to a recent report published by the National Health Service (NHS) Kidney Care,

costs with CKD case management exceeds those of breast, lung, colon and skin cancers

combined [4].

Urolithiasis can be influenced directly by the types of formed crystals, as each

crystal has its own different ability to bind to the renal epithelium and to initiate the

formation of the stones. Calcium oxalate (CaOx) crystals make up about 70% of urinary

stones [5], and they are found in three different forms: monohydrate (COM), dihydrate

(COD) and trihydrate (COT). The COM crystal is thermodynamically stable, exhibiting

monocyclic geometric morphology (which, in optical microscopy, can be visualized with

a rectangular shape) and showing greater affinity with the renal epithelium. For these

reasons, they are found in kidney stones at a frequency twice as high as that of COD

crystals [6,7]. The COD are commonly found in the urine of asymptomatic persons for

urolithiasis and present a characteristic tetrahedral bipyramidal morphology, which can

be easily visualized by light microscopy [7]. COT crystals show drusiform morphology.

They are unstable, and therefore are rarely found in the urine of patients [8].

The formation of these CaOx crystals is derived from a physico-chemical process

divided into 3 phases: nucleation, aggregation and crystal growth. The preponderant

condition for crystal formation (nucleation) is urinary supersaturation, as due to the high

concentration of ions in a solution, the condensation of these salts occurs forming tiny

crystals, which are called nuclei. The aggregation occurs by the union of one or more

growing nuclei, which form crystals of larger dimension and mass that can precipitate in

the renal epithelium, being able to adhere or to be internalized by cells [9]. The growth

of the kidney stone occurs by aggregation of preformed crystals or by secondary

nucleation of a crystal adhered to the intrarenal structures [10]. The thickness of a crystal

is related to the proportion of its faces. The CaOx crystals have three growth faces, and

each face differs in size and naming according to the type of crystal referred to: faces

28


(101), (010) and (121) for the COM crystals (Figure 1A); and faces (100), (101) and (011)

for the COD crystals (Figure 1B).

The changes in the CaOx crystal’s face are caused by the other molecules present

in the urinary tract, taking into account the fact that the concentration of ions in healthy

persons and lithogenic patients is practically the same. These molecules, such as citrate,

pyrophosphate, glycosaminoglycans and glycoproteins, present in the renal and urinary

ducts, have the function of stabilizing CaOx crystals, mainly in the COD morphology [11,

12].

Recently, seaweeds sulfated polysaccharides (SPs) also begun to be evaluated for

their anti-lithic capacity. In a recently published review [13], it was verified that these

polymers can inhibit the formation of CaOx renal stone formation in different ways: they

inhibit crystallization (both in the nucleation phase [14,15,16] and in growth phase [16,

17]); they inhibit the aggregation phase [14, 15, 16, 17, 18]; they inhibit the occurrence

of COM crystals and the transformation of COM to COD [14, 16, 17, 18]; and they inhibit

renal tubular cell injury [19,20,21].

The Brazilian coast has a wide diversity of marine seaweed, especially in the

Northeast coast; however, few species had their SPs characterized and evaluated for their

therapeutic potential. The SPs from Caulerpa cupressoides var. flabellata have been

studied by our research group for almost a decade.

Initially, we have shown that SPs-rich extract from C. cupressoides exhibited

several biological activities, including anticoagulant, antiproliferative and antioxidant

activities [22]. Later, four different high molecular weight SPs, named CCB-0.3, CCB-

0.5, CCB-1.0 and CCB2.0 were obtained from this SPs-rich extract. The SPs CCB-F0.3,

CCB-F0.5 exhibited antioxidant activity and significant iron chelating ability [23].

However, the effect of these SPs on the formation of CaOx crystals has not yet been

described. It is noteworthy that, so far, no SP of green seaweed had its action on evaluated

crystal formation. Based on these considerations, the objective of the present study was

to obtain SPs of C. cupressoides (CCB-F0.3, CCB-F0.5, CCB-F1.0 and CCB-F2.0) and

to evaluate their effect on crystallization of CaOx in vitro, as well as from the different

morphological characteristics of the formed CaOx crystals to propose a model of

interaction between the populations of SPs obtained with the CaOx crystals.

29


2. Materials and Methods

2.1 Sulfated polysaccharides extraction from green seaweed C. cupressoides

After collection, the green seaweed Caulerpa cupressoides var. flabellata

Børgesen was cleaned with running water and oven dehydrated at 45 °C. They were

sprayed and then treated four times with two volumes of ethanol for depigmentation and

delipidation of the material. Two volumes of 0.25 M NaCl were added to the powder

obtained, with the pH being adjusted to 8.0. The proteolytic enzyme maxatase (60 °C, for

18 h) was added to this material. The suspension was then centrifuged at 10.000 g for 20

minutes. The precipitate was discarded and the volume of the supernatant was measured

fractionated with increasing volumes of acetone, obtaining SPs according to a method

established by Costa and collaborators, 2012 [23].

2.2 Chemical and Physicochemical Analysis

The total sugars were determined according to Dubois and collaborators (1956)

by the phenol–H2SO4 reaction [24]. The sulfate content was determined based on the

gelatin/barium method [25]. Protein quantification was determined using Spector’s

method [26]. The SPs were submitted to electrophoresis in order to evaluate the presence

of sulfated polysaccharides and to identify the different populations of these [27].

2.3 Fourier Transformed Infrared (FT-IR) Spectroscopy Analysis

The infrared spectra of the CaOx crystals controls formed after incubation with

the SPs of C. cupressoides were obtained using infrared spectroscopy via Fourier

transform (IRAffinity-1 spectrometer, Shimadzu Corp., Kyoto, Japan) equipped with the

IRsolution 1.20 software. SPs and crystals samples (5 mg) were completely mixed with

dried potassium bromide powder (KBr) and then compressed. The sweep frequency range

was 4000–400 cm−1. Thirty-two scans at a resolution of 4 cm−1 were evaluated and

referenced against air. The Infrared Spectroscopy Analysis was carried out in the

Department of Chemistry of the Federal University of Rio Grande do Norte.

2.4 Calcium Oxalate Crystallization Assay

The CaOx crystals can be formed in vitro from Ca2+ and oxalate to a mixture of

calcium chloride (8 mM/L), sodium oxalate, sodium chloride (200 mM/L) and sodium

acetate (10 mM/L) as final concentrations of this solution from physiological

concentrations of urine. The formation of the CaOx crystals was evaluated in the presence

30


or absence (control) of the Caulerpa’ polysaccharides [15]. After formation of these, the

solutions consisting of these crystals were centrifuged at 5.000 x g and the supernatant

was discarded. The precipitate is composed mainly of crystals of CaOx, and was

resuspended in 0.5 mL of water, and a solution of 0.1 mL was placed on a histological

slide and observed under an optical microscope (600x). Images of 10 different fields were

obtained for each slide, then the crystal diameters and sizes were analyzed using the NIS

Elements AR Ver4.30.01 DU1 64-bit software, year 2014 (Melville, NY, USA). The

conclusions about the measurements of the CaOx analyzes were obtained after a trial of

distinct experiments, being repeated four times.

2.5 Scanning electron microscopy (SEM) and Dispersive Energy Spectroscopy

(EDS)

To observe the superstructure and composition of generated crystals in the

presence of the SPs of C. cupressoides, a scanning electron microscopy (Hitachi Tabletop

Microscope TM-3000 model, with 5 kV voltage acceleration, 50/60Hz of frequency,

image magnification from 15 to 30000) and dispersive energy spectroscopy (Swift ED

TM-3000, Oxford Instruments detector) images were generated with 1280x960 pixels

resolution. The Scanning Electron Microscopy was carried out in the Department of

Materials Engineering of the Federal University of Rio Grande do Norte.

2.6 Zeta Potential (ζ) Measurements

After 30 minutes of crystal formation in the presence and absence of the

polysaccharides [15], the solutions were centrifuged (5000 x g). The crystal concentrate

was then suspended in 1.5 mL of water, and the zeta potential of the ζ samples was

obtained using a Zeta Plus® analyzer (active temperature control between -5°C at 110°C,

±0.2°C, 1 at 4 s/cycle, pH range: 2 at 12, conductivity: 0 at 7.5 mS/cm, intensity of the

electric field: 0 at 3.2 kV/m), Brookhaven instruments, Holtsville, NY, USA.

31


2.7 Light microscopy and fluorescence microscopy for the analysis of CaOx crystal

morphology

To better understand the morphology and arrangement of the SPs in the CaOx

crystals, polysaccharide was labeled with fluorescein isothiocyanate (FITC). 5 mg of each

polysaccharide was added to 0.1 mL of phosphate buffer (PBS) at pH 7.0 containing 0.1

mg FITC. The solution was kept in an environment with reduced brightness, at room

temperature for 1 hour. The material was then labeled with deionized water in membranes

with pores of 6 kDa, and then lyophilized. Samples without SPs, as well as samples of

FITC-labeled polysaccharides were not subjected to new production of CaOx crystals and

slides, assembled according to item 2.4. Images were captured from different fields under

a fluorescent microscope (TE-Eclipse 300, Nikon, Melville, NY, USA). We performed

three different experiments.

2.8 Statistical Analysis

All of the data are expressed as the mean ± standard deviation (n = 3). The

ANOVA test was performed to check the difference between results. The Student–

Newman–Keuls test (p < 0.05) was used to solve similarities found by ANOVA. All tests

were performed in GraphPad Prism 5 (GraphPad Softwares, La Jolla, CA, USA).

3. Results and Discussion

3.1 C. cupressoides’s sulfated polysaccharides extraction, chemical and

physicochemical analysis

In a previous study [23], our group extracted four populations of sulfated

polysaccharides from the C. cupressoides seaweed. These populations were named as

CCB-F0.3, CCB-F0.5, CCB-F1.0, and CCB-F2.0, respectively. In this work, using the

same previously described methodology, we also obtained four polysaccharide

populations from C. cupressoides. These samples were subjected to agarose gel

electrophoresis in PDA buffer. In figure 2 we showed an electrophoresis slide stained

with toluidine blue, the toluidine blue has affinity for SPs. We can see the four populations

stained with toluidine blue, indicating that they are constituted of SPs. Different

electrophoretic mobility can also be verified: CCB-F0.3, CCB-F0.5 are polysaccharides

of low mobility in comparison with the others and CCB-F1.0 presented intermediate

mobility, whereas CCB-F2.0 was the population with higher mobility. By comparing our

32


data with the electrophoresis slide presented by Costa and

colleague [23], we can confirm the identity of the polysaccharides obtained.

As observed previously [23], in all extracted SPs, the protein contamination was

below 0.1% (Table 1), confirming the efficacy and reproducibility of the extraction

methodology.

Table 1 revealed a higher sulfate/sugar ratio for CCB-F0.3 (~1.10) and the lowest

ratio for CCB-F2.0 (~ 0.72). These values differ from those previously observed [23];

these authors found CCB-F0.5 with higher sulfate/sugar ratio and CCB-F1.0 with the

lowest ratio of the four fractions. These data were not surprising, since many authors

report changes in the chemical compositions of polysaccharides extracted from the same

species of seaweed when collected at different sites [28, 29]. However, seaweed, both in

Costa and colleges [23] as in here, were collected at the same site (6 ° 1'8.19 "S - 35 °

6'33.40" W), which discards this possibility, even though they were collected in different

years, which takes seaweed to be exposed to different environmental conditions, leading

to changes in the SPs composition. This effect has already been reported by different

authors, but these variations are not homogeneous; each species of seaweed responds

differently to the environmental changes in the collection site, like the green seaweed

Ulva fasciata’s SPs varied in yield, monosaccharide composition and sulfate amount [30];

on the other hand, Delesseria sanguínea’s SPs’ monosaccharide composition and sulfate

content have not been affected by the seasonality [31].

In order to evaluate whether the chemical composition differences of SPs in this

study interfered with the biological activity described earlier [23], we choose the

antioxidant test of iron chelation to verify if the activity of SPs obtained here was similar

to that previously described. As seen in Table 1, the iron chelating activity of the samples

varied from 34 ± 2% (CCB-F1.0) to 53 ± 1% (CCB-F0.5). These values are similar to

those observed by Costa and colleagues [23] and indicates that, despite the differences

between sulfate/sugar ratios found in this study and previously published [23], we believe

that these variations promote only subtle differences in polysaccharide structure, and

these differences are not sufficient to affect the activities of SPs of C. cupressoides. This

led us to continue our studies with the obtained polysaccharides.

33


3.2 Effect of sulfated polysaccharides C. cupressoides on the formation of calcium

oxalate crystals

It is possible to mimic the formation of these crystals in vitro [15] and to evaluate

the effect of compounds on the formation of these crystals. Based on light field optical

microscopy, we can infer the effect of SPs on the formation of crystals by quantification

of each type of crystal. The data obtained are summarized in Table 2.

When we analyze the data showed in the table, we can see that the presence of

polysaccharides increased the amount of crystals formed. In the presence of CCB-F0.3,

the amount of crystals increased 12-fold, followed by CCB-F1.0, which increased by

approximately 9-fold. However, despite the increase in the amount of crystals, the size of

the crystals formed when treated with C. cupressoides seaweed SPs was reduced about

four-fold on average, reaching, in some cases, about 1 µm after treatment (Table 2). The

CCB-F0.3 again stood out by decreasing 7 times the size of COM and 5 times the size of

COD.

Wesson and collaborators [32] proposed that anionic compounds tend to increase

the number of crystals and decrease their size since these negatively charged compounds

interact more with the rich calcium faces of both COM and COD crystals, blocking their

growth [32]. In addition, the anionic compounds induce repulsions between the formed

nuclei (the nano/micro crystals), preventing the aggregation process, as they are

associated with the crystals. These two factors prevent the formation of larger

crystals. With respect to the large number of crystals formed in the presence of anionic

compounds, we believe that as the ions are not completely consumed during nuclei

formation, these are available for the formation of new nuclei. These factors together

justify the observation of the large number of small crystals formed in the presence of C.

cupressoides SPs.

Also, in Table 2, we can see that all obtained SPs (except CCB-F2.0) induced the

formation of an amount of COD-type larger than COM-type, and, again, CCB-F0.3 was

highlighted, as in the presence of CCB-F0.3, the number of crystals was 209 ± 12.3 units

of COD type, much higher than the number of COM type (48 ± 10.9 units), making a

ratio of approximately 4 COD for each COM.

It is important to emphasize that COM-type crystals, when formed in vivo, can be

concentrated in the renal tubular fluid and interact with kidney tubular epithelial cells,

giving them enough time to grow on the cell surface or to aggregate mutually so as to

form large crystals, which finally leads to the formation of kidney stones. On the other

34


hand, COD crystals, due to their morphology, have an area of contact with the minimal

renal epithelium and, therefore, bind the cell membrane in a smaller amount [33].

Other SPs of different seaweeds showed the ability to inhibit the formation of

COM crystals or to stabilize the dihydrate form of the CaOx crystal (COD). For example,

Ouyang and colleagues [14] worked with the edible seaweed Laminaria japonica and

found that its native and modified SPs induced the formation of only COD crystals

[14]. However, SPs from Dictiopteris justii are able to inhibit the formation of CaOx

crystals. In addition, its sulfated glucan was also able to stabilize CaOx crystals in the

COD form [16]. There is no hypothesis that explains how SPs stabilize CODs. However,

studies with carboxylated polymers with the same net charge showed that they did not

influence the COD stability in the same way, and it was proposed that the distribution of

the charges around the molecule was a more important factor than the charge alone during

this process of stabilization [34]. So, this should probably be an important factor for SPs

as well.

3.3 Morphology of the crystals formed in the presence of Caulerpa polysaccharides

For a more detailed analysis of the morphological characteristics of the formed

crystals, the images were obtained by scanning electron microscopy (SEM) and were

analyzed subsequently. The results are summarized in Figure 3.

The images of Figure 3 corroborate with the results of Table 2, showing that there

is an increase in the amount of formed crystals, but decreasing their sizes. Still in

agreement with Table 2, there are more COD-type crystals in the crystal images formed

after incubation with CCB-F0.3, CCB-F0.5 and CCB-F1.0 (Fig. 3C to 3G) when

compared to Control crystals (Figures 3A and 3B). The crystals that were formed without

the incubation with the SPs (control) had the morphological characteristic of COM and

COD, marked by isotropic growth on all the faces of the crystals. Moreover, we observed

control only in the crystals of the COT-type (arrow tipped diamond, Figure 3B), i.e., all

treatments with seaweed SPs C. cupressoides inhibited the formation of this crystal.

Regarding the COD crystals, after treatment, they showed a more rounded shape

(without well-defined tips) than the control group. CCB-F0.3 (Figure 3C) induces the

formation of nearly round, spherical crystals with the tiny (100) faces, evidenced by the

arrow in Figure 3D. The CCB-F2.0 and CCB-F1.0 polysaccharides induce the formation

of COD, which assumes the tetragonal bipyramid shape, but with the tips slightly

rounded. CCB-F0.5 induces the formation of crystals with tetragonal bipyramid structures

35


with thicker (100) faces (white arrow with arrowhead, Figure 3E and 3F); besides, these

crystals have rounded edges when compared to COD formed in the control.

Still regarding COD crystals, we also observed that both CCB-F0.5 and CCB-F1.0

induces the formation of a mixture of COD crystals, which, in addition to the already

described types, also forms dumbbell-shaped crystals. These crystals showed two

hemispheres connected with a rod (Figure 3F). COD crystals with traditional bipyramidal

structure have the face (101) as the dominant face. According to Thomas and colleagues

[35], the COD crystals only take the form of dumbbells when the ratio between faces

{100} and {101} is greater than 1, that is, the face (100) grows much more than the face

(101). These authors also demonstrated that the presence of polyacrylate (PAA) also

promotes the formation of COD crystals in the form of dumbbells. This indicates that

COD crystals in the form of dumbbells can be formed in the presence of some anionic

compounds. However, we still do not know what SP’ structural features are required for

this to occur.

Regarding COM crystals, when formed after treatment with C. cupressoides's

SPs, they were smaller and had structure with the faces {100} dominating. There was an

increase in growth in the direction [010] rather than growth in the direction [100]. This

makes COM have edges and tips with less sharp angles, as they are shaped like an ellipse

(oval in the form of a plaque). Thus, the morphological characteristic assumed by COM

crystals after incubation with C. cupressoides's SPs is advantageous, as while leaving the

edges of their faces with a slight angle (rounded), the possible interaction of these crystals

in renal epithelium can be decreased, which will consequently favor its passage in the

urinary tract, thus reducing the formation of kidney stones. In agreement with our

hypothesis, there are studies that analyzed the crystals of CaOx contained in the urine of

lithogenic patients, and they verified that the COM crystals had edges and tips with sharp

angles and concluded that these are important factors for the anchorage of these crystals

in the renal epithelium [33, 36].

3.4 FT–IR spectrum analyses

The COM and COD crystal FT-IR spectra are showed in Figure 4. We can observe

that the main difference between these spectra is the region between 3040 and 3500 cm-

1. While in COM crystal spectra, there are at least five prominent bands (Figure 4) in this

same region. In COD crystal spectra, there is only one great intensity band around 3485

cm-1 (Figure 4 – COD spectra). Other typical signal of COM crystal were observed around

36


947, 885 and 663 cm-1 (Figure 4 – COM spectra), whereas characteristic COD signals

were observed at 916 and 609 cm-1. Both spectra are similar with those previously

described for COM and COD crystals [37, 38].

Since we observed that more COD crystals were formed in the presence of CCB-

F03 and more COM crystals were formed in the presence of CCB-F2.0, these samples

were chosen to perform infrared analysis. In these spectra of the crystals formed in the

presence of CCB-F0.3 and CCB-F2.0, there are bands around 1232-1256 cm-1, which

indicate asymmetric vibration S=O [39], and the bands around 1150 e 1025-1033 cm-1

are indicative of vibration associated to C-O-S-O [40]. These bands were also observed

in the spectra of the crystals obtained in the presence of the other SPs (data not shown)

and confirm the presence of SPs CCB-F0.3, CCB-F0.5, CCB-F1.0 and CCB-F2.0 in

CaOx crystals.

Spectra analysis also allows noticing that, in the presence of CCB-F0.3, there is

a predominant formation of COD crystals, since a single band was observed in the region

between 3040 and 3500 cm-1. However, in the CCB-F2.0 spectra, it was possible to

observe multiple bands in this region, indicating that SPs are mainly complexed with

COM crystals. These data thus corroborate with those obtained by microscopic analyzes.

3.5 Zeta potential

The zeta potential (ζ) of crystals formed in the presence of C. cupressoides’ SPs

was measured in order to verify if changes in the number and/or morphology of crystals

were associated with changes in the surface charge of these crystals. The results are shown

in Table 3.

The ζ mean value of untreated CaOx crystals was + 8.85 ± 3.30 mV. This positive

profile of crystal charge surface can be mainly related with the presence of calcium ions

in crystal structure. All SPs decreased the zeta potential of CaOx crystals, ranging from -

25.82 ± 6.36 mV in the presence of CCB-F0.3 to -68.70 ± 12.01 mV in the presence of

CCB-F2.0.

An interesting fact is that the increase of crystals ζ did not correspond to the

sulfate/sugar ratio (Table 1), that is, polysaccharide with high amounts of sulfate groups

did not promote formation of crystals with lower ζ value. Other authors have already

noted this fact [15, 16], and it is proposed that SP associated with crystal tends to assume

a conformation that allows it to have higher or lower exposition of its charged groups.

Therefore, it is a situation where, in a negatively charged polysaccharide, it may assume

37


a conformation in which its charges are not so exposed in the crystal surface, resulting in

ζ of the crystal being closer to zero.

The ζ increases with the presence of SPs of C. cupressoides, which explains part

of the formation of many small crystals. The elevated negative charge on crystal surface

would lead to repulsion of other crystals and would block crystal aggregation and

growth. This interference in crystal aggregation/growth has also been observed by other

authors while working with other seaweeds SPs [15, 16]. In those works, the authors also

report formation of many small crystals and elevated crystal negative charge.

Zeta potential analyzes provide us great indications that SPs interact with CaOx

crystals structure. However, there are no studies that actually prove direct binding

between SPs from seaweed and CaOx crystals. Despite, this connection is well described

for other molecules that also have negative charges in their structure, such as polyacrylate

[35], osteopontin [41, 42], glycosaminoglycans [43], and citrate [44], for example.

Therefore, it can be confirmed, based on the data presented here and compared with those

in the literature, that SPs seaweeds are able to alter the surface charge of CaOx crystals

and thereby modify their crystallization dynamics.

3.6 Fluorescence and flow cytometry analyzes of CaOx crystals and FITC-labeled

SPs.

In another set of experiments, the SPs were covalently conjugated to the FITC, as

described in Methods (item 2.7), and used as fluorescent probes with goal to observe

binding of SPs to the formed crystals. To this end, FITC-labeled polysaccharides were

incubated with the supersaturated solutions inducing the formation of CaOx crystals, and

the crystals resulting from this incubation were analyzed by flow cytometry and

fluorescence microscopy. As control, crystals formed in the presence of fluorescently

unlabeled SPs were used.

As expected, crystals formed in the presence of unfluorescent SPs were not

detected by flow cytometry. However, using this technique, we verified that 90% of

crystals formed in the presence of fluorescent SPs showed positive FITC signal.

When those crystals were analyzed by fluorescence microscopy, we noticed that

they appear marked by FITC (green) in almost all of their totality (Figure 5); besides, we

were able to differentiate formed COM and COD crystals. These data give evidence that

SPs are interacting with entire crystalline network, probably with calcium present in these

crystals.

38


3.7 Stabilization of COD crystals

The data presented so far showed that, with the exception of CCB-F2.0, all other

SPs of C. cupressoides induce higher amount of COD crystals in comparison to the

positive control. In addition, SPs gave COD crystals’ higher stability. This behavior has

already been observed by Escobar and colleagues [45], in the presence of other sulfated

(dermatan sulfate, oversulfated heparin and keratan sulfate) and phosphorylated

(phosphorylated chitosan) polysaccharides. Interestingly, there was a greater formation

of COM crystals when these authors used desulfated dermatan, chitosan and hyaluronic

acid (carboxylated, but not sulfated polysaccharide). These results show the importance

of sulfate groups in polysaccharides for the stabilization of COD crystals [45].

However, not every sulfated polysaccharide promotes stabilization of COD

crystals, as shown by Melo and colleagues [16] and also by our results, since CCB-2.0

induces more COM formation. This shows that the SP’s COD stabilizing effect is not

merely a charge effect, but it also depends on how charges are distributed across the

polysaccharide chain. Moreover, these data show that COD formed in the presence of the

different C. cupressoides’s SPs may take different forms, which seems to indicate that

SPs can stabilize COD in different ways, perhaps because they associate with crystals in

different faces.

In order to confirm this hypothesis, the surfaces of the COD crystals obtained after

the incubation with the SPs studied here had their atomic composition characterized

through the spectroscopic chemical microanalysis of X-rays by energy dispersion (EDS).

The sulfur atoms were quantified at different points of the crystals: apex, face and base

(Figure 6A). The sulfur found on the surface of the crystals should represent the sulfate

(SO42-) groups of SPs, since CaOx is composed only of calcium, carbon and oxygen, so

some considerations have been made.

The results from analyzes are summarized in the table of Figure 6B. We observed

in this table that three situations occur in the sulfur distribution: the first occurs in the

crystals formed with CCB-F0.3 and CCB-F2.0, where there is twice the amount of sulfur

in the region of the base than in the other portions; The second occurs with the crystals

formed with CCB-F0.5, where there are almost ten times more sulfur at the base of these

crystals than at the apex or face; The last situation is when there is a different distribution

at the base, face and apex, with the amount of sulfur at the base being twice as large as

the apex, as is the case with CCB-F1.0.

39


We found no other articles that have done this type of analysis; therefore, it was

not possible to compare our results with those of other authors. However, some

considerations have been made.

Is it necessary for the SPs to be distributed differently throughout the crystal so

that there is a greater stabilization of the COD crystals? It does not seem so, since CCB-

F1.0 has this type of distribution, but the COD:COM ratio obtained with this sample was

only 2: 1 (Table 2).

The other SPs were more concentrated on the base. However, there is a subtle

relationship between the amount of sulfur that should be at the base, apex and face. If

there is a lot of sulfur in the base (about ten times more) than in the other points, as

occurred with the presence of CCB-F0.5, there is stabilization of COD, but with only a

COD: COM ratio of 3: 1 (Table 2). The ideal distribution for COD stabilization seems to

be that observed with CCB-F0.3, since the COD: COM ratio was 5:1. The CCB-F0.3

concentrates more on the base, but its amount is only twice of that observed in the other

points; in addition, the amount of this SP at the apex and face are similar. This distribution

profile was also observed with CCB-F2.0. However, we found that the crystals formed

with this polysaccharide have twenty times less sulfur than the crystals formed with CCB-

F0.3.

4. Conclusions

Four sulfated polysaccharides were extracted from the seaweed Caulerpa

cupressoides and were named CCB-F0.3; CCB-F0.5; CCB-F1.0; and CCB-F2.0. These

SPs interact in vitro with calcium oxalate crystals, making their surface’s more negative.

They also induce the decrease in the size and number of formed crystals. This effect did

not depend on the amount of sulfate groups present in SPs. With the exception of CCB-

F2.0, SPs induce the formation of a greater amount of COD crystals compared to the

control group, with CCB-F0.3 being the most efficient. This occurred because CCB-F0.3

was distributed in the base:apex:face of the crystal in a ratio of 2:1:1. We believe that this

balance/proportion between SPs at the interaction points is crucial to avoid dehydration

of the COD crystal to COM, which guarantees their stability.

Acknowledgements

The authors wish to thank Conselho Nacional de Desenvolvimento Científico e

Tecnológico (CNPq), Coordenação de Aperfeiçoamento Pessoal de Nível Superior

40


(CAPES) and also Ministério de Ciência, Tecnologia, Inovações e Comunicações

(MCTI) for the financial support. Hugo Rocha is an honored CNPq fellowship researcher.

Dayanne Lopes Gomes, Karoline Rachel Theodósio Melo and Moacir Queiroz Fernandes

Neto have a Ph.D. scholarship from CAPES. The Infrared Spectroscopy Analysis was

carried out in the Department of Chemistry of the Federal University of Rio Grande do

Norte, and the Scanning Electron Microscopy was carried out in the Department of

Materials Engineering of the Federal University of Rio Grande do Norte. The authors

would like to thank and acknowledge the permission to use the facilities of these

laboratories. This research was presented at Programa de Pós-Graduação em Ciências da

Saúde at Universidade Federal do Rio Grande do Norte, as part of the Ph.D. thesis of

Dayanne Lopes Gomes.

Conflicts of Interest

The authors declare no conflict of interest.

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[41] B. Grohe, J. O'young, D. Ionescu, et al., “Control of calcium oxalate crystal growth

by face-specific adsorption of an osteopontin phosphopeptide”, Journal of the

American Chemical Society, vol. 129, no. 48, pp. 14946–14951, 2007.

[42] Y-C. Chien, D. L. Masica, J. J. Gray, S. Nguyen, H. Vali, and M. D. McKee,

“Modulation of calcium oxalate dihydrate growth by selective crystal-face binding

of phosphorylated osteopontin and polyaspartate peptide showing occlusion by

sectoral (compositional) zoning”, Journal of Biological Chemistry, vol. 284, no. 35,

pp. 23491–23501, 2009.

[43] S. E. R. Hernandez and N. H. Leeuw, “Effect of Chondroitin 4-Sulfate on the Growth

and Morphology of Calcium Oxalate Monohydrate: A Molecular Dynamics Study”,

Crystal Growth & Design, vol. 15, no. 9, pp. 4438−4447, 2015.

[44] S. R. Qiu, A. Wierzbicki, C. A. Orme, et al., “Molecular modulation of calcium

oxalate crystallization by osteopontin and citrate”, Proceedings of the National

Academy of Sciences, vol. 101, no. 7, pp. 1811−1815, 2004.

[45] C. Escobar, A. Neira-Carrillo, M. S. Fernández, and J. L. Arias, “Role of sulfated

macromolecules in urinary stone formation”, Biomineralization: From Paleontology

to Materials Science, pp. 343–358, 2007.

46


Tables

Table 1. Chemical characterization and Chelating ability of SPs from C. cupressoides. D-

galactose and sodium sulfate were used as standards for sugar and sulfate quantification,

respectively. The concentration of each SP used to perform the iron chelation test was 1.5

mg/mL. Data are expressed as the mean of three determinations ± standard deviation.

Different letters indicate significant difference for the same amount of SPs (p <0.05).

Polysaccharide (SO4)/total sugar

(%/%) Protein

(%) Chelating ability

(%) CCB-F0.3 1.10 ± 0.02 a

0.06 ± 0.01 a 36 ± 4 a

CCB-F0.5 0.86 ± 0.03 b

0.05 ± 0.01 b 53 ± 1 b

CCB-F1.0 0.85 ± 0.02 b 0.11 ± 0.02 c 34 ± 2 a

CCB-F2.0 0.72 ± 0.01 c 0.11 ± 0.01 c 13 ± 4 c

a, b, c Different letters indicate a significant difference between the SPs of C. cupressoides (p <0.05)

47


Table 2. Number and average size of the crystals formed by treatment with sulfated

polysaccharides from C. cupressoides. COM - monohydrate calcium oxalate crystals;

COD - dihydrate calcium oxalate crystals; COT - trihydrate calcium oxalate crystals.

COTs were only found in the control (1 ± 0.9 units).

Total amount

of crystals

(units)

COM (units) COM size

(µm)

COD

(units)

COD size

(µm)

Control CaOx 21 ± 4.4 13 ± 4.1 11.9 ± 0.16 7 ± 2.0 12.8 ± 0.89

CaOx + CCB-F0.3 257 ± 4.2 48 ± 10.9 1.7 ± 0.08 209 ± 12.3 2.55 ± 0.19

CaOx + CCB-F0.5 77 ± 8.3 20 ± 6.9 4.2 ± 0.64 58 ± 9.8 4.9 ± 0.12

CaOx + CCB-F1.0 184 ± 10.8 66 ± 9.1 4.4 ± 0.29 118 ± 9.8 4.7 ± 0.79

CaOx + CCB-F2.0 32 ± 9.4 23 ± 7.3 4.8 ± 0.96 9 ± 3.3 6.2 ± 0.83

.

48


Table 3. Zeta potential of crystals formed in the presence of polysaccharide fractions of

C. cupressoides seaweed at 25 °C.

a, b, c, d Different letters indicate a significant difference (p <0.05) in Zeta Potential between CaOx formed

with the presence or absence of polysaccharides.

ζ (mV)

CaOx + 8.85 ± 3.30a

CaOx + CCB-F0.3 - 25.82 ± 6.36b

CaOx+ CCB-F0.5 - 43.87 ± 8.63c

CaOx + CCB-F1.0 - 51.50 ± 2.14c

CaOx + CCB-F2.0 - 68.70 ± 12.01d

49


Figure Legends

Figure 1. Representation of the faces of the calcium oxalate crystals. (A) Crystals COM;

(B) Crystals COD. The numbers presented between "parentheses" represent each face of

a crystal; The numbers displayed between “brackets” represent the direction of growth of

each face of a crystal; And, the numbers presented between "braces" represent the set of

same faces of a same crystal.

Figure 2. Electrophoretic mobility of the sulfated polysaccharides from C. cupressoides

seaweed. 50 μg of the Caulerpa SPs were applied on agarose gel prepared with 1,3-

diaminopropane-acetate buffer 0.05M, pH 9.0, and then subjected to electrophoresis at

90 V/cm for 60 min. The gel was maintained in 0.1% cetyltrimethylammonium bromide

for 2 h, dried and subsequently stained with 0.1% toluidine blue (in 50% ethanol and 1%

acetic acid in water) for 15 min. The gel was unstained with the same staining solution

without the dye.

Figure 3. Scanned Electron micrographs of the crystals formed morphotypes after

incubation with polysaccharides of green seaweed C. cupressoides. The CaOx crystals

were formed in a stable CaOx solution target (1 mM) in the absence (A and B images)

and in the presence of SPs C. cupressoides (0.25 mg/mL). (C and D) CCB-F0.3; (E and

F) CCB-F0.5; (G) CCB-F1.0; (H) CCB-F2.0. Shows the COM shape;

Shows the COD form and shows the COT form.

Figure 4. Infrared spectrum of COM, COD and CaOx crystals formed after incubation

with Caulerpa SPs. FT-IR with main signals found in infrared spectra of CaOx crystals

formed in the presence and absence (control) of CCB-F0.3 and CCB-F2.0. Signs in red

represent the unique peaks of COM; Signals in blue represent the unique COD peaks;

Pink signs represent the fashion hybrids for COM and COD; Signs in green represent the

sugar or sulfate peaks.

Figure 5. Fluorescent crystal analyzes by flow cytometry and fluorescence microscopy.

(A) Fluorescence quantification of crystals labeled by fluorescent SPs and detected by

flow cytometry, expressed as percentage; (B) Comparison between crystals in light field

microscopy (non-fluorescent), observed in the upper line, and the SPs-FITC-conjugated

crystals (fluorescent in green), observed in the bottom line. The assays were performed

twice (n = 3).

50


Figure 6. Crystals surfaces’s atomic composition characterization by EDS. (A)

Representation of selected sites for quantification of sulfur atoms (apex, face and base);

(B) Percentage of sulfur distribution in different parts of CaOx and CaOx treated with

polysaccharide; (C) Calcium (red dots) and oxygen (blue dots) marking in CaOx with the

presence of CCB-F0.5; (D) Increase in sulfur marking (green dots) in CaOx with the

presence of CCB-F0.5.

51


Figures

Figure 1

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Figure 2

53


Figure 3

54


Figure 4

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Figure 5

56


Figure 6

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5.2. ARTIGO 2

58


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76


6. COMENTÁRIOS, CRÍTICAS E SUGESTÕES

A busca para elucidar um possível mecanismo de interação com os PS e

os cristais de CaOx nos fez recorrer a disversas técnicas, o que conferiu um

caráter interdisciplinar ao trabalho. Realizamos testes em departamentos de

diversas áreas da UFRN (Química, Engenharia de Materiais e Instituto do

Cérebro). Portanto, o desenvolvimento deste estudo me proporcionou a

oportunidade de interagir com diversos ramos da pesquisa científica e considero

esse fato imprescindível não só para conseguir boas publicações como também,

e principalmente, para formação profissional e científica.

Ao longo do desenvolvimento desta pesquisa, o cronograma de atividades

seguiu dentro dos padrões esperados e todas as dificuldades encontradas foram

superadas. No 3º mês após o ingresso no programa fiquei grávida. Porém, isso

não interferiu no cumprimento das disciplinas. O fato de já estar inserida no grupo

da pesquisa há 11 anos facilitou para que eu dinamizasse a pesquisa no retorno

da licença maternidade. Não posso deixar de citar os colaboradores do próprio

laboratório (BIOPOL/UFRN), que de forma séria porém ao mesmo tempo

bastante descontraída nos ajudam a vencer os obstáculos no decorrer dos anos.

É importante ressaltar que durante esse período além da condução da

minha pesquisa de doutorado fiz algumas orientações às pesquisas dos alunos

de iniciação científica e também colaborações com outros projetos de mestrado

e doutorado. Alguns desses trabalhos já foram publicados, outros estão em

processo de construção e submissão/aprovação dos artigos. Dos já publicados

nos últimos quatro anos estão a produção de 6 artigos publicados em revista

científicas, sendo um como primeira autora, dois como segunda autora e os

demais dividindo co-autoria. As revistas foram: International Journal of Biological

Macromolecules, Evidence-Based Complementary and Alternative Medicine,

Marine Drugs, Molecules e Artificial Organs. Além disso, também fiz parte de 5

(cinco) bancas examinadora de trabalhos de conclusão de curso (monografia).

Durante esse processo de formação também participei de alguns

Congressos, Simpósios e Encontros científicos internacionais e nacionais como

autora ou em co-autoria de 18 resumos científicos, como por exemplo nos 42º e

45º Annual Meeting of the Brazilian Society for Biochemistry and Molecular

Biology (SBBq), 2013 e 2016. Destaco a minha participação no IV CURSO DE

77


FÉRIAS DE MORFOLOGIA: UM ELO ENTRE OS DISCENTES DA FACISA E

DO ENSINO MÉDIO ministrando a palestra “Mitos e Verdades sobre Cálculos

Renais” no ano de 2016. Essa iniciativa de passar o meu conhecimento científico

para a comunidade foi bastante enriquecedor e retrata o real papel da

Universidade.

Como projeto para o futuro, planejamos continuar com os estudos na

mesma linha de pesquisa, em um futuro pós-doutorado para finalizar alguns

questionamentos levantados ao longo da pesquisa que prometem excelentes

resultados.

78


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81


APÊNDICES

82


8.1. ARTIGO 3

Baccharis trimera (Less.) DC Exhibits an Anti-Adipogenic Effect by

Inhibiting the Expression of Proteins Involved in Adipocyte Differentiation

Periódico: Molecules




83


84


8.2. ARTIGO 4

Bothrops jararaca and Bothrops erythromelas Snake Venoms Promote

Cell Cycle Arrest and Induce Apoptosis via the Mitochondrial

Depolarization of Cervical Cancer Cells

Periódico: Evidence-Based Complementary and Alternative Medicine (Print)


ISSN: 1741-427X (Printed version)


85


86


8.3. ARTIGO 5

Fucan-coated silver nanoparticles synthesized by a green method induce

human renal adenocarcinoma cell death

Periódico: International Journal of Biological Macromolecules




87


88


8.4 ARTIGO 6

Evaluation of Anti-Nociceptive and Anti-Inflammatory Activities of a

Heterofucan from Dictyota menstrualis

Periódico: Marine Drugs


ISSN: 1660-3397


89


90


ANEXOS

91


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92


93


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or surgical procedure used, as well as evidence that all possible steps were taken

to avoid animal suffering at each stage of the experiment.

universidade federal do rio grande do norte · obrigada por me abençoar muito mais do que mereço....

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