Quinases dependentes de ciclinas (CDKs) compreendem uma família de proteínas que podem ser subdivididas em dois grupos funcionais majoritários baseados na sua função no ciclo celular e/ou controle transcricional. Já foram identificadas mais de 30 CDKs humanas. A CDK10 é uma proteína quinase dependente de ciclina pertencente ao grupo de quinases relacionadas à Cdc2. CDK10 é essencial na fase G2/M do ciclo celular, possivelmente no progresso dessa fase, monitorando a replicação completa do DNA e permitindo que as células passem desse ponto de restrição. Essa proteína é um importante determinante de resistência à terapia endócrina para câncer de mama. Portanto, é um alvo potencial para o desenvolvimento de inibidores, uma vez que está presente em células cancerosas. O estudo da estrutura e função da CDK10 deverá ser realizado após sua clonagem, expressão e purificação. O cDNA da CDK10 foi amplificado por reação em cadeia da polimerase (PCR), e posteriormente, o produto foi aplicado em gel de agarose para análise e purificação. O vetor de clonagem recombinante foi obtido, o qual foi clonado em células competentes e sequenciado. A obtenção do plasmídeo recombinante para expressão deu-se pela inserção do DNA nos vetores de expressão pET28a(+) e pET23a(+) (Novagen). A proteína foi expressa em células competentes e analisada por eletroforese em gel de poliacrilamida. Em seguida, a proteína obtida foi purificada em coluna de afinidade por níquel e em coluna de afinidade por ATP. Com a otimização dos resultados obtidos, será possível, futuramente, caracterizar bioquimicamente a CDK10 e elucidar suas estruturas secundária e terciária. O estudo da CDK10 irá contribuir para o entendimento da sua relação estrutura-função e sua relação com oncogenes e supressores de tumor, e o estudo estrutural para o desenvolvimento de inibidores químicos de baixo peso molecular que possa inibir especificamente a CDK10. / Cyclin-dependent kinases (CDKs) comprise a family of proteins that can be subdivided into two major groups based on their functional role in cell cycle and / or transcriptional control. Over 30 human CDKs have been identifies. CDK10 is a cyclin-dependent kinase protein that belongs to the cdc2-related kinases group. CDK10 is essential in phase G2 / M of the cell cycle, possibly in the progress of this phase, monitoring the complete DNA replication and allowing the cells to pass through this restriction point. This protein is an important determinant of resistance to endocrine therapy for breast cancer. Therefore, it is a potential target for the development of inhibitors, since it is present in cancerous cells. The study of the structure and function of CDK10 must be performed after its cloning, expression and purification. cDNA of CDK10 was amplified by polymerase chain reaction (PCR) and then the product was applied into agarose gel for analysis and purification The cloning vector was obtained, which was cloned into competent cells and sequenced. The obtaining of the recombinant plasmid for expression was due to the insertion of DNA into expression vectors pET28a(+) and pET23a(+) (Novagen). The protein was expressed in competent cells and analyzed by electrophoresis on polyacrylamide gel. Then, the obtained protein was purified by nickel affinity column and ATP affinity column. With the optimization of the results obtained, it will be possible, in the future, to biochemically characterize CDK10 and elucidate its secondary and tertiary structures. The study of CDK10 will contribute to the understanding of its structure-function relationship and its relationship with oncogenes and tumor suppressors, and the structural study for the development of low molecular weight chemical inhibitors that can specifically inhibit CDK10. The structural studies will contribute to the development of chemical inhibitors of low molecular weight that may this and other CDKs.

Quinases dependentes de ciclinas (CDKs) compreendem uma família de proteínas que podem ser subdivididas em dois grupos funcionais majoritários baseados na sua função no ciclo celular e/ou controle transcricional. Já foram identificadas mais de 30 CDKs humanas. A CDK10 é uma proteína quinase dependente de ciclina pertencente ao grupo de quinases relacionadas à Cdc2. CDK10 é essencial na fase G2/M do ciclo celular, possivelmente no progresso dessa fase, monitorando a replicação completa do DNA e permitindo que as células passem desse ponto de restrição. Essa proteína é um importante determinante de resistência à terapia endócrina para câncer de mama. Portanto, é um alvo potencial para o desenvolvimento de inibidores, uma vez que está presente em células cancerosas. O estudo da estrutura e função da CDK10 deverá ser realizado após sua clonagem, expressão e purificação. O cDNA da CDK10 foi amplificado por reação em cadeia da polimerase (PCR), e posteriormente, o produto foi aplicado em gel de agarose para análise e purificação. O vetor de clonagem recombinante foi obtido, o qual foi clonado em células competentes e sequenciado. A obtenção do plasmídeo recombinante para expressão deu-se pela inserção do DNA nos vetores de expressão pET28a(+) e pET23a(+) (Novagen). A proteína foi expressa em células competentes e analisada por eletroforese em gel de poliacrilamida. Em seguida, a proteína obtida foi purificada em coluna de afinidade por níquel e em coluna de afinidade por ATP. Com a otimização dos resultados obtidos, será possível, futuramente, caracterizar bioquimicamente a CDK10 e elucidar suas estruturas secundária e terciária. O estudo da CDK10 irá contribuir para o entendimento da sua relação estrutura-função e sua relação com oncogenes e supressores de tumor, e o estudo estrutural para o desenvolvimento de inibidores químicos de baixo peso molecular que possa inibir especificamente a CDK10. / Cyclin-dependent kinases (CDKs) comprise a family of proteins that can be subdivided into two major groups based on their functional role in cell cycle and / or transcriptional control. Over 30 human CDKs have been identifies. CDK10 is a cyclin-dependent kinase protein that belongs to the cdc2-related kinases group. CDK10 is essential in phase G2 / M of the cell cycle, possibly in the progress of this phase, monitoring the complete DNA replication and allowing the cells to pass through this restriction point. This protein is an important determinant of resistance to endocrine therapy for breast cancer. Therefore, it is a potential target for the development of inhibitors, since it is present in cancerous cells. The study of the structure and function of CDK10 must be performed after its cloning, expression and purification. cDNA of CDK10 was amplified by polymerase chain reaction (PCR) and then the product was applied into agarose gel for analysis and purification The cloning vector was obtained, which was cloned into competent cells and sequenced. The obtaining of the recombinant plasmid for expression was due to the insertion of DNA into expression vectors pET28a(+) and pET23a(+) (Novagen). The protein was expressed in competent cells and analyzed by electrophoresis on polyacrylamide gel. Then, the obtained protein was purified by nickel affinity column and ATP affinity column. With the optimization of the results obtained, it will be possible, in the future, to biochemically characterize CDK10 and elucidate its secondary and tertiary structures. The study of CDK10 will contribute to the understanding of its structure-function relationship and its relationship with oncogenes and tumor suppressors, and the structural study for the development of low molecular weight chemical inhibitors that can specifically inhibit CDK10. The structural studies will contribute to the development of chemical inhibitors of low molecular weight that may this and other CDKs.

No description available.

Our laboratory delineated a role for alternative pre-mRNA splicing (AS) in triple negative breast cancer (TNBC). We found the translational regulator cytosolic polyadenylation element binding protein 2 (CPEB2) which has two isoforms, CPEB2A and CPEB2B, is alternatively spliced during acquisition of anoikis resistance (AnR) and metastasis. The splicing event which determines the CPEB2 isoform is via inclusion/ exclusion of exon four in the mature mRNA transcript. The loss of CPEB2A with a concomitant increase in CPEB2B is required for TNBC cells to metastasize in vivo. We examined RNAseq profiles of TNBC cells which had CPEB2 isoforms specifically downregulated to examine the mechanism by which CPEB2 isoforms mediate opposing effects on cancer-related phenotypes. Downregulation of the CPEB2B isoform inhibited pathways driving the epithelial-to-mesenchymal transition (EMT) and hypoxic response, whereas downregulation of the CPEB2A isoform did not have this effect. Specifically, CPEB2B functioned as a translational activator of TWIST1 and HIF1a. Functional studies showed that specific downregulation of either HIF1α or TWIST1 inhibited the ability of CPEB2B to induce AnR and drive metastasis. The mechanism governing inclusion/ exclusion of exon 4 was determined to be serine/ arginine-rich splicing factor 3 (SRSF3). Binding of SRSF3 to a consensus sequence within CPEB2 exon 4 promoted its inclusion in the mature mRNA, and mutation of this sequence abolished association of SRSF3 with exon 4. SRSF3 expression was upregulated in TNBC cells upon acquisition of AnR correlating with a reduction in the CPEB2A/B ratio. Importantly, downregulation of SRSF3 by siRNA in these cells induced the exclusion of exon 4. Downregulation of SRSF3 also reversed the CPEB2A/B ratio in a wild-type CPEB2 exon 4 minigene construct, but not a mutant CPEB2 minigene with the SRSF3 RNA cis-element ablated. Physiologic studies demonstrated SRSF3 downregulation ablated AnR in TNBC cells, and was “rescued” by ectopic expression of CPEB2B. Importantly, biostatistical analysis of The Cancer Genome Atlas database showed a positive relationship between alterations in SRSF3 expression and lower overall survival in TNBC. Overall, this study demonstrates that SRSF3 modulates CPEB2 AS to induce the expression of the CPEB2B isoform that drives TNBC phenotypes correlating with aggressive human breast cancer.

Le récepteur de la vitamine D (VDR) est un facteur de transcription activé par la forme active de la vitamine D3. VDR est une cible thérapeutique potentielle pour de multiples pathologies telles que les maladies auto-immunes et neurodégénératives et certains cancers. VDR module l’expression de gènes par le recrutement sélectif de corégulateurs. Les données structurales disponibles à ce jour pour des complexes de récepteur nucléaire-corégulateurs sont très limitées. Cette étude se focalise sur l’architecture du complexe formé par VDR et un grand fragment du coactivateur MED1, une sous-unité du complexe Médiateur qui fait le lien entre les récepteurs nucléaires et la machinerie basale de transcription. Les résultats obtenus nous sont permis de caractériser l'interaction du récepteur avec le coactivateur et de révéler l'architecture globale du complexe. Ce travail fournit une base solide pour la détermination structurale d’autres complexes impliqués dans le contrôle de la transcription. / The vitamin D nuclear receptor (VDR) is a transcription factor activated by the biologically active form of vitamin D3. VDR is a potential candidate to treat neurodegenerative and autoimmune disorders, and cancer. VDR modulates the expression of vitamin D3-regulated genes by selective recruitment of coregulators of transcription which are, in turn, attractive targets in epigenetic-oriented drug discovery. Available structural data for receptor-coregulator complexes are limited; investigation of such complexes is highly important. The present work focuses on the architecture of the complex between VDR and a large part of the coactivator MED1, a subunit of the Mediator complex linking nuclear receptors to the basal transcription machinery. Obtained results revealed important details of the interaction, as well as the overall organization of the complex. This work provides a solid background for the structural investigation of similar complexes involved in the transcriptional control.

Orientador: Michel Georges Albert Vincentz / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T12:39:15Z (GMT). No. of bitstreams: 1 Duarte_GustavoTurqueto_D.pdf: 11219627 bytes, checksum: b885f7a08525b694c5783c91f122c6c1 (MD5) Previous issue date: 2012 / Resumo: As plantas, sendo organismos sésseis, desenvolveram um conjunto de mecanismos que possibilitam a adaptação a condições ambientais adversas visando à manutenção da homeostase energética para o desenvolvimento e propagação. Tais respostas valem-se da integração entre a biossíntese de hormônios, ativação de vias gênicas de resposta a estresse e um balanço adequado do uso da energia disponível. Os açúcares, além de serem fontes de carbono e energia, também atuam como moléculas sinalizadoras podendo agir conjuntamente com sinais hormonais na adaptação a estresses bióticos e abióticos e no controle do desenvolvimento. Nesse contexto, diversos estudos apontam para uma importante relação entre o ácido abscísico (ABA), um dos principais hormônios relacionados à resposta a estresses, e a glicose. A sinalização por ABA, além de atuar sobre a regulação da transcrição, é conhecida por envolver fatores de controle de estabilidade do RNAm. Contudo, a participação destes mecanismos em respostas mediadas por glicose ainda é pouco explorada. Num primeiro momento, o presente trabalho visou avaliar o potencial das participações de regulações pós-transcricionais em resposta a ABA e glicose em Arabidopsis thaliana, através da determinação do perfil de expressão de RNAm após a inibição da transcrição. Um modelo experimental com condições de inibição de transcrição otimizadas foi estabelecido e utilizado para análise de transcriptoma por microarranjos CATMA em resposta à glicose e ABA. Um total de 962 genes foi identificado como diferencialmente expresso após os tratamentos, sugerindo uma possível regulação pós-transcricional por glicose sobre 204 transcritos, por ABA sobre 245 e pela combinação dos dois sinais sobre 513 transcritos. Esses genes foram classificados de acordo com o Gene Ontology, sugerindo uma relação importante com respostas adaptativas a condições de estresse. Aparentemente, as respostas mediadas por glicose e ABA seguem estratégias opostas, sendo que as respostas pós-transcricionais por ABA podem também atuar como um mecanismo rápido de retro-regulação negativa sobre a via central de sinalização desse hormônio, uma forma de dessensibilizar e reiniciar as respostas da via. Na segunda parte deste trabalho, levando em consideração as evidências do envolvimento do controle de estabilidade de RNAm na sinalização por glicose, foi avaliada a participação da via de regulação por microRNAs (miRNAs) em respostas mediadas por esses sinal durante os estágios iniciais de desenvolvimento da planta. Os mutantes ago1-25 e hyl1-2, deficientes em atividade e biossíntese de miRNAs, respectivamente, apresentaram hipossensibilidade à glicosedurante um determinado período do desenvolvimento da planta, entre a germinação e o estabelecimento. Tal resultado levanta a possibilidade de que a via dos miRNAs participa do atraso do desenvolvimento mediado por glicose. Visando compreender quais miRNAs poderiam estar envolvidos, análise de expressão em larga escala por reação em cadeia da polimerase em tempo real (qRT-PCR) de 200 precursores de miRNAs (pri-miRs) em resposta a glicose foi conduzida, apontando para uma potencial regulação sobre 38 deles, vários dos quais já são conhecidos por participarem direta ou indiretamente do controle de desenvolvimento da planta. Aparentemente, a deficiência na maquinaria de miRNAs leva a um desbalanço nas regulações de genes responsivos à glicose durante os primeiros estágios de desenvolvimento / Abstract: Plants, as sessile organisms, have developed a set of mechanisms that allow efficient adaptation to adverse environmental conditions. These processes rely on the integration of hormone biosynthesis, activation of stress-responsive pathways and on a balanced use of the available energy. Sugars, besides their role as carbon and energy sources, may also function as signaling molecules that may act together with hormonal signals to trigger adaptive responses to biotic and abiotic stresses. In this context, several studies have indicated an important relation between abscisic acid (ABA), one of the major hormones related to abiotic stresses responses, and glucose. ABA signaling, besides its function over transcription control, is known to involve factors regulating the stability of mRNAs. However, the importance of glucose-mediated mRNA decay control is essentially unknown. Our work intended to evaluate the potential of the participation of post-transcriptional regulations in response to ABA and glucose in Arabidopsis thaliana, by determining mRNA profile alteration in response to these signals after transcription inhibition. An experimental model which optimizes the conditions for transcription inhibition was established and used for transcriptome profiling with CATMA microarrays. A total of 962 genes were found to be differentially expressed after the treatments, suggesting a possible post-transcriptional control acting upon 204, 245 and 513 transcripts in response to glucose, ABA and the combination glucose + ABA, respectively. The genes were classified by their functions according to Gene Ontology, suggesting a close relation with adaptive response to stress conditions. Apparently, ABA- and glucose-mediated control of mRNA stability follows two opposite strategies, while ABA post-transcriptional responses may also act as a fast negative feedback mechanism over its own core signaling pathway, as a way to desensitize and reset the pathway responses. The second part of this work focused on the participation of microRNAs (miRNAs) pathway in responses mediated by glucose during plant early developmental stages. The mutants ago1-25 and hyl1-2, which are deficient in miRNA activity and biogenesis, respectively, showed hyposensitivity to glucose during a narrow time window of early plant development, between germination and seedling establishment. Such result raises the possibility that miRNA pathway may be involved in the glucose-mediated delay of early seedling development. To obtain further evidences about which miRNAs could be involved, the expression profile of 200 pri-miRs was evaluated by large-scale quantitative real-timepolymerase chain reaction (qRT-PCR) profiling, indicating that 38 pri-miRNA are potentially regulated by glucose, several of which are known to participate directly or indirectly in plant development. The data indicate that deficiency in miRNA machinery leads to an imbalance on glucose control over gene expression during early seedling development / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

Thèse réalisée en cotutelle avec l'Université Pierre et Marie Curie, Paris VI, France / Le contrôle de la transcription constitue le principal niveau de la régulation de l’expression des gènes dans les cellules eucaryotes. Dans le génome de ces derniers, les éléments régulateurs peuvent être localisés à de très grandes distances du gène qu’ils régulent. Le laboratoire a identifié 6 familles indépendantes avec un diabète insipide néphrogénique (DIN) lié à l’X portant de grandes délétions en amont du gène de l’AVPR2. Dans chacune de ces familles, les gènes AVPR2 et AQP2 ont été retrouvés intacts et les hommes sont atteints de DIN lié à l’X dans sa forme rénale « classique ». Le séquençage et l’analyse de 30 et 31 kilobases en amont et en aval de l’AVPR2 ont permis l’identification de 6 zones délétées chez 6 familles indépendantes, dont 5 zones de taille supérieure à 7 kilo bases, et une zone, de 102 paires de bases, commune à l’ensemble des délétions. Chez le patient porteur de cette délétion, l’osmolalité urinaire ne répond pas au dDAVP. Contrairement à ce qui est observé chez les patients atteints de DIN avec mutations de l’AVPR2, celui-ci présente des réponses hémodynamiques et de coagulation, normales. Ceci indique que les récepteurs V2 ne sont pas exprimés dans le tubule collecteur mais le sont au niveau des cellules endothéliales. Le but de notre travail est donc de tenter de comprendre les mécanismes régulateurs du locus de l’AVPR2, et plus précisément d’étudier l’expression « tissu spécifique » de ce gène. Les études réalisées in vivo, dans le système Hprt, confirment le rôle activateur de la séquence de 102 pb : coloration intense des tubules collecteurs avec la construction comportant la zone délétée et absence avec la construction ne la contenant pas. Cependant, les expériences menées in vitro semblent indiquer que cet effet dépende du contexte extracellulaire, isotonique ou hypertonique, de la nature des cellules, du tubule proximal ou collecteur, ainsi que du promoteur de l’AVPR2. L’identification des protéines liant potentiellement l’une des extrémités de la délétion a révélé la présence, soit de protéines régulatrices, soit de séquences inconnues, toutes exprimées dans le rein. À terme, ces études, ainsi que celles en découlant, permettront de positioner l’AVPR2 comme une cible de choix dans le traitement des diabètes insipides, centraux et néphrogéniques, par thérapie génique. / Transcriptional control is the primary means of regulating genes expression in eukaryotes cells. In the genome of the latter, regulatory elements can be localised with very long distance from the gene which they control. The laboratory identified six independent families with X-linked nephrogenic diabete insipidus (NDI) bearing large deletions upstream of the AVPR2 gene leaving intact AVPR2 and AQP2 coding sequences. Males bearing these deletions have classical renal X-linked NDI. The sequencing and analysis of 30 and 31 kilo bases upstream and downstream, respectively, encompassing the AVPR2 gene had led to identify 6 deletions in 6 ancestrally independent families including, 5 larger than 7 kilo bases and one of 102 base paires shared by the other deletions. In male patient bearing the 102 bp upstream deletion, urinary osmolality was unresponsive to dDAVP but, unlike patients with mutations in the coding sequence, their coagulation and hemodynamic responses to dDAVP were normal. This suggests that V2 receptors are not expressed in renal collecting duct cells but normally expressed in endothelial cells. Our goal is thus to understand the regulatory mechanism controlling the AVPR2 locus and more precisely the tissu specific expression of this gene.. The studies carried out in vivo, in the Hprt system, confirm the enhancer role of the sequence of 102 bp: intense coloration of the collecting tubules with construction comprising the deleted zone and abscence with construction not containing it. However, in vitro undertaken experiments seem to indicate that this effect depends on the extracellular context, isotonic or hypertonic, of the nature of the cells, of the tubule proximal or collecting duct, as well as promoter of the AVPR2. The identification of proteins potentially binding one of the ends of the deletion revealed the presence, either of regulating proteins, or of unknown sequences, all expressed in the kidney. In the long term, these studies, like those while rising, will make it possible to position the AVPR2 gene like a target of choice in the treatment of the diabetes insipidus, central and nephrogenic, by genic therapy.

Le contrôle de la transcription constitue le principal niveau de la régulation de l’expression des gènes dans les cellules eucaryotes. Dans le génome de ces derniers, les éléments régulateurs peuvent être localisés à de très grandes distances du gène qu’ils régulent. Le laboratoire a identifié 6 familles indépendantes avec un diabète insipide néphrogénique (DIN) lié à l’X portant de grandes délétions en amont du gène de l’AVPR2. Dans chacune de ces familles, les gènes AVPR2 et AQP2 ont été retrouvés intacts et les hommes sont atteints de DIN lié à l’X dans sa forme rénale « classique ». Le séquençage et l’analyse de 30 et 31 kilobases en amont et en aval de l’AVPR2 ont permis l’identification de 6 zones délétées chez 6 familles indépendantes, dont 5 zones de taille supérieure à 7 kilo bases, et une zone, de 102 paires de bases, commune à l’ensemble des délétions. Chez le patient porteur de cette délétion, l’osmolalité urinaire ne répond pas au dDAVP. Contrairement à ce qui est observé chez les patients atteints de DIN avec mutations de l’AVPR2, celui-ci présente des réponses hémodynamiques et de coagulation, normales. Ceci indique que les récepteurs V2 ne sont pas exprimés dans le tubule collecteur mais le sont au niveau des cellules endothéliales. Le but de notre travail est donc de tenter de comprendre les mécanismes régulateurs du locus de l’AVPR2, et plus précisément d’étudier l’expression « tissu spécifique » de ce gène. Les études réalisées in vivo, dans le système Hprt, confirment le rôle activateur de la séquence de 102 pb : coloration intense des tubules collecteurs avec la construction comportant la zone délétée et absence avec la construction ne la contenant pas. Cependant, les expériences menées in vitro semblent indiquer que cet effet dépende du contexte extracellulaire, isotonique ou hypertonique, de la nature des cellules, du tubule proximal ou collecteur, ainsi que du promoteur de l’AVPR2. L’identification des protéines liant potentiellement l’une des extrémités de la délétion a révélé la présence, soit de protéines régulatrices, soit de séquences inconnues, toutes exprimées dans le rein. À terme, ces études, ainsi que celles en découlant, permettront de positioner l’AVPR2 comme une cible de choix dans le traitement des diabètes insipides, centraux et néphrogéniques, par thérapie génique. / Transcriptional control is the primary means of regulating genes expression in eukaryotes cells. In the genome of the latter, regulatory elements can be localised with very long distance from the gene which they control. The laboratory identified six independent families with X-linked nephrogenic diabete insipidus (NDI) bearing large deletions upstream of the AVPR2 gene leaving intact AVPR2 and AQP2 coding sequences. Males bearing these deletions have classical renal X-linked NDI. The sequencing and analysis of 30 and 31 kilo bases upstream and downstream, respectively, encompassing the AVPR2 gene had led to identify 6 deletions in 6 ancestrally independent families including, 5 larger than 7 kilo bases and one of 102 base paires shared by the other deletions. In male patient bearing the 102 bp upstream deletion, urinary osmolality was unresponsive to dDAVP but, unlike patients with mutations in the coding sequence, their coagulation and hemodynamic responses to dDAVP were normal. This suggests that V2 receptors are not expressed in renal collecting duct cells but normally expressed in endothelial cells. Our goal is thus to understand the regulatory mechanism controlling the AVPR2 locus and more precisely the tissu specific expression of this gene.. The studies carried out in vivo, in the Hprt system, confirm the enhancer role of the sequence of 102 bp: intense coloration of the collecting tubules with construction comprising the deleted zone and abscence with construction not containing it. However, in vitro undertaken experiments seem to indicate that this effect depends on the extracellular context, isotonic or hypertonic, of the nature of the cells, of the tubule proximal or collecting duct, as well as promoter of the AVPR2. The identification of proteins potentially binding one of the ends of the deletion revealed the presence, either of regulating proteins, or of unknown sequences, all expressed in the kidney. In the long term, these studies, like those while rising, will make it possible to position the AVPR2 gene like a target of choice in the treatment of the diabetes insipidus, central and nephrogenic, by genic therapy. / Thèse réalisée en cotutelle avec l'Université Pierre et Marie Curie, Paris VI, France

Bacterial pathogens dramatically affect host cell transcription programs for their own profit, however the underlying mechanism in most cases remain elusive. While investigating the effects of listeria monocytogenes on histone modifications, we discovered a new transcription regulatory machanism by which the expression of genes is repressed, during infection. Upon infection by L. monocytogenes, the secret virulence factor, InlB, binds the c-Met receptor and activates signaling through PI3K/Akt. This signaling platform is necessary for causing the relocalization of the histone deacetylase, SIRT2, to the nucleus and associating to chromatin.In characterizing the mechanism governing SIRT2 nuclear relocazing during infection, our results have demonstrated that SIRT2 undergoes a post-translational modification. SIRT2 undergoes dephosphorylation at a novel N-terminal phospho-site. SIRT2 is recruiter to the transcription star sites of genes repressed during inection leading to H3K18 deacetylation and transcriptional repression.finnaly, my results demonstrate that SIRT2 is hijacked by L monocytogenes and promotes an increase in intracellular bacteria. Together, these data uncover a key role for SIRT2 mediated H3K18 deacetylation during infection and characterize a novel mechanisme imposed by a pathogenic bacteriomto reprogram the host cell.

De nombreuses bacteries pathogènes sont capables d'affecter les programmes transcriptionnels de la cellule hôte pendant l'infection. Cependant, les mécanismes contrôlant ce processus restent largement méconnus. En investigant les effets de la Listerai monocytogenes sur les modifications des histones de l'hôte, nous avons mis en évidence un nouveau mecanisme de régulation de transcription nécessaire pour la répression de certains gènes, pendant l'infection. Lors de l'infection par L. monocytogenes, le facteur de virulence sécrété, InlB, se lie au récepteur c-Met et active la signalisation par les intermédiaires PI3K et Akt. cette plateforme de signalisation est nécessaire pour la relocalisation de la deacetylase d'histone, SIRT2, au noyau et l'association à la chromatine.En caractérisant me mécanisme gouvernant la relocalisation nucléaire de SIRT2 lors de l'infection, nous avons démontrés que SIRT2 subit une modification post-traductionnelle. SIRT2 est déphosphorylée à un nouveau site de phosphorylation localisé à la partie terminale de la protéine. SIRT2 est recrutée au site de démarrage de la transcription des gènes réprimés lors de l'infection menant à la deacetylation de H3K18 et la répression transcriptionnelle. Nous avons mis en évidence que SIRT2 est détournée par L. monocytogenes et provoque une croissance des bactéries intracellulaires. Ces résultats démontrent un clef de SIRT2 en provoquant la deacetylation de H3K18 mors de l'infection et dévoilent un nouveau mécanisme imposée par les bactéries pathogènes dans le but de reprogrammer la cellule hôte. / Bacterial pathogens dramatically affect host cell transcription programs for their own profit, however the underlying mechanism in most cases remain elusive. While investigating the effects of listeria monocytogenes on histone modifications, we discovered a new transcription regulatory machanism by which the expression of genes is repressed, during infection. Upon infection by L. monocytogenes, the secret virulence factor, InlB, binds the c-Met receptor and activates signaling through PI3K/Akt. This signaling platform is necessary for causing the relocalization of the histone deacetylase, SIRT2, to the nucleus and associating to chromatin.In characterizing the mechanism governing SIRT2 nuclear relocazing during infection, our results have demonstrated that SIRT2 undergoes a post-translational modification. SIRT2 undergoes dephosphorylation at a novel N-terminal phospho-site. SIRT2 is recruiter to the transcription star sites of genes repressed during inection leading to H3K18 deacetylation and transcriptional repression.finnaly, my results demonstrate that SIRT2 is hijacked by L monocytogenes and promotes an increase in intracellular bacteria. Together, these data uncover a key role for SIRT2 mediated H3K18 deacetylation during infection and characterize a novel mechanisme imposed by a pathogenic bacteriomto reprogram the host cell.