A técnica de cultura de tecidos permite a propagação rápida e maciça de propágulos geneticamente semelhantes, isentos de doenças, sendo amplamente empregada para a obtenção de gemas adventícias e embriões somáticos visando principalmente, a multiplicação clonal sob ação de reguladores de crescimento. Resultados satisfatórios foram obtidos com a espécie Bactris gasipaes Kunth. por meio da regeneração direta de gemas adventícias e de embriões somáticos, no entanto, as consequências decorrentes da prolongada manutenção in vitro de espécies perenes como a pupunheira, não estão elucidadas, sendo que as pesquisas mais expressivas ocorrem com espécies anuais e restritas a órgãos específicos. Considerando que o tempo de cultivo pode promover a senescência e a habituação de determinados tecidos aos reguladores de crescimento, afetando consideravelmente o potencial morfogênico, o objetivo principal do presente trabalho foi investigar a ocorrência destes processos em folhas, raízes e bases caulinares de plântulas e microplantas com um e oito anos de cultivo, respectivamente. Para tanto, o processo de senescência foi monitorado por meio de análises histológicas, ultraestruturais e histoquímicas à detecção de substâncias ergásticas e à fragmentação do DNA, ao passo que o processo de habituação, foi monitorado por meio de análises morfofisiológicas, histológicas e histoquímicas. Os resultados pertinentes ao processo de senescência evidenciaram a ocorrência de intenso processo de morte celular programada nas células de diversos tecidos nas estruturas analisadas das microplantas, sendo que estes eventos foram escassos e limitados às bases caulinares nas plântulas. Além disso, foi observada a presença elevada de plastoglóbulos no interior dos cloroplastos e de compostos fenólicos nas estruturas foliares e radiculares das microplantas. Já, em relação aos resultados obtidos à detecção do processo de habituação nestas microplantas, quando comparados às plântulas, foram detectados problemas relacionados ao alongamento da parte aérea e do sistema radicular, bem como alterações morfológicas nas raízes e uma pronunciada redução no potencial morfogênico das células pré-procambiais em relação às plântulas. Estes resultados evidenciam que a manutenção in vitro de pupunheiras por longos períodos promoveu o envelhecimento dos propágulos em decorrência à senescência generalizada, bem como provavelmente os conduziu ao processo de habituação aos reguladores de crescimento ANA e/ou BAP, inviabilizando a propagação em grande escala desta espécie. / The tissue culture technique allows rapid and massive spread of propagules genetically similar, free from diseases, being the technique widely employed to obtain adventitious buds and somatic embryos mainly targeting the clonal multiplication under the action of growth regulators. Satisfactory results have been obtained with the specie Bactris gasipaes Kunth. through direct regeneration of adventitious buds and somatic embryos, however, the consequences from prolonged in vitro maintenance of perennial species such as peach palm are not clear, and the most significant research occur with annual species and restricted to specific organs. Whereas the cultivation time can promote senescence and habituation of certain tissues to the growth regulators, affecting considerably the morphogenic potential, the main objective of this study was to investigate the occurrence of these processes in leaves, roots and stem bases of seedlings and microplants with one and eight years of cultivation, respectively. Thus, the senescence process was monitored by histological, ultrastructural and histochemical detection of ergastic substances and DNA fragmentation, whereas the habituation process was monitored by analyses histologic, histochemic and morpho-physiological. The relevant results from the senescence process showed the occurrence of an intensive process of programmed cell death in cells of various tissues of the analised microplants structures, and these events were rare and limited to the stem bases in the seedlings. Furthermore, it was observed the high presence of plastoglobules inside chloroplast and phenolic compounds in the leaf and root structure of microplants. Already, the results obtained in relation to the detection of the habituation process in these microplants, when compared to the seedlings, were detected problems related to the elongation of shoots and roots, as well as morphological changes in roots and a pronounced reduction in the morphogenic potential of pre procambial cells compared to seedlings. These results demonstrate that the in vitro maintenance of peach palm for long periods promoted the aging of seedlings due to senescence widespread and probably led to the habituation process, to the growth regulators NAA and/or BAP, difficulting the propagation on a large scale of this species.

No description available.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / After the progressive telomere shortening in successive cell divisions, normal somatic cells undergo a growth arrest called cellular senescence that occurs due to incomplete DNA replication. Senescence can also be activated by various types of stressful stimuli, including aberrant oncogenic signaling, oxidative stress and DNA damage. Senescent cells have limited proliferative capacity and seems to play an important role in tumorigenesis. They are also involved in the inflammation associated with aging and cancer progression. The process of senescence vary significantly between cells, but the different paths for the aging, however, converge to p53 and pRB. The network simulation is based on the model proposed by Porath using a Boolean model to represent the state of activation of genes involved, including the p16-pRb and p53-p21 pathways. The simulation includes 23 nodes representing the genes of the regulatory network where one of them represents the activation of the senescent state as a result of network processing. Experiments with human fibroblasts indicate that inactivation of both genes, p53 and pRB is necessary to block senescence. The simulations confirms that these pathways are able to trigger senescence independently. The simulation shows that pRb is essential to maintain the senescent state even when p16 and p53 are switched off, but the simultaneous inactivation of both p53 and pRB blocks senescence. In addition, the simulation shows that inactivation of the p16-pRb pathway is not essential to preserve the senescent state, however when p53-p21 pathway is inactivated, the senescent state is preserved. / Após o progressivo encurtamento dos telômeros em sucessivas divisões celulares, as células somáticas normais se submetidas a uma parada do crescimento chamado senescência celular que ocorre devido à replicação incompleta do DNA. A senescência também pode ser ativada por diversos tipos de estímulos estressantes, incluindo sinalização oncogênica aberrante, estresse oxidativo e danos ao DNA. Células senescentes têm capacidade proliferativa limitada e parecem desempenhar um papel importante na tumorigênese. Elas também estão envolvidas na inflamação associada com o envelhecimento e progressão do câncer. As vias de senescência variam significativamente entre as células, mas os caminhos diversos para a senescência, no entanto, convergem para p53 e pRb. A simulação é baseada no modelo proposto por Porath usando um modelo booleano para representar o estado de ativação dos genes envolvidos, incluindo as vias p16-pRb e a p53-p21. A simulação inclui 23 nós representando os genes da rede regulatória onde um deles representa o estado celular senescente que pode assumir estados Verdadeiro ou Falso como resultado do processamento de rede Experiências com fibroblastos humanos indicam que a inativação de ambos os genes, p53 e pRb, é necessária para bloquear a senescência. As simulações confirmam que essas vias são capazes de acionar a senescência independentemente. A simulação mostra que pRb é essencial para a manutenção do estado senescente mesmo se p16 e p53 forem desligados, no entanto a inativação simultânea de ambos p53 e pRb bloqueia senescência. Além disso, a simulação mostra que a inativação da via p16-pRb não é essencial para preservar o estado senescente, no entanto, quando a via p53-p21 é inativada, o estado senescente é preservado.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Handroanthus chrysotrichus is a Brazilian native forest species which presents several important aspects both under the economic and ecological points of view, because its ability to provide timber products as well as for being indicated for degraded and permanent preservation areas recovery. Its seeds present problems concerning loss of viability when subjected to storage. This way, micropropagation emerges as an alternative for the species propagation. However, some characteristics inherent in in vitro grown plants may promote physiological and anatomic changes in tissues under that condition. Thus, the aim of this study was to evaluate aspects related to micropropagation and leaf anatomy of H. chrysotrichus. Firstly, health and germination tests were applied to seeds from different lots of that species. As for in vitro multiplication of H. chrysotrichus explants, different sources and concentrations of cytokines combined or not with auxin were tested, as well as the influence of successive subculture execution on multiplication rates. Aspects concerning leaf senescence of sprouts maintained in vitro in Woody Plant Medium (WPM) were also assessed. Concerning rooting in vitro, different concentrations of 3-Indolebutyric acid (IBA) combined with the presence or absence of vermiculite in ½ WPM were tested and, later, the ex vitro acclimatization of plants micropropagated was assessed. Finally, the test was conducted in order to compare the leaf anatomy of H. chrysotrichus plants grown in vivo, in vitro and acclimatized. The seed lots of H. chrysotrichus showed differences in health quality and geminal capacity of the seeds. As for in vitro multiplication of H. chrysotrichus, the use of cytokinins was not considered necessary. High rates of in vitro multiplication were obtained, when considered in vitro formation of buds. As for leaf senescence, the species showed great potential, remaining long periods in the same nutrient medium. There was rooting in vitro in more than 50% of the shoots cultivated in the presence of vermiculite, regardless IBA concentration. It was not possible to satisfactorily promote the acclimatization of micropropagated plants, as only 7.12% of the seedlings survived. Differences in leaf anatomy of plants grown in vivo, in vitro or acclimatized were verified. The results can contribute with information about H. chrysotrichus, concerning aspects related to its in vitro culture and leaf anatomy of the tissues grown in different environments. / Handroanthus chrysotrichus é uma espécie florestal nativa do Brasil, que apresenta diversos aspectos importantes tanto sob o ponto de vista econômico, pela produção de produtos madeiráveis, como, também, ecologicamente, uma vez que é indicada para a recuperação de áreas degradadas e de preservação permanente. As sementes desta espécie apresentam problemas referentes à perda da viabilidade quando submetidas ao armazenamento. Sendo assim, a micropropagação emerge como uma alternativa de propagação para a espécie. No entanto, algumas características inerentes às plantas cultivadas in vitro podem promover alterações fisiológicas e anatômicas nos tecidos mantidos nessas condições. Desse modo, o objetivo deste estudo foi avaliar aspectos relacionados à micropropagação e à anatomia foliar de H. chrysotrichus. Primeiramente, foram realizados testes de sanidade e germinação nas sementes provenientes de diferentes lotes desta espécie. Já para a multiplicação in vitro de explantes de H. chrysotrichus foram testadas diferentes fontes e concentrações de citocininas combinadas ou não à auxina, bem como a influência da execução de sucessivos subcultivos nas taxas de multiplicação. Também foram avaliados aspectos relacionados à senescência foliar de brotações desta espécie conservadas in vitro em meio nutritivo WPM. Em relação ao enraizamento in vitro, testaram-se diferentes concentrações de Ácido 3-Indolbutírico - AIB combinadas à presença ou ausência de vermiculita no meio nutritivo ½WPM e, posteriormente, avaliou-se a aclimatização ex vitro das plantas micropropagadas. Por fim, foi realizado o ensaio visando comparar a anatomia foliar de plantas de H. chrysotrichus cultivadas in vivo, in vitro ou aclimatizadas. Os lotes de sementes de H. chrysotrichus apresentaram diferenças na qualidade sanitária e capacidade geminativa das sementes. Na multiplicação in vitro de H. chrysotrichus, o uso de citocininas foi considerado dispensável. Foram obtidas elevadas taxas de multiplicação in vitro em H. chrysotrichus, quando considerada a formação in vitro de gemas. Quanto à senescência foliar, a espécie demonstrou grande potencial, podendo permanecer longos períodos no mesmo meio nutritivo. Houve rizogênese in vitro em mais de 50% das brotações cultivadas na presença de vermiculita, independentemente das concentrações de AIB. Não foi possível promover a aclimatização das plantas micropropagadas de maneira satisfatória, sendo que apenas 7,12% das mudas sobreviveram. Foram verificadas diferenças na anatomia foliar de plantas desenvolvidas in vivo, in vitro ou aclimatizadas. Os resultados obtidos poderão contribuir com informações a respeito de H. chrysotrichus quanto a aspectos relacionados ao seu cultivo in vitro e anatomia foliar dos tecidos cultivados em diferentes ambientes.

Nos travaux montrent qu’au cours de la fibrillation atriale (FA), les microparticules (MP) reflètent et contribuent à un état d’hypercoagulabilité et pro-inflammatoire. Leurs concentrations similaires dans les deux oreillettes de patients en FA témoignent d’une absence de différence de statut pro-thrombotique entre ces deux cavités cardiaques. Au cours des procédures d’ablation de FA, les concentrations de MP évoluent parallèlement à l’augmentation de l’activation cellulaire et plaquettaire. Nous avons également montré dans l'altération tissulaire des oreillettes en FA, l'importance de la sénescence qui évolue avec la progression du trouble du rythme. Nous avons caractérisé un modèle cellulaire de sénescence réplicative de cellules endothéliales auriculaires de porc permettant d'identifier l'apparition d'un phénotype pro-thrombotique, pro-inflammatoire, pro-adhésif et de mieux comprendre la physiologie de la cellule endothéliale atriale sénescente et le rôle majeur du système rénine-angiotensine dans ces mécanismes. / Our data evidence that during atrial fibrillation (AF), microparticles (MP) contribute to an enhanced hypercoagulable and pro-inflammatory state. Similar concentrations of MP measured in left and right atria of AF patients highlight the absence of chamber-specific enhanced thrombogenic status. During AF ablation procedures, MP concentrations progress in parallel with cell and platelet activation. We also showed that AF progression is strongly related to human atrial senescence burden pointing toward a possible network that links in human atrium, senescence burden, endothelial dysfunction, thrombogenicity and atrial remodeling. We also developed a model of left atrium endothelial cell replicative senescence providing compelling evidences indicating that atrial endothelial senescence promotes thrombogenicity, inflammation and proteolysis. These data underline the major role of renin-angiotensin system in endothelial atrial cell senescence.

Le cancer colorectal est un problème de santé publique majeur pour lequel la recherche de nouveaux traitements est indispensable. Notre équipe a démontré l’efficacité d’une immunothérapie par un lipide A dans un modèle de cancer du colon chez le rat. Lorsque les rats sont porteurs de petites carcinomatoses, le lipide A induit la guérison de 95% des rats. L’étude des mécanismes d’action de cette immunothérapie nous a permis de montrer que l’effet antitumoral du lipide A est dépendante de la cytotoxicité induite par le granzyme B produit par les neutrophiles intratumoraux. En effet, nous avons montré que, dans le microenvironnement tumoral, les neutrophiles produisent du granzyme B et présentent un phénotype de type N2 protumorigène. Lorsque les rats sont traités par lipide A, il y a modification du phénotype des neutrophiles en type N1 antitumoral et libération du granzyme B qui induit l’apoptose des cellules tumorales. Lorsque les rats développent des tumeurs beaucoup plus volumineuses, l’efficacité du lipide A est diminuée et seul 40% des animaux sont guéris. L’injection préalable d’oxaliplatine permet alors de maintenir l’efficacité de l’immunothérapie par le lipide A. Nous avons montré que l’oxaliplatine induit la sénescence des cellules tumorales, générant ainsi un microenvironnement propice au recrutement au sein des tumeurs des neutrophiles, lesquels sont activables par l’immunothérapie subséquente. L’association de l’induction de la sénescence et de l’activation des cellules immunitaires par immunothérapie est une approche efficace et originale sur laquelle les recherches doivent se poursuivre. / Colorectal cancer is a major public health concern in France. Resistance to standard chemotherapy requires development of novel therapeutic approaches. In the past decades, our team showed the immunotherapeutic properties of lipid A in a model of colon cancer in rats. 95% of rats bearing small carcinomas were cured following treatment by lipid A. The study of mechanisms underlying this immunotherapy allowed us to show that the antitumor effect of lipid A was dependent on cytotoxicity induced by granzyme B produced by intratumoral neutrophils. Indeed, we have shown that, in the tumor microenvironment, neutrophils produced granzyme B and had a pro-tumorigenic N2 phenotype. When rats were treated with lipid A, neutrophils shifted to an antitumor N1 phenotype and released granzyme B, thus inducing apoptosis of tumor cells. In rats bearing advanced carcinoma, the effectiveness of lipid A was reduced and only 40% of animals were cured. An injection of oxaliplatin prior to lipid A treatment allowed sustaining the effectiveness of lipid A immunotherapy. In the present study, we showed that oxaliplatin injection induced tumor cell senescence. The microenvironment produced by senescent cells enabled then the recruitment of neutrophils within tumors, subsequently activated by lipid immunotherapy.Combining the induction of tumor cells senescence and activation of immune cells by an immunotherapeutic agent constitute an original and interesting therapeutic approach, but still studies must be carrying out to better understand underlying mechanisms.

Comprendre les mécanismes de sénescence ou développer de nouveaux anti-cancéreux impose d’utiliser des modèles biologiques divers et complémentaires. De par sa facilité de mise en œuvre, la culture cellulaire est une des méthodes les plus employées. Cependant, travailler sur des cellules isolées ne permet pas toujours d’extrapoler les résultats à des conditions plus complexes comme un organisme entier. Les biologistes disposent alors de modèles intégrés plus ou moins évolués : levures, poissons (zebrafish …), grenouille (xénope …), insectes (drosophile …), rongeurs (souris, rats …), chien, primates, etc. Cependant leur mise en œuvre est souvent difficile, tant d’un point de vue administratif que technologique.L’objectif de notre travail a été d’initier le développement de deux nouveaux modèles biologiques intégrés (Caenorhabditis elegans et Hypsibius dujardini), beaucoup plus facilement manipulables, pour des études en oncopharmacologie et sénescence.Le potentiel de Caenorhabditis elegans a été évalué au travers de deux axes : (i) nous avons utilisé le nématode pour déterminer les potentiels toxiques, mutagènes et cancérogènes de gaz candidats au remplacement du gaz SF6 (ii) à l’aide du Seahorse XF24 Analyzer, nous avons mis au point un test fonctionnel original : la mesure de la respiration mitochondriale du nématode entier.Nous avons choisi de développer comme second modèle, Hypsibius dujardini, beaucoup moins travaillé que C. elegans. C’est un tardigrade connu pour sa résistance aux conditions extrêmes, notamment à la dessication. Afin de mieux comprendre cette résistance, nous avons choisi d’analyser le fonctionnement mitochondrial de cet organisme en sortie d’anhydrobiose, en utilisant des marquages mitochondriaux et en mesurant sa respiration à l’aide du Seahorse XF24 Analyzer. / Biological models are necessary to understand how organisms works, how evolution of living run, to test new treatments or to perform toxicological assessments as well. Cellular culture is one of the methods employed because it is easy to use. However, working on isolated cells doesn’t always allow to challenge of the results with more complex conditions as found in full organisms. Biologist needs to develop new biological models for new assessments but the new model choice can be a problem. Murine model, frog, drosophila, yeast, zebrafish,… have each other some benefits and limits. Their choice is directly linked with their use and with the type of research we intend to make.The aim of our work was to develop biological models to oncopharmacology and aging studies.The nematode model with Caenorhabditis elegans was used in several projects. One study was made on gases. They were tested in terms of toxicity, mutagenicity and cancerogenicity. On the other hand, a new tool was developed for prospective studies on either toxicology or mechanistic with the mitochondrial respiration measure with the Seahorse XF24 Analyzer device.The second biological model studied is the tardigrade and more exactly Hypsibius dujardini. Tardigrades are extremely resistant organisms to harshest conditions. They can resist to desiccation. To gain insights on tardigrade resistance, we have choice to analyze the mitochondrial dynamics in the course of anhydrobiosis exit by using mitochondrial dyes and respiration measurements.

Grâce à leurs propriétés de différenciation, les cellules stromales mésenchymateuses (CSM) constituent aujourd’hui un outil en médecine régénérative. La moelle osseuse reste une des plus utilisées. Une diminution de la capacité de prolifération et de différenciation des CSM-MO, au cours des passages, a été montrée. En parallèle, certaines études montrent que l'impact de l'âge du donneur sur les propriétés de CSM-MO reste encore controversé. Le but de notre étude était de mieux comprendre l'effet de l'âge du donneur mais aussi des passages en culture sur la capacité de prolifération et de différenciation des CSM de moelle osseuse. Les échantillons ont été séparés en 4 groupes en fonction de l’âge des donneurs (<20 ans; 20-40 ans; 40-60 ans; >60 ans) et les analyses ont été réalisés lors de la culture de cellules pendant 5 passages. Les résultats obtenus montrent que la capacité de prolifération de CSM-MO obtenues à partir de donneurs jeunes est supérieure à celle de cellules des donneurs âgés. De plus, cette capacité de prolifération diminue en fonction des passages en culture. En parallèle, la capacité des cellules à former des colonies, mesurée par le test CFU-F, diminue légèrement en fonction de l’âge des donneurs mais de façon importante en fonction du passage. Enfin, la capacité de différenciation des CSM-MO vers les trois types cellulaires étudiés, diminue en fonction des passages de cellules mais également en fonction de l’âge des donneurs. Notre étude montre que les propriétés des CSM issues de moelle osseuse sont modifiées lors de l’amplification in vitro mais aussi en fonction de l’âge des donneurs / Today with their properties of differentiation into specific cells types, mesenchymal stromal cells (MSC) can be used in regenerative medicine. Bone marrow (BM) is the better characterized one. The researchers have proven that with increasing passage number in culture the proliferation and differentiation potential of MSC decrease. In parallel many researchers have showed the impact of donor age on MSC properties remains controversial. The aim of our study was to better understand the effect of donor age but also culture passages on the proliferation and differentiation ability of bone marrow mesenchymal stromal cells. The samples were separated into 4 groups depending on the donor age (<20 years; 20-40 years; 40-60 years; > 60 years) and The samples were cultured for 5 passages. The results obtained show that the MSC proliferative capacity obtained from young donors is greater than that of cells from older donors. In addition, the proliferative capacity decreases with increasing passage number in culture. In parallel, the ability of colony-forming unit-fibroblast, measured by the CFU-F assay, decreases slightly depending on the age of the donors but significantly depending on the passage. Finally, the MSC differentiation ability decreases according to the passage of the cells but also depending on the donor age. Our study shows that the properties of bone marrow derived MSC are modified not only during amplification in vitro but also in terms of donor age

En dehors du cadre pathologique, la notion de temps est la base essentielle dans le processus de vieillissement de l’organisme et des systèmes associés. Ces derniers vont progressivement présenter un déclin de leur(s) fonction(s) où de nombreux mécanismes complexes vont intervenir à différents niveaux. Parmi les premiers constats proposés, le vieillissement est la conséquence d’un processus inéluctable, d’une succession d’agressions au niveau cellulaires qui pourraient être réparées voire évitées, ouvrant ainsi la voie à de futures études qui permettront à terme de proposer une explication détaillée, complète et claire de ce processus. Pour exemple, les travaux que nous avons menés tentent d’apporter un élément de réponse afin d’établir un lien entre sénescence et vieillissement, avec pour base de générer par reprogrammation cellulaire des cellules hiPSCs à partir de cellules issues de biopsies de patients jeunes et âgées, sénescentes ou prolifératives. Outre la caractérisation de leur état pluripotent comparable à celui des cellules souches embryonnaires, nous avons également mis en évidence après une différenciation spécifique en fibroblastes, que les caractéristiques cellulaires de ces fibroblastes présentaient un effacement des marques du vieillissement, signe d’une plasticité cellulaire possible au cours du vieillissement. A présent, étendre une telle étude au modèle tissulaire cutané par la mise en place de protocoles de différenciation dans le lignage épidermique permettra à l’avenir de mieux comprendre pour mieux appréhender les pathologiques associées au vieillissement, et ainsi pouvoir offrir aux patients une médecine appropriée et concrète. / Beside the pathological context, time is the essential basis in the aging process of the body and associated systems. These will gradually introduce a decline in function(s) where many complex mechanisms will take part at different levels. Among proposed findings, aging is the result of an inevitable process, a succession of cellular stress that could be prevented or repaired, opening the way for future studies that will eventually offer an explanation detailed, clear and complete which occur. For example, our work provide a response element to establish a link between senescence and aging, based on the generation of hiPSCs by cellular reprogramming of cells from biopsies of young, older, senescent and proliferating cells patients. Further characterization of their pluripotent state comparable to that of human embryonic stem cells, we have also showed by a specific differentiation into fibroblasts, that cellular characteristics of those fibroblasts had erased aging features. Next step, is to extend such study in cutaneous tissue model by the introduction of differentiation protocols in the epidermal lineage which will able us to better understand aging-associated diseases, and thus bring the ability to propose an appropriate and cocnrete medicine to aged patients.

Les télomères sont des séquences d’ADN, généralement répétées en tandem, localisées à l’extrémité des chromosomes linéaires. Une des fonctions principales des télomères est de différencier l’extrémité des chromosomes des cassures double-brin, et ainsi de prévenir l’activation des voies de réparation de l’ADN. Chez les mammifères, cette fonction est plus spécifiquement assurée par le complexe shelterin. Il s’agit d’un complexe hétérogène composé de six protéines distinctes : TRF1, TRF2, POT1, RAP1, TPP1 et TIN2, qui interagit spécifiquement avec l’ADN télomérique. Au sein de ce complexe, les protéines RAP1 et TRF2 coopèrent afin d’empêcher l’extrémité des chromosomes d’être perçue comme un dommage de l’ADN, ce qui autrement aboutirait à des fusions inter-chromosomiques suite au processus de réparation. La protéine TRF2 se lie directement à la molécule d’ADN dans laquelle elle s’enroule de façon spécifique. Cette propriété est primordiale pour générer une structure d’ADN en forme de boucle, appelée t-loop, et dont le bon fonctionnement des télomères dépend. Les travaux effectués au cours de cette thèse ont mis en évidence deux scenarii indépendants dans lesquels la protéine RAP1 assure un rôle critique dans la stabilité des télomères. Premièrement, RAP1 peut prévenir les fusions inter-chromosomiques dans des cellules exprimant une forme altérée de TRF2 incapable de former des t-loops. Deuxièmement, l’inhibition de RAP1 dans des cellules en sénescence réplicative conduit à l’activation des voies de réparation de l’ADN et à la formation de fusions inter-chromosomiques. Ces observations font écho à des résultats précédents obtenus dans des cellules HeLa traitées avec l’inhibiteur de la télomérase BIBR1532, et dont l’expression de la protéine RAP1 était abolie par shRNA. De plus, j’ai montré que les fusions interchromosomiques engendrées par la perte de RAP1 sont dépendantes de la ligase IV, qui est un acteur principal de la voie de réparation de l’ADN par recombinaison non-homologue (NHEJ). Dans l’ensemble, ces travaux démontrent l’importance de la protéine RAP1 dans la stabilité des télomères lorsque la protéine TRF2 est non fonctionnelle, mais aussi dans des situations physiologiques telles que la sénescence réplicative. / In mammals, the shelterin complex is the guardian of telomere stability. It operates through a set of six proteins (TRF1, TRF2, POT1, RAP1, TPP1 and TIN2) that binds telomeric DNA and protects it from being recognized as DNA double-strand breaks and therefore control DNA repair and DNA damage response pathways. Among them, RAP1 and TRF2 cooperate and together protect chromosome extremities from end-to-end fusions. TRF2 is seen as a major factor to control telomere DNA topology by wrapping DNA around itself in a right handed manner. This property of TRF2 is required to promote the formation of t-loops, special DNA structures at telomeres that are considered as protective barriers to DNA damage response and fusion. Here we demonstrate two independent situations where RAP1 dysfunction is critical for telomere protection. First, in cells expressing a wrapping-deficient TRF2 allele that cannot form t-loops, RAP1 appears as a backup anti-fusion mechanism. Second, RAP1 downregulation in replicative senescent cells leads to telomere fusions and DNA damage response activation. This is consistent with similar observations in HeLa cells treated with the telomerase inhibitor BIBR1532, and in which RAP1 expression was abolished by an inducible shRNA system. In addition, we show that fusions triggered by RAP1 loss are dependent upon ligase IV, which is a key player of the classical non-homologous end-joining (c-NHEJ) repair pathway. Altogether, these results indicate that RAP1 takes over telomere protection when TRF2 cannot properly function or in the normal physiological situation, such as replicative senescence.