Diferentes abordagens para a descoberta de novos alvos terapêuticos contra a doença de Chagas
Ano de defesa: | 2019 |
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Autor(a) principal: | |
Outros Autores: | |
Orientador(a): | , |
Banca de defesa: | , , , |
Tipo de documento: | Tese |
Tipo de acesso: | Acesso aberto |
Idioma: | por |
Instituição de defesa: |
Universidade do Estado do Rio de Janeiro
|
Programa de Pós-Graduação: |
Programa de Pós-Graduação em Microbiologia
|
Departamento: |
Centro Biomédico::Faculdade de Ciências Médicas
|
País: |
Brasil
|
Palavras-chave em Português: | |
Palavras-chave em Inglês: | |
Área do conhecimento CNPq: | |
Link de acesso: | http://www.bdtd.uerj.br/handle/1/19538 |
Resumo: | Trypanosoma cruzi is the causative agent of Chagas disease. The parasite needs to enter in the host cell to grow and expand infection. Trypomastigotes are classicaly infective forms of T. cruzi, and they are able to invade host cell. After the invasion of the mammalian cell, parasites start amastigogenesis process, in which trypomastigotes forms differentiate into amastigotes that proliferate.Those steps of intracelular invasion and amastigogenesis are essential to parasite. Currently, two drugs are used to treatment: benznidazol and nifurtimox. Both drugs show side adverses effects that causes treatment interruption by the patient, due to of the low efficacy in chronic phase of Chagas diseases, making important the development of new drugs. Basically, two aproaches are applied in drugs development: phenotype and targed-based. In this work, we used two strategies in the targed-based approach. The first was to study the parasite biology relationship with host cell. In this context, we search ROS relation, especifically H2O2, in the intracelular infection. Once ROS are produced in the phagolysosome and in this compartment has signaling to amastigogenesis, we investigated the H2O2 influence in this process.We observed that H2O2 increased 30% of differentiation after two hours of induction by low pH. However, among antioxidant molecules tested, only PEG-catalase, enzyme that neutralizes directly H2O2 inhibited amastigogeneses improvement ROS-induced. We observed that H2O2 increased a serine/threonine phosphatase activity, as well as PP2A inhibitor okadaic acid (OKA) reverted H2O2 effect upon amastigogenesis. Corroborating ROS involvement, observed that differentiaton induced by acid pH produced H2O2 after one hour, being reduced later. When we evaluated the superoxide dismutase gene expression of T. cruzi, we observed that mitochondrial isoform (Fe-SODA) were up regulated after 60 minutes and 90 minutes differentiation induction. Furthermore, we detected a reduction in the oxygen consumption and maximal eletron tranference system capacity, as well as loss in the mitochondrial membrane potential (ΔΨm) after 60 minutes and 120 minutes of amastigogenese induction. In consideration of these results, wherein mitochondrial dysfunction is followed by loss of ΔΨm, H2O2 production observed can be from mitochondrial by mitochondrial permeability transition pore (mPTP). Indeed, mPTP inhibitor cyclosporin and mitochondrial antioxidant mitoTEMPO reduced H2O2 generation induced by low pH. Together, this results showed that H2O2 act as signaling molecule to differentiation. The second strategy were trypomastigote surface screening by phage display library. After biopanning, were identified two high affinity clones, N3 and N1. The peptide express in N3 clone were synthetized and in vitro bound assays showed peptide specificity to infective forms of T. cruzi. Furthermore, peptide were active inhibiting Vero cells invasion. Thus, N3 peptide bound in a surface molecule of T. cruzi that is important to cell invasion. |
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Paes, Marcia Cristinahttp://lattes.cnpq.br/7463829190927034Nogueira, Natália Pereira de Almeidahttp://lattes.cnpq.br/6320509465681227Dias, Silvia Amaral Gonçalveshttp://lattes.cnpq.br/6104190112253764Silva, José Roberto Machado ehttp://lattes.cnpq.br/6430514515393384Fernandes, José Roberto Meyerhttp://lattes.cnpq.br/8471949500798737Galina Filho, Antôniohttp://lattes.cnpq.br/8940530306495736http://lattes.cnpq.br/9150050143205166Paula, Jéssica Isis Oliveira dejessicaisisopaula@gmail.com2023-05-09T16:24:37Z2019-11-01PAULA, Jéssica Isis Oliveira de. Diferentes abordagens para a descoberta de novos alvos terapêuticos contra a doença de Chagas. 2019. 129 f. Tese (Doutorado em Microbiologia) – Faculdade de Ciências Médicas, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, 2019.http://www.bdtd.uerj.br/handle/1/19538Trypanosoma cruzi is the causative agent of Chagas disease. The parasite needs to enter in the host cell to grow and expand infection. Trypomastigotes are classicaly infective forms of T. cruzi, and they are able to invade host cell. After the invasion of the mammalian cell, parasites start amastigogenesis process, in which trypomastigotes forms differentiate into amastigotes that proliferate.Those steps of intracelular invasion and amastigogenesis are essential to parasite. Currently, two drugs are used to treatment: benznidazol and nifurtimox. Both drugs show side adverses effects that causes treatment interruption by the patient, due to of the low efficacy in chronic phase of Chagas diseases, making important the development of new drugs. Basically, two aproaches are applied in drugs development: phenotype and targed-based. In this work, we used two strategies in the targed-based approach. The first was to study the parasite biology relationship with host cell. In this context, we search ROS relation, especifically H2O2, in the intracelular infection. Once ROS are produced in the phagolysosome and in this compartment has signaling to amastigogenesis, we investigated the H2O2 influence in this process.We observed that H2O2 increased 30% of differentiation after two hours of induction by low pH. However, among antioxidant molecules tested, only PEG-catalase, enzyme that neutralizes directly H2O2 inhibited amastigogeneses improvement ROS-induced. We observed that H2O2 increased a serine/threonine phosphatase activity, as well as PP2A inhibitor okadaic acid (OKA) reverted H2O2 effect upon amastigogenesis. Corroborating ROS involvement, observed that differentiaton induced by acid pH produced H2O2 after one hour, being reduced later. When we evaluated the superoxide dismutase gene expression of T. cruzi, we observed that mitochondrial isoform (Fe-SODA) were up regulated after 60 minutes and 90 minutes differentiation induction. Furthermore, we detected a reduction in the oxygen consumption and maximal eletron tranference system capacity, as well as loss in the mitochondrial membrane potential (ΔΨm) after 60 minutes and 120 minutes of amastigogenese induction. In consideration of these results, wherein mitochondrial dysfunction is followed by loss of ΔΨm, H2O2 production observed can be from mitochondrial by mitochondrial permeability transition pore (mPTP). Indeed, mPTP inhibitor cyclosporin and mitochondrial antioxidant mitoTEMPO reduced H2O2 generation induced by low pH. Together, this results showed that H2O2 act as signaling molecule to differentiation. The second strategy were trypomastigote surface screening by phage display library. After biopanning, were identified two high affinity clones, N3 and N1. The peptide express in N3 clone were synthetized and in vitro bound assays showed peptide specificity to infective forms of T. cruzi. Furthermore, peptide were active inhibiting Vero cells invasion. Thus, N3 peptide bound in a surface molecule of T. cruzi that is important to cell invasion.O Trypanosoma cruzi é o agente causador da doença de Chagas. O parasito obrigatoriamente necessita entrar na célula para replicar e expandir a infecção no hospedeiro vertebrado. Os tripomomastigotas são classicamente descritos como as formas infectantes do T. cruzi, e possuem a capacidade invadir a célula hospedeira. Após a invasão na célula do vertebrado, o parasito sofre o processo de amastigogênese, no qual formas tripomastigotas se diferenciam para as formas amastigotas, que proliferam. As etapas de invasão intracelular e amastigogênese são essenciais para o estabelecimento do parasito, sendo alvos de pesquisa para desenvolvimento de drogas. Atualmente são utilizados dois fármacos para o tratamento: benznidazol e nifurtimox. Ambos os fármacos apresentam efeitos adversos que provocam a interrupção do tratamento pelo paciente, além de terem baixa eficácia na fase crônica da doença de Chagas, tornando importante o desenvolvimento de novas drogas. Existem basicamente duas abordagens no desenvolvimento de novos fármacos: a fenotípica e a baseada em alvo. Nesse trabalho foi utilizada a abordagem baseada em alvo. Dentro dessa abordagem foram empregadas duas estratégias diferentes. A primeira consistiu em entender a biologia da relação do parasito e da célula hospedeira. Nesse contexto, estudamos o papel das ROS, mais especificamente o H2O2, na infecção intracelular. Uma vez que é observada a produção de ROS no fagolisossomo e que nesse compartimento ocorre a sinalização para a amastigogênese, investigou-se a influência de H2O2 nesse processo. Foi observado que o H2O2 aumentou em 30% a diferenciação após duas horas de indução por pH ácido. No entanto, dentre os antioxidantes testados, apenas PEG-catalase, uma enzima que dismuta diretamente H2O2 em O2 e H2O, foi capaz de inibir a indução da amastigogênese por estas ROS. Observou-se que o H2O2 foi capaz de ativar uma serina/treonina fosfatase, bem como, o ácido ocadáico (OKA) inibidor de PP2A, reverteu o efeito deste ROS na amastigogênese. Corroborando a participação de ROS, observou-se que a indução à diferenciação por pH ácido promoveu a produção de H2O2 após 1 hora, sendo essa reduzida posteriormente. Ao avaliar a expressão gênica da superóxido dismutase do T. cruzi, observou-se que a isoforma mitocondrial (Fe-SODA) foi modulada positivamente após 60 minutos e 90 minutos de indução à diferenciação. Além disso, foi observada a redução do consumo de oxigênio e capacidade máxima de respiração, bem com a redução do potencial de membrana mitocondrial (ΔΨm) após 60 e 120 minutos de indução à amastigogênese. Considerando esses resultados, em que a disfunção mitocondrial é acompanhada pela perda de ΔΨm, a produção de H2O2 observada pode ser de origem mitocondrial promovida pela abertura do poro de transição de permeabilidade mitocondrial (mPTP). De fato, o inibidor de mPTP, ciclosporina, e o antioxidante mitocondrial, mitoTEMPO, foram capazes de inibir a produção de H2O2 induzida pelo pH ácido. Em conjunto, esses resultados demonstraram que o H2O2 atuou como molécula sinalizadora para diferenciação de formas tripomastigotas para amastigotas em T. cruzi. A segunda estratégia consistiu na varredura da superfície de tripomastigotas com uma biblioteca de fagos. Após o biopanning, foram identificados dois clones com afinidade para as formas tripomastigotas, N3 e N1. O peptídeo expresso no clone N3 foi sintetizado e ensaios in vitro de ligação demonstraram a especificidade do peptídeo com as formas infectantes do T. cruzi. Além disso, o peptídeo mostrou-se bioativo, inibindo a invasão em células Vero. Desta forma, o peptídeo N3 se liga à uma molécula de superfície de T. cruzi que é importante para a invasão celular.Submitted by Heloísa CB/A (helobdtd@gmail.com) on 2023-05-09T16:24:37Z No. of bitstreams: 1 Tese - Jéssica Isis Oliveira de Paula - Completa - 2019.pdf: 13045711 bytes, checksum: e5f74d166bc94e4ebdb2fffb9c20448b (MD5)Made available in DSpace on 2023-05-09T16:24:37Z (GMT). No. of bitstreams: 1 Tese - Jéssica Isis Oliveira de Paula - Completa - 2019.pdf: 13045711 bytes, checksum: e5f74d166bc94e4ebdb2fffb9c20448b (MD5) Previous issue date: 2019-11-01application/pdfporUniversidade do Estado do Rio de JaneiroPrograma de Pós-Graduação em MicrobiologiaUERJBrasilCentro Biomédico::Faculdade de Ciências MédicasTrypanosoma cruziAmastigogenesisROSPhage displayBiopanningTarget-based approachTrypanosoma cruziAmastigogêneseROSPhage displayBiopanningAbordagem baseada em alvoCIENCIAS BIOLOGICAS::BIOQUIMICA::BIOQUIMICA DOS MICROORGANISMOSDiferentes abordagens para a descoberta de novos alvos terapêuticos contra a doença de ChagasDifferents approachs in target-based drug discovery against Chagas Diseaseinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccessreponame:Biblioteca Digital de Teses e Dissertações da UERJinstname:Universidade do Estado do Rio de Janeiro (UERJ)instacron:UERJORIGINALTese - Jéssica Isis Oliveira de Paula - Completa - 2019.pdfTese - Jéssica Isis Oliveira de Paula - Completa - 2019.pdfapplication/pdf13045711http://www.bdtd.uerj.br/bitstream/1/19538/2/Tese+-+J%C3%A9ssica+Isis+Oliveira+de+Paula+-+Completa+-+2019.pdfe5f74d166bc94e4ebdb2fffb9c20448bMD52LICENSElicense.txtlicense.txttext/plain; charset=utf-82123http://www.bdtd.uerj.br/bitstream/1/19538/1/license.txte5502652da718045d7fcd832b79fca29MD511/195382024-02-26 15:59:53.338oai:www.bdtd.uerj.br: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Biblioteca Digital de Teses e Dissertaçõeshttp://www.bdtd.uerj.br/PUBhttps://www.bdtd.uerj.br:8443/oai/requestbdtd.suporte@uerj.bropendoar:29032024-02-26T18:59:53Biblioteca Digital de Teses e Dissertações da UERJ - Universidade do Estado do Rio de Janeiro (UERJ)false |
dc.title.por.fl_str_mv |
Diferentes abordagens para a descoberta de novos alvos terapêuticos contra a doença de Chagas |
dc.title.alternative.eng.fl_str_mv |
Differents approachs in target-based drug discovery against Chagas Disease |
title |
Diferentes abordagens para a descoberta de novos alvos terapêuticos contra a doença de Chagas |
spellingShingle |
Diferentes abordagens para a descoberta de novos alvos terapêuticos contra a doença de Chagas Paula, Jéssica Isis Oliveira de Trypanosoma cruzi Amastigogenesis ROS Phage display Biopanning Target-based approach Trypanosoma cruzi Amastigogênese ROS Phage display Biopanning Abordagem baseada em alvo CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOQUIMICA DOS MICROORGANISMOS |
title_short |
Diferentes abordagens para a descoberta de novos alvos terapêuticos contra a doença de Chagas |
title_full |
Diferentes abordagens para a descoberta de novos alvos terapêuticos contra a doença de Chagas |
title_fullStr |
Diferentes abordagens para a descoberta de novos alvos terapêuticos contra a doença de Chagas |
title_full_unstemmed |
Diferentes abordagens para a descoberta de novos alvos terapêuticos contra a doença de Chagas |
title_sort |
Diferentes abordagens para a descoberta de novos alvos terapêuticos contra a doença de Chagas |
author |
Paula, Jéssica Isis Oliveira de |
author_facet |
Paula, Jéssica Isis Oliveira de jessicaisisopaula@gmail.com |
author_role |
author |
author2 |
jessicaisisopaula@gmail.com |
author2_role |
author |
dc.contributor.advisor1.fl_str_mv |
Paes, Marcia Cristina |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/7463829190927034 |
dc.contributor.advisor2.fl_str_mv |
Nogueira, Natália Pereira de Almeida |
dc.contributor.advisor2Lattes.fl_str_mv |
http://lattes.cnpq.br/6320509465681227 |
dc.contributor.referee1.fl_str_mv |
Dias, Silvia Amaral Gonçalves |
dc.contributor.referee1Lattes.fl_str_mv |
http://lattes.cnpq.br/6104190112253764 |
dc.contributor.referee2.fl_str_mv |
Silva, José Roberto Machado e |
dc.contributor.referee2Lattes.fl_str_mv |
http://lattes.cnpq.br/6430514515393384 |
dc.contributor.referee3.fl_str_mv |
Fernandes, José Roberto Meyer |
dc.contributor.referee3Lattes.fl_str_mv |
http://lattes.cnpq.br/8471949500798737 |
dc.contributor.referee4.fl_str_mv |
Galina Filho, Antônio |
dc.contributor.referee4Lattes.fl_str_mv |
http://lattes.cnpq.br/8940530306495736 |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/9150050143205166 |
dc.contributor.author.fl_str_mv |
Paula, Jéssica Isis Oliveira de jessicaisisopaula@gmail.com |
contributor_str_mv |
Paes, Marcia Cristina Nogueira, Natália Pereira de Almeida Dias, Silvia Amaral Gonçalves Silva, José Roberto Machado e Fernandes, José Roberto Meyer Galina Filho, Antônio |
dc.subject.eng.fl_str_mv |
Trypanosoma cruzi Amastigogenesis ROS Phage display Biopanning Target-based approach |
topic |
Trypanosoma cruzi Amastigogenesis ROS Phage display Biopanning Target-based approach Trypanosoma cruzi Amastigogênese ROS Phage display Biopanning Abordagem baseada em alvo CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOQUIMICA DOS MICROORGANISMOS |
dc.subject.por.fl_str_mv |
Trypanosoma cruzi Amastigogênese ROS Phage display Biopanning Abordagem baseada em alvo |
dc.subject.cnpq.fl_str_mv |
CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOQUIMICA DOS MICROORGANISMOS |
description |
Trypanosoma cruzi is the causative agent of Chagas disease. The parasite needs to enter in the host cell to grow and expand infection. Trypomastigotes are classicaly infective forms of T. cruzi, and they are able to invade host cell. After the invasion of the mammalian cell, parasites start amastigogenesis process, in which trypomastigotes forms differentiate into amastigotes that proliferate.Those steps of intracelular invasion and amastigogenesis are essential to parasite. Currently, two drugs are used to treatment: benznidazol and nifurtimox. Both drugs show side adverses effects that causes treatment interruption by the patient, due to of the low efficacy in chronic phase of Chagas diseases, making important the development of new drugs. Basically, two aproaches are applied in drugs development: phenotype and targed-based. In this work, we used two strategies in the targed-based approach. The first was to study the parasite biology relationship with host cell. In this context, we search ROS relation, especifically H2O2, in the intracelular infection. Once ROS are produced in the phagolysosome and in this compartment has signaling to amastigogenesis, we investigated the H2O2 influence in this process.We observed that H2O2 increased 30% of differentiation after two hours of induction by low pH. However, among antioxidant molecules tested, only PEG-catalase, enzyme that neutralizes directly H2O2 inhibited amastigogeneses improvement ROS-induced. We observed that H2O2 increased a serine/threonine phosphatase activity, as well as PP2A inhibitor okadaic acid (OKA) reverted H2O2 effect upon amastigogenesis. Corroborating ROS involvement, observed that differentiaton induced by acid pH produced H2O2 after one hour, being reduced later. When we evaluated the superoxide dismutase gene expression of T. cruzi, we observed that mitochondrial isoform (Fe-SODA) were up regulated after 60 minutes and 90 minutes differentiation induction. Furthermore, we detected a reduction in the oxygen consumption and maximal eletron tranference system capacity, as well as loss in the mitochondrial membrane potential (ΔΨm) after 60 minutes and 120 minutes of amastigogenese induction. In consideration of these results, wherein mitochondrial dysfunction is followed by loss of ΔΨm, H2O2 production observed can be from mitochondrial by mitochondrial permeability transition pore (mPTP). Indeed, mPTP inhibitor cyclosporin and mitochondrial antioxidant mitoTEMPO reduced H2O2 generation induced by low pH. Together, this results showed that H2O2 act as signaling molecule to differentiation. The second strategy were trypomastigote surface screening by phage display library. After biopanning, were identified two high affinity clones, N3 and N1. The peptide express in N3 clone were synthetized and in vitro bound assays showed peptide specificity to infective forms of T. cruzi. Furthermore, peptide were active inhibiting Vero cells invasion. Thus, N3 peptide bound in a surface molecule of T. cruzi that is important to cell invasion. |
publishDate |
2019 |
dc.date.issued.fl_str_mv |
2019-11-01 |
dc.date.accessioned.fl_str_mv |
2023-05-09T16:24:37Z |
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info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
PAULA, Jéssica Isis Oliveira de. Diferentes abordagens para a descoberta de novos alvos terapêuticos contra a doença de Chagas. 2019. 129 f. Tese (Doutorado em Microbiologia) – Faculdade de Ciências Médicas, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, 2019. |
dc.identifier.uri.fl_str_mv |
http://www.bdtd.uerj.br/handle/1/19538 |
identifier_str_mv |
PAULA, Jéssica Isis Oliveira de. Diferentes abordagens para a descoberta de novos alvos terapêuticos contra a doença de Chagas. 2019. 129 f. Tese (Doutorado em Microbiologia) – Faculdade de Ciências Médicas, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, 2019. |
url |
http://www.bdtd.uerj.br/handle/1/19538 |
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por |
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por |
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info:eu-repo/semantics/openAccess |
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openAccess |
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application/pdf |
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Universidade do Estado do Rio de Janeiro |
dc.publisher.program.fl_str_mv |
Programa de Pós-Graduação em Microbiologia |
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UERJ |
dc.publisher.country.fl_str_mv |
Brasil |
dc.publisher.department.fl_str_mv |
Centro Biomédico::Faculdade de Ciências Médicas |
publisher.none.fl_str_mv |
Universidade do Estado do Rio de Janeiro |
dc.source.none.fl_str_mv |
reponame:Biblioteca Digital de Teses e Dissertações da UERJ instname:Universidade do Estado do Rio de Janeiro (UERJ) instacron:UERJ |
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Universidade do Estado do Rio de Janeiro (UERJ) |
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UERJ |
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UERJ |
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Biblioteca Digital de Teses e Dissertações da UERJ |
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Biblioteca Digital de Teses e Dissertações da UERJ |
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http://www.bdtd.uerj.br/bitstream/1/19538/2/Tese+-+J%C3%A9ssica+Isis+Oliveira+de+Paula+-+Completa+-+2019.pdf http://www.bdtd.uerj.br/bitstream/1/19538/1/license.txt |
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MD5 MD5 |
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Biblioteca Digital de Teses e Dissertações da UERJ - Universidade do Estado do Rio de Janeiro (UERJ) |
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bdtd.suporte@uerj.br |
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