La sénescence cellulaire est un mécanisme de suppression cellulaire caractérisé par un arrêt irréversible du cycle cellulaire. Les cellules sénescentes ont également un secrétome spécifique qui influe sur les cellules voisines, pouvant induire leur entrée prématurée en sénescence, l'apoptose, la prolifération cellulaire et le développement tumoral. Dans ce contexte, l'objectif de ma thèse était d'identifier une nouvelle protéine sécrétée par les fibroblastes sénescents et de caractériser ses fonctions dans les cellules sénescentes et dans les cellules tumorales. Nous avons mis en évidence la protéine WNT16B. Elle est surexprimée dans plusieurs modèles de sénescence cellulaire obtenus à partir de fibroblastes pulmonaires. Dans un modèle de souris transgénique, WNT16B est également associée avec l'accumulation de cellules sénescentes dans les lésions précancéreuses pulmonaires. WNT16B est impliqué dans le mécanisme de sénescence cellulaire. En effet, l'inhibition de WNT16B prévient l'entrée en sénescence, en empêchant l'activation de p53 et l'expression de p21/WAF1, et donc l'arrêt du cycle cellulaire.Nous avons finalement associé l'expression de WNT16B avec la présence de cellules sénescentes dans des tumeurs prélevées chez des patients atteints de carcinomes pulmonaires non à petites cellules. Cette expression est corrélée avec une meilleure survie pour les patients ayant reçu un traitement chimiothérapeutique. Ce dernier résultat fait donc le lien entre traitement, sénescence cellulaire et survie, et illustre le rôle potentiel des marqueurs des cellules sénescentes pour le suivi des patients. Globalement, ces travaux ont donc non seulement permis d'identifier WNT16B comme nouveau marqueur des cellules sénescentes, mais ils ont également ouvert des perspectives d'utilisation de ce marqueur pour l'identification, le traitement et le suivi des patients atteints d'un cancer. / Cellular senescence is a tumor suppression mechanism consisting of an irreversible cell cycle arrest. Senescent cells also express a specific secretome that plays a role in the microenvironment. It may induce premature senescence, apoptosis, cellular proliferation or tumor development in the neighboring cells. In this context, my thesis aimed to identify and characterize a new senescence-associated secreted protein, before highlighting its role in tumorigenesis. We identified the WNT16B protein. It was overexpressed in various cellular senescence models obtained from lung fetal fibroblasts. Moreover, WNT16B expression was associated with the accumulation of senescent cells in precancerous lesions in a transgenic mice model. WNT16B is also involved in the senescence machinery. Indeed, WNT16B inhibition prevented the senescence onset through inactivation of the p53/p21 pathway, thus stopping the cell cycle progression. Finally, we observed a correlation between the WNT16B expression and the occurrence of senescent tumoral cells in NSCLC samples. This expression was also correlated with a better patient outcome after chemotherapeutical treatment. Thus, this last result linked the treatment efficiency, the occurrence of senescence and the patient survival. Overall, my thesis established elevated WNT16B levels as a novel biomarker for senescence that might be useful for the identification, the treatment follow-up and the prognosis of patient outcome.

Senescence is a cell stress program characterized by a stable cell cycle arrest and thus aims to protect against replication of potentially harmful cells. In oncogene-induced senescence (OIS) the cell cycle arrest is brought about by activation of an oncogene. This in turn initiates a DNA damage response and subsequently, the DDR induces p53-p21 and RB tumour suppressor pathways. The metabolism of senescent cells is highly altered, notably there is increased secretion of proteins and increased functional activity of certain metabolic enzymes. There have been many recent studies investigating the role of specific metabolic pathways in OIS and how they may be targeted for therapeutic benefit. This thesis aims to identify novel metabolic regulators of OIS, by combining high throughput RNAi screening and LC-MS based methods. This thesis has identified and validated 17 essential OIS metabolic genes; in this list, there was enrichment for genes involved in lipid biosynthetic processes. Lipid metabolism was an attractive focus for this thesis as it has not been extensively studied in current literature. Next, ceramide synthase 4 (CERS4) was extensively validated as a key enzyme for both OIS and replicative senescence. Using LC-MS based lipidomics, CERS4-driven rewiring of lipid metabolism in OIS was revealed and this corresponded with an accumulation of ceramides due to increased de novo ceramide synthesis. It was then confirmed OIS-related ceramide is mechanistically linked to cell cycle via the PP1-RB-E2F axis. Ceramide activates PP1, which physically binds to RB in a CERS4-dependent manner. PP1 is then able to dephosphorylate and activate RB, which inhibits transcription of E2F targets (cell cycle genes). Overall, this thesis identifies a metabolic checkpoint that links altered lipid metabolism with OIS.

La sénescence constitue la dernière étape du cycle de vie de certains organes des plantes. Elle permet leur dégradation tout en réallouant les constituants des tissus sénescents vers d’autres organes. Dans le contexte de la nodulation symbiotique fixatrice d’azote entre certaines plantes légumineuses et des bactéries rhizobia, un processus de sénescence a été décrit. Cependant, les connaissances sur les mécanismes de régulation de la sénescence des nodosités symbiotiques sont limitées. Au sein du laboratoire, le facteur de transcription MtNAC969 a été identifié comme un régulateur de la sénescence des nodosités. L'objectif de cette thèse est d'identifier et de caractériser de nouveaux régulateurs de la sénescence de l'organe symbiotique. Nous avons développé : (i) une approche visant à identifier des facteurs de transcription corégulés avec MtNAC969 ou avec une cystéine protéase MtCP6 utilisée comme marqueur de la sénescence des nodosités ; et (ii), une approche avec "à priori" se focalisant sur la fonction des différentes voies de signalisation des cytokinines. Cette thèse a permis d'identifier deux facteurs de transcription, MtbHLH107 et MtNAC009 et de décrypter le rôle des cytokinines dans la sénescence des nodosités. Cette thèse a permis d'identifier d'une part, deux nouveaux gènes potentiellement régulateurs de la sénescence nodositaire, MtbHLH107 et MtNAC009; et d’autre partde décrypter le rôle des cytokinines dans la sénescence de cet organe symbiotique. / Senescence is the last step of plant organ lifespan and allows their degradation in order to remobilize components from senescent tissues toward others organs. In the nitrogen fixing symbiosis nodulation occurring between legume plants and rhizobia bacteria, a senescence process has been described. However, limited knowledge about regulatory systems controlling senescence in the symbiotic nodule is available. In the laboratory, the MtNAC969 transcription factor was identified as a regulator of nodule senescence. The aim of this PhD project is to identify and characterize new regulatory factors involved in nodule senescence. We developed two independent approaches : (i) the identification of genes coregulated with MtNAC969 or a cystein protease MtCP6 used as nodule senescence marker ; and (ii), targeted approach focused on the role of cytokinin signaling pathways in nodule senescence. This project allowed us to identify two regulator transcription factors, MtbHLH107 and MtNAC009 ; and to decipher the cytokinin role in the senescence of the symbiotic organ. This PhD thesis allowed us to identify two new potential regulators of nodule senescence, MtbHLH107 and MtNAC009; and to decipher the role of cytokinins in the senescence of this symbiotic organ.

Premature cellular senescence is the process of permanent cell cycle arrest in response to various inducers, such as DNA damage, oxidative stress, chemotherapy agents, and irradiation. Senescent cells produce and secrete numbers of cytokines, chemokines, growth factors, which compose specific senescence-associated secretory phenotype (SASP). Senescence is considered to be an important barrier against tumor progression. On the other hand, senescent cells can also exert protumorigenic effects in their microenvironment. Based on this concept, the major aim of this thesis was to determine tumor cells senescence in terms of different inducers, namely chemotherapeutic agent docetaxel (DTX) and cytokines IFNγ and TNFα, and to demonstrate the role of immunotherapy in senescent cells elimination. Our results show that DTX-induced senescent cells can exert a tumor-promoting effect when co-injected with proliferating cells in mice. Importantly, we demonstrate that IL-12-based immunotherapy suppresses senescence-accelerated tumor growth. These results suggest that IL-12-based immunotherapy can be effectively used in anti-tumor therapy mainly in a case when the microenvironment is altered by the presence of tumor senescent cells. On the other hand, the data we obtained in vitro show that bystander or...

Aging is a complicated and multifactorial phenomenon. Model systems involving the induction of replicative senescence in cultured cells have been indispensable in elucidating some of the mechanisms underlying this complex process. An understanding of how and why cellular senescence occurs is thus critical to the field of aging research. While there is much correlative evidence to suggest a connection between poly(ADP-ribose) (PAR) and mammalian longevity, no studies have been done to explore a possible role for PARP-1 — the enzyme responsible for synthesis of 90% of cellular PAR — in mechanisms of senescence. Furthermore, many techniques currently used for analysis of protein poly(ADP-ribosyl)ation are fraught with imprecision. We therefore sought to address these issues both by developing methods for the unambiguous analysis of poly(ADP-ribosyl)ation by mass spectrometry, and by exploring the role of PARP-1 in nicotinamide-mediated cellular lifespan extension. Due to the challenges introduced by PAR's biochemical characteristics, successful mass spectrometric analysis of poly(ADP-ribosylation) will require the use of techniques to reduce the mass, charge, and heterogeneity of the polymer, as well as methods to enrich for poly(ADP- ribosyl)ated protein. To this end, we evaluated the effectiveness of several approaches, including ammonium sulfate fractionation, boronate affinity chromatography, snake venom phosphodiesterase digestion, manipulation of PARP-1 reaction conditions, and immobilized metal affinity chromatography (IMAC) for the preparation of poly(ADP-ribosyl)ated protein samples prior to MS analysis using both MALDI-TOF and Q-TRAP LC-MS. Based on this work, we developed a three-tiered scheme that may provide the first ever identification of poly(ADP- ribosyl)ated peptides from full-length wild-type PARP-1 by mass spectrometry. Past work in our laboratory has demonstrated that nicotinamide (NAM), a component of vitamin B3, significantly extends the replicative lifespan of human fibroblasts. In order to help elucidate the role of PARP-1 in cellular senescence, we then analyzed the poly(ADP-ribosyl)ation response of aging cells undergoing NAM-mediated lifespan extension. While NAM is a known PARP-1 inhibitor, we found that oxidative stress-induced poly(ADP- ribosyl)ation is increased, not decreased, in NAM-treated cells. We propose that supplemented NAM is taken up by the NAD salvage pathway, ultimately leading to increased cellular NAD and extending replicative lifespan by both preventing PARP-mediated NAD depletion and upregulating SIRT1. We further propose that the demonstrated protective effects of NAM treatment in a number of disease models are due not to PARP-1 inhibition as is commonly assumed, but to upregulation of NAD salvage. / Ph. D.

Explaining the origin and maintenance of biodiversity is a central goal in ecology and evolutionary biology. Some of the most important, theoretical explanations for this diversity centre on the evolution of life histories. Comparative studies on life history evolution, have received significant attention in the zoological literature, but have lagged in plants. Recent developments, however, have emphasised the value of comparative analysis of data for many species to test existing theories of life history evolution, as well as to provide the basis for developing additional or alternative theories. The primary goal of this study was to explore existing theories of life history evolution using a dataset of demographic information in the form of matrix population models for a large number of plant species. By projecting average matrix population models for 207 plant species, life tables and fecundity schedules were obtained and, in turn, were used to estimate relevant life history parameters. These parameters were then used to explore the i) lability of life history traits in plants ii) their continuum of life history variation, iii) the evolution of senescence and iv) the significance of demographic entropy in population ecology. Elasticities and sensitivities of life history traits showed significant phylogenetic signal compared to other life history traits, although, all the values of phylogenetic signal observed were < 1 indicating that life history traits are generally labile. Eighty one percent of species in the datset had mortality curves that increased with age compared to one hundred percent of species that showed a reproductive value curve that decreases with age at the end of life. In particular, the parameters that measured pace and duration were inversely related suggesting in general, the presence of senescence in our data set. Finally, the tenets of the directionality theory based on demographic entropy were generally not confirmed. This study provides an important contribution to the life history evolution of iteroparous perennial plants and confirms existing theories on life history evolution.

Senescence is the age-related progressive deterioration of physiological processes leading to an increased likelihood of death and is a phenomenon that occurs nearly universally throughout all the world’s organisms. This thesis initially investigated the impact of ageing on the adult Drosophila melanogaster cellular immune response and demonstrated that the cellular immune response in D. melanogaster adults did experience an age-dependent decline in function. There was a striking reduction in haemocyte ability to phagocytose foreign particles with up to 30% less phagocytosis occurring in four week old flies compared to one week olds. Haemocyte number also declined in female flies by up to 32% across these ages. An exploration into the mechanisms that could underlie these observed senescent declines in haemocyte number and function revealed that the age-dependent reduction in the circulating haemocyte population occurred regardless of whether flies were unharmed, wounded or infected. The loss of phagocytosis ability in haemocytes in ageing flies was shown to be a cell autonomous process; there was an equal age-dependent decline (~13%) in haemocyte phagocytic activity in both in vivo and ex vivo assays. However, an attempt to identify phagocytic receptor systems that drove senescence in haemocyte function was unsuccessful. The contribution of the cellular immune response in determining survival following a fungal infection was not conclusively demonstrated, however flies with reduced Dif expression had significantly increased pathogen susceptibility. Although pathogen resistance can decline due to immune senescence, disease defence may also be enhanced as an animal’s life progresses through the formation of immunological memories of prior microbial encounters. This thesis revealed that the cellular immune response in D. melanogaster provides a strong, broadly specific and relatively long-lasting immunological priming response. Haemocytes phagocytosed up to 33% more microbes per cell during a secondary encounter, and up to 50% more if flies had received two homologous primes. This was not general immune upregulation as a heterologous microbial encounter caused a reduction in the phagocytic ability of haemocytes compared to controls. The level of enhancement in the phagocytic ability of haemocytes also declined with the age of the fly, meaning that the ability to develop a primed response senesced. These results are unprecedented in Drosophila and challenge our conventional interpretation of immune senescence because individual immune history has been shown to shape later cellular immune responses. Ageing is a complex and variable process. Some of the differences observed in ageing rates between populations can be due to different selection pressures. Natural selection acts on genetic variation within a population to increase fitness whereas host-parasite interactions predominantly influence genes related to immune parameters. Many genes have pleiotropic effects as well as there being potential trade-offs between investment in longevity, reproduction and immunity. To explore potential genetic variation in immune and life history traits and whether variation in immune parameters negatively influenced other life history traits related to ageing, a panel of outcrossed genotypes of D. melanogaster were assessed. As the flies were derived from individuals originally sourced from a natural population, the results suggest that a striking amount of genetic variation in immune and life history traits is present in wild populations. However no significant correlations between genetic variation in ageing and genetic variation in investment in immunity were identified. Though, perhaps not surprisingly, no key biomarker of ageing in D. melanogaster was identified; this thesis has contributed some significant findings on the effects of ageing on adult D. melanogaster especially relating to their cellular immune response.

Death domain-associated protein 6 (DAXX) is a multifunctional protein involved in diverse cellular processes. It acts as a histone chaperone or regulator of transcription and apoptosis, in which is its role quite controversial. DAXX also participates in regulation of cell DNA damage response (DDR). DAXX co-creates and stabilizes complex with Mdm2, which negatively regulates the stability of p53, an important tumor suppressor, which is a part of signalling node in the DDR. If DNA damage is not lethal for the cell and unables it to proliferate, the irreversible state of cell cycle called cellular senescence takes place. Under physiological conditions, induction of senescence can prevent the development of tumorigenesis. Therefore, the description of mechanisms involved in the induction of senescence has potential clinical significance. In my thesis, I aimed to determine changes in the level of DAXX protein in senescent cells and to characterize the manner of its regulation. In tumor cells MCF-7 and primary BJ fibroblasts, I observed decrease in DAXX protein level and its regulation. I tested the hypothesis according to which an increase in DAXX level before DNA damage canprevent induction of cellular senescence, or increase in its expression during senescence can cause recovery of cell proliferation....

Breast cancer is the most common cancer in the UK and Basal-like breast cancer (a highly aggressive subtype) accounts for approximately 8-22% of all cases depending on ethnicity. Unlike most human malignancies and indeed other PAM50 breast cancer subtypes, the vast majority of Basal-like tumours are positive for wild type p16. This p16 signature is associated with a particularly poor prognosis and p16-positive Basal-like breast cancer remains the most clinically challenging subtype and is the focus of this project. Pro-senescence therapies are gaining momentum as attractive strategies for the treatment of those breast cancers with current unmet clinical need. To identify targets for pro-senescence therapy in p16-positive Basal-like breast cancer, a genome‐wide siRNA screen and two subsequent validation screens using two p16-positive cancer cell lines were performed. Screening revealed 20 siRNAs that induced senescence within both cancer cell lines. Strikingly, 11 of these 20 siRNAs targeted ribosomal proteins, implicating disrupted ribosomal biosynthesis in senescence activation in p16-positive Basal-like breast cancer. Importantly, subsequent experiments in normal human mammary epithelial cells established that specific ribosomal protein knockdown is well tolerated by normal cells. Analysis of the METABRIC data set showed a high degree of ribosomal dysregulation in Basal-like tumours and revealed that all 11 ribosomal hits identified were frequently overexpressed in p16-positive Basal-like breast cancers. Kaplan Meier analysis confirmed that elevated expression of six of the 11 ribosomal proteins correlates with a reduced overall survival in these women, further supporting a role for these proteins as drivers of disease. These six ribosomal hits, associated with the poorest patient survival, were prioritised for further validation. Senescence induction was found to be highly stable, and associated with dramatic changes to nucleolar morphology, reminiscent of the nucleolar signature observed upon premature senescence induction in normal human mammary epithelial cells. In addition, siRNA rescue experiments indicated that senescence initiation is dependent on p16 and p21 expression and is accompanied by p16 nuclear translocation and p21 degradation. Further, ribosomal protein silencing in MDA-MB-231 cells (p16-null Basal-like breast cancer cell line) resulted in a 'death-like' phenotype, partially dependent on p21 expression suggesting that, within a cancer context, ribosomal protein silencing may induce a differential response depending on the status of p16. In conclusion, it is proposed that these six ribosomal candidates may form the basis of a novel pro-senescence therapy for p16-positive Basal-like breast cancer. They may also represent novel prognostic biomarkers for this disease subset and may help to improve disease stratification and future directed personalised therapies.

Senescence is classically defined as an irreversible cell cycle arrest. There is now convincing evidence that senescent cells accumulate during human ageing, potentially driving age-related dysfunction through depletion of mitotically active cells and stimulation of chronic inflammation. Recently, a landmark paper demonstrated that removal of p16INK4A (p16)-positive senescent cells in mice prolonged healthy lifespan, suggesting a direct link between senescence and age-related dysfunction. As such, restraining the senescent pool or slowing their rate of accumulation presents an attractive therapeutic strategy for extending healthspan. Previously, our laboratory has demonstrated that siRNA transfection can reverse deep senescence in p16-positive primary adult human mammary epithelial cells using a panel of senescence markers. Subsequent siRNA screening revealed 28 hits which strongly induced reversal in the deeply senescent HMECs. In this project, siRNA knockdown of p16 combined with p21WAF1/CIP1 (p21) was found to reverse deep replicative senescence in primary adult human mammary fibroblasts, as defined by a panel of senescence markers. This discovery provided the opportunity to screen for novel siRNAs which induce reversal in both cell types. Screening in the deeply senescent HMFs of the 28 shortlisted candidates and 33 protein interactors identified using bioinformatics revealed 45 siRNAs which significantly increased cell number compared to the negative siRNA control. Subsequent immunofluorescence staining and high content analysis of the top 14 candidates identified 10 hit siRNAs which induced senescence reversal as defined by a panel of markers. Interestingly, these 10 hits were enriched for cytoskeletal and cell adhesion processes, suggesting an interplay between external forces and senescence induction. The top siRNA hit, early growth response 2 or EGR2, a transcription factor, was validated and selected for further exploration as a novel driver of senescence. Bioinformatics analysis revealed an enrichment for EGR2 binding sites within genes dynamically expressed in HMEC senescence, including p16, p21, and nine hits identified to reverse senescence in HMFs and HMECs. Furthermore, deeply senescent HMFs and HMECs were found to have a significantly increased nuclear EGR2 foci number compared to proliferating cells, and this was significantly decreased in reversed HMFs. In conclusion, it is proposed that EGR2 may represent a novel driver of both HMF and HMEC senescence.