To understand whether integrin β4 is involved in vascular endothelial cell (VEC) senescence, we examined integrin β4 level changes, as well as P53 and reactive oxygen species (ROS) levels and alterations of phosphatidylcholine-specific phospholipase C (PC-PLC) activity before and after knocking-down integrin β4 by small interfering RNA. We found integrin β4, P53 and ROS levels increased significantly, while Ca2+-independent PC-PLC activity obviously decreased during VEC senescence. On the other hand, integrin β4 down-regulation attenuated the senescence phenotype and reversed Ca2+-independent PC-PLC activity, and P53 and ROS levels. The data suggested that integrin β4 might mediate VEC senescence through depressing Ca2+-independent PC-PLC and elevating the levels of P53 and ROS.

Les Microvésicules (MVs) sont des marqueurs circulants de l’activation cellulaire au cours de la dysfonction du greffon et l’athérothrombose. Les MVs de la cellule bêta ou intratissulaires vasculaires, sont peu connues. Le suivi longitudinal de 19 patients transplantés d’îlots pancréatiques par les MV- PSA-NCAM+ sanguines témoigne d’une altération précoce du greffon avant l’identification des marqueurs clinique et biologique de la perte du greffon. La cinétique de libération des MVs leucocytaires, endothéliales et hépatiques suggère leur intérêt pour l’identification de la cause de la perte du greffon et pour la surveillance de l'immunosuppression. Après validation d’une méthode d’extraction douce des MVs tissulaires sur des plaques d’athérome, nous avons mesuré l’accumulation de MVs pro-sénescentes dans l’aorte de rats âgés, qui étaient réduite par l’ingestion d’EPA : DHA (6 :1), avec une baisse des propriétés pro-sénescentes identifiée sur les cellules endothéliales d’artères coronaires en culture. Le contrôle des MV nocives par cytoprotecteur du vaisseau réduirait la sénescence endothéliale. / Microvesicles (MVs) are circulating markers that reflect cellular activation during graft dysfunction and atherothrombosis. Data on graft tissue MVs or vessel are scarce. The longitudinal follow-up of 19 patients with pancreatic islets transplants showed that circulating MV-PSA-NCAM+ typifying the early graft loss of islet graft prior its detection using the clinical and biological markers of graft loss. In addition, the kinetics release of leukocyte, endothelial and hepatic MV suggest their interest in identifying the cause of graft loss and in monitoring of immunosuppression. Using a new tissular MV mild extraction process validated with arteriosclerotic plaques, we evidenced an accumulation of pro-senescent MVs in the aorta of old rats that was significantly reduced by EPA: DHA (6 :1) intake as well as their pro-senescent properties on coronary artery endothelial cell cultures. Altogether, the pharmacological control of the release of noxious MVs using vessel cytoprotectors would limit the consequences of endothelial senescence.

Le cannibalisme d’une cellule vivante par une autre cellule vivante représente une nouvelle modalité de mort cellulaire non autonome. Mes travaux de thèse ont permis d’identifier et de caractériser les acteurs impliqués et d’apprécier l’influence de ce processus sur le devenir de la cellule cannibale. Nous avons ainsi révélé que l’activation d’une signalisation cellulaire impliquée dans la régulation du cycle cellulaire va causer une libération d’ATP qui stimulera les récepteurs purinergiques P2Y2 de la cellule de manière autocrine. Cette étape sera suivie d’une augmentation de l’exposition de la protéine d’adhérence E-cadhérine à la membrane plasmique et de réarrangements du cytosquelettes médiés par la kinase ROCK, et permettra ainsi à une cellule vivante de cannibaliser une autre cellule vivante. Ce phénomène aussi connu sous le nom de « cellule dans une cellule » est fréquemment observé dans les biopsies tumorales. De plus, nous révélons au cours de ces travaux la capacité des cellules internalisées à être éliminées par un processus qui implique la protéine de l’autophagie ATG5 et les protéines pro-apoptotiques BAK et BAX. Ce processus est associé au déclenchement d’une instabilité génétique et d’un stress oxydatif au niveau des cellules cannibales et va déclencher la sénescence de ces cellules que nous avons appelé « entescence ». Cette nouvelle modalité d’induction de la sénescence participe à la suppression des tumeurs in vivo et semble prédire la réponse des patients aux traitements néoadjuvants anticancéreux. À l’opposé, l’échappement à l’entescence favorise la progression tumorale et est associé à une mauvaise réponse des patients aux traitements. L’ensemble de ces travaux met en lumière l’existence d’une nouvelle modalité d’induction de la sénescence cellulaire qui survient à la suite du cannibalisme cellulaire. Une meilleure compréhension des mécanismes impliqués dans son déclenchement et son exécution pourrait selon nous participer au développement de nouvelles approches thérapeutiques afin de lutter contre le cancer. / Cannibalism of live cells by other live cells is a new modality of non-autonomous cell death. This investigation led to the characterization of the molecular mechanisms implicated as well as the identification of the consequences of this process on the fate of the cannibal cell.We revealed that the activation of a signaling pathway involved in the regulation of the cell cycle can trigger a release of ATP that will stimulate the activity of the P2Y2 purinergic receptor in an autocrine manner. These events will lead to the increase of E-cadherin membrane exposition and change the organisation of the cytoskeleton in a ROCK-dependent manner, allowing this live cell to eat another live cell. This process called "cell in cell structure" is frequently observed in tumoral biopsies. Then, we revealed that the internalized cell will be eliminated by a process dependent on the autophagy protein ATG5 and the pro-apoptotic proteins BAX and BAK. These events are associated to the triggering of genomic instability and an oxidative stress in the cannibal cell leading these cells to a new senescence program that we called "entescence".This new senescence program seems to be a tumor suppressor mechanisms in vivo and is correlated to a better response of patient to neoadjuvant anticancer treatments. Moreover, escaping entescence seems to favor tumor growth and is associated to a bad response to anticancer treatments.Taken together, these results highlight the existence of a new senescence program that is initiated by cellular cannibalism. A better understanding of the molecular mechanisms regulating its initiation and its execution may lead to develop new innovative anticancer therapeutical approaches.

Environ, 60 % des personnes atteintes d’un cancer auront au moins une séance de radiothérapie au cours de la prise en charge thérapeutique de leur maladie. Les doses de radiothérapie sont limitées en raison du risque important de fibrose séquellaire des tissus sains. Les rayonnements ionisants (RI) peuvent induire différents types de mort cellulaire y compris l'apoptose et la sénescence. Les cellules sénescentes ont une sensibilité réduite à l'apoptose et un phénotype sécrétoire inflammatoire. De plus, les RI peuvent induire la production d’espèces réactives de l’oxygène (ERO) qui provoquent des lésions de l'ADN dans les tissus non ciblés, et des effets systémiques associés à l'inflammation. Différentes équipes ont proposé des tests prédictifs de la radiosensibilité individuelle des patients basés sur l’évaluation du taux d'apoptose radio-induite des lymphocytes T CD4+/CD8+ (LT). Cependant, l’impact des différences de sensibilité à l’apoptose/sénescence des sous-populations de LT sur le taux d’apoptose n’a pas été étudié. Notre hypothèse est que la sensibilité à l’apoptose/sénescence radio-induite des LT circulants est associée à la sur/sous-représentation de sous-populations particulières de LT CD4+ dont les fonctions sont en rapport avec la survenue de fibrose. Nos résultats chez le donneur sain montrent que les LT CCR6+Th17 pro-fibrogéniques sont moins sensibles à l’apoptose et plus sensibles à la sénescence que les LT CCR6negTh et les Treg. Cette sénescence peut être préjudiciable car les lymphocytes CCR6+Th17 situés dans les tissus irradiés peuvent sécréter de l'IL-8 et du VEGF-A. La modulation des voies ERO/MAPK ou mTOR pourrait être une cible potentielle pour la prévention de la radiotoxicité induite par les CCR6+Th17 sénescents. Enfin, le ratio de cellules circulantes H2A.J+CCR6+Th17 sénescentes / CCR6+Treg pourrait être utilisé comme marqueur potentiel de la radiosensibilité individuelle. / On average, 60% of cancer patients have at least one radiation session during their care throughout the history of their disease. The doses of radiotherapy are limited because of the high risk of fibrosis-type side effects of healthy tissues. Ionizing radiation can induce a variety of cell death responses including apoptosis, but also senescence. Senescent cells have reduced sensitivity to apoptosis, and a pro-inflammatory secretory phenotype. In addition, ionizing radiations can induce the production of reactive oxygen species (ROS) that cause DNA damage in non-target tissues, and systemic effects associated with inflammation. In order to improve the personalization of radiotherapy, different teams proposed predictive tests of the individual radiosensitivity of patients by establishing a relationship between a low rate of radio-induced apoptosis of CD4+/CD8+ T lymphocytes (LT) and a high risk of secondary fibrosis. However, the impact of the differences in individual cell sensitivity to radiation-induced senescence on the ratio between LT cell subpopulations has not been studied. Our results on healthy donors show that pro-fibrogenic CCR6+ Th17 cells are less sensitive to apoptosis and more susceptible to senescence compared to CCR6neg LT. This senescence can be detrimental as irradiated CCR6+Th17 lymphocytes located in the irradiated tissue can secrete IL-8 and VEGF-A. Modulation of ROS/MAPK or mTOR signaling pathways could be potential targets for the prevention of this CCR6+Th17-induced radiotoxicity. Finally, the ratio of circulating H2A.J+ senescent CCR6+ Th17/CCR6+Treg cells may be used as a potential marker of individual radiosensitivity.

L'autophagie est importante pour le recyclage et la mobilisation des éléments nutritifs dans les plantes. Plusieurs lignées sur-exprimant les gènes AtATG8a-i d’Arabidopsis ont été sélectionnées afin de déterminer l'effet de l'augmentation de l'activité autophagique sur l'efficacité de la remobilisation de l'azote. Les lignées sur-expresseur ont présenté une meilleure remobilisation de l'azote des feuilles de la rosette jusqu'aux graines qque le contrôle, mais uniquement dans des conditions de culture en nitrate pléthorique. Les lignées sur-exprimant ATG8a et ATG8g ont été les plus performantes. Une vaste collection de mutants atg, comprenant les mutants atg8a-i, a ensuite été utilisée pour mesurer la remobilisation de l’azote des rosettes, aux tiges et aux semences, afin de déterminer quels gènes ATG sont essentiels pour la remobilisation. Une attention particulière a été accordée à la famille de gènes ATG8a-i afin de déterminer si un membre de la famille ATG8 pourrait être plus spécifiquement dédié au recyclage de l'azote lors de la sénescence des feuilles pour la remobilisation. Bien que les divers mutants atg8 n’aient pas présenté de différence majeure par rapport au sauvage, l'un d'entre eux a montré un léger phénotype de sénescence précoce, suggérant que cette isoforme pourrait être plus spécialisée dans la remobilisation de l'azote. Afin d'améliorer l'activité de l'autophagie chez l'orge, nous avons surexprimé HvATG5 chez l'orge et testé la sensibilité des sur-expresseurs à plusieurs conditions de stress. Nous avons constaté que les sur-expresseurs de HvATG5 étaient plus tolérants à une faible alimentation en nitrate, à une faible teneur en soufre, et surtout moins sensibles aux effets de l'obscurité prolongée. Afin d'estimer les rôles respectifs des protéases induites par la sénescence et de l'autophagie, plusieurs mutants de protéase (sag12, rd21A, cathB3) ont été croisés avec des mutants d'autophagie (atg5, atg7. Leur contribution à la remobilisation de l'azote a été mesurée par marquage 15N. / Autophagy is important for nutrient recycling and mobilization in plants. Several Arabidopsis AtATG8a-i overexpressing lines were selected in order to determine the effect of increasing autophagy on nitrogen remobilization efficiency. The overexpressing lines remobilized more nitrogen from the rosette leaves to the seeds but only when cultivated under high nitrate conditions. The lines overexpressing ATG8a and ATG8g were the most performant. A large collection of atg mutants including the atg8a-i mutants was then used to monitor N-remobilization from the rosettes to the stems and seeds in order to determine which ATG genes are essential for N remobilization. A special focus was dedicated to the ATG8a-i gene family in order to determine whether a member of the ATG8 family could be more specifically dedicated to N-recycling during leaf senescence for remobilization. Although the various atg8 mutants were not different from wild-type, one of them presented slight early senescence phenotype, suggesting this isoform could be more specialized in N remobilization. In order to enhance autophagy activity in barley, we overexpressed HvATG5 in barley and tested sensitivity of over-expressors to several stress conditions. We found that barley HvATG5 over-expressors were more tolerant to low nitrate supply, to low sulfur, and especially less sensitive to dark-stress effects. In order to estimate the respective roles of autophagy and senescence induced proteases, several protease mutants (sag12, rd21A, cathB3) were crossed with autophagy mutants (atg5, atg7) in order to monitor their respective contributions to nitrogen remobilization.

No description available.

The A-type lamins are intermediate filament proteins that constitute a major part of the eukaryotic nuclear lamina—a tough, polymerized, mesh lining of the inner nuclear membrane, providing shape and structural integrity to the nucleus. Lamin A (LA) filaments also permeate the nucleoplasm, providing additional structural support, but also scaffolding numerous tethered molecules to stabilize, organize, and facilitate molecular interactions to accomplish critical functions of cellular metabolism. Over the past 2 decades, much attention has been focused on roles of LA in maintenance of nuclear structural integrity. Only since the late 1990s have scientists discovered the devastating effects of LA gene (LMNA) mutations, as they have associated hundreds of LMNA mutations to a large group of diseases, called laminopathies, with a broad spectrum of phenotypes, ranging from skeletal, muscular, and neurological defects, to defective lipid storage, to accelerated aging phenotypes in diseases called progerias. Recent advances demonstrate LA regulatory functions include cell signaling, cell cycle regulation, transcription, chromatin organization, viral egress, and DNA damage repair. Amidst the flurry of fascinating research, only recently have researchers begun to focus attention on the different isoforms that exist for LA, a precursor form among them. LA is initially synthesized as Prelamin A (PreA), and undergoes a series of modifications that truncate the protein to produce “mature” LA. Existence of the precursor form, and its complex maturation pathway, have puzzled researchers since their realization. With a pattern of expression related to cell cycle phase, we hypothesized a role for PreA in cell cycle control. To investigate, we have performed array studies to assess gene expression effects at the levels of transcript expression, protein expression, and phosphorylation modification status. Here, we present evidence for a PreA-mediated program of cell cycle regulatory gene and protein expression modulation. Implicated pathways include RB-E2F, p53, p27Kip1, FoxOs, p300, and the Cyclins, with additional evidence indicating a role for the Pin1 prolyl isomerase in mediating PreA regulation of the cell cycle.

T cells in chronic viral infections are featured by premature aging with accelerated telomere erosion, but the mechanisms underlying telomere attrition remain unclear. Here, we employed human CD4 T cells treated with KML001 (a telomere-targeting drug) as a model to investigate the role of telomere integrity in remodeling T cell senescence. We demonstrated that KML001 could inhibit cell proliferation, cytokine production, and promote apoptosis via disrupting telomere integrity and DNA repair machineries. Specifically, KML001-treated T cells increased dysfunctional telomere-induced foci (TIF), DNA damage marker γH2AX, and topoisomerase cleavage complex (TOPcc) accumulation, leading to telomere attrition. Mechanistically, KML001 compromised telomere integrity by inhibiting telomeric repeat binding factor 2 (TRF2), telomerase, topoisomerase I and II alpha (Top1/2a), and ataxia telangiectasia mutated (ATM) kinase activities. Importantly, these KML001-induced telomeric DNA damage and T cell senescent phenotype and machineries recapitulated our findings in patients with clinical HCV or HIV infection in that their T cells were also senescent with short telomeres and thus more vulnerable to KML001-induced apoptosis. These results shed new insights on the T cell aging network that is critical and essential in protecting chromosomal telomeres from unwanted DNA damage and securing T cell survival during cell crisis upon genomic insult.

Background: T cells play a key role in controlling viral infections; however, the underlying mechanisms regulating their functions during human viral infections remain incompletely understood. Here, we used CD4 T cells derived from individuals with chronic viral infections or healthy T cells treated with camptothecin (CPT) - a topoisomerase I (Top 1) inhibitor - as a model to investigate the role of DNA topology in reprogramming telomeric DNA damage responses (DDR) and remodeling T cell functions. Results: We demonstrated that Top 1 protein expression and enzyme activity were significantly inhibited, while the Top 1 cleavage complex (TOP1cc) was trapped in genomic DNA, in T cells derived from individuals with chronic viral (HCV, HBV, or HIV) infections. Top 1 inhibition by CPT treatment of healthy CD4 T cells caused topological DNA damage, telomere attrition, and T cell apoptosis or dysfunction via inducing Top1cc accumulation, PARP1 cleavage, and failure in DNA repair, thus recapitulating T cell dysregulation in the setting of chronic viral infections. Moreover, T cells from virally infected subjects with inhibited Top 1 activity were more vulnerable to CPT-induced topological DNA damage and cell apoptosis, indicating an important role for Top 1 in securing DNA integrity and cell survival. Conclusion: These findings provide novel insights into the molecular mechanisms for immunomodulation by chronic viral infections via disrupting DNA topology to induce telomeric DNA damage, T cell senescence, apoptosis and dysfunction. As such, restoring the impaired DNA topologic machinery may offer a new strategy for maintaining T cell function against human viral diseases.

Accurate estimates of parameters and demographic rates of wildlife populations are crucial to management and conservation. One parameter of interest is age because it influences multiple demographic rates and behaviors including reproduction, dispersal, migration, and disease transmission. There are a number of methods used to estimate ages of wildlife. One of the most common methods used for mammals is evaluation of patterns in dental eruption and tooth wear. In the first chapter, we evaluate the accuracy associated with estimates of age from dental eruption and patterns of tooth wear for mule deer (Odocoileus hemionus). In the second chapter, we use age estimates validated within the first chapter to explore how age and other maternal factors may influence number of offspring born per litter in mule deer. We further test relative support for two common hypotheses--terminal investment and reproductive restraint--regarding the relationship between age and reproductive investment in this species. Our results will inform conservation, management, and research efforts associated with mule deer.