Dissertations / Theses on the topic 'Septimus'

Septinas pertencem a uma família de proteínas de ligação a GTP e são encontradas em diversos eucariontes, participando de diferentes processos celulares citoesqueléticos. As septinas apresentam um domínio central de ligação a GTP (domínio G) flanqueado por uma região amino-terminal e outra carboxi-terminal. As septinas se caracterizam por interagirem entre si formando heterocomplexos que se polimerizam, constituindo filamentos. A única estrutura resolvida de um complexo de septinas é de um hexâmero, composto por duas subunidades de três septinas humanas diferentes: SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7. Esta estrutura revelou que a formação do filamento envolve interações conservadas entre os domínios G, estando o restante da estrutura desordenado no cristal. Além disso, mostrou que dois tipos de interface se alternam ao longo do filamento, as chamadas interfaces G (que incluem a região de ligação do nucleotídeo de duas subunidades) e interfaces NC (que incluem as regiões N e C-terminais do domínio G). Várias evidências sugerem que as regiões C-terminais da proteína sejam as principais responsáveis pela seleção do parceiro correto de interação para montagem dos heterocomplexos. Nesse contexto, buscou-se avaliar a importância das regiões C-terminais na seleção das parceiras SEPT6 e SEPT7 para formar a interface NC, frente ao domínio G. Inicialmente, uma septina quimérica foi produzida de forma a conter o domínio G de SEPT2 e o C-terminal de SEPT6, gerando SEPT2G6C. As proteínas SEPT7GC, SEPT6GC, SEPT2GC e SEPT2G6C foram expressas e purificadas separadamente. Análises de estabilidade térmica e de afinidade proteína-proteína dos pares indicou que a quimera foi capaz de interagir com SEPT7GC, gerando o heterodímero SEPT7GC-SEPT2G6C, mas este não se mostrou tão estável quanto o heterodímero fisiológico. Foi também avaliada a importância da ligação do nucleotídeo para formação da interface G e, para isso, foram construídos os mutantes SEPT2GT78M e SEPT2GD185N, cujos resíduos importantes para hidrólise e ligação do nucleotídeo, respectivamente, foram alterados. A análise de oligomerização por cromatografia de exclusão molecular mostrou deslocamento no volume de eluição das proteínas expressas sozinhas e coexpressas com SEPT6G, indicando que a formação do dímero via interface G depende da ligação do nucleotídeo, mas não da sua hidrólise. Finalmente, foi avaliada a estabilidade térmica e estrutural e a propensão à formação de amilóides do heterodímero SEPT6G-SEPT2G, o qual apresentou maior estabilidade estrutural quando comparado ao homodímero de SEPT2G, mas ainda exibiu alteração de sua estrutura para um estado capaz de ligar Thioflavina-T, sugerindo a formação de amilóides. Entretanto, isso foi observado em temperaturas cerca de 30 ºC acima daquela observada para o homodímero, confirmando a maior estabilidade do heterodímero e sugerindo que a formação da interface G com o parceiro correto pode ser um fator importante na prevenção da formação de estruturas amilóides à temperaturas fisiológicas.<br>Septins belong to a family of GTP binding proteins and are found in several eucaryotes, participating in different cytoskeletal cell processes. The septins have a central GTP binding domain (G domain) flanked by an amino-terminal and a carboxy-terminal regions. The septins are characterized by the ability to interact with each other forming heterocomplexes which polymerize themselves, forming filaments. The only solved structure of a septin complex is a hexamer, formed by two subunits of three different human septins: SEPT7-SEPT6-SEPT2- SEPT2-SEPT6-SEPT7. This structure revealed that the filament arrangement involves conserved interaction between G domains, being the remainder of the structure disordered in the crystal. Moreover, two types of interface alternate along the filament were shown, socalled G interfaces (which include the nucleotide binding region of the two subunits) and NC interfaces (which include the N- and C- terminal regions of G domain). Plenty of evidences suggest that C-terminal regions of the protein are the main responsible for the selection of the correct interaction partner to assembly of heterocomplexes. In this context, it was sought to evaluate the importance of the C-terminal regions in the selection of the partnerships SEPT6 and SEPT7 to form the NC interface, against the G domain. For this, a chimerical septin was designed so that contains the G-domain of SEPT2 and the C-terminal of SEPT6, creating SEPT2G6C. The SEPT7GC, SEPT6GC, SEPT2GC and SEPT2G6C proteins were expressed and purified individually. Thermal stability and protein-protein affinity analysis of the pairs indicated that the chimera was able to interact with SEPT7GC, forming the heterodimer SEPT7GC-SEPT2G6C, which, however, did not show as stable as the physiological heterodimer. The importance of nucleotide binding to the interaction through G interface was also evaluated and, for that, SEPT2 mutants on GTP-domain were constructed, SEPT2T78M and SEPT2D185N, whose important residues in the hydrolysis and linking of nucleotide, respectively, were changed. Oligomerization analysis by size exclusion chromatography showed a shift in the elution volume of proteins expressed alone and coexpressed with SEPT6, indicating that the complexation of proteins to form G interface depends on the nucleotide binding, but not on its hydrolysis. Finally, the thermal and structural stability and the propensity to amyloid formation of heterodimer SEPT6G-SEPT2G were evaluated, which showed greater structural stability when compared to SEPT2 homodimers, but still exhibited alteration of its structure to a state that was able to bind Thioflavin-T, suggesting amyloid formation. However, this was observed at temperatures around 30 ºC above that observed for the homodimer, confirming the greater conformational stability of the heterodimer and suggesting that the formation of G interface with the right partner can be an important factor of the amyloid filament prevention at physiological temperatures.

Orientador: João Alexandre Ribeiro Gonçalves Barbosa<br>Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia<br>Made available in DSpace on 2018-08-15T21:46:33Z (GMT). No. of bitstreams: 1 Souza_TatianadeArrudaCamposBrasilde_D.pdf: 2667585 bytes, checksum: 67a64b614fe46e32c061e0f43f20406e (MD5) Previous issue date: 2010<br>Resumo: Para que a divisão celular ocorra é necessário que uma célula passe por algumas etapas que possibilitem esta divisão. Ao conjunto destes processos denomina-se ciclo celular. A regulação espacial e temporal destes processos é fundamental para a preservação celular e do material genético. Falha neste processo pode levar à morte celular ou a alterações genéticas causando divisão desregulada e crescimento de tumores. Dentre diversas proteínas envolvidas no ciclo celular, encontram-se as septinas. Até o momento, foram encontrados em humanos 14 diferentes genes para septinas. Septinas são proteínas ligadoras de GTP, primeiramente caracterizadas em leveduras, que estão associadas a eventos biológicos importantes em eucariotos. Neste trabalho foram selecionadas três septinas humanas para estudos funcionais e estruturais: septina 6, septina 8 e septina 10. Nossos resultados deram origem a três artigos que descrevem: I) estratégias de clonagem, expressão, purificação e caracterização preliminar da septina humana 8; II) a capacidade das septinas 2, 6, 8 e 11 em ligar e hidrolisar GTP, mas com diferentes níveis de atividade GTPásica, sendo a septina 2 humana capaz de hidrolizar GTP mais rapidamente que as demais septinas e III) a interação entre a septina 10 humana e a proteína Promyelocytic leukemia zinc finger (PLZF), cuja expressão em linhagens celulares hematopoiéticas resultam na supressão do crescimento e interrupção do ciclo celular. A interação da septina 10 com a proteína PLZF foi confirmada in vitro por experimento de pull-down e a localização celular da septina 10 mostra que esta septina é expressa no citoplasma da célula. Além disso, resultados preliminares mostram a relação da ligação de GTP e formação de filamentos pela septina 6 e diversas estratégias para a cristalização das septinas estudadas como: microseeding, streak-seeding, variações de pH, precipitantes e aditivos, metilação de lisinas e screening de tampões, cujos resultados não foram satisfatórios para a resolução de uma estrutura de septina humana<br>Abstract: A cell passes through some steps to enable for cell division and all these processes are called Cell Cycle. The spatial and temporal regulation of this process is crucial to maintaining cellular and genetic material. Failure in this process can lead to cell death or genetic changes causing division and unregulated growth of tumors. Among several proteins involved in cell cycle, there are the septins. To date, 14 different human genes coding for septins were found. Septins are GTP binding proteins first characterized in yeast, which are associated with important biological events in eukaryotes. In this study we selected three human septins to study functionally and structurally: septin 6, septin 8 and septin 10. Our results led to three articles that describe: i) strategies for cloning, expression, purification and preliminary characterization of human septin 8; ii) the ability of septin 2, 6, 8 and 11 to bind and hydrolyze GTP, but with different levels GTPase activity, human septin 2 is capable of hydrolyzing GTP faster than the other septins and III) the interaction between the human septin 10 and promyelocytic leukemia zinc finger protein (PLZF), whose expression in hematopoietic cell lines results in growth suppression and cell cycle arrest. The interaction between septin 10 and PLZF was confirmed by in vitro pull down assay and the cellular localization of septin 10 shows that this septin is expressed in the cell cytoplasm. Furthermore, our studies preliminary results show the relation of GTP binding and filament formation of the septin 6 and several strategies for the crystallization of septins as micro-seeding, streak-seeding, variations in pH, precipitants and additives, methylation of lysine and screening of buffers, but the results were not satisfactory for the resolution of a human septin structure<br>Doutorado<br>Genetica de Microorganismos<br>Doutor em Genetica e Biologia Molecular

Septinas são proteínas que ligam GTP e interagem entre si formando heterocomplexos, os quais formam filamentos e estruturas de maior nível de organização. Tais filamentos, além de se mostrarem importantes durante a citocinese também podem estar envolvidos em outros processos celulares tais como determinação da polaridade celular e reorganização do citoesqueleto. As septinas, inicialmente descobertas em Saccharomyces cerevisiae, já foram identificadas também em fungos, algas-verdes, mamíferos, porém nunca em plantas. Tipicamente, septinas apresentam três domínios estruturais compostos por um domínio central de ligação ao GTP flanqueado por um N-terminal variável e um C-terminal que pode conter sequências do tipo coiled-coil. Este trabalho teve como objetivo a caracterização do domínio de ligação a GTP da septina 8 humana (SEPT8G) por meio de estudos biofísicos. Nesse sentido, SEPT8G foi eficientemente produzida em E. coli, tendo seu estado dimérico em solução confirmado por cromatografia de exclusão molecular. Diferentemente das outras septinas já reportadas, mesmo dimérica a SEPT8G apresentou-se na forma apo. Ensaios de atividade GTPásica foram realizados, confirmando a incapacidade dessa septina em hidrolisar o GTP. Ainda, análises de estabilidade térmica por Dicroísmo Circular revelaram que a presença do íon magnésio leva à diminuição de sua estabilidade estrutural. Baseando-se em resultados prévios de interação obtidos pela técnica do duplo-híbrido em leveduras, estudos voltados à análise da interação entre as septinas 7 e 8 e, posteriormente, entre as septinas 5, 7 e 8 foram realizados. A co-purificação do complexo formado pelas septinas 7 e 8 mostrou-se dependente da região C-terminal completa de SEPT7 de modo que, quando ausente, a interação mostrou-se expressivamente prejudicada. Já o complexo formado pelas septinas 5/7/8 foi obtido contendo as três proteínas em frações equimolares e solúveis, disponibilizando assim um novo heterocomplexo de septinas para ensaios funcionais e estruturais.<br>Septins are GTP-binding proteins that interact with each other to form heterocomplexes, which form filaments and higher order structures. These filaments are important for cytokinesis and may be involved in other cellular processes such as the determination of cell polarity and cytoskeleton reorganization. The septins, initially discovered in Saccharomyces cerevisiae, have also been identified in fungi, green algae, mammals but not in plants. Typically, septins have three structural domains comprising a central GTP binding domain flanked by a variable N-terminal and a C-terminal which can contain coiled-coil structures. This work sought to characterize the GTP-binding domain of the human septin 8 (SEPT8G) through biophysical studies. Accordingly, SEPT8G was efficiently produced in E. coli, and its dimeric state in solution was confirmed by size exclusion chromatography. Compared to other septins previously reported, the dimeric SEPT8G presented itself as an apo-protein. GTPase activity assays were performed, confirming the inability of this septin to hidrolyse GTP. Additionally, thermal stability analyses by Circular Dichroism showed that the presence of the magnesium ion leads to a decrease of structural stability. Considering previous results of septins interactions from yeast two-hybrid experiments, we have analyzed the interaction between the septins 7, 8 and subsequently among septins 5, 7 and 8. Co-purification of the complex formed by the septins 7 and 8 showed to be dependent on the complete SEPT7 C-terminal region so that, when absent, the interaction was significantly impaired. The obtained complex formed by septins 5/7/8 contained the three proteins in soluble and equimolar fractions, providing a new septin heterocomplex for functional and structural studies.

A família das septinas caracteriza-se pela capacidade de ligar nucleotídeos de guanina e de se associarem formando filamentos. Diversas funções biológicas têm sido reportadas para esses filamentos e sua dissociação pode estar relacionada a patologias. A septina 12 humana expressa especificamente em testículos, foi identificada em filamentos que compõem o annulus do espermatozoide, cuja integridade está relacionada com a morfologia deste. Embora muitos estudos tenham sido reportados, vários aspectos das bases moleculares e fisiológicas de sua função e automontagem permanecem desconhecidos. Neste trabalho, procurou-se obter informações estruturais para o domínio de ligação ao nucleotídeo da SEPT12 (SEPT12G), do mutante SEPT12GT89M e do heterodímero SEPT7-SEPT12. A expressão destas proteínas foi realizada em células de E. coli da linhagem Rosetta(DE3) pelos vetores de expressão pET28a(+) e pETDuet-1. As etapas de purificação foram cromatografia de afinidade e exclusão molecular. A proteína SEPT12G foi submetida à avaliação do estado oligomérico, fluorescência intrínseca, ensaios de conteúdo de nucleotídeo, atividade GTPásica e transição térmica. O heterodímero SEPT7-SEPT12 foi submetido à avaliação do estado oligomérico e conteúdo de nucleotídeo. Ensaios de cristalização foram realizados para todas as proteínas. A coleta de dados realizada na linha I24 do Diamond Light Source (Didcot, Inglaterra) resultou em conjuntos de dados de alta resolução para a SET12G, SEPT12GT89M e baixa resolução para a SEPT7NGc. Os estudos biofísicos mostraram que a SEPT12G foi obtida em sua forma nativa ou, seja, capaz de ligar e hidrolisar o nucleotídeo GTP e que o heterodímero obtido apresentou ambas as proteínas. As estruturas cristalográficas foram resolvidas e permitiram realizar observações importantes para o grupo I das septinas humanas (SEPT3, SEPT9 e SEPT12). Para a SEPT12 pôde-se observar como a mudança que ocorre no motivo G4 pode alterar a estabilidade da interface G. No contexto do grupo I esta estrutura permitiu concluir que todas as proteínas deste subgrupo podem formar duas interfaces NC, dos tipos aberta e fechada. Além disso, reforçou a observação da orientação diferencial da hélice &alpha;5\', cuja função ainda não está esclarecida, mas sem dúvidas é um diferencial que caracteriza este grupo, possivelmente relacionado com a ancoragem da região polibásica na conformação aberta. A estrutura cristalográfica do mutante SEPT12T89M permitiu observar que a mutação levou a uma alteração na primeira esfera de coordenação do íon Mg2+, mudança que interrompe o mecanismo do switch universal e deixa a proteína catalíticamente inativa. Por fim, o estudo cristalográfica do complexo entre a SEPT12 e SEPT7 não foi possível, uma vez que todas as tentativas resultaram em cristais contendo apenas a SEPT7, o que pode ser consequência da precipitação da SEPT12 ou da condição de cristalização utilizada, que desestabiliza o heterodímero.<br>The septin family of proteins is characterized by their ability to bind guanine nucleotides and associate into filaments. Several biological functions have been reported for these filaments and their dissociation may be related to pathologies. Human septin is 12 specifically expressed in testes and has been identified in filaments that form the sperm annulus, whose integrity is related to its morphology. Although many studies have been reported, the molecular and physiological bases of septin filament function and self-assembly have yet to be completely elucidated. This study aims to obtain structural information for the nucleotide binding domain of SEPT12 (SEPT12G), the SEPT12GT89M mutant and the SEPT7-SEPT12 heterodimer. Expression of these proteins was performed in E. coli Rosetta(DE3) strain using the pET28a (+) and pETDuet-1 expression vectors. Purification was performed by affinity and size exclusion chromatography. The SEPT12G protein was submitted to an evaluation of its oligomeric state, intrinsic fluorescence, nucleotide content, GTPase activity and thermal transition. The oligomeric state and nucleotide content of SEPT7-SEPT12 was also evaluated. Crystallization assays were performed for all proteins. Data collection on line I24 of the Diamond Light Source (Didcot, England) resulted in high-resolution data sets for SET12G and SEPT12GT89M but only low resolution data for the SEPT7NGc. Biophysical studies showed that SEPT12G was obtained in its native form or, in other words, capable of binding and hydrolyzing GTP and that the purified heterodimer presented both proteins. The crystallographic structures were solved by molecular replacement allowing the identification of features characteristics of the group I septins (SEPT3, SEPT9 and SEPT12). The structure also confirmed that all the proteins of this group are able to form two different NC interfaces: open and closed. In addition, it reinforced the observation that the &alpha;5\' helix assumes a different orientation, whose function has not yet been clarified, but without doubt is a characteristic of this group which may be related to anchoring the polybasic regions whilst in the open conformation. The SEPT12T89M mutant crystal structure shows that the first shell coordination of the Mg2+ ion is altered, leading to an interruption of the universal switch mechanism and a consequent lack of catalytic activity. Finally, structural studies of the interaction between SEPT12 and SEPT7 were not possible, since all attempts resulted in crystals containing only SEPT7. This may be a consequence of SEPT12 precipitation or the crystallization condition used, destabilizing the heterodimeric interface.

Septinas são proteínas pertencentes à família das GTPases que estão envolvidas em uma variedade de funções celulares. Verificamos através de análises bioinformáticas que Schistosoma mansoni, um dos principais agentes etiológicos da esquistossomose, possui quatro genes que codificam septinas (SmSept5, SmSept10, SmSept7.1 e SmSept7.2). O objetivo deste trabalho foi a produção heteróloga das proteínas codificadas por estes genes, visando estudos estruturais e funcionais das mesmas. As septinas SmSEPT5 e SmSEPT10 foram expressas em sistema recombinante e foi possível a obtenção de formas solúveis destas proteínas. Experimentos de gel filtração e cross-linking mostraram que elas são diméricas em solução e estáveis em ampla faixa de pH, embora agregados proteicos tenham sido observados com o aumento da temperatura. Ambas as proteínas foram capazes de ligar GTP e GDP, embora apenas SmSEPT5 tenha apresentado atividade GTPásica. Mg2+ se mostrou essencial para a ligação de GTP a ambas as proteínas, enquanto a ligação do GDP foi independente da presença deste cofator. Ensaios de cristalização com o domínio GTPase de SmSEPT10 resultaram em cristais de ótima qualidade cuja difração resultou na obtenção da estrutura com melhor resolução alcançada até o momento para septinas: 1.9 &Aring; para a forma ligada a GDP e 2.1 &Aring; para a forma ligada a GTP. A análise da sobreposição das estruturas obtidas resultou na observação do deslizamento de uma fita &beta; em relação às demais, que acreditamos estar envolvido com o mecanismo de associação destas proteínas à membranas. Um sistema de coexpressão foi construído em que SmSEPT5, SmSEPT10 e SmSEPT7.2 foram coexpressas e copurificadas, resultando na verificação da formação de hetero-oligômeros e filamentos por estas proteínas. Tratamento de diversas fases do ciclo de vida do parasito com um composto (FCF) que afeta a dinâmica de filamentos de septina, resultou em um fenótipo reversível de paralisia no parasito. Estudos de imunolocalização revelaram a colocalização de septinas e actina em fibras musculares do parasito, sugerindo que a interação entre filamentos de septina e actina pode ter um papel importante nas funções motoras do parasita. A imunolocalização revelou ainda a presença de septinas em placas epiteliais ciliadas de miracídios, células germinativas de miracídios e esporocistos e protonefrídios de cercárias. Os resultados apresentados aqui constituem a primeira descrição de septinas em platelmintos e nos possibilitam o estabelecimento de correlações estruturais e funcionais entre septinas de S. mansoni e complexos análogos de septinas de outros organismos, contribuindo para a elucidação da função desta família de proteínas.<br>Septins belong to the GTPase family of proteins and are involved in a variety of cellular processes. We have identified four genes encoding septins in Schistosoma mansoni, one of the main causative agents of schistosomiasis, these genes were named SmSept5, SmSept10, SmSept7.1 e SmSept7.2. The aim of this study was to clone the cDNA of these genes in order to subject the resulting proteins to a set of biophysical and functional studies. Biophysical characterizations were undertaken with SmSEPT5 and SmSEPT10 because of the higher solubility of these proteins, which were dimeric in solution and stable over a wide range of pH, although increasing temperatures promoted the aggregation of these proteins. The nucleotide binding assays revealed that both were capable of binding GTP and GDP, although only SmSEPT5 presented GTPase activity. Mg2+ has shown to be essential for GTP binding in both proteins while GDP binding was independent of this cofactor. Crystallization assays with the GTPase domain of SmSEPT10 have resulted in crystals of high quality and consequently high resolution structures were obtained: 1.9 &Aring; to the GDP bound form and 2.1 &Aring; to the GTP bound form, the best resolution achieved to date for any septin member. The sobreposition of the structures obtained enabled us to observe a strand slippage in &beta;3 strand suggesting that it might be part of an activation mechanism involved in the association of these proteins to membranes. A coexpression system was produced, in which SmSEPT5, SmSEPT10 and SmSEPT7.2 were coexpressed and copurified. These proteins were able to assemble into heterocomplexes that further polymerize into filaments. Functional studies performed with a drug (FCF) that affects the dynamics of septin filaments have resulted in a reversible paralysis phenotype in the parasite. Immunolocalization experiments revealed the colocalization of septins and actin in muscular structures of the parasite, suggesting that the interaction of septin and actin filaments might have an important role in the motor activity of the parasite. The immunolocalization also revealed the presence of septins in the ephitelial plates of miracidia, germ cells of miracidia and sporocysts and protonephridia of cercariae. The results presented here constitute the first description of septins in phatyhelmintes and enabled us the establishment of structural and functional relaltionships among septins from S. mansoni and analogous septin complexes in other organisms, contributing to elucidate the function of this family of proteins.

Septinas compreendem uma conservada família de proteínas de ligação a nucleotídeo de guanina, capazes de formar filamentos. No entanto, apesar de sua importância em vários eventos celulares, ainda há pouca informação disponível no que diz respeita aos detalhes de suas funções moleculares e o mecanismo que conduz a formação de hetero-oligômeros. O objetivo desta tese foi a clonagem, co-expressão e cristalização de septinas e seus complexos (SEPT9-SEPT11-SEPT7; SEPT2-SEPT6-SEPT7-SEPT9; SEPT4-SEPT6-SEPT7 e SEPT4- SEPT8-SEPT7) seguidas por análise estrutural comparativa. Os cDNAs que codificam para as proteínas SEPT9, SEPT6, SEPT2, SEPT11, SEPT8, SEPT4, SEPT9GC&alpha;0 (resíduos 262-568) e SEPT9GC (resíduos 279-568) foram clonadas com sucesso em vetores de transferências ou de expressão. O complexo que foi melhor caracterizado, foi o composto por SEPT2-SEPT6-SEPT7-SEPT9GC&alpha;0, o qual foi confirmado por LC-MS/MS após digestão tríptica, revelando a produção in vitro de um complexo de septina tetramérico. Além disso, caracterizou-se o hetero dímero formado por SEPT7NGc-SEPT9GC. No entanto, as proteínas que foram mais plenamente investigadas foram as construções SEPT9GC&alpha;0 e SEPT9GC que foram estudados individualmente para caracterizações biofísicos e estruturais. SEPT9GC&alpha;0 apresentou-se bastante instável, porém, SEPT9GC foi eficientemente produzida e caracterizada. O estado oligomérico da proteína (monômero) foi confirmado por cromatografia de exclusão molecular. A proteína foi produzida na forma apo, e foi incapaz de hidrolisar GTP. A finidade de SEPT9GC pelos nucleotídeos GDP e GTP&gamma;S, foi avaliada por Calorimetria de Titulação Isotérmica -ITC, revelando que SEPT9GC possui uma maior afinidade por GTP&gamma;S, com Kd de 25,9 &micro;M, o qual é dependente do íon Mg2+. Foram realizados estudos de cristalização dessa proteína, que resultou em cristais de alta resolução, com a proteína complexada a GDP e GTP&gamma;S. Interessantemente, a estrutura do complexo de SEPT9GC com o nucleotídeo GTP&gamma;S foi obtido por soaking de um cristal previamente obtido complexado com GDP. Este é o primeiro relatório da aplicação deste método para septinas. Finalmente, obtivemos três conjuntos de dados cristalográficos, dois para SEPT9GC-GDP, com resolução de 2.8 &Aring; e 2.1&Aring; e um conjunto de dados de SEPT9GC-GTP&gamma;S obtido a 2.8 &Aring;. Dentre as principais características observadas na estrutura de SEPT9GC, tem-se a interface NC responsável pela polimerização dos filamentos, a qual se mostrou diferente dependendo do tipo de nucleotídeo ligado, com encurtamento do filamento na presença de GTP&gamma;S. Isso indica a comunicação entre as interfaces consecutivas G e NC ao longo do filamento. A mudança conformacional na interface envolve o rearranjo dos resíduos que são altamente conservados em todas as septinas, bem como alguns que são específicos do grupo de SEPT9 e podem ter implicações para a montagem de filamentos e de associação da membrana.<br>Septins belong to a highly conserved family of guanine nucleotide binding proteins, capable of forming filaments. Despite their importance for several critical cellular events including cell division, little information is available about their molecular functions and the mechanism which leads to the formation of hetero-oligomers. The aim of this thesis was the cloning, co-expression and crystallization of septins and their complexes (SEPT9-SEPT11-SEPT7; SEPT2-SEPT6-SEPT7-SEPT9; SEPT4-SEPT6-SEPT7 and SEPT4-SEPT8-SEPT7), followed by comparative structural analysis. The cDNAs coding for the proteins SEPT9, SEPT6, SEPT2, SEPT11, SEPT8, SEPT4, SEPT9GC&alpha;0 (residues 262-568) and SEPT9GC (residues 279-568) were successfully cloned into transferable or expression vectors. The best characterized complex was that composed of SEPT2-SEPT6-SEPT7-SEPT9GC&alpha;0, which was confirmed by LC-MS/MS analysis after triptic digestion, unveiling the in vitro production of a tetrameric septin complex. Additionally, we characterized the hetero-dimer formed by SEPT7NGc-SEPT9GC. However, the most fully investigated proteins were the constructs SEPT9GC&alpha;0 and SEPT9GC which were studied individually for biophysical and structural characterizations. SEPT9GC&alpha;0 was highly unstable contrasting with SEPT9GC that was efficiently produced and characterized. The presence of the oligomeric state of SEPT9GC (monomer) was confirmed by molecular exclusion chromatography. The protein was produced in the apo form and was capable of hydrolyzing GTP. The affinity of SEPT9GC for GDP and GTP&gamma;S nucleotides was evaluated by Isothermal Titration Calorimetry (ITC) revealing that SEPT9GC has a higher affinity for GTP&gamma;S, with a Kd of 25.9 &micro;M, which is dependent on the presence of Mg2+. Crystallization studies of this protein were performed, obtaining high quality crystals of protein complexes with GDP and GTP&gamma;S. Interestingly, the structure of the complex of SEPT9GC with the nucleotide GTP&gamma;S was obtained by soaking a previously grown crystal of the GDP complex. This is the first report of the application of this method for septins. Finally, we have obtained three sets of crystallographic data, two for SEPT9GC-GDP, at resolutions of 2.8 &Aring; and 2.1 &Aring;, and one set of data for SEPT9GC-GTP&gamma;S at 2.8 &Aring;. Among the main characteristics observed in the SEPT9GC structure is the NC interface responsible for filament polymerization, which is shown to vary according to the type of bound nucleotide, with foreshortening of the filament in the presence of GTP&gamma;S. This indicates communication between consecutive G and NC interfaces along the filament. The conformational change at the interface involves the rearrangement of residues which are highly conserved in all septins as well as several which are SEPT9 group specific and may have implications for filament assembly and membrane association.

Septinas constituem uma família conservada de proteínas de citoesqueleto pertencentes à superclasse das P-loop GTPases. Tais proteínas estão envolvidas em vários processos celulares. Em humanos, algumas septinas também estão relacionadas a casos de patologia. Suas seqüências são divididas em três domínios: domínio N-terminal, domínio GTPase e domínio C-terminal, que geralmente possui predição de coiled coil. A principal característica da família está na capacidade de seus membros formarem filamentos compostos por septinas diferentes. Em 2007, Sirajuddin et al apresentaram a primeira e única estrutura cristalográfica de um complexo de septinas, formado pelas septinas 2, 6 e 7. Embora os domínios C-terminal estivessem presentes, eles não apresentaram densidade eletrônica. Assim, a estrutura não trouxe informação estrutural sobre tais domínios. Atualmente, existem quatro estruturas de septinas depositadas no PDB: complexo 2/6/7 e três estruturas SEPT2 sem o C-terminal. Dada a inexistência de informações estruturais a nível atômico para os domínios C-terminal e baixa qualidade das poucas estruturas existentes, propusemos a obtenção de informações bioquímicas e estruturais dos domínios C-terminal de septinas humanas e a obtenção da estrutura cristalográfica de SEPT3-GC (GTPase mais C-terminal). Vale ressaltar que SEPT3 pertence ao único grupo de septinas que possui predição de não apresentar um coiled coil no C-terminal e para o qual não há nenhuma estrutura disponível. Expressamos e purificamos os domínios C-terminal de SEPT2, SEPT6 e SEPT7 (SEPT2-C, SEPT6-C e SEPT7-C). Mostramos que eles formam homo dímeros e que SEPT6-C e SEPT7-C formam um hetero dímero (KD 15,8 nM), nomeado por SEPT67-C. Tanto SEPT6-C quanto SEPT7-C tendem a precipitar, ao passo que SEPT67-C e SEPT2-C (KD 4 &mu;M) são estáveis à altas concentrações. Tentamos, sem sucesso, cristalizar SEPT2-C e SEPT67-C. Via ressonância magnética nuclear, vimos que SEPT2-C possui duas regiões dinamicamente diferentes, uma central, em &alpha;-hélice, e duas extremidades desestruturadas. Neste ponto, planejamos construções para as regiões centrais dos domínios C-terminal, nomeadas SEPT2-CC, SEPT4-CC, SEPT6-CC e SEPT7-CC, referente às septinas 2, 4, 6 e 7. Obtivemos cristais para SEPT2-CC, SEPT4-CC e SEPT6-CC. Contudo, resolvemos apenas a estrutura de SEPT4-CC, mostrando que a construção forma um coiled coil anti-paralelo. Então, propusemos, pela primeira vez, um possível mecanismo de formação de ligações cruzadas entre filamentos de septinas. Por outro lado, obtivemos cristais para uma construção contendo os domínios GTPase e C-terminal de SEPT3 (SEPT3-GC) e resolvemos sua estrutura (2,9 &Aring;). Vimos que SEPT3-GC forma filamentos no cristal, utilizando as mesmas duas interfaces já descritas anteriormente para as outras estruturas. Comparamos a estrutura obtida com estruturas da literatura e observamos diferenças significativas em algumas regiões, além de diferença em relação à orientação das duas subunidades adjacentes. Deve ser notado que a estrutura de SEPT4-CC é a primeira estrutura de um coiled coil de septina e que a estrutura de SEPT3-GC é a primeira estrutura de uma septina do grupo I. Em conclusão, o presente trabalho apresentou um conjunto de resultados os quais auxiliará no entendimento desta intrigante família de proteínas, inclusive em relação à formação de filamentos e as interações entre estes.<br>Septins are a conserved family of cytoskeletal proteins belonging to the superclass of the Ploop-GTPases, involved in various cellular processes. In humans, some septins are also linked to cases of pathology. Their sequences are divided into three domains: an N-terminal domain, a GTPase domain and a C-terminal domain, which usually is predicted to form a coiled coil. The main feature of the family is the ability of its members to form filaments composed of different septins. In 2007, Sirajuddin et al presented the first and only crystal structure of acomplex of septins, formed by septins 2, 6 and 7. Although the C-terminal domains were present, they showed no electron density, indicating disorder. Thus, this structure provides little structural information concerning their nature. Currently, there are four septin structures deposited in the PDB: the 2/6/7 complex and three structures of SEPT2 lacking the Cterminal domain . Based on the absence of structural information at atomic resolution about the C-terminal domains and the low quality of the few existing structures we set out to characterize both biochemically and structurally the C-terminal domains of selected human septins as well as to obtain the crystal structure of SEPT3-GC (a construct containing the GTPase and C-terminal domains). It is noteworthy that SEPT3 belongs to the only group of septins that are predicted to lack the C-terminal coiled coil and for which no crystal structure is available. We have expressed and purified the C-terminal domains of SEPT2, SEPT6 and SEPT7 (SEPT2-C, SEPT6-C and SEPT7-C). We show that they form homo-dimers and that SEPT6-C and SEPT7-C form a hetero-dimer (KD 15.8 nM), SEPT67-C. Both SEPT6-C and SEPT7-C were unstable, but SEPT67-C and SEPT2-C (KD 4 &mu;M) were both stable at high concentrations. NMR measurements showed that SEPT2-C has two dynamically different regions, a central one which is -helical, and two extremities which are unstructured. Constructs were designed for the central regions of the C-terminal domains of septins 2, 4, 6 and 7 (SEPT2-CC, SEPT4-CC, SEPT6-CC and SEPT7-CC). We have obtained crystals of SEPT2-CC, SEPT4-CC and SEPT6-CC and have solved the structure of SEPT4-CC which unexpectedly is observed to form an anti-parallel coiled coil. We use this observation to propose, for the first time, a possible mechanism for the formation of cross-links between septin filaments. Crystals were obtained for SEPT3-GC its structure solved at 2.9 &Aring;. We observe that SEPT3-GC forms filaments in the crystal, employing the same two interfaces previously described for other structures. We compare the structure obtained with those from the literature and observe significant differences in some regions of the molecule as well as differences in the relative orientation of the subunits. It should be noted that the structure of SEPT4-CC is the first structure of a septin coiled coil and that the structure of SEPT3-GC is the first structure of a septin from group I. In conclusion, this study presentsm results which will assist in the understanding of this intriguing protein family, including observations pertinent to filament formation and cross-linking.

La division cellulaire est un processus essentiel requis pour la prolifération des organismes unicellulaires, pour le développement des organismes multicellulaires, ainsi que pour le renouvellement cellulaire dans les tissus et organes. Un contrôle défectueux de la division cellulaire peut conduire à la mort cellulaire ou à une hyper-prolifération cellulaire et contribuer à la progression du cancer. La division cellulaire est donc sous le contrôle de mécanismes de régulation très stricts. Sa dernière étape, la cytocinèse, nécessite un anneau contractile acto-myosique qui se contracte pour permettre le clivage en deux cellules filles. Parallèlement à l'assemblage de l’anneau contractile, des modifications de la composition lipidique de la membrane plasmatique ont lieu avec un impact fonctionnel sur la cytokinèse. S. pombe a été utilisé comme modèle pour disséquer l’impact des lipides dans d`assemblage de l’anneau contractile. Dans la première partie de ma thèse, mon objectif était d’étudier comment les lipides pourraient réguler le positionnement de l’anneau contractile chez la levure S. pombe. J’ai combiné une approche génétique à de l’imagerie sur cellules vivantes de l'assemblage de l’anneau contractile à partir des nœuds précurseurs de l’anneau pour comprendre comment le niveau d'ergostérol affecte le positionnement du plan de division. J'ai constaté que l'augmentation du niveau d'ergostérol empêchait l'assemblage d’actine-F par la formine Cdc12 à partir de nœuds précurseurs, bloquant leur compaction en anneau fin. Comme la stabilité des câbles d’actine n’est pas perturbée globalement et que le phénotype peut être partiellement compensé par inhibition du complexe Arp2/3, en compétition avec les formines, nous proposons que l'augmentation du niveau d'ergostérol dans la membrane plasmique pourrait inhiber l'activité de la formine Cdc12. En plus de l’anneau contractile acto-myosique, la cytocinèse implique un réseau supplémentaire de cytosquelette, les septines, qui forment des filaments au niveau du site de division. Les septines constituent une famille conservée de protéines liant le GTP dont la suppression conduit à des défauts de cytocinèse. Elles servent également d’échafaudages pour les interactions protéine-protéine et/ou de barrières de diffusion pour la compartimentation des protéines pendant la cytocinèse et au-delà. Chez S. pombe, contrairement à S. cerevisiae, les septines arrivent tardivement au site de division et sont seulement impliquées dans les étapes tardives de la cytocinèse, pour promouvoir la séparation des deux cellules filles. Dans la deuxième partie de ma thèse, j’ai décidé d’explorer le comportement dynamique des septines au cours de la progression de la cytocinèse et leurs mécanismes de régulation. En utilisant l’imagerie sur cellules vivantes et des déterminants temporels précis de progression du cycle cellulaire, j’ai identifié une nouvelle étape de recrutement des septines sur le cortex cellulaire à proximité de l’anneau contractile acto-myosique, en un large réseau qui se compacte ensuite en anneau fin. J'ai également trouvé des preuves que Mid2, une protéine de la famille de l'anilline, favorise la compaction des septines et peut agir comme un « bundler » de septines. Cependant, l’analyse de mutants bloqués en mitose indique que cette protéine n’est pas suffisante pour accomplir cette tâche. De plus, j'ai déterminé qu’une activité de Cdk1 élevée permet le recrutement et l’assemblage des septines mais que la voie du SIN est aussi nécessaire pour promouvoir leur recrutement au milieu de la cellule et favoriser leur compaction. Enfin, j’ai trouvé que les niveaux de PIP2 influaient non seulement sur la quantité de septines et de Mid2 associés au cortex médian et leur compaction mais aussi sur le « timing » du recrutement des septines sur le site de division. Ceci démontre que l’organisation des septines dépend d’une série de régulations complexes coordonnées par la machinerie du cycle cellulaire<br>Cell division is an essential process required for the proliferation of unicellular organisms, for the development of multicellular organisms, as well as for cell renewal within tissues and organs. Defective control of cell division can either lead to cell death, or to cell hyper-proliferation and contribute to cancer progression. Cell division is therefore under the control of very tight regulatory mechanisms. In animals and fungi, its final step, cytokinesis, requires an acto-myosin-based contractile ring that constricts to promote sister cell cleavage. Modifications in the lipid composition of the plasma membrane take place during cell division, with functional impact on cytokinesis.Fission yeast is a simple single cell eukaryotic organism which has been extensively used to study cell division because of its stereotyped rode shape, easy genetics and short generation time. In particular, this model system has proved very powerful for the molecular dissection of contractile ring assembly. However remarkably little is known on how membrane lipids regulate contractile ring assembly. In the first part of my PhD, my objective was to study how lipids could regulate contractile ring positioning in fission yeast. I have combined fission yeast genetics with live-cell imaging of contractile ring assembly from precursor nodes to understand how ergosterol levels affect division plane positioning. I have found that increased ergosterol levels prevent F-actin assembly from cytokinetic precursor nodes by the formin Cdc12, avoiding their compaction into a medially placed contractile ring. Since the stability of F-actin cables was not altered altogether and the phenotype could be partially rescued by inhibition of the Arp2/3 complex which competes with formins, we propose that increasing ergosterol levels in the plasma membrane may inhibit the activity of the formin Cdc12.In addition to the contractile ring, cytokinesis involves an additional component of the cytoskeleton, the septins, which form filaments at the division site. Septins are a family of conserved GTP binding proteins whose deletion leads to cytokinetic defects. They also serve as scaffolds for protein-protein interactions and/or as diffusion barriers for protein compartmentalization in cytokinesis and beyond. In fission yeast, in contrast to budding yeast, septins are late at the division site and are only involved in late stages of cytokinesis, to promote sister cell separation. In the second part of my PhD, I decided to explore the dynamic behavior of septins and decipher how they are regulated. Using live cell imaging and precise cell cycle timers, I have identified a new step in the recruitment of septin to the cell cortex in the proximity of the contractile acto-myosin ring, in a broad meshwork that then compacts into a tight ring. I have also found evidence that the anillin-like protein Mid2 is necessary to promote this compaction and may act as a bundler for septin filaments. However, analysis of mutants blocked in mitosis shows that this protein is not sufficient to accomplish this task. Moreover, I have determined that high Cdk activity allows septins and Mid2 initial recruitment and assembly, but the SIN pathway also plays a role in promoting their recruitment at the cell middle and is then required to drive their compactio. Additionally, I have found that PIP2 levels influence not only the amount of septins and Mid2 filaments associated at the medial cortex together with their compaction but also the timing of septin recruitment to the division site. This demonstrates that septin assembly relies on complex regulations coordinated by the cell cycle machinery

As septinas constituem uma conservada família de proteínas de ligação a nucleotídeos de guanina e formação de heterofilamentos. Em mamíferos, tais proteínas estão envolvidas em uma variedade de processos celulares tais como citocinese, exocitose e tráfego de vesículas. A SEPT4 tem sido identificada em depósitos filamentosos e inclusões citoplasmáticas em Alzheimer e doença de Parkinson, respectivamente. A SEPT4 é a única proteína em associação com proteínas aberrantes em depósitos em ambos os tipos de doenças Assim, estudos adicionais de propriedades bioquímicas estruturais e funções fisiológicas para a SEPT4 podem promover importantes insights em relação ao mecanismo das doenças neurodegenerativas citadas acima. O desenovelamento térmico do domínio GTPase do SEPT4-G revelou um intermediário que agrega rapidamente na forma de fibras tipo amilóide em condições fisiológicas. Neste estudo a análise cinética da agregação do SEPT4-G foi monitorada utilizando fluorescência extrínseca e dicroísmo circular. Com os resultados e análises realizados durante este trabalho de mestrado foi possível compreender com mais detalhes a cinética de formação de agregados tipo amilóide do domínio SEPT4-G. Este trabalho também descreve a cristalização, coleta de dados, resolução e refinamento do modelo cristalográfico para a enzima Glutationa-S-Transferase de Xylella fastidiosa. Tal enzima está associada à patogenicidade da bactéria X. fastidiosa, responsável por várias doenças em plantas economicamente importantes, incluindo a Clorose Variegada dos Citros (CVC) ou Amarelinho. Algumas análises também foram realizadas após a obtenção do modelo cristalográfico demonstrando as diferenças estruturais entre GSTs bacterianas.<br>The septins are a conserved family of guanine nucleotides-binding and hetero-filament forming. proteins. In mammals they are involved in a variety of cellular processes, such as cytokinesis, exocytosis, and vesicle trafficking. SEPT4 has been reported to accumulate in tau-based filamentous deposits and cytoplasmic inclusions in Alzheimers and Parkinsons diseases respectively. Sept4 is unique in its association with the aberrant protein depositions in both types of diseases. Further studies on the biochemical, structural properties and physiological functions of Sept4 may therefore provide important insights into the common mechanism underlying diverse neurodegenerative disorders. Thermal unfolding of the GTPase domain of SEPT4 (SEPT4-G) revealed an unfolding intermediary which rapidly aggregates into amyloid-like fibers under physiological conditions. In this study, the kinetic analysis of aggregation of SEPT4-G was monitored using extrinsic fluorescence and circular dichroism spectroscopy. The aggregates have the ability to bind specific dyes such as Thioflavin-T (ThT), suggesting that they are amyloid in nature. Fibrils formation was monitored by the increase in ThT emission and electron microscopy as a function of temperature, pH, metal ions and protein concentration. Glutathione S-transferases (GSTs) form a group of multifunctional isoenzymes that catalyze the glutathione-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GST from Xylella fastidiosa (XfGST) was crystallized by the vapour-diffusion method and its crystallography structure was solved for molecular replacement and refined. Afterwards, sequential and structural analyses were carried out for XfGST and others GSTs.

Septinas são GTPases consideradas como um novo componente do citoesqueleto. Essas proteínas interagem entre si para formar heterocomplexos filamentosos e estruturas de alta ordem que são importantes para a citocinese e uma variedade de outros processos celulares. Existem muitos aspectos mecânicos dessas proteínas que não são totalmente compreendidos, incluindo a forma como os heterocomplexos se agrupam corretamente. Em humanos, há 13 genes que codificam septinas, classificadas em quatro grupos quanto à similaridade em relação à estrutura primária. O complexo hexamérico SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7 foi o melhor caracterizado, com uma estrutura cristalina resolvida à 4 &Aring;. Segundo às Regras de Kinoshita, as septinas desse complexo podem ser substituídas nesse arranjo por outras pertencentes ao mesmo grupo. Neste trabalho utilizamos a técnica de microscopia eletrônica de transmissão com análise de partícula única para estudar dois complexos de septinas. Um dos complexos estudados neste projeto é formado por septinas humanas, para as quais atualmente não há informações estruturais disponíveis. O complexo SEPT5-SEPT6-SEPT7 foi expresso heterólogamente em E. coli e purificado em alta concentração salina para evitar a polimerização. A análise de partículas únicas de imagens por contrastação negativa mostrou a presença de partículas alongadas de aproximadamente 25 nm de comprimento, compostos por seis monômeros, como esperado. Com o objetivo de localizar a posição da SEPT5 no complexo, foi realizada uma fusão com MBP (Maltose Binding Protein) e imunomarcação com anticorpo monoclonal anti-SEPT5, concluindo que a SEPT5 está localizada na extremidade do complexo hexamérico. Porém, a SEPT5 pertence ao mesmo grupo da SEPT2, que foi relatada estar localizada no centro do hexâmero. Este resultado possibilitou uma nova discussão sobre a maneira que as septinas formam os complexos e, como a sensibilidade à concentração salina está relacionada com a fragilidade da interface NC, análogo ao observado em complexos de Saccharomyces cerevisiae. Um complexo de Ciona intestinalis incluindo a SEPT2, SEPT6, SEPT7 e SEPT9, também expresso heterólogamente em E. coli, foi preparado por contrastação negativa. A análise de partícula única das imagens coletadas mostrou um heterocomplexo aparentemente hexamérico, embora fosse esperado um octâmero devido à presença das quatro septinas diferentes, sendo uma pertencente à cada um dos quatro grupos. Os resultados deste trabalho proporcionaram um avanço na compreensão da formação de heterocomplexos de septinas e como essas proteínas interagem umas com as outras nesta montagem.<br>Septins are GTPases that appear to be a novel component of the cytoskeleton. These proteins interact with each other to form filamentous heterocomplexes and high order structures which are important for cytokinesis and a variety of other cellular processes. There are many mechanistic aspects of these proteins that are not fully understood, including how the heterocomplexes correctly assemble. In humans, there are 13 genes encoding septins, classified in four groups based on primary structure. The SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7 hexameric complex was the best characterized, with a crystalline structure solved at 4 &Aring;. According to Kinoshita´s Rules, the septins of this complex can be replaced in this arrangement by others belonging to the same group. In this work, we used transmission electron microscopy with single particle analysis to study two septin complexes. One of the complexes studied in this project is composed of three human septins, for which there is currently no structural information available. The SEPT5-SEPT6-SEPT7 complex was heterologously expressed in E. coli and purified at high salt concentration to avoid polymerization. Single particle analysis of negatively stained samples showed the presence of elongated particles of approximately 25 nm in length. To locate SEPT5 in the complex, a fusion with MBP (Maltose Binding Protein) and immunoblotting with anti-SEPT5 monoclonal antibody was performed, concluding that SEPT5 is located at the end of the hexameric complex. However, SEPT5 belongs to the same group as SEPT2, which was reported to be located in the center of the hexamer. This result allowed for a new discussion on the way that septins form heterocomplexes and also, on how the sensitivity of the NC interface in related to salt concentration, analogous to that observed in the heterocomplex of Saccharomyces cerevisiae. A Ciona intestinalis complex including SEPT2, SEPT6, SEPT7 and SEPT9, also expressed heterologously in E. coli, was prepared by negative staining. The single particle analysis of the collected images showed an apparently hexameric heterocomplex, although an octamer was expected due to the presence of the four different septins, one belonging to each of the four groups. The results of this work represent advances in the understanding of the formation of septin heterocomplexes and how these proteins interact with each other during assembly.

Septinas são proteínas que pertencem a super família das GTPases e que foram inicialmente identificadas em Saccharomyces cerevisae, mas logo em seguida, também em eucariotos superiores, exceto em plantas. Estas proteínas estão envolvidas em uma variedade de processos celulares tais como segregação de cromossomos, polaridade celular, dinâmica da membrana, tráfego de vesículas, exocitose, apoptose, entre outros. Mutações ou alterações no padrão de expressão de septinas são associadas com vários cânceres e doenças neurológicas. Objetivando contribuir com informações funcionais sobre tais proteínas, as septinas humanas 1, 5 e 7 foram usadas como iscas em ensaios de duplo híbrido em leveduras visando à identificação de seus parceiros protéicos. Após a varredura de bibliotecas de cDNA de leucócitos e cérebro fetal humano, os parceiros protéicos predominantemente encontrados foram outras septinas de grupos diferentes aos das iscas. As interações septina-septina envolveram o domínio de ligação a GTP. Ainda, outros parceiros, diferentes de septinas, foram também identificados nas bibliotecas e estes se mostraram funcionalmente relacionados à endocitose, à regulação da atividade de GTPases, ao tráfego intracelular, aos ciclos de sumoilação, à manutenção da placa metafásica e à maturação do centrossomo. Algumas destas funções são inéditas e foram pela primeira vez relacionadas às septinas. Este trabalho também esteve voltado à caracterização biofísica das septinas 3 e 5 (SEPT3 e SEPT5). Muitos protocolos diferentes foram desenvolvidos na tentativa de obter amostras homogêneas de SEPT5, mas não foram bem sucedidos. Por outro lado, SEPT3 recombinante, destituída do domínio amino-terminal (SEPT3GC) foi eficientemente produzida em E. coli. O estado monomérico de SEPT3GC em solução foi confirmado por cromatografia de exclusão molecular e espalhamento de raios-X a baixos ângulos (SAXS). SEPT3GC mostrou-se ativa e capaz de hidrolizar GTP in vitro. A afinidade de SEPT3GC por GTP&gamma;S e GDP foi avaliada por calorimetria de titulação isotérmica (ITC), sendo que o KD de SEPT3GC para GTP&gamma;S foi de 5,43 &mu;M e a ligação para este nucleotídeo foi dependente de Mg2+. A ligação para GDP não foi detectável. Agregados de SEPT3GC induzidos por temperatura foram capazes de ligar a sonda fluorescente tioflavina-T, sugerindo uma natureza amilóide para tais estruturas.<br>Septins are proteins that belong to the superfamily of GTPases, which were initially identified in Saccharomyces cerevisiae, and then in higher eukaryotes, except plants. These proteins are involved in a variety of cellular processes such as chromosome segregation, cell polarity, membrane dynamics, vesicle trafficking, exocytosis, apoptosis, among others. Mutations or changes in the expression of septins have been associated with various cancers and neurological diseases. Aiming to provide functional information about these proteins, the human septins 1, 5 and 7 were used as baits in yeast two-hybrid assay in order to identify their protein partners. After screening cDNA libraries from human leukocytes and fetal brain, the protein partners predominantly found were septins from others groups. The septin-septin interactions involved the GTP binding domain. Others non-septins interactors have also been identified in the libraries and were functionally related to endocytosis, the regulation of the GTPase activity, intracellular trafficking, sumoylation, maintenance of metaphase plate and centrosome maturation. Some of these functions are new and were related to the septins for the first time. This work also focused on the biophysical characterization of the septins 3 and 5 (SEPT3 and SEPT5). Many different protocols were developed aiming to obtain homogeneous samples of SEPT5, but were not successful. On the other hand, recombinant SEPT3, without the amino-terminal domain (SEPT3GC), was produced in a homogeneous form in E. coli. SEPT3GC is monomeric in solution as confirmed by size exclusion chromatography and small-angle X-ray scattering (SAXS). Also, SEPT3GC has shown to be active and able to hydrolyze GTP in vitro. The SEPT3GC affinity by GTP&gamma;S and GDP were evaluated by isothermal titration calorimetry (ITC). The KD for GTP&gamma;S was about 5,43 &mu;M and it was observed to be Mg2+ dependent. The binding to GDP was not detectable. SEPT3GC aggregates induced by temperature were able to bind the thioflavin-T fluorescent probe, suggesting an amyloid nature for such structures.

Neste trabalho estudamos por espalhamento de raios-X a baixos ângulos (SAXS) quatro diferentes sistemas de interesse biológico. Visamos investigar a auto-agregação de proteínas e de complexos proteicos que darão origem a fibras amilóides, interação proteína-proteína, simulando ambientes altamente concentrados, interação proteína-membrana simulando vesículas de matriz extracelular (MVs) de sistemas de biomineralização e interações proteína-agentes desnaturantes. No caso de formação de amilóides, investigamos a agregação do domínio GTPase da septina 6 (SEPT6G) e do complexo formado com o domínio GTPase da septina 2 (SEPT2G-SEPT6G). A temperaturas de até 15°C, tanto SEPT6G quanto SEPT2G-SEPT6G apresentam-se predominantemente diméricas em solução. Já a 25°C, o heterodímero SEPT2G-SEPT6G permanece estável enquanto agregados maiores de SEPT6G evoluem e coexistem em solução com SEPT6G-SEPT6G dimérica, sendo que a proporção de dímeros diminui com a temperatura. No estudo das MVs, mostramos que miméticos lipossomais de DPPC e DPPC:DPPS (9:1) possuem as mesmas características estruturais na ausência e presença de cálcio na solução. A interação da proteína anexina V humana (A5), envolvida em processos de biomineralização, impacta na membrana modelo induzindo a formação de nanoporos. A adição da fosfatase alcalina tecido não-específico (TNAP) não altera as propriedades estruturais do proteolipossomo na presença de A5. A ação do surfactante dodecil sulfato de sódio (SDS) a 30 mM não altera a conformação da albumina soro bovina (BSA), de maneira que é observada a formação de micelas de SDS coexistindo com a proteína livre em solução. Já a adição de 50 mM de SDS induz um desenovelamento parcial da proteína, identificado pela análise das curvas de SAXS via modelo de \"colar de pérolas\". A ação de uréia a 3 M e 8 M promove um desenovelamento parcial e total da BSA, respectivamente, com subsequente agregação de proteína dependente da temperatura (T > 30°C). A adição de 6 mM de SDS em proteínas parcialmente desenoveladas pela ação da uréia promove um desenovelamento mais acentuado. O potencial efetivo resultante da interação entre duas proteínas distintas, BSA e lisozima a concentração total de 100 mg/mL em solução, pH 7.0, foi obtido da análise de curvas de SAXS. Para isto, utilizou-se uma análise simplificada (em primeira aproximação) considerando um potencial efetivo de interação entre BSA-BSA, lisozima-lisozima e lisozima-BSA. Variamos a razão molar BSA:LISO até 1:42. No pH estudado, BSA tem uma carga residual superficial de -11e, enquanto a lisozima possui +9e. Conforme variamos a razão molar BSA:LISO, observamos dois regimes para o potencial efetivo resultante: i) até BSA:LISO 1:2, a carga efetiva do sistema é praticamente nula com um potencial resultante de caráter atrativo e ii) para razões entre BSA:LISO 1:3 a 1:42, a carga efetiva aumenta e o potencial resultante tem caráter repulsivo. Assim, lisozima e BSA coexistem sem agregar, através de um delicado balanço de forças atrativas e repulsivas no sistema.<br>In this work we have used small-angle x-ray scattering (SAXS) to study four systems of biological interest. We aim to investigate the self aggregation of proteins and protein complexes that would form amyloid fibers; protein/protein interaction, simulating high concentrations; protein/cell-membrane interaction, simulating extracellular matrix vesicles (MVs) from biomineralizing systems; and protein/denaturating-agents interactions. On the case of amyloid formation, we have investigated the aggregation of G-domain of septin-6 (SEPT6G) and the protein complex formed with G-domain of septin-2 (SEPT2G-SEPT6G). At temperatures lower than 15°C, both SEPT6G and SEPT2G-SEPT6G were found predominantly as dimers. At 25°C, SEPT2G-SEPT6G heterodimer is still stable while aggregates of SEPT6G grow. Both coexist in solutions of SEPT2G-SEPT6G dimers, with the percentage of dimers decreasing the higher the temperature. As for the study of MVs, we have shown that DPPC and DPPC:DPPS (9:1) liposomal mimetics have the same structural characteristics at the absence or presence of Calcium. The interaction with human annexin V protein (A5), related to biomineralization processes, affects the model membrane by the creation of nanopores. The addition of tissue-nonspecific alkaline phosphatase (TNAP) does not change the structural properties of the proteoliposome when A5 is present. The addition of SDS surfactant (30 mM) does not alters the conformation of bovine serum albumin (BSA), and we have observed the formation of SDS micelles coexisting with free protein in solution. The addition of 50 mM of SDS, on the other hand, induces the partial unraveling of the protein, as seen by the analysis of SAXS data via the pearl necklace\'\' model. The effect of adding 3M and 8M urea is, respectively, the partial and total unraveling of BSA, with ensuing aggregation of the protein dependent on the temperature (T > 30°C). The introduction of SDS 6mM promotes further unraveling in proteins that were previously partially unraveled by urea. The resulting effective potential for the interaction between BSA and lysozyme at total concentration of 100mg/ml and 7.0 pH has been obtained from the analysis of SAXS curves. In order to obtain this result we have used a simplified analysis (first order approximation) in which were considered the effective potentials for the interactions between BSA-BSA, lysozyme-lysozyme and lysozyme-BSA. We have varied the BSA:LISO molar ratio up to 1:42. At the studied pH, BSA has a surface residual charge of -11e, and lysozyme has +9e. As we changed the BSA:LISO molar ratio, we have found two regimens for the resulting effective potential: i) up to BSA:LISO 1:2, the effective charge of the system is virtually zero and the resulting potential is attractive; and ii) for BSA:LISO between 1:3 and 1:42 the effective charge increases, and the resulting potential is repulsive. Therefore, both lysozyme and BSA coexist without forming aggregates, by a delicate balance of attractive and repulsive forces.

Les septines forment une famille de GTPases conservée chez les champignons et dans les cellules animales [Kinoshita et al., 2003]. Pendant la division cellulaire, elles se localisent aux sites de cytocinèse et sont essentielles pour ce processus dans la levure bourgeonnante, les embryons de drosophile et les cellules de mammifère en culture [Faty et al., 2002]. Dans les levures bourgeonnantes, les septines, composées de réseaux parallèles de 1laments [Byers et al., 1976], forment un anneau au cou mère-fille. Cet anneau est étroitement associé à la membrane plasmique et constitue un échafaudage pour le recrutement de la myosine II et d'autres facteurs cytocinétiques au futur site de clivage [Longtine et al., 2003]. De plus, l'anneau de septines contribue à la formation d'une barrière de diffusion latérale sur la membrane plasmique, qui aide à maintenir les facteurs de la polarité cellulaire dans le bourgeon [Barral et al., 2000; Takizawa et al., 2000]. Chez les métazoaires, les septines sont aussi requises pour la compartimentation du cortex cellulaire [Schmidt et al., 2004; Joo et al., 2005] et sont impliquées dans une myriade de processus cellulaires, y compris l'assemblage et l'orientation du corps polaire du fuseau [Kusch et al., 2002; Spiliotis et al., 2005], l'exocytose et le transport vésiculaire [Hsu et al., 1998;Beites et al., 1999], la migration cellulaire [Finger et al., 2003], et l'apoptose Larisch et al., 2000; Gottfried et al., 2004].[...]Dans ce mémoire, après avoir passé en revue les fonctions essentielles des septines chez la levure bourgeonnante Saccharomyces cerevisiae, je présente une étude dans laquelle j’ai démontré que UNC-59 et UNC-61 forment un complexe hétérotétramérique capable de s’associer en structures d’ordre supérieur et en filaments dans les cellules et que le complexe hétérotétramérique de septines est assemblé à partir d’hétérodimères UNC-59/UNC-61. De plus, au cours d’un projet visant à développer un outil moléculaire pour l’étude des fonctions des septines in vivo dans des cellules sauvages de Saccharomyces, j’ai mis au point une méthode d’isolement d’intrabodies à partir de la banque d’anticorps recombinants humains en phage display ETH-2 et démontré leur activité d’inhibition de la formation de l’anneau de septines au col du bourgeon dans les cellules de Saccharomyces cerevisiae. L’ensemble des résultats présentés met en lumière l'arrangement moléculaire des monomères dans le complexe de septines et suggère que les complexes de septines s’assemblent dans les filaments et les structures d'ordre supérieur au col du bourgeon chez Saccharomyces cerevisiae. Ainsi, nous proposons que les septines forment la quatrième composante du cytosquelette<br>Septins form a family of GTPases conserved in fungi and animal cells [Kinoshita etal., 2003]. During cell division, they localize at cytokinesis sites and are essential for this process in budding yeast, Drosophila embryos and cultured mammalian cells [Fatyet al., 2002].In budding yeasts, septins, composed of parallel networks of filaments [Byers et al.,1976], form a mother-daughter neck ring. This ring is closely associated with the plasma membrane and constitutes a scaFold for the recruitment of myosin II and other cytokinetic factors at the future cleavage site [Longtine et al., 2003]. In addition, the septin ring contributes to the formation of a lateral diffusion barrier on the plasma membrane, which helps maintain the factors of cell polarity in the bud [Barral et al.,2000; Takizawa et al., 2000].In metazoans, septins are also required for compartmentalization of the cellular cortex [Schmidt et al., 2004; Joo et al., 2005] and are involved in a myriad of cellular processes, including assembly and orientation of the polar body of the spindle [Kusch etal., 2002; Spiliotis et al., 2005], exocytosis and vesicular transport [Hsu et al., 1998;Beites et al., 1999], cell migration [Finger et al., 2003], and apoptosis [Larisch et al.,2000; Gottfried et al., 2004].[...]The set of results presented highlights the molecular arrangement of the monomersin the septin complex and suggests that the septin complexes assemble in filaments and higher order structures at the bud neck in Saccharomyces cerevisiae. Thus, wepropose that septins form the fourth component of the cytoskeleton

Le choc septique est une pathologie fréquente associée à un taux de mortalité dépassant les 50%. Il s’agit de la première cause de mortalité en réanimation. Malgré les avancées des connaissances physiopathologiques sur le sepsis, le taux de mortalité reste très élevé. La majorité des recherches pré-cliniques sont réalisées sur des animaux jeunes et sains et sans antécédents cardiovasculaires, alors qu’il est bien établi que l’incidence et le taux de mortalité du sepsis augmente sévèrement avec l’âge. L’objectif de cette étude est la mise au point d’un modèle animal de sepsis ayant plusieurs antécédents cardiovasculaires, et l’évaluation des effets de la combinaison de deux maladies (hypertension et infarctus du myocarde (IDM)) chez des rats septiques sur la fonction cardiovasculaire et sur les voies de signalisation responsables de la réponse inflammatoire et apoptotique. L’étude a été réalisée sur des rats normotendus Wistar-Kyoto (WKY) et des rats Hypertendus Spontaneously Hypertensive (SHR) qui ont subi l’induction d’un IDM par ligature de l’artère coronaire gauche suivie ou non par l’induction d’un sepsis par ligature et perforation du caecum. Nous avons montré que les rats polypathologiques SHR, ayant subi un IDM préalable au choc septique, présentent une diminution drastique des paramètres cardiaques et hémodynamiques par rapport aux rats WKY ayant subi seulement un choc septique. En effet, le débit cardiaque, la pression artérielle moyenne et la fraction d’éjection sont diminuées respectivement de 70 %, 60 % et 67 % chez les rats SHR polypathologiques par rapport aux rats WKY ayant seulement un sepsis. On a observé également une hypo-réactivité vasculaire aux vasopresseurs plus importante chez les rats SHR-IDM-CLP comparée à celle des rats WKY-CLP. L’expression génique des récepteurs adrénergiques alpha-1 est fortement diminuée chez les rats SHR-IDM-CLP par rapport aux rats WKY-CLP. L’étude du taux d’expression des protéines impliquées dans l’apoptose révèle une surexpression des protéines caspase 3, caspase 8 et Pp38 chez les rats SHR polypathologiques des rats SHR-IDM-CLP par rapport aux rats WKY-CLP ayant seulement un sepsis. De plus, on remarque une forte diminution de l’expression des protéines eNOS et Akt chez les rats SHR-IDM-CLP comparés aux rats WKY-CLP. L’hypertension artérielle associée à l’insuffisance cardiaque a conduit à une plus grande sensibilité des rats au sepsis. Les rats SHR polypathologiques ont développé une insuffisance cardiaque aggravée par rapport aux rats WKY ayant seulement un sepsis. Le modèle de rats SHR ayant subi un IDM préalable au sepsis se rapproche d’avantage des situations cliniques observées quotidiennement dans les services de réanimation. Ce modèle pourrait servir de bon candidat pour les études pré-cliniques<br>Septic shock is considered as the most common cause of death among patients admitted in medical intensive care units. Mortality remains very high (50%) despite advances in physiopathological knowledge of this disease. In fact, most experimental studies are often conducted on young and healthy animals, whereas it is well established that the incidence and mortality rates dramatically increase with age. The goal of this study was to evaluate the effect of the combination of two diseases (hypertension and myocardial infarction) in a septic rat model, on cardiovascular function, cell survival and the inflammatory response. Therefore, we used normotensive Wistar-Kyoto rats (WKY) and Spontaneously Hypertensive Rats (SHR) in which myocardial infarction was induced by left coronary descending artery ligation, followed or not by cecal ligation and perforation-induced sepsis. Our results showed that the polypathological SHR rats in which a myocardial infarction and sepsis were induced, exhibited a significant decrease in cardiac and hemodynamic parameters compared to WKY rats that have sepsis (CLP) only. Indeed, cardiac output, mean arterial pressure and ejection fraction were reduced by 70%, 60% and 67% respectively in the polypathological SHR rats versus the WKY rats with sepsis. We observed also that the vascular hyporeactivity in the polypathological SHR rats was higher than for the WKY CLP rats. A mesure of gene expression level of alpha1 adrenergic receptor shows a relatively low expression level in the rats from SHR IDM CLP rats compared to WKY CLP that could explain the observed vasodilation. The protein level of caspase3 and 8, Pp38 and proteins involved in apoptosis, is upregulated in SHR IDM CLP rats compared to WKY CLP rats. However, the polypathological SHR rats show a significant decrease in the expression of eNOS and Akt protein compared to WKY CLP rats. The combination of hypertension with myocardial infarct is associated with higher sensitivity of the rats to sepsis. The polypathological SHR rats developed a severe heart failure compared to WKY rats having sepsis only. Our animal model, the polypathological SHR rats is very close to observed clinical situations in intensive care units. This model could serve as a good candidate for pre-clinical studies

Le sepsis sévère est un problème majeur de santé publique mondiale. Sa mortalité élevée résulte d’une réponse dérégulée de l’hôte au sepsis, associant hyper inflammation et immunodépression. Le choc septique est la forme la plus grave du sepsis, impliquant une défaillance cardiovasculaire à laquelle peuvent se surajouter d’autres défaillances d’organes. Le foie, organe majeur impliqué dans la défense et la réponse au stress induit par la septicémie peut être victime de cette réponse inflammatoire exagérée. Une dysfonction hépatocellulaire (DHC) peut survenir et évoluer jusqu’à la défaillance d’organe. Dans ce cas, l’existence d’une insuffisance hépatique est associée à un mauvais pronostic dans le choc septique à court terme. A partir d’une grande cohorte prospective de patients en choc septique, nous avons montré dans ce travail que cette DHC était associée à une surmortalité à long terme. Malgré, une meilleure connaissance de la physiopathologie du sepsis et en particulier des altérations du foie, l’impact des thérapeutiques utilisées au cours du choc septique, telles que les catécholamines (adrénaline, noradrénaline), reste indéterminé. Les travaux préliminaires de notre équipe avaient permis de montrer l’effet pro-inflammatoire de l’adrénaline sur un modèle de culture hépatocytaire. Dans ce travail, nous avons cherché à évaluer l’influence des cellules de Küpffer acteur de l’environnement péri-hépatocytaire. Pour cela nous avons utilisé un modèle de co-culture d’hépatocytes (HepaRG) et de macrophages (THP1 différenciés par PMA), stimulé par le lipopolysaccharide (LPS) et/ou l’adrénaline. L’analyse de la réponse d’expression génique et de production de cytokines a permis d’identifier l’adrénaline comme facteur capable de modifier la réponse immune vers un état pro-inflammatoire même en présence d’un mécanisme anti-inflammatoire développé par les macrophages, indiquant ainsi un rôle potentiellement délétère de l’adrénaline sur les mécanismes de défenses du foie<br>Severe sepsis is a major health problem. Its high mortality rate over the world is the result of a dysregulated host response to sepsis including an exaggerated inflammation response and immune suppression. Septic shock is the most severe expression of sepsis, including cardiovascular failure and other organ failure. The liver, a major organ involved in the defense and stress response induced by sepsis may also be a victim of this inflammatory response to infection. A hepatocellular dysfunction (HCD) can develop and evolve to the organ failure. In this case, the liver failure is associated with poor prognosis in septic shock in the early course of sepsis. Here, we have shown in a large prospective cohort of patients with septic shock that the HCD was associated with long-term mortality. Despite a better understanding of the pathophysiology of sepsis, especially liver changes, the impact of treatment used during septic shock, such as catecholamines (epinephrine, norepinephrine), remains unknown. The preliminary work of our team had demonstrated the proinflammatory effect of adrenaline on a hepatocyte culture model. In this work, we studied the influence of hepatocyte environment especially Küpffer cells. Thus, we used a co-culture model including hepatocytes (HepaRG) and macrophages (differentiated THP1 PMA) stimulated by lipopolysaccharide (LPS) and / or adrenaline. The gene expression and the cytokine profile analysis allowed to identify adrenaline as a factor able to shift the immune response to a proinflammatory state even if macrophages developed an anti-inflammatory response, indicating a deleterious effect of adrenaline on liver defense mechanisms

Septinas compreendem uma conservada família de proteínas de ligação a nucleotídeo de guanina e formação de heterofilamentos. Em termos estruturais, elas possuem uma organização comum: um domínio GTPase central, uma região N-terminal e um domínio C-terminal, este último é predito para formar estruturas em coiled coil. Atualmente, o heterocomplexo de septinas humanas (SEPT2/SEPT6/SEPT7) mais bem caracterizado revela a importância do domínio GTPase na formação do filamento, todavia a ausência de densidade eletrônica para os domínios C-terminais faz com que sua função permaneça obscura. Estudos com septinas de mamíferos, e de outros organismos como C. elegans e S. cerevisea sugerem que alguns grupos de septinas (por exemplo, II e IV em mamíferos) interagem através de seus domínios C-terminais, e estes poderiam atuar de modo determinante para a montagem correta do filamento. Assim, o presente projeto objetivou estudar a afinidade homo/heterotípicas para os domínios C-terminais das septinas humanas dos grupos II (SEPT6C/8C/10C/11C) e IV (SEPT7C), investigando se esses domínios contribuem para preferência das septinas interagirem com proteínas de grupos distintos durante a formação do heterofilamento. Os domínios C-terminais foram expressos em E. coli e purificados. Foram conduzidos estudos de ultracentrifugação analítica e espectropolarimetria de dicroísmo circular, que permitiram identificar maior afinidade e estabilidade da associação heterotípica comparada à homotípica. Foram obtidas constantes de dissociação aparente para homodímeros em torno de baixo &micro;M, enquanto que para heterodímeros os dados já existentes no grupo revelaram constante de dissociação na ordem de nM. Para entender os fatores no nível atômico responsáveis pela significativa predileção na interação entre os domínios C-terminais dos grupos II e IV foram realizados estudos utilizando modelagem e análise das sequências primárias. As análises sugerem a presença de um alto número de resíduos carregados na posição a do coiled coil como responsável pela seletividade. Consequentemente, o heterodímero seria favorecido em virtude do menor efeito repulsivo proveniente do intercalamento dos resíduos carregados em a. Desse modo, os resultados indicaram a atuação decisiva ou cooperativa dos domínios C-terminais na organização preferencial das septinas durante a formação do filamento, favorecendo a interface NC entre septinas dos grupos II e IV.<br>Septins comprise a conserved protein family that binds guanidine nucleotide and forms heterofilaments. In structural terms they have a common organization: a central GTPase domain, a N-terminal domain and a C-terminal domain, this last one is predicted to form coiled coil structures. Currently, the human septin heterocomplex best characterized (SEPT2/SEPT6/SEPT7) reveals the importance of the GTPase domain in filament assembly, however the absence of electron density for the C-terminal domains makes its function still unknown. Studies with mammals septins, and of others organisms like C. elegans and S. cerevisea suggests that some septins groups (e.g. II e IV in mammals) interact via its C-terminal domains and this could act in a determinative way to correct filament assembly. In this way, this project aimed to study the homo/heterotypical affinity for the C-terminal domains of human septins belonging to groups II (SEPT6C/8C/10C/11C) e IV (SEPT7C), investigating whether this domain contributes with the preference of septins to interact with proteins of different groups during assembly of the heterofilament. The C-terminal domains were expressed in E. coli and purificated. It was carried out studies using analytical ultracentrifugation and circular dichroism spectropolarimetry tecniques which allowed identification of major affinity and stability in the heterotypical association compared to homotypical. It was measured apparent dissociation constants for homodimers of low &micro;M range while for heterodimers our group\'s data shows dissociation constants in the nM range. To understand at atomic level the factors responsible for this significant preference in the C-terminal domains interaction between groups II and IV was performed molecular modelling studies and analysis of the primary sequence. These analysis suggests the presence of a high number of charged residues in position a of the coiled coil as responsible for selectivity. Consequently, the heterodimer would be therefore favoured because of the minor repulsive effect coming from the staggered of charged residues in a. Thus, these results indicate the crucial or cooperative action of C-terminal domains in preferential organization of septins during filament assembly, favouring the NC interface between septins of groups II and IV.

Septinas abrangem uma família conservada de proteínas que ligam e hidrolisam GTP e formam heterofilamentos, anéis e redes para realizar as suas funções. Apresentam três domínios estruturais: o domínio N-terminal contendo uma sequência polibásica (para ligar membranas), o domínio de ligação ao nucleotídeo (G) e o domínio C-terminal que inclui uma sequência predita de formar um coiled-coil. Em humanos, as 13 septinas são classificadas em quatro grupos (I, II, III e IV) baseadas nas sequências de aminoácidos. O único filamento caracterizado estruturalmente, até hoje, é o formado por SEPT2-SEPT6-SEPT7, mostrando que as subunidades interagem através de duas interfaces (chamadas G e NC). Os determinantes estruturais da montagem correta do filamento são pouco conhecidos, sendo o estudo limitado pela complexidade em purificar e cristalizar complexos triméricos ou tetraméricos. Uma abordagem alternativa é estudar interfaces individuais de um filamento (G e/ou NC) por separado. Assim, o presente projeto objetivou estudar, utilizando uma abordagem biofísica e estrutural, a interface G formada por SEPT5 e SEPT8 para elucidar os fatores importantes em determinar a sua especificidade. Os domínios GTPase de SEPT5 e SEPT8 foram clonadas em vetor de expressão bicistrônico pET-Duet, co-expressas e co-purificadas. Estudos de análise do estado oligomérico e homogeneidade foram conduzidos utilizando cromatografia de exclusão molecular, espalhamento dinâmico de luz e ultracentrifugação analítica, revelando um complexo dimérico e monodisperso. O complexo apresenta uma mistura aproximadamente equimolar de nucleotídeos (GTP e GDP) ligados enquanto SEPT8(G) sozinha é incapaz de ligar qualquer um dos dois. Além disto o complexo apresenta uma termoestabilidade maior que SEPT8(G), verificado por um aumento em Tm de 5&deg;C. Com o intuito de observar os determinantes estruturais da especificidade, ensaios de cristalização foram conduzidos e assim, cristais do complexo SEPT5-SEPT8(G) que difrataram apenas a muito baixa resolução foram obtidos. Na ausência de uma estrutura cristalográfica, modelagem por homologia foi realizada para analisar as interfaces G entre diferentes combinações de septinas. Identificamos uma interação entre aminoácidos característicos (aminoácidos únicos para cada grupo de septinas) para o complexo formado entre membros do grupo III, (incluindo SEPT5) e membros do grupo II, (incluindo SEPT8). Esta interação entre Phe131 (grupo III) e Thr19 (grupo II) pode explicar a especificidade na formação de uma interface G entre septinas destes grupos durante a formação do filamento e além disso, a importância da presença do GTP ligado ao septina do grupo II. Com isto, propomos pela primeira vez uma explicação plausível da relevância da perda de atividade catalítica das septinas deste grupo, um fato inexplicado até o momento. Mutação dos resíduos identificados levou a uma mudança no seu perfil de eluição do complexo durante purificação por exclusão molecular indicando alterações na formação do complexo mutante.<br>Septins are a conserved family of proteins that bind and hydrolyze GTP and form heterofilaments, rings and networks in order to carry out their functions. They have three structural domains: an N-terminal domain containing a polybasic sequence (for membrane binding), a nucleotide-binding (G) domain and a C-terminal domain including a sequence predicted to form a coiled-coil. In humans, 13 septins have been classified into four groups (I, II, III and IV) based on their amino acid sequences. The only structurally characterized filament described to date is formed by SEPT2-SEPT6-SEPT7, which reveals that the subunits interact through two different interfaces (G and NC). The structural determinants of correct filament assembly are poorly known, and this is limited by the complexity of purifying and crystallizing trimeric or tetrameric complexes. An alternative approach is to study a single filament interface (G or NC) on its own. Here, we aimed to study, using biophysical and structural approaches, the G interface formed between SEPT5 and SEPT8 to elucidate the factors relevant to determining its specificity. The GTPase domain of SEPT5 and SEPT8, were cloned into the bicistronic expression vector pET-Duet, co-expressed and co-purified. Studies to determine the oligomeric state and homogeneity of the complex were conducted using size exclusion chromatography, dynamic light scattering and analytical ultracentrifugation, revealing a monodisperse dimer for SEPT5-SEPT8(G). The complex elutes with an approximately equimolar mixture of bound nucleotides (GTP and GDP) whereas SEPT8(G) alone is shown to be unable to bind either. Furthermore, the complex has a greater thermostability than SEPT8(G), demonstrated by an increase of 5&deg;C in Tm. In order to determine the structural determinants of specificity, crystallization trials were conducted and crystals of the SEPT5-SEPT8(G) complex were obtained, but these diffracted to only very low resolution. In the absence of a crystal structure, homology modeling was performed to analyze the potential G interfaces between different septin combinations. An interaction between characteristic amino acids (those which are unique to given septin group) was identified for the complex formed between group III septins (including SEPT5) and group II septins (including SEPT8). This interaction, between Phe131 (group II) and Thr19 (group III) may explain the specificity in the formation of a G interface between septins of these groups during filament formation and furthermore the importance of GTP bound to the group II septin. These observations allow us to propose for the first time a plausible explanation for relevance of the loss of catalytic activity by this septin group, an unexplained fact up until now. Mutation of the identified residues resulted in a change in the elution profile of the complex from the size exclusion column suggesting structural alterations in the mutants.

Le cancer du sein est une cause importante de mortalité féminine en France et l’émergence de chimiorésistance, en particulier avec les taxanes, dont le paclitaxel (Taxol®), est une limite importante à l’emploi de ces molécules. Comme de nombreux anticancéreux, les taxanes ciblent les microtubules (MTs), des polymères de tubuline intervenant dans de nombreuses fonctions cellulaires. Ces derniers alternent entre des phases de croissance et de désassemblage, leur conférant ainsi un caractère dynamique. En se liant à la beta-tubuline, le Taxol stabilise les MTs et bloque la mitose, conduisant la cellule vers l’apoptose. La résistance au Taxol est un processus multifactoriel impliquant des mécanismes tels que la surexpression de pompes d’efflux (P-glycoprotéine), des mutations des gènes d’alpha- et de beta-tubuline ou une expression altérée de protéines associées aux MTs (MAPs) telles que MAP4 (stabilisatrice des MTs) ou la stathmine (dépolymérisante), contribuant ainsi à la restauration de la dynamique microtubulaire.Le projet repose sur l’emploi de la lignée tumorale mammaire MDA-MB 231 rendue résistante par paliers à 25nM de Taxol dans des conditions de blocage des pompes d’efflux. L’objectif étant d’explorer les variations protéiques de l’environnement des MTs associées au phénotype chimiorésistant, nous avons comparé le protéome d’extraits microtubulaires de cellules sensibles (Tv) et résistantes au Taxol (T8). Parmi les 112 protéines statistiquement enrichies dans une des deux populations de MTs, on retrouve notamment une augmentation des tubulines beta III et IV, une augmentation et une diminution respectives des moteurs moléculaires kinésine-1 et dynéine ainsi que l’enrichissement de plusieurs septines (SEPT2, 8, 9 et 11) dans les fractions de MTs T8. Les septines sont des GTPases associées en complexes hétéro-oligomériques impliquées dans la cytocinèse et l’organisation du cytosquelette de MTs et d’actine. Le recrutement de ces protéines candidates sur les MTs des cellules T8 a été validé par Western blot (Froidevaux-Klipfel et al., 2011) et immunofluorescence. Dans la littérature, les septines sont décrites comme étant localisées soit au niveau de l’actine soit au niveau des MTs dans les cellules de mammifères. De façon intéressante, dans nos cellules MDA-MB 231, nous observons un recrutement partiel de SEPT2 sur les fibres d’actine dans les cellules Tv alors qu’elle colocalise avec les MTs des cellules T8. Bien que la dépolymérisation de l’actine dans les cellules Tv n’entraine aucun déplacement des septines vers les MTs, cette localisation différentielle des septines sur les MTs des cellules résistantes pourrait néanmoins participer au phénotype chimiorésistant.Par ailleurs, il a été décrit dans la littérature que SEPT2 se lie aux MTs polyglutamylés afin de faciliter le transport vésiculaire à la membrane de protéines impliquées dans la polarisation cellulaire. La polyglutamylation est une modification post-traductionnelle permettant la formation de chaines latérales d’un à plusieurs résidus glutamate sur les tubulines alpha ou beta, régulant ainsi les interactions entre MTs et MAPs. Nos résultats montrent que l’accumulation des septines sur le réseau de MTs des cellules T8 s’accompagne d’une augmentation de la polyglutamylation mais aussi de la tyrosination de la tubuline. De plus, des tests de viabilité cellulaire ont mis en évidence que l’inhibition partielle par RNAi des septines ainsi que des polyglutamylases et de la tubuline tyrosine ligase, comme la surexpression d’enzymes responsables de la déglutamylation de la tubuline, permettent de restaurer une certaine sensibilité au Taxol des cellules T8. Inversement, la surexpression de certaines enzymes responsables de la polyglutamylation et de la tyrosination de la tubuline dans les cellules Tv permet d’instaurer une résistance au Taxol des cellules sensibles.La compilation de nos résultats permet de proposer un nouveau mécanisme de résistance au Taxol des cellules cancéreuses mammaires MDA-MB 231 : une augmentation du niveau de tyrosination de l’alpha-tubuline serait à l’origine de l’allongement des chaînes polyglutamylées sur le MT entraînant une diminution de la liaison de la protéine stabilisatrice MAP4 ainsi que le recrutement des septines sur les MTs. Ces modifications favoriseraient alors le recrutement du facteur de sauvetage CLIP-170 et de kinésines dépolymérisantes telles que MCAK à l’extrémité en croissance du MT des cellules résistantes T8, permettant une certaine restauration de la dynamique microtubulaire et contribuant ainsi à l’apparition du phénotype chimiorésistant.Ces études sont indispensables pour établir les bases d’un nouveau mécanisme de résistance au Taxol impliquant les septines, ainsi qu’un lien de causalité avec la tyrosination et la polyglutamylation. Au-delà, la recherche de ces modulations clés pourra être réalisée sur des cancers mammaires sensibles et résistants au Taxol, issus de biopsies de patients. Ainsi, il sera possible à terme, de déterminer si les septines, la tyrosination et la polyglutamylation de la tubuline ont une véritable importance fonctionnelle dans la résistance de cancers du sein au Taxol<br>Breast cancer remains the leading cause of women mortality in France, and chemoresistance emergence, in particular to taxanes, including paclitaxel (Taxol®), is an important limitation to the use of these molecules. As do many anticancer drugs, taxanes target microtubules (MTs), tubulin polymers involved in many cellular functions. These alternate between growing and shrinking stages, thus providing dynamic instability. By binding to beta-tubulin, Taxol stabilizes MTs and prevents successful mitosis, leading to apoptosis. Taxol resistance is a multifactorial process including mechanisms such as overexpression of drug efflux pumps (P-glycoprotein), mutations in the genes for alpha- and beta-tubulin or altered expression of MT-associated proteins (MAPs) like the MT-stabilizing MAP4 or the depolymerizing stathmin, therefore contributing to MT dynamics restoration.The current project relies on the use of the breast carcinoma cell line MDA-MB 231 made gradually resistant to 25nM of Taxol under blocking conditions of efflux pumps. To get a broader insight into the protein modifications from the MT environment associated with the chemoresistant phenotype, we compared the proteomic profiles of total MT fractions from Taxol-sensitive (Tv) and Taxol-resistant (T8) cells. Among the 112 differentially enriched proteins found in one of the two populations, we evidenced increased levels of betaIII and betaIV-tubulins, a slight increase and a decrease of molecular motors kinesin-1 and dynein, respectively, and the enrichment of several septins (SEPT2, 8, 9 and 11) in MT fractions of T8 cells. Septins are GTPases that associate into hetero-oligomeric complexes involved in cytokinesis and in MT and actin cytoskeleton organization. Recruitment of these candidate proteins on MTs of T8 cells has been validated by Western blot analysis (Froidevaux-Klipfel et al., 2011) and immunofluorescence experiments.In the literature, septins localize either on actin or MTs in mammalian cells. Interestingly, in our MDA-MB 231 cells, SEPT2 is recruited partially on actin stress fibers in sensitive Tv cells, whereas it colocalizes with MTs in T8 ones. Although actin depolymerization in Tv cells does not induce any shift towards MTs, this differential localization of septins on MTs of resistant cells could nevertheless participate in the chemoresistant phenotype.Furthermore, it has been described in the literature that SEPT2 binds to polyglutamylated MTs to facilitate vesicular transport to the plasma membrane of proteins implicated in cell polarization. Polyglutamylation is a post-translational modification allowing the formation of side-chains of several glutamate residues on alpha- or beta-tubulins, thus regulating interactions between MTs and MAPs. Our results show that septin accumulation on the MT network of T8 cells is associated with an increase in polyglutamylation but also tyrosination of tubulin. In addition, cell viability assays showed that partial inhibition by RNAi of septins as well as polyglutamylases and tubulin tyrosine-ligase, but also overexpression of enzymes responsible for tubulin deglutamylation, could restore Taxol sensitivity of T8 cells. By contrast, overexpression of enzymes responsible for tubulin polyglutamylation and tyrosination in Tv cells could induce Taxol resistance of sensitive Tv cells.The compilation of our results enables us to provide a new mechanism of Taxol resistance of the breast cancer cells MDA-MB 231: an increased level of alpha-tubulin tyrosination would induce the lengthening of polyglutamylated chains on the MT, resulting in a reduced binding of the stabilizing protein MAP4 as well as the recruitment of septins on MTs. These modifications would promote the recruitment of the rescue factor CLIP-170 and that of depolymerizing kinesins such as MCAK to the growing end of the MT of resistant T8 cells, leading to a restoration of MT dynamics, thus contributing to the emergence of the chemoresistant phenotype.These studies are essential to lay the basis for a new mechanism of Taxol resistance involving septins, and a causal relationship to tyrosination and polyglutamylation. Beyond, the search for these key modulations will be performed on Taxol sensitive and resistant breast cancers, from patients’ biopsies. Thus, it will be eventually possible to determine whether septins, tubulin tyrosination and polyglutamylation have a real functional importance in breast cancer resistance to Taxol

Le choc septique est la première cause de mortalité en réanimation. Des techniques extracorporelles de purification sanguine sont aujourd'hui proposées pour améliorer le pronostic de cette pathologie. Leur mode d'action est basé sur l'immunomodulation de la réponse inflammatoire systémique de l'hôte, obtenue principalement par épuration nonsélective des médiateurs de l'inflammation. Nous rapportons les résultats de différentes études in vitro, animales et cliniques ayant évalué les techniques de purification sanguine suivantes :hémofiltration à haut débit, hémofiltration en cascade, hémofiltration hautement adsorbante,filtration et adsorption couplée, hémoadsorption, et hémodialyse à haute perméabilité.Ce travail de recherche translationnelle montre que les techniques de purification sanguine sont non seulement capables d'épurer les médiateurs de l'inflammation mais aussi l'endotoxine pour l'hémofiltration hautement adsorbante et l'hémoadsorption. Nous démontrons par ailleurs la faisabilité technique, la sécurité d'application et les intérêts del'hémofiltration en cascade et de l'hémodialyse continue à haute perméabilité. Les effets hémodynamiques bénéfiques des techniques de purification sanguine sont également retrouvés. Pour les années à venir, il conviendra d'optimiser les techniques les plus performantes en tenant compte de leurs avantages et inconvénients respectifs. Sur le planphysiopathologique, l'effet plus direct de ces thérapies sur les leucocytes sera à approfondir. Il semble maintenant admis que convection, diffusion et adsorption ne doivent plus être opposés mais plutôt être considérés comme des mécanismes complémentaires

As septinas compõem o quarto componente do citoesqueleto das células eucarióticas, atrás da actina, miosina e filamentos intermediários. São proteínas filamentosas que se arranjam em forma de fibras e anéis, desempenhando um papel estrutural na célula. Os seres humanos expressam 13 septinas, que são divididas em 4 grupos diferentes de acordo com sua estrutura primária: grupo I (SEPT3, SEPT9, SEPT12); grupo II (SEPT6, SEPT8, SEPT10, SEPT11, SEPT14); grupo III (SEPT1, SEPT2, SEPT4, SEPT5) e grupo IV (SEPT7), sendo que SEPT13 foi caracterizada como um pseudogene de SEPT7. O filamento fisiológico mais bem estudado é composto por SEPT2-SEPT6-SEPT7-SEPT9 (nesta exata sequência), e é usado como a base para a descrição da formação canônica, onde se acredita que septinas do mesmo grupo ocupam o mesmo lugar no filamento. Entretanto, ensaios de duplo-híbrido identificaram muitas interações não canônicas inesperadas entre septinas como SEPT9-SEPT6 e SEPT9-SEPT8, sugerindo estes também possam existir in vivo. Além destes, estudos mostraram a existência de interações entre septinas do grupo I e grupo II, e especialmente no caso SEPT11-SEPT12, a interação deixa de existir ao inserir uma mutação sítio-dirigida na interface G destas proteínas. O presente trabalho investiga a interação entre SEPT3, uma septina do grupo I, com todas aquelas do grupo II. Esta interação foi estudada por análises de coexpressão e copurificação em resina de afinidade ao cobalto, onde apenas a SEPT3 possuía uma extensão de seis histidinas em seu N-terminal. Esta primeira análise mostrou que SEPT3 não foi copurificada com todos os membros do grupo II dando uma clara evidência de variação de afinidade dentro do grupo. Usando esta abordagem, SEPT6, SEPT10 e SEPT14 não mostraram interação com SEPT3, enquanto SEPT8 e SEPT11 copurificaram com SEPT3, mas não em concentrações estequiométricas. Para os complexos SEPT3-SEPT8 e SEPT3-SEPT11, uma segunda etapa de purificação foi realizada por meio de cromatografia de exclusão molecular, onde um pico de grande variância em relação à média indicou um valor de massa molecular entre monômeros e dímeros. Os mesmos, quando avaliados por espalhamento de luz a múltiplos ângulos mostraram variação na massa molecular ao longo do pico de eluição conforme ele era eluído. Tal variação era compatível com a eluição de dímeros no início até monômeros no final. Os estudos da interação entre SEPT3-SEPT8 por ultracentrifugação analítica indicou uma tendência de associação em altas concentrações das proteínas, compatível com a constante de dissociação determinada por termoforese em microescala, na ordem de dezenas de micromolar. Tais resultados levantaram questões acerca da relevância fisiológica destes complexos e reforçam a importância de um estudo mais aprofundado na formação dos complexos não canônicos de septinas para o desenvolvimento celular.<br>The septins are accepted to be the fourth cytoskeleton component of the eukaryotic cells, after actin, myosin and intermediate filaments. They are filament forming proteins that are organized in fibers and rings, having a structural role in the cell. Humans express 13 septins, which are divided into 4 different groups according to their primary structure: group I (SEPT3, SEPT9, SEPT12); group II (SEPT6, SEPT8, SEPT10, SEPT11, SEPT14); group III (SEPT1, SEPT2, SEPT4, SEPT5) e group IV (SEPT7). SEPT13 was later characterized as a SEPT7 pseudogene. The best characterized filament is built up from SEPT2-SEPT6-SEPT7-SEPT9 (in this exact sequence), and is used as a basis for the description of the so-called canonical arrangement, which accepts that septins from the same group can occupy the same position within the filament. However yeast two-hybrid assays identified several unexpected interactions such as SEPT9-SEPT6 and SEPT9-SEPT8, raising the possibility that these could also exist in vivo. Furthermore, studies have shown the existence of interactions between group I and group II, and especially in the SEPT11-SEPT12, the interaction dissolves when a mutation in the G interface is inserted. The present work investigates the interaction between SEPT3, a group I septin, with all of those from group II. This interaction was studied through co-expression and co-purification methods using metal affinity chromatography, where only the SEPT3 contained the six histidines extention. This initial analysis showed that SEPT3 did not co-purify with all group II members, clearly pointing to variability in the affinity within group. Using this approach SEPT6, SEPT10 e SEPT14 showed no interaction with SEPT3, whilst SEPT8 and SEPT11 co-purified with SEPT3, but not in stoichiometric concentrations. For the SEPT3-SEPT8 and SEPT3-SEPT11 complexes, a second purification stage was performed using size exclusion chromatography, where a broad peak was observed corresponding to a molecular mass value which was intermediate between a dimer and a monomer. The same complexes, when evaluated by multiple angle light scattering revealed a variation in the molecular mass across the peak as it eluted. Such variation was compatible with elution of dimers at the beginning and monomers at the end. Studies for the SEPT3-SEPT8 interaction via analytical ultracentrifugation suggested a trend to associate in high protein concentration, consistent with the dissociation constant found by microscale thermophoresis, which was of the order of ten micromolar. The results raise questions concerning the physiological relevance of these complexes and reinforce the importance of further studies on the non-canonical assembly of septin complexes for cellular development.

La protéine TAT1 (Testis Anion Transporter 1 ; SLC26A8) appartient à la famille des SLC26, une famille de transporteurs d’anions qui contribuent dans différents épithelia à l’homéostasie cellulaire. La protéine TAT1 s’exprime exclusivement dans les cellules germinales mâles, chez l’homme et chez la souris. Sur le spermatozoïde mature, la protéine TAT1 est localisée à la jonction des pièces intermédiaire (PI) et principale (PP) du flagelle, au niveau de l’annulus, une structure en forme d’anneau composée de différents polymères de Septines (1, 4, 6, 7 et 12).Le modèle murin d’invalidation du gène Tat1 présente une infertilité mâle par asthénozoospermie totale (absence de mobilité des spermatozoïdes) et des défauts de capacitation associés à des anomalies structurales du flagelle (plicature du flagelle, disjonction entre la PI et la PP, atrophie de l’annulus). Ce modèle indique que la protéine TAT1 pourrait avoir un rôle structural dans le maintien de l’annulus et dans la mise en place du flagelle. Par ailleurs, la protéine TAT1 possédant une activité de transport d’anions, il est vraisemblable qu’elle puisse influer directement sur la régulation de la mobilité et de la capacitation puisqu’il est bien établi que les échanges ioniques sont essentiels au contrôle de ces deux processus.En effet, les ions chlorure, bicarbonate et calcium participent à l’activation de la voie de signalisation AMPc/PKA, au cours des processus de mobilité et de capacitation (i.e. processus de maturation ayant lieu dans le tractus génital féminin et conférant au spermatozoïde un mouvement hyperactivé et la capacité à interagir avec l’ovocyte).Plusieurs travaux ont montré une interaction physique et fonctionnelle des membres de la famille SLC26 avec le canal chlorure/bicarbonate CFTR (Cystic Fibrosis Transmembrane conductance Regulator) dont les mutations sont responsables de la mucoviscidose. De manière intéressante des données récentes ont montré l’expression de CFTR dans le spermatozoïde et son rôle dans la régulation des flux de chlorure au cours de la capacitation. Au cours de ma thèse, nous avons testé la coopération entre les protéines TAT1 et CFTR ; nous avons pu montrer que la protéine TAT1 est capable d’interagir physiquement avec CFTR et de stimuler son activité de transport d’anions, suggérant qu’in vivo les deux protéines forment un complexe moléculaire impliqué dans la régulation des flux de chlorure et de bicarbonate dans le spermatozoïde.Tout comme TAT1, plusieurs membres de la famille SLC26 ont une expression tissulaire spécifique. Par ailleurs, les mutations génétiques de certains SLC26 sont associées à des pathologies humaines (surdité, diarrhée chlorurée congénitale et chondrodysplasie). De par le phénotype du modèle murin Tat1 et l’importance des SLC26 en pathologie humaine, TAT1 constitue un bon candidat dans la recherche des causes génétiques des asthénozoospermies humaines.Le laboratoire a mis en place au cours de ma thèse, un projet de recherche de mutations du gène TAT1 dans les asthénozoospermies humaines. Le séquençage des régions codantes du gène TAT1 dans une cohorte de 147 hommes infertiles par asthénozoospermie a ainsi permis d’identifier des variations de séquence inédites du gène chez 7 sujets. L’étude in vitro de certains variants indique pour trois d’entre eux une instabilité des formes mutantes associée à un défaut de stimulation du canal CFTR, in vitro. Par ailleurs, les spermatozoïdes de ces patients présentent d’importantes anomalies flagellaires dans la mise en place de la pièce intermédiaire, compatible avec un rôle de la protéine TAT1 et de ses partenaires (les septines) dans la genèse du flagelle<br>TAT1 (Testis Anion Transporter 1 ; SLC26A8) belongs to the SLC26 family of anion transporters, which is implicated in cellular homeostasis of different epithelia. TAT1 is exclusively expressed in male germ cells, in human and mouse. On mature spermatozoa, TAT1 is located at the annulus, a ring-shaped structure composed of different septins polymers (1, 4, 6, 7 and 12), at the junction of the midpiece (MP) and principal piece (PP) of the flagellum.The knock-out mouse model of Tat1 gene shows a male infertility by complete asthenozoospermia (lack of sperm motility) and capacitation defects combined with flagellar structural abnormalities (flagella bending, MP and PP disjunction and atrophy of the annulus). This model suggests that the TAT1 protein could fulfill structural roles in the annulus and during flagellum biogenesis. Moreover TAT1 displayind an anion transport activity, it could also be implicated in the control of sperm motility and capacitation by regulating anions exchannges, which are well known to be essential for both processes.Indeed, chloride, bicarbonate and calcium ions are involved in the activation of the cAMP/PKA pathway, controlling sperm motility and capacitation processes (i.e. maturation events occuring in the female genital tract and providing the spermatozoa an hyperactivation movement and the ability to interact with oocyte).Several publications have reported a physical and functionnal interaction between SLC26 family members and the chloride/bicarbonate CFTR channel (Cystic Fibrosis Transmembrane conductance Regulator), which mutations are responsible of cystic fibrosis. Interestingly, recent data showed CFTR expression in spermatozoa and its role in the regulation of chloride fluxes during capacitation. During my thesis, we tested TAT1 and CFTR cooperation; we showed that TAT1 can interact physically with CFTR and stimulate its anion transport activity, suggesting that in vivo they form a molecular complex involved in the regulation of chloride and bicarbonate fluxes during sperm capacitation.Like TAT1, several SLC26 family members have a tissue specific expression. Furthermore genetic mutations in several SLC26 members result in human pathology such as deafness, congenital chloride diarrhea and chondrodysplasia. According to the phenotype of the KO Tat1 mouse model and the role of SLC26 members in human pathology, TAT1 constitutes a good candidate for the search of genetic causes of human asthenozoospermia.During my thesis, the laboratory has set up, a research project aiming at identifying mutations in the TAT1 gene that are responsible for human asthenozoospermia.Sequencing of the TAT1 gene coding regions in a cohort of 147 infertile men presenting with asthenozoospermia allowed us to identify several new sequence variations in in the TAT1 gene. In vitro study of these variants shows that 3 of them are associated with protein instability and abrogate CFTR stimulation. Besides, patients sperm show important flagellar abnormalities in the midpiece, consistent with a role of TAT1 and its partners (septins) in flagellum biogenesis

L’intérêt des faibles doses d’hydrocortisone (HC) et de fludrocortisone (FC) dans le traitement du choc septique est aujourd’hui débattu. Nous avons évalué les effets biologiques de l’association HC+FC dans le choc septique, et les effets biologiques et hémodynamiques des deux molécules, administrées seules ou en association, chez le volontaire sain et chez le rat normal et en choc endotoxinique. Chez les malades en choc septique, l’association HC+FC a exercé les effets biologiques gluco- et minéralo-corticoïdes attendus parallèlement à leur effet bénéfique sur la mortalité. Chez le volontaire sain, en administration unique et aux doses utilisées dans le choc septique, l’HC a entraîné un effet minéralo-corticoïde plus marqué et plus précoce que la FC et a induit des effets hémodynamiques transitoires non retrouvés avec la FC, et l’HC et la FC ont diminué la réponse pressive à la phényléphrine avec un effet additif. Chez le rat, la FC et l’HC ont augmenté la pression artérielle, cet effet étant plus marqué chez les rats en choc endotoxinique que chez les rats normaux, et ont modifié la réactivité vasculaire d’anneaux d’artère mésentérique à la phényléphrine, le sens et l’amplitude de ces effets dépendant de la dose et des conditions expérimentales (animaux normaux ou en choc). La FC a montré qu’elle pouvait induire des effets biologiques et sur la réactivité vasculaire, chez le volontaire sain et le rat normal ou en choc endotoxinique, similaires à ceux de l’HC. Ces effets dépendent de la dose utilisée et des conditions expérimentales. Une recherche de la dose optimale à utiliser dans le choc septique semble aujourd’hui nécessaire avant de réévaluer son efficacité<br>The interest of low doses of hydrocortisone (HC) and fludrocortisone (FC) in the treatment of septic shock is controversial. We investigated the biological effects of the combination of HC+FC in septic shock, and the biological and hemodynamic effects of the two drugs, given alone or in combination, in healthy volunteers and in normal and endotoxemic rats. In septic shock patients, HC+FC induced the expected gluco- and mineralo-corticoid effects and these effects were observed simultaneously to the improvement of patient’s outcome. In healthy volunteers, using the same single doses than those used in septic shock, HC induced a more marked and earlier mineralo-corticoid effect than FC and induced transient hemodynamic effects whereas FC did not, and HC and FC decreased the pressor response to phenylephrine with additive effects. In rats, FC and HC increased blood pressure, this effect being more marked in endotoxemic than in normal rats, and modified the contractile response of mesenteric artery rings to phenylephrine, the direction and magnitude of these effects depending on the dose and experimental conditions (normal or endotoxemic animals). Our studies showed that FC could induce biological effects and modify vascular reactivity, in healthy volunteers and in normal and endotoxemic rats, in a similar way than HC. These effects depend on the dose and experimental conditions studied. Defining the optimal dose to be used in septic shock patients seems necessary before re-assessing its efficacy

Twelve commercial vaccines against clostridiosis were evaluated having in their composition Clostridium septicum toxoids and/or bacterial cells, have been evaluated for potency by mice serum neutralization tests using rabbit or bovine sera and challenge test in guinea-pig. The vaccines, coded as T1, T10 and T11, elicited alpha antitoxin titers in rabbits which were superior to the minimum test level of 2.5 lU/mL, as recommended for quality control, and the vaccines T2 and T4 eliciting titers of 2.0 and 2.5 lU/mL. Similar results were obtained as detected in bovine sera against vaccines T1, T2, T4 and T10. The T11 vaccine was not evaluated in bovines. According to the challenge tests in guinea pigs no vaccine met the minimum requeriments for approval. Clostridium septicum vaccines were in general inefficient for stimulating the immune response as evaluated by the recomendaded tests.<br>Foram avaliadas quanto à eficiência 12 vacinas comerciais contra clostridioses, que continham toxóides e/ou bacterinas de Clostridium septicum, pelo teste de soroneutralização em camundongos a partir de soros de coelhos e bovinos vacinados e pelo teste de desafio direto em cobaias. As vacinas codificadas como T1, T 10 e T11 apresentaram, em coelhos, títulos de antitoxina alfa superiores ao nível mínimo de teste de 2,5 Ul/mL recomendado pelo controle deste produto e as vacinas T2 e T4 títulos de 2.0 e 2.5 Ul/mL. Resultados semelhantes foram obtidos nos soros de bovinos em relação às vacinas T1, T2, T4 e T10. A vacina T11 não foi testada em bovinos. Pelo método do desafio direto em cobaias, nenhuma vacina atendeu aos requisitos mínimos. As vacinas contra Clostridium septicum, em sua maioria, foram ineficientes em estimular resposta compatível com níveis de teste.

Les septines constituent une nouvelle classe de protéines du cytosquelette. Les septines de levure s’auto-assemblent en filaments non-polaires liés à la face interne de la membrane plasmique à travers des interactions spécifiques avec le L- α-phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2. Les septines sont localisées au niveau de sites de constriction durant la cytokinèse et ont un impact sur le remodelage de la membrane. Nous avons utilisé une plage d’outils biomimétiques in vitro pour examiner comment les septines de levure se comportent sur des membranes courbées et déformables. Des tests in vitro utilisant des Vesicules Unilamellaires Géantes (GUVs) sont des outils pertinents pour obtenir des informations sur l’interaction protéine-lipide, la mécanique de membrane et la sensibilité à la courbure. Obtenir des GUVs dopées au PI(4,5)P2 peut être difficile. Nous avons d’abord optimisé l’incorporation de PI(4,5)P2 dans des GUVs en étudiant l’interaction entre les septines et les GUVs. Nous montrons que cette interaction est plus spécifique qu’en utilisant un rapporteur usuel (phospholipase C1). Nous avons montré que l’électro-formation sur fils de platines est la méthode la plus appropriée pour atteindre une interaction septines- lipides optimale. De plus, nous avons montré que les GUVs contenant du PI(4,5)P2 doivent être utilisées quelques heures après leur préparation. En effet, avec le temps, le PI(4,5)P2 est éjecté de la membrane des GUVs et la concentration de PI(4,5)P2 dans la bicouche diminue. Ensuite, nous avons analysé comment les septins peuvent contrôler les propriétés mécaniques de la membrane et analysé comment les déformations de la membrane pouvaient être induites par la sensibilité des septines à des courbures spécifiques. En effet, nous avons montré que les septines reforment la membrane de GUVs avec l’apparition de piques périodiques. Nous avons montré que ces déformations sont associées à la préférence de courbure des filaments de septine. En se liant à des bicouches supportées posées sur des substrats possédant un motif ondulé présentant des courbures positives et négatives, les filaments de septine restent droits et perpendiculares à la courbure aux endroits convexes et se plient négativement pour suivre les géométries concaves. En se basant sur ces résultats, nous proposons un modèle théorique qui décrit quantitativement les déformations et la sensibilité à la courbure micrométrique observée in vitro. Le modèle capture la réorganisation des filaments de septines durant la cytokinèse in vivo, fournissant des informations sur la mécanique de la division cellulaire<br>Septins constitute a novel class of cytoskeletal proteins. Budding yeast septins self-assemble into non-polar filaments bound to the inner plasma membrane through specific interactions with L- α-phosphatidylinositol-4,5- bisphosphate (PI(4,5)P2). Septins localize at constriction sites during cytokinesis and impact membrane remodeling processes. We have analyzed a range of in vitro biomimetic tools to examine how yeast septins behave on curved and deformable membranes. In vitro assays using Giant Unilamellar Vesicles (GUVs) are relevant tools to reveal insights in proteins-lipids interactions, membrane mechanics and curvature sensitivity. GUVs doped with PI(4,5)P2 are challenging to prepare. We first optimized the incorporation of PI(4,5)P2 lipids into GUVs by probing the proteins-PI(4,5)P2 GUVs interactions. We show that the interaction between budding yeast septins and PI(4,5)P2 is more specific than using usual reporters (phospholipase C1). We have shown that electro-formation on platinum wires is the most appropriate method to achieve an optimal septin-lipid interaction. Besides, we have shown that PI(4,5)P2 GUVs have to be used within a few hours after their preparation. Indeed, over time, PI(4,5)P2 is expelled from the GUV membrane and the PI(4,5)P2 concentration in the bilayer decreases. Next, we analyzed how septins can control the mechanical properties of membranes and analyzed how membrane deformations could be induced by a specific curvature sensitivity of septins. Indeed, we have shown that septins reshape the membranes of Giant Unilamellar Vesicles with the formation of periodic spikes. We have shown that membrane deformations are associated to septin filament curvature arrangement preferences. When binding to bilayers supported on custom-designed periodic wavy patterns displaying positive and negative micrometric radii of curvatures, septin filaments remain straight and perpendicular to the curvature of the convex parts, while bending negatively to follow concave geometries. Based on these results, we propose a theoretical model that quantitatively describes the deformations and micrometric curvature sensitivity observed in vitro. The model captures the reorganizations of septin filaments throughout cytokinesis in vivo, providing mechanistic insights into cell division

Ce travail visait à étudier l’activation des polynucléaires neutrophiles (PNNs) au cours du choc septique. Après une introduction sur la physiopathologie du choc septique, de la coagulation intravasculaire disséminée (CIVD) et des PNNs, nous rapportons différents résultats expérimentaux et cliniques. Dans une étude prospective sur 100 patients, nous avons montré que l’activation des PNNs, mesurée par leur fluorescence en cytométrie en flux pourrait représenter un marqueur diagnostique original de la CIVD du choc septique. Ensuite, nous avons mis en évidence un des mécanismes de l’activation des PNNs, la NETose, à l’aide de marqueurs sériques. Afin de confirmer la relevance de ce phénomène, nous avons visualisé les NETs spécifiquement dans le sang de patients en choc septique avec CIVD en immunofluorescence. Enfin, nous avons identifié des protéines associées à l’activation du PNN dans la CIVD du choc septique par une étude protéomique, afin d’en envisager les mécanismes. Le PNN pourrait ainsi être un acteur incontournable de la dérégulation de l’immunothrombose au cours de la CIVD, et être envisagé comme cible thérapeutique<br>This PhD thesis focused on the activation of polymorphonuclear granulocytes (PMNs) during septic shock. After an introduction including an overview on pathophysiology of septic shock, disseminated intravascular coagulation (DIC) and PMNs, original experimental and clinical data are reported. In a prospective study, we enrolled 100 patients and showed that neutrophils’ activation measured by neutrophil fluorescence using flow cytometry could represent an original biomarker of septic shock-induced DIC. Furthermore, we highlighted a mechanism of neutrophil’s activation: NETosis, through assessment of indirect serum’s markers associated specifically to DIC during septic shock. In order to confirm the relevance of this result, we reported a direct visualization of circulating NETs in blood of DIC-patients using immunofluorescence. Finally, we performed a proteomic study and identified some proteins involved in neutrophils’ activation during septic shock-induced DIC. Thus, PMN could represent an important actor of immunothrombosis’ deregulation during DIC, and could be considered as a potential therapeutic target

Cette thèse a permis de repréciser le pronostic actuel des malades cirrhotiques en réanimation, en particulier ceux en choc septique. Nous avons démontré que ce pronostic s’est amélioré, y compris pour les malades en choc septique sous ventilation mécanique ou dialysés, les malades cirrhotiques ayant bénéficié des progrès de la réanimation constatés chez les malades de réanimation sans cirrhose. Nous avons également retrouvé que le pronostic des malades cirrhotiques en réanimation était beaucoup plus en rapport avec les défaillances d’organes aiguës qu’avec la gravité de la cirrhose sous-jacente. La revue de la littérature que nous avons effectuée retrouve des taux de mortalité, rapportés par les études les plus récentes, comparables aux nôtres. La plupart de ces études confirment que les scores de défaillance d’organes prédisent mieux le pronostic que les scores généraux de réanimation et bien mieux que les scores spécifiques de cirrhose calculés le jour de l’admission. Nous avons également retrouvé que, parmi les défaillances d’organes, la défaillance de la coagulation évaluée par le taux de plaquettes par le score SOFA, n’a pas d’impact sur le pronostic des malades cirrhotiques en réanimation. De plus, ces défaillances d’organes prédisent mieux le pronostic lorsqu’elles sont évaluées au 3ème jour de réanimation. Concernant les malades en choc septique, nous avons retrouvé que les marbrures et la baisse de la StO2 au niveau du genou apparaissent plus tardivement chez les malades cirrhotiques qui vont décéder. Ces signes prédisent dès la 6ème heure la mortalité au 14ème jour avec une excellente spécificité mais une sensibilité faible<br>In this manuscript, we reassessed the actual outcome of cirrhotic patients admitted to intensive care units, in particular in those with septic shock. We found that this outcome dramatically improved, including for patients with septic shock, placed under mechanical ventilation or receiving renal replacement therapy. We identified that the organ failures scores better predict the outcome than the liver-specific scores. We performed a review of literature; the most recent studies are in accordance with our results in term of mortality rates and in term of accuracy of the different scores to predict the outcome in these particular patients. We also identified that, among the different organ failures, the coagulation failure, defined by the platelet level according to the SOFA score, was not associated with a worse outcome. Moreover, the organ failures better predict the outcome when assessed 3 days after the admission to the intensive care unit. Regarding cirrhotic patients with septic shock, we demonstrated that mottling and the decrease in StO2 in the knee area appear later in patients with cirrhosis than in patients without. Those signs assessed 6 hours after the admission, predict the mortality at day 14 with an excellent specificity, but a relatively low sensibility

Septins are highly conserved components of the cytoskeleton found in animals and fungi. They play a variety of roles in key cellular processes including cell division, cell migration and membrane trafficking. During host-pathogen interactions, septins inhibit bacterial infection by forming cage-like structures around pathogens such as Shigella. In addition, two recent genome-wide RNAi screens demonstrated that septins play an undefined role during vaccinia virus replication. Utilizing cell-based assays and microscopy I set out to determine the role of septins in vaccinia infected cells. I found that septins are recruited to vaccinia virus immediately following its fusion with the plasma membrane during viral egress. Live cell imaging reveals that septins are lost from beneath the virus once the virus stimulates Arp2/3 complex-dependent actin polymerization to enhance its cell-to-cell spread. Virus-induced actin polymerization involves the phosphorylation of the viral protein A36, leading to the recruitment of Cdc42, Nck, Grb2, WIP and N-WASP, which activate the Arp2/3 complex. Chemical or genetic inhibition of A36 phosphorylation dramatically increases the number of virus particles co-localizing with septins. Further experiments demonstrate that the recruitment of Nck and subsequently dynamin, but not Grb2, WIP:N-WASP or the Arp2/3-complex, promote the loss of septins from virions. RNAi-mediated depletion of septins increases virus release, accelerates cell-to-cell spread, and induces more robust actin tails. Collectively, my results demonstrate that septins limit the spread of vaccinia infection in cell monolayers and the recruitment of dynamin downstream of Nck enables the virus to overcome septin-mediated restriction. This is the first example of septins having an anti-viral effect and my work identifies a new role for septins in host defence.

En dépit de l'amélioration des thérapeutiques anti-infectieuses, le choc infectieux ou septique s'accompagne d'une mortalité extrêmement élevée. Dans l'évolution de la maladie, l'apparition d'une dysfonction myocardique est un élément de mauvais pronostic. Celle-ci est habituellement attribuée aux effets délétères des différents médiateurs libérés lors de la réponse inflammatoire. Les processus d'apoptose sont également impliqués dans la physiopathologie de la dysfonction myocardique septique. Dans un premier temps, notre travail a consisté à caractériser in vivo et in vitro un modèle de défaillance myocardique septique. Le modèle in vivo a permis de vérifier l'intensité de la réponse inflammatoire systémique et myocardique par l'étude des voies d'activation cellulaire NFkB dépendantes. Le modèle in vitro a permis d'établir le lien fonctionnel entre l'activation du processus apoptotique induit par des molécules pro-apoptotiques telles que la sphingosine et l'apparition d'une anomalie contractile de la cellule cardiaque. Ces résultats nous ont conduits à démontrer par quels mécanismes le processus apoptotique pouvait altérer la fonction contractile. Nos travaux ont montré que les processus enzymatiques et mitochondriaux d'apoptose sont rapidement activés mais que l'activation des caspases effectrices, enzymes protéolytiques, ne conduit pas à la dégradation nucléaire terminale et à la mort cellulaire. En revanche, les caspases effectrices, à l'origine d'une désorganisation des sarcomères et d'une perturbation de l'homéostasie calcique, sont directement responsables de la défaillance cardiaque. Ainsi, les connaissances fondamentales acquises au cours de ce travail de thèse seront directement valorisables par la mise au point de nouvelles stratégies thérapeutiques du choc septique humain<br>Despite numerous advances in medical treatment, infectious shock also called septic shock is associated with an extremely high mortality rate. Sepsis-induced myocardial dysfunction is greatly associated with a fatal outcome. Most of the sepsis-induced myocardial dysfunction has been attributed to the deleterious effects of mediators released during the inflammatory response. Apoptosis processes are also involved in the pathogenesis of sepsis-induced myocardial dysfunction. The first aim of our study was to characterize both an in vivo and an in vivo model of sepsis-induced myocardial dysfunction. First, in the in vivo model, we checked that NFkB-dependent pathways, which represent a hallmark of the inflammatory response, were activated. In the in vitro model, the use of sphingosine, a pro-apoptotic molecule, unravelled a possible link between apoptotic pathway activation and cardiac cell contractile dysfunction. These results required further studies in order to explain how apoptotic processes may lead to myocardial dysfunction. We showed that mitochondria and apoptotic proteases are rapidly activated. Interestingly, although effector caspases are not associated with terminal nuclear degradation and cell death these proteolytical enzymes lead to sarcomeric destruction and calcium homeostasis alterations. Such modifications are responsible for contractile dysfunction Thus, these new fundamental mechanisms could be useful to develop new therapeutic strategies in the human septic shock

As proteínas pertencentes à família das septinas foram originalmente descobertas em 1971 em decorrencia de estudos genéticos em células mutantes. Essas proteínas encontradas em fungos e animais, mas não em plantas apresentam como principais características a presença de um domínio conservado de ligação aos nucleotídeos de guanina (GTP) e a formação de filamentos homo- e hetero-oligoméricos, que são estruturas altamente organizadas. Estudos filogenéticos e moleculares em humanos identificaram 14 septinas que são divididas em 4 grupos (I, II, III e IV). Estas moléculas associam-se com membranas celulares, actina, microtúbulos do citoesqueleto e estão envolvidas em inúmeros processos que ocorrem no córtex celular e requerem organização espacial, tais como: citocinese, ciclo celular, formação de barreiras de difusão, alinhamento de fuso. Alterações na expressão das septinas estão associadas a vários tipos de tumores e a doenças de Parkinson e Alzheimer. Neste trabalho, com o objetivo de obter informações estruturais e bioquímicas das septinas 7 e 9 humanas. Este projeto é parte de um esforço conjunto coordenado pelo Prof. Dr. Richard C. Garratt e conhecido informalmente como Septimoma. As construções recombinantes SEPT 7, SEPT 7G, e SEPT 9G foram expressas em Escherichia coli e as proteínas recombinantes obtidas. As análises em eletroforese SDS-Page e em gel nativo indicam que essas proteínas foram purificadas com sucesso. A atividade GTPase e o estado oligomérico na forma dimérica foram verificados. Estudos de dicroísmo circular e fluorescência determinaram que esses recombinantes são formados por uma mistura de estruturas secundárias &alfa; e &beta;, e também que o C e o N terminais aumentam a estabilidade das proteínas. Foram obtidos cristais da SEPT 7G e, por meio da técnica de raios-X, foi determinado um modelo tridimensional da proteína com resolução de 3,4o.<br>Proteins belonging to the septin family were originally discovered in 1971 through genetic studies of mutant cells. These proteins found in fungi and animals, but not in plants present, as their main characteristics, a conserved guanine nucleotide-binding domain (GTP) and they also form homo and hetero-oligomeric filaments that are highly organized structures. Phylogenetic and molecular studies in humans have identified 14 septins which are divided into 4 subfamilies (groups I, II, III and IV). These molecules associate with cell membranes, actin, cytoskeleton microtubules and they are related to a number of processes that take place in the cell cortex and that require spatial organization, such as cytokinesis, cell cycle, diffusion barrier formation and spindle alignment. Alterations in the expression of septins are associated with several types of tumors and with Parkinsons and Alzheimers diseases. In this work, with the goal of obtaining structural and biochemical information of human septins 7 and 9, the recombinants SEPT 7, SEPT 7G and SEPT 9G were expressed in E. coli. Analyses both in SDS-Page electroforesis and in native gel suggest that these proteins were purified successfully for they are soluble and homogeneous. GTpase activity has been verified in all of these recombinants, which shows that these proteins are present in native form and that additional molecules are not needed for this activity. It was possible to determine through different techniques such as molecular exclusion chromatography and SAXS that all the molecules in solution are grouped as dimeric form. Circular dichroism and fluorescence spectroscopic studies have determined both that such recombinants are formed by means of a mixture of &alfa; and &beta; secondary structures and that the C and N-terminals increase the stability of proteins. Protein stability studies under different pH and temperature conditions show that the raise of the latter produces a greater molecular aggregation. Measurements of fluorescence emissions have indicated that the SEPT 7, SEPT 7G and SEPT 9G form structures of amyloid-like filaments found in many septins. Crystal structures of SEPT 7G have been obtained and, by means of the X-ray technique, a 3-D model of the protein has been determined with a resolution of 3.4o. It has been possible to predict, with molecular modeling studies, regions formed by loops that showed low electronic density in the GTPase crystallographic model. Therefore, it has been possible to add more structural information to this domain and to form the complete polypeptide without cuts.