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Menopause is characterized by prolonged lifespan beyond the point of reproductive cessation. Defined so that at least 25% of adulthood is nonreproductive, humans and some toothed whale species are the only groups that have been found to exhibit menopause. Menopause is a puzzling trait that seems to contradict classical evolutionary theory that equates selection operating on reproduction to selection operating on survival. I created two computational models to gain better understanding of the evolution of menopause. The first model explored why menopause is not observed in elephants despite their being characterized by key features in common with menopausal species, specifically offspring care from older females and longevity. Simulations allowed testing the effects of varying age at reproductive cessation and levels of offspring care, modeled by decreases in interbirth intervals. I found that hypothetical populations with greatest post-reproductive lifespans, characterized by longer interbirth intervals and earlier reproductive cessation, were most likely to be out-competed by contemporary elephants. Conversely, hypothetical populations that were most reproductively competitive, those with shorter interbirth intervals and older ages of reproductive cessation, returned post-reproductive lifespans that failed to meet the 25% post-reproductive lifespan criterion for menopause. I identified a small region in the parameter space where populations that were both menopausal and reproductively competitive evolved, but the majority of that region corresponds to biologically unrealistic scenarios. The scenario that is most feasible involves an interbirth interval of 4 years and an age at reproductive cessation of 40 years. The second model studied how menopause might have evolved in humans through a behavioural strategy of ending reproduction early to avoid risk of aneuploidy later in life and diverting resources toward extant kin. I found that populations that ceased reproduction earlier and exhibited greater post-reproductive lifespan returned lower reproductive success. The model also demonstrated that the aneuploidy avoidance behaviour is most successful when reproduction ends at approximately age 50. These concepts have never been explored computationally before, so these experiments contribute a novel simulation-based perspective to the growing body of knowledge surrounding the origin and evolution of menopause. / Thesis / Master of Science (MSc) / Menopause can be defined generally for a group as a life history characterized by prolonged post-reproductive lifespan. Defined specifically so that at least 25% of adulthood is nonreproductive, menopause has been recorded in only humans and some species of toothed whales. This trait presents an evolutionary puzzle, as it appears to contradict classical evolutionary theory, which suggests that reproduction should continue until the end of life. In this thesis, I use computational modeling to explore why elephants have not evolved menopause despite sharing key features with menopausal species and how aneuploidy might have contributed to the evolution of menopause in humans.

<p>The ends of eukaryote chromosomes, a region called the telomeres, plays a critical role in maintaining genome stability. With each round of mitotic division the telomeres erode until a critically short length is reached, which signals the cell to permanently stop dividing. This is recognized as a contributing factor to ageing and the onset of age related diseases. Telomere repeat binding factor 2 (TRF2), is an important telomere DNA binding protein that has an essential role in protecting telomeres from being recognized as DNA breaks, however it has also been implicated in other aspects of telomere maintenance, such as telomere replication and telomere transcription. TRF2 acts as a protein hub for the recruitment of a number of telomere associated proteins involved in telomere maintenance, and it has been shown to be heavily modified by numerous types of post-translational modification. We demonstrate that TRF2 is methylated on arginine residues in its N-terminal region by protein arginine methyltransferase 1 (PRMT1) and that this arginine methylation is important for proper telomere maintenance. We further demonstrate that methylated TRF2 is a component of the nuclear matrix and has a distinct staining pattern in senescent cells. The importance of telomeres to ageing is exemplified by previously reported observations that defects in telomere maintenance are a common characteristic to numerous premature ageing disorders. We show that the premature ageing disorder, Cockayne Syndrome has an underlying defect in telomere maintenance. Approximately 80% of Cockayne Syndrome patients have mutations in the Cockayne syndrome group B (CSB) protein. We identified a novel interaction between TRF2 and CSB. The work presented in this thesis characterizes these novel interactions and gives new insight into the function of TRF2 in telomere maintenance.</p> / Doctor of Science (PhD)

The Gadd45 family of proteins (Gadd45a, Gadd45b, and Gadd45g) has been shown to act as stress sensors in response to various physiological and environmental stressors, including oncogenic stress. However, the role of Gadd45b in senescence remained unclear. Here, we show for the first time that primary mouse embryo fibroblasts (MEFs) from Gadd45b null mice proliferate slowly; accumulate increased levels of DNA damage, and senesce prematurely. Notably, this is in contrast with Gadd45a null MEFs that show enhanced growth rate and escape senescence. This difference in growth rate increases with increasing passage number, suggesting that senescence results from exposure to environmental stressors. The impaired proliferation and increased senescence in Gadd45b null MEFs can be partially reversed by culturing cells at physiological oxygen levels, indicating that in the absence of Gadd45b, primary MEFs are less able to cope with elevated levels of oxidative stress. Interestingly, in contrast to other senescent MEFs, which arrest at G1 phase of cell cycle, Gadd45b null MEFs arrest at the G2/M phase of cell cycle. Furthermore, FACS analysis of Gadd45b null MEFs showed less phospho-histone H3-positive cells compared to wild type MEFs indicating that Gadd45b null MEFs are arrested in G2 phase rather than M phase. Interestingly, other stressors such as sub-lethal H2O2 and UV irradiation, that are known to increase oxidative stress, triggered increased premature senescence in Gadd45b null MEFs compared to wild type MEFs. By staining embryos for SA-β-gal gal, we also show that embryos from Gadd45b null mice exhibit increased SA-β-gal gal staining compared to wild type embryos, thus providing in vivo evidence for increased senescence in Gadd45b null mice. Finally, investigating the effect of loss of Gadd45b on senescence related diseases, we show that loss of Gadd45b promotes senescence and aging phenotypes in the skin as well as increased senescence and attenuated fibrotic response to CCl4 induced liver fibrosis. Together, these results highlight a novel and significant role for Gadd45b in the senescence response of cells to stress. / Molecular Biology and Genetics

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24655号 / 農博第2538号 / 新制||農||1097(附属図書館) / 学位論文||R5||N5436(農学部図書室) / 京都大学大学院農学研究科農学専攻 / (主査)教授 白岩 立彦, 教授 中﨑 鉄也, 教授 丸山 伸之 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Yes / Abdominal aortic aneurysm (AAA) is a silent, progressive disease with high mortality and increasing prevalence with aging. Smooth muscle cell (SMC) dysfunction contributes to gradual dilatation and eventual rupture of the aorta. Here we studied phenotypic characteristics in SMC cultured from end-stage human AAA (5cm) and cells cultured from a porcine carotid artery (PCA) model of early and end-stage aneurysm. Human AAA-SMC presented a secretory phenotype and expressed elevated levels of differentiation marker miR-145 (2.2-fold, P<.001) and senescence marker SIRT-1 (1.3-fold, P<.05), features not recapitulated in aneurysmal PCA-SMC. Human and end-stage porcine aneurysmal cells were frequently multi-nucleated (3.9-fold, P<.001 and 1.8-fold, P<.01 respectively, versus control cells) and displayed aberrant nuclear morphology. Human AAA-SMC exhibited higher levels of the DNA damage marker H2AX (3.9-fold, P<.01 vs. control SMC). These features did not correlate with patients’ chronological age; and are therefore potential markers for pathological premature vascular aging. Early-stage PCA-SMC (control and aneurysmal) were indistinguishable from one another across all parameters. The principal limitation of human studies is tissue availability only at end-stage disease. Refinement of a porcine bioreactor model would facilitate study of temporal modulation of SMC behaviour during aneurysm development and potentially identify therapeutic targets to limit AAA progression. / Supported in part by a grant from the Leeds Teaching Hospitals Charitable Foundation (9R11/8002)

Yes / Increased cardiovascular morbidity and mortality in individuals with type 2 diabetes (T2DM) is a significant clinical problem. Despite advancements in achieving good glycaemic control, this patient population remains susceptible to macrovascular complications. We previously discovered that vascular smooth muscle cells (SMC) cultured from T2DM patients exhibit persistent phenotypic aberrancies distinct from those of individuals without a diagnosis of T2DM. Notably, persistently elevated expression levels of microRNA-145 co-exist with characteristics consistent with aging, DNA damage and senescence. We hypothesised that increased expression of microRNA-145 plays a functional role in DNA damage signalling and subsequent cellular senescence specifically in SMC cultured from the vasculature of T2DM patients. In this study, markers of DNA damage and senescence were unambiguously and permanently elevated in native T2DM versus non-diabetic (ND)-SMC. Exposure of ND cells to the DNA-damaging agent etoposide inflicted a senescent phenotype, increased expression of apical kinases of the DNA damage pathway and elevated expression levels of microRNA-145. Overexpression of microRNA-145 in ND-SMC revealed evidence of functional links between them; notably increased secretion of senescence-associated cytokines and chronic activation of stress-activated intracellular signalling pathways, particularly the mitogen-activated protein kinase, p38a. Exposure to conditioned media from microRNA-145 overexpressing cells resulted in chronic p38a signalling in naïve cells, evidencing a paracrine induction and reinforcement of cell senescence. We conclude that targeting of microRNA-145 may provide a route to novel interventions to eliminate DNA-damaged and senescent cells in the vasculature and to this end further detailed studies are warranted.

Yes / (1) Abdominal aortic aneurysm (AAA) is a silent, progressive disease with significant mortality from rupture. Whilst screening programmes are now able to detect this pathology early in its development, no therapeutic intervention has yet been identified to halt or retard aortic expansion. The inability to obtain aortic tissue from humans at early stages has created a necessity for laboratory models, yet it is essential to create a timeline of events from EARLY to END stage AAA progression. (2) We used a previously validated ex vivo porcine bioreactor model pre-treated with protease enzyme to create "aneurysm" tissue. Mechanical properties, histological changes in the intact vessel wall, and phenotype/function of vascular smooth muscle cells (SMC) cultured from the same vessels were investigated. (3) The principal finding was significant hyperproliferation of SMC from EARLY stage vessels, but without obvious histological or SMC aberrancies. END stage tissue exhibited histological loss of α-smooth muscle actin and elastin; mechanical impairment; and, in SMC, multiple indications of senescence. (4) Aortic SMC may offer a therapeutic target for intervention, although detailed studies incorporating intervening time points between EARLY and END stage are required. Such investigations may reveal mechanisms of SMC dysfunction in AAA development and hence a therapeutic window during which SMC differentiation could be preserved or reinstated. / This research was funded in part by The Leeds Teaching Hospitals Charitable Foundation (R11/8002). E.R.C. was supported by a PhD studentship from the Engineering and Physical Sciences Research Council (EPSRC; EP/F500513/1). R.J.H. was the recipient of an Intercalated Batchelor of Science Degree in Science award from the Royal College of Surgeons of England. M.A.B.(FS/18/12/33270 and FS/12/54/29671), K.I.B. (FS/12/26/29395), and K.J.G. (FS/11/91/29090) were supported by BHF Clinical Research Training Fellowships.

Le cancer de l’ovaire (COv) est le cancer gynécologique le plus létal chez la femme et les traitements existants, chirurgie et chimiothérapie, ont peu évolué au cours des dernières décennies. Nous proposons que la compréhension des différents destins cellulaires tels que la sénescence que peuvent choisir les cellules du cancer de l’ovaire en réponse à la chimiothérapie pourrait conduire à de nouvelles opportunités thérapeutiques. La sénescence cellulaire a été largement associée à l’activité de la protéine TP53, qui est mutée dans plus de 90% des cas de cancer de l’ovaire séreux de haut grade (COv-SHG), la forme la plus commune de la maladie. Dans nos travaux, à partir d’échantillons dérivés de patientes, nous montrons que les cultures primaires du cancer de l’ovaire séreux de haut grade exposées au stress ou à des drogues utilisées en chimiothérapie entrent en senescence grâce à l’activité d’un isoforme du gène CDKN2A (p16INK4A). Dans ces cellules, nous avons évalué les caractéristiques fondamentales de la sénescence cellulaire tels que les altérations morphologiques, l’activité béta galactosidase associée à la sénescence, les dommages à l’ADN, l’arrêt du cycle cellulaire et le phénotype sécrétoire associé à la sénescence. En utilisant des micromatrices tissulaires construites à partir d’échantillons humains de COv-SHG pré- et post-chimiothérapie, accompagnées de leurs données cliniques, nous avons quantifié des marqueurs de sénescence incluant une diminution de la prolifération cellulaire quelques semaines après chimiothérapie. De façon intéressante, l’expression de p16INK4A dans les échantillons de COv-SHG prétraitement corrèle avec une survie prolongée des patientes suite au traitement. Ceci suggère ainsi pour la première fois un impact biologique bénéfique pour la présence de cellules cancéreuses qui sont capable d’activer la sénescence, particulièrement pour le traitement du cancer de l’ovaire. Dans le but de complémenter les thérapies actuelles avec des approches de manipulation pharmacologique de la sénescence, nos résultats suggèrent qu’il serait important de déterminer l’impact positif ou négatif de la sénescence induite par la thérapie sur la progression de la maladie et la survie, pour chaque type de cancer de façon indépendante. / Human ovarian cancer (OvCa) is the deadliest gynecologic malignancy and existing surgical/chemotherapeutic treatment options have been relatively static for decades. We propose that understanding OvCa cell fate decisions taken in response to chemotherapy could guide new therapeutic opportunities. Damage-induced cellular senescence is often associated with TP53 activity, which is heavily mutated in high grade serous (HGS) OvCa (>90%), the most common form of this disease. Here, using patient derived tissues, we show that primary HGS-OvCa cultures predominantly trigger CDKN2A- associated (p16INK4A isoform) senescence following exposure to stress or chemotherapy. Key senescence hallmarks including altered morphology, senescence-associated-Betagalactosidase, DNA damage, cell cycle arrest and the senescence-associated secretory phenotype were evaluated and detected in damaged cells. Using tissue microarrays built from pre- and post-treatment human HGS-OvC tissue samples with accompanying clinical data, we quantified post-treatment hallmarks of senescence including reduced cell proliferation weeks after chemotherapy. Importantly, p16INK4A expression in pretreatment HGS-OvC samples correlated with increased patients survival, suggesting for the first time that senescence-competence in human cancer cells may have a beneficial impact on treatment outcomes for patients. In order to guide the potential improvement of existing human therapies via pharmacological senescence manipulation, our results suggests that it is important to determine for many types of human cancer whether treatment-induced senescence positively or negatively impacts disease progression and patient survival.

The promyelocytic leukemia protein (PML) is a tumour suppressor. It has been reported that PML interaction with the p53 protein is involved in the activation of cell cycle checkpoints and, when persistent, may lead to the premature onset of cellular senescence. Cellular senescence is a state of permanent cell growth arrest that is associated with characteristic morphological and metabolic changes and persistent DNA damage signalling. Importantly, PML nuclear bodies coassociate with persistent DNA damage foci in senescent cells; however, the role of this interaction is still obscure. My goal was to characterize the role of PML in DNA damage response (DDR) and the induction of premature cellular senescence after genotoxic stress, namely X-radiation, using both siRNA-mediated PML knock down (PML KD) and complete PML knock out (PML KO) in human cells. The dynamics of DNA damage foci, levels of various proteins involved in DDR, and proliferation rate were measured in both PML KD and KO cells. No significant changes in the formation of DNA damage foci, activated DDR (p53 and Chk2), activated p21CIP1/WAF1 cyclin-dependent kinase inhibitor, senescent morphology, and SA-β-galactosidase activity in PML KO cells were observed. However, PML KO cells displayed higher levels of retinoblastoma protein (Rb) and...