Les thérapeutiques antirétrovirales ont permis d’augmenter la survie des personnes vivant avec le VIH (PVVIH). Cependant, de nombreuses comorbidités non liées au VIH émergent et sont une préoccupation majeure dans la prise en charge des patients. L’activation, l’inflammation et l’immunosénescence pourraient jouer un rôle majeur dans ce processus. De nombreux marqueurs existent pour mesurer ces dysfonctionnements et ils ont souvent été considérés sans prendre en compte leurpossible interdépendance. Les objectifs de cette thèse était i) de proposer une combinaison de ces marqueurs, ii) d’évaluer l’association entre la combinaison de ces marqueurs et la présence des comorbidités, et iii) d’évaluer l’association entre la combinaison de ces marqueurs, et le risque de survenue des comorbidités et de la mortalité chez des PVVIH inclus dans la sous étude CIADIS de la cohorte ANRS CO3 Aquitaine. Nous avons identifié deux scores : le score CIADIS cellulaire et soluble. Le score cellulaire était plutôt associé à la multimorbidité et à la survenue d’une nouvelle comorbidité quelle qu’elle soit. Le profil des dysfonctionnements immunitaires sous-jacent était différent lorsque l’on s’intéressait aux comorbidités séparément. Ces résultats soutiennent l’hypothèse que différents profils d’activation, d’inflammation et de sénescence sous-jacents pourraient être impliqués dans le développement de différentes comorbidités. Nos résultats montrent que des analyses intégrant de nouveaux biomarqueurs pourraient accroître la compréhension des comorbidités. Nous allons continuer de travailler sur l’identification des profils de dysfonctionnements immunitaires pour des comorbidités spécifiques. / Antiretroviral therapies have improved the survival of HIV-infected people. However, many non-HIVrelated comorbidities occur and represent a major concern in patient care. Activation, inflammation and immunosenescence could play a major role in this process. Many markers can measure those dysfunctions and they are often used without accounting for their possible interdependency. The objectives of this thesis were i) proposing a combination of those markers, ii) assessing the association between the combination of markers and the presence of comorbidities and iii) assessing the association between the combination of markers and the risk of occurrence of comorbidities and mortality in HIV-infected patients included in the sub-study CIADIS from cohort ANRS CO3 Aquitaine. We identified two scores: the cellular and the soluble CIADIS scores. The cellular score was mostly to multimorbidity and occurrence of any kind of new comorbidity. The profile of underlying immune dysfunctions was different when looking separately at the comorbidities. These results support the assumption that several underlying profiles of activation, inflammation and senescence could be involved in the development of different comorbidities. Our results show that integrating new biomarkers in analyses could improve the understanding of comorbidities. We will continue to work on the identification of profiles of immune dysfunctions for some specific comorbidity.

Cell signaling is central to integration of internal and external cues that regulate development and homeostasis. Most development is thought of as pre-adult, but limited developmental processes occur in adults. Gametogenesis incorporates elements of both these facets, with a distinct developmental plan for gamete synthesis which is regulated by integration of homeostatic inputs such as nutrient status, and environmental cues. Signaling pathways integrate and transduce information from these cues to evoke a response. A decline in homeostasis and subsequent cues occurs over time, in the case of reproductive tissues leading to a progressive loss of fertility. The Janus Kinase and Signal Transducer and Activator of Transcription or Jak/Stat signaling pathway is conserved between vertebrates and invertebrates and is necessary for numerous functions needed to maintain organism and reproductive homeostasis, as well as contributing to various developmental events. The pathway in the fruit fly Drosophila melanogaster, is composed of a single receptor, Domeless, one Janus kinase, Hopscotch, one known effector, Stat92E, and the Unpaired family of ligands consisting of Upd, Upd2, and Upd3. Jak/Stat signaling is highly pleiotropic in both sexes with involvement in homeostasis and reproduction, making it an ideal model for studying the role of signaling in reproductive aging. Reduction of pathway activity in females results in a higher proportion of unfertilized eggs, which increases with age, and in males leads to a premature onset of infertility. Central to both is integration through cell signaling to evoke an appropriate response. This dissertation explores two of the requirements for Jak/Stat signaling: the pleiotropic requirement for Jak/Stat activity during oogenesis and male reproductive maintenance. Jak/Stat functions from the beginning of oogenesis, in the stem cell niche. From there it participates in multiple functions including specification of a subset of somatic cells called the border cells through the polar cells, a pair of cells at either pole of the egg. Pathway stimulation in the border cells drives their migration with the polar cells to the oocyte boundary, where the polar cells each form an extension in a coordinated manner into the micropyle, the means for sperm entrance during fertilization. Loss of Jak/Stat activity in the border cells prevents border cell migration. While border cell migration has been well studied, polar cell involvement after completion of border cell migration is less well known. To investigate the requirements for polar cell activity and Jak/Stat activity after the completion of border cell migration, we reduced Jak/Stat signaling in the polar cells which, while having no effect on border cell migration, results in blocked micropyles due to loss of coordination of extensions during their outgrowth. Reduced function in the polar cells did not significantly affect expression of adhesion molecules. But, the loss of Stat92E is phenocopied by loss of DE-cadherin. Hence, these results indicate a previously unknown autocrine requirement for Jak/Stat activity in the polar cells. The testes also have a continuous requirement for Jak/Stat activity for stem cell maintenance and differentiation of the germline into mature sperm. Reproductive maintenance not only requires sustained production of gametes, but reproductive tissues are also subject to deterioration of homeostatic functions that contribute to organismal aging. Males from thirty-nine lines of the Drosophila Genetic Reference Panel (DGRP), a panel of inbred, fully sequenced lines, were screened for age at infertility. Data were used to perform a genome-wide association study (GWAS) to identify the genetic architecture of reproductive aging. Candidate variants associated with cell signaling regulators, genes with functions in maintaining cell homeostasis, and organism behavior were uncovered. Notably, several SNPs fell in and near Ptp61F, a negative regulator of Jak/Stat activity. While variants in the primary components of the Jak/Stat pathway were not identified, the general classes of candidate loci functions reflect the requirements for homeostasis, metabolism, and development that have been shown by other studies examining the genetics of aging and fecundity. Thus, we show that Jak/Stat has an amazing amount of pleiotropy that encompasses both the real-time functions of fertility and the time related process of aging.

COPD is characterized by tissue inflammation and impaired remodeling that suggests fibroblast maintenance of structural homeostasis is dysregulated. Thus, we performed in vitro wound healing experiments on normal and diseased human lung fibroblasts and developed an ABM of fibroblasts closing a scratched monolayer using NetLogo to evaluate differences due to COPD or cigarette smoke condensate exposure. This ABM consists of a rule-set governing the healing response, accounting for cell migration, proliferation, death, activation and senescence rates; along with the effects of heterogeneous activation, phenotypic changes, serum deprivation and exposure to cigarette smoke condensate or bFGF. Simulations were performed to calibrate parameter-sets for each cell type using in vitro data of scratch-induced migration, viability, senescence-associated beta-galactosidase and alpha-smooth muscle actin expression. Parameter sensitivities around each calibrated parameter-set were analyzed. This model represents the prototype of a tool designed to explore fibroblast functions in the pathogenesis of COPD and evaluate potential therapies.

Thesis (M.Sc. (Biochemistry)) -- University of Limpopo, 2011 / Soybean is one of the most cultivated legume plants in developing countries. Nodule senescence is a major limitation in producing high yields of soybean as it coincides with the pod filling stage. Delaying nodule senescence could be a way of increasing the yield of soybean therefore determination of the role of cysteine protease in soybean is of vital importance. In this study, soybean plants were grown under controlled temperature and light conditions. Leaves and root crown nodules were collected at 4, 6, 10, 12 and 16 weeks of age. In a comparative 1-dimensional SDS-PAGE analysis of soybean nodule proteomes as the plant matured, it showed differences in proteins expressed as shown by different banding patterns with less variation between the younger soybean nodule extracts (4, 6 and 10 weeks old) as compared to the older ones (12 and 16 weeks old). As determined by azocasein assay and protease zymography, the protease activity of the nodule extracts generally decreased with an increase in the age of the nodules whereas that of the leaves increased as the plants grew older. Cysteine proteases in the soybean nodule extracts readily cleaved the Z-Arg-Arg-AMC substrate with the highest activity shown in the younger nodules as compared to the older ones. In the leaf extracts, cysteine protease activity increased with age of the leaves. DCG-04, a biotinylated irreversible inhibitor, proved to be an effective label in profiling of activity of cysteine proteases in 1-dimensional and 2-dimensional systems. The labelling was inhibited specifically by cysteine protease inhibitor, E-64. In root nodules, the DCG-04 probing demonstrated that the expression of cysteine proteases is higher in early stages of development of the soybean nodules as compared to the later stages whereas in the leaves, there is higher expression of cysteine proteases in the old leaves (16 weeks). Using 2-dimensional polyacrylamide gel electrophoresis, five cysteine protease isoforms were visualised with the size ranging from approximately 25 to 30 kDa and a pI range of 4-6. In older nodules (12 and 16 weeks old) the higher pI isoforms are down-regulated with the 26 kDa and pI 4.5 protease being the predominant isoform. Affinity precipitation of the cysteine proteases yielded a strong band with the size of about 26 kDa. All assays used show that while in leaves, the expected trend of high expression of cysteine proteases in senescing leaves is observed, in soybean nodules the expression of cysteine proteases decreases with senescence. There is, therefore, no correlation between senescence and cysteine proteases in nodules. The highly expressed cysteine protease in young nodules could play a developmental or regulatory role during the early stages of development.

Cheatgrass is an invasive weed that has come to dominate large areas of the western United States. Once an ecosystem has been converted to a cheatgrass monoculture, it is extremely difficult to restore native vegetation. Cheatgrass negatively impacts wildlife and increases wildfire frequency and intensity. Understanding how cheatgrass so effectively invades western ecosystems is essential to turning the tide of invasion. One possible key to cheatgrass’ success is alteration of soil nutrient cycling. The goal of this study is to explore how nitrogen (N) may accumulate in cheatgrass soils via redistribution of N within soil N pools. To accomplish this we investigated soil N cycling in soils underneath cheatgrass and crested wheatgrass. We used a 15N isotope tracer to determine the contribution of root exudates to soil N pools. During the 1-week 15N tracer experiment, cheatgrass roots exuded more than twice as much N (0.11 mg N kg-1 soil d-1) as crested wheatgrass roots (0.05 mg N kg-1 soil d-1). We propose that exudation of high N content root exudates leads to the changes in soil N pool size and transformation rates commonly observed in soils under cheatgrass. This research uses a simple and relatively inexpensive isotope tracer to shed light on mechanisms by which invasive plants may alter soil processes. By understanding these mechanisms we may be able to develop strategies for better managing cheatgrass invasion.

Proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. Similarities and differences between the proteases expressed in different species may give valuable insights into their physiological roles and evolution. Systematic comparative analysis of the available sequenced genomes of two model organisms led to the identification of an increasing number of protease genes, giving insights about protein sequences that are conserved in the different species, and thus are likely to have common functions in them and the acquisition of new genes, elucidate issues concerning non-functionalization, neofunctionalization and subfunctionalization. The involvement of proteases in senescence and PCD was investigated. While PCD in woody tissues shows the importance of vacuole proteases in the process, the senescence in leaves demonstrate to be a slower and more ordered mechanism starting in the chloroplast where the proteases there localized become important. The light-harvesting complex of Photosystem II is very susceptible to protease attack during leaf senescence. We were able to show that a metallo-protease belonging to the FtsH family is involved on the process in vitro. Arabidopsis knockout mutants confirmed the function of FtsH6 in vivo.

Cellular immortalization is a major hallmark of cancer and is a multi-step process that requires numerous cell-type specific changes, including inactivation of control mechanisms and stabilization of telomere length. The telomeres at the chromosome ends are essential for genomic stability, and limit the growth potential of most cells. With each cell division, telomeres are shortened. Short telomeres may induce an irreversible growth arrest stage called senescence, or a growth crisis stage characterized by high genomic instability and cell death. Only very rarely do cells escape from crisis and become immortal, a stage that has been associated with the activation of the telomerase enzyme which can elongate and stabilize the telomeres. The processes leading to senescence bypass, growth crisis escape and finally immortalization are only beginning to be elucidated. Most of our knowledge of the immortalization process is based on analyses of human fibroblast and epithelial cell cultures immortalized by genetic modification. In this thesis, spontaneously immortalized human T lymphocytes derived from patients with Nijmegen Breakage Syndrome and a healthy individual were used to identify critical events for senescence bypass and immortalization. Genetic analysis showed a clonal progression and non-random genetic changes including the amplification of chromosomal region 2p13-21 as an early event in the immortalization process. Telomere length gradually shortened at increasing population doublings and growth crisis was associated with critically short telomeres. The clone(s) that escaped growth crisis demonstrated a logarithmic growth curve, very short telomeres and, notably, no increase in telomerase activity or expression of the telomerase catalytic gene, hTERT. Instead, upregulation of telomerase activity and telomere length stabilization were late events in T lymphocyte immortalization. Escape from crisis was associated with downregulation of DNA damage response genes and altered expression of cell cycle regulators and genes controlling the cellular senescence program. These data indicated that a number of layers of regulation are important in the process of immortalization and to provide further mechanistic detail, epigenetic analysis was carried out. Genome wide methylation array analysis identified early and step-wise methylation changes during the immortalization process. Interestingly, applying these findings to tumors of T cell origin revealed commonly methylated CpG sites in transformed cells. Deregulated gene expression of the polycomb complexes may have contributed to the epigenetic changes observed. Taken together, our analysis of spontaneously immortalized T cell cultures identified several steps in the immortalization process including genetic, epigenetic, gene expression and telomere/telomerase regulatory events, contributing further insights to the complexity of cancer cell immortalization.

<p>Recent changes in the timing of annual events are a sign that climate change is already impacting ecosystems. Carbon sequestration by forests increases with longer growing seasons. Biodiversity can be affected by mis-timing of events through shading interactions and frost damage. Projecting forests' ability to provide these ecosystem services in the future requires an understanding of trees' phenological responses to a new climate. I begin by proposing a first order definition of an `optimal' phenological response to warming: that the mean temperature following budburst should remain essentially constant. Analogously, the temperature preceding senescence can serve the same role. </p><p>To understand which environmental cues will drive future changes in phenology, I assimilate clues from observational and experimental literature. For budburst in woody plants, spring warmth, over-winter chilling and light drive nearly all behavior, but species' responses vary widely. Species using chilling or light as safety mechanisms against budburst during mid-winter thaws are thought to be less able to phenologically track a warming climate. However, I show that even species cued solely by spring warmth are likely to under-track temperature changes. Fall cues are more idiosyncratic, and a plant's driver of senescence is likely to vary from year to year. </p><p>Models are a tempting method to untangle species budburst cues and forecast phenology under warmer climate scenarios. I tested two models' ability to recover parameters used to simulate budburst data. The simpler model was cued only by spring warmth while the complex one modulated warmth requirements with chilling exposure. For the simple model, parameters could be recovered consistently from some, but not all, regions of parameter space. The complex model's parameters were largely unrecoverable. To understand the consequences of parameter uncertainty, I applied both models to an 18 year phenological record of 13 deciduous tree species. While a few species fell into identifiable regions of the simple model's parameter space, most did not, and projected budburst dates had wide parameter-derived uncertainty intervals. These bands were wider still under a 5°C warming scenario. Even greater uncertainty resulted from the complex model.</p><p>To better understand plants' potential for growing season extension I subjected seedlings to warmer climates in a series of open-topped chambers in sites at each end of the eastern deciduous biome. Soil and air were heated to 3 or 5°C above ambient, or left unheated. For nearly all species, warming hastened budburst and germination and delayed senescence. However, these events failed to track temperature changes, happening at warmer temperatures in hotter chambers. Individual species showed a remarkable variability of all events' dates within treatments, and even within chambers. Because phenological traits are heritable, this offers a potential for evolutionary response to climate change.</p><p>This research has shown that while individual trees extend their growing seasons under warmer temperatures, they typically under-respond to the magnitude of warming, suggesting forests' capacity for increased carbon sequestration may reach a limit. However, within populations, trees vary substantially in their phenological responses, forming a possibility for evolutionarily adaptation to changing cues.</p> / Dissertation

Flavonoids are a large class of dietary polyphenols that have emerged as candidate agents for chemoprevention in prostate cancer. Despite the large number of known flavonoids (over 9000), only a few have been studied in prostate cancer to date. The work presented in this thesis describes the identification of novel anti-proliferative flavonoids, their molecular effects on cell cycle and related proliferation and survival pathways, and their chemopreventive properties in a murine model of prostate carcinogenesis. We identified several novel flavonoids with potent anti-proliferative effects in human prostate cancer cells in vitro. Non-prostate cell lines were generally resistant to the effect of these flavonoids. Two of the most potent flavonoids identified, 2,2-dihydroxychalcone (DHC) and fisetin, induced S and G2 phase cell cycle arrest in LNCaP and PC3 prostate cancer cells. Gene expression studies employing oligonucleotide microarray demonstrated profound down-regulation in gene expression of 75 key cell cycle (predominantly G2 and M phase) genes by DHC and fisetin, and the enhanced expression of 50 stress-response genes with important roles in cell proliferation and survival. DHC and fisetin induced apoptosis, but not accelerated senescence, in prostate cancer cells. The chemopreventive effect of 4 flavonoids identified from the in vitro studies was examined in an autochthonous murine model of prostate cancer (TRAMP). Mice were administered diets supplemented with 1% DHC, 1% fisetin or a combination of flavonoids (0.25% DHC, 0.25% fisetin, 0.25% quercetin, 0.25% luteolin) for 32 weeks. We demonstrated a significant reduction in genitourinary weight, and a reduction in prostate cancer grade in mice administered 1% DHC and combination diets. Flavonoid supplementation was, however, associated with gastrointestinal toxicity in some mice. Liquid chromatography-mass spectrometry demonstrated the accumulation of high levels of flavonoid in the prostates of TRAMP mice. These findings lay the foundation for further studies of flavonoids in clinical chemoprevention trials.

In most eukaryotes, chromosomes ends are protected by telomeres which are formed by repetitive DNA, specialized binding proteins, and higher order structures. Telomeres become shorter following replication due to the positioning and degradation of terminal RNA primers, as well as resection by nucleases. Extensive telomere shortening over many cell cycles elicits a DNA damage checkpoint that culminates in senescence or, in the absence of tumor suppressor pathways, apoptosis. These effects block the expansion of cells with unstable genomes, but can also precipitate disease in tissues that rely on regeneration for function. In many unicellular eukaryotes and proliferative human cells including cancer cells, telomeres can be maintained by the telomerase reverse transcriptase (TERT) and its associated RNA (TR). The elongation of telomeric DNA by telomerase depends on the telomerase essential N-terminal (TEN) and C terminal reverse transcriptase (RT) domains. We found that human TEN interacted with single-stranded telomeric DNA and restored function, in trans, to an hTERT mutant lacking hTEN. Telomerase required hTEN residues for activity, telomere maintenance, and extension of cellular replicative lifespan. Two inactive hTERT variants bearing mutations in TEN and RT domains, respectively, cooperated to regenerate telomerase activity in vitro. hTEN interacted with several regions of hTERT suggesting that dimerization may occur via TEN-TERT interactions. The in vivo defect of certain hTEN mutants may involve an inability to interact with factors that recruit the enzyme to the telomere and/or stimulate activity. Human homologs of the S. cerevisiae recruitment factor Est1 interacted with telomerase in a species-specific manner. The TPR domain of hEST1A interacted with the N-terminus of hTERT. The TPR domain of ScEst1 was required for telomere length maintenance by telomerase, and, paradoxically, also negatively regulated telomere length. In preliminary experiments, hTERT interacted with hPOT1/hTPP1. This interaction may stimulate the elongation of telomeres by telomerase. The DNA and protein interactions described herein expand our knowledge of telomerase and present new targets for the manipulation of telomerase function in human disease.