The myelodysplastic syndrome (MDS) is a group of hematopoietic clonal disorders resulting in the inefficient production of myeloid lineage blood cells, with the prevalence of patients older than 65 years. One of the possible treatment options for MDS is 5- azacytidine and 5-aza-2'-deoxycytidine therapy. These compounds have been shown to cause the induction of cell-cycle arrest, cell differentiation and/or apoptosis. The in vitro experiments with 5-aza-2'-deoxycytidine indicated that this compound causes the premature cellular senescence, a state of the irreversible cell-cycle arrest. We have asked, whether 5-azacytidine, as a molecule with similar structure, is capable of causing the same effect. This treatment strategy could be beneficial in case that the negative pro- inflammatory effect of senescent cells on their surroundings can be nullified. In this thesis we have shown that 5-azacytidine induces DNA damage response, which is described as a fundamental event for the onset of the cell senescence. We tested 5- azacytidine treated HeLa cells for several markers of the cell senescence - the increase of the β-galactosidase activity, the PML and PML nuclear bodies and the formation of persistent DNA damage signaling lesions - albeit all these markers were positive, it was the very low increase in...

Organisms such as mammals need tissue renewal as an important process for maintenance of their viability. Because proliferation is essential also for tumourigenesis, cells need tumour-suppressor mechanisms to protect organism against cancer. Cellular senescence, the permanent state of cell-cycle arrest, features one of these intrinsic barriers against tumourigenesis after DNA damage and understanding of this process may lead to finding of novel therapeutic targets and to optimization of chemotherapy for patients with cancer. In the first part of the PhD thesis, we investigated activation of JAK/STAT signalling pathway in drug-induced senescence. We used genotoxic drugs like aphidicolin, camptothecine, 5-bromo- 2'-doexyuridin, etoposide or thymidine to induce premature senescence in normal and cancer cells. All this chemicals were able to persistently activate JAK/STAT signalling in monitored cells. Activation of STATs was accompanied with up-regulation of expression of interferon-stimulated genes (ISGs), such as MX1, IRF1, IRF7 and PML. Since IRF1 and IRF7 can be directly involved in stimulation of the IFN genes, we show activated expression as well as secretion of IFNbeta and IFNgamma, but not IFNalpha in drug-induced senescent cells. Furthermore, an inhibition of JAK1 as a major kinase of STAT...

In Krebszellen induzieren Onkogene und Chemotherapie zelluläre Seneszenz. Dabei handelt es sich um einen terminalen Wachstumsarrest, bei dem durch Trimethylierung der Aminosäure Lysin an Position 9 (K9) des Histons H3 (H3K9me3) die Aktivierung S-Phase-relevanter Gene epigenetisch blockiert ist. Obwohl Therapie-induzierte Seneszenz (TIS) das Gesamtüberleben von Mäusen mit einer Lymphomerkrankung verbessert, stellt eine Elimination seneszenter Zellen aufgrund weiterhin bestehender und neu erworbener tumorigener Eigenschaften ein wichtiges therapeutisches Ziel dar. Die Arbeit zeigt anhand des transgenen Eμ-myc Mausmodells, in dem TIS in Abhängigkeit von der H3K9-Histonmethyltransferase Suv39h1 durch Chemotherapie induziert wird, dass TIS-Zellen in vitro und in vivo einer metabolischen Reprogrammierung unterliegen, die therapeutisch genutzt werden kann. TIS-kompetente Lymphome erhöhen im Gegensatz zu TIS-kompromittierten, Suv39h1- defizienten Lymphomen den Glukoseumsatz und die Produktion von ATP. Diese Umstellung des Stoffwechsels erfolgt als Antwort auf eine erhöhte Proteotoxizität, die durch Bestandteile des Seneszenz-assoziierten sekretorischen Phänotyps (SASP) hervorgerufen wird. SASP-Faktoren lösen in TIS-Zellen erhöhten Stress im endoplasmischen Retikulum (ER) aus und forcieren die Fehlfaltung von Proteinen, die nach vermehrter Ubiquitinierung durch Autophagie unter Energieverbrauch abgebaut werden. Deshalb sind stark SASP-exprimierende TIS-Lymphome im Vergleich zu genetisch durch die Inhibition des Transkriptionsfaktors NfκB veränderten und dadurch SASP-supprimierten TIS-Lymphomen empfindlich gegenüber der Inhibition des Glukosestoffwechsels oder der Blockade von Autophagie. Beides führt zur Elimination von TIS Zellen durch Caspase-12- und Caspase-3-abhängige, ER-initiierte Apoptose. Folglich erwirkt die pharmokologische Inhibition dieser veränderten Stoffwechselbedürfnisse nach TIS Induktion eine Tumorregression und ein verbessertes Gesamtüberleben in vivo. Zusammenfassend zeigen diese Ergebnisse eine katabole Stoffwechsellage seneszenter Zellen, die therapeutisch durch konzeptionell neue „synthetisch-letale“, metabolische Therapien eliminiert werden können. Damit wird erstmals in der Krebstherapie ein Tumor-selektives Seneszenzprogramm zusammen mit der Blockade von Stoffwechselwegen genutzt. / Cellular senescence is a terminal growth arrest of viable cells characterized by S-phase entry-blocking histone 3 lysine 9 trimethylation (H3K9me3) in response to oncogene activation and anticancer chemotherapy. Although therapy-induced senescence (TIS) improves long-term outcome, senescent tumor cells acquire potentially harmful characteristics. Therefore, their quantitative elimination presents a therapeutic opportunity. In this thesis the Eμ-myc transgenic mouse lymphoma model, in which TIS depends on the H3K9 histone methyltransferase Suv39h1, is used to show mechanism and therapeutic exploitation of senescence-related metabolic reprogramming in vitro and in vivo. After senescence-inducing chemotherapy, TIS- competent lymphomas but not TIS-incompetent Suv39h1- lymphomas displayed increased glucose turnover and higher ATP production. The thesis demonstrates that this was due to massive proteotoxic stress, which is a consequence of the enhanced production of secretory proteins - referred to as the senescence-associated secretory phenotype (SASP) - of senescent cells. Consequently, SASP-producing TIS cells exhibited endoplasmic reticulum stress, an unfolded protein response (UPR), and increased ubiquitination, thereby targeting toxic proteins for autophagy in an acutely energy-consuming fashion. Accordingly, TIS lymphomas, unlike senescence models that lack a strong SASP response, for example due to the inhibition of the transcription factor NfκB, were more sensitive to blocking glycolysis or autophagy, which lead to their selective elimination through caspase-12- and caspase-3-mediated endoplasmic reticulum-related apoptosis. Consequently, pharmacological targeting of these metabolic liabilities upon TIS induction in vivo prompted tumor regression and improved treatment outcome further. These findings unveil the hypercatabolic nature of TIS that is therapeutically exploitable by synthetic lethal metabolic targeting. Thus, this treatment approach for the first time combines the inhibition of a tumor-specific senescence program with the interference of a metabolic pathway.

Face à la menace des pathogènes présents dans l’environnement, l’immunité représente une fonction cruciale pour la survie des organismes. Cependant, cette fonction représente divers coûts de développement et d’utilisation, et le caractère limité des ressources dans l’environnement impose des compromis d’allocation entre différentes fonctions (immunité, croissance, reproduction). Sur le long-terme, ces choix peuvent avoir de lourdes conséquences sur les probabilités de se reproduire et de survivre à chaque âge. L’objectif de cette thèse a été de décrire les variations avec l’âge du phénotype immunitaire d’un mammifère longévif, le chevreuil (Capreolus capreolus) et de mieux comprendre les compromis régissant l’allocation de ressources entre l’immunité et les autres grandes fonctions de l’organisme. Cette thèse a été menée au sein de deux populations naturelles, permettant de tester l’influence de conditions environnementales contrastées sur ces variations. Nous avons montré qu’une croissance rapide pendant les premiers mois de vie du chevreuil n’imposait pas de coûts en terme de développement du phénotype immunitaire sur la même période (niveaux des traits innés et acquis), ni sur le long-terme. Nous avons aussi montré que le développement de l’immunité des jeunes n’était pas dépendant de l’âge de leur mère, mais était fortement influencé par la condition corporelle de celle-ci. Chez les adultes, nous avons décrit les variations avec l’âge d’une dizaine de traits reflétant l’immunité innée et adaptative. Cela a permis de mettre en évidence de profondes modifications du profil immunitaire aux âges avancés, i.e. une augmentation de la production de marqueurs inflammatoires (haptoglobine, beta-globulines) et une diminution de la réponse adaptative (lymphocytes). L’augmentation parallèle avec l’âge de la charge parasitaire des individus appuie l’idée que le chevreuil est sujet à l’immunosenescence. Enfin, nous avons montré que la longueur des télomères leucocytaires varie avec l’âge. Nous n’avons pas trouvé d’associations entre la longueur des télomères et les proportions de chaque forme leucocytaire (neutrophiles, monocytes, lymphocytes). Cependant, nous avons montré que de forts niveaux de certains marqueurs inflammatoires (beta- et alpha1-globulines) semblent être associés à des télomères courts dans les cellules immunitaires. Ces résultats ouvrent de nombreuses pistes pour une meilleure compréhension des mécanismes physiologiques à la base du vieillissement / Immunity determines an organism’s sensitivity to pathogens and parasites and thus represent a crucial function that affects survival of individuals in the wild. However, this function represents several energy costs for development and use, and in natural conditions, resources are limited. Organisms consequently face energy allocation trade-offs between costly functions such as immunity, growth or reproduction. On the long term, these allocations are supposed to have serious consequences on probability of individuals to reproduce and to survive at each age.The aim of this thesis was to describe age-related variations of immune phenotype in a wild and long-lived mammal, the roe deer (Capreolus capreolus), and to provide a better understanding of energy trade-offs between immune function and other life-history traits. This thesis was conducted in roe deer of both sexes and from two natural populations, which allow to test the influence of sex and contrasting environmental conditions on these variations.We first described that rapid growth did not impair the development of young roe deer immune phenotype (levels of innate and adaptive traits), neither on the short-term (during growth), neither on the long-term (during adulthood). We also proved that immune development of juveniles was not dependent of maternal age, but was strongly influenced by maternal body condition. In adult roe deer, we have described the precise patterns of age-related changes in ten immune traits reflecting both innate and adaptive immunity. It revealed that roe deer are subjected to profound changes in their immune profile with increasing age, i.e. an increase in the production of inflammatory markers (haptoglobin, beta-globulin) and a decrease in the adaptive response (lymphocytes). In the same individuals, the parallel increase with age of parasite load supports the idea that deer are subject to immunosenescence. Finally, we described age-related changes in leukocyte telomere length. We found no associations between telomere length and proportions of each leukocyte form (neutrophils, monocytes, lymphocytes). However, we observed that high levels of some inflammatory markers (beta- and alpha1-globulin) tend to be associated with short telomeres in immune cells. These results open many avenues for a better understanding of the physiological mechanisms underlying aging

De nombreuses études soulignent une relation directe entre la prévalence de la fibrillation auriculaire (FA) et le vieillissement. La senescence cellulaire et le phénotype sécrétoire associé semblent jouer un rôle central dans le développement de l'inflammation auriculaire. Cette inflammation est à l’origine d’un remodelage auriculaire délétère (stress oxydant, fibrose) favorable à la perpétuation et au maintien de la FA. Par ailleurs, il est connu que la FA favorise la coagulation locale et systémique. Cependant, l'impact des facteurs de la coagulation, notamment la thrombine, sur la FA est peu connu. L’objectif de cette étude était de déterminer le lien entre la sénescence des cellules endothéliales atriales et le phénotype pro-inflammatoire et pro-adhésif, la fibrose et le remodelage auriculaire tout en évaluant l’impact de la coagulation, et en particulier le rôle de la thrombine. / Many studies documented strong relationship between ageing and development of atrial fibrillation (AF). Moreover, it has been found that senescence and senescence-associated- secretory-phenotype play an important role in development of overall atrial inflammation which can ultimately ends up in atrial structural remodeling paving the way to AF perpetuation and maintenance. Moreover, it has been known for decades that AF has been associated with the activation of local and circulating coagulation factors. However, little is known about the impact of coagulation-derived factors, in particular thrombin, on the onset of AF. The aim of the present study was to determine the link between atrial endothelial cells (AECs) senescence and the induction of pro-inflammatory, pro-adhesive, pro-fibrotic and pro-remodelling AECs patterns and also to evaluate the contribution of coagulation derived-factors such as thrombin.

Ce travail scientifique a abordé la problématique de la communication cellulaire entre le pancréas exocrine et endocrine dans la mucoviscidose. La contribution des infections pulmonaires chroniques et des traitements immunosuppresseurs sur la dégénerescence pancréatique a été aussi étudiée. Les résultats obtenus ont montré que le LPS disséminé par des infections récurrentes peut cibler les cellules pancréatiques exocrines, en conduisant à la formation des microparticules membranaires qui sont nuisibles pour la survie et le fonctionnement des cellules endocrines. Dans cette communication intercellulaire, la protéine CFTR est un médiateur essentiel de la sévérité des signaux délivrés par les microparticules et de la réponse cellulaire à l'inflammation du pancréas, en participant à l'équilibre de la sécrétion d'insuline des cellules endocrines. En outre, les données ont mis en évidence que l'administration prolongée d’immunosuppresseurs chez les patients greffés pourrait différemment induire l'apoptose de manière dépendante de la mitochondrie, cela en favorisant l'entrée des cellules en sénescence prématurée, un état métabolique du dysfonctionnement cellulaire. / This scientific work tackled the issue of the communication between exocrine and endocrine pancreas in cystic fibrosis. Also, the contribution of recurrent lung infections and immunosuppressive therapy to the pancreatic cell degenerescence was studied in vitro. Results obtained showed that disseminated LPS released by recurrent infections could target pancreatic exocrine cells, leading to the formation of membrane microparticles that are deleterious for endocrine cell survival and function. In this intercellular cross-talk, CFTR is a critical mediator for the severity of the MP-delivered signals and for the pancreatic cell response to inflammation, also participating to the balance of insulin secretion of endocrine cells. Furthermore, data evidenced that long-term administration of immunosuppressive drugs in grafted patients may differently induce apoptosis in a mitochondrial-dependent fashion, possibly favoring cells to enter premature senescence, which is a metabolic state of cellular dysfunction.

Medicago truncatula est une Légumineuse établissant une interaction symbiotique avec une bactérie tellurique de la famille des Rhizobiacées, Sinorhizobium meliloti. Cette interaction induit l’organogénèse racinaire d’un nouvel organe, la nodosité dans laquelle s’établit un microenvironnement propice à la différenciation de S. meliloti en bactéroïde fixateur du diazote atmosphérique. Ce dernier réduit ainsi le N2 atmosphérique en ammonium, assimilé ensuite par la plante hôte. Cette réduction étant très endergonique M. truncatula fournit aux bactéroïdes des substrats carbonés issus de la photosynthèse. Cependant, cette interaction n’est pas pérenne, du fait de la mise en place d’un processus de sénescence ; processus conduisant à la lyse des bactéroïdes et des cellules hôtes végétales. Cependant, à l’heure actuelle, ce processus de rupture symbiotique reste largement méconnu. Afin de mieux caractériser ce processus de sénescence, nous avons développé de nouveaux outils cytologiques permettant par microscopie confocale de suivre in vivo la viabilité, mais également le fonctionnement des bactéroïdes au sein de la cellule hôte végétale. Ces nouvelles approches cytologiques pourraient ainsi offrir de nouvelles perspectives pour une caractérisation plus précise du déroulement du processus de sénescence nodositaire. Dans le cadre de ce travail de thèse, nous avons également cherché à déterminer l’implication de deux cystéines protéases dans la mise en place du processus de sénescence nodositaire. Une des caractéristiques de ce processus de sénescence est une hausse de l’activité protéolytique, notamment des activités cystéine protéases. L’analyse transcriptomique par cDNA-AFLP du processus de sénescence nodositaire (Van de Velde et al. 2006) a pu mettre en évidence 508 gènes différentiellement exprimés dont deux cystéines protéases, MtCP6 et MtVPE. L’analyse spatio-temporelle de MtCP6 et MtVPE, par fusion transcriptionnelle avec le gène rapporteur GUS, a permis de mettre en évidence l’induction de ces deux gènes lors du processus de sénescence nodositaire aussi bien développementale qu’induit par un traitement abiotique ou lors d’une interaction symbiotique non efficace. De plus, nous avons pu démontrer, par génétique inverse, que la diminution de l’expression de ces deux protéases retarde la mise en place du processus de sénescence, alors que leur expression précoce conduit à la promouvoir. Enfin, l’étude par microscopie confocale de la localisation subcellulaire de ces protéases par fusion traductionnelle avec la GFP, démontre leur adressage aux bactéroïdes. Nos données tendent donc à démontrer le rôle clef de MtCP6 et de MtVPE dans le processus de sénescence nodositaire, où ces protéases pourraient participer directement au déclenchement d’une dégradation des bactéroïdes. / Medicago truncatula is a leguminous plant establishing a symbiotic interaction with the bacteria Sinorhizobium meliloti. This symbiosis leads to the de novo development of root nodules involved in biological nitrogen fixation. However, this symbiotic interaction is time limited and an early senescence appears in mature nodule entailing the formation of a senescence zone (zone IV). This degradation process occurs earlier in comparison to senescence of the whole plant. During nodule developmental senescence of plant host cells, a gradual degradation process induces a loss of vacuole and peribacteroid membrane (PBM). But this nodule degradation process still remains to be unravelled. To increase our understanding of the nodule senescence process, we developed new cytologic tools allowing an in vivo assessment of the viability and functioning of bacteroids within plant host cells. Therefore, these new tools provide a new insight of the nodule senescence process which may help for a finer characterization of the nodule senescence. In the M. truncatula model, a previous cDNA-AFLP analysis enlightens an upregulation of several cysteine proteinases during the transition from nitrogen fixing nodule to a senescent one; including an early expression of an SPG31-like peptidase known to be involved in leaf senescence (MtCP6) and a Vacuolar Processing Enzyme described as a plant caspase-like protein (MtVPE) involved in mechanisms similar to hypersensitive response in A. thaliana. In planta spatiotemporal analysis of the expression of these two cysteine proteinases using promoter:reporter gene GUS confirmed their expression during natural senescence at the junction between the nitrogen fixing zone (zone III) and the senescence zone (zone IV). Therefore, to acquire a better insight into the role of these cysteine proteases during the senescence program, we knocked down by RNAi the expression of each gene specifically at the interzone III-IV. Depletion of these transcripts induced a drastic increased of N2 fixation and nodule size. Conversely, overexpression of both genes in the zone III of nodule leads to an extension of the senescence zone. Confocal microscopy images of protein:GFP fusions showed that both proteinases are addressed to bacteroids within plant host cells. Our data revealed that MtCP6 and MtVPE are key players of the nodule senescence process and may be directly involved in symbiosome degradation.

Sperm phenotype is an essential indicator of the male ejaculate quality and may have a significant impact on male reproductive success. Sperm phenotypes are considerably variable across species but variation is also found among males within species. This thesis examines (1) variation in sperm phenotypes among males in barn swallows (Hirundo rustica), (2) changes in male ejaculate quality with age, (3) relationships between sperm morphology and motility, (4) effects of sperm phenotypes (morphology and motility) on male fertilization success, using a large dataset of 174 observation for 130 males, and (5) the influence of female reproductive environment on sperm motility. From the tested variables, only midpiece length correlated with male age. Older males had shorter midpiece but no relationship between male age and reproductive success was found. Sperm length negatively affected sperm motility and, simultaneosly, relative midpiece length posively correlated with sperm motility. No correlation was found between the male reproductive success and sperm motility, presence of abnormalities, length of outermost tail feathers or age. Males with shorter relative midpiece were more successful in within-pair paternity, and males with shorter sperms but longer relative midpiece were more successful in...

La sénescence endothéliale est une étape précoce menant à la dysfonction endothéliale, laquelle favorise la pathogénèse de maladies cardiovasculaires lors du vieillissement physiologique et, de manière prématurée, chez le sujet diabétique. La première étude indique que le stress oxydant favorise l’induction du système angiotensine local menant à la sénescence et la dysfonction endothéliale dans les cellules endothéliales en culture. La deuxième étude indique que l’expression redox-sensible des co-transporteurs sodium-glucose 2 (SGLT2) entraîne via le système angiotensine la sénescence et la dysfonction endothéliale en réponse au high glucose. De plus, les observations indiquent que l’Empagliflozine, un inhibiteur sélectif de SGLT2, et les anthocyanes du jus de cassis sont capables d’inhiber l’induction de la sénescence endothéliale au glucose. De ce fait, le système angiotensine local et le SGLT2 sont des cibles intéressantes pour retarder le vieillissement vasculaire. / Endothelial senescence is an early step to endothelial dysfunction, known to favor the development of cardiovascular diseases during ageing, or its accelerated form in diabetes. The first in vitro study shows that the activation of the local angiotensin system is favored by the oxidative stress and leads to endothelial senescence and dysfunction. The second study indicates that endothelial senescence and dysfunction in response to high glucose are driven by the redox-sensitive expression of sodium-glucose co-transporters SGLT-2, via the angiotensin system. Moreover, data also indicate that empagliflozin, a SGLT2 selective inhibitor, and anthocyans from black-currant juice can inhibit the glucose-induced endothelial senescence. Therefore, the local angiotensin system and SGLT2 are promising targets to stunt vascular ageing.

Cellular senescence has been proposed to be equivalent to organismal aging and is one of the outcomes of the cell fate decision process in response to DNA damage that occurs in cells. When a cell encounters DNA damage, the cell cycle is immediately halted to evaluate which decision to take in response to genomic insult. The choices are between repairing the damage and continue division, or enter a non-replicative but viable state called senescence or to die if damage is severe (Figure 1). The signaling cascade, which detects this damage and regulates the cell fate decision, is collectively called as DNA damage response (DDR). However, the exact mechanism of how delineation for each decision happens is still not clear. Since DNA damage works as a mediator for cell fate decision, my work aimed to study senescence as a DNA damage response. In addition, the role of free radicals like ROS in cellular senescence is not very clear because though an increase in their concentrations is recorded in aged cells, it is not evident if the increase seen the cause or the effect of aging, primarily because they themselves capable of causing DNA damage. This conundrum have always led to confounding observations wrt role of free radicals in the cellular senescence process and if the senescence is caused through agents which rely on ROS to cause DNA damage, ROS becomes absolutely integral to the aging process. To understand this aspect formed the first line of investigation in my work along with identification of the sensor of DNA damage, which drives various cell fates. During organismal ageing there is an accumulation of senescent cells, which could be the major reason for functional decline of tissues and organs with age. However, to study changes associated with signaling molecules with respect to ageing, a cellular model system for senescence driven through DNA damage was needed, using which interplay between senescent / aged cells and cellular niche can be established. Studying the spatial and temporal alterations in signaling dynamics, within the cell as well as with the neighbouring niche during the senescence process in anticipated to provide us better understanding about the complex process of ageing. For this, the objectives were defined to establish and characterize the DNA damage induced senescence model using various parameters, and especially study the signaling dynamics of GPCR mediated signaling in senescence. The role of chemokine receptor, CXCR4 and its ligand, CXCL12 mediated signaling was chosen for the study. The following sections describe the findings that were obtained from the various objectives studied during the course of this study. Section 1. Development and characterization a model system to study cellular senescence as a DNA damage response. In this part of the study, I characterized genotoxic stress induced cellular senescence model using 5-Bromodeoxyrudine as the DNA damaging agent. BrdU, owing to its property of being a thymidine analogue, is incorporated in dividing cells, and this incorporation is recognized as DNA damage. This triggers ‘persistent’ DNA damage response signaling, including activation of ATM kinase, one of the primary DNA damage sensor. As anticipated, the DDR response detected was directly proportional to the dose of BrdU treatment and so was Reactive Oxygen Species (ROS) levels, a known senescence mediator. Using this model system of direct DNA damage mediated DDR activation and induction of cellular senescence, the growth-arrested cells were extensively characterized for presence and quantum of most of the senescence associated markers known in literature. BrdU treated cells, which became senescent showed presence of DNA damage, morphological changes like flat, enlarged, granule rich appearance, expression of senescence associated molecular markers like p21, IL8, showed senescence associated beta galactosidase activity, refractiveness to growth factor for division, increased ROS levels, Golgi dispersion, etc. The secretome of the treated cells also showed increased secretion of inflammatory cytokines which are attributed to a senescence phenotype, called as Senescence Associated Secretory Phenotype (SASP), which triggered proliferative and migratory effect on cancer cells. Overall, in this part of the study, it was established that BrdU can cause DNA damage and induce senescence as one of the cell fate in response to the intermediate dose of damage. The senescent cells generated in the model system was established to be akin to senescence observed by replicative exhaustion of normal cells, thereby making our model applicable to the physiological studies as well. Section 2. Insights into the role of ATM-ROS axis during senescence initiation and maintenance using DDR mediated cellular senescence model. While the BrdU model system for generating senescent cells was being developed and characterized, it was observed that there is an increase in ATM activation as well as ROS production concomitant to the a dose of BrdU. At the same time it was also observed that senescent cells showed persistent DDR signaling and high levels of ROS. Using this premise, in the second objective of my study I aimed to identify if ATM and ROS are critical during initiation of senescence, when the cells are insulted with the DNA damaging agent or during the maintenance of senescent state of the cells. By quenching ROS during the initiation state, I recorded that ROS is not critical for inducing senescence and perhaps the increase in ROS levels in senescent cells is due to their higher metabolic activity. By inhibiting ATM activation during DNA damage, it was observed that BrdU induces senescence through direct DNA damage, and active ATM and DDR signaling is absolutely critical for the senescence initiation. It was also established that ATM is not just a DNA damage sensor but also a redox regulator in the senescence model system. Prevention of ATM activation in presence of DNA damage blocked senescence initiation and also triggered increased ROS levels in the cells affecting their long term viability, suggesting ATM regulates ROS levels as well in addition to sensing DNA damage. In order to study the role of ATM-ROS axis in the maintenance of senescence state, already senescent cells were subjected to ROS quenching and/ or ATM inhibition and it was identified that both these signaling molecules are essential for maintaining the viability of senescent cells. The findings from these study thereby show that senescence can be divided into two temporally distinct stages, initiation or early senescence stage and second, maintenance stage of senescence. Overall, I was able to characterize the presence of temporally linked ROS – dependent and ROS – independent events in cellular senescence, which are independently mediated by ATM kinase (Figure 1). Dose of Genotoxic Stress damage DDR Senescence initiation Repair Cell cycle ATM arrest kinase Death Growth arrest Senescence maintenance Senescence Cell ROS viability Elevated metabolism Figure 1. Signaling cascades regulating senescence onset and maintenance mediated through DDR. Cells enter senescence state in response to DNA damage, depending on the dose of insult, through an ATM dependent and ROS independent pathway. Unlike this ATM-ROS axis is critical for the maintenance of senescent state of damaged but viable cells. Section 3. Understanding the role of CXCR4 – CXCL12 mediated signaling in senescence. Age dependent changes in cellular signaling are less explored and I was specifically interested in understanding how presence of senescent cells affects its microenvironment or vice versa i.e. how microenvironment affects senescent cells. In this premise the third objective of this study was defined towards identifying role of a GPCR, CXCR4 mediated signaling in cellular senescence and associated inflammation. CXCR4 is a ubiquitously expressed GPCR and it’s only known ligand is CXCL12/ SDF1 (stromal derived factor ), which is a homeostatic chemokine (i.e. its levels does not change under most physiological conditions). During characterization of DNA damage induced senescence model system, it was observed that this receptor expression is induced during DNA damage ells, which was also found to be so from data available from other gene expression studies as well. During the course of my work, I identified that senescent cells show CXCR4 up regulation in response to DNA damage, mediated through activation of ATM kinase - HIF1 axis and plays a critical role in enhancing the senescence associated inflammatory response in presence of its ligand, CXCL12. This CXCL12 dependent enhanced inflammatory response in damaged cells was determined to be sensitive to the pertussis toxin treatment and hence dependent on G protein activation. Further downstream analysis revealed the pro-inflammatory effect of the CXCR4 receptor activation was due to cAMP level suppression post activation by the Gi subunit. Given that cAMP levels are antagonistic to inflammatory phenotype, using a library of pharmacological compound library, I also discovered that cAMP specific PDE, phophodiesterase 4A, is also involved in regulating inflammatory response during the initiation stage of cellular senescence. The screen also confirmed the involvement of previously identified molecular components such as p38 MAPK and leukotrienes in the senescence associated inflammatory phenotype. The examination of the role of the CXCR4- CXCL12 axis in the deeply senescent cells surprisingly revealed that deeply senescent cells are refractory to CXCL12 stimulation in terms of inflammatory response, which was experimentally determined to be associated with impaired calcium release. Overall, the findings from this part of the study revealed a novel signaling cascade where CXCR4 up regulation is a part of the DDR response in cells, which utilizes the Local Excitation Global Inhibition (LEGI) mechanism to enhance the sensitivity of the damaged cells to its ligand CXCL12. This enhanced sensitivity mediates the CXCL12 dependent inflammatory response, which aids in attracting immune cells for clearance of these damaged cells. Once the cells have entered the senescent state, the axis is physiologically down modulated and the senescent cells showed refractiveness to CXCL12 stimulation, probably to prevent persistent acute inflammation, if the senescent cells are not cleared (Figure 2). Figure 2. CXCL12-CXCR4 axis in cellular senescence. During senescence initiation stage, when cells encounter DNA damage (Step 1), there is induction of CXCR4 receptor (Step 2), which enhances of CXCL12 mediated signaling for increased inflammatory response (Step 3). In the maintenance stage, where the cells are not cleared (Step 4), the axis is suppressed (Step 4), thereby bringing the levels of inflammatory secretome down, and thereby preventing damage to the cells (Step 5).