Endocytosis is a biological event well described in where macromolecules are cytofarynx complex and organelles found at the posterior region of the and contain lysosomal enzymes, such as cruzipain. produce polyclonal or monoclonal antibodies (mAbs) against proteins identified in proteomic analyzes of reservosomes (cruzipain and Tc to label organelles of the endocytic pathway and amastigotes. Recombinant proteins were expressed in into Balb/c mice. Reactivity of the anti extracts of T. cruzi and recombinant protein antibodies anti-TcHIP showed this protein in the Golgi apparatus confirmed by co-localization with GFP recombinant cruzipain recognized the epimastigotes. For production of mAbs, splenocytes with recombinant cruzipain were fused with the P3X63Ag8.653 myeloma cell line (ATCC CRL-1580). Hybridomas were immunofluorescence. The most stable hybridoma was selected for limiting dilution and one clone against recombinant cruzipain (mAb CZP reservosomes. This antibody co-localization of uptaken transferrin with cruzipain. It was also possible to demonstrate for the first time by flow cytometry of Trypanosoma cruzi Trypanosoma cruzi ingested via the flagellar pocket and/or then targeted to reservosomes. Reservosomes are cell body that store ingested molec Aim of this dissertation TcHIP), and use the to study endocytosis in E. coli, purified and inoculated eactivity anti-sera was confirmed by western blot proteins. Immuno-localization using polyclonal of T. cruzi GFP-TcRab7. Polyclonal antibodies against protein in the Golgi and reservosomes of obtained from a mouse immunized then screened by ELISA, western blot and CZP-315.D9) was obtained, specific was then used as a tool to investigate in amastigotes the the endocytic activity in T. cruzi amastigotes. / Approved for entry into archive by Renata Fontoura (comunicaicc@fiocruz.br) on 2014-12-04T12:19:07Z (GMT) No. of bitstreams: 1 Dissertação Cassiano Martin Batista (1).pdf: 5056433 bytes, checksum: 4df99de041c7ead17fee43aacb621dc6 (MD5) / Made available in DSpace on 2014-12-04T12:19:08Z (GMT). No. of bitstreams: 1 Dissertação Cassiano Martin Batista (1).pdf: 5056433 bytes, checksum: 4df99de041c7ead17fee43aacb621dc6 (MD5) Previous issue date: 2014 / Fundação Oswaldo cruz, Instituto Carlos Chagas. Curitiba, PR, Brasil / A endocitose é um evento biológico já bem descrito em formas epimastigotas de Trypanosoma cruzi, onde macromoléculas são internalizadas através da bolsa flagelar e/ou do complexo citóstoma/citofaringe e direcionadas aos reservossomos. Reservossomos são grandes organel estocam moléculas ingeridas e cruzipaína. O objetivo desta dissertação foi produzir anticorpos policlonais ou monoclonais (mAbs) contra proteínas identificada (cruzipaína e TcHIP), e utilizá estudos de endocitose em formas amastigotas de foram expressas em E. coli reatividade dos anti-soros foi confirmada por cruzi e contra proteína recombinante. Imunolocalização utilizando anticorpos policlonais anti-TcHIP mostrou que esta proteína está presente no complexo de Golgi em T. cruzi, o que foi confirmado por co anticorpos policlonais obtidos contra cruzipaína recombinante demonstraram esta proteína no Golgi e em reserv mAbs, esplenócitos de um camundongo imunizado com cruzipaína recombinante foram fusionados com células de mieloma da linhagem P3X63Ag8.653 (ATCC CRL Os hibridomas foram triados p hibridoma mais estável foi cruzipaína recombinante (mAb CZP Este anticorpo foi utilizado como ferramenta pa transferrina ingerida com a cruzipaína em formas amastigotas. Foi também possível demonstrar pela primeira vez por citometria de fluxo a atividade endocítica em formas amastigotas de T. cruzi. Trypanosoma cruzi, organelas localizadas na região posterior do parasito que contém enzimas lisossomais, como por exemplo a identificadas na proteômica de reservossomos utilizá-las como marcadores de organelas da via endocítica e em T. cruzi. Proteínas recombinantes coli, purificadas e inoculadas em camundongos Balb/c. A western blot contra extrato total de, co-localização com TcRab7-GFP. Por outro lado, reservossomos de epimastigotas de T. cruzi. / Endocytosis is a biological event well described in where macromolecules are cytofarynx complex and organelles found at the posterior region of the and contain lysosomal enzymes, such as cruzipain. produce polyclonal or monoclonal antibodies (mAbs) against proteins identified in proteomic analyzes of reservosomes (cruzipain and Tc to label organelles of the endocytic pathway and amastigotes. Recombinant proteins were expressed in into Balb/c mice. Reactivity of the anti extracts of T. cruzi and recombinant protein antibodies anti-TcHIP showed this protein in the Golgi apparatus confirmed by co-localization with GFP recombinant cruzipain recognized the epimastigotes. For production of mAbs, splenocytes with recombinant cruzipain were fused with the P3X63Ag8.653 myeloma cell line (ATCC CRL-1580). Hybridomas were immunofluorescence. The most stable hybridoma was selected for limiting dilution and one clone against recombinant cruzipain (mAb CZP reservosomes. This antibody co-localization of uptaken transferrin with cruzipain. It was also possible to demonstrate for the first time by flow cytometry of Trypanosoma cruzi Trypanosoma cruzi ingested via the flagellar pocket and/or then targeted to reservosomes. Reservosomes are cell body that store ingested molec Aim of this dissertation TcHIP), and use the to study endocytosis in E. coli, purified and inoculated eactivity anti-sera was confirmed by western blot proteins. Immuno-localization using polyclonal of T. cruzi GFP-TcRab7. Polyclonal antibodies against protein in the Golgi and reservosomes of obtained from a mouse immunized then screened by ELISA, western blot and CZP-315.D9) was obtained, specific was then used as a tool to investigate in amastigotes the the endocytic activity in T. cruzi amastigotes.

Dissertação (mestrado)—Universidade de Brasília, Faculdade em Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2016. / Submitted by Albânia Cézar de Melo (albania@bce.unb.br) on 2017-02-21T12:58:13Z No. of bitstreams: 1 2016_NilzadoCarmoFontes.pdf: 5507563 bytes, checksum: a852350264b81411483c9906f8dccb46 (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2017-03-27T15:30:17Z (GMT) No. of bitstreams: 1 2016_NilzadoCarmoFontes.pdf: 5507563 bytes, checksum: a852350264b81411483c9906f8dccb46 (MD5) / Made available in DSpace on 2017-03-27T15:30:17Z (GMT). No. of bitstreams: 1 2016_NilzadoCarmoFontes.pdf: 5507563 bytes, checksum: a852350264b81411483c9906f8dccb46 (MD5) / Os distúrbios congênitos de glicosilação tipo II (CDG II) são doenças graves, multissistêmicas, devidos a mutações deletérias em genes que codificam proteínas que mantêm a integridade do complexo de Golgi e de outros componentes que participam do processo de N-glicosilação. Este representa modificação pós-traducional de proteínas que dá origem às estruturas de N-glicanos, que são essenciais para o funcionamento normal do organismo. Para avaliação do desempenho da espectrometria de massa no diagnóstico dos CDG II, foi realizada análise e comparação por espectrometria de massa, de estruturas de N-glicanos das moléculas de glicoproteinas plasmáticas de indivíduos portadores e não portadores de distúrbios congênitos de glicosilação tipo II e verificou-se que as diferenças entre os dois grupos foram em maior parte quantitativas. Entretanto, um subtipo de CDG II (MAN1B1-CDG) levou a produção e acúmulo de N-glicanos diferenciais que permitem um direcionamento do diagnóstico molecular. Através de diferenças quantitativas, a análise e estudo das estruturas de N-glicanos acumuladas estreita o número de genes candidatos ao diagnóstico molecular, auxiliando na identificação destes distúrbios, que não apresentam sintomas clínicos nem glicobiomarca específicos, e se confundem clinicamente com outras doenças metabólicas. Este trabalho é pioneiro no Brasil e evidenciou a importância da espectrometria de massa no auxílio ao diagnóstico de portadores de distúrbios congênitos de glicosilação tipo II. / Congenital Disorders of Glycosylation type II (CDG II) comprise a group of severe, multisystem diseases caused by mutations in genes responsible for maintaining the integrity of Golgi apparatus and of other components involved in N-glycosylation processing, i.e, post-translational modifications, which result in N-glycan structures. We performed analyses of N-glycan structures of plasma glycoproteins of CDG II patients and healthy controls, in order to evaluate the performance of mass spectrometry in the diagnosis of these diseases. Most of the differences between both groups of individuals were quantitative. One of the CDG II subtypes (MAN1B1-CDG) led to the accumulation of abnormal N-glycans, which allowed diagnosis, not yet confirmed by molecular methods. Analysis and characterization of accumulated N-glycan structures narrow the number of candidate genes to be considered in the diagnosis. This helps in the identification of these diseases, because the definite diagnostic is not possible by clinical features and there are no specific biomarkers. To the best of our knowledge, this is the first mass spectrometry-based study in Brazil aiming the diagnosis of these disorders. It also highlights the importance of mass spectrometry in the diagnosis of CDG II.