L’ataxie de Friedreich est une maladie neurodégénérative sévère causée par un défaut d’expression de la frataxine (FXN), une petite protéine mitochondriale impliquée dans la biogenèse des centres fer-soufre (Fe/S), des groupement prosthétiques aux fonctions cellulaires essentielles. Chez les mammifères, il a été montré que la frataxine stimule la synthèse in vitro de centres Fe/S sur la protéine d’échaffaudage ISCU, grâce à l’augmentation de la production d’ions sulfures par le complexe NFS1-ISD11-ISCU. Cependant, le mécanisme par lequel la frataxine active la biogenèse des centres Fe/S n’a pas encore été défini. Nous avons étudié les effets de FXN sur les cinétiques de formation et de réduction des persulfures, des intermédiaires clés de la production d’ions sulfures, générés par la cystéiene désulfurase NFS1, à l’aide d’un test de détection des persulfures basé sur l’utilisation de composés synthétiques peptide-maléimide et de la spectrométrie de masse. Nous avons montré que FXN active deux réactions très similaires : la réduction du persulfure de NFS1 par des réducteurs à thiols comme le DTT, la L-cystéine et le glutathion et le transfert de soufre de NFS1 vers ISCU, conduisant à l’accumulation de persulfure sur la cystéine C104 d’ISCU. Nous avons constaté que la vitesse de réduction du persulfure d’ISCU par les thiols n’est pas affectée en présence de FXN et que ce persulfure est réduit plus lentement que celui de NFS1. Nous avons corrélé l’activation par FXN de la réduction du persulfure de NFS1 par les thiols à une stimulation de l’assemblage d’un centre Fe/S sur ISCU. Dans nos conditions expérimentales, l’atome de soufre du persulfure d’ISCU n’est pas incorporé dans le centre Fe/S synthétisé, mais nos résultats ne permettent pas d’exclure que ce persulfure puisse être réduit par une réductase dédiée, encore non identifiée. L’ensemble de nos données indiquent que le rôle de la frataxine est de contrôler la réduction du persulfure de NFS1, en augmentant les vitesses de transfert de soufre vers ISCU et de réduction du persulfure de NFS1 par les thiols. / Friedreich ataxia is a severe neurodegenerative disease caused by reduced expression of frataxin (FXN), a small mitochondrial protein involved in iron-sulfur (Fe/S) cluster biogenesis which are prostetic groups with essential cellular functions. It has been shown in vitro that mammalian FXN activates Fe/S cluster synthesis on the scaffold protein ISCU, by rising up suflide ion production by NFS1-ISD11-ISCU complex. However, the mechanism by which frataxin stimulates Fe/S cluster biogenesis has not been yet defined. We have studied the effect of FXN on the kinetics of formation and reduction of persulfides that are key intermediates of sulfide ion production generated by NFS1, using mass spectrometry and a new detection assay for persulfide based on gel-mobility shift following alkylation by maleimide-peptide compounds. We demonstrate that frataxin activates two similar reactions : sulfur transfer from cysteine desulfurase NFS1 to ISCU leading to accumulation of a persulfide on ISCUcysteine C104 and reduction of NFS1 persulfide by thiol reducers such as DTT, L-cysteine and glutathion. We have observed that FXN does not stimulate the rate of ISCU persulfide reduction by thiols and that this persulfide is reduced much more slowly than NFS1 persulfide. We have then correlated the reduction of NFS1 persulfide with Fe/S cluster assembly. Under our experimental conditions, the sulfur from ISCU persulfide is not incorporated into the Fe/S cluster. However, we cannot exclude that an as yet not identfiied reductase could reduces ISCU persulfide and trigger Fe/S cluster assembly. Overall, our data point to a regulatory function of FXN as an enhancer of persulfide reduction, stimulating the rates of sulfur transfer to ISCU and NFS1 persulfide.

Chlorophyll spielt eine unabdingbare Rolle für die lichtabhängige Reaktion der Photosynthese. Die adäquate Versorgung mit Chlorophyll wird dabei durch die Tetrapyrrolbiosynthese (TBS) gewährleistet. In den letzten Jahrzehnten wurde eine Vielzahl von Proteinen identifiziert, welche an der Anpassung der TBS an wechselnde (a)biotische Wachstumsbedingungen der Pflanze beteiligt sind. Allerdings konnte bislang nicht zweifelsfrei geklärt werden, wie die TBS mit der Integration von Chlorophyllen in die Photosysteme koordiniert wird. Vor einigen Jahren wurde ein Interaktionspartner der Protochlorophyllid-Oxidoreduktase (POR) in Synechocystis identifiziert, welcher als potenzieller Faktor dieser Koordination in Frage kommt. Das POR-INTERACTING TPR-Protein (Pitt) stabilisiert POR an der Thylakoidmembran und interagiert auch mit dem Vorstufenprotein des D1. Pitt gehört zur Familie der tetratricopeptide repeat (TPR) Proteine, deren Vertreter vorrangig für die Vermittlung von Protein-Protein-Interaktionen zuständig sind. Aus diesem Grund war, neben der Identifikation des potenziellen Pitt-Homologs im Modelorganismus Arabidopsis thaliana, die Analyse von anderen Vertretern dieser Proteinklasse ein vielversprechender Ansatz bei der Identifikation von weiteren Regulatoren der TBS oder Photosynthese. Von den fünf ausgewählten TPR-Proteinen aus Arabidopsis thaliana mit einer hohen Sequenzähnlichkeit zu Pitt waren vier in der Lage, physisch mit POR zu interagieren. Von diesen vier Kandidaten ist das durch das Gen At1g78915 kodierte, membranintegrale TPR-Protein (TPR1) der beste Kandidat des putativen Pitt-Homologs in Arabidopsis. Vergleichbar zu Pitt interagiert TPR1 mit POR und stabilisiert das Enzym an den plastidären Membranen. Die Stabilisierung von POR durch TPR1 spielt eine entscheidende Rolle während der Etiolierung und Ergrünung von Keimlingen. Darüber hinaus steht TPR1 im Zusammenhang mit der schnellen Inaktivierung der 5-Aminolävulinsäuresynthese. / Chlorophyll plays an indispensable role in the light reaction of the photosynthesis. The adequate supply of chlorophyll is ensured by tetrapyrrole biosynthesis (TBS). Within the last decades, multiple proteins were identified, which are involved in adjusting the TBS-pathway to changing (a)biotic plant growth conditions. Nevertheless, it is not fully understood how the TBS-pathway is coordinated parallel to the assembly of the photosystems and the integration of chlorophylls into the pigment-binding subunits of the photosystems. Several years ago, an interaction partner of the protochlorophyllide-oxidoreductase (POR) was identified in Synechocystis which was proposed to be involved in the coordination of these mechanisms. The POR-INTERACTING TPR-Protein (Pitt) binds and stabilizes POR at the thylakoid membranes and interacts with the precursor protein of D1. Therefore, Pitt could facilitate the incorporation of chlorophylls into the plastid-encoded nascent photosynthetic subunits. Pitt belongs to the tetratricopeptide repeat (TPR) protein family, whose members mediate protein-protein-interactions. Besides the identification of the potential Pitt-homolog in the model organism Arabidopsis thaliana, analysis of additional members of the TPR-protein superfamily was a promising approach for the identification of further posttranslational regulators of TBS and photosynthesis. Five Arabidopsis thaliana TPR-proteins with a high sequence similarity to Pitt were selected. Four of those proteins are able to interact physically with POR. Among them, the TPR-protein encoded by the gene At1g78915 (TPR1) was the best candidate to represent a putative Pitt homolog in Arabidopsis. Similar to Pitt, TPR1 is a plastid-localized integral membrane protein, which interacts with POR at the thylakoid membranes. The stabilizing effect of TPR1 on POR is especially needed during etioliation and greening. Additionally, TPR1 is required for a inactivation of the 5'-aminolevulinic acid synthesis.

Inborn errors of metabolism represent a heterogenous group of rare conditions, most having an incidence of less than 1 in 100,000 births. Because of their low prevalence, they are on the margin of attention of general research and even more so of large pharmaceutical companies. Study of rare diseases is the only way to design therapeutic options in order to improve quality of life of affected patients. Present Thesis particularly focuses on disturbances in mitochondrial energy metabolism. The main goals were the characterization of mitochondrial biogenesis within foetal development, as well as in childhood and adulthood. Another aim was to define clinical, biochemical and molecular aspects of mitochondrial optic neuropathies in childhood and adulthood. This work supported the earlier observations that gestational week 22 is the edge of viability, which has to be taken into account in upcoming discussions about guidelines on resuscitation of preterm neonates. Secondly, over last four years, we managed to examine and describe large cohort of patients with optic neuropathies based on a mitochondrial dysfunction. We have managed to characterize the biochemical and molecular-genetic background in more than 200 patients, and both selected cases (LHON/MELAS overlap syndrome) and cohort studies (MELAS,...

Les cellules sont la plus petite forme de vie individuelle qui forme un organisme. La structure et la santé de tous les organismes est essentiellement définie par le nombre, le type et la taille de leurs cellules. Composé d'environ 30 trillions de cellules, l'homme possède des cellules aux fonctions et aux tailles remarquablement variées, allant d'un neurone pouvant atteindre un mètre à une cellule lymphoïde d'environ 16 µm de diamètre. Il est connu que la taille est fondamentalement l'équilibre entre la croissance cellulaire et la division cellulaire. Néanmoins, les questions sur les réseaux moléculaires qui contrôlent et déterminent le maintien de la taille optimale des cellules restent à déchiffrer. D'innombrables travaux ont caractérisé mTORC1 comme une voie régulatrice majeure de la croissance cellulaire jouant un rôle central, intégrant des stimuli intra et extracellulaires. Ce travail porte sur l'investigation et la caractérisation des acteurs moléculaires et des processus qui orchestrent la taille des cellules humaine déterminées par l'épistase chimique. J'ai entrepris une bibliothèque CRISPR / Cas9 à inactivation prolongée (EKO) dans NALM-6 (lignée cellulaire de lymphome pré-B), suivie d'un fractionnement de la taille des cellules par élutriation à contre-courant en présence de rapamycine (inhibiteur de mTOR), et comparé aux données non publiées données du laboratoire utilisant les mêmes méthodes sans rapamycine. Cette analyse de l'étude indique que dans le contexte amont de mTOR, la perte de gènes liés à la détection des nutriments entraîne une perte de taille en présence d'inhibition de mTOR. En outre, plusieurs knockouts géniques dans la biogenèse des ribosomes et l'homéostasie du calcium ont conduit à une perte ou un gain de taille, montrant un rôle pivot possible de ces processus dans le contrôle de la taille des cellules d'une manière dépendante de mTOR. Ce travail a fourni des informations sur les gènes et réseaux connus et inconnus qui peuvent réguler la taille des cellules d'une manière dépendante de mTOR. Ces résultats doivent être validés et approfondis. / All organisms are essentially structured and fitness defined by cell number, type and size. Composed of around 30 trillion cells, humans have cells with remarkably varied functions and size, ranging from a neuron that can reach one meter in length to a lymphoid cell that is around 16 μm in diameter. At a fundamental level, size is determined by the balance between cell growth and cell division. The molecular networks that control and maintain optimal cell size are yet to be deciphered. The mTORC1 pathway is a major regulator of cell growth that plays a central role in integrating intra- and extra-cellular stimuli. This study addresses the investigation and characterization of the molecular players and processes that orchestrate cell size in human cells, as determined by chemical-genetic size screens and epistasis analysis. I undertook a CRISPR/Cas9 extended-knockout (EKO) genome-wide library screen in the NALM-6 pre-B lymphoma cell line, followed by cell size fractionation by counter flow elutriation in the presence of the mTOR inhibitor rapamycin, and compared the screen data to a similar screen performed in the absence of rapamycin. The analysis indicates that upstream of mTOR, the loss of genes that are related to nutrient sensing, results in size changes in the presence of mTOR inhibition. Also, several gene knockouts in ribosome biogenesis and calcium homeostasis led to size alterations, suggesting a possible a pivotal role of these processes in cell size control in a mTOR-dependent fashion. This study provides insights into the genetic networks that regulate cell size in a mTOR-dependent fashion and establishes new hypotheses for future experimental tests.

In mammalian cells, homeostasis and fate during development relies on the proper transport of membrane-bound cargoes to their designated cellular locations. The hetero-tetrameric adaptor protein complexes (APs) are required for sorting and concentration of cargo at donor membranes, a crucial step during targeted transport. AP2, which functions at the plasma membrane during clathrin-mediated endocytosis, is well characterized. In contrast, AP1 a clathrin adaptor mediating the delivery of lysosomal hydrolases via mannose 6-phosphate receptors (MPRs) and AP3 an adaptor ensuring the proper targeting of lysosomal membrane protein are difficult to study by classic cell biology tools. To gain new insights on these APs, our lab has previously designed an in vitro system. Reconstituted liposomes were modified with small peptides mimicking the cytosolic domains of bona fide cargoes for AP1 and AP3 respectively and thereby enabling the selective recruitment of these APs and the identification of the interacting protein network. In the study at hand we utilize above-described liposomes to generate supported lipid bilayers and Giant Unilamellar Vesicles (GUVs), large-scale membrane systems suited for analysis by fluorescence microscopy. By using cytosol containing fluorescently-tagged subunits, we visualized clathrin coats on artificial membranes under near physiological conditions for the first time. Moreover, we demonstrated clathrin-independent recruitment of AP3 coats on respective GUVs. Presence of active ARF1 was sufficient for the selective assembly of AP1-dependent clathrin coats and AP3 coats on GUVs. By using dye-conjugated ARF1, we show that ARF1 colocalized with AP3 coats on GUVs and that increased association of ARF1 with GUVs coincided with AP1-dependent clathrin coats. Our previous study identified members of the septin family together with AP3 coats on liposomes. Here we show on GUVs, that active ARF1 stimulated the assembly of septin7 filaments, which may constrain the size and mobility of AP3 coats on the surface. Subsequent cell biology studies in HeLa cells linked septins to actin fibers on which they may control mobility of AP3-coated endosomes and thus their maturation. An actin nucleation complex, based on CYFIP1 was identified together with AP1 on liposomes before. Here we show on GUVs, that CYFIP1 is recruited on the surface surrounding clathrin coats. Upon supply of ATP, sustained actin polymerization generated a thick shell of actin on the GUV surface. The force generated by actin assembly lead to formation of long tubular protrusions, which projected from the GUV surface and were decorated with clathrin coats. Thereby the GUV model illustrated a possible mechanism for tubular carriers formation. The importance of CYFIP1-reliant actin polymerization for the generation of MPR-positive tubules at the trans-Golgi network (TGN) of HeLa cells was subsequently demonstrated in our lab. The notion that tubulation of artificial membranes could be triggered by actin polymerization allowed us to perform a comparative mass spectrometry screen. By comparing the abundance of proteins on liposomes under conditions promoting or inhibiting actin polymerization, candidates possibly involved in stabilization, elongation or fission of membrane tubules could be identified. Among the proteins enriched under conditions promoting tubulation, we identified type I phosphatidylinositol-4-phosphate 5-kinases. Their presence suggested an involvement of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in tubule formation. By cell biology studies in HeLa we show, that down regulation of these enzymes altered the dynamics of fluorescently-tagged MPRs, illustrating the importance of locally confined PI(4,5)P2 synthesis during formation of coated carriers at the TGN. Bin–Amphiphysin–Rvs (BAR) domains are known to sense membrane curvature and induce membrane tubulation. Among various BAR domain proteins, Arfaptin2 was enriched under conditions allowing tubulation of liposomes. By microscopy studies on HeLa cells we show, that Arfaptin2 as well as its close paralog Arfaptin1 were present on AP1-coated MPR tubules emerging from the TGN. We further show, that tubule fission occurred at regions were Arfaptin1 is concentrated and that simultaneous down regulation of both Arfaptins lead to increased number and length of MPR tubules. Since fission of coated transport intermediates at the TGN is poorly understood, our findings contribute a valuable component towards a model describing the entire biogenesis of coated post-Golgi carriers. In conclusion, combining artificial membrane systems and cell biology studies allowed us to propose new models for formation as wall as for fission of AP1-coated transport intermediates at the TGN. Further we gained new insights on AP3 coats and the possible involvement of septin filaments in AP3-dependent endosomal maturation.

Mgr. Daniela Fornuskova PhD thesis Biochemical and molecular studies of cytochrome c oxidase and ATP synthase deficiencies ABSTRACT The mammalian organism fully depends on the oxidative phosphorylation system (OXPHOS) as the major energy (ATP) producer of the cell. Disturbances of OXPHOS may be caused by mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). One part of the thesis is focused on the role of early and late assembled nuclear-encoded structural subunits of cytochrome c oxidase (CcO) as well as Oxa1l, the human homologue of the yeast mitochondrial Oxa1 translocase, in the biogenesis and function of the human CcO complex using stable RNA interference of COX4, COX5A, COX6A1 and OXA1L, as well as expression of epitope-tagged Cox6a, Cox7a and Cox7b, in HEK (human embryonic kidney)- 293 cells. Our results indicate that, whereas nuclear- encoded CcO subunits Cox4 and Cox5a are required for the assembly of the functional CcO complex, the Cox6a subunit is required for the overall stability of the holoenzyme. In OXA1L knockdown HEK-293 cells, intriguingly, CcO activity and holoenzyme content were unaffected, although the inactivation of OXA1 in yeast was shown to cause complete absence of CcO activity. In addition, we compared OXPHOS protein deficiency patterns in mitochondria from skeletal...

ATP synthase represents the key enzyme of cellular energy provision and ATP synthase disorders belong to the most deleterious mitochondrial diseases affecting pediatric population. The aim of this thesis was to identify nuclear genetic defects and describe the pathogenic mechanism of altered biosynthesis of ATP synthase that leads to isolated deficiency of this enzyme manifesting as an early onset mitochondrial encephalo-cardiomyopathy. Studies in the group of 25 patients enabled identification of two new disease-causing nuclear genes responsible for ATP synthase deficiency. The first affected gene was TMEM70 that encodes an unknown mitochondrial protein. This protein was identified as a novel assembly factor of ATP synthase, first one specific for higher eukaryotes. TMEM70 protein of 21 kDa is located in mitochondrial inner membrane and it is absent in patient tissues. TMEM70 mutation was found in 23 patients and turned to be the most frequent cause of ATP synthase deficiency. Cell culture studies also revealed that enzyme defect leads to compensatory-adaptive upregulation of respiratory chain complexes III and IV due to posttranscriptional events. The second affected gene was ATP5E that encodes small structural epsilon subunit of ATP synthase. Replacement of conserved Tyr12 with Cys caused...

Le cancer du pancréas est la quatrième cause de décès par cancer au Canada. Avec des mutations activatrices de KRas présentes dans près de 90% des lésions bénignes et tumeurs, ce cancer arbore une activation de la voie MAPK très tôt dans son développement. Or, peu de littérature existe sur les étapes clés de la progression et sur le rôle précis de cette signalisation dans le passage des lésions bénignes (PanIN) au stade avancé (PDAC). Depuis plusieurs années, notre laboratoire s’intéresse aux kinases ERK1/2, actives en aval de Ras, des acteurs centraux du programme de sénescence cellulaire, soit un programme antitumoral intrinsèque aux cellules. L’hypothèse centrale des présents travaux est donc que les mutations de KRas acquises dès le stade PanIN induisent une sénescence qui agit comme barrière à la progression tumorale, et que l’atténuation du signal de ERK est impliquée dans le contournement de ce mécanisme. La première partie de cette thèse montrera donc les avancées que nous avons faites sur la caractérisation de la progression entre le stade bénin et le stade avancé, de laquelle l’acquisition d’un caractère souche, la transition épithélio-mésenchymateuse et le développement d’une dépendance mitochondriale semblent être des déterminants. Ensuite, nous présenterons nos découvertes sur le rôle des kinases ERK1/2, de la sénescence cellulaire et du stress nucléolaire dans une nouvelle approche visant à restaurer un mécanisme de suppression tumorale inspiré des lésions bénignes et impliquant une altération de la biogenèse ribosomique. Finalement, pour bonifier cette nouvelle stratégie, nous présenterons les résultats d’un criblage CRISPR-Cas9 génome-entier nous ayant permis d’identifier les composantes d’une stratégie « one-two punch » basée sur l’induction de sénescence dans les cellules PDAC combinée à l’inhibition de la Glutathion peroxydase 4 (GPX4), de façon à promouvoir une sénolyse efficace dans ce contexte. Dans leur ensemble, les travaux présentés dans cette thèse montrent un avancement significatif dans la compréhension de la biologie des cancers pancréatiques en identifiant à la fois des vulnérabilités intrinsèques et inductibles afin de générer de nouvelles idées thérapeutiques pour ce cancer hautement fatal. / Pancreatic cancer is the fourth leading cause of death by cancer in Canada. With frequent activating mutations in KRas in up to 90% of benign lesions and tumors, this cancer possesses an early activation of the MAPK pathway. However, key events of its progression from the PanIN stage to the PDAC stage and the precise role of MAPK signaling in it are still poorly understood. For many years, our laboratory has taken interest in the ERK1/2 signaling pathway, activated downstream of oncogenic Ras and a key mediator of cellular senescence. Cellular senescence is considered an intrinsic antitumor mechanism due to its ability to stably halt the cell cycle. The central hypothesis of this work is then that KRas mutations that are acquired at the PanIN stage induce cellular senescence which acts as a barrier against tumor development. Still, this powerful mechanism can be circumvented as cells tend to attenuate the ERK1/2 signaling to promote progression and acquisition of more aggressive features. Thus, the first part of this thesis will present our most recent advances in characterizing the progression events between PanIN and PDAC stages, during which stem cell features acquisition, epithelial-mesenchymal transition and mitochondrial dependency seem to occur. Next, we will present our discoveries regarding the implication of ERK1/2 kinases, cellular senescence and nucleolar stress in a new approach to restore a tumor suppression mechanism inspired by the PanIN stage and based on ribosome biogenesis alteration. Finally, to potentiate this strategy, we will show the results of a genome-wide CRISPR-Cas9 screen that identified the components of a “one-two punch” approach to induce cellular senescence in PDAC cells and to efficiently eliminate them by GPX4 inhibitors-mediated senolysis. Globally, the work presented in this thesis show significant progress in the field of pancreatic cancer, identifying previously unknown vulnerabilities of those cancer cells and paving the way for the development of new therapeutic combinations.

No description available.

Environ 30% des cancers humains ont une mutation gain de fonction dans l’oncogène RAS, menant à une prolifération cellulaire accrue et une expansion clonale. Cependant, il est bien établi qu’une hyperactivation soutenue de cette voie mène au phénotype inverse, soit la sénescence cellulaire, définie par un arrêt stable de la prolifération. Ce destin cellulaire caractérise les lésions bénignes et la progression vers une tumeur maligne est associée à son contournement. Toutefois, les mécanismes moléculaires permettant aux cellules de distinguer entre une signalisation normale et oncogénique par RAS afin de les engager vers la sénescence plutôt que la prolifération demeurent inconnus. Ainsi, l’hypothèse à la base de ces travaux est que la décision d’engagement vers la sénescence implique une reprogrammation transcriptionnelle qui précède l’établissement des phénotypes caractéristiques de la sénescence, tel le phénotype sécrétoire (SASP) (Article 1). Nous avons ainsi identifié un point de restriction (SeRP) critique pour l’engagement des cellules vers la sénescence en réponse à l’oncogène HRASG12V. Ce SeRP intègre l'intensité et la durée du stress oncogénique, tout en gardant une mémoire des stress antérieurs, en modulant l’accessibilité à la chromatine via l’induction d’un réseau auto-régulé de facteurs de transcription comprenant notamment ETV4 et RUNX1 (Article 2). Notre modèle actuel nous porte à croire que cette augmentation d’accessibilité à la chromatine impliquerait principalement une décondensation de l’hétérochromatine périnucléolaire. Ceci mènerait à l’induction du SASP et aux défauts de ribogénèse observés dans la sénescence. Nous montrons d’ailleurs via la génération d’un modèle murin transgénique que l’induction de tels défauts de ribogénèse à l’échelle systémique mène à un phénotype de vieillissement prématuré suggérant une sénescence des cellules souches (Article 3). Les cellules souches ayant des niveaux particulièrement élevés de ribogénèse et étant très sensibles à des altérations de leur niche tels que l’inflammation chronique, nous pensons que, de manière fortuite, ce modèle reproduit en quelque sorte les conséquences du SeRP. En somme, l’ensemble des travaux présentés dans cette thèse permettent une meilleure compréhension des mécanismes moléculaires régulant l’engagement vers la sénescence. À termes, ces nouvelles notions permettraient de concevoir des stratégies thérapeutiques permettant de faire pencher la balance vers la sénescence dans un contexte de cancers mutés en RAS. / Around 30% of human cancers have a gain-of-function mutation in the RAS oncogene, resulting in increased cell proliferation and clonal expansion. However, it is well established that a sustained hyperactivation of this same pathway leads instead to the opposite phenotype, namely cellular senescence, which is defined by a stable proliferation arrest. This cell fate characterizes benign lesions and progression to malignancy is associated with its bypass. However, the molecular mechanisms allowing cells to distinguish between normal and oncogenic RAS signaling in order to commit them to senescence rather than proliferation remain unknown. Thus, the hypothesis underlying the present work is that this decision to commit to senescence involves a transcriptional reprogramming that precedes the establishment of the senescence-characteristic phenotypes such as the secretory phenotype (Article 1). We have thus identified a restriction point (SeRP) critical for the commitment of cells towards senescence in response to HRASG12V oncogene. This SeRP integrates both the intensity and duration of oncogenic stress while keeping a memory of previous stresses. This integration is achieved by modulating chromatin accessibility via the induction of a self-regulated network of transcription factors including among others ETV4 and RUNX1 (Article 2). Our current model leads us to believe that this increase in chromatin accessibility during the SeRP would mainly involve decondensation of perinucleolar heterochromatin. This would lead to the induction of the pro-inflammatory secretome of senescent cells (SASP) and the ribogenesis defects observed in senescence. Besides, we show via the generation of a transgenic mouse model that the induction of such ribogenesis defects at the systemic scale leads to a premature aging phenotype suggesting stem cells senescence (Article 3). Stem cells having particularly high levels of ribogenesis and being very sensitive to alterations of their niche such as chronic inflammation, we believe that serendipitously, this model somehow reproduces the consequences of the SeRP. In short, all the work presented in this thesis allows for a better understanding of the molecular mechanisms regulating the commitment to senescence. Ultimately, these new notions would allow to design therapeutic strategies to tip the balance towards senescence in the context of RAS-mutated cancers.