Die Aufrechterhaltung der sogenannten failsafe Programme ist ein wichtiges Kennzeichen des zellulären Lebens, da das genaue Gleichgewicht zwischen Proliferation und Wachstumsarrest es sicherstellt, dass die Zelle sich selber vor potentiell gefährlichen Mutationen schützen kann. Apoptose und Seneszenz bewahren die Zellhomeostase und sind von aüsserster Bedeutung dafür, die Zelle vor maligner Transformation zu bewahren. Die Seneszens zeichnet sich dabei durch Veränderungen des Heterochromatins bei Genen aus, welche für den Eintritt in die DNA-Synthese-Phase des Zellzyklus verantwortlich sind. Insgesamt wurde die Bedeutung epigenetischer Modifikationen bei malignen Erkrankungen in den letzten Jahren immer deutlicher. So konnte gezeigt werden, dass die Rb-assoziierte Histon-Methyltransferase Suv39h1 ein entscheidender Vermittler der Seneszenz in Ras-induziertem Maus Lymphom-Modell ist. In dieser Arbeit wurde gezeigt, dass die transgenen Eµ-myc Maüse (ähnlich dem Burkitt''s Lymphom im Menschen), wenn sie mit Suv39h1-defizienten Maüsen gekreuzt werden. Die Überexpression von Myc, wie sie in humanen und murinen Tumoren zu beobachten ist, induziert primär ein apoptotische Antwort mit dem zentralen Vermittler p53. Allerdings bewirkte die Suv39h1 Defizienz, obwohl sie die Lebenserwartung signifikant verkürzte, keine Veränderung der Apoptose-Rate in diesem Modell. Dieses Ergebnis demonstriert die Tumor-suprimierende Wirkung des Suv39h1 in Myc-getriebener Lymphomagenese. Weiterhin wurde in dieser Arbeit ein zusätzlicher Mechanismus zur Vermittlung Myc-induzierter Seneszenz unter Einbeziehung des Zytokin-Signaling identifiziert. Im Falle der Expression von intaktem Suv39h1 in der Tumorzelle kann TGF-beta die Myc-induzierte Seneszenz in vivo beeinflussen. Schliesslich ergaben die Untersuchungen, dass die Suv39h1-defizienten Lymphome über eine beeinträchtigte Therapie-Anwort verfügen, da sie keine Therapeutika-induzierte Seneszenz ausführen können. / Maintenance of cellular failsafe pathways (apoptosis and senescence) is one of the hallmarks of life, as fine equilibrium between proliferation and growth arrest ensures that the cell can protect itself from the potentially dangerous mutations. Senescence is a mechanism distinguished by heterochromatin modifications leading to silencing of the genes responsible for the entry to the DNA synthesis [S] phase of the cell cycle. Involvement of epigenetic modifications in cancer development has been subject of a more intense research in the past few years. It was shown that the Rb-associated histone methyltransferase Suv39h1 is a critical mediator of senescence in a Ras-induced mouse lymphoma model. Additional mechanisms of the senescence regulation are currently being under investigation. In this thesis Eµ-myc transgenic mice, crossed to mice deficient for Suv39h1, were shown to succumb to the same type of B-cell lymphoma (similar to Burkitt''s lymphoma in humans). Overexpression of Myc, as seen in human and mouse tumors will primarily induce an apoptotic response using p53 as a mediator of this response. In Eµ-myc, Suv39h1-/- mouse model deficiency in Suv39h1 has significantly shortened life expectancy, but it did not affect spontaneous apoptosis rates. This finding established Suv39h1 as a tumor suppressor in Myc-lymphomagenesis. Further, an additional mechanism of mediating oncogene-induced senescence involving cytokine signaling was identified and reported here. When intact Suv39h1 is present in the tumor, TGF-beta is able to mediate oncogene-induced senescence in vivo. Finally, in the treatment studies it was shown that Suv39h1 deficient lymphomas have an impaired response to chemotherapy, caused by their inability to execute drug-induced senescence. This finding can be of use for the design of novel cancer therapies.

Zelluläre Seneszenz wird als terminaler Zellzyklusarrest definiert, der mit dem Altern und funktionellen Verlust von Geweben verknüpft ist. Eine Seneszenzreaktion wird ebenso durch Onkogene und zytotoxischen Stress verursacht. Die Ausführung des Seneszenzprogramms wird durch eine zeitlich hochdynamische Aktivität von Transkriptionsfaktoren (TF) bedingt. Interessanterweise kann die Zelllinienzugehörigkeit einer Zelle durch die Expression von linien-aberranten TF überschrieben werden. Die vorliegende Arbeit untersucht Chemotherapie-induzierte Seneszenz (TIS) in Bcl-2 überexprimierenden, deshalb vor Apoptose geschützten, murinen Eµ-Myc B-Zell Lymphomen in An- oder Abwesenheit der Seneszenz-essentiellen Histonmethyltransferase Suv39h1. Analysen auf Transkriptom- und auf Proteinebene ergeben dabei, dass in einer Seneszenz-spezifischen Weise die TF AP-1, PU.1 und C/EBPβ induziert werden, welche normalerweise für die Funktion und Entwicklung myeloischer Zelllinien bedeutend sind. Dementsprechend korreliert der Seneszenzzustand mit Transkripten, Oberflächenmarkern und einer enzymatischen Funktion der myeloischen Linie. Indem die identifizierten TFs heruntergeschaltet oder überexprimiert werden, wird ihre direkte Beteiligung an der Linienuntreue der TIS Lymphome demonstriert. TIS-Kapazität wird als für den Erfolg von Krebstherapie günstige Eigenschaft betrachtet, da sie zu einem Wachstumsblock führt. Nichtsdestotrotz können sich verweilende TIS Zellen krebsbiologisch auch nachteilig auswirken. Anhand von murinen und humanen, klinisch annotieren Transkriptomdatensätzen kann hier in beiden Spezies ein myeloisch verschobenes, Linienuntreue anzeigendes Genexpressionsprofil mit einer besseren Überlebensprognose korreliert werden. Die vorliegenden Befunde legen nahe, dass die Modulation von TF Aktivitäten in Seneszenz einen potentiellen therapeutischen Angriffspunkt darstellt, um den für den Therapieerfolg nützlichen Zweig des TIS Phänotyps zu befördern. / Cellular senescence is regarded as an irreversible cell cycle arrest associated with tissue aging and its functional decline. A senescence response is also evoked by oncogenic and cytotoxic stress. The execution of the senescence program relies on a highly dynamic sequence of transcription factor (TF) activities. Interestingly, cell lineage commitment can be overridden by the expression of lineage-aberrant TFs. This thesis examines chemotherapy-induced senescence (TIS) in Bcl-2 overexpressing, thus apoptosis-protected, murine Eμ-Myc B-cell lymphomas with or without the senescence-essential histone methyltransferase Suv39h1. Transcriptome as well as protein level analyses reveal senescence-specific induction of the TFs AP-1, PU.1 and C/EBPβ which are typically crucial for myeloid lineage commitment and function. Correspondingly, the senescent state associates with myeloid lineage transcripts, surface markers and enzymatic function, reminiscent of, but not equal to a transdifferentiation phenotype. By knocking down and overexpressing the identified TFs, we demonstrate their direct involvement in the lineage infidelity of TIS lymphomas. TIS-capacity is viewed as beneficial to cancer therapy outcome due to its block on proliferation. However, lingering TIS cells can also be detrimental due to the acquisition of latent stemness properties or tumor-protective remodeling of their microenvironment. By interrogating murine and human, clinical course-annotated transcriptome data sets, an association between a myeloid-skewed, lineage infidelity indicating gene expression profile and better tumor prognosis is established in both species. The presented findings suggest that modulation of the senescent TF activities could be therapeutically exploited to foster the cancer patient-beneficial branch of the TIS phenotype.

Die schlechte Prognose der meisten disseminierten Tumorerkrankungen ist häufig in einer vorbestehenden oder erworbenen Resistenz gegenüber Zytostatika begründet. Da die meisten Zytostatika mit zellulären Strukturen interagieren, war lange angenommen worden, dass der antineoplastische Effekt unmittelbar durch massive Zellschädigung bewirkt wird. Hieraus folgte, dass Chemoresistenz auf Mechanismen beruhen müsse, welche das Zytostatikum an der Wechselwirkung mit seiner intrazellulären Zielstruktur hindern. Arbeiten der letzten Jahre haben jedoch gezeigt, dass die meisten Zytostatika indirekt über DNA-Schädigung ein relativ uniformes, genetisch kodiertes Zelltod-Programm auslösen, demzufolge postuliert wurde, dass auch Apoptosedefekte für "Multi-Drug-Resistenz" verantwortlich sein könnten. Allerdings ist der tatsächlich Beitrag zytostatika-induzierter Apoptose am Therapieerfolg nicht geklärt, wobei der Wahl geeigneter Testsysteme eine wesentliche Bedeutung für diese Kontroverse zuzukommen scheint. Gegenstand der vorliegenden Arbeit ist daher die Etablierung eines transgenen Lymphom-Modells, in welchem chemotherapeutische Effekte an spontan entstandenen Tumoren mit definierten genetischen Läsionen in ihrer natürlichen Umgebung untersucht werden können. Hierbei konnte gezeigt werden, dass Mutationen in apoptose-relevanten Genloci wie p53, INK4a/ARF oder bcl2 sowohl die Manifestation myc-transgener Lymphome dramatisch beschleunigen, als auch den Therapieerfolg kompromittieren. Neben Apoptose wurde darüberhinaus prämature Seneszenz, ein terminaler Zellzyklus-Arrest, als prognose-relevantes Chemotherapie-Effektorprogramm identifiziert. Damit dokumentiert die vorliegende Arbeit einen wichtigen Zusammenhang von Gendefekten, die während der Tumorigenese erworben wurden, und später evidenter Chemoresistenz, wobei manche Mutationen bereits vor Zytostatika-Exposition Resistenz begründen können. Die Identifikation und pharmakogenomische Charakterisierung potentiell resistenz-vermittelnder Gene und Mutationen in relevanten Testsystemen wird für die Entwicklung spezifischerer, aber weniger toxischer "targeted Therapeutics" von großer Bedeutung sein. / Intrinsic or acquired chemoresistance is the major cause for the adverse outcome of disseminated malignancies. The fact that most anticancer agents bind to subcellular targets prompted the assumption that drug-induced cytotoxicity must be a direct consequence of severe cellular damage. Hence, chemoresistance was thought to arise from mechanisms that prevent or disrupt the drug-target interaction. By contrast, more recent data suggested that DNA damage caused by most, if not all, anticancer agents may trigger a relatively uniform, genetically encoded cell death program. In turn, defects in the apoptotic machinery should account for multi-drug resistance as well. However, due to technical limitations of current test systems, it has been difficult to assess the overall contribution of apoptotic cell death to treatment outcome. In the studies presented here, a transgenic mouse lymphoma model was established in order to exploit drug responses of spontaneously developed malignancies growing at their natural sites but harboring defined genetic defects. Using this model, alterations in apoptosis-related gene loci such as p53, INK4a/ARF or bcl2 result in both dramatic acceleration of myc-driven lymphomagenesis and compromised treatment responses. Importantly, not only apoptosis, but premature senescence, a terminal cell-cycle arrest, was found to impact on treatment outcome. In essence, this work describes and important connection between cancer genes and cancer therapy, i.e. genetic defects acquired during tumorigenesis may already co-select for chemoresistance prior to any drug encounter. The identification and pharmacogenomic evaluation of resistance conferring candidate genes and mutations using adequate test systems is likely to play a key role in the development of novel, more specific but less toxic so called "targeted Therapeutics".

We studied the dynamic of nine populations of Alpine ibex ( Capra ibex ibex ) in five protected areas. We showed a strong effect of age on demographic parameters, with a marked decrease of survival after 10-12 years of age. We also found a high variability of old females' reproduction between populations. Contrary to what is expected for a highly dimorphic species like ibex, males survived as well as females except for old individuals. Winter harshness had a negative impact on survival of old individuals but not on reproduction and survival of young. We conclude that ibex have evolved a highly conservative life-history tactic compared to other ungulates studied to now. From a management viewpoint we also showed that under some circumstances ground counts might provide reliable estimates of ibex population trends.

The aim of this thesis was to investigate morphogenesis in the fruit fly Drosophila melanogaster using three in vitro tissue culture systems. Primary embryonic cultures derived from Drosophila melanogaster were used to study the effect of the moulting hormone ecdysone on cells in culture. The hypothesis was that the effect of ecdysone on these primary embryonic cells would parallel events which occur during metamorphosis in vivo and therefore the primary embryonic cultures could be used as an ‘in vitro’ model system. Transgenic fly lines expressing GFP were used to visualise and identify specific cell types and it was shown that cells in primary embryonic cultures respond to ecdysone morphologically. However due to the variability of cultures it was concluded that this culture system was not suitable for use as a model system. As defined cell types were observed the development of a protocol suitable for use with the primary embryonic culture system using dsRNA in order to demonstrate RNA interference was undertaken. Although this was unsuccessful, as cells in the primary embryonic cultures appeared to be resistant to dsRNA, some technical avenues remain to be explored. The Drosophila melanogaster cell line, Clone 8+, was used to investigate cell adhesion in tissue culture. Statistical analyses were carried out and it was established that derivatives of the parent cell line, Clone 8+, showed differential adhesion and proliferation characteristics. Analysis of microarray data was carried out in order to identify genes which may be responsible for the loss of cell adhesion in Clone 8+ cell lines and the potential roles of these genes in adhesion were discussed. A gene of interest, glutactin, was identified which may be responsible for loss of cell adhesion. Antibody staining was used to establish the expression of the protein glutactin in the Clone 8+ cell lines. The expression of glutactin suggested that the Clone 8+ cell line had maintained properties of the wing disc epithelial cell-type and disruption of cell polarity was considered as a possible mechanism. It was shown that f-actin colocalised with glutactin and the role of the cytoskeleton in glutactin secretion was discussed. It was concluded that glutactin was not responsible for loss of cell adhesion in the Clone 8+ cell lines. Further analysis of the microarray data revealed potential genes that could be responsible for the loss of cell polarity in the Clone 8+ cell lines and the possibility of cellular senescence was considered. It was hypothesised that the properties of adhesion and proliferation related to their ‘in vitro’ age. In the final investigation the movement of epithelial cells in Drosophila melanogaster third instar larval imaginal discs during morphogenesis was investigated. Firstly a lumen was identified in fixed imaginal disc tissue in association with cells expressing f-actin. This result was discussed in relation to the process of dorsal closure and wound healing. Further investigations involved live imaging of the dynamic process of evagination in the imaginal wing disc using transgenic flies expressing moesin-GFP. It was concluded that the lumen was not associated with the process of wound healing and it was concluded that the lumen appeared to be the mechanism directing peripodial epithelium contraction during morphogenesis of the imaginal wing disc. Dorsal closure and the process of invagination in relation to morphogenesis of the imaginal wing disc were discussed.

Le facteur de transcription E4F1 fût initialement identifié comme une cible cellulaire de l'oncoprotéine virale E1A au cours de l'infection par l'adénovirus sérotype V. E4F1 est une protéine multifonctionelle essentielle au cours du développement embryonnaire précoce et joue des rôles importants dans l'équilibre entre prolifération/survie de différents types cellulaires, notamment des cellules souches. Au niveau moléculaire, E4F1 possède des activités transcriptionelles intrinsèques mais possède également une activité ubiquitine E3 ligase atypique dirigée contre d'autres facteurs de transcription tel que le suppresseur de tumeur p53. Récemment, il a été démontré qu'E4F1 régule les voies oncogéniques impliquant p53 et Rb qui jouent un rôle essentiel au cours de la sénescence cellulaire. La sénescence, qui est définie par un arrêt irréversible du cycle cellulaire, est considéré comme un mécanisme suppresseur de tumeurs essentiel au cours des phases précoces du développement tumoral. L'objectif de ma thèse a été d'évaluer le rôle d'E4F1 au cours de la sénescence cellulaire. Au travers d'études menées sur des fibroblastes humains primaires et des fibroblastes embryonnaires murins dérivés de souris génétiquement modifiées pour le gène E4F1, j'ai examiné comment la perturbation des activités d'E4F1 module l'initiation ou le maintien de la sénescence prématurée induit par l'oncogène RAS, la déplétion du membre de la famille polycomb Bmi1, ou par les dommages à l'ADN. Mes résultats suggèrent que la déplétion d'E4F1 protège partiellement contre l'induction de la sénescence alors que l'expression ectopique d'E4F1 accélère la sénescence par son implication dans la voie INK4A/ARF-p53. L'ensemble de mes résultats supportent la notion qu'E4F1 est un régulateur important de la sénescence cellulaire. / E4F1 was originally identified as a cellular target of the viral oncoprotein E1A during adenoviral infection. E4F1 is a multifunctional protein that is essential during early embryogenesis and plays important roles in the proliferation/survival balance of different cell types including stem cells. At the molecular level, E4F1 exhibits intrinsic transcriptional activities but also an ubiquitin E3 ligase function that targets other transcription factors, including the p53 tumor suppressor. Recent studies indicate that E4F1 impinge on several pathways, including the Rb and p53 pathways, that are known to influence cellular senescence, an irreversible state of cell cycle arrest that is considered to be an essential tumor suppressor mechanism during early steps of tumorigenesis. The objective of my thesis was to evaluate the roles of E4F1 during cellular senescence. Using human primary fibroblasts and mouse embryonic fibroblasts derived from genetic ally engineered mouse models, I investigated how perturbations of E4F1 activities modulated the initiation or the maintenance of premature senescence induced by oncogenic Ras, depletion of the polycomb member Bmi1 or DNA damage. My results suggest that E4F1 depletion partly protects from the induction of cellular senescence whereas ectopic expression of E4F1 accelerates premature senescence through its implication in the Ink4a/ARF-p53 pathway. Altogether, my results support the notion that E4F1 is an important regulator of cellular senescence.

La sénescence cellulaire est une réponse aux stresses selon laquelle des cellules pouvant proliférer optent pour entrer dans un arrêt du cycle cellulaire en réponse à une variété de stimulations intrinsèques et extrinsèques telle que, par exemple, le raccourcissement des télomères, un stress oxydatif, des dommages à l’ADN ou l’activation constitutive d’oncogènes. Toutes ces stimulations ont en commun le potentiel d’initier ou de promouvoir une transformation néoplasique qui peut dégénérer en cancer. En fait, la sénescence est maintenant acceptée comme un mécanisme cellulaire autonome pour empêcher le développement du cancer et elle est reconnue, particulièrement dans les cellules humaines, pour s’établir et se maintenir à l’aide d’au moins deux voies de suppression tumorale majeures : les voies de p53/p21CIP et de p16INK4A/pRB. Ces deux voies sont capables d’activer et d’augmenter l’expression d’un autre suppresseur tumoral : la protéine PML. En tant que suppresseur de tumeur, PML est suffisant pour induire la sénescence dans des cellules normales, mais il n’arrive généralement pas à activer une réponse complète de sénescence dans les cellules cancéreuses. Considérant ces faits, le premier objectif de cette thèse était d’étudier le mécanisme de résistance des cellules cancéreuses contre la sénescence induite par PML. Nous avons trouvé que, dans des cellules normales, la surexpression de CDK4 ou de CDK6 (CDK4/6) est suffisante pour contourner la sénescence induite par PML. De même dans les cellules cancéreuses, l’expression de ces kinases, souvent retrouvées augmentées dans de nombreux cancers, prévient probablement l’induction d’une sénescence par PML. Effectivement, grâce à l’inhibition de l’expression et/ou de la fonction kinase des CDK4/6, nous avons réussi à restaurer un programme de sénescence dans des cellules cancéreuses. En fait, l’utilisation de palbociclib, un inhibiteur spécifique de CDK4/6 maintenant en essais cliniques, permet d’augmenter l’habileté de PML à induire un arrêt de croissance plus fort et plus durable dans des cellules en cultures ainsi qu’une meilleure réduction de la progression de tumeurs dans des souris. Cette sénescence plus complète corrèle avec une augmentation de la présence de marqueurs d’autophagie, une meilleure répression des gènes cibles des E2F et une signature d’expression de gènes correspondant à l’inhibition de la méthylation de l’ADN. Ce dernier point découle du fait que l’inhibition de CDK4/6 par le palbociclib promeut une dégradation par autophagie de la DNA méthyltransférase DNMT1. Nous avons aussi démontré que CDK4 est capable d’interagir avec DNMT1 et de le phosphoryler in vitro. Ces résultats soulignent la valeur potentielle des inhibiteurs de CDK4/6 en tant que modulateurs épigénétiques pour faciliter l’activation de la sénescence dans des cellules cancéreuses. La sénescence induite par PML est fortement liée aux modifications post-traductionnelles. Parmi ces dernières, la SUMOylation joue un rôle important dans la fonction d’échafaudeur de PML et dans la formation des corps de PML. Les corps de PML sont des structures nucléaires dynamiques stimulées par des stresses, comme l’activation d’oncogènes menant à la sénescence, et dont la formation permet la séquestration de protéines spécifiques pour leur régulation et/ou pour leur modification post-traductionnelle. À travers le recrutement de protéines, les corps de PML régulent de nombreuses fonctions cellulaires telles que la sénescence, l’apoptose, la réponse antivirale, la réponse aux dommages à l’ADN et la régulation de l’expression de gènes. Compte tenu de cela, le deuxième objectif de cette thèse était de caractériser le rôle de la SUMOylation dans la sénescence induite par un oncogène, soit par l’expression de l’oncogène RAS. À l’aide d’une analyse du protéome de SUMO3 dans les cellules sénescentes versus des cellules en croissances, nous avons pu identifier 25 sites de SUMOylation dans 23 protéines dont l’incidence était significativement régulée par la sénescence. Il est à noter que la plupart de ces protéines (un tiers) sont connues pour être associées au corps de PML. Curieusement, UBC9 (la seule enzyme E2 pour la SUMOylation) a été retrouvée plus SUMOylée dans la sénescence sur sa Lys-49. Des études fonctionnelles d’un mutant d’UBC9 pour la Lys-49 ont démontré une diminution de son association aux corps de PML et la perte de la capacité d’UBC9 surexprimé à retarder la sénescence. De plus, la localisation forcée d’UBC9 dans les corps de PML gêne la sénescence induite par PML ou RAS. Ces résultats nous permettent de proposer des fonctions pro- et anti-sénescence de la SUMOylation des protéines, particulièrement pour UBC9. Mots-clés : Sénescence, PML, CDK4 et CDK6 (CDK4/6), méthylation de l’ADN, palbociclib, corps de PML, SUMOylation, UBC9 / Cellular senescence is a stress response wherein proliferating competent cells undergo a stable cell cycle arrest in response to a variety of intrinsic and extrinsic stimuli, including telomere shortening, oxidative stress, DNA damage or the constitutive activation of oncogenes among others. All these stimuli have in common the potential to initiate or promote neoplastic transformation that can degenerate in cancer. Senescence, particularly in human cells, is established and maintained by at least two major tumor suppressor pathways: the p53/p21CIP and p16INK4A/pRB pathways and is now accepted as a potent cell-autonomous mechanism for suppressing the development of cancer. Both pathways are able to activate and increase the expression of the tumor suppressor protein PML. As a tumor suppressor, PML is sufficient to induce senescence in normal cells; however, upon the same stimuli, cancer cells fail to engage a complete senescence response. Given this, the first aim of this thesis is to investigate the resistance mechanisms of cancer cells to PML-induced senescence. We found that overexpression of the CDK4 and CDK6 (which are often up-regulated in cancer) are sufficient to bypass PML-induced senescence in normal cells. In cancer cells the expression of these kinases impairs the PML-induced senescence. By inhibiting the expression and/or function of CDK4/6 we were able to restore the senescence program in cancer cells. Also, the specific CDK4/6 inhibitor palbociclib (currently used in clinical trials) increased the ability of PML to regulate a stronger and more permanent growth inhibition in cell culture and decreased tumor progression in mice. This complete senescence response correlated with an increase in autophagy markers, repression of E2F target genes and a gene expression signature of blocked DNA methylation. Furthermore, CDK4/6 inhibition by palbociclib promotes autophagy-dependant degradation of the DNA methyltransferase DNMT1. More important, we were able to demonstrate that CDK4 directly interacts and phosphorylates DNMT1 in vitro. These results highlight the potential value of CDK4/6 inhibitors as epigenetic modulators to facilitate activation of cellular senescence in cancer cells. PML-induced senescence is tightly regulated by post-translational modifications (PTMs). Among these PTMs, SUMOylation plays an important role in the scaffold function of PML and the formation of the PML-NBs (PML-Nuclear Bodies). PML-NBs are dynamic structures triggered by stress such as oncogene-induced senescence, and its formation allows the sequestration of target proteins for their regulation and/or its post-translational modification. By protein recruitment, PML-NBs regulate several cellular functions such as senescence, apoptosis, antiviral response, DNA repair and gene regulation. Given this; the second aim of this thesis is to characterize the role of SUMOylation in oncogene mediated cellular senescence, specifically by the expression of the oncogene RAS. By a SUMO3 proteome analysis of senescent cells we were able to identify 25 SUMO sites in 23 proteins that were significantly regulated during senescence. Importantly, most of these proteins were PML-NB associated. Interestingly, UBC9 (the only SUMO E2 enzyme), was found more SUMOylated in senescence on its Lys-49. Functional studies of a UBC9 mutant in Lys-49 showed a decreased association to PML-NBs and the loss of UBC9’s ability to delay senescence. Moreover, forced localization of UBC9 into PML-NBs counteracted RAS and PML-induced senescence. These results allowed us to propose a pro- and an anti-senescence function of protein SUMOylation, specifically for UBC9. Keywords: Senescence, PML, CDK4/6, DNA methylation, palbociclib, PML-NBs, SUMOylation, UBC9

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.