As teorias existentes para o estudo do fenômeno de envelhecimento biológico são divididas basicamente em duas categorias: teorias bioquímicas e teorias evolucionárias. As teorias bioquímicas associam o envelhecimento a danos que podem ocorrer nas células, tecidos, órgãos e às imperfeições dos mecanismos bioquímicos responsáveis pela manutenção da vida. As teorias evolucionárias, por sua vez, explicam o envelhecimento sem recorrerem a mecanismos bioquímicos específicos. Elas são de natureza hipotético-dedutiva associando o envelhecimento ao resultado de uma história de vida, ajustada pelo processo de seleção natural que garante a perpetuação de uma espécie. Por apresentar estas características, as teorias evolucionárias são mais adequadas à utilização dos métodos da Física. Todo nosso trabalho será desenvolvido à luz destas teorias. Na primeira parte deste trabalho fazemos uma rápida discussão acerca das dificuldades em se determinar com rigor, propriedades biológicas que possam ser usadas com eficiência no processo de quantificação do envelhecimento. Mostramos que uma das formas mais eficientes para a detecção do envelhecimento é por meio da análise das taxas de mortalidade, realizadas com a ajuda de tabelas atuarias. Estas tabelas apontam para a existência de uma lei de mortalidade, responsável por padrões específicos de mortalidade, em população nas quais se observa o envelhecimento. Expomos as hipóteses centrais sobre as quais se baseiam tanto as teorias bioquímicas quanto as teorias evolucionárias e, ainda, os mecanismos de envelhecimento utilizados por estas duas teorias. Propomos um modelo para populações estruturadas por idade contendo os principais ingredientes das teorias evolucionárias de envelhecimento a saber, mutações benéficas e deletérias, hereditariedade, taxas reprodutivas e seleção natural. Encontramos uma solução exata para este modelo e mostramos que o mesmo não apresenta envelhecimento. Calculamos as probabilidades de sobrevivência médias e o expoente de crescimento Malthusiano cujos resultados indicam que o modelo pode exibir extinção populacional. Acreditamos que este modelo possa ser aplicado no estudo de população de protozoários e celenterados. Por meio de um formalismo matricial, encontramos uma solução exata para a evolução temporal do modelo de Partridge e Barton na presença do vínculo pleiotrópico, mutações somáticas e fecundidades arbritárias. São determinados valores de estado estacionário para as probabilidades de sobrevivência médias e para o expoente de crescimento Malthusiano. A idade média da população também é calculada e exibe um decaimento tipo lei de potências. Por último estudamos o modelo de envelhecimento proposto por Heumann e Hötzel. Por meio de pequenas modificações neste modelo, mostramos, que ao contrário do que se acreditava, ele é capaz de sustentar populações com mais de três idades. Além disso, nossas simulações mostram que este modelo apresenta uma grande quantidade de resultados interessantes, tais como, senescência catastrófica, lei de mortalidade de Grompertz e a influência que diferentes estratégias reprodutivas têm sobre a longevidade da população. / There are two kinds of aging theories: biochemical and evolutionary. Biochemical theories invoke damage to cells, tissues, and organs and connect senescence with imperfections of the biochemical processes responsible for the maintenance of life. The evolutionary theories, on the other hand, explain senescence without any especific biochemical mechanisms. Aging evolutionary theories are hypothetico-dedutive and assume that senescence is a consequence of na optimal life history, controlled by natural selection, which guarantees perpetuation of the species. Such characteristics make the evolutionary theories more suited for the application of Physics methods. In our work, we will consider only this kind of theory. In the first part of this thesis, we present a brief discussion on the difficulties to obtain rigorously biological properties which can be efficiently used in the quantificaion of the aging process. One way to measure senescence is through an analysis of the so called table of life. These tables indicate the existence of a mortality Law which is responsible for a specific mortality pattern. We explain the main ideas on which the biochemical and evolutionary theories are based. We propose a simple age-structured population model containing all the relevant features of the evolutionary aging theories: beneficial and deleterious mutations, reproductive rates, and natural selection. An exact solution for this model is found and, to our surprise, it does not exhibit senescence. Average survival probabilities and Malthusian growth exponents are calculated and they indicate that the system may have a mutational meltdown. We believe that this model is a good candidate to appropriately describe some coelenterate and prokaryote groups. In the presence of the pleiotropic constraint and deleterious somatic mutations, the time evolution of the Partridge-Barton model is exactly solved for na arbitrary fecundity using a matricial formalism. The steady state values for the mean survival probabilities and the Malthusian growth exponent are obtained. The mean age of the population shows a Power Law decay. Finally, we study the aging model proposed by Heumann and Hötzel. By introducing a minor change in this model, we show that it is able to keep many age intervals in disagreement with previous ideas. Moreover, our numerical simulations show a plethora of new interesting features, namely catastrophic senescence, the Gompertz Law and the effects that different reproductive strategies may have on life expectancy.

Ribosome biogenesis is an essential biological process that is required for cell division and growth. Cancer cells alter their physiology in order to meet their excessive growth demands and therefore maintain abnormal metabolism and homeostasis. Under normal conditions, ribosome biogenesis is tightly regulated to maintain adequate ribosomal content of the cell. However, several oncogenes promote this process and elevated ribosome biogenesis is often found in cancer cells, where it can support the high biosynthetic demand of these cells. Hence, ribosome biogenesis is a process that might provide candidate targets for therapeutic intervention. The main aim of this research was to assess whether inhibition of late stage biogenesis of the 60S ribosomal subunit would result in tumour suppressive responses in normal and cancer cells. We focused upon two GTPases, EFL1 and LSG1, that catalyse the last two cytoplasmic reactions in the maturation of the 60S subunit. We observed that RNAi-based silencing of the GTPases in human lung fibroblasts triggered growth arrest and senescence, which was mediated by the p16 and p53 pathways. Inhibition of these pathways revealed that loss of p53 could bypass the senescence response. However, when cells were plated at low density, knockdown of LSG1 conferred a tumour suppressive response, even in the absence of p53. Knockdown of LSG1 in MCF-10A mammary epithelial cells that lack the p16 locus also induced a robust senescence response and this was also observed in transformed derivatives of MCF-10A cells. Preliminary data obtained in a 3D mammosphere culture model also revealed that inhibition of 60S maturation could elicit an antiproliferative response. Taken together, these data indicate that at least some cancer cells would be responsive to a therapy based upon inhibition of 60S subunit biogenesis. We further characterised the senescence response that was obtained through knockdown of LSG1 by performing gene expression analysis. This revealed a minimal Senescence-Associated Secretory Phenotype (SASP) that was restricted to members of the TGF-β family and lacked the canonical pro-inflammatory cytokines and chemokines that are found in the SASP of cells undergoing oncogene-induced senescence (OIS). Surprisingly, we also observed a dramatic increase in expression of multiple genes in the cholesterol biosynthesis pathway, although inhibition of this pathway indicated that cholesterol biosynthesis was not required for the senescence response. Further insight into the mechanisms of induction of the ribosomal stress senescence response was sought through pilot CRISPR screen and reverse phase protein array (RPPA) analyses. These revealed some interesting leads that will direct future studies.

Telomere attrition is a natural process that occurs due to inadequate telomere maintenance. Once at a critically short threshold, telomeres signal the cell to cease division and enter a cell fate termed senescence. Telomeres can be elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Mutations in the telomerase complex or telomere-related genes give rise to the premature aging disorder Dyskeratosis Congenita (DC). DC provides a unique model system to study human aging in relation to telomerase insufficiency and the subsequent accelerated telomere attrition. In this thesis, skin fibroblasts as well as keratinocytes and T-cells were analyzed from patients with Autosomal Dominant Dyskeratosis Congenita (AD DC) caused by a single allele mutation in the telomerase RNA component (TERC) that leads to telomerase haploinsufficiency. These cells were determined to have a severe proliferative defect and extremely short telomeres. It is demonstrated that the short telomeres in AD DC cells initiate a DNA damage response transduced by the p53/p21WAF/CIP pathway which mediate an elevation in steady-state levels of mitochondrially-derived superoxide and oxidative stress. Exogenous expression of the catalytic reverse transcriptase component of telomerase (TERT) activated telomerase in DC fibroblasts but resulted in reduced activity (~50% compared to control fibroblasts); however telomeres were successfully maintained, albeit at a short length. Simultaneous expression of both TERT and TERC led to robust telomerase activity and elongation of telomeres, indicating that TERC haploinsufficiency is a rate-limiting step in telomere maintenance in DC cells. Reconstitution of telomerase activity in AD DC cells ameliorated the proliferative defects, reduced the p53/p21WAF/CIP response and decreased oxidative stress. Increased superoxide and slow proliferation found in DC cells could also be mitigated by inhibiting p21WAF/CIP or by decreasing the oxygen tension to which the cells are exposed. Together, these results support the hypothesis that the insufficient telomerase leads to critically short telomeres which signal the activation of p21WAF/CIP, leading to increased steady-state levels of mitochondrial superoxide and metabolic oxidative stress as a means to engage senescence. These studies provide insight into mechanisms whereby shortened telomeres lead to premature aging in a humans and point to potential strategies to reduce the effects of tissue dysfunction in DC patients.

Due to the duration of human ageing, researchers must rely on models such as animals and cells. Replicative senescence and stress-induced premature senescence (SIPS) are two cellular models sharing many features. Although telomeres play a major role in replicative senescence, their involvement in SIPS is unclear. In this work, we first wanted to investigate how accurate models of ageing are. We published a new model of the evolution of human ageing, which offers a refined view of the evolution of ageing in humans and suggests that human models should be favoured. Though studying other mammals, reptiles, and birds may also be useful, we conclude that lower life forms such as yeast and invertebrates are not representative of the human ageing process. Secondly, we wanted to elucidate the importance of telomeres in SIPS and study gene expression and regulatory networks. Using a telomerase-immortalized cell line, we found no evidence that damage specific to the telomeres is at the origin of SIPS. In our published model, neither the TGF-â1 pathway nor telomeres appear to play a crucial role in SIPS. We suggest that widespread damage to the DNA causes SIPS and propose a rearrangement of gene expression networks as a result of stress. Moreover, we advise caution in using telomerase in anti-ageing therapies since telomerase expression may alter the normal cellular functions and promote tumorogenesis. Lastly, we published strategies to integrate the modern computational approaches to research ageing. Although we find it unlikely that a full understanding of ageing may be achieved within a near future, we argue that understanding the structure and finding key regulatory genes of the human ageing process is possible.

Sperm senescence, a decline in sperm quality caused by male ageing and by sperm ageing before or after copulation, may have fitness costs manifested as infertility or lowered genetic quality of offspring. This thesis tested the distinct evolutionary roles of sperm senescence using a laboratory-adapted population of Drosophila melanogaster. We developed a practical approach to avoid confounding male age with sperm age by standardizing pre-copulatory sperm age and mating history in young and old male age groups. Applying this approach, we documented sperm senescence in D. melanogaster and discussed its potential evolutionary importance. First, ageing males declined in fitness as evidenced by the reduction in fertilization potential of their ejaculates but not by decreased offspring fitness (the ability that a fly can survive to adulthood, successfully mate and produce viable offspring). This suggests a decline in the quality or quantity of seminal fluid or spermatozoa, with no decline in the genetic quality of sperm that actually fertilized ova. Second, post-copulatory sperm senescence has significant negative impacts on offspring fitness, indicating degraded genetic integrity of the spermatozoa stored in females. In both cases, male ageing and sperm ageing had similar fitness impact on male and female offspring, different from what has been suggested by previous work. In addition, We demonstrated that female fecundity, fertility, and length of the fertile period after a single mating were positively associated with the concentration of yeast in their food, and were negatively associated with the duration of yeast restriction in their diet, which suggested that sperm storage is affected by the nutritional status of the females. By revealing the significance of sperm senescence on male and female fertilization success and the fitness of the next generation, this thesis sheds light on a number of evolutionary and applied issues, and provokes new questions for future research on sperm senescence. / Thesis (Ph.D, Biology) -- Queen's University, 2014-03-07 10:38:12.879

Hepatitis B virus (HBV) is a significant global threat to human health due to its ability to cause chronic infections that can lead to hepatocellular carcinoma (HCC). While the process by which HBV increases the risk of HCC is unclear, evidence suggests that the hepatitis B X protein (HBx) may be a contributing factor. Cellular senescence is an important barrier to tumorigenesis, blocking the proliferation of cells that harbor excessive DNA damage or contain activated oncogenes. Autophagy is a non-proteasomal degradative pathway used by cells to recycle cytoplasmic contents under periods of nutrient starvation. This pathway is induced in response to a wide range of cellular stress factors, and has also been characterized as an effector mechanism for the establishment of cellular senescence. In this study, retroviral transduction of HepG2 cells with HBx resulted in the induction of cellular senescence and autophagy. The mechanism by which HBx can induce senescence is unclear. However, an increase in the accumulation of DNA damage was observed. HBx did not modulate the levels of the anti-apoptotic proteins Bcl-2, Bcl-xL, or Mcl-1, which can inhibit autophagy through interactions with the autophagy regulator Beclin 1. As well, the activity and phosphorylation status of JNK/SAPK, an inducer of autophagy via Bcl-2 phosphorylation, was unchanged. These results suggest that senescence may act as a barrier to HBx-induced oncogenesis, and may offer some explanation as to why HBx does not function as a more potent oncogene. Also, we propose that HBx modulates autophagy through a mechanism other than Bcl-2 phosphorylation or expression over the time course of this study.

As animals age, they are expected to invest successively more energy in reproduction as they have fewer subsequent chances to reproduce (the “restraint” hypothesis). Conversely, the oldest animals may show restraint in reproduction because even a small increase in energy expended during reproduction may lead to death. Alternatively, both young and very old animals may lack the ability to maintain high levels of energy expenditure (the “constraint” hypothesis), leading to reduced reproductive success. Many studies have observed an increase in reproductive success with age followed by a reduction at the end of life, but fewer studies have examined the proximate mechanisms, which provide a context for understanding ultimate causes. I examined over 30 behavioural and physiological metrics of aging in two species of free-living long-lived seabirds (thick-billed murres Uria lomvia and black-legged kittiwakes Rissa tridactyla) and a short-lived passerine (tree swallows Tachycineta bicolor). For all species, reproductive success was high at intermediate ages. In support of the “restraint” hypothesis, when birds were stressed glucocorticoid hormones, which direct energy away from reproduction and towards survival, were higher in young birds (swallows) and both young and very old birds (kittiwakes and murres). When birds were handicapped older birds expended more energy. When challenged exogenously, there was no change in hormone levels with age, implying that they were “choosing” to be restrained. Resting metabolic rate (RMR) declined linearly with age in both seabird species. T3, which I show is indicative of RMR in birds, also declined with age, demonstrating that the reduction in metabolism was strategic and not due to changing body composition. In contrast, daily energy expenditure in both seabird species during breeding was constant with age while antioxidant capacity became elevated during middle age, and further increased with age. Several measures of behavioural performance did not vary with age. I conclude that hormonal cues lead to greater investment in adult’s energy stores over its offspring’s energy reserves (restraint hypothesis) at the start of life. At the end of life, both hypotheses were supported; energy expenditure was constrained by senescence, leading to increased restraint in investing additionally in offspring.

Genetic improvement of a number of crops including soybean (Glycine max L. [Merr]) has been associated with stay-green. Research on stay green genes has focused primarily on genes involved with photosynthesis and chlorophyll degradation. The current study explores the impact of a group of developmental genes, known as the E gene series, on the rate of soybean leaf senescence. The objective of this experiment was to determine the role of E-genes in the control of leaf senescence in soybean. The experiment was conducted in a split-plot design with three replications. The main plots were two photoperiods imposed following R1; i) natural day length (Amb) and ii) incandescent day length extension of 3 hours (Amb+3). The split plots were five E-gene near-isogenic lines (NILs), planted on different dates to obtain synchronous flowering. Phenology, photosynthesis, normalized difference vegetative index (NDVI) and fluorescence measurements were taken including, dark adapted photosynthetic efficiency (Fv/Fm), electron transport rate (ETR), and leaf chlorophyll concentration (SPAD). Leaf tissues were also analyzed for gene expression patterns among Harosoy isolines. Yield parameters like dry matter accumulation, harvest index and grain yields were recorded. The leaf net photosynthesis was more closely related to ETR than to SPAD values, suggesting that visual observation of stay-green may not be as effective in evaluating functional senescence as measurement of ETR. Cultivars with the dominant E1 allele maintained functional photosynthesis for longer, such that full senescence was delayed by 10-15 days in these cultivars. This phenomenon was observed under both photoperiod treatments and irrespective of the genetic background (Clark and Harosoy) in which the alleles appeared. Maintenance of functional photosynthesis by the E1 dominant allele can be attributed to maintenance of high ETR, and Fv/Fm, as well as delayed decline in leaf chlorophyll concentrations. Expression of senescence related genes were delayed in the isoline which had delayed leaf senescence phenotype. Consistent with the effect on leaf senescence, the dominant alleles also reduced the rate of phenological development, such that R5 occurred later in genotypes with dominant alleles and under the Amb+3 treatment. Cultivars with the dominant E1 allele under extended photoperiod treatment accumulated more biomass and had decreased apparent harvest index which caused no change in grain yields. The dominant E allele may delay leaf senescence directly or indirectly, through its delay of reproductive development.

Leaf senescence is the final stage of leaf development, during which the leaf relocates most of itsvaluable nutrients to developing or storing parts of the plant. As this process progresses, leaves losetheir green color and their capacity to perform photosynthesis. Shade and darkness are well-knownas factors inducing leaf senescence and it has been proposed that senescence can be initiated byreductions in photosynthesis, photomorphogenesis and transpiration. However, despite the fact thatthe signaling mechanisms regulating each of these processes have been extensively described,particularly in seedlings, their contribution to the initiation of senescence in mature leaves stillremains unclear. Furthermore, the use of different experimental systems to study shade-inducedleaf senescence has yielded several divergent results, which altogether complicate the overallunderstanding of leaf senescence. To address this, darkened plants and individually darkened leaves, which show different rates of leafsenescence, were studied. Comparing the transcriptome and metabolome of these two darktreatmentsrevealed that they differed distinctly with regard to their metabolic strategies. Wholedarkened plants were severely carbohydrate-starved, accumulated amino acids and slowed downtheir metabolism. In contrast, individually darkened leaves showed continued active metabolismcoupled to senescence-associated degradation and relocation of amino acids. This knowledge was used to set up a new system to study how shade affects leaf senescence in themodel plant Arabidopsis thaliana. Use of this system revealed that different senescence-associatedhallmarks appeared in response to different intensities of shade. Some of these hallmarks werefurther shown to be part of both leaf senescence and photosynthetic acclimation to low light. Finally, using this system on phytochrome mutants revealed that loss of phytochrome A increasedthe loss of chlorophyll under shade, without increasing the expression of senescence-associatedgenes. Together, these findings suggest that shade-induced leaf senescence, which is generally perceived asa single process, is actually an intricate network of different processes that work together tomaintain an optimal distribution of nutrients within the plant.

No description available.