L’élimination programmée d’ADN spécifique de la lignée germinale pour former un nouveau noyau somatique a été décrite chez les eucaryotes. Ces réarrangements sont initiés par l’introduction de cassures double brin (CDB) de l’ADN et la préservation de l’intégrité du génome requiert une réparation efficace. Chez Paramecium tetraurelia, le génome est largement réarrangé pendant le développement du nouveau noyau somatique, après l’introduction de milliers de cassures double brin programmées par la transposase domestiquée PiggyMac (Pgm)Ces réarrangements consistent en l’excision précise de dizaines de milliers de séquences uniques et non codantes (IES) qui interrompent 47% des gènes dans la lignée germinale ; et l’élimination hétérogène de séquences répétées qui mène à des délétions internes de taille variable ou à la fragmentation des chromosomes avec addition de télomères aux extrémités.L’implication de la voie du Non Homologous End Joining (NHEJ) dans l’excision précise des IES a été prouvée. Dans des cellules déplétées de Ligase IV ou XRCC4, les cassures aux bornes des IES sont introduites normalement mais il n’y a pas de jonctions d’excision formées et les extrémités cassées s’accumulent sans être dégradées. Mais la voie de réparation impliquée dans les réarrangements imprécis est encore inconnue. L’hypothèse d’une réparation par la voie NHEJ alternative (alt-NHEJ), indépendante de Ku et impliquant la résection des extrémités et l’utilisation de microhomologie, a été émise. C’est pourquoi pendant ma thèse je me suis intéressé à ma thèse au rôle des protéines Ku.Deux gènes KU70 et trois gènes KU80 ont été identifiés dans le génome de la paramécie. KU70a et KU80c sont spécifiquement induits pendant les réarrangements programmés du génome et les protéines localisent dans les noyaux somatiques en développement. Des expériences d’extinction de ces gènes par ARN interférence ont prouvé que ces gènes étaient indispensables. Au niveau moléculaire, l’ADN non réarrangé est amplifié dans les cellules déplétées de Ku. De plus, les cassures double brin programmées ne sont pas introduites aux bornes des IES.Mes résultats suggèrent que Ku fait partie d’un complexe de pré-excision, avec la transposase domestiquée Pgm, et est nécessaire pour l’introduction des cassures double brin programmées pendant les réarrangements programmés du génome. / Programmed elimination of germline specific DNA has been described in several eukaryotic organisms. These rearrangements are initiated through introduction of DNA double strand breaks (DSB). To ensure genome integrity, efficient repair is needed. In Paramecium tetraurelia, the genome is widely rearranged during development of a new somatic nucleus after introduction of tens of thousands of DSBs by the domesticated transposase PiggyMac (Pgm)These rearrangements consist in: the precise excision of thousands of unique and non coding sequences called IESs that interrupt 47% of genes in the germline; and the heterogeneous elimination of repeated sequences. It leads to internal deletions of variable sizes or to chromosome fragmentation with telomere addition at DNA ends.Implication of the Non Homologous End Joining Pathway (NHEJ) in precise IES excision has been proved. In cells depleted for Ligase IV or XRCC4, DSBs at IES boundaries are introduced normally but broken DNA ends accumulate without being repaired nor degraded. The repair pathway implicated in heterogeneous rearrangements is still unknown. An hypothesis would be that heterogeneous rearrangements involve a Ku independent alternative NHEJ (alt-NHEJ) pathway characterized by end resection and use of microhomologies. During my thesis I studied the role of Ku proteins in programmed genome rearrangements.Two KU70 genes and three KU80 genes has been identified in the Paramecium genome. KU70a and KU80c are specifically induced during programmed genome rearrangements. Encoded proteins localize in developing somatic nuclei. Gene extinction by RNA interference experiments proved that these genes are necessary for programmed genome rearrangements. At molecular level, non rearranged DNA is amplified in cells depleted for Ku. And more surprisingly, no programmed DSBs are introduced at IES boundaries in these cells.My results indicate that Ku is a part of a pre excision complex with the domesticated transposase Pgm and necessary for the introduction of programmed DSB during programmed genome rearrangements.

La réplication des chromosomes est altérée par les facteurs endogènes et/ou exogènes qui perturbent la progression des fourches de réplication. Les cellules doivent donc coordonner la synthèse d’ADN avec des mécanismes assurant la stabilité et le rétablissement des fourches bloquées. La recombinaison homologue (RH) est un mécanisme universel qui permet la réparation de l’ADN et participe au maintien de la réplication des chromosomes. Néanmoins, les mécanismes qui régulent la RH, notamment la RH ectopique versus la RH allélique, restent mal compris. Un autre mécanisme essentiel assurant la stabilité des génomes est l’assemblage de l’ADN néo-synthétisé autour de nucléosomes, conduisant à la constitution de fibres chromatiniennes nécessaires à l’organisation structurale du matériel génétique. Chez Saccharomyces cerevisiae, des défauts d’assemblage de la chromatine conduisent à une instabilité des fourches de réplication et augmentent le taux de RH. Sachant que les chaperonnes d’histones jouent un rôle crucial durant l’assemblage de la chromatine, j'ai décidé de me concentrer sur le rôle de la chaperonne d’histones H3-H4 appelé Chromatin Assembly Factor 1 (CAF-1) dans les mécanismes de RH, chez Schizosaccharomyces pombe. En effet, la RH est associée à une étape de synthèse de l’ADN, et peu de choses sont connues sur l’assemblage de la chromatine au cours de cette synthèse. Mes résultats ont exclu un rôle de CAF-1 dans la recombinaison allelique et le maintien de la stabilité des fourches de réplication. Par contre, CAF-1 joue un rôle important dans les mécanismes de recombinaisons ectopique et dans la formation de réarrangements chromosomiques induits par des blocages de fourches. Mes données suggèrent un modèle selon lequel CAF-1 permet la stabilisation d’intermédiaires de recombinaison précoces (D-loop), via le dépôt de nucleosomes au cours de l’extension par polymérisation de ces intermédiaires. Ainsi CAF-1 neutralise la dissociation des intermédiaires de recombinaison précoces par l’ADN helicase Rqh1. CAF-1 ferait partie d'un équilibre qui règle la stabilité/dissociation des intermédiaires de recombinaison précoces. J'ai montré que le rôle de CAF-1 dans cet équilibre a une importance toute particulière pendant la recombinaison non-allelique, révélant ainsi un nouveau niveau de régulation des mécanismes de RH par l'assemblage de la chromatine. / The replication of chromosomes can be challenged by endogenous and environmental factors, interfering with the progression of replication forks. Therefore, cells have to coordinate DNA synthesis with mechanisms ensuring the stability and the recovery of halted forks. Homologous recombination (HR) is a universal mechanism that supports DNA repair and the robustness of DNA replication. Nonetheless, mechanisms regulating HR pathways, such as ectopic versus allelic recombination, remain poorly understood. Another essential pathway for genome stability is the wrapping of newly replicated DNA around nucleosomes, leading to the constitution of a chromatin fibre, which allows the structural organization of the genetic material. In Saccharomyces cerevisiae, deficiencies in chromatin assembly pathways lead to replication forks instability and consequent increase in the rate of HR. Histone chaperones play a crucial role during chromatin assembly, thus I decided to focus on the H3-H4 histone chaperone Chromatin Assembly Factor 1 (CAF-1), to study its role in HR processes in Schizosaccharomyces pombe. Indeed, HR includes a DNA synthesis step and little is known about the associated chromatin assembly. My data excluded a role for CAF-1 in allelic recombination and in the maintenance of forks stability. However, CAF-1 was found to play an important role during ectopic recombination, in promoting chromosomal rearrangements induced by halted replication forks. My data support a model according to which CAF-1 allows the stabilization of early recombination intermediates (D-loop), via nucleosome deposition during the elongation of these intermediates. Doing so, CAF-1 counteracts the dissociation of early recombination intermediates by the helicase Rqh1. Therefore, CAF-1 appears to be part of an equilibrium that regulates stability/dissociation of early steps of recombination events. Importantly, I found that the role of CAF-1 in this equilibrium is of particular importance during non-allelic recombination, revealing a novel regulation level of HR mechanisms and outcomes by chromatin assembly.

Cette recherche clinique vise à explorer le retentissement de l'hypofertilité masculine et de la fécondation in vitro (FIV) sur le fonctionnement psychique des épouses dans le premier centre de consultation FIV en Egypte (1986), en contexte culturel égyptien. Près de 60% des couples qui consultent au Centre FIV présentent le diagnostic d'infertilité d'origine masculine. Nous nous intéressons à comprendre ce qui constitue la spécificité du fonctionnement psychique de ces femmes dans ce contexte culturel où l'infertilité masculine est une pathologie taboue, suscitant la honte familiale, et où seule la FIV homologue- utilisant les gamètes d'un couple marié- est autorisée par la loi. La première hypothèse renvoie à l'existence d'une souffrance psychique chez l'épouse liée au maintien du secret de l'hypofertilité masculine. La seconde hypothèse suppose que le diagnostic et les traitements médicaux sont accompagnés chez l'épouse d'une idéalisation oedipienne de ses propres parents. Notre échantillon est composé de dix femmes âgées de 20 à 40 ans inscrites dans un protocole FIV. Sur le plan méthodologique, nous nous sommes basés sur des entretiens de recherche semi-directifs et sur l'inventaire abrégé de dépression de Beck BDI-13. L'analyse des résultats met en évidence la présence chez les épouses d'un discours de plainte adressé envers leurs conjoints, en même temps que l'expression d'une idéalisation de leurs propres parents. La présence de symptômes de dépression variables est relevée dans l'échantillon. Enfin, la manière dont les femmes s'approprient subjectivement l'expérience de la FIV est un indicateur pertinent de leur équilibre psychique. Pour conclure, cette étude se veut être une recherche-action visant à mettre en place un dispositif clinique au service des femmes et des couples dans une institution médicale, et qui soit adapté à ce contexte culturel particulier. / This study aims at exploring the impact of male subfertility and In Vitro Fertilization (IVF) on the psychological functioning of a group of female spouses, in the first Egyptian IVF center in Egypt (1986), within the Egyptian cultural context. Approximately 60% of couples consulting at the Egyptian Center for IVF carry the diagnosis of male factor infertility. We are interested in understanding what constitutes the specificity of the psychological functioning of these women within this cultural context, where male infertility is a taboo pathology provoking family shame, and where only homologous IVF -using a married couple's gametes- is allowed by the law. The first hypothesis states that there is a specific female suffering related to the maintenance of the secret of male subfertility. The second hypothesis assumes that for the wife, the diagnosis disclosure and medical treatments are followed by an oedipal idealization of the her parents. Our sample is composed of ten women aged between 20 and 40 years old, and undergoing an IVF protocol. On the methodological aspect, we have used semi-structured interviews and the 13-Item Beck Depression Inventory. Results indicate the existence of a complain discourse among the wives, addressed towards their husbands, and at the same time an idealization of their own parents. Variable depression symptoms have been observed in our sample. Finally, the way women integrate psychologically the IVF experience is a relevant indicator of their psychological balance. As a conclusion, this study aims at being a research-action, which objective is to elaborate a clinical intervention at the service of women and couples, and suitable to this particular cultural context.

BRCA1 est un suppresseur de tumeur majeur jouant un rôle dans la transcription, la réparation de l’ADN et le maintien de la stabilité génomique. En effet, des mutations dans le gène BRCA1 augmentent considerablement le risque de cancers du sein et de l’ovaire. BRCA1 a été en majorité caractérisé pour son rôle dans la réparation de l’ADN par la voie de recombinaison homologue (HR) en présence de bris double brins, par example, induits par l’irradiation gamma (IR). Cependant, la fonction de BRCA1 dans d’autres voies de réparation de l’ADN, comme la réparation par excision de nucléotides (NER) ou par excision de base (BER), demeurent toutefois obscures. Il est donc important de comprendre la régulation de BRCA1 en présence d’agents génotoxiques comme le méthyle méthanesulfonate (MMS) ou l’UV, qui promouvoient le BER et le NER respectivement. Nos observations suggèrent que BRCA1 est dégradée par le protéasome après traitement avec le MMS ou les UV, et non avec l’IR. Par ailleurs, cette dégradation semble compromettre le recrutement de Rad51, suggérant que la voie de HR est inhibée. Nos résultats suggèrent que la HR est inhibée afin d’éviter l’activation simultanée de multiples voies de réparation. Nous avons aussi observé que la dégradation BRCA1 est réversible et que la restauration des niveaux de BRCA1 coïncide avec le recrutement de Rad51 aux sites de dommages. Cela suggère que la HR est réactivée tardivement par les bris double brins générés suite à l’effondrement des fourches de réplication. Ayant observé que BRCA1 est hautement régulé par l’ubiquitination et est ciblé par le protéasome pour dégradation, nous avons émis une hypothèse que BRCA1 est régulé par des déubiquitinases. Cela amène à caractériser plus en profondeur par un criblage en déplétant les déubiquitinases individuellement par RNAi et en observant leur effet sur le recrutement de BRCA1 et des protéines reliées à cette voie. Un criblage préliminaire nous a permi d’identifié candidats potentiels tel que BAP1, CXORF53, DUB3, OTUB1 et USP36. / BRCA1 is a tumour suppressor involved in transcription, DNA repair and maintenance of genomic stability. Indeed, BRCA1 mutation carriers have an exceptionally higher risk of breast and ovarian cancers. BRCA1 is mainly known for its role in homologous recombination repair (HR) by recruiting HR proteins to chromatin upon double strand break (DSBs) formation, e.g., following treatment with ionizing irradiation (IR). However, the function of BRCA1 in other DNA repair pathways such as nucleotide excision repair (NER) or base excision repair (BER) is still obscure. It is thus of fundamental and clinical importance to investigate BRCA1 function following exposure to diverse genotoxic agents. Using human cultured cell, we observed that BRCA1 is downregulated by the proteasome upon treatment with MMS or UV, but not with IR. Moreover, this downregulation prevents Rad51 recruitment to chromatin following exposure to MMS. Given that DNA damage induced by UV and MMS trigger NER and BER pathways respectively, this implies that HR could be inhibited in order to prevent competition between independent DNA repair pathways. We also found that BRCA1 downregulation is reversible and the recovery of BRCA1 levels correlates with the reappearance of BRCA1 and Rad51 on chromatin. This implies that the HR has been reactivated at the late stage of DNA damage for the repair of double strand breaks generated by replication fork collapse. Since BRCA1 stability is highly regulated by ubiquitination and is downregulated following MMS treatment, one would expect that a deubiquitinase is responsible for relieving this downregulation to promote the reactivation of the HR pathway. To characterize this aspect further, we conducted DUB RNAi screens in which a particular DUB is depleted and the localization of BRCA1 and other related proteins were observed. According to a preliminary screen, a few DUBs (BAP1, CXORF53, DUB3, OTUB1, and USP36) were identified as potential regulators of the stability and localization of BRCA1 and proteins involved in homologous recombination.

No description available.

The transplantation of allogeneic cells and tissues for the treatment of human disease has been a life-saving procedure for many thousands of patients worldwide. However, to date, neither solid organ transplantation nor bone marrow transplantation have reached their full clinical potential. Significant limitations to the advancement of clinical transplantation stem from our current inability to prevent the rejection of allogeneic tissues by the immune system of the host. Similarly, in patients that receive allogeneic bone marrow transplants, we cannot permanently prevent the engrafted immune system from mounting a response against the patient. This problem, termed graft versus host disease is the most prevalent cause of morbidity and mortality in recipients of allogeneic bone marrow transplants. Clinically, we rely on lifelong immunosuppression to prolong survival of allogeneic tissues within the host. Our currently available therapeutics burden patients with side-effects that range from being unpleasant to life-threatening, while in most cases offering only a temporary solution to the problem of alloimmunity. Efforts are underway to develop protocols and therapeutics that more effectively prevent the pathology associated with alloimmunity. To minimize patient risk, extensive pre-clinical studies in laboratory animals are conducted to predict clinical responses. In the case of immunologic studies, many of these pre-clinical studies are carried out in murine models. Unfortunately, studies of murine immunity often do not predict outcomes in the clinic. One approach to overcome this limitation is the development of a small animal model of the human immune system. In this dissertation, we hypothesized that NOD-scid IL2rγnull mice engrafted with human peripheral blood mononuclear cells (PBMC), termed the hu-PBMC-NOD-scid IL2rγnull model, would provide a model that more accurately reflects human immunity in vivo than other models currently available. To investigate this possibility, we first investigated whether NOD-scid IL2rγnull mice were able to support the engraftment of human PBMC. We found that NOD-scid IL2rγnull mice engraft with human PBMC at much higher levels then the previous gold standard model, the NOD-scid mouse. We then investigated the kinetics of human cell engraftment, determined the optimal cell dose, and defined the influence of injection route on engraftment levels. Even at low PBMC input, NOD-scid IL2rγnullmice reproducibly support high levels of human PBMC engraftment. In contrast to previous stocks of immunodeficient mice, we observed low intra- and interdonor variability of engraftment. We next hypothesized that the human PBMC engrafted in NOD-scid IL2rγnull mice were functional and would reject transplanted allogeneic human tissues. To test this, human islets were transplanted into the spleen of chemically diabetic NOD-scid IL2rγnull mice with or without intravenous injection of HLA-mismatched human PBMC. In the absence of allogeneic PBMC, the human islets were able to restore and maintain normoglycemia. In contrast, human islet grafts were completely rejected following injection of HLA-mismatched human PBMC as evidenced by return to hyperglycemia and loss of human C-peptide in the circulation. Thus, PBMC engrafted NOD-scid IL2rγnull mice are able to provide an in vivomodel of a functional human immune system and of human islet allograft rejection. The enhanced ability of NOD-scid IL2rγnull mice to support human cell engraftment gave rise to the possibility of creating a model of graft versus host disease mediated by a human immune system. To investigate this possibility, human PBMC were injected via the tail vein into lightly irradiated NOD-scid IL2rγnull mice. We found that in contrast to previous models of GVHD using human PBMC-injected immunodeficient mice, these mice consistently (100%) developed GVHD following injection of as few as 5x106PBMC, regardless of the PBMC donor used. We then tested the contribution of host MHC in the development of GVHD in this model. As in the human disease, the development of GVHD was highly dependent on host expression of MHC class I and class II molecules. To begin to evaluate the extent to which the PBMC-engrafted NOD-scid IL2rγnull humanized mouse model of GVHD represents the clinical disease, we tested the ability of a therapeutic in clinical trials to modulate GVHD in these mice. In agreement with the clinical experience, we found that interrupting the TNFα signaling cascade with etanercept delayed the onset and severity of disease in this model. In summary, we conclude that humanized NOD-scid IL2rγnull mice represent an important surrogate for investigating in vivo mechanisms of both human islet allograft rejection and graft versus host disease.

Species of the Bordetella genus cause the highly contagious whooping cough disease in humans (B. pertussis, B. parapertussis) and related respiratory diseases in other mammals (B. bronchiseptica, B. parapertussis). One of the virulence systems of Bordetellae is the type III secretion system (T3SS) employed for translocation of effector proteins directly from bacterial cytosol into the cytosol of host cells. The T3SS protein BopN protein has been categorized as a Bordetella effector protein. Nevertheless, the homologous proteins in other gram-negative bacteria function in establishing the secretion hierarchy through T3SS and some of them block T3SS secretion in high calcium environments before bacteria-host cell contact has been established. In this thesis I examined the function of the BopN protein and the role of calcium ions in T3SS activity of B. bronchiseptica. Two independent methods have been used for determination of T3SS secretion activity. Addition of 2 mM calcium ions into bacterial media decreased secretion of the T3SS reporter, while no such effect was observed in a B. bronchiseptica strain lacking the bopN gene. Mass spectrometry data confirmed the inhibition of T3SS activity in the presence of calcium ions. Enhanced calcium levels resulted in decreased mobilization and secretion of...

Made available in DSpace on 2015-07-22T20:49:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-10-29. Added 1 bitstream(s) on 2015-08-11T03:25:34Z : No. of bitstreams: 1 Publico-018.pdf: 1910669 bytes, checksum: a40b61267560276f1db47642341c6e56 (MD5) / Introdução: Polimorfismos genéticos estão associados com um aumento do risco de tromboembolismo venoso (TEV) e outras doenças cardiovasculares. Estudos prévios sugerem que a doença venooclusiva hepática (DVOH), que se desenvolve intra transplante de medula óssea pode ser atribuída à polimorfismos gênicos. Objetivos: Avaliar a correlação entre polimorfismos genéticos e o risco de doença venooclusiva hepática no transplante de medula óssea e a associação com a presença de variáveis como o uso de vancomicina, drogas citotóxicas usadas no regime de condicionamento, presença de disfução hepática anterior ao transplante, presença de infecção por citomegalovírus, e grupos de doenças avaliáveis. Pacientes e métodos: Este estudo caracteriza-se por um estudo clinico observacional multicêntrico. Foram avaliados 120 pacientes, submetidos a transplante alogênico de medula óssea. Cada paciente foi avaliado propectivamente para DVOH, confirmado pelo critério de Seattle modificado, idade variando de 2 a 65 anos. Fatores de risco para DVOH severo foram analisados usando modelos estatisticos de avaliação. Detectamos simultaneamente através de PCR seguido de digestão e detecção através de eletroforese em gel de agarose, mutação do HFE C282Y, H63D, S65C; MTHFR C677T; Fator V de Leiden, Protrombina 20210A e glutationa S-tranferase. Resultados: Neste estudo observacional de pessoas, submetidas a transplante de medula óssea alogênico, a presença de disfução hepática pré transplante foi um fator de risco significante para DVOH. Nós tambem observamos uma associação entre a presença de mutação HFE S65C e disfunção hepática. Conclusões: Em resumo, nossos dados sugerem que disfunção hepatica observada pré transplante de medula óssea é um fator de risco estatisticamente significante para DVOH e que a presença da mutação do HFE S65C apresenta uma tendência ao desenvolvimento de dano hepático. Contudo, não evidenciamos associação dos polimorfismos estudados com DVOH. / TEDE

Tolerance-based stem cell transplantation using sub-lethal conditioning is being considered for the treatment of human disease, but safety and efficacy remain to be established. In order to study these two issues, we first established that mouse bone marrow recipients treated with sub-lethal irradiation plus transient blockade of the CD40-CD154 costimulatory pathway develop permanent hematopoietic chimerism across allogeneic barriers. Our conditioning regimen of 6 Gy irradiation, a short course of anti-CD154 mAb and 25 million fully allogeneic BALB/c bone marrow cells consistently produced long-term, stable, and multilineage chimerism in C57BL/6 recipients. Furthermore, chimeric mice displayed donor-specific transplantation tolerance, as BALB/c skin allografts were permanently accepted while third-party CBA/JCr skin allografts were promptly rejected. We next determined both the safety and efficacy of this protocol by infecting chimeric mice with lymphocytic choriomeningitis virus (LCMV) either at the time of transplantation or at several time points afterwards. Infection with LCMV at the time of transplantation prevented engraftment of allogeneic, but not syngeneic, bone marrow in similarly treated mice. Surprisingly, infected allograft recipients also failed to clear the virus and died. Post-mortem study revealed hypoplastic bone marrow and spleens. Hypoplasia and death in these mice required the combination of 6 Gy irradiation, LCMV infection on the day of transplantation, and an allogeneic bone marrow transplant but did not require the presence of anti-CDl54 mAb. Allochimeric mice infected with LCMV 15 days after transplantation were able to survive and maintain their bone marrow graft, indicating that the deleterious effects of LCMV infection on host and graft survival are confined to a narrow window of time during the tolerization and transplantation process. The final section of this thesis studied the mechanisms of graft rejection and death in sublethally irradiated recipients of allogeneic bone marrow and infection with LCMV at the time of bone marrow transplantation. Infection of interferon-α/β receptor knockout mice at the time of transplantation prevented the engraftment of allogeneic bone marrow, but the mice survived. Therefore, IFN-αβ is involved in the development of marrow hypoplasia and death, whereas a second mechanism is involved in blocking the development of chimerism in these mice. Through the use of depleting mAb's and knockout mice we demonstrate that three types of recipients survived and became chimeric after being given sublethal irradiation, anti-CD154 mAb, an allogeneic bone marrow transplant and a day 0 LCMV infection: mice depleted of CD8+ T cells, CD8 knockout mice, and TCR-αβ knockout mice. Our data indicate that the mediator of bone marrow allograft destruction in LCMV-infected mice treated with costimulatory blockade is a radioresistant CD8+ NK1.1- TCRαβ+ T cell. We conclude that a non-cytopathic viral infection at the time of transplantation can prevent engraftment of allogeneic bone marrow and result in the death of sub-lethally irradiated mice treated with costimulation blockade. The abrogation of allogeneic bone marrow engraftment is mediated by a population of CD8+ NK1.1- TCRαβ+ T cells and the mediator of hypoplasia and death is viral induction of IFN-αβ.

Notre génome est continuellement endommagé par des agents provoquant des lésions de l’ADN. Les cassures doubles brins de l’ADN (CDBs) sont les lésions les plus dangereuses. En effet, une CDB mal réparée peut mener à des aberrations de l’ADN pouvant conduire à l’apparition d’un cancer. Dans le but d’éviter les effets délétères des CDBs, nos cellules ont développé une voie de signalisation, nommée réponse aux dommages de l’ADN (RDA), permettant la détection des cassures et l’activation des points de contrôle du cycle cellulaire afin d’arrêter le cycle pendant la réparation des CDBs. Une des caractéristiques principales de la RDA est l’accumulation d’un grand nombre de facteurs sur l’ADN autour de la cassure, formant un foyer visible en microscopie. Cependant, l’efficacité de réparation de l’ADN est entravée par la structure condensée de la chromatine environnante. Les mécanismes de réparation de l’ADN surmontent ce problème en recrutant de nombreuses protéines permettant le réarrangement de la chromatine afin de faciliter la réparation. Le but de mon travail de thèse est d’identifier de nouvelles protéines impliquées dans le remodelage de la chromatine autour des CDBs. D’une part nous avons pour but d’identifier le protéome complet d’un foyer de réparation de l’ADN grâce à la technique PICh (Proteomics of Isolated Chromatin loci). D’autre part, nous étudions le rôle de l’oncoprotéine SET/TAF-1β, que nous avons identifié lors d’un criblage siRNA réalisé dans le but de découvrir de nouveaux facteurs chromatiniens impliqués dans la réparation des CDBs. / Various DNA damaging agents, that can cause DNA lesions, assault constantly our genome. The most deleterious DNA lesions are the breaks occurring in both strands of DNA (Double stand breaks: DSBs). Inefficient repair of DSBs can lead to aberrations that may induce cancer. To avoid these deleterious effects of DSBs, cells have developed signalling cascades which entail detection of the lesions and spreading of the signal that leads to arrest in cell cycle progression and efficient repair. A major characteristic of DNA damage response (DDR) is the accumulation of a vast amount of proteins around the DSBs that are visible in the cell as DNA damage foci. However, efficient DNA repair is hampered by the fact that genomic DNA is packaged into chromatin. The DNA repair machinery overcomes this condensed structure to access damaged DNA by recruiting many proteins that remodel chromatin to facilitate efficient repair. The aim of my PhD work is to identify novel proteinsinvolved in the DDR and/or the remodelling of chromatin surrounding DSBs. On one hand, we take advantage of the PICh (Proteomics of Isolated Chromatin loci) technique and we aim to identify the entire proteome of DNA repair foci. On the other hand, we study the role of the oncogene SET/TAFIβ, a major hit of a siRNA screen performed to identify novel chromatin related proteins that play role in repair of DSBs.