L’adressage des protéines vers leur correct emplacement est crucial pour la cellule. L’information d’adressage est fournie sous la forme d’une séquence signale par le polypeptide lui-même. Chez Escherichia coli, les protéines membranaires sont adressées vers la membrane de façon co-traductionnelle via la particule de reconnaissance du signal (SRP) tandis que les protéines sécrétées suivent la voie de translocation post-traductionnelle caractérisée par les protéines SecB et SecA qui sont impliquées dans le processus d’adressage. Ces deux voies convergent au niveau du canal de translocation des protéines SecYEG. Chose intéressante, SecYEG a la possibilité de recruter les domaines accessoires SecDF-YajC et YidC et ainsi former le complexe holotranslocon (HTL). La recherche actuelle sur la translocation des protéines se concentre principalement sur la structure et fonction du canal de translocation des protéines hétérotrimérique bactérien SecYEG qui est conservé. Peu de choses sont connues concernant la structure et la fonction des composants additionnels SecD, SecF et YidC formant la machinerie de translocation et qui sont essentiels pour E. coli. Ceci est dû principalement à l’absence d’un complexe holotranslocon SecYEG-DF-YidC (HTL) recombinant purifié. En conséquence, une analyse biophysique et structurale minutieuse de ce large complexe transmembranaire composé de sept sous-unités est toujours en suspens.En utilisant un nouveau système d’expression pour des complexes multi-protéiques basé sur la recombinaison de vecteur chez E. coli, nous avons avec succès surproduit l’holotranslocon SecYEG-DF-YajC-YidC et son sous-complexe composé de SecDF-YajC-YidC (DFYY). Nous avons également réussi à solubiliser avec l’aide de détergents et à purifier ces complexes. L’holotranslocon purifié a ensuite été utilisé afin de caractériser de façon biochimique le complexe et de déterminer la structure de l’holotranslocon. Premièrement, le complexe HTL semble être plus compétent pour l’insertion co-traductionnelle des protéines membranaires comparé à SecYEG isolé. Concernant la translocation post-traductionnelle d’une protéine de la membrane externe à tonneau β, dépendante de la présence de SecA et d’ATP, l’influence de la force proton motrice sur ce processus est augmentée. De plus, la présence du domaine accessoire semble améliorer l’attachement du ribosome au translocon. En utilisant des cellules déplétées de SecDF et YajC, nous avons identifié des substrats possibles de HTL qui doivent maintenant être confirmés et analysés manière plus approfondie par des expériences de translocation in vitro.Par la suite, nous avons résolu la structure de l’holotranslocon par cryo-microscopie électronique (ME) et analyse des particules isolées. En comparant les reconstructions de ME8du complexe HTL avec le sous-complexe de domaine accessoire SecDF-YajC-YidC, nous avons été capable de localisé le complexe principal SecYEG. La structure de HTL par cryo-ME a pu être affinée jusqu’à une résolution de 10.5 Å. Cette structure permet le placement des structures à haute résolution disponibles de SecYEG, SecDF et YidC afin de générer un modèle quasi-atomique de l’holotranslocon. Les jeu de données ainsi obtenus sont volumineux et souffrent d’un taux élevé de « faux positifs », probablement dû à des réactions de réticulation inter-complexe. C’est pourquoi ils nécessitent une évaluation minutieuse et les résultats intéressants devraient être confirmés par une méthode indépendante. Dans le futur, des études structurales du complexe ribosome-HTL par cryo-ME ainsi qu’une reconstitution de HTL dans des nanodisques vont être menées pour révéler la conformation de HTL en cours de translocation dans un environnement plus physiologique. Des études biochimiques complémentaires sur le mécanisme de co- et post-translocation par HTL et son spectre substrats abordent la question du rôle physiologique de l’holotranslocon dans la cellule. / Targeting of proteins to their proper location in the cell is crucial to the cell. The targeting information is provided in form of a signal sequence by the polypeptide itself. In Escherichia coli, membrane proteins are targeted co-translationally via the signal recognition particle (SRP) to the membrane whereas secretory proteins follow the post-translational translocation pathway characterized by the proteins SecB and SecA involved in the targeting process. Both pathways converge at the protein-conducting channel SecYEG. Interestingly, SecYEG has the possibility to recruit accessory domains SecDF-YajC and YidC, forming the holotranslocon (HTL) complex. Current research on protein translocation mostly focuses on the structure and function of the conserved bacterial heterotrimeric protein conducting channel SecYEG. Not much is known about the structure and function of the additional components of the translocation machinery SecD, SecF and YidC which are essential for E. coli. This is largely due to the lack of a purified, recombinant SecYEG-DF-YidC holotranslocon (HTL) complex. Accordingly, a thorough biophysical and structural analysis of this large, seven-membered transmembrane complex is still pending.Using a new recombineering-based vector system for expression of multi-protein complexes in E. coli, we successfully over-produced the SecYEG-DF-YajC-YidC holotranslocon and its subcomplex consisting of SecDF-YajC-YidC (DFYY). We also succeeded in detergent-solubilising and purifying these complexes. The purified holotranslocon was used to biochemically characterize the complex and to determine the structure of the holotranslocon. First of all, the HTL seems to be more competent for co-translational membrane proteins insertion compared to SecYEG alone. Regarding the post-translational translocation of a β-barrel outer-membrane protein, driven by SecA and ATP, the proton-motive force dependence of this process is increased. Furthermore, the presence of the accessory domains seems to enhance the binding of the ribosome to the translocon. By using cells depleted of SecDF and YajC, we identified possible HTL-substrates which have to be confirmed and further analyzed yet by in vitro translocation experiments.Subsequently, we solved by cryo-electron microscopy (EM) and single particle analysis the structure of the holotranslocon. By comparing the EM reconstructions of the HTL complex with the subcomplex of the accessory domains SecDF-YajC-YidC, we were able to localize the core complex SecYEG. The HTL cryo-EM structure could be refined to a resolution of 10.5 Å. This structure allows the placement of the available high–resolution crystal structure of SecYEG, SecDF, and YidC to generate a quasi-atomic model of the holotranslocon.6In order to confirm our quasi-atomic model, we made use of different crosslinking- and mass spectroscopy-based approaches (CLMS) to characterize the protein-protein interactions within the holotranslocon complex. These CLMS data sets are large and suffer from a high rate of ‘false positives’, possibly caused by inter-complex crosslinks. Thus, they need to be carefully evaluated and interesting fits should be confirmed by an independent method. In the future, structural studies of the ribosome-HTL complex by cryo-EM together with reconstitution of the HTL into nanodiscs will be undertaken to reveal the conformation of the actively translocating HTL in a more physiological environment. Additional biochemical studies on the molecular mechanism of co- and post-translocation by HTL and its substrate spectrum are addressing the question about the physiological role of the holotranslocon in the cell.

abstract: Over the last century, X-ray crystallography has been established as the most successful technique for unravelling the structure-function relationship in molecules. For integral membrane proteins, growing well-ordered large crystals is a challenge and hence, there is room for improving current methods of macromolecular crystallography and for exploring complimentary techniques. Since protein function is deeply associated with its structural dynamics, static position of atoms in a macromolecule are insufficient to unlock the mechanism. The availability of X-ray free electron lasers presents an opportunity to study micron-sized crystals that could be triggered (using light, small molecules or physical conditions) to capture macromolecules in action. This method of ‘Time-resolved serial crystallography’ answers key biological questions by capturing snapshots of conformational changes associated with multi-step reactions. This dissertation describes approaches for studying structures of large membrane protein complexes. Both macro and micro-seeding techniques have been implemented for improving crystal quality and obtaining high-resolution structures. Well-diffracting 15-20 micron crystals of active Photosystem II were used to perform time-resolved studies with fixed-target Roadrunner sample delivery system. By employing continuous diffraction obtained up to 2 A, significant progress can be made towards understanding the process of water oxidation. Structure of Photosystem I was solved to 2.3 A by X-ray crystallography and to medium resolution of 4.8 A using Cryogenic electron microscopy. Using complimentary techniques to study macromolecules provides an insight into differences among methods in structural biology. This helps in overcoming limitations of one specific technique and contributes in greater knowledge of the molecule under study. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2018

Na,K-ATPase é uma proteína de membrana que tem como função manter o equilíbrio osmótico nas células pela hidrólise de ATP. A ouabaína (OUA) se liga a Na,K-ATPase e é capaz de ativar cascatas de sinalização. As subunidades a da Na,K-ATPase possuem 4 isoformas que são distribuídas de forma diferenciada nos tecidos. As células da glia são importantes na resposta contra lesões no cérebro e também controlam a inflamação. Dados na literatura mostram que a OUA tem efeito protetor em alguns tipos de dano. O objetivo do estudo é avaliar a função da isoforma a2 na cultura de células da glia em resposta à OUA e ao LPS. Nós investigamos a ação da OUA em diversas concentrações e LPS (1g/mL) na viabilidade celular (LDH) e proliferação celular (MTT). O LPS foi utilizado como modelo de inflamação e uma das perguntas era se o tratamento prévio com OUA, seria capaz de reverter a ativação do fator de transcrição NF-kB que está envolvido com inflamação. O pré-tratamento com OUA diminuiu a ativação do NF-kB induzida pelo LPS. Após, nós silenciamos a isoforma a2 das células da glia com RNAi. Os nossos dados mostram que o pré-tratamento com OUA reverte o efeito na ativação do NF-kB causado pelo LPS. Provavelmente, a isoforma a2 está relacionada com alguma via de sinalização que interage com a via do LPS. / Na,K-ATPase is a conserved membrane protein which maintains the osmotic balance in the cell by the hydrolysis of ATP. Ouabain (OUA) binds to Na,K-ATPase and it can activate signaling pathways. The a subunits of Na,K-ATPase have 4 isoforms which are distributed in a different pattern in the tissues. Glial cells have an important role in the response against injury and they also control inflammation. Some data have reported that OUA can protect against some types of injury. The aim of this study is to evaluate the role of a2 isoform in glial cells in response to OUA and LPS stimulus. We investigated the action of OUA and LPS in cell viability (LDH) and cell proliferation (MTT). LPS was used as a model of inflammation and one of our questions was if the treatment with OUA before LPS was capable of reduce the activation of the transcription factor NF-kB which is involved in inflammation. The pre-treatment with OUA decreased the NF-kB activation induced by LPS. We also silenced the a2 isoform in culture glial cells with iRNA. Taken together our data showed that OUA pretreatment reversed the NF-kB activation induced by LPS in primary cultures of glial cells from mice. Probably,the a2 isoform is related with some signaling pathway that interacts with the LPS pathway.

Orientadores: Aureo Tatsumi Yamada, Wirla Maria da Silva Cunha Tamashiro / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T04:32:24Z (GMT). No. of bitstreams: 1 Bizinotto-Assuncao_MarciaCristina_D.pdf: 17669234 bytes, checksum: 7623e53be29f1a69dba9c95b19066e0e (MD5) Previous issue date: 2006 / Resumo: O fenômeno da gestação em mamíferos de placentação hemocorial, onde o feto alogênico, apresentando metade dos genes de origem paterna, desenvolve um íntimo contato com o tecido materno sem desencadear uma resposta de rejeição pelo sistema imune materno, é ainda uma questão da imunologia da reprodução não esclarecida completamente. O diálogo na interface materno-fetal do útero gestante envolve uma gama de citocinas da categoria Th1fTh2, bem como de várias populações celulares, onde se destacam as células Natural KiUer uterinas (uNK). As uNK são populações linfocitárias transitórias que migram a partir de progenitores localizados em órgãos linfóides secundários como linfonodo e baço para o útero gestante e, neste proliferam e diferenciam ao longo da gestação. A presença destas células no útero gestante tem sido relacionada com o reconhecimento dos antígenos HLA-G e HLA-E expressos pelos trofoblastos, resultando na inibição tanto da resposta imune inata quanto da adaptativa. Esta atuação peculiar das uNK sugere que a modulação destas células no útero gestante esteja relacionada com a expressão de receptores específicos não compartilhada com células NK circulantes (eNK). Em uNK de camundongos, a lectina DBA (Doliehos biflorus agglutin) identifica glicoconjugados contendo N-acetil D-galactosamina presentes na superfície destas células e que não são expressos por outros linfócitos, corroborando com a hipótese de que as uNK expressam receptores específicos não comuns às eNK Neste trabalho, nós propusemos avaliar a possível expressão de moléculas específicas na superfície das células uNK de camundongos e identificar as proteínas candidatas através de técnicas proteômicas. Para tanto foram inicialmente utilizadas células uNK isoladas e purificadas de úteros de camundongos prenhes como in6culos antigênicos em machos da mesma espécie, para avaliar a capacidade de indução de uma resposta imune com produção de anticorpos reativos a esses antígenos. Outra estratégia utilizada foi o isolamento de proteínas de homogenados uterinos, de células uNK e da membrana destas células, na tentativa de isolar e identificar aquelas reativas à lectina DBA. Como resultado das inoculaçães de células uNK, os camundongos receptores produziram anticorpos séricos que reconheciam células uNK através de reações imunocitoquímicas. Este resultado confirma a expressão de moléculas específicas em células uNK, que por sua vez as caracteriza como uma subpopulação de NK específica da gestação. Dos dois animais com maior nível de anticorpos séricos foram coletados os baços para o preparo das células empregadas em experimentos de fusão celular para a obtenção de hibridomas secretores de anticorpos monoclonais. Um dos anticorpos monoclonais obtidos reconheceu moléculas presentes na superfície e no citoplasma das células uNK de várias linhagens de camundongos, assim como moléculas em células presentes no endométrio de ratas prenhes e no endométrio gestacional de humanos, sugerindo que moléculas homólogas são expressas em uNK de outras espécies. No entanto, este anticorpo monoclonal anti-uNK de camundongo (mamuNK1) apresentou reação cruzada com componentes citoplasmáticos de outras células, sugerindo não serem específicos para uNK Em relação ao isolamento dos glicoconjugados lectina DBA reativos observouse que através da cromatografia de gel filtração o isolamento mostrou-se pouco eficiente para o "pool" de proteínas contidas no homogenado uterino, ou mesmo de homogenado de células uNK, em decorrência da existência de múltiplas frações de proteínas capazes de ligar à lectina DBA. Restringindo-se o "pool" de proteínas àquelas obtidas apenas da membrana de células uNK e, através da eletroforese de duas dimensões pode-se delimitar as proteínas de interesse, as quais foram submetidas à análise proteomica. A identificação das proteínas isoladas das membranas de células uNK através de técnicas proteomicas sugeriram as proteínas Conserved oligomeric Golgí complex component 4 (COG4) e Heat shock 70-líke proteín 1 (Hsp 70 L1) como potenciais candidatas relacionadas com as atividades das células natural kíller no útero gestante / Abstract: The phenomenon of pregnancy in mammalians with hemochorial type placenta ions where the allogeneic fetus presenting the half of gene from paternal origins growth in tight contact with maternal tissues, without triggering the rejections responses of maternal immune system is still a non-solved question of immunology of reproduction. The cross-talk at maternal-fetal interface in the pregnant uterus involves a range of Th1rrh2 cytokines, as well as, several cell populations with accentuated participation of uterine Natural Killer (uNK) Iymphocytes. The uNK .are transient leukocyte population that migrate from precursor cells localized at secondary Iymphoid organs such as Iymph nodes and spleen into the pregnant uterus and proliferate and differentiate during the gestation. The presence of these cells in the pregnant uterus has been related to recognition of the HLAG and HLA-E antigen present in the trophoblast, which results in inhibition of both innate and adaptive immune response. This peculiar activity of uNK suggests that the modulation of such cells in the pregnant uterus should be related with the expression of specific receptors not shared with peripheral blood circulating NK (cNK) cell. In the mouse uNK, the DBA (Dolichos biflorus agglutin) lectin identify n-acetyl D-galactosamine containing glycoconjugates present on the surface of these cells not expressed by other Iymphocytes, which corroborate the hypothesis of uNK expressing specific receptors not shared by cNK. The present work aimed to evaluate the presumed expression of specific molecules on mouse uNK cell surface and identify the candidate proteins by proteomic methods. Initially were used uNK cells isolated and purified from pregnant mice uteri as antigen to be inoculated in the male mice and evaluate the capacity of induction of immune response with production of antibodies reactive to these antigens. Other strategy was to realize procedures of protein isolation from uterine homogenates, uNK cells and the membrane of these cells, in the attempt to isolate and identify those reactive to DBA lectin. As results of inoculation of uNK cells were verified responses in the inoculated males by presence of serum antibodies that recognized uNK with immunocytochemistry. These results confinn the expression of specific molecules in uNK cells which characterize these cells as specific NK subset of pregnancy. From two animaIs with highest levei of antibodies in 1he serum were collected the spleen to isolate the Iymphocytes clones for monoclonal antibodies obtantion. One of these antibodies recognized antigen molecules found on the surface and cytoplasm of the uNK cells of several mice strains, as well as, molecules in cells present in the pregnant rats and human beings endometrium, which suggests the expression of homologous molecules in uNK of other species. However, this mouse anti-mouse uterine NK (mamuNK1) was reactive with citoplasmatic components the other cells, suggesting not be specific to uNK. About the isolation of DBA lectin reactive glycoconjugates by purification in gel filtration chromatography revealed low efficiency to the pool of proteins contained in the uterine homogenate, or even in the homogenate of isolated uNK cells due to the multiple DBA lectin positive protein fractions. By restriction of protein pool to those obtained from the uNK cells membrane and by two dimension electrophoresis was delimited the target proteins that were submitted to the proteomic analysis. The identification of isolated proteins of the membranes of uNK cells with proteomics methods suggested the Conserved proteins oligomeric Golgi complex Component (4 COG4) and Heat shock 7D-like protein 1 (Hsp 70 L1) as potential candidates related to the activity of the natural to killer cells in the pregnant uterus / Doutorado / Histologia / Doutor em Biologia Celular e Estrutural

Orientador: Eneida de Paula / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T08:33:29Z (GMT). No. of bitstreams: 1 Cunha_ElizabethSousada_M.pdf: 3228323 bytes, checksum: 545a2be4ec32cc1a223c4b4337dc369a (MD5) Previous issue date: 2008 / Resumo: A mitocôndria é uma organela vital, pois em células aeróbicas, ela é responsável pela produção da maior parte da energia química necessária para a célula. A síntese de ATP ocorre por fosforilação oxidativa na Fo,F1-ATPase, usando a energia gerada pela cadeia de transporte de elétr ons, uma série de enzimas presentes na membrana mitocondrial interna. A análise de proteínas de membrana pode ser feita pela técnica de eletroforese, tanto em condições nativas quanto desnaturantes. Neste trabalho analisamos a solubilização de proteínas da membrana interna de mitocôndria de músculo de rato, através do uso de surfatantes zwiteriônicos (ASB-14, ASB-16, CHAPS), não-iônicos (Digitonina, C12E8, Triton X- 100) e aniônico (Colato de sódio) na separação por eletroforese em gel nativo e bidimensional. Desenvolvemos um novo protocolo para preparo de eletroforese em gel nativo, mantendo o tampão de amostra descrito no procedimento de BN-PAGE e adaptando as demais etapas da técnica de acordo com o protocolo de Laemmli et al (1970). Os géis assim preparados em temperatura ambiente mostraram melhor resolução que os pelo método BN-PAGE, foram obtidos menor tempo de corrida (1- 2 horas), sem troca de tampões e com emprego de reagentes de menor custo. Quanto a eficiência dos diferentes surfatantes, usados em uma mesma concentração (43,2 mM), o zwiteriônico ASB-16 foi o melhor solubilizador das proteínas de MMI, tanto em relação a quantidade total de proteína solubilizada como em relação a número de ¿spots¿ visualizados na eletroforese bidimensional, seguido pelo não-iônico Triton X-100. O surfatante não-iônico dodecil maltosídeo foi usado na preparações dos géis 2D em concentrações menores inferiores (20%) do que as indicadas na literatura (10% ou 195,8 mM), sem prejuízo da eficiência de solubilização protéica. Os resultados obtidos com géis nativos mostraram que todos os surfatantes estudados podem ser usados para separar os complexos protéicos da MMI, mas apresentam seletividade específica pelos complexos da Cadeia Respiratória, devendo esse parâmetro ser melhor estudado futuramente, para a determinação de protocolos específicos para isolamento dos diferentes tipos de complexos / Abstract: The majority of the chemical energy produced inside aerobic cells is a result of the oxidative phosphorylation of ATP, inside the mitochondria. A great part of mitochondria proteins are organized into complexes located in the inner mitochondria membrane. Isolation and identification of those proteins have been conducted by electrophoresis, both in native as in denaturant condition. In this work we analysed solubilization of the inner mitochondrial membrane proteins of rat¿s gastrocnemius muscles, through the use of zwitterionic (ASB-14, ASB-16, CHAPS), non-ionic (Digitonin, C12E8, Triton X-100) and anionic (sodium cholate), by native and two-dimensional electrophoresis. We have developed a new protocol for the native gel electrophoresis, using the buffer sample described in BN-PAGE but changing other steps of the technique according to the protocol of Laemmli et al (1970). With this novel protocol, the gels prepared at ambient temperature had a better resolution than the classic BNPAGE technique; with low costs and short-time runs (1-2 hours). As for the effectiveness of surfactants studied to solubilize inner mitochondrial membrane proteins, when used at the same concentration (43.2 mM), the zwitterionic ASB-16 was the best, both accordingly to the total amount of solubilized protein, as for the number of "spots" detected in the two-dimensional electrophoresis, followed by non-ionic Triton X-100. The non-ionic surfactant dodecyl maltoside was used in the 2D electophoresys preparation at lower concentrations (20%) than that recommended in the literature (10% or 195.8 mM) without prejudice in the efficiency of protein solubilization. The results with native gels proved that all the studied surfactants can be used to separate the proteins of MMI, but with different selectivity for each enzymatic complex. This specificity should be better studied in the future, looking for the determination of specific protocols for better isolation of each types of MMI complex / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2017-08-04T16:47:05Z No. of bitstreams: 2 Dissertação - Juliana Santana de Curcio - 2014.pdf: 2001377 bytes, checksum: 2f9ef22cd0a300cc93ee483fab0fb9c1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-08-07T15:54:46Z (GMT) No. of bitstreams: 2 Dissertação - Juliana Santana de Curcio - 2014.pdf: 2001377 bytes, checksum: 2f9ef22cd0a300cc93ee483fab0fb9c1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-08-07T15:54:46Z (GMT). No. of bitstreams: 2 Dissertação - Juliana Santana de Curcio - 2014.pdf: 2001377 bytes, checksum: 2f9ef22cd0a300cc93ee483fab0fb9c1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-02-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Paracoccidioides spp. represents thermodimorphic fungi that cause paracoccidioidomycosis, the most widespread systemic mycosis in Latin America. During the establishment of the infection, proteins located in cell membranes are responsible for many essential processes for pathogen survival as transport of nutrients, accession, signaling and energy production. Cell membranes are basically composed of a lipid bilayer with proteins associated. The proteins can be associated with the membrane through transmembrane domains, posttranslational added modifications or through electrostatic interactions. Therefore the knowledge of the constitution of the proteins of membranes is important to understand some processes developed by Paracoccidioides sp. for its growth and survival within the host. To this end, two techniques of separation and identification of proteins were employed. The first methodology used two-dimensional electrophoresis and mass spectrometry, where the extract of membranes proteins was obtained after steps of ultracentrifugation and the second methodology used liquid chromatography coupled with mass spectrometry, to this methodology three protocols were employed to obtain the extract of membranes proteins. The first methodology employed ultracentrifugation, and the second e third methodology used ultracentrifugation/sonication and ultracentrifugation with elevation of pH respectively. After these steps, the extract of membranes proteins was obtained through of ultracentrifugation and elevation of pH with sodium carbonate. Transmission Electron Microscopy (TEM) was performed to confirm the quality of the sample, a fraction enriched in cell membranes of Paracoccidioides sp.. Posteriorly the proteins obtained by standard methodology were subjected to tryptic digestion and analyzed by NanoUPLC-MSE.. In silico analyzes of the identified proteins were performed to determine the location and possible association of the proteins with the cell membranes. From total of proteins identified, one hundred thirty -three proteins were identified as belonging the cell membranes of Paracoccidioides sp.. Eighty-eight proteins showed at least one transmembrane domain and thirteen identified proteins showed a signal peptide at the N-terminus, beyond the transmembrane domain. Different forms of association of the proteins with membranes of Paracoccidioides sp. were detected, including domain transmembrane, GPI anchor, prenylation, myristoylation, palmitoylation. The most of the of integral membrane proteins identified in proteome were annotated in the genome of Paracoccidioides sp. Together these results show the establishment of an experimental protocol for the extraction of membranes proteins of Paracoccidioides spp., as well identify proteins from membrane fractions this fungus. / Paracoccidioides spp. representa um gênero de fungos termodimórficos, agentes etiológicos da paracoccidioidomicose, a micose sistêmica mais comum na América Latina. Durante o processo infeccioso proteínas localizadas em membranas celulares são responsáveis por muitos processos essenciais para a sobrevivência do patógeno, como transporte de nutrientes, adesão, sinalização e produção de energia. Basicamente membranas celulares são constituídas de uma bicamada lipídica com proteínas associadas. As proteínas de membranas associam-se a bicamada lipídica através de domínios transmembrana, por adição de uma modificação pós traducional e por meio de interações eletrostáticas. Portanto, o conhecimento da constituição proteica das membranas de Paracoccidioides sp. permite entender alguns dos processos desenvolvidos pelo fungo para sobrevivência dentro do hospedeiro. Desta forma o presente trabalho teve como objetivo à determinação da metodologia para obtenção de proteínas de membranas e a consequente descrição dessas proteínas. Para este fim, duas técnicas de separação e identificação de proteínas foram empregadas. A primeira metodologia baseava-se em eletroforese bidimensional e espectrometria de massas onde o extrato proteico de membranas foi obtido após duas etapas de ultracentrifugação e a segunda metodologia utilizava cromatografia líquida acoplada à espectrometria de massas, para esta metodologia três protocolos foram empregados no intuito de obter extratos proteicos de membranas. O primeiro protocolo emprega ultracentrifugação e o segundo e terceiro protocolo empregava ultracentrifugação/sonicação e ultracentrifugação combinado com elevação do pH respectivamente. Após as etapas de padronização do protocolo, o extrato proteico de membranas foi obtido através de ultracentrifugação e solubilização com carbonato de sódio em pH elevado. A microscopia eletrônica de transmissão (MET) foi realizada para confirmar a qualidade da amostra enriquecida com a fração de membranas de Paracoccidioides sp.. Posteriormente o extrato proteico de membranas proveniente da metodologia padronizada foi submetido à digestão tríptica e analisado por NanoUPLC-MSE. Análises in silico das proteínas identificadas foram realizadas a fim de se determinar a localização destas proteínas e possíveis associações com as membranas. Do total de proteínas identificadas cento e trinta e três proteínas foram classificadas como pertencentes a membranas celulares de Paracoccidioides sp., oitenta e oito proteínas apresentaram ao menos um domínio transmembrana e treze proteínas, além de domínio transmembrana apresentaram um peptídeo sinal na porção N-terminal da proteína. Diferentes formas de associação com as membranas de Paracoccidioides sp. foram identificadas neste trabalho, incluindo domínio transmembrana, âncora de GPI, prenilação, palmitoilação e miristoilação. A maioria das proteínas integrais de membrana identificadas no proteoma já foram anotadas no genoma deste fungo. Juntos estes resultados demonstram o estabelecimento de um protocolo experimental para a extração de proteínas de membranas de Paracoccidioides spp., bem como identificam as proteínas de frações de membranas do fungo.

Made available in DSpace on 2014-08-20T13:32:50Z (GMT). No. of bitstreams: 1 dissertacao_nubia_seyffert.pdf: 4325642 bytes, checksum: 2836f7bcdef6a962ef1e00c38936c413 (MD5) Previous issue date: 2007-02-05 / Leptospiral immunoglobulin-like (Lig)proteins are exposed on membrane surface of pathogenic Leptospira and may play a role in host cell attachment and invasion during infection.A pair of Ligs,named LigA and LigB,with identical and non-identical polypeptide regions has recently been identified.The two proteins elicit a strong humoral immune response during acute host infection and have been suggested as targets for development of vaccines and diagnostic tests for leptospirosis.This study reports the production and characterization of monoclonal antibodies (Mabs)against recombinant non-identical fragments of LigA (rLigANI)and LigB (rLigBNI).The recombinant fragments were used for mice immunization and screening hybridomas by indirect ELISA.Four Mabs obtained against rLigANI and six Mabs obtained against rLigBNI were isotyped and evaluated regarding their potential for use in studies of immunoprotection and diagnostic test development.The Mabs were of the IgM (1),Ig 2b (3)and Ig 1 (6)isotypes and reacted with the native proteins from a pathogenic strain of Leptospira i terroga s serovar Copenhageni L1 130 in an indirect ELISA and immunofluorescence.Mabs affinity constants felt between 4x10 7 M -1 and 2x10 8 M -1 ,and epitope mapping by additive ELISA has shown that each Mab either react with the same epitope in the recombinant molecule or cause steric hindrance. Tissue sections from kidneys of hamsters experimentally infected with leptospires and probed with the Mabs reacted positively by immunohistochemistry.These findings suggest that the Mabs obtained can be useful for the studies of immunoprotection and development of diagnostic tests. / As proteínas leptospiral immu oglobuli -like (Lig)estão expostas na superfície da membrana das leptospiras patogênicas e podem estar envolvidas no ataque e penetração às células do hospedeiro durante a infecção.Recentemente,foi identificado um par de Ligs,nomeadas LigA e LigB,com regiões polipeptídicas idênticas e não-idênticas.As duas proteínas estimulam uma resposta humoral forte durante a infecção aguda no hospedeiro e têm sido sugeridas como alvos para o desenvolvimento de vacinas e testes diagnósticos para leptospirose.Este estudo relata a produção e caracterização de anticorpos monoclonais (Mabs)contra fragmentos recombinantes não idênticos de LigA (rLigANI)e LigB (rLigBNI).Os fragmentos recombinantes foram utilizados para a imunização dos camundongos e triagem dos hibridomas por ELISA indireto.Quatro Mabs obtidos contra rLigANI e 6 Mabs contra rLigBNI foram isotipados e avaliados quanto ao seu potencial para uso em estudos de imunoproteção e desenvolvimento de testes diagnósticos.Os Mabs foram dos isotipos IgM (1),Ig 2b (3)and Ig 1 (6)e reagiram com as proteínas nativas de Leptospira i terroga s sorovar Copenhageni cepa L1 130 em ELISA indireto e imunofluorescência.A constante de afinidade dos Mabs manteve-se entre 4x10 7 M -1 and 2x10 8 M -1 ,e o mapeamento de epitopos por ELISA de aditividade demonstrou que cada Mab reage com o mesmo epitopo na molécula recombinante ou causa um impedimento espacial (steric hi dra ce ).Finalmente,os Mabs reagiram positivamente em testes imuno-histoquímicos de seções do tecido renal de hamsters experimentalmente infectados com leptospiras.Esses dados sugerem que os Mabs obtidos podem ser usados para estudos de imunoproteção e desenvolvimento de testes de diagnóstico de leptospirose.

Les protéines de la famille Mep/Amt/Rh sont largement conservées dans l’évolution. Cette famille comprend les facteurs Rhésus dont les antigènes Rh humains sont les membres les plus notoires. Le rôle des protéines de type Mep/Amt/Rh en tant que transporteurs d’ammonium a largement été décrit chez les bactéries, les champignons et les plantes. Néanmoins, leur mécanisme de fonctionnement demeure élusif et la régulation de leur activité a été peu abordée chez les organismes eucaryotes. En utilisant comme modèles de la famille Mep/Amt/Rh les trois transporteurs d’ammonium de la levure Saccharomyces cerevisiae, nous avons tenté de comprendre les mécanismes de fonctionnement et de régulation de cette famille de protéines membranaires.<p>Nous montrons qu’un résidu aspartate, conservé dans la famille Mep/Amt/Rh et situé à proximité d’un vestibule cation-attractif, joue un rôle structural dans la reconnaissance de l’ammonium chez le transporteur Mep2. De plus, un résidu histidine très conservé dans le pore hydrophobe des protéines Mep/Amt/Rh est substitué par un aspartate chez un sous-groupe de transporteurs d’ammonium fongiques. Cette substitution permet de définir deux sous-familles fonctionnelles possédant des propriétés bien distinctes.<p>Nous montrons également que la kinase Npr1 intervient dans la modulation de l’activité intrinsèque des trois protéines Mep qui demeurent inactives mais stables à la membrane plasmique en absence de la kinase. <p>Hormis leur rôle dans le transport d’ammonium en tant que source d’azote, nous montrons que l’activité des protéines Mep est requise pour différentes réponses physiologiques. Une diminution d’entrée d’ammonium en absence des protéines Mep ou de leur régulateur positif Npr1 entraîne une dérépression des gènes soumis à la répression catabolique azotée ainsi qu’un défaut dans le repompage de l’ammonium catabolique excrété durant la croissance en présence d’autres sources azotées. Un rôle supplémentaire de senseur d’ammonium avait été attribué au transporteur Mep2 dans l’induction de la croissance filamenteuse en réponse à une limitation en ammonium. Nous montrons que l’état d’activité de la protéine Mep2 est étroitement lié à sa capacité à induire le développement filamenteux. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Doctorat en Sciences / info:eu-repo/semantics/nonPublished