La réplication de l'ADN, qui doit assurer à chaque cycle cellulaire une copie fidèle du génome pour que les cellules filles héritent du même génome, est un processus hautement régulé, faisant intervenir son organisation en chromatine mais aussi sa dynamique au sein de l'architecture nucléaire. Le vieillissement cellulaire, qu'il soit physiologique, pathologique ou induit in vitro par des conditions de culture sub-optimales, est accompagné de modifications de l'organisation du génome en chromatine, susceptibles de modifier la régulation spatiotemporelle du programme de réplication. Dans quelle mesure ces modifications sont réversibles et s'accompagnent d'une restauration du programme de réplication sont des questions que nous avons abordées. Notre étude a donc consisté dans un premier temps à analyser les modifications du programme de la réplication, dans différentes situations de vieillissement cellulaire, afin de vérifier notre hypothèse. Nous avons analysé sur l'ensemble du génome l'organisation spatiale (le long du génome) et temporelle des domaines de réplication, le timing, de cellules prolifératives ou approchant de la sénescence réplicative, de donneurs jeunes, âgés ou encore atteints de vieillissement accéléré ou progéria. Nous avons pu observer que certains domaines de timing permettent de distinguer des cellules jeunes de cellules âgées, ou des cellules prolifératives de pré-sénescentes. Afin d'explorer la réversibilité de ces processus, nous avons utilisé la reprogrammation en cellules souches pluripotentes induites ou iPS, suivie d'une redifférenciation fibroblastique. Nous avons pu démontrer que les iPS produites présentaient toutes les mêmes profils de timing, correspondant à celui d'une cellule pluripotente, indiquant que les modifications liées à l'âge ou à la sénescence pouvaient être reprogrammées. Ceci a ensuite été confirmé par une redifférenciation de ces iPS en cellules fibroblastiques dont les profils de timing de réplication ont pu être associés à ceux de fibroblastes jeunes. Cette étude nous a permis de mettre en évidence l'extrême plasticité de l'organisation spatio-temporelle de la réplication, révélant la possibilité de restaurer une dynamique de réplication altérée avec le vieillissement et l'entrée en sénescence, en manipulant le destin cellulaire vers un état indifférencié. Cette étude de la dynamique des domaines de réplication qui accompagne les modifications épigénétiques de la vie cellulaire a été complétée par l'étude à l'échelle moléculaire du rôle d'une histone déméthylase, Jarid1C/KDM5C, dans la réplication au sein des clusters d'origines. Ensemble, ces résultats apportent de nouveaux éléments sur l'interdépendance des dynamiques chromatiniennes et de réplication au cours de la vie cellulaire. / DNA replication allows at each cell cycle the exact copy of the genome that will be transmitted to daughter cells. Thus, the replication process is highly regulated in concert with its chromatin organization but also its dynamics in the nuclear architecture. Cellular ageing, be it physiologic, pathologic or induced in vitro by sub-optimal culture conditions, is accompanied by modifications of the chromatin organization of the genome. This could lead to spatio-temporal modifications of the replication program. We studied to what extent these modifications are reversible and could lead to the recovery of the replication program. In a first step, we analyzed modifications of the replication program upon several ageing situations to test our hypothesis. We analyzed the whole genome spatio-temporal organization of replication domains, the timing, of proliferating or near-senescent cells, of young, old or progeria (a premature ageing disease)-affected donors. We observed that young cells could be distinguished from old cells, and proliferative from near-senescent, by looking at some particular timing domains. To explore the reversibility of these processes, we used reprogramming to induce pluripotent stem cells (iPS cells) followed by fibroblastic re-differentiation. We were able to demonstrate that the derived iPS cells have similar timing profiles corresponding to pluripotent cells profiles: ageing- and senescence-related modifications of the replication timing could be reprogrammed. It was confirmed by re-differentiating these iPS into fibroblastic cells which timing profiles could be associated to young fibroblasts ones. By manipulating cell fate toward an undifferentiated state, this study shows the extreme plasticity of the DNA replication spatio-temporal organization and highlights a chance to restore the replication dynamics when altered by ageing and senescence. This study of the replication dynamics linked to the epigenetic modifications of cells life was completed by a study at the molecular scale of the Jarid1C/KDM5C histone demethylase influence on replication within origin clusters. Together, these results bring new insights into the interdependency of chromatin and replication dynamics during cell fate modifications.

abstract: An in vitro model of Alzheimer’s disease (AD) is required to study the poorly understood molecular mechanisms involved in the familial and sporadic forms of the disease. Animal models have previously proven to be useful in studying familial Alzheimer’s disease (AD) by the introduction of AD related mutations in the animal genome and by the overexpression of AD related proteins. The genetics of sporadic Alzheimer’s is however too complex to model in an animal model. More recently, AD human induced pluripotent stem cells (hiPSCs) have been used to study the disease in a dish. However, AD hiPSC derived neurons do not faithfully reflect all the molecular characteristics and phenotypes observed in the aged cells with neurodegenerative disease. The truncated form of nuclear protein Lamin-A, progerin, has been implicated in premature aging and is found in increasing concentrations as normal cells age. We hypothesized that by overexpressing progerin, we can cause cells to ‘age’ and display the neurodegenerative effects observed with aging in both diseased and normal cells. To answer this hypothesis, we first generated a retrovirus that allows for the overexpression of progerin in AD and non-demented control (NDC) hiPSC derived neural progenitor cells(NPCs). Subsequently, we generated a pure population of hNPCs that overexpress progerin and wild type lamin. Finally, we analyzed the presence of various age related phenotypes such as abnormal nuclear structure and the loss of nuclear lamina associated proteins to characterize ‘aging’ in these cells. / Dissertation/Thesis / Masters Thesis Bioengineering 2017

<div>As aging proceeds, the occurrence of cardiovascular diseases increases independent of other risk factors. At atherosclerotic sites, the rise in the senescent cell population was also observed. Patients with Hutchinson Gilford Progeria Syndrome (HGPS) also showed accelerated aging syndromes and extensive atherosclerosis progression, which was due to missense mutations on the LMNA gene that led to the production of progerin, an aberrant lamin A isoform instead of regular lamin A protein. Lamins act as structural and functional components in nuclear lamina, and recent findings suggested that the ectopic expression of mutant lamin A or lamin A precursor (prelamin A) not only caused defects in cell mechanics but also disturbed mechanotransduction pathways involving lamin A, both of which may contribute to vascular dysregulation. Moreover, the observation of the accumulation of prelamin A in normal aged vascular cells further suggests shared dysregulations involving lamin A in the vascular system between aged people and HGPS patients.</div><div>In the vascular system, endothelial cells were well regulated by hemodynamic forces in vivo to maintain vascular homeostasis. Endothelial dysfunction, including impaired vasodilation and increased permeability, was regarded as the initial marker of atherosclerosis. Despite recent advancements and discussions about the potential mechanisms of progerin-induced vascular disorders, how progerin triggers endothelial dysfunction in a mechanical environment as an early event during atherosclerotic lesion formation has not been studied intensively.</div><div>To help answer the gap question, we first set our goal to understand the effect of laminar flow at arterial levels on endothelial lamins as part of the aging process. Spatial and temporal changes in lamin A/C expression were observed as cell passage went up without flow present. As shear stress was applied, lamin A/C expressions were modulated on both transcriptional and translational levels, which were also dependent on PDL. To further examine how progerin was involved in EC functions with a particular focus on the flow effects, we next generated a stable endothelial cell line that expressed progerin as our EC aging model. Endothelial wound repair under laminar flow at different rates was characterized, and differential cell proliferation activities, as well as migration deficiencies in progerin-expressing ECs during the process, were also recognized. Furthermore, we also showed the overactivated mTORC2 pathway and unusual actin polymerization activities in these cells after flow application. Our results reported changes in cell migration by progerin with flow application for the first time and provided potential candidate pathways that were disturbed by progerin under arterial flow, which may help explain the high occurrence of atherosclerotic lesions in HGPS vasculature, even at straight portion. The reported progerin-induced wound recovery defects in endothelial cells in the presence of physiological flow may also suggest a mechanism of how progerin disturbs endothelial integrity and functions under mechanical stimuli in the development of vascular pathologies.</div><div>Further extended studies may help to understand the roles of progerin in initiating atherosclerosis, which will aid in the development of potential therapies for those suffering from prelamin A-associated accelerated aging syndromes.</div>