Human Cytomegalovirus (HCMV), the prototypic β-herpesvirus, is the most common cause of congenital infections as well as morbidity and mortality in immunocompromised patients. The anti-HCMV drugs currently available have a number of drawbacks (i.e. detrimental side-effects and/or the appearance of drug resistant strains), which limit their clinical usefulness. Therefore, a better understanding of host-virus interactions is important to develop new, safe and effective ways to treat HCMV. HCMV has evolved various strategies to make the host cell more conducive for the replication process, many of these involve modulation of host signalling pathways through proteins or non-coding RNAs. The focus of this thesis is on the regulation of one class of non-coding RNA, microRNAs (miRNA) by HCMV as well as murine CMV (MCMV). miRNAs are short ~22 nucleotide RNA sequences, which negatively regulate the stability and translational efficiency of specific target messenger RNAs (mRNAs). It has been previously shown that three host-encoded miRNAs, miR-199-3p, miR-199-5p and miR-214, are down-regulated in both MCMV and HCMV infected cells. Despite the biological and genomic differences between the two viruses, this down-regulation occurs in both infections, suggesting a possible conserved antiviral role of the miRNAs in mouse and human cells. Consistent with this, miR-199-3p and miR-214 manifest antiviral properties against MCMV and HCMV when over-expressed in vitro. This thesis investigates two hypotheses: 1) CMV down-regulates the expression of these host miRNAs through a mechanism involving viral factors, 2) The down-regulation of miR-199-3p leads to the up-regulation of its targets and this influences the cell in a way that favours some aspect of the viral life cycle. The first part of this project examined the regulation of miR-199-3p, miR-199-5p, and miR-214, which derive from a single primary transcript (pri-miRNA). The down-regulation of all three miRNAs was found to occur at the transcriptional level by 4 hours post infection. The promoter of the miR-199a/214 cluster was therefore cloned into a reporter vector in order to interrogate the factors regulating transcription of pri-miRNA in infection; this was carried out in the murine model based on availability of reagents. The reduction in the pri-miRNA was found to correlate with a decrease in the transcriptional activity of miR-199a/214 promoter in infected cells. Further analysis revealed the presence of a sequence between -421 to -273 relative to the transcription start site (TSS) that was critical for promoter activity. This sequence contains a putative serum response element (SRE), which includes two binding sites for the SRF dimer (serum response factor) and a binding site for a molecule of TCF (ternary complex factor), ELK-1. Initial knock-down studies suggest that these transcription factors are required for basal activity but it remains unknown whether they are involved in the differential expression of miR-199a/214 observed during infection. Another binding site for the transcription factor TWIST-1 was found outside this region, which is known to regulate the miR-199a/214 cluster in other cell types. Western blot analysis showed reduced expression of TWIST-1 in cells infected with HCMV and MCMV infections, by 24 and 48 hours, respectively, suggesting a role of TWIST-1 in regulating miR-199a/214 cluster during these infections. This regulation seems to be dependent on viral gene expression, as a replication deficient viral mutant fails to repress the promoter function and subsequent pri-miRNA production. Taken together, these results suggest an active viral mechanism for transcriptional repression of the miR-199a/214 promoter. To understand the antiviral function of miR-199-3p, the second part of this thesis examined whether miR-199-3p regulates host signalling pathways important for CMV replication and/or the life cycle. A microarray analysis was carried out with samples from cells transfected with miR- 199-3p mimic versus inhibitor. This revealed 198 genes significantly down-regulated by the miRNA. From the 198 genes, Ingenuity pathway analysis (IPA) software identified several host pathways with a potential role in HCMV infection including: PI3K/AKT signalling, the ERK-MAPK cascade, and prostaglandin production. This thesis examined the role of miR-199-3p in regulating the PI3K/AKT pathway in HCMV infection. It was found that miR-199-3p modulates the phosphorylation of the central regulator of PI3K/AKT signalling, AKT. Transfection of miR-199-3p before the infection impedes the complete phosphorylation of AKT, which is known to be required for the immediate early viral gene expression and replication. This provides an explanation for the antiviral function of miR-199-3p, through its ability to modulate AKT phosphorylation. An open question, however, is how the natural down-regulation of miR-199-3p from 24 to 72 hours post infection naturally affects AKT phosphorylation. Several predicted targets of miR-199-3p, such as PIK3CB, ITGA3, and ITGA6 were shown to be up-regulated at these late time points, correlating with the miR-199-3p down-regulation. The interaction of miR-199-3p with target sites in the 3′UTRs of PIK3CB and ITGA3 was validated by luciferase reporter assays and western blotting and qRT-PCR results indicated that protein and mRNA levels of ITGA6 were regulated by miR-199-3p mimic transfection. However, the knock-down of these three targets did not result in a significant decrease of the viral growth, and thus cannot alone explain the antiviral function of miR-199-3p. Overall, this study suggests that the transcriptional repression of miR- 199a/214 is likely a strategy employed by CMV to support its own growth through attenuating the biological effect of miR-199-3p within the host cell.

Phosphatidyl D-myo-inositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] contributes to the activation of protein kinase B (PKB) by interacting with the PKB PH domain. PKB is known to be up-regulated in several cancer cell types. Compounds that can display selective inhibition of this kinase have promising chemotherapeutic potential, and inhibition of the PH domain of PKB represents a realistic means by which to achieve this. Analysis of the X-ray crystal structures of apo PKBαPH and PKBαPH bound to D-myo-inositol 1,3,4,5-tetrakisphosphate [InsP4, the inositol head group of PtdIns(3,4,5)P3] led to the design of PtdIns(3,4,5)P3 and InsP4 analogues as potential PKB PH domain inhibitors. The synthesis of PtdIns(3,4,5)P3 analogues modified at the C-4 position was investigated, but it was discovered that such compounds were prone to migration of the 1-position phosphate. Subsequently, a range of racemic InsP4 analogues, modified at the C-1 or C-4 position, were successfully synthesised. Advanced progress has also been made towards the synthesis of enantiomerically pure analogues of InsP4.

Objective: The mechanisms by which lipopolysaccharide (LPS) pretreatment induces cardioprotection following ischaemia/reperfusion (I/R) have not been fully elucidated. We hypothesized that activation of phosphoinositide 3-kinase (PI3K)/Akt and high mobility group box 1 (HMGBx1) signaling plays an important role in LPS-induced cardioprotection. Methods: In in vivo experiments, age- and weight-matched male C57BL/10Sc wild type mice were pretreated with LPS before ligation of the left anterior descending coronary followed by reperfusion. Infarction size was examined by triphenyltetrazolium chloride (TTC) staining. Akt, phospho-Akt, and HMGBx1 were assessed by immunoblotting with appropriate primary antibodies. In situ cardiac myocyte apoptosis was examined by the TdT-mediated dUTP nick-end labeling (TUNEL) assay. In an in vitro study, rat cardiac myoblasts (H9c2) were subdivided into two groups, and only one was pretreated with LPS. After pretreatment, the cells were transferred into a hypoxic chamber under 0.5% O2. Levels of HMGBx1 were assessed by immunoblot. Results: In the in vivo experiment, pretreatment with LPS reduced the at risk infarct size by 70.6% and the left ventricle infarct size by 64.93% respectively. Pretreatment with LPS also reduced cardiac myocytes apoptosis by 39.1% after ischemia and reperfusion. The mechanisms of LPS induced cardioprotection involved increasing PI3K/Akt activity and decreasing expression of HMGBx1. In the in vitro study, pretreatment with LPS reduced the level of HMGBx1 in H9c2 cell cytoplasm following hypoxia. Conclusion: The results suggest that the cardioprotection following I/R induced by LPS pretreatment involves PI3K/Akt and HMGBx1 pathways.

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. We have previously reported that glucan phosphate (GP) significantly increased survival in a murine model of cecal ligation and puncture (CLP)-induced sepsis. In the present study, we examined the effect of GP on cardiac dysfunction in CLP-induced septic mice. GP was administered to ICR/HSD mice 1 h before induction of CLP. Sham surgically operated mice served as control. Cardiac function was significantly decreased 6 h after CLP-induced sepsis compared with sham control. In contrast, GP administration prevented CLP-induced cardiac dysfunction. Macrophage migration inhibitory factor (MIF) has been implicated as a major factor in cardiomyocyte apoptosis and cardiac dysfunction during septic shock. CLP increased myocardial MIF expression by 88.3% (P < 0.05) and cardiomyocyte apoptosis by 7.8-fold (P < 0.05) compared with sham control. GP administration, however, prevented CLP-increased MIF expression and decreased cardiomyocyte apoptosis by 51.2% (P < 0.05) compared with untreated CLP mice. GP also prevented sepsis-caused decreases in phospho-Akt, phospho-GSK-3β, and Bcl-2 levels in the myocardium of septic mice. These data suggest that GP treatment attenuates cardiovascular dysfunction in fulminating sepsis. GP administration also activates the phosphoinositide 3-kinase/Akt pathway, decreases myocardial MIF expression, and reduces cardiomyocyte apoptosis.

The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca 2+]i, Ca2+ transients and membrane Ca2+ current, ICa, in cultured murine HL-1 cardiomyocytes. LY294002 (120 μM), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca 2+]i, Ca2+ transients and ICa. We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 28 nM); ? (TGX-221; 100 nM) and γ (AS-252424; 100 nM), to determine the contribution of specific isoforms to HL-1 [Ca 2+]i regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca2+]i, and inhibited Ca 2+ transients. Triciribine (120 μM), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca2+]i, and Ca 2+ transients and ICa. We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca2+]i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca 2+]i required for excitation-contraction coupling in cardiomyoctyes.

L'activation séquentielle des Kinases Dépendantes des Cyclines (CDK), associées à leur sous-unité régulatrice la cycline, contrôle la progression des cellules eucaryotes dans le cycle cellulaire. L'activité des complexes CDK/cycline est notamment régulée par une balance entre phosphorylation inhibitrice (Wee, Myt) et déphosphorylation activatrice par les phosphatases CDC25. Dans les cellules humaines, trois phosphatases à double spécificité CDC25A, B et C sont impliquées dans la régulation de ces complexes en différents points du cycle cellulaire. CDC25A agit à la transition G1/S alors que CDC25C contrôle l'entrée en mitose. Par contre, CDC25B agirait en phase S ainsi qu'à la transition G2/M. L'existence de trois variants de CDC25B (B1, B2 et B3) issus d'un épissage alternatif pourrait expliquer cette controverse. Afin d'étudier les rôles et les implications de chaque variant de CDC25B, nous avons d'abord étudié leur régulation par la protéine kinase CK2, kinase qui pourrait jouer un rôle dans le contrôle de la transition G2/M. Nos études in vitro ont démontré que CK2 phosphoryle les trois variants de CDC25B mais pas la protéine CDC25C. Une analyse par spectrométrie de masse de CDC25B indique qu'au moins deux résidus, les sérines 186 et 187, sont phosphorylés in vitro par CK2. De plus, CDC25B interagit avec CK2 in vitro et in vivo dans des cellules humaines et d'insectes. Enfin, la phosphorylation de CDC25B par CK2 augmente son activité phosphatase in vitro ainsi qu'in vivo. CK2 est donc un régulateur positif de l'activité catalytique des CDC25B. Au cours du cycle cellulaire ou en réponse aux points de contrôle, CDC25B est également régulée au niveau de sa localisation intracellulaire. En effet, la phosphatase réalise une navette entre le cytoplasme et le noyau, navette qui peut être régulée notamment par phosphorylation. Nous avons montré que la protéine kinase AKT/PKB phosphoryle in vitro CDC25B sur la sérine 353 et qu'elle provoque son accumulation dans le cytoplasme. L'activation d'AKT/PKB par le peroxyde d'hydrogène reproduit la relocalisation de CDC25B. Par contre, si la mutation de la sérine 353 abolit sa phosphorylation par AKT/PKB, elle n'induit qu'un retard dans la relocalisation cytoplasmique de CDC25B ce qui indique que d'autres mécanismes participent à ce phénomène. Ainsi nos différents travaux ont permis d'identifier deux nouveaux régulateurs de CDC25B, CK2 qui régule son activité catalytique et AKT/PKB qui participe au contrôle de sa localisation intracellulaire, et nous permettent de mieux comprendre la régulation de cette phosphatase même si de nombreux partenaires doivent encore être identifiés.

Les cassures double-brin (DSB) sont des dommages dangereux de l’ADN et représentent un facteur de risque pour la stabilité du génome. Le maintien de l'intégrité du génome est essentiel pour les cellules souches adultes, qui sont responsables de la régénération des tissus endommagés et de l'homéostasie tissulaire tout au long de la vie. La régénération musculaire chez l'adulte repose sur les cellules souches musculaires (cellules satellites, SCs) qui possèdent une remarquable capacité de réparation des DSB, mais dont le mécanisme sous-jacent reste inconnu. Ce projet de thèse consistait à étudier comment la différenciation musculaire est affectée lorsque la réparation des DSB est altérée, et quels sont le(s) mécanisme(s) et les conséquences de ce défaut de réparation sur la régénération musculaire. Au cours de cette étude, il est apparu de façon originale que les facteurs de réparation des DSB peuvent affecter la myogenèse, indépendamment de leur fonction dans la réparation de l'ADN. La présente étude a porté sur le rôle de la protéine kinase dépendante de l'ADN (DNA-PK), un facteur crucial pour la réparation non-homologue des DSBs (NHEJ), au cours de la différenciation musculaire chez la souris. L’étude a ciblé l'activation des SCs et la régénération musculaire in vitro et in vivo et a également abordé la régulation de cette kinase. Le rôle "canonique" de la DNA-PK, et donc du NHEJ, dans les SCs a également été étudié en présence de lésions de l'ADN radio-induites. Le rôle d’ATM, une kinase qui orchestre les réponses cellulaires aux DSB, a également été abordé dans le contexte de la régénération musculaire. Ces résultats confirment la notion émergente du rôle multifonctionnel des protéines de réparation de l’ADN dans d’autres processus physiologiques que la réparation elle-même, ce qui m’a également permis de réaliser une étude bibliographique. Ce travail i) identifie de nouveaux régulateurs de la myogenèse et ii) contribue à la compréhension de la résistance des cellules souches musculaires au stress génotoxique. Ces résultats pourraient avoir des implications dans l'amélioration des thérapies cellulaires de la dysfonction musculaire en agissant sur les régulateurs nouvellement découverts. / DNA double-strand breaks (DSBs) are dangerous DNA damages and a risk factor for genome stability. The maintenance of genome integrity is crucial for adult stem cells that are responsible for regeneration of damaged tissues and tissue homeostasis throughout life. Muscle regeneration in the adult relies on muscle stem cells (satellite cells, SCs) that have a remarkable DSB repair activity, but the underlying mechanism is not known. The aims of the present PhD project were to investigate how muscle differentiation is affected when DSB repair is impaired, and which are the mechanism(s) and the consequences on muscle regeneration. During this study, a novel possibility has arisen, namely that DSB repair factors affects myogenesis independently of their DNA repair activity, suggesting a novel function, not previously anticipated, of these factors. The present study has addressed the role of DNA-dependent protein kinase (DNA-PK), a crucial factor in non-homologous end-joining (NHEJ) repair of DSBs, in muscle differentiation in the mouse. Studies have targeted SC activation and muscle regeneration in vitro and in vivo and also addressed the regulation of this kinase. In parallel the more “canonical” role of DNA-PK, and thereby of NHEJ, has been investigated in SCs via radiation-induced DNA damage. The role of ATM, a kinase that orchestrates cellular responses to DSBs in muscle regeneration has also been addressed. These results support the emerging notion of multifunctional repair proteins in a variety of physiological processes beyond the repair process itself, on which I have conducted a bibliographical study. This work i) identifies novel regulators of myogenesis, and ii) helps understanding the resistance of muscle stem cells to genotoxic stress. It has potential implications for improving cellular therapies for muscle dysfunction by acting on the newly discovered regulators.