UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE · 2019-05-26 · Não sei se somos almas gêmeas,...

MINISTÉRIO DA EDUCAÇÃO

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

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE

EFEITO IMUNOMODULADOR E ATIVIDADE ANTIMICROBIANA DE HETEROFUCANAS DE Sargassum filipendula

CINTHIA BEATRICE DA SILVA TELLES

NATAL/RN 2015



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 2015

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MINISTÉRIO DA EDUCAÇÃO

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

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Aprovada em: 26/06/ 2015

Banca Examinadora:

Presidente da Banca:

Prof. Dr. Hugo Alexandre de Oliveira Rocha (UFRN)

Membros da Banca

Prof. José Veríssimo Fernandes

Prof. Valter Ferreira de Andrade Neto

Prof. Toshiyuki Nagashima Junior

Prof. Danilo José Ayres de Menezes

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Dedico esta obra

A Deus.

Pela minha existência e por me oferecer condições para realizar essa conquista.

Ao meu filho, Davi.

Você é minha VIDA, tem me ensinado a cada dia o que é o amor incondicional, a

minha maior alegria é a sua felicidade. É o seu sorriso, a sua alegria de viver, seus

beijos e abraços apertados que me proporcionam força para continuar nessa

caminhada. Te amo muito.

Ao meu marido, Jean.

Não sei se somos almas gêmeas, afinal temos tantas diferenças, mas tenho a certeza

que nos completamos… você é a alegria que falta nas minhas manhãs, é a

sensibilidade dos meus dias… Obrigada por estar sempre ao meu lado.

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Dedico esta obra

Aos meus pais, Ana e Carlos.

Por serem exemplo de amor incondicional, presentes em todos os momentos da

minha vida, com certeza essa conquista foi construída graças a todo amor, apoio e

incentivo dedicado a mim durante todos esses anos. Espero poder retribuir todo o

investimento, e ainda proporcionar muito orgulho.

Ao meu irmão, Agusto.

Exemplo de homem… hoje tenho certeza que o que sou devo muito a você, meu

irmão mais novo, mas que sempre foi o mais maduro de nós dois… Agradeço o seu

amor, carinho, compreensão, apesar de não usarmos muito as palavras para

expressar todo o nosso amor, nos amamos muito.

A Hugo Rocha,

Amigo e orientador, exemplo de amor pela profissão… obrigada pela paciência, pelos

ensinamentos, pelos momentos de descontração e principalmente por estar ao meu

lado na conclusão desse trabalho, a caminhada foi longa, mas, com certeza, só

consegui realizar com o seu incentivo e confiança.

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Agradecimentos especiais

Ao meu marido. Jean Gouveia, meu amigo e companheiro. Obrigada por dividir a sua

vida comigo, que nosso amor supere todos os obstáculos.

Ao meu filho Davi, que Deus abençoe seus caminhos, e lhe dê muita saúde e

sabedoria, estarei sempre ao seu lado, lutando pela sua felicidade. Te amo muito.

À minha família, em especial, meus pais Ana Lúcia e Carlos Roberto e ao meu irmão Augusto César que me incentivaram em todos os momentos;

Ao amigo e orientador, Hugo Rocha, por estar ao meu lado nessa caminhada.

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) e do DBQ (UFRN), aos coordenadores do

programa de pós-graduação (PPGCSa) e às secretárias do programa.

Aos membros da banca de qualificação: Profa. Luciana Guimarães, que desde a qualificação do mestrado ofereceu valiosas sugestões para melhorar a minha pesquisa e a

e a Jailma Almeida uma profissional exemplar e sempre dedicada.

Aos meus amigos de laboratório pela colaboração e ajuda nos meus experimentos e pelos

momentos de descontração:

A Jadrilma minha amiga, madrinha, anjo… obrigada por fazer parte da minha vida

profissional e pessoal, devo muito da minha “transformação” ao seu carinho e presença na minha vida e na vida do meu filho… obrigada por tudo.

A Dayn exemplo de perseverança, mãe, mulher e profissional exemplar. Desejo muito que

o seu futuro profissional seja brilhante, tenho um grande carinho por você.

A Ruth e a Rafael, amigos que admiro muito, a Leandro (ou será rosa) hoje faz parte da família, você é um exemplo de profissional.

A Mariana ou Santana, amiga do lab, da turma do mestrado, hoje amiga para a vida, você

é um exemplo de dedicação, de amor pelo que faz obrigada pela ajuda sempre que precisei te adoro!

Agradeço muito à família BIOPOL, todos vocês são essenciais nessa trajetória: Gabriel,

Moacir, Joanna, Pablo, Raniere, Ivan, Leonardo Nobre (Leo), Letícia, Arthur, Vinicius, Max, Rony, Marília, Monique, Larisse, Mônica, Danielle, Almino Afonso, Profa.

Fabiana Lima, Ana Karina, Sara.

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Ao Prof. Marcelo Silva e a amiga Sônia Pestana do Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa que durante os seis meses vividos em Lisboa,

Portugal, compartilharam comigo seus conhecimentos e proporcionaram meses maravilhosos;

A Profa. Tiana Tasca e todas as alunas da Faculdade de Farmácia da Universidade

Federal do Rio Grande do Sul, em especial a aluna Amanda Picolli, pelo acolhimento e

disposição na realização dos ensaios.

Agradeço a Carol pela ajuda na realização dos experimentos com Leishmania e todos do

Instituto de Medicina Tropical da UFRN que ajudaram na realização desse trabalho.

Agradeço, de forma especial, àqueles que contribuíram de forma diferencial para minha formação como pessoa, os meus amigos de SEMPRE: Tathiana, Raiane, Gabriela, Ana Paula, Isabel, Deborah, Nilmara e Isabelli. Sem o apoio de vocês tudo seria mais difícil

Agradeço aos meus sogros: Benigna e Gouveia. Sem a dedicação incondicional de vocês a criação do meu filho, nosso bebe, a realização dessa tese seria bem mais difícil. Muito

obrigada pela ajuda e pelo carinho. Também dedico esta tese a vocês!

Agradeço a todos os amigos que trabalham comigo na EAJ: Lígia, Welson, Ângela, André, Afrânio, Rose, etc. vocês são exemplos de profissionais, tenho aprendido muito

ao lado de vocês. E, em especial, agradeço as substitutas que são insubstituíveis, Maiara e Louize, uma das conquistas durante esse período na EAJ com certeza foi a amizade de

vocês.

Agradeço aos professores e amigos do IFRN campus Currais Novos, em especial, a

Andreilson, Itála, Jane, Jana, Dayana, Maura, Mayara, Lívia, Jonas, Marcelo, Danúbia,

Cris, dentre tantos outros que mesmo com uma palavra de incentivo me deram força para

a conclusão desse trabalho.

Obrigada a todos pelo apoio, pelo carinho, pelo consolo nas frustrações. Agradeço demais

a todos que contribuíram de forma direta ou indireta para a realização desse trabalho.

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“A vida é um sonho, torne-o real”.

(Madre Teresa)

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RESUMO

Macroalgas marinhas constituem uma fonte extremamente rica de compostos

bioativos, dentre eles, polissacarídeos sulfatados. Sargassum filipendula (SF),

alga pertencente à ordem Phaeophycea, é fonte de heterofucanas conhecidas

pela capacidade de modular uma série de funções biológicas. Considerando a

necessidade de encontrar fármacos mais eficientes no combate a infecções

microbianas, as heterofucanas de SF foram avaliadas como agentes

imunomoduladores e antimicrobianos. As heterofucanas SF0.5V, SF0.7V e

SF1.0V apresentaram uma forte atividade imunomoduladora intensificando a

liberação de óxido nítrico (NO) por macrófagos murinos (RAW 264.7), bem

como, por macrófagos originados de monócitos primários humanos. Além

disso, quando macrófagos humanos foram infectados por Leishmania infantum

e tratados com SF0.5V, SF0.7V e SF1.0V ocorreu um aumento significativo na

liberação extracelular de NO. Como a citotoxicidadede de macrofágos contra a

forma intracelular de leishmanias é mediada pela produção de NO, avaliamos a

atividade leishmanicida sobre a forma amastigota intracelular de L. infantum e

observamos que macrófagos infectados e tratados com SF0.5V, SF0.7V e

SF1.0V se tornaram menos susceptíveis à infecção. As heterofucanas que se

mostraram com capacidade de induzir a atividade anti-leishmania também

apresentaram melhor taxa de produção de NO, porém os dados de correlação

levaram a observação que este não é o principal mecanismo de ação das

fucanas de SF no combate a esse protozoário. A heterofucana SF0.5V também

apresentou atividade inibitória da formação de biofilme (~ 50%) frente a

bactéria Staphylococcus epidermidis. Já SF0.7V e 1.0V inibiram quase que

totalmente a replicação do protozoário Trichomonas vaginalis. Resultados

como esse refletem o espectro de ação desses polissacarídeos sulfatados

obtidos de SF e mostram o seu potencial como agentes imunomoduladores e

microbicidas.

Palavras chaves: Imunomodulação; Oxido nítrico; Leishmania infatum,

Trichomonas vaginalis, antibiofilme; macrófagos; fucanas.

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LISTA DE ABREVIATURAS E SIGLAS

µL Microlitros

g Grama

h Hora

kDa Kilodalton

KH2PO4 Fosfato monopotássico

M Molar

mg Miligrama

Min. Minutos

mL Mililitros

mM Milimolar

mm Milímetros

NaCl Cloreto de sódio

NaOH Hidróxido de sódio

nm Nanômetros

PA Para análise

RAEC Linhagem de células endoteliais de aorta de coelho

Raw264.7 Linhagem de células de macrófagos murinos

SF0.5 Fração precipitada com 0,5 volumes de acetona


SF1,5 Fração precipitada com 1,5 volumes de acetona


SF2,0 Fração precipitada com 2,0 volumes de acetona

xii

LISTA DE FIGURAS Figura 1. Esquema representataivo da metodologia de extração e caracterização físico-

química e farmacológica das heterofucanas da alga S. filipendula....................... 25

xiii

SUMÁRIO

RESUMO................................................................................................................................... x LISTA DE ABREVIATURAS E SIGLAS..................................................................................... xi LISTA DE FIGURAS.................................................................................................................. xiii 1. INTRODUÇÃO....................................................................................................................... 15

1.1. SISTEMA IMUNOLÓGICO……………………………………………………………… 15 1.2. PARASITAS VERSUS SISTEMA IMUNOLÓGICO………………………………...... 16

1.2.1. Leihmania sp………………………………………………………………… 16 1.2.2. Trichomonas vaginalis……………………………………………………… 17 1.2.3. Staphylococcus epidermidis e Klebsiella pneumina…………………..... 18

1.3. MODIFICADORES DA RESPOSTA BIOLÓGICA……………………………………. 19 1.4. FUCANAS: POLISSACARÍDEOS SULFATADOS DE MACROALGAS

MARRONS………………………………………………………………………………... 19

2. JUSTIFICATIVA.................................................................................................................... 20 3. OBJETIVOS........................................................................................................................... 21

3.1. OBJETIVO GERAL................................................................................................. 21 3.2. OBJETIVOS ESPECÍFICOS.................................................................................. 21

4. MÉTODOS............................................................................................................................. 22 4.1. MATERIAIS BIOLÓGICOS..................................................................................... 22

4.1.1. Algas...................................................................................................... 22 4.2. OBTENÇÃO DAS HETEROFUCANAS DE Sargassum filipendula....................... 22

4.2.1. Obtenção do pó cetônico..................................................................... 22 4.2.2. Proteólise.............................................................................................. 22 4.2.3. Fracionamento do extrato bruto com concentrações crescentes

de acetona............................................................................................. 23

4.3. ANÁLISES QUÍMICAS........................................................................................... 23 4.3.1. Dosagem de açúcares totais............................................................... 23 4.3.2. Dosagem de sulfato……………………………………………................ 23 4.3.3. Dosagem de proteínas……….............................................................. 24 4.3.4. Determinação da composição monossacarídica……....................... 24

5. ARTIGOS PRODUZIDOS...................................................................................................... 26

5.1. ARTIGO 1 (SUBMETIDO)...................................................................................... 27 5.2. ARTIGO 2 (PUBLICADO)…................................................................................... 53

6. COMENTÁRIOS, CRÍTICAS E SUGESTÕES...................................................................... 72 7. REFERÊNCIAS..................................................................................................................... 74

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Telles, C.B.S. PPGCSA/CCS

1. INTRODUÇÃO

1.1. Sistema imunológico

O sistema imunológico é uma rede interativa de órgãos linfóides, células,

fatores humorais, citocinas, quimiocinas, dentre outros elementos, que

trabalham em conjunto para defender o organismo do ataque desses

“invasores” [1]. A resposta imunológica é dividida em dois tipos de acordo com

a velocidade e especificidade da reação, respostas inatas e adaptativas. A

imunidade inata abrange os elementos do sistema imune (Neutrófilos,

monócitos, macrófagos, complemento, citocinas e proteínas de fase aguda)

que fornecem a defesa imediata do hospedeiro [2]. A adaptativa consiste de

reações específicas contra o antígeno através do recrutamento de linfócitos T e

linfócitos B [2].

Durante o processo infeccioso, ou seja, no momento da invasão e

multiplicação do patógenoé estabelecido um processo inflamatório que é a

resposta protetora do corpo contra a infecção [3]. O início do processo

inflamatório é a detecção pelo sistema imune inato de elementos que são

comuns várias classes de agentes infecciosos, conhecidos como padrões

moleculares associados a patógenos (PAMPs) como, por exemplo, o

lipopolissacarídeo bacteriano (LPS); ou pela identificação dos padrões

moleculares associados ao dano (DAMPs), moléculas endógenas que

sinalizam o dano do tecido às células do sistema imune inato [4]. Após o

reconhecimento de PAMPs ou DAMPs por receptores celulares, como os

receptores transmembrana Toll-like (TLR), expresso na superfície de células

fagocíticas, ocorre uma cascata de sinalização intracelular que promove a

expressão de citocinas pro-inflamatórias como interleucina 1 β (IL-1β),

interleucina 6 (IL-6) e 12 (IL-12), fator de necrose tumoral (TNF), interferon

(INF-γ)e óxido nítrico sintase induzível (iNOS) [5,6], que, justamente com

quimiocinas e moléculas coestimulatórias, induzem o recrutamento de células

efetoras da inflamação como os monócitos e neutrófilos para o sítio do dano. A

resposta imune adaptativa também participa do processo inflamatório,

principalmente através dos linfócitos T auxilares, T CD4+, que quando

estimulados por células apresentadoras de antígeno, podem se diferenciar em

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diferentes tipos de células efetoras: células Th1, pró-inflamatórias; Th2, anti-

inflamatórias; células T regulatórias, Tregs; e células Th17 [7].

As células Th1 são críticas durante a resposta pró-inflamatórias, induzem a

liberação de INF-γ, IL-2 e TNF-α, fudamentais na ativação de macrófagos. As

células Th2 regulam a resposta imune humoral, proliferação de células B,

respostas alérgicas e proteção contra infecções de helmintos, promovendo a

produção de citocinas anti-inflamatórias IL-4, IL-5, IL-10 e IL-13. As células

Treg promovem a supressão da ativação, proliferação e funções efetoras de

várias células imunes como as células T, células NK, células B e células

apresentadoras de antígeno [8]. Secretam citocinas imunossupressoras IL-10 e

TGF-β que inibem a proliferação da resposta Th1 e Th2 para minimizar o dano

tecidual [9]. As células Th17 estão envolvidas na resposta contra

microparasitas extracelulares que necessitam de uma forte resposta

inflamatória e que não são adequadamente tratadas pelas respostas Th1 ou

Th2 [10].

Apesar de o sistema imunológico atuar fielmente na defesa do

organismo, alguns agentes invasores podem transpor a resposta imune e, na

ausência de um sistema imunológico eficaz, mesmo infecções menores podem

se estabelecer e tornarem-se fatais.

1.2. Parasitas “versus” Sistema Imunológico

Os parasitas são um grupo altamente diversificado de organismos que

desenvolveram diferentes estratégias para infectar seus hospedeiros.

1.2.1. Leishmania sp.

As leishmanioses são antropozoonoses consideradas causa de

morbidade e mortalidade em áreas subtropicais e tropicais. É uma doença com

um amplo espectro de manifestações clínicas, que incluem as formas: cutânea,

mucocutânea e visceral [11]. O agente causador da leishmaniose pertence ao

gênero Leishmania [12], família Trypanosomatidae e infecta o homem através

da picada do vertebrado fêmea da subfamília Phlebotominae [13]. Este parasita

tem um ciclo de vida digenético, multiplicando-se no intestino médio de

flebotomíneos na forma promastigota flagelada e como amastigotas não

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flageladas dentro fagócitos de mamíferos [14], sendo considerado um parasita

intracelular obrigatório.

Os macrófagos, células alvo das formas amastigotas de Leishmania,

possuem diversas formas de combater microorganismos invasores, dentre

elas, a ativação da enzima NADPH oxidase que induz a formação de espécies

reativas do oxigênio; a alteração do pH das vesículas fagocíticas levando a

desnaturação proteica [15]; a ativação da enzima óxido nítrico sintase induzida

(iNOS), que quando ativada sintetiza óxido nítrico (NO), molécula efetora no

combate às infecções [16]. Porém, mesmo diante do arsenal imunológico

disponível contra microorganismos invasores, as espécies de Leishmania são

capazes de evadir a resposta imunológica do hospedeiro vertebrado desde o

momento da infecção. A primeira resposta evitada é a lise mediada pelo

sistema complemento, as metaloproteses de superfície majoritárias (MSP),

presente na superfície das formas promastigotas infectantes, atuam clivando o

componente C3 em seus subprodutos e convertendo C3b em sua forma inativa

C3bi, que permanecem aderidas a superfície do protozoário, opsonizando-as e

facilitando sua fagocitose através da ligação aos receptores CR1 e CR3 dos

macrófagos essa ligação leva à diminuição da expressão de INF-γ, IL-12 e,

aumento na secreção de IL-4, contribuindo para a sobrevivência das

promastigotas [17]. Os macrófagos, definem o curso da infecção dependendo

da maneira como são ativadas. As células T auxiliares apresentam papel

fundamental nessa ativação, a expansão de clones Th1 leva a um controle da

doença, enquanto a expansão de clones Th2 leva a uma piora no quadro da

doença [15].

O tratamento das leishmanioses é baseado em antimoniais

pentavalentes (PVAs), cuja ação curativa foi descoberta pelo brasileiro Gaspar

Vianna em 1912 [18]. No entanto, estes fármacos, que vêm sendo utilizadas há

mais de seis décadas, estão longe de serem satisfatórios devido aos seus altos

custos de produção, toxicidade, surgimento de resistência medicamentosa

frente a todas as espécies de leishimania em várias partes do mundo [11], além

de apresentarem reconhecidos efeitos colaterais [19].

1.2.2. Trichomonas vaginalis

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Trichomonas vaginalis é um protozoário flagelado parasita que causa

tricomoníase, a mais comum doença sexualmente transmissível não viral [20].

A aderência e a citotoxicidade exercidas pelo parasita sobre as células do

hospedeiro são controladas por fatores de virulência, como adesinas, cisteino-

proteinases, integrinas, cell-detaching factor (CDF) e glicosidases. A aderência

do parasita as células epiteliais modula a expressão gênica de proteínas

funcionais das células do hospedeiro, como aquelas associadas à manutenção

da estrutura da matriz extracelular, moléculas pró apoptóticas e pró-

inflamatória. Um mecanismo de escape do sistema de defesa do hospedeiro é

a capacidade de T. vaginalis se auto-revestir de proteínas presente no plasma

do hospedeiro, impedindo, assim a identificação deste pelo sistema imune [21].

Metronidazole e Tinidazole são os únicos fármacos recomendados para

o tratamento da infecção por T. vaginalis [22]. No entanto, alguns estudos vêm

mostrando a resistência do T. vaginalis a esses fármacos [23], o que dificulta

ainda mais o controle da infecção.

1.2.3. Staphylococcus epidermidis e Klebsiella pneumonia

As bactérias são os microorganismos que mais frequentemente causam

infecções no homem [24]. Estes microorganismos são capazes de se replicar

tanto no interior das células do hospedeiro como em ambientes extracelulares

[25]. As infecções causadas por bactérias extracelulares são as mais

frequentes. Nesses casos os mecanismos de defesa estão relacionados

principalmente com as barreiras naturais do hospedeiro, a resposta imune

inata, a ativação do complemento e a produção de anticorpos. Nas bactérias

intracelulares a característica principal é a capacidade de sobrevivência dentro

dos macrófagos, podendo estimular as células TCD4+ através da expressão de

antígeno associado ao MHC classe II, os quais produzem citocinas ativadoras

de TCD8+ que reconhecem e destroem as células infectadas que expressam

antígenos associados a moléculas do MHC classe I. A ativação de células

TCD4+ leva à secreção de IFN-γ, que ativa os macrófagos levando à produção

aumentada de NO e destruição da bactéria [24].

Independente do tipo de parasita causador da infecção, o sistema imune

é fundamental para manutenção da homeostase do organismo infectado,

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portanto, torna-se um alvo importante para o desenvolvimento de estratégias

de tratamento. Dentre as abordagens incluem estratégias como vacinação

terapêutica com o designer de adjuvantes para conduzir determinados tipos de

resposta imune [26] e a busca por moléculas imunomoduladoras [27].

1.3. Modificadores da resposta biológica

O crescimento dos conhecimentos de imunologia clínica tem revelado

que a fisiopatologia de doenças pode ser causada tanto por exacerbação como

imunodeficiências da resposta imune. A terapia imunológica moderna é dividida

em dois grupos de imunomoduladores: o dos imunoestimuladores, que

conduzem ao aumento da imunidade inata e adaptativa, e o dos

imunossupressores, que diminuem a atividade do sistema imune [28].

Modificadores da resposta biológica (BRMs) são moléculas que atuam

como ativadores ou supressores da resposta de células do sistema imune [29].

Os principais efeitos biológicos promovidos pelos BRMs são atividades

anticoagulante, antitumoral, antifúngica, antibacteriana e antiparasitária [30-35].

Polissacarídeos de diferentes origens e com características estruturais variadas

vêm sendo estudados quanto as suas potenciais aplicações biológicas [36-40].

1.4. Fucanas: Polissacarídeos sulfatados de macroalgas marrons

As fucanas são polímeros que têm como característica principal à

presença da L-fucose sulfatada na sua estrutura [39,41], são os principais

polissacarídeos sulfatados obtidos das Phaeophyceas ou algas marrons.

O gênero Sargassum C. Agardh (Sargassaceae) constitui um dos mais

representativos dentre os 41 gêneros da ordem Fucales (Phaeophyceae,

Heterokontophyta), é amplamente distribuído nas regiões tropicais e

subtropicais do globo e é considerado um importante componente da flora

marinha [42]. As espécies desse gênero são reconhecidas fontes de fucanas

com uma variedade de atividade biológica: Sargassum horneri [43]; S.

tenerrimum [44]; S. patens [45]; S. stenophyllum [46], S. wightii [47], S. vulgare

[48], S. siliquosum [49].A alga marrom Sargassum filipendula além de possuir

extratos ricos em nutrientes que são utilizados na preparação de produtos

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cosméticos [50], apresenta fucanas ampla capacidade antioxidante e antimural

[51,52].

2. JUSTIFICATIVA

Diante da patogenicidade das infecções causadas pelos microorganismos

avaliados no presente trabalho e, devido ao limitado arcenal terapêutico no

controle desses patógenos, além do efeito adverso dos fármacos disponíveis,

que possuem um alto custo e o surgimento de resistência medicamentosa, vem

sendo travada uma verdadeira batalha entre a criatividade humana na

produção de drogas antimicrobianas cada vez mais potentes e de amplo

espectro de ação.

Substâncias produzidas a partir de produtos naturais, como as macroalgas

marinhas, podem oferecer uma oportunidade para superar esses efeitos

adversos, pois apresenta como vantagens o suprimento sustentável e baixo

custo.

Macroalgas marinhas do grupo das Phaeophyceas são potentes fontes

de moléculas bioativas, dentre elas, destacam-se as fucanas. O gênero

Sargassum é reconhecido por apresentar na constituição das suas espécies

fucanas com ampla atividade biológica. Recentemente, o nosso grupo de

pesquisa demonstrou que a espécie Sargassum filipendula é fonte de cinco

heterofucanas com alta atividade antioxidante e antitumoral. Contudo, apesar

da forte atividade biológica encontrada para as fucanas obtidas desta alga

marron, as atividades imunomuladoras e antimicrobianas destes compostos

ainda não foram estudadas. Assim, este trabalho surge como o primeiro passo

para se entender a possível ação imunomuladora e antimicrobiana das fucanas

de Sargassum filipendula.

21


3. OBJETIVOS

3.1. GERAL

Avaliar o potencial imunomudulador e antimicrobiano das heterofucanas

de S. filipendula frente aos microorganismos Leishmania infantum,

Trichomonas vaginalis, Staphylococcus epidermidis e Klebsiella pneumonia

(KPC).

3.2. ESPECÍFICOS

• Extrair polissacarídeos sulfatados da alga marrom Sargassum filipendula;

• Caracterizar quimicamente as heterofucanas obtidas;

• Avaliar o efeito das heterofucanas sobre macrófagos murinos (Raw 264.7)

quanto à produção de NO, Il-6 e TNF-α;

• Investigar a atividade anti-leishmania das heterofucanas sobre macrófagos

infectados com Leishmania infatum.

• Avaliar o efeito das heterofucanas nos ensaios anti-Trichomonas vaginalis;

• Investigar se as heterofucanas possuem efeito antibacteriano e antibiofilme

contra Staphylococcus epidermidis e Klebsiella pneumonia (KPC).

22


4. MÉTODOS

Serão descritos a seguir apenas os métodos que necessitam de um

maior detalhamento para compreensão, os demais métodos encontram-se

claramente descritos nos artigos.

4.1. MATERIAIS BIOLÓGICOS

4.1.1. Algas

A alga marinha marrom Sargassum filipendulafoi coletada na Praia de

Búzios, município de Nísia Floresta (litoral sul do Rio Grande do Norte), em

marés baixas entre 0,0 a 0,2 metros a uma temperatura situada entre 28-30 °C.

Após serem coletadas, as algas foram trazidas ao laboratório de

Biotecnologia de Polímeros Naturais (BIOPOL) da Universidade Federal do Rio

Grande do Norte (UFRN), onde foram lavadas e retiradas as epífitas e

inclusões calcárias. Em seguida foram secadas em estufa a 45 ºC, trituradas e

guardadas em recipientes apropriados.

4.2. OBTENÇÃO DAS HETEROFUCANAS DA ALGA Sargassum filipendula

4.2.1. Obtenção do pó cetônico

A alga seca e pulverizada foi suspensa em dois volumes de acetona PA,

durante 24 horas para despigmentação e delipidação do material. A mistura foi

decantada e o resíduo colocado para secar a 45 °C sob aeração e denominado

de “pó cetônico”. Esse pó foi utilizado em seguida na proteólise.

4.2.2. Proteólise

A 100 gramas de pó cetônico foram adicionados dois volumes de NaCl a

0,25 M e o pH ajustado para 8,0 com NaOH. A esse material foi adicionado a

enzima proteolítica prozima (15 mg/g de pó cetônico). Essa suspensão

23


permaneceu em banho-maria a 60 °C por 24 h. Depois, foi filtrado e o

sobrenadante submetido a uma centrifugação 10.000 x g por 15 minutos a

temperatura de 4 °C. Após a centrifugação, o sobrenadante foi denominado de

extrato bruto.

4.2.3. Fracionamento do extrato bruto com concentrações crescentes de

acetona

O extrato bruto obtido foi fracionado com volumes crescentes de

acetona, obtendo-se as frações polissacarídicas. Adicionou-se um volume de

acetona, sob agitação leve, necessário para que se visualizasse uma turvação

da solução, essa solução foi mantida em repouso a 4 ºC durante 18 h, o

precipitado foi coletado por centrifugação a 8.000 x g por 15 minutos a 4 ºC e

seco a pressão reduzida.

Em seguida, esse procedimento foi repetido até que não se visualizasse

mais a formação de precipitado [37]. As frações obtidas foram denominadas

conforme o volume de acetona no qual foram precipitadas (SF0.5V, SF0.7V,

SF1.0V, SF1.5V e SF2.0V) (Figura 01).

4.3. ANÁLISES QUÍMICAS

4.3.1. Dosagem de açúcares totais

Açúcares totais de cada extrato polissacarídico e de cada fração

polissacarídica obtida foram determinados pelo método do fenol/ácido sulfúrico,

utilizando-se como padrão galactose, sendo as leituras realizadas a 490 nm

[53].

4.3.2. Dosagem de sulfato

O teor de sulfato total de cada extrato polissacarídico e de cada fração

polissacarídica obtida foi quantificado, após uma hidrólise ácida com 4 N de

HCl por 6 horas à temperatura de 100 °C, por turbidimetria pelo método da

gelatina-bário, tendo-se como padrão o sulfato de sódio [54].

24


4.3.3. Dosagem de proteínas

O teor de proteína correspondente de cada extrato polissacarídico e de

cada fração polissacarídica obtida foi determinado com o reagente de comassie

blue R 250 e a leitura realizada a 595 nm [55].

4.3.4. Determinação da composição monossacarídica

A composição monossacarídica dos polissacarídeos sulfatados das

algas Sargassum filipendula e Dyctiopteris delicatula foi determinada através

de cromatografia liquida de alta performance (HPLC) contendo um detector de

índice refrativo modelo L-2490. Utilizou-se uma coluna LichroCART® 250-4

(250 mm × 40 mm) contendo como pré-coluna a Lichrospher® 100 NH2 (5 μm).

Os polímeros foram hidrolisados (2 M HCl, 100 °C, 2 h) e posteriormente os

seus monossacarídeos foram analisados. Como referências, os seguintes

açucares foram utilizados como padrões: arabinose, galactose, glicose, fucose,

manose, ramnose, ácido glucurônico, ácido manurônico, N-acetil glicosamina e

xilose. A fase móvel consistiu de uma mistura de 0,1 mol/l de KH2PO4 (pH 10)-

acetonitrila (80:20). O fluxo foi de 1.0 mL/min e a temperatura da coluna foi de

80 °C.

25


Figura 01. Esquema representataivo da metodologia de extração e caracterização físico-química e farmacológica das heterofucanas da alga S. filipendula.

26


5. ARTIGOS PRODUZIDOS

5.1. Artigo 1 (SUBMETIDO)

Immunomodulatory effect and antimicrobial activity of Sargassum

filipendula heterofucanas

Periódico: Marine Drugs

Fator de impacto (2015): 3.512

ISSN: (Printed version)

ISSN: (Online version)

Qualis: Medicina II – A2

Indexada: PubMed – indexado por MEDLINE

5.2. Artigo 2 (PUBLICADO)

Methanolic Extracts from Brown Seaweeds Dictyota cilliolata and Dictyota

menstrualis Induce Apoptosis in Human Cervical Adenocarcinoma HeLa

Cells

Periódico: Molecules

Molecules. 2015 Apr 13;20(4):6573-91

Fator de impacto (2015): 2.095

ISSN: 1420-3049 (Printed version)

ISSN: 1420-3049 (Online version)

Qualis: Medicina II – A2

Indexada: PubMed – indexado por MEDLINE

5.3. Capítulo de livro

Chapter: Carrageenans

Periódico: Biochemistry and Molecular Biology in the Post Genomic Era

Binding: ebook Pub. Date: 2015 ISBN: 978-1-63483-002-7

27


5.1. ARTIGO 1 (SUBMETIDO)

Mar. Drugs 2015, 13, 1-x manuscripts; doi:10.3390/md130x000x

marine drugs ISSN 1660-3397

www.mdpi.com/journal/marinedrugs

Article

Immunomodulator effects and antimicrobial activity of heterofucanas from Sargassum filipendula

Cinthia Beatrice Silva Telles 1,2

, Carolina Mendes-Aguiar 3, Gabriel Pereia Fidelis

1,

Amanda Piccoli Frasson4, Leandro Silva Costa

1,5, Tiana Tasca

3,; Selma Maria

Bezerra Jeronimo1,2

and Hugo Alexandre Oliveira Rocha 1,2,

*

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, Brasil; E-Mails: [email protected]

(C.B.S.T.); [email protected] (L.S.C.), [email protected]

(G.P.F.); [email protected] (H.A.O.R) 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, Brasil. 3 Instituto de Medicina Tropical do Rio Grande do Norte, Departamento de Bioqiímica

Universidade Federal do Rio Grande do Norte (UFRN), Natal, Rio Grande do Norte-

RN 59078-970, Brasil; E-Mail: [email protected] (C.M.A.);

[email protected] (S.M.B.J.) 4 Faculdade de Farmácia da Universidade Federal do Rio Grande do Sul, Av. Ipiranga,

2752, Porto Alegre, RS, 90610-000, Brasil. E-Mails: [email protected] (T.T);

[email protected] (A.P.F.) 5 Intituto Federal de Educação, Ciência e Tecnologia do Rio Grande do Norte (IFRN),

Santa Cruz, Rio Grande do Norte-RN 59200-000, Brasil.

* Author to whom correspondence should be addressed; E-Mail: [email protected];

Tel.: +55-84-3215-3416 (ext. 207); Fax: +55-84-3211-9208.

Academic Editor:

Received: / Accepted: / Published:

OPEN ACCESS

28


Abstract: Fucans, sulphated polysaccharides that contain L-fucose in its

constitution, obtained from species of Phaeophyceae of the Sargassum kind,

display several biological activities. Heterofucans from Sargassum

filipendula are bioactive molecules that contain strong antiproliferative and

antioxidant activity. However, their immunomodulatory and antimicrobial

activities have not yet been examined. In this context, the aim of this

research was to evaluate the heterofucans as for their immunomodulatory

capacity and antimicrobial action against Leishmania infantum,

Trichomonas vaginalis, Staphylococcus epidermidis and Klebsiella

pneumonia (KPC). The five heterofucans obtained from S. filipendula show

activities that are distant as stimulants of the immune system and microbial

agent. The SF0.5V, SF0.7V amd SF1.0V heterofucans were capable of

acting in the activation of murine and human macrophages. In addition to

that, SF0.5V has shown antibiofilm activity of S. epidermides and SF0.7V

and 1.0V almost completely inhibited the survival of the protozoan T.

vaginalis. Results such as this one, reflect the broad range of action of the

sulphated polysaccharides obtained from seaweeds, especially from the

species S.filipendula

Keywords: Immunomodulation; Nitric oxide; Leishmania infatum,

Trichomonas vaginalis, antibiofilm; macrophages; fucans.

1. Introduction

The immune response performs a fundamental role in the defense against infectious

agents and it constitutes the most important prevention against the occurrence of

infection spreads that are normally associated with a high mortality rate [1]. The

establishment of an infection, in a vulnerable host, covers several mechanisms; one of

the most relevant is the way that the microorganism interacts with the immune system

and its response against the invader [2].

The parasites are highly diversified of organisms that developed different strategies

to infect their hosts. These infectious agents vary from single-celled organisms such as

bacteria and protozoans to multi-celled organisms, such as nematodes and helminths

(worms). The bacteria are microorganisms capable of replicating inside the host cells as

much as in extracellular environments, blood circulation, intestinal lumen, airways,

amongst others [2]. On the other hand, protozoans are infectious intracellular agents that

normally infect the host for long periods of time [1]. The immune mechanisms involved

29


in fighting the bacterial infections and caused by protozoans vary; generally,

intracellular parasites are eliminated through mechanisms mediated by cell and the

extracellular ones via mechanisms that mainly involve the complement system and

antibodies [3, 4].

The drugs available for the treatment of parasite related diseases are far from

satisfactory due to their high production costs, toxicity, as well as the appearance of

resistance [5-8].

The increase in knowledge about clinical immunology has been revealing that the

physiopathology of illnesses can be caused by exacerbation, as well as by

immunodeficiencies of the immune response. The modern immunologic therapy is

divided in two basic groups of immunomodulators: the immunostimulants, which lead

to the increase of innate and adaptive immunity, and the immunosuppressives that

diminish the activity on the immune system [9].

Natural products are important sources of innovative therapeutic agents for infectious

diseases, cancer, lipid disorders and immunomodulation [10].

Sulphated polysaccharides are a complex group of bioactive macromolecules in

which some of the hydroxyl groups from sugar waste are replaced by sulphate groups.

Seaweeds are the main non-animal sources of obtainment of these anionic

polysaccharides; in this group, we find the Fucans, which are a family of

polysaccharides that contain L-fucose in its constitution [11].

Over the last few years, a variety of groups reported that sulphated polysaccharides

obtained from Phaeophytas species, from the genus Sargassun showcase various

biological activities: Sargassum horneri [12]; S. tenerrimum [13]; S. patens [14]; S.

stenophyllum [15], S. wightii [16], S. vulgare [17], S. siliquosum [18]. Our group

assessed the heterofucans from Sargassum filipendula, common seaweed along the

northeastern cost of Brazil, and demonstrated that these polymers are bioactive

molecules presenting strong antiproliferative and antioxidant activity. However, the

immunomodulator and antimicrobial purified activities of the sulphated polysaccharides

from S. filipendula have not yet been examined. In this context, the objective of this

study was to obtain sulphated polysaccharides from S. filipendula and evaluate their

immunomudulating and antimicrobial activities facing Leishmania infantum,

Trichomonas vaginalis, Staphylococcus epidermidis and Klebsiella pneumonia (KPC).

2. Results and Discussion

Chemical characteristics of heterofucanas SF0.5V, SF0.7V, SF1.0V, SF1.5V and

SF2.0V obtained from seaweed S. filipendula are presented in Table 1. The

heterofucans SF0.5V and SF2.0V were the ones that presented the lowest ratio

sugar/sulphate, indicating that the amount of sulphate by sugar residue is bigger when

30


compared to the other heterofucans. The monosaccharides fucose, galactoses, glycoses

and xyloses were found in different amounts in all polysaccharides. Mannoses and

glucuronic acid are also present in almost all of the heterofucans, except for SF1.5V,

that does not have mannoses residues and SF2.0V that does not have glucuronic acid in

its structure. Comparing the data described on table 1 with the data published by Costa

and collaborators [19], it is possible to verify that both are very similar, which indicates

that the heterofucans extracted are the same obtained by Costa and collaborators [19].

These fucans present antioxidant and antitumor activities, however other activities have

not been assessed yet, therefore, we verified weather these fucans had

immunomodulator and antimicrobial effect.

Table 1. Chemical characteristics of the heterofucans from S.filipendula.

2.1. Imunomodulator effect of the sulphated heterofucans from S. filipendula in murine

macrophages (RAW 264.7)

Macrophages are essential for the maintenance of homeostasis and play a main role

in the defense of the host against pathogens. In this process, activated macrophages

release some immunomodulator factors, such as NO, IL-6, TNF-α, amongst others [21].

Therefore, initially, we evaluated the effect of heterofucans in murine macrophages

(RAW 264.7) in culture and isolation conditions.

2.1.1. Production of NO

The production of NO by RAW 264.7 cells, stimulated by the different heterofucans

(0.125, 0.25 and 0.5 mg/mL) is demonstrated on Figure 01. After 24 hours of treatment,

the level of NO in the cultures stimulated with the heterofucans SF1.5V and SF2.0V

was similar to that observed for the non-stimulated cells (control group). On the other

hand, it was observed that the amount of NO present on the supernatant of the

macrophages increased considerably when the cells were incubated with SF0.5V,

SF0.7V and SF1.0V, this effect presents a tendency to be dose-dependent. It is

important to stress that the RAW cells, in the presence of SF0.5V (0.5 mg/mL) made

the amount of NO in extracellular environment similar to the one found in the

31


extracellular environment of the cells that were treated with positive control, which

suggests a strong immunostimulating action of the macrophages by this heterofucan.

Other groups also proved that different fucans have distinct potentials as

immunostimulating agents, e.g. fucans obtained from the weeds Ascophyllum nodosum

and Fucus vesiculosus increased the amount of NO released to the extracellular

environment when in contact with RAW cells. However, the Ascophyllum fucana was

six times more potent than Fucus [22]. The presented information clearly indicates that

the potency of stimulation of the RAW cells carried out by the fucans in order to release

NO is not the same and depends on the properties of each fucana, as was observed with

the heterofucans from S filipendula.

Amongst the properties featured as important for a fucan to interfere in the amount of

NO releases, it is important to mention the degree of sulphatation. Nevertheless, the

results obtained with the heterofucans (SF0.5V and SF2.0V) were intriguing; despite the

two present similar ratio sugar/sulphate, they presented different effects in the

immunostimulation of macrophages and releasing of NO, which means that one of them

is a stimulator (SF0.5v), whereas the other one (SF2.0v) does not affect the amount of

NO in the environment.

According to Leiro and collaborators [23] the sulphate groups are important points so

that a sulphated polysaccharide is able to stimulate RAW 264.7 cells to release NO to

the extracellular environment. These authors presented that sulphated polysaccharides

of the Ulva rígida weed were stimulating agents of the RAW cells, however when they

were disulphated, their activity was lost. Jiang and collaborators [24] also demonstrated

that when disulphating ascophylan, a homofucan obtained from the weed Ascophyllum

nodosum, its RAW macrophages stimulating activity was significantly decreased when

compared to the native ascoplylan.

Despite many authors presenting that the amount of sulphate groups present in the

polysaccharide is important to its action, the position in which these groups are

distributed in the molecule is a much more determining factor so that a sulphated

polysaccharide present higher or lower activity [25-28]. Therefore, we believe that this

would be the reason that makes SF0.5v and SF2.0V, that present similar ratio

sugar/sulphate, present distinct activities.

32


Figure 1. Effect of the heterofucans from S. filipendula over the release of

NO by RAW264.7 cells. The data are presented as average ± standard

deviation (n = 3). The letters a, b indicate a significant difference (p <0,05)

between the concentration of the heterofucans. # Indicates the significant

difference (p <0,05) between the concentration of the heterofucana and

positive control. * Indicates significant difference (p <0,05) between the

concentration of the heterofucana e negative control. NC: Negatice control;

PC: Positive Control (LPS 2 µg/mL).

2.1.2. Production of the cytokines TNF-α and IL-6

On Figure 02 we observe that the cells RAW 264.7, when exposed to the

heterofucans SF1.5V and SF2.0V, did not promote alteration on the level of TNF-α and

IL-6 in the extracellular environment, proving, alongside with the previous data, that

these polymers possibly do not act as immunomodulator agents.

The heterofucana SF1.0V also did not induce the cells RAW 264.7 to produce and

release to the extracellular environment significant amount of TNF-α and IL-6. This

characteristic is not entirely of these fucans, other heterofucan, extracted from the

seaweed Dictyota menstrualis, was also not able to interfere on the production of these

two cytokines [29]. The presented data lead to the observation that the

immunomodulator mechanism of SF1.0V would be centred in its capacity to interfere

on the production and release of NO.

The fucans that altered the highest amount of TNF-α and IL-6 were SF0.5V and

SF0.7V, in both the cases the TNF-α was the cytokine that was most affected by the

presence of the fucans. As to SF0.5V, it was observed that the presence of 0.25 mg/mL

33


of this fucan increased in about a thousand times the amount of TNF-α in the

extracellular environment in comparison with the amount of TNF-α found in the control

group. This result was very expressive, since other fucans of Fucus vesiculosus [22] and

Ascophyllum nodosum [30], in the concentration of 0.2 mg/mL, were only capable of

increasing the amount of TNF-α in the extracellular environment in 20 times.


TNF-α and IL-6 by RAW264.7 cells. The data are present as average ±

standard deviation (n = 3). The letters a, b indicate a significant difference

(p <0,05) between the concentration of the heterofucans. # Indicates

significant difference (p <0,05) between the effect of the heterofucana and

positive control. * Indicates significant difference (p <0,05) between the

effect of the heterofucan and negative control. NC: Negative control; PC:

Positive control (LPS 2µg/mL).

Do and collaborators [31], evaluating the effect of the homofucan extracted from the

brown weed Fucus vesiculosus in the induction of the production of NO by

macrophages (RAW 264.7 and primary peritoneal cells), observed that this fucan made

the cells that were being studied increase the release of NO to the extracellular

environment, as well as TNF-α. These authors suggest that TNF-α has a synergic effect

and increases the release of NO to the extracellular environment even more, which

would explain the effect of SF0.5V and SF0.7V, in other words, the way these fucans

stimulate the release of TNF-α induces a higher release of NO. And also explains why

SF1.0V stimulates the release of a smaller amount of NO, because that fucan does not

affect the release of TNF-α.

34


Different groups that study fucans state that they are immunomodulators because

they induce the activation in vitro of murine macrophages (RAW 264.7) leading to the

increase of the production of NO and cytokines such as TNF-α and IL-6 [22,32-34].

Which leads us to propose that S. filipendula synthetizes imunomodulator fucans.

2.1.3. Cytotoxicity of the heterofucans related to RAW 264.7 cells

With the objective of evaluating weather the increase in the production of these

chemical mediators, NO and the cytokines (TNF-α and IL-6) was not a response arising

from the toxicity of the heterofucans, we evaluated the cytotoxicity of these

heterofucans facing the same lineage used in previous trials. The data are on Figure 4.

Figure 4. Effect of the heterofucans from S. filipendula over the

proliferation of RAW264.7 cells. The data are present as average ± standard

deviation (n = 3). There were no meaningful differences in all of the tested

concentrations.

By analysing Figure 04, it was possible to observe that under all of the conditions

tested, the heterofucans did not compromise the cellular viability of the phagocytic

cells, suggesting that these polymers do not have cytotoxic effect over RAW264.7 cells.

Considering the cited aspects, we suggest that the increase in the release of NO, TNF-α

and IL-6 result from the immunomodulating capacity of the heterofucans facing the line

of macrophages.

2.2. Antimicrobial activity

Considering that heterofucans from S. filipendula proved to be immunomodulator

agents, we also evaluated the effectiveness of these heterofucans facing various

35


parasites, for instance: Leishmania infatum; trichomonas vaginalis; Klebsiella

pneumoniae (KPC) and Staphylococcus epidermidis.

2.2.1. Infection caused by Leishmania infatum in human macrophages

Considering that our group have not yet standardized the methodology for infection

of RAW cells with leishmania, we chose to work with macrophages originated from

primary human monocytes, as described in methods.

2.2.1.1. Production of NO

Macrophages originated from primary human monocytes exposed for 24 and 48

hours to the heterofucans from S. filipendula, in the concentration of 0.5 mg/mL,

increased distinctly the amount of NO in the extracellular environment (Figure 5A).

The heterofucans SF0.5V, SF0.7V and SF1.0V (Fig 5A) promoted meaningful increase

in the release of NO to the extracellular environment in all of the periods analysed when

compared to the untreated macrophages. In addition, the release of NO by macrophages

stimulated by SF0.5V and SF0.7V in the period of 48 hours of exposure was about 2

times higher than that presented by untreated macrophages. The other polysaccharides

(SF1.5V and SF2.0V) did not present meaningful effect over the production of NO by

macrophages. These data is similar to that obtained during the treatment of murine

macrophages (RAW 264) (Figure 02), that demonstrated the polysaccharides SF0.5V,

SF0.7V and SF1.0V presented an effective immunomodulating action.

A second cultivation of phagocytic human cells was carried out. However, this time

these cells were initially infected with the amastigote form (intracellular form of

leishmania) and posteriorly incubated with the heterofucans from S. filipendula with the

intent of evaluating the effect of the fucans in the release of NO by the infected cells.

On Figure 5B we can observe that the human macrophages infected and incubated

with the heterofucans from S. filipendula (0.5 mg/mL), also distinctly increased the

amount of NO in the extracellular environment. The production of NO after the

treatment for 24 and 48 hours with the heterofucana SF2.0V was not altered; it kept the

level of NO similar to that observed with the untreated observed cells. On the other

hand, all of the other heterofucans induced an increment in the production of NO

(Figure 5B). The heterofucans SF0.5V and SF0.7V were the best activators of the

immune response, and presented time depending effect. Within 48 h these fucans

elevated the amount of NO in about five times, comparing to the control.

36


37



NO by human macrophages. Production of NO by human macrophages after

treatment with the heterofucans (A); Production of NO by macrophages

infected by L. infatum and treated with the heterofucans (B). The data are

presented as averages ± standard deviation (n = 3). The letters a, b indicate

a significant difference (p <0,05) in the release of NO on different periods. *

Indicates significant difference (p <0,05) between different heterofucans and

the negative control.

2.2.1.2. Leishmanicidal activity

After the evaluation of the effects of the heterofucans over the production of NO, we

evaluated the leishmanicidal activity over the amastigote intracellular form of

Leishmania infantum.

The results on Figure 6 demonstrate that the highest percentage of cells infected by

amastigotes can be found in the control group cells. It is also possible to observe that

there is no significant difference between the percentage of cellular infection in the

control group and in the group treated with the heterofucan SF2.0V. The other fucans, at

least, in one of the conditions tested diminished significantly the rate of cellular

infection by amastigotes.

38


Figure 6. Leishmanicidal activity of the heterofucans from S. filipendula.

The results are expressed in average ± standard deviation (n = 3) of the

percentages of the macrophages infected with L. infantum * Indicates

significant difference (p <0,05) between the concentration of the

heterofucan and the CN. CN: Negative control – Untreated macrophages

infected with L. infatum.

In a second experiment, the macrophages were plated over a cover slip and infected

in the presence or not of the heterofucans (0.5 mg/mL), posteriorly they were fixed and

coloured, and the number of amastigotes that were inside the cells was determined. The

data are on Figure 7. In the control group and in the group treated with SF2.0V, the

number of intracellular parasites was similar and corresponded to approximately 4

amastigotes per macrophage. During the treatment with the other heterofucans (SF0.5V,

SF0.7, SF1.0V and SF1.5V) the number of amastigotes was smaller, about 3

amastigotes/macrophages. (Figure 07A).

Figure 7. Leishmanicidal activity of the heterofucans from S. filipendula.

Number of amastigotes of L.infatum per infected macrophages (A).

Micrographs of the parasites internalized in the macrophages (B). The cover

slips were processed by optical microscopy, coloured with Panotic e

photographed using a 100X zoom. The arrows show internalized parasites.

39


Figure 07B illustrates micrographs that represent the preparation of macrophages

infected by Leishmania (L.) infatum, treated or not with the heterofucans from S.

filipendula. On all of the preparations, we observed the presence of amastigotes in the

interior of the cells, indicated by arrows. We observed a slight decrease in the number

of amastigotes after treatment with the heterofucans when compared to the untreated

cells (control group)

Altogether, the presented data demonstrates that the fucans (mainly SF0.5V, SF0.7V

and SF1.0v) decreased in approximately 20% the percentage of macrophage infection

(Figure 06). This percentage is low if compared to the activity of fucoidan of F.

vesiculosus, which promotes a decrease of about 90% of the number of human

macrophages infected by Leishmania [35]. That indicates that the fucans from

Sargassum are not candidates for the treatment of leishmaniasis, but, due to their

immudomodulator capacity, they can act as supporting agents in the treatment of this

illness, and is raw material for the production of nanoparticles that carry

antileishmanicidal drugs.

2.2.2. Anti-trichomonas vaginalis activity

Heterofucans from the seaweed S. filipendula were also analysed against

trophozoites of T. vaginalis (Figure 08). Triage revealed that the heterofucans SF0.5V

and SF2.0V did not present inhibitory activity facing trophozoites. Whereas SF0.7V,

SF1.0V and SF1.5V presented anti-T. vaginalis activity after 24h of treatment.

Figure 8. Effect of different heterofucans from S. filipendula (2 mg /mL)

against isolated of T. vaginalis sensitive to metronidazol. Control:

Trophozoites in untreated culture with the heterofucans. The data represent

averages ± standard deviation of at least three experiments. . # Indicates

significant difference (p <0,05) between the concentration of the

heterofucan and control.

40


The heterofucans SF0.7V and SF1.0V that presented best inhibitory capacity facing

trophozoites of T. vaginalis (approximately 90% of inhibition) present ratio

sugar/sulphate of approximately 4.5, in counterpart, SF0.5 and SF2.0V present smaller

values (4,06 and 3,72) did not have any activity in this essay, which leads us to suggest

that the degree of sulphatation should not be a preponderant factor to a satisfactory anti-

T. vaginalis activity.

The action of the heterofucans as strong inhibitory agents of the flagellate protozoan

Trichomonas vaginalis is of key importance since trichomoniasis, sexually transmitted

disease (STD), is an infirmity present all over the world [36] and its treatment is

essentially based in the use of the drug 5-nitroimidazole [37]. However, some reports

have demonstrated the appearance of resisting strains [38]. Therefore, it is necessary to

search for a new therapeutic arsenal. Besides, there is not a study that has demonstrated

the activity of polysaccharides in the fighting of T. vaginalis, so this is the first study

that brings up that polysaccharides obtained from seaweed (SF0.7V, SF1.0V and

Sf1.5V) have cytotoxic action against this pathogenic protozoan.

2.2.3. Antibacterial and Antibiofilm activity

All of the heterofucans from S. filipendula were evaluated as to their antibacterial

and inhibitory capacity concerning the formation of bacterial biofilms (Table 02). In this

study, no heterofucana of S. filipendula was proved effective in the fighting of bacterial

growth, also did not considerably inhibit the formation of biofilms, promoted by the

association of bacteria of the species Klebsiella pneumoniae (KPC). The heterofucans

also did not have antibacterial activity against Staphylococcus epidermidis. However,

SF0.5V presented antibiofilm activity, inhibiting about 50% of biofilm formation by

these bacteria.

Table 2. Antibacterial and antibiofilm activity of the heterofucans from S.filipendula. a

The data are the average value of three determinations ± DP; b

The results were

obtained in the concentration of 2 mg/mL, other tested concentrations did not have

significant activity; c rifampicin (Sigma-Aldrich Co., St. Louis, MO, USA) was used as

control antibiotic.

41


The difference in the activity of SF0.5V in the formation of biofilms between the two

species of bacteria studied, may result from the structural difference presented by this

two bacterial groups; since the bacterial strains of the species S. epidermides are gram

positive, whereas KPC is a gram negative bacteria and, therefore, has a membrane

external to the bacterial cellular wall, this way they have binders distinct from the ones

presented by S. epidermides.

Considering that the heterofucana SF0.5V does not have bactericidal activity (Table

02), its antibiofilm activity of S. epidermidis should be mediated by other mechanism

unrelated to the inhibition of bacterial growth. Some studies show that the charge of the

surface is an important parameter to the formation of biofilms. Positively charged

surfaces promote a stronger bacterial adhesion, probably due to attractive electrostatic

forces [39]. This way, sulphated polysaccharides or with carboxylic groups, when bond

to the surfaces, would provide negative charges to the surface and would affect the

bacterial adhesion by electrostatic repulsion. As an example we have the effect of two

ulvanes (sulphated polysaccharides) extracted from the green seaweeds Ulva rotundata

and Ulva compressa. They were efficient in the reduction of the colonization of titanium

substrate by S. epidermides, reducing in 96% the initial adhesion [40]. However, we do

not completely agree with this hypothesis, since only one of the five tested heterofucans

presents antibiofilm activity. Probably, as in other sulphated polysaccharides activities,

the distribution of negative charges by the molecule is a factor that is more important

than the simple fact of having negative charge for a polysaccharide to present

antibiofilm activity.

Another possibility that cannot be overruled would be that of the ability of the fucan

to bond to the bacterial surface. Some studies indicate that the acting mechanism of

some polysaccharides happens through a competitive inhibition of the carbohydrate-

protein interactions. As an example, we have Zinger-Yosovich and Gilboa-Garber [41]

who observed that the adhesion that depends on the lectin of P. aeruginosa to the

human cells is effectively inhibited by galactomannans. That way, antibiofilm

polysaccharides would block the sugar binding proteins present on the surface of

bacteria, or adhesins present in fimbria and pili [41]. It is intended, in the future, to

produce antibodies anti-SF0.5V and use them as a tool to discover its molecular

antibiofilm target.

42


This antibiofilm activity presented by the heterofucana SF05V is fundamentally

important since biofilms hamper the arrival of antimicrobial drugs and even phagocytic

cells to the infection site. It can be harmful to health, as in the case of bacterial pellicles

that develop on teeth - originating cavities - and other problems related to mouth, lungs,

urinary catheters and contact lenses, which can originate serious infections on tissues

(osteomyelitis and endocarditis) and rejection to prosthetic material[42,43].

2.3. Heterofucans from S.filipendula versus biological activity

In this study we analysed five heterofucans from the seaweed S. filipendula regarding

their immunomodulating and antimicrobial capacity. On table 3, we attributed positive

(+) and negative (-) scores to identify which heterofucan presented the best performance

on the trials carried out in this project. We observed that the heterofucan SF2.0V

followed by SF1.5 presented the worst responses on the tests performed. On

counterpart, SF0.5V and SF0.7V were the most promising. In addition, we noticed that

none of the fucans is effective in all kinds of tests because each polymer presents a

unique structure responsible for the specificity of a response during the different trials.

In the future, it is intended to structurally characterize these fucans and propose

correlations between their structures and the activities they perform.

Table 3. Biological activity of heterofucans from S. filipendula score.

43


3. Experimental Section

3.1. Materials

Bromide of 3- (4,5-dimetiltiazol-2-il) -2-5-diphenyltetrazoliumbromide (MTT),

Griess reagent, Histopaque 1077, Methanol PA, MCSF (Stimulating factor of

macrophages colony), medium of bacterial cultivation Luria Bertani (LB), Serum AB

(SIGMA) and metronidazole were acquired from Sigma Chemical Company, St. Louis,

MO, USA. Medium of cellular culture (RPMI 1640) (Developed by Roswell Park

Memorial Institute) and DMEM (Dulbecco's Modified Eagle's Medium), trypsin and

Bovine Fetal Serum (BFS), were obtained from CULTILAB (Campinas, Brazil). L-

glutamine, gentamicin, penicillin, streptomycin, sodium bicarbonate, HEPES, sodium

pyruvate and saline solution tamponed with phosphate buffered saline (PBS) were

acquired from Invitrogen Corporation (Burlington, ON, USA). ELISA Kits (for the

TNF-α, and IL-6) were acquired from BD Biosciences. Ficoll-Hypaque was acquired

from GE Healthcare. Panoptic dye was acquired from NewProv. Entellan was acquired

from Merck. Half Schneider’s insect was obtained from Gibco. All of the other solvents

and chemical products were obtained from analytic degree.

3.2. Biological material

The cell line of murine macrophages RAW 264.7 (ATCC number TIB-71) was

donated by Dr. Carmen Ferreira (Biochemistry department, UNICAMP, Brazil). The

protozoan Trichomonas vaginalis (ATCC number 30236) and Leishmania infatum were

given, respectively, by Dr. Tiana Tasca (Clinical Analysis and Toxicology Laboratory,

Faculdade de Farmácia, UFRGS, Brazil) and by Dr. Selma Jerônimo (Institute of

Tropical Medicine of Rio Grande do Norte, Biochemistry Department, UFRN, Brazil).

The bacterial strains Staphylococcus epidermidis (ATCC 35984) and the clinical

isolate of Klebsiella pneumoniae were given by Dr. Alexandre Jose Macedo

(Biotecnology center and Faculdade de Farmácia, UFRGS, Brazil).

3.3. Maintenance of cell lineages

The lineage of murine macrophages (RAW 264.7) were cultivated in supplemented

DMEM with 10% of Bovine Fetal Serum (BFS) and antibiotics (100 U / mL of

penicillin and 100 µg / mL of streptomycin). The cells were maintained as cultures in

monolayers at a humidified atmosphere of 5% of CO2 at 37 ° C.

Tricomonas vaginalis were cultivated in vitro in the TYM (trypticase-yeast extract-

maltose) medium, pH 6.0, supplemented with 10% (v/v) of serum inactivated by heat,

and incubated at 37 ° C [44]. The organisms in logarithmic growing phase, displaying

44


more than 95% of viability and normal morphology were retracted, centrifuged and

suspended again in medium TYM for utilizing in tests.

The promastigotes of Leishmania Infantum were kept at 25oC in Schneider’s insect

medium, supplemented with 10% BFS, 200 IU/mL of penicillin and 200 µg/mL of

streptomycin, and grew until stagnant phase.

Staphylococcus epidermidis and the clinic isolate of Klebsiella pneumoniae (KPC)

were utilized as biofilm former bacterial models. S. epidermidis and KPC were

cultivated in Luria Bertani medium (LB) at 37 ° C under agitation of 150 rpm (Shaker

Série Excella E25; New Brunswick Scientific) and adjusted to an OD600 equivalent to

108 CFU / mL for utilization in antibacterial and antibiofilm trials.

3.4. Obtainment of peripheral human blood mononuclear cells

For the obtainment of peripheral human blood mononuclear cells (PBMC), 15 mL of

venous blood from six donors were obtained aseptically in heparinized tube. The tubes

containing the blood were diluted in sterile saline solution (v/v). The PBMCs were

obtained through the Ficoll-Hypaque gradient and adjusted to the concentration of 2 x

106 cells/mL in RPMI 1640 complete (100 L/mL of gentamicin, L-glutamine 2mM,

30mM HEPES), containing 10% of AB Serum (Life technologies GIBCO BRL,

Gaithersburg, MD).

After that the PBMCs were incubated at 37 ° C, 5% CO2 for 4 h in 6 and 24 well

plates, for the adhesion of the monocytes. To the 6 and 24 well plates, were added,

respectively, 2 and 3 mL/well of cell suspension. The 6 well plates were prepared with 4

cover slips round/well, for posterior microscopic analysis of the cells. After 4 h of

incubation, the medium - with the non-adherent cells - was removed. The wells were

washed with RPMI medium at room temperature. The adherent cells were incubated in

complete RPMI medium (10% of serum AB, 5 ng/mL of MCSF (Macrophage Colony

Stimulating Factor) for 6 days at 37 ° C, 5% of CO2.

3.5. Extraction of sulphated polysaccharides (Heterofucans)

The Phaeophyta Sargassum filipendula was collected at Búzios beach, Nísia

Floresta, Rio Grande do Norte, Brazil. The heterofucans SF0.5V, SF0.7V, SF1.0V,

SF1.5V and SF2.0V of S. filipendula were obtained utilizing the methodology described

by Costa and collaborators. [19].

3.6. Production of nitric oxide (NO)

The production of NO was analysed through quantification of nitrite production by

Griess reaction [45]. To measure the production of nitrite, aliquots of 100 µL - obtained

45


from supernatants of the cultures to be dosed - were incubated with equal volume of

Griess reagent and were incubated at room temperature for 10 minutes. The analysis

was carried out in the microplate reader Multiskan Ascent (Thermo Labsystems,

Franklin, MA, USA) with absorbance to 540 nm. Utilizing as standard curve the

generated per NaNO2.

3.7. Cytokine analysis

RAW 264.7 (3 × 105 cells/mL) cells treated with the different heterofucans in the

concentrations of 0.125, 0.25, 0.5 mg/mL, were cultivated in 24 well plates. After 24 h,

the supernatants of the culture were collected. Following, they were centrifuged at 4000

rpm for 5 min. The levels of TNF-α and IL-6 were determined utilizing specific ELISA

kits (immunoabsorbent enzymatic test), the negative control consists of untreated cells

with the heterofucans and positive control those in the presence of LPS (2µg/mL). The

plate was read at 450 nm, with remediation at 570 and 590 nm.

3.8. Cytotoxicity tests in macrophages

The cytotoxicity in RAW 264.7 cells were measured through the MTT test as

described previously by Telles and collaborators [26]. The cells were cultivated in 96

well plates to a density of 5 x 103 cells/well with the heterofucans in different

concentrations (0.125, 0.25, 0.5 and 1.0 mg/mL) for 24 hours at 37 ° C and 5% of CO2.

After the incubation, 100 uL of MTT were added to each well, incubated during 4 h at

37 ° C and 5% of CO2, in the dark. The product MTT-formazano, dissolved in 100 mL

of ethanol was estimated through the measurement of the absorbance to 570 nm

Multiskan Ascent (Thermo Labsystems, Franklin, MA, USA) microplate reader.

3.9. Leishmanicidal activity

Cultures of Leishmania infantum at stagnant phase were resuspended in 1 mL of

complete RPMI medium and the concentration was adjusted to 107

parasites/mL. 6 well

plates cultivated with human macrophages were incubated with 3 mL of suspension of

parasite/well at 37 ° C, 5% of CO2 for 2 h for the infection of macrophages. The

medium containing parasites that did not adhere was retracted and the wells were

washed with RPMI medium at room temperature. The different heterofucans (0,5

mg/mL) were added in supplemented RPMI medium (10% of AB serum) to the wells

with macrophages infected with the parasite and the treatment was held for 24h.

To evaluate the percentage of macrophages infected with amastigotes of L. infatum,

the covers slips were removed after 24h, washed with PBS, fixed in methanol PA for 3

min at room temperature and dyed with Panotic. The plates were set with Entellan on

46


clean plate. 200 total macrophages were counted and the quantity of amastigotes per

infected macrophage was counted.

3.10. Anti-T. vaginalis assay

Heterofucans from S. filipendula were analysed against T. vaginalis trophozoites

(ATCC 30236). In 96 well microplates, 50 µL/well of solutions containing the different

heterofucans and150 µL/well of the suspension of trophozoites were added, resulting in

a final volume of 200 µL containing 2,5 x 105 trophozoite/mL and 2.0 mg/mL of the

heterofucan to be tested. In control cultures, heterofucans samples were substituted by

distilled water. The plates were incubated for 24 h at 37 ° C. After that period, 20 µL of

a solution of resaurzurin at 0.1 mg/mL in Phosphate Buffered Saline was added in each

well. After 1 hour of incubation at 37° C the fluorescence of each well was read in a

fluorescence spectrophotometer (Spectramax Gemini XS – Molecular Devices

Cooperation, Sunnyvale, CA, USA), the quantification of viable trophozoites was

carried out as described by Duarte and collaborators [46].

3.11. Antibiofilm assay

Antibiofilm activity was measured as described by Melo-Silveira and collaborators

[47]. 80 uL of bacterial suspension (Staphylococcus epidermidis ATCC-35984 and a

clinic isolate of KPC Klebsiella pneumonia type 174), 80 µl of the heterofucans (0,5;

1,0; 1,5; and 2,0 mg/mL) and 40 mL of tryptone soy broth (TSB) (Oxoid Ltd., England)

were added to the 96 well plate and incubated (37 °C for 24 h). The rest of the adhered

bacteria was fixed at 60 ° C for 1h. The biofilm formed was dyed with 0.4% of crystal

violet for 15 minutes at room temperature. The crystal violet bonded to the cells/biofilm

was solubilized with 99,5% of DMSO (Sigma-Aldrich Co., St. Louis, MO, USA) and

read at 570 nm (Spectramax M2e multimode Microplate Reader, Molecular Devices,

Sunnyvale, CA , USA). The controls were considered as 100% of the formation of

biofilm and the values obtained for the extract were the average of three experiments.

3.12. Bacterial inhibition assay

The experiment was carried out as described by Melo-Silveira e collaborators [47].

The bacterial growth of S. epidermidis (ATCC 35984) and a clinic isolate of KPC was

evaluated through the difference of absorbance measured at DO 600 nm in the end and

in the beginning of the incubation time, in 96 well polystyrene microtitration plates.

Different concentration of the heterofucans (0.5; 1.0; 1.5; and 2.0 mg/mL) were

incubated in the presence of each bacterial strain. The control with distilled water was

considered as 100% of bacterial growth. All of the experiments were carried out in

triplicate.

47


3.13. Statistical analysis

All the data were expressed in average ± standard deviation. The analysis was carried

out by analysis of variance. Student-Newman-Keuls post-tests were done for multiple

comparison by group. In all the cases, statistical significance was established at p <0,05.

4. Conclusions

The heterofucans from S. filipendula presented distant activities as stimulators of the

immune system and antimicrobial agents. The heterofucans SF0.5V, SF0.7V and

SF1.0V were able to act in the activation of murine and human macrophages promoting

increase in the release of the chemical mediators that are important for fighting

intracellular parasites. In addition, SF0.5V presented antibiofilm activity facing the

strain of S. epidermides whereas SF0.7V and 1.0V inhibited almost completely the

survival of the protozoan T. vaginalis.

All five heterofucans obtained from the seaweed S. filipendula presented

different/specific levels of activities in the trials carried out, making again evident that

their biological activities depend on their structural characteristics. Results such as this

reflect the great spectrum of action of these sulphated polysaccharides, but also

intensify the need of posterior studies looking to elucidate the complete structure of

these polysaccharides, configuration of glycosidic bonds, their position as well as the

position of sulphate groups and ramification points.

Acknowledgments

Research was supported by Ministério de Ciência, Tecnologia e Informação

(MCTI), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq),

Brazil. Hugo A O Rocha is a CNPq fellowship honored researcher. Cinthia Telles

had a Ph.D. scholarship from CAPES.

Author Contributions

Conceived and designed the experiments: C.B.S.T, T.T, L.S.C and H.A.O.R.

Performed the experiments: C.B.S.T., C.M.A and A.P.F. Analyzed the data: C.B.S.T. and

H.A.O.R. Contributed reagents/materials/analysis tools: G.P.F, L.S.C., T.T., S.J., L.F.D.P

and H.A.O.R. Wrote the paper: C.B.S.T. and H.A.O.R.

Conflict of Interest

The authors declare no conflict of interest.

48


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53


5.1. ARTIGO 2 (PUBLICADO)

72

Almeida-Lima J. PPGCSA/CCS

6. COMENTÁRIOS, CRÍTICAS E SUGESTÕES

Há décadas nosso grupo de pesquisa tem realizado uma estensiva

busca de atividades biológicas/farmacológicas de polissacarídeos sulfatados

obtidos de macroalgas marinhas. Fucanas, obtidas de diferentes espécies de

algas marrons são objetivos de estudo de pesquisas do grupo, dentre elas, as

fucanas obtidas da macroalga marron Sargassum filipendula. No entando,

apesar de algumas atividades biolólogicas já terem sido descritas para esses

polímeros nenhum estudo sobre atividades antimicrobina foi realizado. Este

trabalho de pesquisa teve como ponto positivo a insersão, na gama de ensaios

biológicos realizados pelo grupo, novas metodologias a serem utilizadas no

laboratório (BIOPOL), permitindo a apliação da detecção de outros prováveis

efeitos biológicos das fucanas.

Um momento importante durante a realização do doutorado foi a

possibilidade de realizar doutorado-sanduíche durante seis meses na

Universidade Nova de Lisboa, Instituto de Higiene e Medicina Tropical, Unidade

Clínica de Doenças Tropicais. A orientação do Prof. Dr. Marcelo Sousa Silva e

os integrantes do seu grupo de pesquisa forneceram total apoio para o

desenvolvimento do estágio. No entanto, não adicionamos os resultados das

heterofucanas de S.filipendula frente as metaloproteinases de Trypanosoma

brucei, pois alguns empaces de cunho técnico durante o estabelecimento de

uma das metadologias não permitiu a confirmação dos resultados alcançados.

Ao longo do desenvolvimento desta pesquisa, o cronograma de

atividades não foi totalemente realizado, pois, inicialmente tinha-se como meta

o sub-fracionamento da heterofucana mais eficiente nos ensaios realizados,

mas diante de vários intercursos ao londo do período final do doutorado

(terceiro ano), como o nascimento do meu filho, sendo necessário o

afastamento durante o período de licença maternidade, e o início na carreira

profissional, assumindo o cargo de professora, não obtive tempo hábil para

cumprimento dessa meta.

73


Durante o desenvolvimento deste estudo tive a oportunidade de

aprimorar bastante meus conhecimentos, pois tive a chance de aperfeiçoar das

técnicas laboratórias em diversos ramos da pesquisa científica, como:

imunologia, farmacologia, biologia celular, bioquímica, dentre tantos outros.

Além de interagir com vários grupos de pesquisas como o já citado em

Lisboa/Portugal sob a orientação do Professor Marcelo Sousa Silva, o grupo da

professora Tiana Tasca em Porto Alegre e, vários grupos de pesquisa

localizados na própria UFRN como os das professoras Selma Jerônimo e Katia

Scortecci.

Com relação à produção científica até o momento tenho 6 artigos

publicados em revista científicas, além de 10 resumos publicados em

congressos científicos durante o período do doutorado. Além disso, também fiz

parte na elaboração do capítulo de livro - Carragenan - e de uma banca

examinadora de trabalho de conclusão de curso (monografia).

Após a conclusão dessa etapa acadêmica, tenho como projeto para o

futuro, continuar com os estudos na mesma linha de pesquisa, em um futuro

pós-doutorado. Além de buscar a aprovação como Professora de Instituições

de Ensino Superior, engajando na tríade ensino/pesquisa/extensão.

74


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UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE · 2019-05-26 · Não sei se somos almas gêmeas,...

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