La plupart des virus sont connus pour leur capacité à causer des maladies symptomatiques chez l’Homme et chez les autres animaux. Certains d’entre eux sont des grands virus à ADN nommés Virus à Grand ADN Nucléo-Cytoplasmique (NCLDV), rapportés comme infectant les cellules eucaryotiques. Au début du XXI ème siècle, quatre familles ont été définies par Iyer et al. comme ayant une origine commune (groupe monophylétique) : Asfarviridae, Phycodnaviridae, Irido-Ascoviridae et Poxviridae.En 2003, la description d’Acanthamoeba polyphaga mimivirus a cassé un paradigme dans le monde des virus. Par leur taille de particule (450nm), par leur longueur de génomes(supérieure à 1Mb) et leur contenu génique, leur découverte a changé la définition traditionnelle des virus (Lwoff). Depuis 2013 et notamment par les isolements successifs de Pandoravirus,Pithovirus et Mollivirus, ces virus ont été décrits comme possédant de nouvelles propriétés.Leur découverte a été rendue possible grâce à la méthode de co-culture utilisant des protistes, notamment des cellules du genre Acanthamoeba. Cette méthode a été de nombreuses fois modulée par différentes équipes. Dans notre cas, nous avons combiné différentes stratégies appliquées à notre co-culture : la co-culture a été couplée à la cytométrie en flux pour détecter la lyse des protistes. De plus, la cytométrie a été utilisée avec un marqueur à ADN dans le but d’identifier de façon putative le virus et de discriminer les différentes populations virales. Enfin,nous sommes capables de séparer ces populations en utilisant un appareil FACS trieur.L’ensemble de ces techniques a permis l’isolement de nouveaux virus. / Most viruses are known for their ability to cause symptomatic diseases in humans andother animals. Some of them are large DNA viruses named Nucleo-cytoplasmic Large DNAviruses (NCLDV), known for infecting eukaryotic cells. At the beginning of the 21st centuryfour families were defined by Iyer et al. as having a common origin (monophyletic group):Asfarviridae, Phycodnaviridae, Irido-Ascoviridae and Poxviridae.In 2003, the description of Acanthamoeba polyphaga Mimivirus broke this paradigmin the virus world. Because of their particles size (450 nm), their genome size (up to 1Mb),and their gene contents, their discovery changed the traditional definition of viruses (Lwoff).Since 2013 and the successive isolations of Pandoravirus, Pithovirus and Mollivirus; theseviruses have been characterized as possessing various novel properties.Their discoveries have been possible thanks to the co-culture method using protistnotably Acanthamoeba genus cells. This method went through multiple improvements and isemployed by different teams in different ways. In our case and in order to enhance thismethod we combined strategies applied in our co-culture. Indeed, this method consists inusing flow cytometry to detect lysis of protist cells (after all steps of co-culture enrichment).In addition, the flow cytometry was used with a DNA marker in order to identity viruses anddiscriminate viral populations. Then, we were able, using a FACS sorter device, to separatedifferent viral populations from our supernatants.Altogether these techniques have permitted the isolation of new viruses.

Malignant mesothelioma (MM) is an aggressive malignancy of the pleural and peritoneal surfaces. Australia has the highest reported national incidence of mesothelioma in the world, and rates are increasing (Leigh et al., 2002). The clinical outcome for patients with this disease is extremely poor, with median survival of 9 to 12 months (Rizzo et al., 2001; Carbone et al., 2002). The latest developments in chemotherapy, radiotherapy and radical surgery have done little to improve the overall survival rate (Kindler 2000; Zellos et al., 2002). New approaches to therapy are thus required (Nowak et al., 2002). Cancer therapy using conditionally replicative adenoviruses (CRAds) and attenuated measles virus (vaccine strain MV-Edm) are novel and promising approaches to cancer treatment. CRAds strategy relies on selective viral replication in tumour cells but not normal cells. Major efforts have been directed toward achieving selective replication by the deletion of viral functions dispensable in tumour cells or by the regulation of viral genes with tumour-specific promoters (Alemany et al., 2000). However, the major clinical limitation of viral therapy has been lack of efficacy rather than safety concerns. In this study, I constructed CRAds in which tumour-specific promoter for Flt-1 (vascular endothelial growth factor receptor) control the essential E1 gene expression, and evaluated the cell-killing efficacy and specificity of CRAds driven by VEGF and Flt-1 promoters in the number of established mesothelioma cell lines and actual primary tumour cells from patients. CRAds with either VEGF or flt-1 promoters showed a strong killeg effect on mesothelioma cells. Co-delivery of CRAds with MMP-9 (matrix metalloproteinase-9) was assessed to determine whether therapeutic efficacy could be improved by reducing tumourassociated fibrosis thereby enhancing viral spread through a tumour mass. Combined therapy did result in greater suppression of tumour growth in vivo. I also identified an immuno-competent murine model of mesothelioma that was permissive for adenoviral replication. Combined viral therapy with immunotherapy (FGK45, an anti-CD40 antibody) in this model resulted in greater effect than Adwt or FGK45 alone and in greatest survival. I evaluated the capacity of MV-Edm to infect human mesothelioma cells to form syncytia, and lead to apoptosis and cell death. I also assessed the mode of death by analysis of markers of apoptosis including caspase-3. In vivo study showed that MVEdm- GFP transduction could be detected in human xenografts in immune deficient mice. Further studies to evaluate the mechanisms and efficacy of anti-tumour immune stimulation induced by tumour cell killing with CRAds and MV-Edm will be discussed in this study. MV-Edm has good killing effect on mesothelioma cells in vitro. In summary the work presented herein provide new insights into stratgies to improve viral therapies for mesothelioma. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1342596 / Thesis (Ph.D.) - University of Adelaide, School of Medicine, 2008

A variety of genes that influence aging have been identified in a broad selection of organisms including Saccharomyces cerevisiae (yeast), Caenorhabditis elegans (worms), Drosophila (fruit flies), Macaca Mulatta (rhesus monkeys), and even Homo sapiens. Many of these genes, such the TOR’s, FOXO’s, AKT’s, and S6K’s are conserved across different organisms. All of these genes participate in nutrient sensing networks. Other conserved genetic networks may similarly affect lifespan. In this thesis, I explored genes from an iron metabolism family and a heat shock protein (HSP) gene family that have been identified, but not confirmed, to influence lifespan. Yeast is a reliable model for mitotic (replicative) aging. Using yeast, I tested whether the FET-genes, encoding a family of iron importer-related genes, are required for mitotic lifespan. I also tested whether another family of genes, the yeast SSA HSP70- encoding genes, related to mammalian HSP70s, influence mitotic aging. I primarily used the replicative lifespan (RLS) assay, in which I measured the mitotic capacity of multiple FET and SSA yeast mutants. I hypothesize that aging occurs when iron transport is misregulated, which may lead to an over-reliance on HSPs for lifespan maintenance. The results presented in this thesis support the hypothesis. First, FET3 was primarily involved in lifespan maintenance under normal conditions (2% glucose), while FET5 was primarily involved in the cellular lifespan extension characteristic of caloric restriction (0.01% glucose), a known anti-aging intervention. In addition, SSA2 appeared to facilitate lifespan maintenance in the absence of FET4, while the presence of SSA1 limited lifespan length. That the aging genes identified in this study are involved in iron metabolism or heat stress suggests that protein aggregation or reactive oxidative species production are common processes through which these genes interact.

Ageing and senescence remain among the most intriguing questions in biology. Saccharomyces cerevisiae has become well established as a fertile model system for the investigation of ageing. Remarkable conservation has been found to exist between interventions extending lifespan in higher animals and yeast – genetic, chemical, and nutritional – suggesting a network of common regulatory pathways controlling large-scale shifts in gene expression involved in senescence. While it has been proposed that epigenetic regulation controls these shifts, evidence remains incomplete. To address this question, novel longevity mutants were isolated in S. cerevisiae using a purpose-designed high-precision screen based on ageing culture outgrowth. A novel long-lived mutant in uncharacterised gene YDR026C was discovered and found to participate in a pathway distinct from TOR signalling, but share epistasis with the histone deacetylase SIR2Δ, a well established regulator of replicative longevity and rDNA maintenance. Through equilibrium density centrifugal separation of culture subpopulations, SIR2Δ and Ydr026cΔ cultures were found to demonstrate reduced and improved maintenance of post-diauxic quiescence respectively, previously shown to underlie chronological survival in strains including snf1Δ. Development of a quantified TUNEL-based assay for genome fragmentation indicated early apoptotic-like behaviour in the SIR2Δ strain. Microdissection experiments and sectored-colony assays of strains containing an rDNA-embedded ADE2 reporter determined that Ydr026cΔ cells also exhibit extended replicative lifespan, and reduced recombination at the rDNA spacer region hotspot, abrogated in SIR2Δ strains. SIR2Δ is well established to repress RNA polymerase II-derived transcripts in the rDNA spacer region, including IGS1-R. Northern analysis determined Ydr026c also silences transcription in the spacer, possibly through preventing termination of the main rRNA transcript, interfering with IGS1-R expression. By transformation with a vector overexpressing IGS1-R, partial reconstitution of the SIR2Δ phenotype was observed, including rDNA hyperrecombination, shortened replicative longevity, and higher-order chromatin structure restoration. These data suggests a model whereby non-coding rDNA spacer transcripts epigenetically determine rDNA maintenance through recombination, leading to physiological phenotypes of replicative and chronological ageing.

Human immunodeficiency virus (HIV) is globally spread virus without available cure. Since its life-long presence, virus is carefully monitored as well as patient's immunological status. Replicative fitness of the virus is one of important aspects which can be taken into account, when monitoring HIV. Here, we are measuring HIV replicative fitness of gag recombinant viruses and comparing the results with replicative fitness of primary isolates. Further, we are comparing our findings of replicative fitness change over time with disease progression in the patient. We found that gag can be major contributor to overall fitness, although not in all cases. Additionally, we observed a correlation of replicative fitness development and slope of patient's CD4+ T cells. Moreover, this relation was even more noticeable in patients with slow disease progression or in carriers of protective alleles. In summary, our results extend the understanding of replicative fitness and its role in disease progression; and pave the way to use the recombinant HIV for replicative fitness measurement in clinical practice. Keywords: HIV, replicative fitness, recombinant virus, HIV disease progression, gag

Ce travail est consacré au rôle pléotropique des microparticules endothéliales dans la réponse et l'homéostasie vasculaire. Un modèle de sénescence réplicative a été caractérisé en utilisant des cellules endothéliales de coronaires de porc en culture primaire. Ce modèle a mis en évidence des changements drastiques du phénotype endothélial avec la production de ROS, la dépolarisation de la membrane mitochondriale et la surexpression de régulateurs clés du cycle cellulaire incluant p53, p21 et p16. La sénescence a transformé le phénotype endothélial vers un statut procoagulant indiqué par la libération de microparticules (MP). L'induction d'activité Facteur Tissulaire (FT) et une réduction drastique de l'inhibition de l'agrégation plaquettaire due à la sécrétion réduite de NO endothélial. Simultanément, une augmentation importante des protéines du système de l'angiotensine à la surface des cellules sénescentes et des MPs qu'elles émettent a été mesurée. D'autres résultats obtenus avec les MPs circulantes de patients transplantés ou atteints de syndrome coronarien suggèrent l'existence dune boucle d'amplification des effets délétères des MPs au travers de la signalisation Redox et l'altération des fonctions vasculaires résultant d'une sénescence exacerbée. En plus de ses qualités reconnues d'immunosuppresseur, la cyclosporine A (CsA) est un inhibiteur puissant de l'ouverture des pores mitochondriaux (mPTP). Certaines études ont présenté le traitement bref et contrôlé par CsA comme un moyen de limiter les dommages vasculaires de l'ischémie reperfusion.Nos données suggèrent une possible modulation de la sénescence endothéliale induite par les MPs grâce au préconditionnement avec des concentrations faibles de CsA. nos résultats suggèrent aussi que de faibles doses de Cs A peuvent avoir un effet bénéfique dans les pathologies cardiovasculaires lorsque la sénescence est exacerbée et contribue les fonctions vasculaires de l’endothélium. / This scientific work has tackled the issue of the pleitropic role mediated by endothelial microparticles function and homeostasis. A replicative model of senescence using coronary endothelial cells was set showing drastic phenotype changes characterized by ROS production, mitochondrial membrane depolarization and the up-regulation of key regulators of cell cycle arrest including p53, p21 and p16.Replicative senescence shifted the coronary endothelial phenotype toward a procoagulant status as evidenced by (i) procoagulant MP shedding (ii) enhanced tissue factor (TF) expression and (iii) a marked decrease in the endothelial NO-mediated inhibition of platelet aggregation. In parallel, a drastic up regulation of the angiotensin system could be evidenced at the surface of senescent cells or derived MP. Results obtained with MPs from patients with acute coronary artery syndrome and from grafted patients,suggested a feedback loop disseminating the deleterious effect of circulating MPs redox signaling and alteration of vascular function owing to exaggerated senescence. In addition to its well-known immunosuppressive properties, cyclosporine A (Cs A) is a potent inhibitor of the opening of the mitochondrial permeability transition pore (mPTP), and several reports have indicated that a brief and timely administration of Cs A can limit ischemia-reperfusion injuries. Our data evidenced the possible pharmacological modulation of endothelial MP-mediated senescence by cell preconditioning with low concentrations of Cs A. our data are thus suggestive of a beneficial effect of CsA in cardiovascular disorders where senescence is altering the endothelial vascular functions.

Les trois complexes SMC Cohésine, Condensine et SMC5/6 sont principalement étudiés pour leurs rôles mitotiques, cependant tous trois sont localisés à proximité des fourches de réplication en condition de stress réplicatif. Au cours de cette thèse, nous nous sommes particulièrement intéressés aux complexes Cohésine et Condensine. Dans une première partie, nous décrivons un nouveau rôle des condensines dans la progression des fourches de réplication en condition de stress réplicatif à l’hydroxyurée (HU) et au Méthyl-Méthane-Sulfonate (MMS). Nos données montrent que dans ces conditions, les condensines limitent l’accumulation de la protéine de liaison à l’ADN simple-brin RPA (Replication Protein A) à proximité des fourches de réplication. Ces résultats révèlent que les condensines limitent l’exposition d’ADN simple brin et pourraient ainsi protéger l’intégrité des fourches de réplication et la stabilité du génome. Dans une seconde partie nous décrivons le mécanisme de recrutement du complexe Cohésine aux fourches de réplication en condition de stress réplicatif. Dans ces conditions, les cohésines renforcent la cohésion des chromatides sœurs afin de faciliter le redémarrage des fourches de réplication par recombinaison homologue. Nous montrons que le complexe SMC-like MRX (Mre11-Rad50-Xrs2), l’histone méthyle-transférase Set1 et l’histone acétyle-transférase Gcn5 sont requis pour le recrutement des cohésines aux fourches de réplication. Nos données révèlent qu’en réponse au stress réplicatif, Gcn5, Set1 et MRX modifient la dynamique des histones. Gcn5 et MRX réduisent la densité d’histone sur l’ADN répliqué alors que Set1 maintient la mobilité des nucléosomes. La modification de la dynamique des histones semble importante pour une réponse cellulaire efficace au stress réplicatif et pour le chargement de complexes SMC aux fourches de réplication. / The three SMC complexes Cohesin, Condensin and SMC5/6 are mainly studied for their role in mitosis, nevertheless they all localize at replication forks in replicative stress conditions. During this thesis, we focused on Cohesin and Condensin. In the first part we describe a new role for the condensin complex in response to replicative stress. In the presence of Hydroxyurea (HU) and Methyl-Methan-Sulfonate (MMS), condensin is required for cell growth and replication fork progression. Moreover, our results show that condensin limits the accumulation of the specific single-strand DNA (ssDNA) binding protein RPA (Replication Protein A) in the vicinity of replication forks under HU treatment, revealing that condensin limits ssDNA accumulation during replicative stress. In this way, Condensin could protect replication fork integrity and genome stability in response to replicative stress. In the second part, we decipher the cohesin recruitment mechanisms at replication fork under replicative stress. In that context, cohesin reinforces sister chromatid cohesion and facilitates homologous recombination (HR) dependent replication fork restart pathways. We show here that the SMC-like MRX complex, the histone methyl transferase Set1 and the histone acetyl transferase Gcn5 are required for cohesin recruitment at stalled replication forks. Our results show that these three proteins affect histone H3 dynamics on replicated DNA in response to replicative stress. Gcn5 and MRX reduce H3 density whereas Set1 maintains nucleosome mobility. These two parameters seem to be important for efficient response to replicative stress and for SMC complexes loading close to stressed replication forks.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder because of a LMNA gene mutation that produces a mutant lamin A protein (progerin). Progerin also has been correlated to physiological aging and related diseases. However, how progerin causes the progeria remains unknown. Here, we report that the large subunit (RFC1) of replication factor C is cleaved in HGPS cells, leading to the production of a truncated RFC1 of ∼75kDa, which appears to be defective in loading proliferating cell nuclear antigen (PCNA) and pol δ onto DNA for replication. Interestingly, the cleavage can be inhibited by a serine protease inhibitor, suggesting that RFC1 is cleaved by a serine protease. Because of the crucial role of RFC in DNA replication, our findings provide a mechanistic interpretation for the observed early replicative arrest and premature aging phenotypes of HPGS and may lead to novel strategies in HGPS treatment. Furthermore, this unique truncated form of RFC1 may serve as a potential marker for HGPS.

Aging is a complicated and multifactorial phenomenon. Model systems involving the induction of replicative senescence in cultured cells have been indispensable in elucidating some of the mechanisms underlying this complex process. An understanding of how and why cellular senescence occurs is thus critical to the field of aging research. While there is much correlative evidence to suggest a connection between poly(ADP-ribose) (PAR) and mammalian longevity, no studies have been done to explore a possible role for PARP-1 — the enzyme responsible for synthesis of 90% of cellular PAR — in mechanisms of senescence. Furthermore, many techniques currently used for analysis of protein poly(ADP-ribosyl)ation are fraught with imprecision. We therefore sought to address these issues both by developing methods for the unambiguous analysis of poly(ADP-ribosyl)ation by mass spectrometry, and by exploring the role of PARP-1 in nicotinamide-mediated cellular lifespan extension. Due to the challenges introduced by PAR's biochemical characteristics, successful mass spectrometric analysis of poly(ADP-ribosylation) will require the use of techniques to reduce the mass, charge, and heterogeneity of the polymer, as well as methods to enrich for poly(ADP- ribosyl)ated protein. To this end, we evaluated the effectiveness of several approaches, including ammonium sulfate fractionation, boronate affinity chromatography, snake venom phosphodiesterase digestion, manipulation of PARP-1 reaction conditions, and immobilized metal affinity chromatography (IMAC) for the preparation of poly(ADP-ribosyl)ated protein samples prior to MS analysis using both MALDI-TOF and Q-TRAP LC-MS. Based on this work, we developed a three-tiered scheme that may provide the first ever identification of poly(ADP- ribosyl)ated peptides from full-length wild-type PARP-1 by mass spectrometry. Past work in our laboratory has demonstrated that nicotinamide (NAM), a component of vitamin B3, significantly extends the replicative lifespan of human fibroblasts. In order to help elucidate the role of PARP-1 in cellular senescence, we then analyzed the poly(ADP-ribosyl)ation response of aging cells undergoing NAM-mediated lifespan extension. While NAM is a known PARP-1 inhibitor, we found that oxidative stress-induced poly(ADP- ribosyl)ation is increased, not decreased, in NAM-treated cells. We propose that supplemented NAM is taken up by the NAD salvage pathway, ultimately leading to increased cellular NAD and extending replicative lifespan by both preventing PARP-mediated NAD depletion and upregulating SIRT1. We further propose that the demonstrated protective effects of NAM treatment in a number of disease models are due not to PARP-1 inhibition as is commonly assumed, but to upregulation of NAD salvage. / Ph. D.

No description available.