A la suite des catastrophes nucléaires d’importantes quantités de radionucléides ont été rejetés dans l’environnement. Le Strontium 90 (90Sr) fait partie de ces rejets. Du fait de sa demi-vie de 29 ans, c’est un polluant persistant qui conduit à la contamination des populations vivant autour des territoires contaminés via l’ingestion chronique de faibles quantités de ce radionucléide. Les études épidémiologiques ont mis en évidence des effets au niveau du système immunitaire, du système hématopoïétique et de la physiologie osseuse chez l’homme. Le 90Sr qui s’incorpore principalement dans l’os pourrait contribuer à l’apparition de ces effets. Le but de ce travail a été de comprendre quels sont les mécanismes d’action du 90Sr qui permettent d’expliquer de tels effets. Un premier modèle in vitro utilisant une lignée de cellules stromales murines (MS5) contaminées par le milieu de culture à 1 ou 10 kBq.ml-1 a été utilisé. Il a permis de montrer que le 90Sr était capable d’induire des cassures double-brin de l’ADN dès 30 minutes d’exposition avec une induction de la senescence et une altération de la fonction de support aux progéniteurs hématopoïétiques. Dans le deuxième modèle in vivo d’effet dose, des souris Balb/c ont été contaminées durant 24 semaines à des concentrations de 90Sr de 4, 20 et 100 kBq.l-1 dans l’eau de boisson. Cette expérience a permis d’observer une augmentation des marqueurs de la résorption osseuse en fonction de la contamination au 90Sr ainsi et une augmentation de l’expression génique des enzymes impliquées dans la défense antioxydante. Une augmentation de p21, marqueur de la senescence et une diminution d’IL-6 ont également été observées. Les implications de ces résultats sur la physiologie osseuse, le système immunitaire et hématopoïétiques sont discutées. Globalement, l’ensemble de ce travail complète les données déjà existantes sur le 90Sr et permet de mieux comprendre les mécanismes d’action du 90Sr sur les cellules stromales médullaires qui sont au centre de la régulation immuno-hématopoïétique. / Abstract : After nuclear disasters, large amounts of radionuclides were released into the environment. Strontium 90 (90Sr) is part of these wastes. Because of its half-life of 29 years, it is a persistent pollutant which leads to the contamination of surrounding populations through the chronic ingestion of low quantities of this radioelement. Epidemiologic studies have demonstrated some effects on immune system, hematopoietic system and bone physiology in humans. 90Sr accumulates mostly in bones and could contribute to the appearance of such effects. The aim of this work is to understand the action mechanisms which could explain the previous observations. In the first in vitro model, a murine stromal cell line (MS5) contaminated through the culture medium with 1 or 10 kBq.ml-1 of 90Sr was used. Thank to this model, an increased number of DNA double-strand breaks in cells after 30 minutes of exposure, a senescence induction and a modification in the support of hematopoietic progenitors were observed. In the second model, Balb/c mice were contaminated during 24 weeks through drinking water containing 90Sr at 4, 20 and 100 kBq.l-1. Both an increase in genic expression of bone resorption markers and in antioxydative enzymes were observed. An increase in p21 expression, marker of senescence, and a decrease in IL-6 were also seen. The implications of these results on bone physiology, immune and hematopoietic systems are discussed. As a whole, all this work completed the preexistent data about 90Sr and contributes to a better understanding of the action mechanisms of 90Sr on marrow stromal cells which have a pivotal function in the regulation of the immune and hematopoietic system.

Therapy resistance of malignant cells represents the main reason responsible for the failure of cancer therapy. The growth of malignant cells at primary tumour sites but most importantly the dissemination of tumour cells and their growth at secondary sites, are the main reasons why patients eventually succumb to the disease. Even novel immune-based therapies find their limitation in most tumour types. The therapy resistance is mediated by the tumour cells but also by other cellular components of the tumour microenvironment. Understanding the tumour cells mechanisms and the tumour microenvironment features responsible for therapy resistance enables the development of novel therapeutic strategies. Here, we show that ionizing irradiation, 5-azacytidine, and IFNγ treatments induced expression of suprabasin (SBSN) and therapy-resistant low-adherent phenotype in cancer cells. Knockdown of SBSN resulted in suppression of the phenotype. Next, we identified aberrantly elevated SBSN in the bone marrow of a subgroup of myelodysplastic syndromes (MDS) patients. SBSN was expressed by myeloid-derived suppressor cells (MDSCs) and showed significant anti-correlation with T cell abundance and CCL2 levels, hence promises a prognostic value in clinical use. We compiled the most of the relevant knowledge of SBSN...

Activée par la chimiothérapie, la sénescence est un mécanisme suppresseur de tumeur qui bloque la progression tumorale. Cependant, des cellules cancéreuses sont capables d’échapper à cette pression ce qui provoque une rechute clinique. Nous avons récemment décrit que les cellules émergentes acquièrent la capacité de résister à l’anoïkis et dépendent de Mcl-1. Cette voie de survie est activée par la kinase Akt qui inhibe la protéine Noxa et l’apoptose. L’une des caractéristiques de la sénescence est l’apparition d’un phénotype sécrétoire appelé SASP qui peut induire des effets délétères aux cellules voisines. Dans cette étude nous avons observé que le sécrétome des cellules persistantes induit la résistance à l’anoïkis, la migration et l’invasion des cellules parentales. Des études de protéomique réalisées au laboratoire ont montré que laTSP-1 est surexprimée dans les stades avancés de tumeurs de patients du sein et du colon. Lors de la persistance, la TSP-1 et son récepteur CD47 sont exprimés plus fortement par les cellules sénescentes. Le blocage de la TSP-1 ou de sa liaison à CD47 augmente l’émergence et induit la formation de sphéroïdes traduisant une augmentation de la proportion de cellules souches. Les facteurs d’auto-renouvellement Nanog etKlf4 sont induits précocement en réponse au traitement. Suite à l’inactivation de CD47 ou à une stimulation avec laTSP-1, l’expression de Nanog est bloquée. L’inhibition de Nanog ou de Klf4 diminue l’émergence. Ainsi, dans les cellules sénescentes, CD47 activerait le mécanisme d’auto-renouvellement et favoriserait l’émergence. En seliant, la TSP-1 bloquerait ces mécanismes et agirait comme un suppresseur de tumeur. / Activated by chemotherapy, senescence is a suppressive mechanism that prevents tumor progression. However some cancer cells can emerge and induce clinical relapse. We have recently described that emergent cells resist toanoikis and depend on Mcl-1. This survival pathway is activated by Akt kinase that inhibits Noxa and apoptosis. One of the caracteristics of senescence is the appearance of the secretory phenotype called SASP that can induce deleterious effects to neighboring cells. In this study, we observed that the secretome of persistant cells induces anoïkis resistance, migration and invasion of parental cells. Proteomics analysis performed at laboratory showed that TSP-1 is over expressed in advanced stages of colon and breast tumors. During persistance, TSP-1 and its receptor CD47 are more expressed by senescent cells. Blockade of TSP-1 or its binding on CD47 increases persistence and induces spheroïds generation showing an increase in the proportion of stem cells. Self-renewal factors Nanog and Klf4 are early expressed following treatment. Following CD47 inactivation or stimulation withTSP-1, the expression of Nanog is blocked. The inhibition of Nanog or Klf4 reduces emergence. So, in senescent cells, CD47 could activate self-renewal and could promote emergence. By linking to its receptor, TSP-1 could block these processes et coud act as a tumor suppressor.

Therapy resistance of malignant cells represents the main reason responsible for the failure of cancer therapy. The growth of malignant cells at primary tumour sites but most importantly the dissemination of tumour cells and their growth at secondary sites, are the main reasons why patients eventually succumb to the disease. Even novel immune-based therapies find their limitation in most tumour types. The therapy resistance is mediated by the tumour cells but also by other cellular components of the tumour microenvironment. Understanding the tumour cells mechanisms and the tumour microenvironment features responsible for therapy resistance enables the development of novel therapeutic strategies. Here, we show that ionizing irradiation, 5-azacytidine, and IFNγ treatments induced expression of suprabasin (SBSN) and therapy-resistant low-adherent phenotype in cancer cells. Knockdown of SBSN resulted in suppression of the phenotype. Next, we identified aberrantly elevated SBSN in the bone marrow of a subgroup of myelodysplastic syndromes (MDS) patients. SBSN was expressed by myeloid-derived suppressor cells (MDSCs) and showed significant anti-correlation with T cell abundance and CCL2 levels, hence promises a prognostic value in clinical use. We compiled the most of the relevant knowledge of SBSN...

Le carcinome ovarien est l'une des tumeurs gynécologiques les plus meurtrières dans le monde et particulièrement au Canada. En effet, il s’agit du troisième cancer de l’appareil reproducteur féminin le plus fréquent au Canada, selon la Société Canadienne du Cancer qui estima que sur 3 000 canadiennes ayant été diagnostiquées avec un cancer de l’ovaire en 2022, environ 1 950 ne survivront pas à la maladie. Les traitements de première ligne pour ce cancer comprennent la chirurgie cytoréductive associée à une chimiothérapie à base de platine et de taxane comme l’association des anticancéreux que sont le Carboplatine et Paclitaxel. Nous retrouvons également comme traitement la radiothérapie, et, plus récemment, les inhibiteurs de la poly (ADP-ribose) polymérase (PARPi) comme l'Olaparib qui sont désormais utilisés en première ligne dans ce type de cancer. Ces traitements peuvent entraîner différentes décisions concernant le devenir des cellules, impliquant non seulement la mortalité ou la survie des cellules cancéreuses, mais aussi un arrêt de la prolifération induit par le traitement appelé TIS pour sénescence induite par la thérapie. Alors que les décisions relatives au devenir des cellules sont déterminantes pour l'issue du traitement du cancer, notre capacité à mesurer le devenir des cellules dans le cancer en temps réel est extrêmement limitée. Pour cette raison, il n'existe pas de modèles de cancer de l'ovaire qui puissent fournir un suivi non invasif du devenir des cellules à des moments spécifiques avec des biomarqueurs adaptés, pour décrypter le rôle des différents devenirs cellulaires, ou pour servir de contrôles expérimentaux précis dans les tests précliniques des stratégies d'intervention basées sur le devenir des cellules. Néanmoins, nous avons démontré qu’un fragment du promoteur du gène de la protéine p21, que nous avons nommé p21SEN, n'est exprimé que pendant la sénescence induite par les radiations, la chimiothérapie et les PARPi dans des lignées d’adénocarcinome ovarien à cellules claires (TOV21G). En effet, nous avons généré des lignées exprimant une protéine fluorescente verte dirigée par le promoteur p21SEN et ainsi, nous avons pu observer et suivre son activation à travers un signal vert durant la sénescence induite par les différents traitements utilisés. De plus, de façon intéressante, nos résultats ont également permis de montrer que cette expression de p21SEN durant la TIS semble être partiellement indépendante du facteur de transcription p53. Ainsi, nous suggérons que le promoteur p21SEN pourrait servir de rapporteur, en partie indépendant de p53, de l'induction de la sénescence dans un modèle utilisant un système de surveillance non invasif des décisions relatives au devenir des cellules dans le cancer de l'ovaire. / Ovarian carcinoma is one of the deadliest gynecological tumors worldwide, and particularly in Canada. In fact, it is the third most common cancer of the female reproductive system in Canada, according to the Canadian Cancer Society, which estimates that out of 3,000 Canadian women diagnosed with ovarian cancer in 2022, around 1,950 will not survive the disease. First-line treatments for this cancer include cytoreductive surgery combined with platinum and taxane chemotherapy such as the combination of the anticancer drugs Carboplatin and Paclitaxel. Other treatments include radiation therapy and, more recently, poly (ADP-ribose) polymerase inhibitors (PARPi) such as Olaparib, which are now used as first-line therapy for this type of cancer. These treatments can lead to different cell fate decisions, involving not only cancer cell death or survival, but also a treatment-induced proliferation arrest called TIS for therapy-induced senescence. While cell fate decisions are critical to the outcome of cancer treatment, our ability to measure cell fate in real time in cancer is extremely limited. For this reason, there are no ovarian cancer models that can provide non-invasive monitoring of cell fate at specific time points with tailored biomarkers, to decipher the role of different cell fates, or to serve as accurate experimental controls in preclinical testing of fate-based intervention strategies. Nevertheless, we have demonstrated that a fragment of the p21 promoter, which we have termed p21SEN, is expressed only during radiation-, chemotherapy-, and PARPi-induced senescence in clear cell ovarian adenocarcinoma cell lines (TOV21G). Indeed, we generated cell lines expressing a green fluorescent protein directed by the p21SEN promoter and thus, we were able to observe and follow its activation through a green signal during the senescence induced by the different treatments used. Moreover, interestingly, our results also showed that this expression of p21SEN during TIS seems to be partially independent of the transcription factor p53. Thus, we suggest that the p21SEN promoter could serve as a partially p53-independent reporter of senescence induction in a model using a non-invasive monitoring system of cell fate decisions in ovarian cancer.

Overwhelming evidence for senescence, the within-individual decline in performance at advanced age, has now been documented in the natural populations of many taxa. As such, the focus of senescence research is shifting from simply documenting its existence, towards understanding the fundamental mechanisms underpinning it and determining which environmental factors give rise to the considerable variation in senescence rates observed in nature. In this thesis I use a wild population of European badgers (Meles meles) to investigate three important traits implicated in, or arising as a direct product of, senescence; immune cell telomere length, pro-inflammatory cytokine response and body mass declines in late life. My work reveals rare longitudinal evidence for the existence of senescence in immune traits in a wild mammal. First, I show that within-individual declines in immune cell telomere length occur with increasing age (Chapter 2). Second, after demonstrating that immune cell telomere length displays repeatable between-individual differences in adulthood, I show that the environmental conditions experienced in early-life contribute to such between-individual variation. Individuals that experienced harsh early-life environmental conditions had shorter immune cell telomere lengths than those that experienced favourable conditions (Chapter 3). Third, I show that within-individual declines in a second immune trait, pro-inflammatory cytokine response, also occur with age (Chapter 4). However, the declines in immune cell telomere length and pro-inflammatory cytokine response occur independently of one another (Chapter 4). Finally I take advantage of a 37 year longitudinal dataset to reveal that sex differences in body mass senescence arise as a consequence of the scale of intra-sexual competition experienced in early adulthood (Chapter 5). Taken together this work provides novel evidence suggesting that age-related declines in immunocompetence can contribute to whole organism senescence in the wild. Furthermore, evidence that early life environmental and social conditions can markedly influence senescence rates has important implications for our understanding of the drivers of variation in senescence rates observed within natural populations.

Plant development is complex and highly regulated. Tens of thousands of genes have been sequenced for the model plant <em>Arabidopsis thaliana</em>, yet few have been functionally annotated and characterized. This thesis describes the expression analysis and characterization of four genes in <em>Arabidopsis</em>. Three of these belong to the eukaryotic translation initiation factor 5A (eIF5A) gene family, and the fourth encodes diacylglycerol acyltransferase 1 (DGAT1). Putative roles for these genes in the development of <em>Arabidopsis thaliana</em> are described. <br /><br /> eIF5A is the only known protein to contain the amino acid hypusine. It has been demonstrated previously that eIF5A acts as a shuttle protein, moving specific mRNAs from the nucleus to the cytoplasm for translation. In <em>Arabidopsis thaliana</em> (At), there are three isoforms of eIF5A, and it is clear from the present study that they each have a unique temporal and spatial expression pattern. AteIF5A-1 and -2 are up-regulated during natural senescence and wounding/pathogenesis, respectively, and it is proposed that they regulate the onset of programmed cell death during these events. AteIF5A-3 is up-regulated in elongating meristem of the root, and it is proposed that this isoform is involved in cell growth. <br /><br /> Over-expression of the individual <em>AteIF5A</em> isoforms <em>in planta</em> resulted in pleiotropic phenotypes. When <em>AteIF5A-1</em> or <em>AteIF5A-2</em> was over-expressed, the phenotypes observed were indicative of their putative roles in the translation of proteins required for programmed cell death. When <em>AteIF5A-3</em> was over-expressed, the phenotypes were indicative of a role for this protein in the regulation of cell and tissue elongation. <br /><br /> Lipid analysis of rosette leaves from <em>Arabidopsis thaliana</em> revealed an accumulation of triacylglycerol with advancing leaf senescence coincident with an increase in the abundance and size of plastoglobuli. The terminal step in the biosynthesis of triacylglycerol in <em>Arabidopsis</em> is catalyzed by DGAT1. When gel blots of RNA isolated from rosette leaves at various stages of development were probed with the <em>Arabidopsis</em> EST clone, E6B2T7, which has been annotated as DGAT1, a steep increase in DGAT1 transcript levels was evident in the senescing leaves coincident with the accumulation of triacylglycerol. The increase in DGAT1 transcript correlated temporally with enhanced levels of DGAT1 protein detected immunologically. Two lines of evidence indicated that the triacylglycerol of senescing leaves is synthesized in chloroplasts and sequesters fatty acids released from the catabolism of thylakoid galactolipids. First, triacylglycerol isolated from senescing leaves proved to be enriched in hexadecatrienoic acid (16:3) and linolenic acid (18:3), which are normally present in thylakoid galactolipids. Second, DGAT1 protein in senescing leaves was found to be associated with chloroplast membranes. These findings collectively indicate that DGAT1 plays a role in senescence by sequestering fatty acids de-esterified from galactolipids into triacylglycerol.

Telomerase is present in over 90% of tumour tissues and immortalized cells and is tightly regulated in most normal somatic cells. This suggests the existence of regulatory mechanisms repressing telomerase in normal cells that somehow have become inactive during cancer development. In this project, I used genetic complementation in the form of microcell-mediated monochromosome transfer (MMCT) to search for chromosomes that repress telomerase activity in a prostate cancer cell line, PC-3. Microcell hybrids generated by introducing normal human chromosome 11 strongly inhibited telomerase. Telomerase is regulated primarily at the level of hTERT transcription, its catalytic subunit. Consequently, endogenous hTERT mRNA levels were measured by quantitative RT-PCR in microcell hybrids generated by transferring normal human chromosomes into a PC-3 sub-clone (PC- 3/hTERT) ectopically expressing hTERT cDNA to prevent senescence. Only hybrids constructed with transferred chromosome 11 showed strong transcriptional repression of hTERT. Next, hybrids were constructed by the MMCT transfer of chromosome 11 fragments (X-ray-induced). FISH analysis of clones with completely silenced endogenous hTERT transcription revealed in all cases a discrete chromosome 11 fragment with both the p-arm and q-arm material. A randomly selected hTERT-repressed clone was treated with ganciclovir to select against the HyTK marker and reverse the phenotype. hTERT expression in majority of GCV-resistant clones returned to levels comparable to the parent PC-3/hTERT cells. Collectively, these results provide strong functional evidence for the presence of a powerful telomerase repressor sequence on the fragment. Transfer of one repressive fragment back into mouse A9 cells was then carried out to facilitate fine-structure mapping of its sequence content. High density STS mapping of the fragment in each of the clones revealed a considerable DNA content heterogeneity across the panel. These content maps, together with a further round of MMCT to confirm hTERTrepressive activity, enabled me to identify three candidate regions on the q-arm of chromosome 11 where the repressor sequence may be located: the first region lies between map positions 64.70Mb to 65.42Mb and the other two regions each flank a single positive STS marker at 69.71Mb and 127.32Mb. KAT5, a histone modifying gene has been identified as a potential candidate for repressing hTERT.

La sénescence et l’apoptose sont deux mécanismes oncosupresseurs induits dans les lésions pré-malignes et capables de s’opposer à la prolifération cellulaire incontrôlée induite par l’activation d’oncogènes mitogéniques. Leur inhibition est nécessaire à la conversion d’une lésion bénigne en tumeur maligne. Nous avons démontré que les gènes embryonnaires TWIST1, TWIST2 et SNAI3 sont fréquemment réactivés dans les cancers humains. De par leur capacité à inactiver fonctionnellement la protéine oncosuppressive p53, ces facteurs embryonnaires permettent d’inhiber la sénescence et l’apoptose induites en réponse à l’activation d’oncoprotéines mitogéniques, de transformer in vitro des fibroblastes primaires murins et de leur accorder un pouvoir tumorigène. Dans les cellules épithéliales, cet échappement aux mécanismes de sauvegarde est associé à une transition épithélio-mésenchymateuse (EMT) et à l’acquisition conséquente de capacités de migration, d’invasion et d’auto-renouvellement. Ces résultats nous ont donc permis de mettre en exergue un lien étroit entre l’inhibition des systèmes de sauvegarde cellulaire et l’EMT et de proposer que la réactivation des gènes embryonnaires tels TWIST ou SNAI3 soit suffisante pour promouvoir la conversion maligne et la dissémination précoce des cellules cancéreuses / Senescence and apoptosis, the two main oncosuppressive mechanisms activated in premalignant tumors, restrict cell proliferation in response to mitogenic oncogene activation. Their inhibition is required for malignant conversion of benign lesions. We demonstrated that TWIST1, TWIST2 and SNAI3 embryonic genes are frequently reactivativated in human cancers. By functionally inactivating the p53 oncosuppressive protein, these embryonic factors override oncogene-induced senescence and apoptosis and cooperate in vitro with mitogenic oncoproteins in murine primary fibroblast transformation, providing cells with tumorigenic potential. In epithelial cells, failsafe program escape is associated with an epithelialmesenchymal transition (EMT), and the consequent acquisition of motility, invasive properties and selfrenewal capabilities. These data highlight an intimate crosstalk between failsafe program escape and EMT and suggest that reactivation of embryonic genes such as TWIST or SNAI3 is sufficient to promote both malignant conversion and early cancer cell dissemination

In this thesis, I explore four different ways in which relatedness affects sexual interactions in the red junglefowl Gallus gallus ssp., and the fruit fly Drosophila melanogaster. First, I show that in both species, inbreeding depression is sex-specific and modulated by parental age and gametic age. However, the sex that suffers higher inbreeding depression was trait- and species-dependent. Second, I examined patterns of inbreeding avoidance. I found no evidence of inbreeding avoidance in the fruit fly, but in the red junglefowl both males and females avoided mating with relatives, independently from sex-ratio of the social group. Third, I investigated whether relatedness amongst members of one sex affects mate choice in members of the opposite sex. Male fruit flies preferentially courted females unrelated to females with whom they had previously mated, while female flies displayed a weak preference for males related to their previous mates. In the red junglefowl, females exposed to male trios of two males related to each other and one unrelated male, displayed a marked preference for mating with the male unrelated to the other two males, and might also bias postcopulatory sperm utilization in favour of the unrelated male. Fourth, I explored the implications of male relatedness on the intensity of male-male competition. Male red junglefowl were less aggressive towards related competitors, but invested more sperm in females that had previously mated with a related male rather than with an unrelated male. In fruit flies, male relatedness had a strong impact on female life-history and offspring viability, although I found no evidence that these effects were modulated by changes in male-male competition. Collectively, the findings of these studies demonstrate the complex relationship between relatedness and other important biological phenomena as such senescence and sexual conflict.