Les concentrations d’oxygène (O2) dans la moelle osseuse hématopoïétique, sont très inférieures à celle de l’air (20% d’O2) puisqu’elles vont de 4% dans les zones juxta-vasculaires à 0,1% près de l’endoste, où siègent les cellules souches hématopoïétiques (CSH), essentiellement quiescentes. Ce paramètre physiologique, rarement pris en compte, est un élément important dans la régulation de l’hématopoïèse. Les effets bénéfiques des faibles concentrations d’oxygène sur le maintien des cellules souches hématopoïétiques sont maintenant bien établis. Par contre, la réponse du compartiment des progéniteurs aux faibles concentrations d’oxygène est moins examinée mais très discutée, certains montrant une différenciation associée à un blocage de la prolifération alors que d’autres montrent leur disparition de la culture probablement par apoptose. C’est dans ce contexte que se place ces travaux qui visent à approfondir les effets des faibles concentrations en oxygène (de 3 à 0,1%) sur ce compartiment. La culture pendant 72h à 0,1% O2 de la lignée murine de progéniteurs hématopoïétiques non leucémiques FDCP-Mix entraîne leur arrêt progressif en quiescence (Ki-67 négatif) de ces cellules sans induction d’apoptose. Cet arrêt est associé à la différenciation granulocytaire d’une majorité de la population. Dans ces mêmes conditions de culture persiste une population restreinte de cellules qui s¹auto-renouvellent lentement et qui sont capables après repiquage en culture à 20% d’O2 de repeupler une culture liquide et de former des colonies en milieu semi-solide. Ces changements fonctionnels sont associés aux modifications de protéines du cycle cellulaire impliquées dans la quiescence cellulaire : p27KIP1, pRb et CDK. Cette caractérisation permet désormais d’utiliser cette lignée comme modèle pour l’étude des équilibres fondamentaux au maintien de l’homéostasie hématopoïétique. / Oxygen concentrations (O2) in hematopoietic bone marrow vary from 4% in capillaries to less than 0.1% in subendosteum, where hematopoietic where mostly quiescent stem cells reside. This physiological factor, rarely investigated, is an essential piece of hematopoiesis regulation. The beneficial effects of low oxygen concentrations on the maintenance of hematopoietic stem cells are now well established. In contrast, the effects of low O2 concentration on the progenitors compartment, were much less explored and are then more controversial: some articles evidence a pro-differentiative effect related to a cell proliferation blockade while others observe their rapid disappearance from cultures probably due to apoptosis. In this particular context takes place this work which aims to investigate the low oxygen concentration effect (from 3% to 0.1%) on this precise compartment. Culture of the murine non-leukemic hematopoietic progenitor cell line FDCP-Mix line at 0.1% O2 during 72h induces a progressive G0 quiescence blockade (Ki-67 negative) without apoptosis increase. This G0 cell cycle arrest is correlated with the granulo-monocytic differentiation of most cells. In the mean time a minor population of self-renewing cells continues to cycle slowly as evidenced by their 5-FU sensitivity in primary culture and by their capacity to give rise to colonies and to repopulate liquid cultures when replated in cultures at 20% O2. G0 quiescence and granulocytic differentiation induced by low O2 concentrations is associated with cell cycle protein modifications: p27KIP1, pRb, CDK. This characterization allows FDCP-Mix usage as model to investigate fundamental balances responsible for hematopoietic long-term maintenance.

A montagem e desmontagem das adesões focais (AF) desempenham um papel fundamental em diversos processos celulares, incluindo migração celular e sobrevivência. Resultados prévios do nosso laboratório mostram que fibroblastos nulos ou silenciados para miosina-Va sofrem um atraso na desmontagem das adesões, sugerindo um papel para a miosinaVa neste processo. Neste trabalho, visamos analisar a dinâmica de montagem das AF em fibroblastos murinos imortalizados NIH3T3, utilizando sondas fluorescentes para visualização de componentes de adesão focal. A formação das AF foi analisada após estímulo por soro de células quiescentes, o que leva a intensa polimerização de actina, reorganização do citoesqueleto e montagem das AF. A cinética de montagem das AF foi observada em ensaios ao longo do tempo, de células fixadas em 0, 5, 15, 30, 120 minutos após estímulo, e marcadas para miosina-Va fosforilada (p-miosina-Va, S1650), FAK fosforilada (p-FAK, Y397), vinculina, dinamina-2, integrina-?1, faloidina, Ki67 e DAPI. Os nossos resultados mostraram um aumento de fluorescência de p-miosina-Va por todo o citoplasma após a estímulo com soro, e revelaram que a p-miosina-Va co-localiza com pFAK nas AF logo após o estímulo, essa localização da p-miosina-Va nas AF diminui ao passar do tempo e retorna após 120 minutos. Isto é consistente com os resultados anteriores de um papel da miosina-Va na dinâmica das AF. Também é possível perceber uma maior concentração de p-miosina-Va e dinamina-2 na região perinuclear, 5 minutos após estímulo, e o espalhamento de ambas as proteínas pelo citoplasma com o passar do tempo. Demonstramos, por Western blotting, que o estímulo por soro não causa alteração na quantidade total de miosina-Va em nenhum dos tempos analisados em relação à condição de quiescência, mas induz, após 5 e 15 minutos, um aumento apreciável de p-miosina-Va, que sofre queda e variações nos tempos posteriores. Para nosso conhecimento, esta é a primeira demonstração de que a fosforilação da miosina-Va aumenta em resposta ao soro e estamos investigando se este evento está ligado à dinâmica das adesões focais em fibroblastos / The assembly and disassembly of focal adhesions (FA) play a critical role in several cellular process, including cell migration and survival. Previous work from our laboratory showed that fibroblasts without myosin-Va show a delay in focal adhesion disassembly, suggesting a role for myosin-Va in this process. In this work, we aim at imaging the dynamics of focal adhesion disassembly and reassembly in cells, with fluorescent probes for visualization of focal adhesion components. Here, we used murine NIH3T3 fibroblasts to analyze FA formation after serum stimulation of quiescent cells, which leads to intense polymerization of actin and reorganization of the cytoskeleton and FA assembly. The kinetics of FA assembly was observed in a time-course assay of cells fixed at 0, 5, 15, 30 and 120 min after serum stimulation, and stained for phosphorylated myosin-Va (p-myosin-Va, S1650), phosphorylated FAK (p-FAK, Y397), vinculin, phalloidin and DAPI. Our results showed an increase of pmyosin-Va staining throughout the cytoplasm upon serum stimulation, and revealed that pmyosin-Va does not colocalize with FAK in FA at early time points. However, colocalization is observed after 30 to 120 min. This is consistent with previous results of a role for myosin-Va in FA disassembly. It is also possible to observe a higher concentration of p-myosin-Va and dynamin-2 in the perinuclear region 5 minutes after stimulation, and the spreading of both proteins in the cytoplasm over time. We demonstrate by Western blotting that serum stimulation does not cause change in total amount of myosin-Va, in any of the times analyzed in relation to the quiescent condition, but induces, after 5 and 15 minutes, an appreciable increase of pmyosin-Va suffering drop and variations in the later times. To our knowledge, this is the first demonstration that phosphorylation of myosin-Va increases in response to serum and we are investigating whether this event is connected to the dynamics of focal adhesions in fibroblasts

As the so-called “Mothership of the Human Genome,” the cell nucleus must keep all vital genetic information safe, but accessible, inside a strong protective envelope. The inner membrane of the nuclear envelope is lined by tough but adaptable proteins called lamins. While lamins polymerize into fibrous structures that hold up the “walls” of the nucleus, they also serve as an internal scaffold for the complex machinery involved in DNA replication and gene expression. It is in this later role that we have been looking for clues to premature and possibly to normal aging. One type of lamins, Lamin A is made through an unusual pathway involving a lipid dependent cleavage of a larger precursor called prelamin A. The functional significance of this processing pathway is that prelamin A cannot assemble and is inhibitory of proper lamina formation. Pathological cases of immature lamin A accumulation include Hutchinson-Gilford progeria syndrome (HGPS) or Progeria characterized by premature aging and Restrictive Dermopathy (RD), a lethal prenatal disease. We have previously shown that accumulation of prelamin A leads to cell cycle arrest and drastic changes in expression of genes involved in cell cycle control, among those, several members of the FoxO family of transcription factors. The goal of this study was to determine the mechanisms by which accumulation of uncleavable prelamin A activates FoxO-mediated cell cycle arrest. Cells expressing an uncleavable form of Lamin A in an inducible manner were used to determine subcellular distribution of FoxO3a upon accumulation of prelamin A. This was done by indirect immunofluorescence and Western blotting. The proliferation rate of these cells and controls expressing wild type Lamin A was also determined by measuring the incorporation of BrdU into DNA. During these experiments, it was hypothesized and observed that overexpression of prelamin A leads to redistribution of FoxO3a from the cytoplasm of the cell to the nucleoplasm. Expression of FoxO3a target genes was accordingly increased, leading to a decrease in cell proliferation. The information obtained from this study could not only be of interest in broadening our knowledge of the mechanisms of quiescence and aging in general, but also could inform the discussion of the use of several therapeutics for the treatment of Progeria and other diseases that result from the accumulation of prelamin A.

Eukaryotic gene expression is a complex process that can be controlled at the level of transcription, post-transcription, translation, or post-translation. In recent years there has been growing interest in understanding the role of the 3'-untranslated region (UTR) in post-transcriptional regulation. The 3'-UTR contains many regulatory sequences, including microRNA (miR) target sites and AU-rich elements (AREs). Although a relatively recent discovery, miRs have been shown to downregulate target gene expression and have important roles in regulating many cellular processes, including cellular growth. Cellular growth consists of two distinct states, proliferation and quiescence. The proliferative state consists of G1, S, G2, and M phases while quiescence is the G0 phase. In response to mitogenic stimuli, quiescent cells enter the proliferative cycle and may transit back to the quiescent state. Reentry into quiescence is essential to prevent aberrant proliferation as well as to protect the cellular life span. Cells that remain in quiescence for an extended period of time lose their ability to proliferate. It has been shown that the redox status of the cells may regulate quiescence and proliferative capacity since overexpression of SOD2 protects the proliferative capacity of quiescent cells. We hypothesized that the redox environment regulates proliferative capacity through miR expression and regulation of miR targets. Early results showed treatment with hydroxytyrosol (HT), an olive-derived catechol, was able to protect the proliferative capacity of quiescent normal human fibroblasts. HT was shown to use hydrogen peroxide and produce superoxide in a catechol-semiquinone-quinone redox cycle. Interestingly, HT also induced SOD2 expression. Further results from microRNA PCR arrays and Taqman PCR assays showed a significant decrease (4-fold) in miR-302 levels during quiescence compared to proliferating normal human fibroblasts, suggesting that miR-302 could regulate cellular growth states. Results from a Q-RT-PCR and dual luciferase-3'-UTR reporter assays identified ARID4a (AT-Rich Interacting Domain 4a, also known as RBP1) and CCL5 (C-C motif Ligand 1) as targets for miR-302. Ionizing radiation, that is well known to induce oxidative stress and delay cell cycle progression, decreased miR-302 levels, which was associated with an increase in its target mRNA levels, ARID4a and CCL5. Such an inverse correlation was also observed in cells treated with hydrogen peroxide, SOD2 overexpressing cells, and HT treated cells. Overexpression of miR-302 suppresses ARID4a and CCL5 mRNA levels, and increased the percentage of S-phase cells. These results identified miR-302 as a redox-sensitive regulator of ARID4a and CCL5 mRNAs as well as demonstrate a regulatory role of miR-302 during quiescence and proliferation.

Dormancy, a state of severely decreased or suspended metabolism, is a widespread survival strategy in nature. In the Foraminifera, one of the most studied groups of marine organisms, its presence had been suggested by circumstantial evidence, but rarely studied directly until recently. Despite the lack of research, stressor-induced dormancy can significantly alter the way in which foraminiferal ecology is understood, especially in marginal environments. In this dissertation, I reviewed the evidence for dormancy in the foraminiferal literature, concluding that evidence for dormancy is widespread across the Phylum. I then explored the role of dormancy in the survival of the diatom-bearing foraminifer Amphistegina gibbosa d’Orbigny when exposed to toxic chemicals, and when kept in dark conditions for extended periods of time. I developed methods for utilizing CellTracker Green™, a fluorescent probe, to explore metabolic activity in symbiont-bearing foraminifers, finding that it can be used in some situations, such as bioassay experiments or other cases of toxic chemical exposure, to distinguish dead from dormant individuals. The results of the associated experiments demonstrated that reduced metabolism occurred in individuals that survived toxic chemical exposure for over two months in darkness, as well as indicating that metabolic recovery can begin to occur within 30 minutes of removal from darkness. Fluorescence microscopy of symbiont autofluorescence also demonstrated that the diatom symbionts are also capable of surviving aphotic conditions, recovering when returned to lighted conditions. Further experiments showed that A. gibbosa and its associated symbionts are capable of surviving up to 20 months in darkness. Although survival decreased as the length of time in darkness increased, 80% of the specimens survived a 20-month treatment. In addition, all treatment lengths showed color recovery, indicating survival of the diatom symbionts, which give A. gibbosa its characteristic golden-brown color. However, patterns of color recovery indicated that extended periods in darkness increased the photosensitivity of the A. gibbosa holobiont, despite entering dormancy.

<p>The present thesis collects results of research applying theory and methods of statistical seismology to the seismicity of Central America, a region with a complex tectonic setting controlled by the interaction of four major plates, namely the Caribbean, Cocos, Nazca and North American plates.</p><p>Three different earthquake catalogues were used for studies focused on stress in a tectonic volume, seismic hazard maps and seismicity patterns (precursors), covering the region 94ºW to 81ºW and 5ºN to 20ºN.</p><p>Variations in the <i>b</i>-value, the parameter in Gutenberg & Richter’s equation Log<i>N</i>=<i>a</i>-<i>bM</i>, as a function of depth in the subduction zone were investigated. High <i>b</i>-values were identified in the upper part of the slab at depths of 80-110km beneath Guatemala-El Salvador and at depths 130-170km beneath Nicaragua. These anomalies may be related to the generation of volcanism occurring above them. Time dependence of the <i>b</i>-value was also studied. Five case studies were selected (events with <i>M</i><i>S</i> ≥7.2) for a detailed analysis. In three out of five cases, it was possible to link <i>b</i>-value minima to the time of occurrence of corresponding large events. </p><p>Seismic quiescence was mapped as a function of time and space by a griding technique. The characteristics of the quiescence were calculated using the statistics <i>Z</i> and <i>ß</i> and for Time Window lengths between 1 and 5 years. Five positive anomalies were found, which can be associated with large earthquakes (<i>M</i><i>S</i>≥7.2).</p><p>Finally, a Monte Carlo approach was utilized to evaluate the ground motion hazard and its uncertainties in northern Central America. A set of new seismic hazard maps exhibiting probabilistic values of peak ground acceleration (PGA) with 50%, 10%, and 5% probabilities of exceedance (PE) in 50 years is presented for a large area of northern Central America, including El Salvador and Guatemala.</p>

Dans les cellules cancéreuses, des défauts affectant l'organisation d'ADN en chromatine sont fréquemment observés. L'étude de facteurs impliqués dans cette organisation est donc essentielle pour mieux appréhender leur implication dans la tumorigénèse. Un facteur particulièrement intéressant dans ce contexte est le facteur d'assemblage de la chromatine, le complexe CAF-1 (Chromatin Assembly Factor 1). CAF-1 est impliqué dans l'assemblage en chromatine de l'ADN lors de la réplication et la réparation de l'ADN. Deux sous-unités de CAF-1 sont sous-exprimés dans les cellules non-proliférantes (quiescentes) et constituent des marqueurs de prolifération dans le cancer. De plus, CAF-1 a un rôle au niveau des régions de chromatine dense proches des centromères, l'hétérochromatine péricentrique, par son interaction avec les protéines HP1 (Heterochromatin Protein 1). Il existe trois isoformes de HP1 dans les cellules mammaires (HP1α, β et γ), dont HP1α est le plus spécifiquement associé aux régions d'hétérochromatine péricentrique, impliqués dans la répression des gènes et la ségrégation des chromosomes. Pendant ma thèse, je me suis penchée sur deux questions majeures: Premièrement, est- ce que l'expression des isoformes de HP1 est régulée d'une façon dépendante de la prolifération et de la tumorigénèse ? En combinant des modèles de lignées cellulaires et des échantillons de tissu humain, j'ai pu montrer que l'expression de l'isoforme HP1α, mais pas HP1β ou γ, est dépendante de la prolifération. La déplétion de HP1α, spécifiquement, affecte le passage de la mitose. De plus, HP1α, mais pas HP1β ou γ, est surexprimé dans de nombreux types de cancer comparé aux tissus sains correspondants. La surexpression de HP1α dans le cancer du sein est corrélée de façon significative à la survie des patientes et la formation de métastases. Ces résultats révèlent HP1α comme un marqueur pronostique dans le cancer du sein et potentiellement dans d'autres types de cancer. Nous proposons que la surexpression de HP1α présente un avantage sélectif pour les cellules cancéreuses, liée à l'organisation de l'hétérochromatine péricentrique et le passage de la mitose. Ces résultats ont donné lieu à un brevet et à une publication dans EMBO Molecular Medecine. La seconde question à laquelle je me suis intéressée est : comment des cellules quiescentes, qui expriment peu de CAF-1, gèrent l'assemblage de la chromatine couplé à la réparation de l'ADN ? En effet, une capacité de réparation différente entre cellules proliférantes (tumorales) et quiescentes (saines) aura un impact majeur sur l'efficacité et la toxicité des traitements génotoxiques comme la chimio- et la radiothérapie. J'ai pu montrer que, dans les cellules quiescentes, les irradiations aux ultra-violets (UV) n'induisent pas l'expression de CAF-1. De plus, la faible quantité de CAF-1 est recrutée aux sites des lésions d'UV, suggérant que sa fonction dans la réparation est conservée hors du cycle cellulaire. Cependant, en quiescence, nous observons une réparation retardée d'un type spécifique de lésions, qui reflète potentiellement une difficulté des cellules quiescentes à gérer la prise en charge de ces lésions au sein de la chromatine. Ces résultats font l'objet d'un manuscrit actuellement en préparation. Dans ces deux projets majeurs, j'ai mis en avant comment des facteurs de l'organisation de la chromatine peuvent être impliqués dans la prolifération, la tumorigénèse et la réparation d'ADN suite à des traitements génotoxiques. De plus, nous avons pu proposer un nouvel outil d'intérêt médical pour le pronostique des cancer du sein.

Understanding natural mechanisms of protection against HIV infection and disease progression are key priorities for informing vaccine and microbicide design. The research presented in this thesis aimed to characterize mechanisms of defence in HIV-exposed seronegative (HESN) individuals, who naturally resist infection by HIV, and HIV-controllers, who are HIV-infected, but suppress viral replication in the absence of treatment. Previous studies have linked resistance to HIV infection with low basal levels of gene transcription and reduced production of inflammatory mediators, suggesting an overall state of immune quiescence in HESN. Immune quiescence may also be protective in HIV-infected individuals, as immune activation drives disease progression. The central hypothesis of this thesis is that immune quiescence protects against HIV infection and disease progression by limiting the pool of activated target CD4+ T cells susceptible to HIV infection. This hypothesis was addressed by evaluating immune function in HESN from the Pumwani commercial sex worker cohort and HIV-controllers from the Manitoba elite controller cohort. In HESN, immune quiescence was marked by low levels of circulating activated T cells and low levels of the proinflammatory mediators IL-1α and IL-8 in the cervical mucosa. Regulatory T cells (Tregs), which suppress T cell activation, were elevated in HESN, and may represent a driver of immune quiescence. Low T cell activation and elevated Tregs were associated with reduced cellular susceptibility to infection in vitro. These data suggest that immune quiescence protects against infection by limiting the activated target CD4+ T cell pool, in support of the central hypothesis. HIV-controllers expressed low levels of the proinflammatory chemokines IP-10 and MCP-1 and low frequencies of activated T cells. These data demonstrate that immune quiescence is not only protective prior to exposure, but is also beneficial following infection. HIV-controllers also had elevated MIP-1α, reduced TGFβ and HIV-specific T cell proliferation responses, which contribute to protection by mechanisms other than immune quiescence. Taken together, these data support a role for immune quiescence in protection from HIV infection and disease progression. Mechanisms of reducing inflammation and target cell activation should be considered during future HIV vaccine and microbicide development.

Understanding natural mechanisms of protection against HIV infection and disease progression are key priorities for informing vaccine and microbicide design. The research presented in this thesis aimed to characterize mechanisms of defence in HIV-exposed seronegative (HESN) individuals, who naturally resist infection by HIV, and HIV-controllers, who are HIV-infected, but suppress viral replication in the absence of treatment. Previous studies have linked resistance to HIV infection with low basal levels of gene transcription and reduced production of inflammatory mediators, suggesting an overall state of immune quiescence in HESN. Immune quiescence may also be protective in HIV-infected individuals, as immune activation drives disease progression. The central hypothesis of this thesis is that immune quiescence protects against HIV infection and disease progression by limiting the pool of activated target CD4+ T cells susceptible to HIV infection. This hypothesis was addressed by evaluating immune function in HESN from the Pumwani commercial sex worker cohort and HIV-controllers from the Manitoba elite controller cohort. In HESN, immune quiescence was marked by low levels of circulating activated T cells and low levels of the proinflammatory mediators IL-1α and IL-8 in the cervical mucosa. Regulatory T cells (Tregs), which suppress T cell activation, were elevated in HESN, and may represent a driver of immune quiescence. Low T cell activation and elevated Tregs were associated with reduced cellular susceptibility to infection in vitro. These data suggest that immune quiescence protects against infection by limiting the activated target CD4+ T cell pool, in support of the central hypothesis. HIV-controllers expressed low levels of the proinflammatory chemokines IP-10 and MCP-1 and low frequencies of activated T cells. These data demonstrate that immune quiescence is not only protective prior to exposure, but is also beneficial following infection. HIV-controllers also had elevated MIP-1α, reduced TGFβ and HIV-specific T cell proliferation responses, which contribute to protection by mechanisms other than immune quiescence. Taken together, these data support a role for immune quiescence in protection from HIV infection and disease progression. Mechanisms of reducing inflammation and target cell activation should be considered during future HIV vaccine and microbicide development.

Regeneration of adult skeletal muscle relies upon a population of quiescent myogenic progenitor cells, called satellite cells (SCs). Upon injury, SCs activate, proliferate, differentiate and fuse to make new myofibers or to repair damaged ones. SCs can also self-renew to repopulate the SC niche. The balance between differentiation and self-renewal is critical to maintain muscle homeostasis and changes in this equilibrium can lead to chronic muscle degeneration. For example, Duchenne’s muscular dystrophy (DMD) is characterized by rounds of muscle degeneration and regeneration leading to increased muscle wasting. One approach to treat DMD is transplantation of SCs. For this treatment to be viable, transplanted cells must contribute to repairing injured muscle and repopulating the SC niche. Here, we show that the transcription factor CCAAT/Enhancer Binding Protein beta (C/EBPβ) regulates SC function. C/EBPβ is down-regulated during differentiation and persistent expression of C/EBPβ inhibits differentiation and expression of the myogenic regulatory factors MyoD and Myogenin. C/EBPβ also promotes Pax7 expression by directly binding to and regulating Pax7 transcription. Using genetic tools to conditionally excise C/EBPβ expression in SCs, we found that C/EBPβ-null SCs lose quiescence and precociously differentiate at the expense of self-renewal. After a single injury, C/EBPβ-deficient SCs failed to self-renew, resulting in impaired muscle repair after a second injury. C/EBPβ-induced quiescence also requires upregulation of caveolin-1. Furthermore, pharmacological manipulation of C/EBPβ expression with the phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX), increased the number of cells available for transplantation into dystrophic muscle and enhanced the expression of stem cell markers in a C/EBPβ-dependent fashion. IBMX treatment improved cell survival and migration, engraftment into the SC niche and repair of dystrophic muscle. Together, these results demonstrate that C/EBPβ is an important regulator of SC function and that pharmacological manipulation of C/EBPβ improves culture conditions for the expansion and selection of SCs available for cell therapy for the treatment of muscular dystrophies.