<p>The capability to control the progression of the cell cycle, including the means to enter into a stable non-proliferative state, is essential for eukaryotic unicellular and multicellular organisms. A quiescent state similar to G0 of higher eukaryotes can be induced by nitrogen starvation of the fission yeast model organism Schizosaccharomyces pombe. Using high-resolution tiling arrays for genome-wide transcriptional profiling we explore the early transcriptional reprogramming on the route to quiescence. Furthermore, we demonstrate that cells carrying a mutation in the high mobility group (HMG) box of the histone lysine demethylase spLsd1 fail to acquire characteristics of quiescent cells and rapidly lose viability under nitrogen-starved conditions. Since no such defect is seen as a result of catalytic inactivation, the HMG domain of spLsd1 seems to confer a function to the protein that is independent of the histone demethylase activity. We show that the HMG domain of spLsd1 is required for transcriptional activation and repression of a large set of genes, both during vegetative growth and on the route to quiescence. We also confirm that spLsd1 is a repressor of antisense transcription, and that this function is at least partially dependent on the HMG domain of the protein.</p>

The capability to control the progression of the cell cycle, including the means to enter into a stable non-proliferative state, is essential for eukaryotic unicellular and multicellular organisms. A quiescent state similar to G0 of higher eukaryotes can be induced by nitrogen starvation of the fission yeast model organism Schizosaccharomyces pombe. Using high-resolution tiling arrays for genome-wide transcriptional profiling we explore the early transcriptional reprogramming on the route to quiescence. Furthermore, we demonstrate that cells carrying a mutation in the high mobility group (HMG) box of the histone lysine demethylase spLsd1 fail to acquire characteristics of quiescent cells and rapidly lose viability under nitrogen-starved conditions. Since no such defect is seen as a result of catalytic inactivation, the HMG domain of spLsd1 seems to confer a function to the protein that is independent of the histone demethylase activity. We show that the HMG domain of spLsd1 is required for transcriptional activation and repression of a large set of genes, both during vegetative growth and on the route to quiescence. We also confirm that spLsd1 is a repressor of antisense transcription, and that this function is at least partially dependent on the HMG domain of the protein.

The present study revealed that FOXO1 is present in the nuclei of non-dividing pituitary cells and in a subset of differentiated cells with highest level of expression in somatotrophs, followed by corticotrophs, thyrotrophs and gonadotrophs throughout development and in adulthood stage. A significant difference in Foxo1 transcript between age-matched males and females at 8-9 weeks of age was demonstrated in the anterior pituitary for the first time. IHC data demonstrating (i) FOXO1 co-localization with p27kip1 (ii) an increase in FOXO1 immunopositive cells within anterior pituitary in p27KO embryos compared to WT (iii) absence of FOXO1 in the nucleus of BrdU positive cells suggested that in absence of p27Kip1 FOXO1 might be important for preventing unbridled cell proliferation. Data suggested that FOXO1 might not be important for initiating pituitary cell differentiation but might be involved with p27kip1 in maintaining pituitary cell quiescence. Increase in nuclear localization of FOXO1 in the pituitary of Foxp3 mutant (lacking insulin signaling) suggested that it might be a down-stream target of insulin/PI3K/PKB pathway in the pituitary as it is in several other tissues.

Development, tissue renewal and longevity of multi-cellular organisms require the ability to switch between a proliferative state and quiescence, a reversible arrest from the cell cycle. The balance of quiescence and proliferation underlies the fundamental feature of generating and maintaining the appropriate number of cells, which is essential for tissue architecture, regeneration, and function. Disruption of quiescence and proliferation balance leads to hypo- or hyper-proliferative diseases. To date, the regulatory mechanism of proliferation has been well established, while cellular quiescence has remained a phenotypic description without a clearly defined molecular control mechanism. Simply, quiescence has long been considered a passive counterpart to proliferation. However, recent findings have revealed that quiescence is an actively maintained state exhibiting a unique gene expression pattern. While quiescence has been traditionally considered as a state (namely G0) outside of the cell cycle, it is in fact a collection of heterogeneous states. In studies conducted in the 70's and 80's using fibroblasts and lymphocytes, it has been observed that the longer the cells were kept under quiescence inducing conditions such as contact inhibition, the deeper the cells moved into quiescence. Deep quiescent cells are still able to reenter the cell cycle upon growth stimulation but they exhibit a longer pre-DNA synthesis phase [1-4]. Shallow quiescent state has also been recently reported in muscle and neural stem cells termed GAlert and "prime" quiescent state, respectively. Heterogeneous quiescent depth entails that cells vary in their sensitivity to growth signals, representing an important yet underappreciated layer of complexity in cell growth control. The cellular mechanisms that control the depth of quiescence remains elusive. In my thesis work, I first investigate the strengths of serum stimulation required for cells to exit deep and shallow quiescence as a determinant of quiescence depth. Through model simulations and experimental measurements, I further demonstrate that various components of the Rb-E2F pathway control quiescence depth with varying efficacy. The Rb-E2F pathway interacts with diverse cellular pathways that respond to environmental signals to jointly modulate quiescence depth. Given that certain circadian clock genes (e.g., Cry) affect key components in the Rb-E2F pathway, I tested the effect of Cry activity on quiescence depth. I found that increased Cry activity resulted in deeper quiescence, contrary to our anticipation based on the literature. Next, we constructed a library of mathematical models that represent possible interactions between Cry and the Rb-E2F pathway. We computationally searched this model library for links that could explain the experimental observations. The modeling search suggested that Cry upregulation may lead to increased expression of cyclin dependent kinase inhibitor (e.g., p21), which in turn drives cells into deeper quiescence. This model prediction was confirmed by my follow-up experiments. Collectively, my thesis work establishes an integrated modeling and experimental framework that will help us to further investigate diverse cellular mechanisms controlling the heterogeneous quiescence depth.

Dans le muscle squelettique adulte, les cellules souches du muscle, nommées les cellules satellites, résident sous la lame basale des fibres musculaires à l’état quiescent jusqu’à ce qu’un dommage musculaire induise leur activation. Après une phase d’activation, les cellules satellites sont capables de proliférer et de se différencier afin de répondre aux besoins des myonucléi au cours de la régénération musculaire. Les cellules satellites, ou au moins une sous-population, sont actuellement considérées comme la principale population de cellules souches du muscle. Des cellules stromales ont été observées au voisinage des cellules satellites, dans un muscle normal et en régénération, incluant les macrophages, les composantes cellulaires des vaisseaux ainsi que les cellules interstitielles de type fibroblastique. Les cellules stromales participent vraisemblablement à la régulation du destin des cellules satellites. Une étude précédente a suggéré la proximité des cellules satellites et du lit capillaire. Nous avons montré que, quelque soit leur statut (quiescente, activée, cyclante), les cellules satellites sont presque toujours localisées à proximité immédiate d’un capillaire, dans un muscle squelettique normal et en régénération. Leur nombre est corrélé avec le nombre des capillaires par myofibre et varie en fonction de la densité des capillaires dans divers situations pathologiques. Les cellules endothéliales stimulent spécifiquement la croissance des cellules myogéniques par l’intermédiaire de facteurs solubles incluant bFGF, HGF, IGF-I, PDGF-BB, VEGF. Inversement, les cellules myogéniques ont un effet proangiogénique sur les cellules endothéliales in vitro, cette activité augmentant avec la différenciation myogénique. Nous proposons qu’il existe des interactions bidirectionnelles entre les cellules myogéniques et les cellules endothéliales, notamment par l’intermédiaire du VEGF sécrété par les cellules endothéliales et les cellules myogéniques différenciées. De plus du VEGF et son récepteur, l’homéostasie vasculaire est essentiellement régulée par un autre système moléculaire, la famille des Angiopoiétines/Tie. Nous avons exploré le rôle du système Angiopoiétine1/Tie-2 dans la régulation du destin des cellules précurseurs myogéniques. Nous avons étudié le rôle de Angiopoiétine1 (Ang1) et son récepteur Tie-2 dans la régulation du destin de cellules précurseurs myogéniques (mpc). Chez l’homme et chez la souris, Tie-2 et Ang1 sont préférentiellement exprimés par les cellules satellites quiescentes in vivo et par les cellules de réserve (RCs) in vitro. La voie de signalisation Ang1/Tie-2, par l’intermédiaire de la voie ERK1/2, induit une diminution de la prolifération et de la différenciation des mpc, une augmentation du nombre des cellules en phase G0 du cycle cellulaire, une augmentation de l’expression des gènes associés au statut de RCs (p130, Pax7, Myf-5, M-cadhérine) et une diminution de l’expression des gènes associés à la différenciation myogénique. L’inhibition de l’expression de Tie-2, par une approche de RNA interférence, a l’effet strictement inverse. Les cellules situées au voisinage des cellules satellites, telles que les cellules musculaires lisses et les cellules interstitielles de type fibroblastique, stimulent l’expression des gènes associés aux RCs par la sécrétion de Ang1, in vitro. In vivo, le blocage de Tie-2, par l’intermédiaire d’anticorps bloquants anti-Tie-2, induit une augmentation du nombre des cellules satellites cyclantes dans le muscle. Inversement, la surexpression de Ang1, par électroporation d’un plasmide dans le muscle, induit une diminution du nombre des cellules satellites cyclantes. / In adult skeletal muscle, the muscle resident stem cells called the satellite cells reside in a sub-laminal location where they stay quiescent until muscle damage triggers their activation. Upon activation, satellite cells have the ability to proliferate, to differentiate and to respond to both the routine turnover of myonuclei and muscle regeneration. Satellite cells, or at least a subset of them, are now considered as the main myogenic stem cells. Several stromal cells are observed in the vicinity of the satellite cells, in both normal and regenerating muscle, including macrophages, vessel cell components and interstitial cells of fibroblastic type. These stromal cells likely participate to the regulation of satellite cell fate. A previous study suggested a proximity of a number of satellite cells to microvessels. We have shown that satellite cells are strikingly close to capillaries, in both human and mouse, whatever their status (activated, cycling, quiescent). The number of satellite cells is correlated with capillarization of myofibers, regardless to their type, in normal muscle and in paradigmatic physiologic and pathologic situations. Endothelial cells specifically enhanced myogenic cell growth through secretion of at least five soluble factors including IGF-1, HGF, bFGF, PDGF-BB and VEGF. Reciprocally, myogenic cells exhibit a proangiogenic effect on endothelial cells in vitro, this activity increasing with myogenic differentiation. We conclude that there are bidirectional interactions between satellite cells and endothelial cells, notably mediated through secretion of VEGF by both endothelial cells and differentiating myogenic precursor cells. Besides VEGF and its receptor, vascular homeostasis is mainly regulated by another molecular system, the Angiopoietin/Tie family. We explored its involvement in the regulation of myogenic precursor cell fate. We studied the involvement of angiopoietin-1 (Ang1) and its tyrosine kinase receptor Tie-2 in myogenic cell fate. Human and mouse satellite cells expressed both Tie-2 and Ang1 in vivo. During in vitro differentiation, Ang1 and Tie- 2 were differentially expressed by human myogenic cells (mpcs): expression was strongly upregulated in reserve cells (RC), a subpopulation of undifferentiated quiescent cells considered as responsible of the self-renewal of the myogenic cell population. Ang1/Tie-2 signalling, through ERK1/2 pathway, decreased mpc proliferation and differentiation, increased the number of cells in G0 phase of the cell cycle, increased expression of RC-associated markers (p130, Pax7, Myf-5, M-cadherin) and downregulated expression of differentiation-associated markers. Silencing Tie-2 had opposite effects. Cells located in the satellite cell neighbourhood (smooth muscle cells, fibroblasts) upregulated RC-associated markers by secreting Ang1 in vitro. In vivo, Tie-2 blockade and Ang1 overexpression increased the number of cycling and quiescent satellite cells, respectively. We propose that Ang1/Tie-2 signalling regulates myogenic cell self-renewal by controlling the return to quiescence of a subset of satellite cells.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23424号 / 医科博第129号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 林 康紀, 教授 伊佐 正, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

SRY (sex determining region Y)-box 2 (SOX2) is one the embryonic stem cell transcription factors that is capable of reprogramming adult differentiated cells into an induced pluripotent cell. SOX2 is amplified in various types of epithelial cancers and its high its expression correlates with poor prognosis and decreased patient survival. Aberrant Wnt signaling drives the colo-rectal carcinogenic process and is a major determinant of the disease outcome. This study demonstrates that SOX2 counteracts Wnt driven tumor cell proliferation and maintains quiescence in a sub-population of Colo-Rectal Cancer (CRC) cells. High SOX2 expression is found in a sub-group of CRC patients with advanced disease. High SOX2 expression coupled with low Wnt activity was found in SW620 metastatic CRC cell line, while the opposite was true for the isogenic SW480 primary tumor cell line. SOX2 silencing increased Wnt activity and enhanced the oncogenic potential of SW620 cells in vitro and in vivo while over-expression had opposite effects in SW480 cells. SOX2 up-regulates the expression of PTPRK and PHLPP2 protein phosphatase genes which in turn attenuates Wnt activity by interfering with Protein Kinase A, B and C mediated beta catenin phosphorylation at Serine 552 and 675 amino acid residues thereby diminishing its nuclear sequestration and transcriptional activation. Thus SOX2 mitigates growth factor mediated Wnt activation in CRC cells and inhibits cellular proliferation so that these cells are forced to change their oncogene addiction. In effect, high SOX2 expression causes clonal evolution of APC mutant CRC cells from a state of high Wnt dependency to a state of low Wnt dependency in the process making such cells resistant to Wnt inhibitor therapy. Enhanced SOX2 transcriptional activity was associated with increased proportion of cancer cells in G0-G1 phase of cell cycle. Changing SOX2 protein levels in cells had a direct correlation with mRNA levels of RBL2-HUMAN and CDKN2B genes, which serve as regulators of G0 and G1 respectively. SOX2 was shown to physically bind and to the promoter region of these two genes and enhance their transcription. Thus high SOX2 expression, up-regulates the expression of key cell cycle inhibitor genes like RBL2 and CDKN2B and keeps cells in a dormant state. This phenomenon allows colon cancer cells to escape from cytotoxic drug therapy directed at rapidly dividing cells and cause treatment failure and disease relapse.

Within the adult mammalian brain, Neural Stem Cell (NSC)s are maintained in distinct neurogenic niches in a mostly quiescent state. Activation of quiescent NSCs first requires re-entry into the cell cycle in order for the pool to proliferate and eventually commit to a neural fate, giving rise to newborn neurons. The canonical Retinoblastoma (Rb)-E2 Promoter Binding Factor (E2f) pathway is not only key in overcoming the Gap 1 Phase (G1)/S-phase restriction, but novelly appears to be involved in adult neurogenesis and NSC activation. I hypothesized that activator transcription factors E2 Promoter Binding Factor 1 (E2f1) and E2 Promoter Binding Factor 3 (E2f3) are crucial for exit from a quiescent state in adult NSCs. The contribution of the activator E2fs in this transition was studied using a Nestin-driven Cre Recombinase-Estrogen Receptor Tamoxifen-2 Ligand Binding Domain (Cre-ERT2) system to induce targeted deletion of E2f1/3 within NSCs in adult mice. We show that loss of E2f1/3 causes significant neurogenic defects, including pro-neural activation and decreased pools of adult NSCs, that preferentially adopt a quiescent profile in the subventricular zone. We employed this model to further isolate subventricular zone-derived NSCs using a Rosa26:Yellow Fluorescent Protein (YFP) reporter and subsequently analysed transcriptional profiles by RNA sequencing. Loss of E2f1/3 shifts NSC transcriptomes towards one overlapping with quiescent neural stem cell signatures (Codega et al., 2014; Basak et al., 2018), further highlighting the requirement of these E2fs for initial activation. A significant portion of these differentially expressed genes are putative E2f targets. Transcriptionally, major pathways involving cell metabolism, cellular signaling, and neural development are perturbed without activator E2f expression. In effect, this combined approach based on in vivo data and bioinformatics analyses offers a method of prospective identification of novel regulators of adult neurogenesis that require the activator E2fs. Preliminary data suggests that AT-Hook Transcription Factor (Akna) is one such target worth pursuing. Cumulatively, this project describes a unique role for E2f1 and E2f3 during NSC exit from quiescence and subsequent activation towards differentiation. As ongoing maintenance of quiescent NSCs is a necessary prerequisite for lifelong neurogenesis, conclusions from this study could determine the therapeutic potential of targeting activator E2fs to combat the niche exhaustion associated with aging, injury, and neurodegenerative diseases.

Motion sensitive operations at sea are conducted in an unpredictable environment. While occasionally these operations can be planned around suitable weather forecast or delayed until smoother motions are apparent, naval ships conducting flight operations may have little liberty in their mission planning and execution. Tools exist to translate the ocean's harsh conditions into discretely defined low motion operational periods. Particularly of interest, the identification of discrete lull periods or quiescence for shipboard helicopter operations can be better defined using a landing period indicator than with the current method of utilizing static deck angle measurements. While few of these systems exist, assessing their operational benefits is difficult due to a lack of well-defined performance metrics. This thesis defines and examines the use of two methodical approaches to evaluating Landing Period Indicators (LPIs) and their subject ship-helicopter dynamic interface system. First a methodology utilizing the comparison of a basic transparent algorithm is detailed and a case study employing this methodology is examined. Second, a system dynamics approach is taken to pilot workload analysis, utilizing a dynamic systems model characterizing a subset of the Dynamic Interface. This approach illustrates the realistic gains in understanding and development that can be accomplished by utilizing system dynamics in the analysis of the Dynamic Interface and LPI insertion. / Master of Science

Notre groupe a montré que l’hypoxie sévère (0.1% O2) induit un arrêt du cycle cellulaire en G0 des cellules humaines CD34+ et des cellules murines FDCP mix. Peu d’études ont exploré l’existence de Cellules Initiatrices de Leucémie (CIL) dans les LAL et leur rôle dans les rechutes. Notre projet s’est focalisé sur l’effet de l’hypoxie sévère sur la quiescence des CIL dans les LAL, qui pourrait être responsable d’un pourcentage de rechutes. En effet dans la niche hématopoïétique, ou sont localisées les Cellules souches hématopoïétiques et probablement les CIL, la concentration d’oxygène avoisinerait 0,1%.Nous avons utilisé la lignée de LAL NALM6 pour explorer les effets de l’hypoxie sévère sur leur survie, leur cycle cellulaire et leur chimiorésistance. Nos résultats ont mis en évidence qu’une culture à 0.1% O2 durant 7 jours de la lignée NALM6: - inhibe leur prolifération sans surmortalité, - révèle une population restreinte de CIL quiescentes et chimiorésistantes capables d’induire une leucémie dans des souris. Nous avons recherché les relations entre l’hypoxie sévère et quelques caractéristiques des cellules primaires de patients atteints de LAL : existence et rôle de CIL résistantes à l’hypoxie et aux agents thérapeutiques conventionnels des LAL ; localisation de ces cellules résiduelles dans la moelle osseuse des souris xénogreffées. Nos résultats suggèrent que certaines rechutes de LAL pourraient être dues à la persistance à long terme de « quiescent/dormant » CIL dans les niches hypoxiques de la moelle osseuse. Ce modèle est intéressant pour explorer les mécanismes in vitro et in vivo de chimiorésistance dans les LAL et le rôle de l’environnement dans ce phénomène. / Our group showed that severe hypoxia (0.1% O2) induces G0 cell-cycle-arrest of human CD34+ cells and of murine FDCP-mix Cells. Few studies explored the existence of quiescent Leukemia Initiating Cells (LIC) in ALL and their role in primary chemoresistance and relapses. Our project is focused on the effect of very low O2 concentrations in the maintenance of quiescent LIC in ALL, that could be responsible of a percentage of relapses. Indeed in bone marrow niches, where hematopoietic stem cells and probably LIC are located, the O2 concentrations are below 0.1%.In the present study we used the NALM-6 ALL cell line to explore the effects of culture at 0.1% O2 on their survival, cell cycle and chemoresistance. Our results evidence that a 7 days culture of NALM-6 cells at 0.1% O2: - inhibits their proliferation without major cell death; - reveals a restricted LIC population of quiescent and chemoresistant LIC; - maintains quiescent chemoresistant LIC that induce leukemia when injected in immunodeficient mice. We investigated the relationships between severe hypoxia and some characteristics of ALL primary cells obtained from patients: existence and role of quiescent chemoresistant LICs in ALL relapses; location of these residual cells inside the bone marrow of engrafted mice. Our results suggest that some ALL relapses could be due to the long term persistence of “quiescent / dormant” LIC in hypoxic bone marrow niches. This model is of interest for exploring the in vitro and in vivo (xenograft) mechanisms of chemoresistance in ALL and the role of the bone marrow environment in this phenomenon.