Thesis (Ph.D.). / Written for the Dept. of Anatomy and Cell Biology. Title from title page of PDF (viewed 2008/05/08). Includes bibliographical references.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Chicago, Pritzker School of Medicine, Department of Molecular Genetics and Cell Biology, June 2002. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, June 2003. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (November 7, 2006) Vita. Includes bibliographical references.

University, Diss., 2001--Kassel. / Lizenzpflichtig.

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.

Abstract The role of vacuolar H+-ATPase (V-ATPase) in exocytic and endocytic membrane transport processes was studied by using its specific inhibitor, bafilomycin A1 (Baf A1), as a tool. On the exocytic pathway, both brefeldin A- and nocodazole-induced retrograde transport of Golgi proteins to the endoplasmic reticulum (ER) were inhibited by Baf A1. Furthermore, p58/ERGIC-53, which normally cycles between the ER, the intermediate compartment (IC), and cis-Golgi, was arrested in pre-Golgi tubules and vacuoles, and the number of p58-positive 80-nm Golgi (COPI) vesicles was reduced, suggesting that the drug inhibits the vesicle-mediated retrieval of the protein from post-ER compartments. The small GTPase rab1p was efficiently recruited to the tubules, accumulating in the presence of Baf A1. In contrast, these tubules showed no enrichment of anterogradely transported proteins, indicating that they participate in retrograde transport. Interestingly, acidic lumenal pH could only be detected in the more central pre-Golgi elements. The forward (anterograde) transport of newly synthesized Semliki Forest virus (SFV) and vesicular stomatitis virus (VSV) glycoproteins from the ER to the cis-Golgi was largely unaffected by Baf A1. However, maturation processes occurring in the trans-Golgi were inhibited, and the amounts of viral glycoproteins appearing at the cell surface were reduced. Newly synthesized VSV glycoprotein accumulated into rab1p-positive Golgi membranes in the presence of Baf A1, indicating that the transport from cis-Golgi was affected. Furthermore, O-glycosylation of the expressed CD8 chimeras and lectin cytochemistry experiments indicate that Baf A1 affects the transport from cis-Golgi. Instead, Baf A1 did not affect the transport of viral glycoproteins from the trans-Golgi network to the cell surface. We propose, that anterograde intra-Golgi traffic may be affected indirectly by Baf A1, as it inhibits retrograde vesicle-mediated transport and thus cisternal maturation. Baf A1 inhibited the entry of SFV into BHK-21 cells. Thus, V-ATPase was responsible for the acidification of the endosomes needed for virus entry. In cells infected with VSV and subsequently treated with Baf A1, virus particles were found to be accumulated in tubular membrane structures, which also contained endocytosed BSA-gold. Neither VSV nor BSA-gold particles were detected in lysosomal glycoprotein (lgp) 120-positive lysosomes, however. Thus, secreted and further endocytosed virus particles accumulate into tubulated endocytic organelles, apparently early endosomes, in Baf A1-treated cells. We conclude that the transport from endosomes to lysosomes is inhibited by Baf A1. The bulk of rab7 GTPase, which participates in vesicle fusion to late endosomes, was localized to the ruffled border (RB) membrane of bone-resorbing osteoclast. This indicates that the membrane has some characteristics of late endosomal membranes and that endocytic membrane transport is oriented towards the RB. Consistently, both endocytosed lumenal horseradish peroxidase and receptor-bound transferrin were delivered to the RB. The delivery of membrane-associated transferrin to the RB further indicates that the RB has some endosomal characteristics and suggests that the endocytic pathway contributes to the maintenance of functional RB. The endocytic pathway could act in balancing the membrane traffic associated with transcytosis from the RB to the basal plasma membrane. Endocytic processes in osteoclasts appeared to be very sensitive to Baf A1. Thus, blocking of the endocytic membrane traffic towards the RB could explain the inactivation of cells by low concentrations of the drug.

La malaria est une maladie infectieuse causant plus de 500 000 morts chaque année. La maladie est causée par un protozoaire de la famille Plasmodium. L’apparition de souches résistantes aux traitements actuels et l’absence de vaccin efficace rendent la découverte de nouvelles cibles thérapeutiques urgente. Le parasite possède un complexe apical, un groupement de vacuoles sécrétoires spécialisées contenant les protéines responsables de l’invasion du globule rouge. Nous nous intéressons aux mécanismes gouvernant le transport intracellulaire de ces protéines et à la biogenèse du complexe apical lors de la formation des nouveaux parasites. Plus particulièrement, nous nous intéressons au rôle des phosphoinositides dans le recrutement des protéines à la membrane de l’appareil de Golgi. Par analyse bio-informatique du génome de P. falciparum, nous avons identifié plusieurs protéines effectrices liant potentiellement les phosphoinositides. Les travaux présentés dans ce mémoire concernent Mal13P1.188, une protéine possédant un domaine Pleckstrin homology. Nous proposons que Mal13P1.188 ait un rôle dans la génération du complexe apical en recrutant les protéines le constituant à la membrane du Golgi par la liaison avec les phosphoinositides. Afin de vérifier nos hypothèses, nous avons généré une lignée de parasite dont le gène de Mal13P1.188 est fusionné avec une GFP et une hémagglutinine. À l’aide de cette lignée de parasite, nous avons pu identifier Mal13P1.188 à proximité de l’appareil de Golgi lorsque les parasites étaient sous la forme schizont du cycle érythrocytaire. D’autres expériences ont permis de confirmer que le domaine Pleckstrin homology de Mal13P1.188 était capable de reconnaître les différentes formes de phosphoinositides. Finalement, d’autres travaux devront être faits sur Mal13P1.188 afin de déterminer si elle est essentielle à la survie du parasite. / Malaria is a deadly infectious disease taking more than 500,000 lives each year. The disease is caused by a protozoan of the Plasmodium family. Resistant strains beginning to spread and the inexistence of an efficient vaccine make the discovery of new targets urgent. The parasite secretes proteins to invade the red blood cell. Those proteins are regrouped in the apical complex, a group of organelles used for the invasion. Our research team focus on the transport mechanisms that drive the formation of the apical complex during the cellular division of new parasite. In other terms, we are interested on the role of phosphoinositide in the recruitment of protein inside the Golgi apparatus. After a bioinformatics analyse the P. falciparum genome, we identified many effectors protein that can bind phosphoinositides. Among them, we focused our work on Mal13P1.188, a protein with a Pleckstrin homology domain. We propose that Mal13P1.188 has a role in the recruitment of the apical proteins to the Golgi membrane using phosphoinositide as a marker on the membrane. To verify that hypothesis, we generated a strain of parasite with endogenous Mal13P1.188 tagged to a GFP and a hemagglutinin. With those parasites, we identified Mal13P1.188 near the Golgi apparatus during the Schizont stage of the blood cycle. Other experiment confirmed that the Pleckstrin homology domain of Mal13P1.188 is able to bind different form of phosphoinositides. Finally, more work has to be done to confirm if Mal13P1.188 is essential to the parasite survival.