Age-related variation in demographic rates has significant consequences for population and evolutionary dynamics, and understanding the processes driving such variation is therefore an important aspect of evolutionary ecology. Reproductive performance may vary over the lifetime of an individual, and this may be the result of both variations in reproductive effort and changes in individual competency. For example, increasing experience is likely to have beneficial effects on reproduction during early life, and senescence, or declines in physiological function, may have negative impacts on the performance of older individuals. The rate at which these changes occur can vary dramatically between species, and even between individuals of the same species. However, understanding the causes and consequences of this variation in the rate of ageing is not always straightforward. As well as the individual-level processes described, the phenotypic composition of successive age classes will contribute to age-related variation observed at the population level. Abrupt changes in performance, such as the poor performance of first time breeders, may be obscured if individuals vary in their age at first reproduction. Population-level patterns may also be influenced by selection; for example, the selective disappearance of low quality individuals from older age classes may mask senescent declines in the performance of longer-lived individuals. Moreover, the physiological mechanisms that underpin within-individual changes in performance are not well understood. Unravelling the drivers of such age-related variation requires longitudinal data, following individuals throughout their lives, which presents challenges for the study of natural populations. Albatrosses are among the longest lived vertebrates. In this thesis, I use data from three species of albatross breeding at Bird Island, South Georgia (54°00’S, 38°03’W) to explore age-related variation. Focusing primarily on the wandering albatross, Diomedea exulans, I characterise the relationship between age and various reproductive traits, and decompose the population-level patterns to reveal effects of experience, senescence and terminal effects across the reproductive lifespan of individuals. I then consider foraging behaviour as a proximate driver of changes in reproductive performance in this species. Using tracking data collected over a 20 year period, I find limited evidence for age-related variation in foraging trips taken throughout the breeding cycle. Going one step further, I explore telomere dynamics in the wandering albatross, examining the potential for telomere length to act as a physiological marker of individual state. Finally, I move on to a species comparison, incorporating data from the black-browed (Thalassarche melanophris) and grey-headed albatross (Thalassarche chrysostoma). I compare the population- and individual-level ageing patterns of these three closely related species, and consider these in light of their differing life history strategies.

Orientador: Carlos Alberto de Souza Costa / Co-orientador: Fernanda Gonçalves Basso / Banca: Gelson Luis Adabo / Banca: Carla Raquel Fontana Mendonça / Resumo:A idadedo indivíduo e a presença de inflamação podem influenciar o metabolismo celular, retardando o processo de reparo tecidual. Diferentes tratamentos, como a laserterapia de baixa potência (LBP) e a aplicação de fatores de crescimento são capazes promover bioestimulação celular através do aumento de sua proliferação e metabolismo. O objetivo deste estudo foi avaliar as atividades de fibroblastos obtidos da gengiva de indivíduos jovens e idosos (Estudo 1), a capacidade de resposta destas células expostas ao fator de necrose tumoral alfa (TNF-α) (Estudo 2), bem como o efeito da LBP ou do fator de crescimento epidérmico (EGF) sobre a viabilidade e metabolismo destas células em cultura (Estudo 3). Culturas primárias de fibroblastos de gengiva de 6 pacientes (3 jovens - J; e 3 idosos - I) foram obtidas e semeadas em meio de cultura completo (DMEM), de acordo com cada protocolo proposto. No Estudo 1, fibroblastos em DMEM completo foram cultivados em placas de 24 compartimentos pelos períodos de 24, 48 ou 72 horas e submetidos a análise da viabilidade celular (MTT, n=36), produção de proteína total (PT, n=36) e síntese de colágeno Sirius Red (SR, n=36). No Estudo 2, após 24 horas do cultivo celular em placas de 96 compartimentos, o meio de cultura completo foi substituído por DMEM sem soro fetal bovino (SFB) acrescido ou não de TNF-α (100 ng/mL) e mantido em contato com as células por 24 horas adicionais. Então, as células foram analisadas quanto a produção de espécies reativas de oxigênio (EROs, n=36), produção de óxido nítrico (NO, n=36) e síntese de quimiocina CCL5 (ELISA, n=36). No Estudo 3, fibroblastos cultivados em placas de 24 compartimentos foram tratados com EGF (100 μM) ou irradiados com laser de baixa potência (LASERTable, InGaAsP - 780 ± 3nm; 25mW). A viabilidade celular (Alamar Blue®, n=12), migração celular (Wound Healing, n=12)...(Resumo completo clicar acesso eletrônico) / Abstract: The age as well as the presence of inflammation can influence the cellular metabolism, slowing the tissue healing process. Different therapies, such as the application of growth factors and low level laser therapy (LLLT) are capable of biostimulating cells by increasing their proliferation and metabolism. The aim of this study was to evaluate: the functions of gingival fibroblasts obtained from young and elderly individuals (Study 1); the response of these cells exposed to tumor necrosis factor alpha - TNF-α (Study 2); and the effect of LLLT or epidermal growth factor - EGF on the viability and metabolism of these connective cell type (Study 3). Primary culture of gingival fibroblasts of 6 patients (3 young - Y and 3 elderly - E) were collected and cultured in complete culture medium (DMEM). In the Study 1 fibroblasts were seeded in 96-well plates in complete DMEM containing 10% of fetal bovine serum (FBS) for 24, 48 or 72 hours and subjected to evaluation of cell viability (MTT, n=36), total protein production (TP, n=36) and collagen synthesis (SR, n=36). In the Study 2, after 24-hour cell seeding, the complete DMEM was replaced by DMEM without FBS supplemented or not with TNF-α (100 ng/mL) which was kept in contact with cells for additional 24 hours. Then, the production of reactive oxygen TNF-α species (ROS, n=36), nitric oxide (NO, n=36) and synthesis of CCL5 chemokine (ELISA, n=36) were assessed. In the Study 3 fibroblasts were seeded in 24-well plates for 24 hours, and then exposed to EGF (100 μM) or submitted to low power laser irradiation (LASERTable device, InGaAsP - 780 ± 3nm; 25mW). Cell viability (Alamar Blue®, n=12), cell migration (Wound Healing, n=12), collagen synthesis (SR, n=12), and synthesis of vascular endothelial growth factor - VEGF (ELISA, n=12) were assessed 72 hours after the treatments. The data were statistical...(Complete abstract electronic access below) / Mestre

Les cellules souche cancéreuses contribuent au développement des sarcomes, mais le manque de marqueurs spécifiques empêche leur caractérisation et la possibilité de cibler ce type de cellules. Nous avons utilisé la séquence régulatrice de la calpaïne-6 dans des systèmes rapporteurs pour identifier les cellules exprimant la calpaïne-6. Ces cellules étaient des cellules initiatrices de tumeurs et se comportaient comme des cellules souche, au sommet de la hiérarchie cellulaire. L'expression de la calpaïne-6 dépend d’un programme génique de cellules souche qui implique Oct4, Nanog et Sox2 et est activée par l'hypoxie. L’inhibition de la calpaïne-6 a bloqué le développement tumoral et a induit la diminution du nombre de cellules souche cancéreuses dans les sarcomes osseux. L’expression de la calpaïne-6 était inversement corrélée à l'expression de marqueurs de sénescence mais était associé à un flux autophagique dynamique. L’inhibition de la calpaïne-6 a induit l'entrée des cellules en sénescence et a supprimé le flux autophagique. Nos résultats révèlent que le calpaïne-6 identifie les cellules souche des sarcomes et joue un rôle important dans le maintien des cellules souche cancéreuses en contrôlant les processus d’autophagie et de sénescence. La calpaïne-6 semble être une cible thérapeutique prometteuse pour éradiquer les cellules souche dans les sarcomes / Cancer stem cells contribute to sarcoma development, but lack of specific markers prevents their characterization and the possibility of targeting. We used the regulatory sequence of calpain-6 in reporter constructions to identify calpain-6–expressing cells. These cells were tumor-initiating cells and behaved like stem cells at the apex of the cellular hierarchy. Calpain-6 expression depended on the stem-cell transcription network that involves Oct4, Nanog, and Sox2 and was activated by hypoxia. Calpain-6 knockdown blocked tumor development and induced depletion of sarcoma stem cells. Calpain-6 was inversely associated with expression of senescence markers but was associated with a dynamic autophagy flux. Calpain-6 knockdown induced cell entry into senescence and suppressed autophagy flux. Our results reveal that calpain-6 identifies sarcoma stem-cell and plays an important role as a regulator of cancer cell fate driving a switch between autophagy and senescence. Calpain-6 may be a promising therapeutic target to eradicate sarcoma stem cells.

Les extrémités des chromosomes portent des structures nommées télomères, nécessaires à la survie des cellules. La sénescence réplicative induite par l’altération des télomères bloque les proliférations cellulaires excessives. Cependant, certaines cellules échappent à ce contrôle et deviennent immortelles. Je me suis intéressée aux mécanismes permettant de reconstituer des télomères fonctionnels par recombinaison chez la levure. Le noyau est divisé en sous-compartiments plus ou moins permissifs pour la recombinaison. J’ai étudié comment la localisation des télomères dysfonctionnels aux pores nucléaires (NPC) conditionne leur réparation et la prolifération des cellules. J’ai montré que les lésions réplicatives au télomères sont relocalisées aux NPC ce qui favorise leur réparation par un mécanisme conservatif. J’ai aussi montré la présence aux NPC de cercles d’ADN télomérique qui pourraient servir de matrice pour allonger les télomères et sortir de la sénescence réplicative. / Chromosome ends are formed by structures called telomeres, which are necessary for genome integrity and cell survival. Upon aberrant proliferation, as in precancerous cells, telomere alterations blocks cell proliferation, a mechanism called replicative senescence. However, some cells escape this control and become immortals. Using budding yeast as model, my work aimed to understand the mechanisms that allow cells to reconstitute functional telomeres using homologous recombination. The nucleus is divided in sub-compartments that are differently permissive for recombination. I investigated how the localization of dysfunctional telomeres to the nuclear pore complex (NPC) determines their repair and favors cell proliferation. I showed that replicative lesions at telomeres relocate to the NPC allowing conservative repair by recombination. I further shed light on the presence at NPC of telomeric DNA circles that may serve as template for telomere elongation and thus escape from senescence.

La sénescence cellulaire est un mécanisme de suppression cellulaire caractérisé par un arrêt irréversible du cycle cellulaire. Les cellules sénescentes ont également un secrétome spécifique qui influe sur les cellules voisines, pouvant induire leur entrée prématurée en sénescence, l'apoptose, la prolifération cellulaire et le développement tumoral. Dans ce contexte, l'objectif de ma thèse était d'identifier une nouvelle protéine sécrétée par les fibroblastes sénescents et de caractériser ses fonctions dans les cellules sénescentes et dans les cellules tumorales. Nous avons mis en évidence la protéine WNT16B. Elle est surexprimée dans plusieurs modèles de sénescence cellulaire obtenus à partir de fibroblastes pulmonaires. Dans un modèle de souris transgénique, WNT16B est également associée avec l'accumulation de cellules sénescentes dans les lésions précancéreuses pulmonaires. WNT16B est impliqué dans le mécanisme de sénescence cellulaire. En effet, l'inhibition de WNT16B prévient l'entrée en sénescence, en empêchant l'activation de p53 et l'expression de p21/WAF1, et donc l'arrêt du cycle cellulaire.Nous avons finalement associé l'expression de WNT16B avec la présence de cellules sénescentes dans des tumeurs prélevées chez des patients atteints de carcinomes pulmonaires non à petites cellules. Cette expression est corrélée avec une meilleure survie pour les patients ayant reçu un traitement chimiothérapeutique. Ce dernier résultat fait donc le lien entre traitement, sénescence cellulaire et survie, et illustre le rôle potentiel des marqueurs des cellules sénescentes pour le suivi des patients. Globalement, ces travaux ont donc non seulement permis d'identifier WNT16B comme nouveau marqueur des cellules sénescentes, mais ils ont également ouvert des perspectives d'utilisation de ce marqueur pour l'identification, le traitement et le suivi des patients atteints d'un cancer.

TTAGGG repeat factor 2 (TRF2) is a protein that plays an important role in capping telomere ends from DNA damage responses. Telomere DNA consists of double strand repeats of the TTAGGG sequence ending with a 3'single-stranded overhang of the guanine strand (the G-strand overhang). TRF2 protects telomeres from being recognized as double-stranded breaks. It is thought that this protection is performed through the formation of T-loop structures and recruitment of proteins into a complex called shelterin. The exact mechanism of T-loop formation is unknown. I show with in vitro biochemical studies that TRF2 specifically interacts with telomeric ss/ds DNA junctions and binding is sensitive to the sequence of the G-strand overhang and double-stranded DNA sequence at the junction. Binding assays with TRF2 truncation mutants suggest that TRF2 interacts with both the double-stranded DNA through the C-terminal DNA binding domain and the G-strand overhang through the N-terminus. Mobility shifts and atomic force microscopy with truncation mutants bound to telomeric DNA also show that a previously uncharacterized "linker" region within TRF2 is involved in DNA-specific TRF2 oligomerization. From these observations, I suggest that TRF2 forms protective loops by oligomerizing through both a previously characterized dimerization domain and the linker region. I propose that loop formation involving the telomere ends is accomplished through direct interactions between TRF2 and the G-strand overhang. In addition to DNA protection, a new role has emerged for TRF2 in sensing DNA damage. TRF2 can be phosphorylated within its dimerization domain by ATM and recruited to DNA damage foci in cells. The inhibition of TRF2 function alone has been shown to induce senescence and apoptosis in vascular endothelial cells. Since the common stimuli for a senescence phenotype is activation of a DNA damage response, I studied the relationship between DNA damage and TRF2 phosphorylation. Ex-vivo characterization of DNA damage-induced changes in vascular smooth muscle cells (VSMC) was undertaken. VSMC treated with H202 induced an increase in reactive oxygen species (ROS), and 8-oxo-guanine accumulation resulting in cell cycle arrest, chromatin condensation and a senescent phenotype. Interestingly phosphorylated TRF2 and ATM were also up regulated. Balloon injury was used to test the connection between phosphorylated TRF2 and senescence during vascular remodeling in rat arteries. Vascular remodeling as judged by neointima formation was associated with accumulation of 8-oxo-guanine, DNA damage signaling, including phosphorylated TRF2, an increase in cell cycle inhibitors and senescence. These events were exaggerated in aged animals and are consistent with a role in telomere dysfunction, and age related diseases.

The transplantation of primary Schwann cells (SC) has been shown to improve nerve regeneration. However, to monitor the survival of transplanted cells within the host, a stable labelling method is required. The in vitro characteristics of green fluorescent protein labelled SC (GFP SC) and their effects in an in vivo peripheral nerve injury model were investigated.   The GFP-SC were readily visualised ex vivo and stimulated significantly better axonal regeneration compared to controls. Clinical use of autologous SC for the treatment of nerve injuries is of limited use due to difficulty in obtaining clinically useful numbers. However, bone marrow mesenchymal stem cells (MSC) can trans-differentiate into SC like cells (dMSC). The in vitro and in vivo differentiation of MSC was explored, and the study extended to include the easily-accessible adipose stem cells (ASC).  In vitro, glial growth factor stimulated MSC express S100, a SC marker, and its expression is maintained following in vivo transplantation.  Similarly, untreated MSC transplanted in vivo also expressed S100, which indicates glial differentiation in response to local cytokines and growth factors. Using an in vitro model, comprising dMSC or dASC co-cultured with adult dorsal root ganglia (DRG) neurons, the capacity of the dMSC and SC like differentiated ASC (dASC) to promote axon myelination was verified: both cell types expressed transcripts for protein zero, peripheral myelin protein-22 and myelin basic protein. The potential of stem cells in nerve repair may be limited by innate cellular senescence or donor age affecting cell functionality thus it was essential to determine the effects of donor age on morphology and functionality of stem cells.  The proliferation rates, expression of senescence markers (p38 and p53) and the stimulation of neurite outgrowth from DRG neurons by stem cells isolated from neonatal, young or old rats were very similar. However, the distribution and ultrastructure of mitochondria in dMSC and dASC from young and old rats were quite different, and seem to indicate physiological senescence of the aged cells.  Given the wide-ranging influence of Notch signalling in cell differentiation, including the neural crest to a glial cell type switch, and self-renewal in mammals, its role in the differentiation of stem cells to SC was investigated. The mRNA for notch-1 and -2 receptors were expressed in the dASC, blockage of notch signaling did not affect the neurotrophic and myelination potential of dASC.  In conclusion, these findings show that GFP labelling has no deleterious effect on SC survival and function. MSC and ASC differentiated into glial-type cells acquire SC morphology, and express characteristic SC markers, and the differentiation process was independent of the Notch signaling pathway. Also, following transplantation into a nerve gap injury dMSC improve regeneration. This study established that following co-culture with DRG neurons, dMSC and dASC were able to express peripheral myelin proteins.  Also, the functional bioactivity of these cells is independent of the donor animal age. Finally, although the glial lineage differentiated aged cells characterized in this study expressed markers typical of senescence they retained the potential to support axon regeneration.

Early events in BRCA1-associated tumorigenesis remain poorly understood. To understand the immediate consequences of BRCA1 loss of function, we modeled BRCA1 loss of function in vitro using normal primary human mammary epithelial cells (HMEC). We have found that in HMEC, loss of BRCA1 results in a novel type of senescence. Loss of BRCA1-induced senescence is not associated with DNA damage or p53 upregulation. We find that p53 protein levels are down regulated due to proteasome-mediated degradation. Although p53 levels are down regulated, we find that BRCA1 loss induced expression of a number of p53-dependent anti-oxidant genes. In particular we uncovered that SESN2, a p53 downstream target gene, inhibits loss of BRCA1 induced ROS and activates autophagy. In contrast to human fibroblasts, we found that loss of BRCA1 induced senescence is p53 independent, and can occur in the absence of ROS upregulation and autophagy induction.

Early events in BRCA1-associated tumorigenesis remain poorly understood. To understand the immediate consequences of BRCA1 loss of function, we modeled BRCA1 loss of function in vitro using normal primary human mammary epithelial cells (HMEC). We have found that in HMEC, loss of BRCA1 results in a novel type of senescence. Loss of BRCA1-induced senescence is not associated with DNA damage or p53 upregulation. We find that p53 protein levels are down regulated due to proteasome-mediated degradation. Although p53 levels are down regulated, we find that BRCA1 loss induced expression of a number of p53-dependent anti-oxidant genes. In particular we uncovered that SESN2, a p53 downstream target gene, inhibits loss of BRCA1 induced ROS and activates autophagy. In contrast to human fibroblasts, we found that loss of BRCA1 induced senescence is p53 independent, and can occur in the absence of ROS upregulation and autophagy induction.

<p>Abstract</p><p>The trefoil factor family of secreted proteins contains three members; trefoil factor 1 or TFF1, trefoil factor 2 or TFF2, and trefoil factor 3 or TFF3. These three proteins share a conserved 42-43 amino acid domain containing 6 cysteine residues resulting in three disulfide bonds that holds the protein in a characteristic three-loop or "trefoil structure" known as the P domain. TFF1 is primarily localized to the stomach and secreted by the gastric mucosa while TFF2 and TFF3 are primarily localized to the colon and duodenum and secreted by the goblet cells. All three of these proteins play a protective role in the gastrointestinal tract where they are normally localized and have been identified as possible tumor suppressors, however, these proteins are also upregulated in cancer within tissues where they are not normally expressed including the breast, pancreas, prostate, and liver. The mechanisms by which two of these factors, TFF1 and TFF3, promote tumorigenesis remain largely undefined. In this dissertation we will attempt to elucidate these mechanisms as well as the regulation of these two proteins in both pancreatic and prostate cancer. Many of the underlying genetic and molecular mechanisms involved in the development of both pancreatic and prostate cancer remain largely unknown and as a result, therapeutic and diagnostic tools for treating these diseases are not as effective as they could be. By deciphering the role of TFF1 and TFF3 in these cancers, they could potentially serve as new therapeutic targets or biomarkers for treating both diseases.</p><p>Chapter 2 of this dissertation will examine the functional role of TFF1 promoting tumorigenesis in pancreatic and prostate cancer. We will show that TFF1 expression is critical for the viability of both pancreatic and prostate cancer cells and that reduction of TFF1 expression in these cells results in decreased tumorigenicity when implanted in immunocompromised mice. It will also be demonstrated that TFF1's function in promoting tumorigenicity is its ability to assist tumor cells overcome the tumor suppressive barrier of senescence. Thirdly, we show that the form of senescence that TFF1 assists in allowing the cells overcome is oncogene-induced senescence (OIS). Lastly, a cell cycle array identifies the potential downstream target p21CIP, a cyclin-dependent kinase inhibitor and OIS marker, whose expression is induced by loss of TFF1 expression.</p><p>In Chapter 3 of this work, we examine the role of another trefoil factor family member, TFF3, and its role in promoting prostate tumorigenesis. Just as with TFF1, it appears that TFF3 3 expression is critical for prostate cancer cell viability and tumorigenicity using the same experimental techniques used in Chapter 2. Using a genetically defined model of prostate cancer, a PI3-kinase-dependent regulatory mechanism of TFF3 emerges in this prostate cancer context. Using this system we begin to see a divergence in both regulation and function of TFF1 and TFF3 in prostate cancer. Finally, a mouse model expressing TFF3 was developed to monitor the histopthological changes associated with expression of this protein. Initial characterization of this model suggests a hyperplastic phenotype coinciding with TFF3 expression in the prostate.</p><p>The two studies in this dissertation establish a role of TFF1 and TFF3 in both prostate and pancreatic tumorigenesis and demonstrate that ablation of expression of both proteins is a potent inhibitor of tumorigenesis. With this knowledge, it is possible that TFF1 and TFF3 may become a potential therapeutic target or diagnostic marker for better treatment of prostate and pancreatic cancer.</p> / Dissertation