Neurofibromatosis type 2 (NF2) is an autosomal dominant inherited condition that predisposes individuals to develop multiple nervous system tumours, primarily schwannoma, meningioma and ependymoma. NF2 is characterised by loss of the tumour suppressor protein Merlin, caused by bi-allelic mutations of the encoding gene NF2 or by loss of heterozygosity. These tumours can occur either sporadically or as part of the NF2 condition. The majority are slow growing and display benign features, but this benignancy renders them largely unresponsive to classic chemotherapeutic agents leaving surgery and radiosurgery as the only remaining treatment options. Depending on their location, NF2-related tumours can cause a number of side effects, including nausea, balance problems, and in some cases hearing and/or vision loss. Phosphorylation is a key regulatory mechanism leading to changes in cell signalling. By identifying phosphoproteins that are significantly activated in tumour cells, novel therapies can be developed aiming to specifically target the phosphorylated status of these proteins thus ‘switching off’ the signalling cascade. The objective of this study is to identify and validate common targets in both Merlin-deficient meningioma and schwannoma to eventually exploit in novel therapeutic approaches. Using phosphoprotein purification followed by mass spectrometry analysis, we identified Signal Transducer and Activator of Transcription 1 (STAT1), phosphorylated at Serine (S) 727 and Tyrosine (Y) 701, PDZ and LIM domain protein 2 (PDLIM2), Heat Shock 70kDa Protein 1A (HSPA1A) and Filamin B (FLNB) as potential common, novel therapeutic targets. We validated these candidates in human primary meningioma and schwannoma tumour cells using a variety of techniques. We also showed that specific 7 knockdown of STAT1 and PDLIM2 was related to a significant decrease in cellular proliferation. Additionally, we performed co-immunoprecipitation using PDLIM2 as the bait protein and identified STAT1, HSPA1A and FLNB as binding partners, suggesting a novel interaction network involving all of the potential targets identified in this study. We also identified activation of several pathways and/ or biological processes in both tumour types that warrant further investigation i.e. endocytosis in schwannoma and the proteasome in meningioma. In conclusion, with our approach we substantially increased the overall body of knowledge regarding the proteome and phosphoproteome of meningioma and schwannoma. We generated a comprehensive set of data that highlighted several potential therapeutic targets and dysregulated pathways which will be further investigated.

Selon l'organisation mondiale de la santé, 21 pays d'Amérique Latine et notamment le Brésil sont touchés par la maladie de Chagas qui est provoquée par le protozoaire Trypanosoma cruzi (T. cruzi). En dépit de très nombreuses études expérimentales, il n'existe aucun traitement efficace contre la maladie de Chagas, et l'identification de nouvelles cibles thérapeutiques est fondamentale pour développer de nouvelles drogues. Dans cette thèse, j'ai revisité 41 protéines chez T. cruzi récemment proposées par Nicolas Carels et al. A partir d'une nouvelle procédure bioinformatique, j'ai trouvé qu'il est possible de prédire les structures 3D de séquences T. cruzi ayant des homologues ou des analogues chez l'homme et très probablement associées aux fonctions trypanothione réductase, cystéine synthase et ATPase, ainsi que des séquences spécifiques à T. cruzi et absentes chez Homo Sapiens associées à des activités 2,4-dienoyl-CoA réductase et leishmanolysine. Les implications de nos modèles raffinées par des dynamiques moléculaires atomistiques (monomère ou dimère) dans leur environnement in vitro (solution aqueuse ou bicouche membranaire) sont discutées à des fins thérapeutiques, et suggèrent que toutes les cibles protéiques, à l'exception de la cystéine synthase, méritent d'autres investigations. / According to the World Health Organization, 21 Latin American countries are endemic for Chagas disease, affecting 10 million people. Chagas' disease, caused by the protozoan Trypanosoma cruzi (T. cruzi), is a parasitic illness endemic mostly in Latin America and particularly in Brazil. Despite many experimental studies, there is no efficient treatment against Chagas disease, and the search for new therapeutic targets specific to T. cruziis critical for drug development. In my thesis, I have revisited 41 protein sequences proposed by the analogous enzyme pipeline, and found that it is possible to provide structures for 33 T. cruzisequences with clear homologs or analogs in H. sapiensand likely associated with trypanothione reductase, cysteine synthase and ATPase functions, and structures for sequences specific to T. cruziand absent in H. sapiens associated with 2,4-dienoyl-CoA reductase, and leishmanolysin activities. The implications of our structures refined by atomistic molecular dynamics (monome or dimer states) in their in vitro environments (aqueous solution or membrane bilayers) are discussed for drug development and suggest that all protein targets, except cysteine synthase, merit further investigation.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13534号 / 論医博第2274号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 滝田 順子, 教授 伊藤 貴浩, 教授 江藤 浩之 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Meduloblastoma é um câncer do sistema nervoso central, altamente invasivo, de origem embrionária, localizado no cerebelo. É mais comum em crianças e corresponde a aproximadamente 20% de todos os tumores intracranianos pediátricos. Os tratamentos mais utilizados são cirurgia e quimioterapia, sendo que a radioterapia é aplicada somente em crianças com mais de 3 anos devido aos seus efeitos colaterais. Diversos estudos têm mostrado o papel do NF-B na regulação de genes envolvidos com o processo neoplásico. NF-B é um fator de transcrição chave na regulação da resposta imune e no processo de inflamação e está envolvido na regulação da transcrição de um grande número de genes relacionados ao processo de tumorigênese, além de ser constitutivamente ativo em diversos tipos de câncer, sendo um importante potencial alvo terapêutico. O DHMEQ (Dehidroximetilepoxiquinomicina) é uma droga que inibe a translocação do NF-B do citoplasma para o núcleo, inibindo assim a sua atuação como ativador transcricional. Vários trabalhos tem mostrado os efeitos antineoplásicos do DHMEQ em inúmeros tipos tumorais, entretanto, não há trabalhos que evidenciem esses efeitos em meduloblastoma. Assim, o presente estudo objetivou avaliar os efeitos dessa droga nas linhagens UW402, UW473 e ONS-76 de meduloblastoma pediátrico através de estudos funcionais e moleculares. Os resultados de proliferação demostraram uma significativa diminuição do crescimento celular nas linhagens de meduloblastoma, inibindo cerca de 80, 70 e 60% nas linhagens UW402, UW473 e ONS-76, respectivamente, na dose de 20 g/mL, e apresentou um IC50 de 10g/mL em 48h para as linhagens UW402 e UW473 e em 72h na linhagem ONS-76. Adicionalmente, elevou o nível de apoptose para 50, 17 e 31% nessas linhagens, respectivamente, inibiu fortemente a capacidade clonogênica, a migração e a invasão celular nas três linhagens e foi sinérgico na combinação com outros quimioterápicos em grande parte dos pontos de combinação, além de radiossensibilizar fortemente as três linhagens. Os resultados são congruentes com o potencial efeito antitumoral de DHMEQ. / Medulloblastoma is a cancer of the central nervous system, highly invasive, of embryonic origin, located in the cerebellum. It is more common among children and accounts for approximately 20% of all pediatric intracranial tumors. The most common treatments are surgery and chemotherapy, and radiotherapy is only to children older than 3 years old due to its side effects. Several studies have demonstrated the role of NF-B in the regulation of genes involved in the neoplastic process. NF-B is a key transcription factor in the regulation of immune response and inflammation process, and it is involved in the transcriptional regulation of a large number of genes related to the tumorigenesis process, and constitutively active in many types of cancer, being an important potential therapeutic target. DHMEQ (Dehidroximetilepoxiquinomicina) is a drug that inhibits the translocation of NF-B from the cytoplasm to the nucleus, thus inhibiting its activity as a transcriptional activator. Several studies have shown the antineoplastic effects of DHMEQ in numerous tumor types, however, there is no surveys that have tested their effects in medulloblastoma. Thus, the present study aimed to evaluate the effects of this drug in UW402, UW473 and ONS-76 pediatric medulloblastoma cell lines through functional and molecular studies. The proliferation test results demonstrated a significant decrease in the cell growth in the medulloblastoma cell lines, inhibiting approximately 80, 70 and 60% for UW402, UW473 and ONS-76, respectively, at a dose of 20g/mL, and showed an IC50 of 10g/mL at 48h for UW402 and UW473 and at 72h in ONS-76. Additionally, increased the level of apoptosis to 50, 17 and 31% in these cell lines, respectively, strongly inhibited the clonogenic capacity, the migration and cell invasion in the three lines and it was synergistic in combination with other chemotherapeutic agents in most combination points, and radiosensitization strongly the three cell lines. The results are congruent with the potential antitumor effect of DHMEQ.

Meduloblastoma é um câncer do sistema nervoso central, altamente invasivo, de origem embrionária, localizado no cerebelo. É mais comum em crianças e corresponde a aproximadamente 20% de todos os tumores intracranianos pediátricos. Os tratamentos mais utilizados são cirurgia e quimioterapia, sendo que a radioterapia é aplicada somente em crianças com mais de 3 anos devido aos seus efeitos colaterais. Diversos estudos têm mostrado o papel do NF-B na regulação de genes envolvidos com o processo neoplásico. NF-B é um fator de transcrição chave na regulação da resposta imune e no processo de inflamação e está envolvido na regulação da transcrição de um grande número de genes relacionados ao processo de tumorigênese, além de ser constitutivamente ativo em diversos tipos de câncer, sendo um importante potencial alvo terapêutico. O DHMEQ (Dehidroximetilepoxiquinomicina) é uma droga que inibe a translocação do NF-B do citoplasma para o núcleo, inibindo assim a sua atuação como ativador transcricional. Vários trabalhos tem mostrado os efeitos antineoplásicos do DHMEQ em inúmeros tipos tumorais, entretanto, não há trabalhos que evidenciem esses efeitos em meduloblastoma. Assim, o presente estudo objetivou avaliar os efeitos dessa droga nas linhagens UW402, UW473 e ONS-76 de meduloblastoma pediátrico através de estudos funcionais e moleculares. Os resultados de proliferação demostraram uma significativa diminuição do crescimento celular nas linhagens de meduloblastoma, inibindo cerca de 80, 70 e 60% nas linhagens UW402, UW473 e ONS-76, respectivamente, na dose de 20 g/mL, e apresentou um IC50 de 10g/mL em 48h para as linhagens UW402 e UW473 e em 72h na linhagem ONS-76. Adicionalmente, elevou o nível de apoptose para 50, 17 e 31% nessas linhagens, respectivamente, inibiu fortemente a capacidade clonogênica, a migração e a invasão celular nas três linhagens e foi sinérgico na combinação com outros quimioterápicos em grande parte dos pontos de combinação, além de radiossensibilizar fortemente as três linhagens. Os resultados são congruentes com o potencial efeito antitumoral de DHMEQ. / Medulloblastoma is a cancer of the central nervous system, highly invasive, of embryonic origin, located in the cerebellum. It is more common among children and accounts for approximately 20% of all pediatric intracranial tumors. The most common treatments are surgery and chemotherapy, and radiotherapy is only to children older than 3 years old due to its side effects. Several studies have demonstrated the role of NF-B in the regulation of genes involved in the neoplastic process. NF-B is a key transcription factor in the regulation of immune response and inflammation process, and it is involved in the transcriptional regulation of a large number of genes related to the tumorigenesis process, and constitutively active in many types of cancer, being an important potential therapeutic target. DHMEQ (Dehidroximetilepoxiquinomicina) is a drug that inhibits the translocation of NF-B from the cytoplasm to the nucleus, thus inhibiting its activity as a transcriptional activator. Several studies have shown the antineoplastic effects of DHMEQ in numerous tumor types, however, there is no surveys that have tested their effects in medulloblastoma. Thus, the present study aimed to evaluate the effects of this drug in UW402, UW473 and ONS-76 pediatric medulloblastoma cell lines through functional and molecular studies. The proliferation test results demonstrated a significant decrease in the cell growth in the medulloblastoma cell lines, inhibiting approximately 80, 70 and 60% for UW402, UW473 and ONS-76, respectively, at a dose of 20g/mL, and showed an IC50 of 10g/mL at 48h for UW402 and UW473 and at 72h in ONS-76. Additionally, increased the level of apoptosis to 50, 17 and 31% in these cell lines, respectively, strongly inhibited the clonogenic capacity, the migration and cell invasion in the three lines and it was synergistic in combination with other chemotherapeutic agents in most combination points, and radiosensitization strongly the three cell lines. The results are congruent with the potential antitumor effect of DHMEQ.

La leucémie aiguë myéloïde (LAM) est une pathologie hématologique dont le pronostic reste très défavorable, malgré les progrès réalisés dans la compréhension des mécanismes physiopathologiques sous-tendant son développement. Identifier de nouvelles stratégies anti-leucémiques représente donc une étape clé dans la concrétisation des avancées thérapeutiques. Grâce à la combinaison de plusieurs approches de criblage génétiques et pharmacologiques, l’objectif de ma thèse a été de définir et valider de nouvelles cibles thérapeutiques dans les LAM. La première partie de ma thèse a eu pour but de transposer en clinique l’inhibition de la Glycogen Synthase Kinase 3 (GSK3). La stabilisation de la β-caténine secondaire à l'inhibition concomitante des deux paralogues de GSK3, représente un obstacle à l’utilisation clinique de cette classe thérapeutique. Mettant à profit la présence d'un «switch» Asp133 à Glu196 dans les domaines de liaison ATP de GSK3, nous avons identifié un inhibiteur sélectif du paralogue GSK3α et mené des études précliniques validant le BRD0705 comme nouveau traitement pro-différenciant dans les LAM. De plus, une combinaison de profilage métabolomique et d'approches de criblage haut débit à l’aide d’une banque de shRNA a permis d'identifier un nouveau lien entre EVI-1, la voie de la créatine kinase et la voie de signalisation GSK3. La deuxième partie de ma thèse a porté sur l'identification de nouvelles cibles thérapeutiques en utilisant une approche de criblage par banque de shRNA dans le modèle murin de LAM porteur de la translocation MLL-AF9. VCP, une AAA-ATPase, a ainsi été identifiée puis validée comme cible thérapeutique. Nous avons montré que VCP orchestre la génération d'une plateforme à ADN simple brin recouverte de RPA, ce qui entraîne l'activation de la kinase ATM et la HR. Dans leur ensemble, nos découvertes permettent une meilleure compréhension de la biologie des LAM et participeront ainsi à l’amélioration des traitements futurs de cette pathologie. / Despite the significant progress made in understanding Acute Myeloid Leukemia oncogenesis over the last decades, this disease remains devastating and the overall five-year survival does not exceed 17%. Developing new translational research strategies focused on the identification of druggable oncogenic targets is critical to continued progress in AML treatment. The goal of this work was to define and validate novel leukemia-specific dependencies using small-molecule inhibitors and RNA-interference-based high-throughput screening methods.The first part of my thesis work aimed at translating Glycogen synthase kinase 3 (GSK3) inhibition into the clinic. Mechanism-based toxicities, driven in part by the inhibition of both GSK3 paralogs and subsequent β-catenin stabilization, were a concern in the clinical translation of this target candidate. Specific knock-down of GSK3α or GSK3β alone does not increase β-catenin, thereby offering a conceptual resolution to GSK3 targeting. The design of selective ATP-competitive inhibitors posed a drug discovery challenge due to the high homology in the GSK3α and GSK3β ATP binding domains. Taking advantage of an Asp133 ® Glu196 “switch” in the GSK3 paralog hinge binding domains, we identified a first-in-class GSK3α selective inhibitor and conducted preclinical studies validating BRD0705 as a promising new differentiation therapy in AML. In addition, a combination of a metabolomic profiling and a pooled shRNA screening method identified a new interplay between the oncogene EVI-1, the creatine kinase pathway and GSK3 signaling. The second part of my studies focused on identification of new therapeutic targets using an in vivo pooled shRNA screening approach in the MLL-AF9-driven AML mouse model. VCP, an AAA-ATPase, was thus identified and validated as a top target. We demonstrated that VCP orchestrates RPA-coated-single-stranded-DNA platform generation, resulting in ATM kinase activation and subsequent HR. Taken together, our discoveries increased our understanding of AML biology and may therefore contribute to novel and more efficacious treatments for this highly aggressive and lethal disease.

Les maladies fibroprolifératives se caractérisent par l’accumulation de constituants de la matrice extracellulaire en réponse à une agression chronique et répétée conduisant à la destruction de l’architecture tissulaire. Les myofibroblaste, sous l’influence du TGFβ, représentent les cellules effectrices majoritaires dans ce processus. Les miARN, régulateurs négatifs de l’expression génique, interviennent dans de nombreux mécanismes physio-pathologiques dont la fibrose tissulaire mais leur mécanismes d’action et la synergie potentielle entre miARN co-régulés au sein d’un même cluster restent mal compris. Nos travaux ont consisté à caractériser l’implication du cluster miR-199/214 généré à partir du LncARN DNM3os, dans la fibrose pulmonaire. Nous avons montré que les miARN de ce cluster participent à l’activation et à la différenciation des fibroblastes en myofibroblastes via la régulation des voies canoniques et non canoniques du TGF-β. Ce cluster agit également en tant qu’inhibiteur de la réparation épithéliale. In vivo, l’inhibition de l’un de ces « fibromiRs », a permis, dans un modèle murin, de réduire significativement les lésions de fibrose. Par ailleurs, de par leur présence et leur stabilité dans les fluides biologiques, les miARN représentent également une nouvelle classe de biomarqueurs diagnostiques ou pronostiques non invasifs. Nous avons ainsi montré que les niveaux sériques de miR-21-5p étaient augmentés chez les patients présentant une fibrose rénale sévère. Ces travaux soulignent l’importance des miARN dans la pathogénèse des maladies fibroprolifératives et montrent qu’ils représentent de nouvelles cibles thérapeutiques ou des biomarqueurs non invasifs. / Fibrotic diseases are characterized by the accumulation of extracellular matrix components in response to chronic aggression leading to the destruction of tissue architecture. Myofibroblasts, controlled by TGFβ, are central effectors in this process. MiRNAs, negative regulator of gene expression, are involved in many patho-physiological mechanisms, including tissue fibrosis but their mechanisms of action and the potential synergistic activity between co-regulated miRNAs within the same cluster remain poorly understood. Our work consisted in characterizing the involvement of the miR-199/214 cluster, generated from LncRNA DNM3os, in pulmonary fibrosis. We have shown that these miRNAs are involved in the activation and the differentiation of fibroblasts to myofibroblasts through the regulation of canonical and non-canonical TGF-β pathways. This cluster also acts as an inhibitor of epithelial repair. In vivo, the inhibition of one of this “FibromiR”, significantly decrease fibrosis, in a murine lung fibrosis model. In addition, because of their presence and their stability in biological fluids, miRNAs also represent a new class of non-invasive diagnostic or prognostic biomarker. We showed that serum levels of miR-21-5p were increased in patients with severe kidney fibrosis. These studies highlight the importance of miRNAs in pathogenesis of fibrotic disorders and show that they represent new therapeutic targets or non-invasive biomarkers.

Introduction: les progrès thérapeutiques pour la prise en charge du carcinome urothélial de la vessie, ou tumeur de vessie infiltrant le muscle (TVIM), sont faibles depuis deux décennies, notamment sur le plan de la chimiothérapie, tant dans les formes localisées que métastatiques. L'identification de nouveaux biomarqueurs pouvant améliorer la prise en charge et le pronostic des patients apparait indispensable, notamment pour prédire la réponse aux traitements systémiques. Notre travail a cherché : (i) à identifier des facteurs biologiques prédictifs de rechute après les chimiothérapies adjuvantes (CA) utilisées actuellement, (ii) à déterminer si les altérations moléculaires, cibles de nouvelles thérapies, identifiées dans la tumeur vésicale sont conservées dans les métastases.Objectifs:(i) Rechercher une association entre la rechute après CA et l'expression de six biomarqueurs, ERCC1, Emmprin, Survivin, MDR1, p53 et topoisomérase IIα, dans la tumeur vésicale et/ou une métastase ganglionnaire(ii) Etudier l'hétérogénéité des mutations de FGFR3 dans les TVIM localement avancéesMatériels et Méthodes: Une cohorte rétrospective de 226 patients traités par cystectomie et CA, et une collection tumorale d'échantillons représentatifs de la tumeur primitive et d'une métastase ganglionnaire ont été constituées. L'expression des six biomarqueurs a été déterminée en immunohistochimie sur ces échantillons. Le statut mutationnel de FGFR3 a été déterminé en SNaPshot sur 84 paires tumeur primitive/métastase ganglionnaire.Résultats: Les survies observées dans la cohorte étaient proches de celles rapportées dans la littérature pour des patients de mêmes stades. La collection tumorale étudiée semble donc représentative des TVIM traitées en France. Aucun des six biomarqueurs n'était retrouvé comme prédictif de rechute après traitement. Une mutation de FGFR3 était retrouvée dans 4 (4,7%) des 84 échantillons appariés sans aucune discordance entre tumeur primitive et métastase ganglionnaire.Conclusions: L'expression de ces six biomarqueurs étudiés en immunohistochimie n'est pas associée à un risque de rechute après CA. Le statut mutationnel de FGFR3 est conservé au cours de la dissémination métastatique, et ses mutations activatrices sont présentes dans près de 5% des TVIM localement avancées. Leur conservation dans le processus métastatique renforce l'intérêt de l'évaluation de nouvelles thérapies ciblant le récepteur FGFR3 muté. Enfin, il est possible de rechercher cette mutation sur la tumeur vésicale ou une métastase avec la même pertinence. / Introduction: In muscle invasive urothelial carcinoma of the bladder (MI UCB), few progresses have been made in the last two decades, particularly in chemotherapy, both in localized and metastatic setting. The identification of new biomarkers that can improve the management and prognosis of patients appears essential. Such biomarkers will be clinically relevant if they can predict the response to systemic treatments. In this thesis, we studied biomarkers in two ways: (i) to identify predictive factors of relapse after cisplatin-based adjuvant chemotherapy (AC), (ii) to confirm that the molecular alterations targeted by new therapies and identified in bladder tumor are present in metastases.Objectives:(i) To examine the relationship between relapse after AC and expression of six biomarkers, ERCC1, Emmprin, Survivin, MDR1 p53 and topoisomerase IIα, in bladder tumor and / or lymph node metastases.(ii) To study the heterogeneity of FGFR3 mutations in locally advanced MI UCBMaterials and Methods: A retrospective cohort of 226 patients treated with cystectomy and AC, and tumor collection of representative samples of the primary tumor and lymph node metastases were constituted. The expression of six biomarkers was determined by immunohistochemistry on these samples and determined by morphometry. FGFR3 mutation status was determined by SNaPshot in 84 paired primary tumors and positive lymph nodes.Results: Survivals observed in the cohort were similar to those reported in the literature for patients of same stages, with tumor collection being representative of the MI UCB treated in France. None of the six biomarkers was found as an independent predictive factor of relapse after AC. FGFR3 mutation was found in 4 (4.7%) of the 84 paired samples with complete concordance between primary tumor and lymph node metastases.Conclusions: The expression of the six studied biomarkers by immunohistochemistry is not associated with risk of relapse following AC. FGFR3 mutation status is maintained during metastases process, and FGFR3 activating mutations are present in about 5% of locally advanced MI UCB. FGFR3 mutation remains a promising therapeutic target in a low subset of patients, and the evaluation of new therapies targeting the mutated FGFR3 receptor are needed. Finally, search for these mutations either in bladder tumor or in metastasis is relevant.

Les bactéries utilisent la communication intercellulaire pour se coordonner afin de réguler des processus bactériens majeurs comme la virulence, la sporulation, la compétence ou la formation de biofilms en fonction de la densité cellulaire. Ce processus repose sur la sécrétion de petites molécules signal appelées phéromones. Chez les bactéries à gram positif, ces phéromones sont de petits peptides qui peuvent être, soit reconnus au niveau de la membrane externe par des systèmes à deux composants dits systèmes indirects, soit être re-internalisés afin de se fixer directement sur un régulateur transcriptionnel cytoplasmique. Dans la recherche de nouveaux agents antimicrobiens, le potentiel thérapeutique d’inhibiteurs ciblant les systèmes de communication intercellulaire est bien étudié chez les bactéries à gram négatif qui utilisent des homosérine lactones comme phéromones. Quelques études s’intéressent aux systèmes indirects de bactéries pathogènes à gram positif mais le potentiel des systèmes directs, plus récemment identifiés, n’a pas encore été explorés.Les récepteurs cytoplasmiques directement régulés par des peptides signal re-internalisés forment la famille des RNPP. Ces récepteurs sont caractérisés par un domaine de fixation du peptide composé d’une répétition de motifs en hélice  appelés TetratricoPeptide Repeats (TPR). La plupart de ces récepteurs sont des régulateurs transcriptionnels contenant un domaine N-terminal de fixation de l’ADN de type Hélice-Tour-Hélice (HTH). Les études précédentes ont montré que, malgré une structure conservée, les modes de régulation des différents membres de la famille RNPP suivent des mécanismes moléculaires distincts. L’un de ces systèmes directs le mieux caractérisé est le système ComRS qui régule la compétence chez les streptocoques. La compétence permet aux bactéries d’internaliser des fragments d’ADN exogènes pour l’acquisition de nouveaux phénotypes tels que la résistance aux antibiotiques ou la virulence. Dans le groupe salivarius, il a été montré que les récepteurs ComR de deux espèces très proches, S. thermophilus et S. vestibularis, ne sont pas capables de coordonner leur état de compétence par échange de leurs peptides ComS respectifs.L'objectif de ma thèse a été d'étudier les déterminants structuraux de la spécificité du système ComRS. J’ai produit, purifié et cristallisé le récepteur ComR de S. vestibularis afin d’en déterminer la structure cristalline, seul et en présence de son peptide signal ComS. La comparaison de ces structures avec celles précédemment résolues chez S. thermophilus, conjointement à une étude fonctionnelle par mutagénèse dirigée réalisée chez nos collaborateurs (P. Hols, UCLouvain, Belgique), a permis d’aller plus loin dans la compréhension du mécanisme de régulation de ComR mais aussi d’identifier les résidus responsables de la spécificité du système ComRS. En parallèle, j’ai également initié la caractérisation structurale de deux paralogues de ComR chez S. salivarius, ScuR et SarF, qui ne sont pas activés par ComS et ne reconnaissent pas les mêmes cibles ADN que ComR malgré des séquences très conservées. Une analyse par SEC-MALS et SAXS m’a permis de montrer que ScuR semble suivre un mécanisme similaire à celui de ComR alors que SarF se comporte différemment en solution. J’ai proposé un modèle par homologie pour ScuR et cristallisé SarF. La résolution de sa structure est en cours. Cette étude permet donc de mieux comprendre la régulation de la compétence chez les streptocoques et ouvre la voie à d’éventuelles applications biotechnologiques ou biomédicales. / Bacteria use intercellular communication to coordinate and regulate major bacterial processes such as virulence, sporulation, competence or biofilm formation, as a function of cell density. This process relies on the secretion of small signal molecules called pheromones. In gram-positive bacteria, these pheromones are small peptides that can be either recognized at the outer membrane by two-component systems called indirect systems, or can be re-internalized to directly interact with a cytoplasmic transcriptional regulator. In the search for new antimicrobial agents, the therapeutic potential of inhibitors targeting intercellular communication systems is well studied in gram-negative bacteria that use homoserine lactones as pheromones. Some studies focus on the indirect systems of gram-positive pathogenic bacteria, but the potential of the more recently identified direct systems has not yet been explored.The cytoplasmic receptors directly regulated by re-internalized signal peptides form the RNPP family. These receptors are characterized by a peptide binding domain consisting of repeats of  helical motifs called TetratricoPeptide Repeats (TPR). Most of these receptors are transcriptional regulators containing an N-terminal DNA binding domain of the helix-turn-helix (HTH) type. Previous studies have shown that, despite a conserved structure, the modes of regulation of the different members of the RNPP family follow distinct molecular mechanisms. One of the best characterized direct systems is the ComRS system that regulates competence in streptococci. Competence allows bacteria to internalize exogenous DNA fragments for the acquisition of new phenotypes such as antibiotic resistance or virulence. In the salivarius group, it has been shown that the ComR receptors of two closely related species, S. thermophilus and S. vestibularis, are not able to coordinate their state of competence by exchange of their respective ComS peptides.The aim of my thesis was to study the structural determinants of the specificity of the ComRS system. I produced, purified and crystallized the ComR receptor from S. vestibularis in order to determine its crystal structure, alone and in the presence of its ComS signal peptide. The comparison of these structures with those previously solved with ComR from S. thermophilus, together with a functional study by directed mutagenesis performed by our collaborators (P. Hols, UCLouvain, Belgium), allowed us to go further in the understanding of the regulatory mechanism of ComR but also to identify residues responsible for the specificity of the ComRS system. In parallel, I also initiated the structural characterization of two ComR paralogs from S. salivarius, ScuR and SarF, which are not activated by ComS and do not recognize the same DNA targets as ComR despite highly conserved sequences. SEC-MALS and SAXS analyses allowed me to show that ScuR seems to follow a mechanism similar to that of ComR whereas SarF behaves differently in solution. I proposed a homology model for ScuR and crystallized SarF. The resolution of its structure is in progress. This study therefore provides a better understanding of the regulation of competence in streptococci and opens the way to potential biotechnological or biomedical applications.

En France, le cancer de la prostate est le premier cancer par son incidence ainsi que la troisième cause de mortalité pour cette pathologie. La progression de la maladie est dépendante des androgènes. Ainsi l’un des traitements majeurs du cancer de la prostate est la déprivation androgénique : le blocage de la production ou de l’action des androgènes conduit à inhiber la croissance tumorale. La plupart des patients répondent à cette thérapie, cependant l’évolution de la pathologie vers un stade d’insensibilité à la castration est inévitable, ce qui est associé à un mauvais pronostic. Au cours de la progression du cancer de la prostate, la voie de signalisation des androgènes demeure active grâce au récepteur des androgènes. Ce récepteur est une cible idéale afin de traiter et de bloquer la progression tumorale. Une telle inhibition du récepteur des androgènes peut être faite par l’utilisation d’un anti-androgènes de seconde génération : l’Enzalutamide. Cette molécule perturbe l’interaction entre le récepteur des androgènes et son ligand, elle peut bloquer la translocation nucléaire du récepteur activé mais elle empêche aussi son interaction avec l’ADN. Bien que l’utilisation de l’Enzalutamide ait contribué à améliorer la survie des patients, son utilisation conduit à l’émergence d’une résistance à l’Enzalutamide, ce qui constitue un défi thérapeutique considérable.L’objectif principal de mes travaux de thèse est d’identifier de nouvelles protéines afin d’améliorer les effets thérapeutiques de l’Enzalutamide ou de surmonter la résistance à ce médicament. Ainsi dans notre étude, nous avons supposé que le traitement à l’Enzalutamide induit l’activation de voies de signalisation spécifiques, pouvant être impliquées soit dans le mécanisme d’action du médicament ou dans la résistance à ce dernier. Cette hypothèse nous a conduit à l’identification des protéines MAPKs p38, qui sont activées lors d’un traitement avec l’Enzalutamide. Nos résultats démontrent que la combinaison d’Enzalutamide et d’inhibiteur de la MAPK p38 a un effet antitumoral significatif aussi bien in vitro que in vivo. Le mécanisme d’action de cet effet cytotoxique et synergique reste à l’étude. Ces données permettraient d’approfondir la compréhension des mécanismes de résistance lors d’un traitement à l’Enzalutamide et contribuer à la potentialisation de l’effet thérapeutique de cet antiandrogènes. / In France, prostate cancer is the most frequently diagnosed male cancer and its progression is tightly associated with the androgen signals. One of the major treatments for prostate cancer is androgen deprivation therapy which is based on blocking the production or action of the androgens to induce a tumor growth inhibition. Most patients respond to this therapy, however they still reach a castration-resistant stage which is associated with a poor prognosis. Since the progression till this late cancer stage is still driven by the androgen signaling pathway, the second-line therapy is focused on targeting the active androgen receptor by using a second-generation anti-androgens: the Enzalutamide. This molecule disrupts the interaction between the androgen receptor and its ligand, it can block the nuclear translocation of the receptor and it also prevents the receptor interaction with DNA. Although Enzalutamide treatment has enhance the patient survival, some drug resistance still arises which is a considerable therapeutic challenge.The main objective of my thesis is to identify new proteins in order to improve the therapeutic effects of Enzalutamide or to overcome resistance to this drug. Thus, in our study, we assumed that Enzalutamide treatment induces the activation of specific signaling pathways which may be involved in the cancer cell response to the treatment. This hypothesis led us to identify the MAPKs p38 proteins, which are activated during treatment with Enzalutamide. Our results show that the combination of Enzalutamide and p38 inhibitor has a significant antitumor effect both in vitro and in vivo. The mechanism of action of this cytotoxic and synergistic effect remains under study. These data would allow a better understanding of the Enzalutamide resistance mechanisms and contribute to the enhancement of the therapeutic effect of this anti-androgen.