The research reported in this PhD thesis provides a novel approach to estimate 3D pitting corrosion kinetics of austenitic stainless steel with exposure to chloride-containing aqueous environments. A quasi-in-situ X-ray computed tomography (X-ray CT) approach was developed, with the aim of providing an experimental methodology to estimate 3D pitting corrosion kinetics under different exposure conditions. The first part summarises a set of preliminary investigations to identify the pitting corrosion behaviour of three austenitic stainless steels (type 303 bar, type 304 plate and type 304L wire) with different inclusion contents. All observed pit densities were related to the inclusion contents, providing confidence in moving to the next stage of the project, for conducting in-situ corrosion studies using X-ray CT. The second section describes the construction of an in-situ electrochemical cell for X-ray CT studies, the aim being to provide an experimental methodology to estimate 3D pitting corrosion kinetics. Pit growth kinetics of individual pits were estimated from segmented 3D X-ray CT data. The evolution of pit current densities, associated pit stability products, and diffusivity parameters over time were obtained. The study also showed that the kinetics of multiple pits could be estimated using this novel approach, based on separating the current response of each pit over time. This was obtained by electrochemical polarisation control and measuring the total current evolution. The third section discusses the effect of plastic strain on 3D pitting corrosion kinetics. Several in-situ X-ray CT experiments were conducted, with a focus on obtaining 3D pit growth, passivation, and re-activation kinetics, to elucidate the effect of applied strain on pit stability and growth. This section explains a possible mechanism for the re-activation of pre-existing corrosion pits, showing that pits grew more rapidly during reactivation than those grown before plastic strain was applied. A marked difference in pit morphology with fractured lacy metal covers was observed with the application of strain. The implications of this observation are discussed in light of stress corrosion crack nucleation mechanisms.

Le virus de l’herpès simplex de type 1 (HSV1) établit une infection latente dans le système nerveux de l'homme, au cours de laquelle un type de transcrits, appelés LATs (pour latency associated transcripts), s'accumule dans les neurones infectés. Le rôle clef des LATs dans le contrôle de la latence virale est reconnu. Cependant, depuis leur découverte dans les années 80, leur mécanisme d'action reste non élucidé.Le gène des LATs est transcrit en un LAT primaire de 8,3kb, qui est épissé, conduisant à la formation de deux LATs stables : le LAT2kb et le LAT1.5kb. De façon remarquable, le LAT2kb et le LAT1.5kb sont des introns. Leur stabilité est la conséquence d'un branchement non canonique qui se traduit par le maintien de la structure en lariat. Par ailleurs, la région du génome codant les LATs contient également le gène RL2 qui code ICP0, la protéine la plus en amont dans la cascade de réactivation du virus. Des études précédentes ont montré qu’au moment de la latence, des transcrits RL2 non épissés, s'accumulent au site principal de la latence (le ganglion de Gasser).Nous avons caractérisé ces transcrits non épissés du gène RL2 dans les tissus infectés de façon latente. Ils contiennent de façon reproductible l’intron 1 et sont d’autant plus abondants dans les tissus infectés de façon latente que les LAT s’accumulent. On peut ainsi distinguer plusieurs types de tissus infectés de façon latente, dont les deux exemples les plus représentatifs sont d’une part le ganglion de Gasser (forte expression des LAT et accumulation de transcrits RL2 non-épissés) et d’autre part le ganglion cervical supérieur (pas d’accumulation de LAT par rapport aux quantités exprimées pendant la phase aiguë de l’infection, et très peu d’expression dans transcrits non-épissés). Dans tous les cas, la réalité du caractère latent de l’infection était confirmé par la présence de génome viral sans expression de transcrits matures de gène viral précoce (représenté par celui de la thymidine kinase) ni tardif (gène UL18). Ces résultats suggèrent une relation entre la présence des LAT et l’accumulation de transcrits RL2 non-épissés, ce qui pourrait être en relation avec le maintien de l’infection à l’état latent dans ces tissus. / The herpes simplex virus type 1 (HSV-1) establishes a latent infection in the nervous system of humans, in which latency associated transcripts (LATs) accumulate in infected neurons. The key role of LATs in the control of viral latency is well established. However, since their discovery in the 80s, their mechanism of action remains unclear.The LAT gene is transcribed into a 8.3 kb primary LAT that is rapidly spliced, leading to the formation of two stable LATs; LAT2kb and LAT1.5kb. Remarkably, the LAT2kb and LAT1.5kb are introns. Their stability is the result of a non-canonical sequence of the branching point, which results in maintaining the lariat structure.Moreover, the region of the genome encoding the LATs also contains the RL2 gene, encoding ICP0 that acts upstream in the cascade of viral reactivation. Previous studies have shown that RL2 unspliced transcripts may accumulate in the main site of HSV-1 latency (trigeminal ganglia). We have characterized these unspliced transcripts RL2 gene in latently infected tissues. They reproducibly contain intron 1 and are particularly abundant in latently infected tissues where LATs also accumulate. We distinguished several types of latently infected tissues, the two most representative examples being the trigeminal ganglion (strong expression of LATs and accumulation of non-spliced transcripts RL2) and, in the opposite, the superior cervical ganglion (no accumulation of LAT compared with the amounts expressed during the acute phase of infection, and little expression in non-spliced RL2 transcripts). In all cases, the reality of the latent nature of the infection was confirmed by the presence of viral genome with no expression of mature transcripts from early viral gene (represented by the thymidine kinase gene) or late (UL18 gene).These results suggest a relationship between the presence of LAT and the accumulation of non-spliced RL2 transcripts, which could be related to the maintenance of latent infection in these tissues.

Dans Escherichia coli la Dam Methyl Transferase (DamMT) est responsable du transfert d’un groupement méthyle sur les adénosines situés au cœur du tétranucléotide GATC; il s’agit donc d’une activité post réplicative. Ainsi, après le passage de la fourche de réplication, le brin d’ADN nouvellement synthétisé est non méthylé – l’ADN est dit hémimethylé. L’ADN reste hémimethylé pendent une brève période - de l’ordre de la minute - avant d’être reméthylé par la DamMT. L’hypothèse de l’implication de la méthylation de l’ADN dans le contrôle général du programme de maintenance de l’ADN repose essentiellement sur cette observation, puisque l’ADN hemimethyle – exception faite de l’origine de réplication et de la région promotrice du gène dnaA – est diagnostique du passage récent de la fourche de réplication. Cette hypothèse, et le criblage phylogénomique qui en a découlé a conduit a l’identification de plusieurs gènes dont les produits sont supposes être impliqués dans la maintenance de l’ADN. yjaG est l’un de ces gènes. Il a été renomme cycC en raison des dérèglements de la progression du cycle cellulaire associés a un mutant nul de ce gène. L’étude effectuée au cours de ma thèse s’attachera à expliquer l’état actuel de nos connaissances sur la protéine CycC et de son implication dans le processus de réplication de l’ADN. Nos résultats montrent que la protéine CycC est impliquée dans la processivité de la réplication lorsqu’il y a un dommage au niveau de l’ADN. CycC spécifie une activité qui conduit à freiner les fourches de réplication, afin de prévenir des avortements des réplisomes. La surexpression de CycC bloque l’initiation de la réplication entre l’ouverture de la molécule d’ADN et le chargement de l’hélicase réplicative. Nous proposons que CycC interagisse avec le complexe réplicative et ralentit les fourches de réplication. Ce ralentissement prévient de nouvelles collisions lorsque les cellules sont dans des conditions de stress-qui cause des arrêts de la réplication. / In Escherichia coli the Dam Methyl Transferase (DamMT) is responsible for the transfer of a methyl group on the adenosine located in tetranucleotide GATC, so this is a post-replicative activity. Thus, after the passage of the replication fork, the newly synthesized DNA strand is unmethylated - DNA is called hemimethylated. DNA remains hemimethylated in a brief period - about a minute - before being reméthylé by DamMT. The hypothesis of the involvement of DNA methylation in the general control of the maintenance program of the DNA is essentially on this observation, since the hemimethylated DNA - except the origin of replication and the region dnaA gene promoter - is diagnostic of the recent passage of the replication fork. This assumption and phylogenomics screening has led to the identification of several genes whose protein are supposed to be involved in the maintenance of DNA. yjaG is one of these genes. It was renamed cycC, the cell cycle progression is deregulated with a null mutant of this gene. The study in my thesis will focus on explaining the current state of our knowledge of the cycC protein and its involvement in the process of DNA replication. Our results show that the CycC protein is involved in the processivity of replication when there is damage into the DNA. CycC specifies an activity that leads to slow replication forks to prevent abortions of replisomes. CycC overexpression blocks the initiation of replication between the open complex of the DNA at oriC and the loading of the replicative helicase. We propose that CycC interacts with the replicative complex and slows replication forks. This slowdown replication prevents new collisions when cells are under stress, causing replication stops.

Le projet de cette thèse était l'évaluation du processus de réactivation et l'étude du site actif de l'AChE inhibée par un agent neurotoxique par des méthode computationnelles. L'objectif était de guider le design rationnel de nouveau réactivateurs. Une étude initiale avec un modèle QM tronqué a indiqué la nécessité de modéliser l'environnement enzymatique pour compenser la charge du Glu334. Elle a aussi confirmé le rôle du trou oxyanionique dans la stabilisation des états de transition de la réactivation. Des simulations QM/MM de la réactivation par le réactivateur classique 2-PAM, ainsi que par deux réactivateurs au coeur aromatique non chargé ont été effectuées. Il a été démontré que le Glu202, un résidu à proximité de la triade catalytique de l'AChE, doit être protoné pour que la réactivation ait lieu. Ces simulations ont aussi montré que le réactivateur peut être déprotoné dans le site actif de l'AChE par His447. Les réactivateurs au coeur aromatique non chargé sont plus nucléophiles que la 2-PAM et l'un d'entre eux est plus aisément déprotoné dans le site actif. Nos résultats indiquent que la capacité d'un réactivateur à être facilement déprotoné est plus importante que sa nucléophilie. Enfin, un mécanisme de migration de protons a été identifié par des calculs QM/MM et EVB. Il implique deux glutamates derrière le site actif, Glu450 et Glu452. La possibilité que ces deux protons soient temporairement protonés et donc impliqués dans une migration de protons a été confirmé par des calculs CpHMD. La migration de proton passe par la N-protonation d'une liaison amide, ce qui constitue un nouveau mécanisme. / The project of this PhD was to investigate the reactivation process and the active site of nerve agent inhibited AChE by computational methodologies to gain insight about the rational design of new reactivators. An initial truncated QM model study provided some insight in the necessary compensation of Glu334 by the enzyme. It also confirmed the role of the oxyanionic hole in the stabilization of the transition state of the reactivation. QM/MM simulations of the reactivation with classical reactivator 2-PAM, as well as two non-pyridinium reactivators, were performed. It was shown that Glu202, a residue near the catalytic triad of AChE, needs to be protonated for the reactivation to occur. Those simulations also showed that the reactivator can be deprotonated in the active site of AChE by His447. Non-pyridinium reactivator were found to have a greater nucleophilicity than 2-PAM and, for one of them, to be easily deprotonated in the active site. Our results indicate that the capacity of a reactivator to be deprotonated in the active site of the enzyme is more important than its nucleophilicity. Finally, a proton relay mechanism was identified through QM/MM and EVB simulations. It involves two glutamate residues, Glu450 and Glu452, positioned behind the active site. The potential for these two residues to be transiently protonated and thus involved in a proton relay was confirmed by CpHMD simulations. This proton relay mechanism relies on the N-protonation of an amide which is a novel mechanism.

The maltreatment of children impacts individuals, communities, states, and societies. One response to the problem is the removal of children from their families, which can cause significant trauma for all involved. Moreover, the financial, legal, and emotional costs increase exponentially when subsequent re-removal, known as reactivation, occurs. Nationwide, the rate of reactivation averages just over 6%; in Arizona, the rate is significantly higher, with 11% of children being reactivated within 2 years of initial reunification. The purpose of this quantitative, non-experimental study was to determine whether poverty, ethnicity, parental substance use, parental employment, marital status, and number of children in the home is predictive of reactivation following reunification. The study was grounded in Bronfenbrenner's ecological theory and Brown's multiple risk factors model. Archival data of 627 family case files from a social service agency were analyzed using logistic regression. Results revealed that number of children was the only significant predictor, with fewer children resulting in higher reactivation rates. The lack of findings for the other predictor variables in light of extant research suggests that further research is needed to determine the unusually high rate of reactivations in this particular region. Further study may thus effect positive social change through findings that may impact educational and social welfare programs, legislative action, and enhancement of family skills training and resources.

Cette étude vise à décrire le rôle de l’héritage et de la segmentation associés au rifting pour la réactivation ainsi qu’à étudier l’importance de l’asymétrie tectonique sur l’évolution thermique syn-rift, en utilisant le système pyrénéo-cantabrique comme laboratoire naturel. L’étude de la jonction entre les segments pyrénéen et cantabrique infirme l’hypothèse d’une faille transformante de Pampelune et met en évidence une zone d’accommodation où les segments de rifts se propagent au nord et au sud des massifs basques, associée à une direction d’extension nord-sud. Lors de la convergence, cette segmentation et le niveau de découplage associé aux évaporites triasiques contrôlent fortement la réactivation ainsi que l’architecture orogénique locale. Enfin, cette étude démontre que l’asymétrie lors de l’hyper-étirement est associée à une évolution thermique asymétrique et diachrone, et souligne l’importance de l’évolution tectonique pour l’architecture thermique. / This study aims to describe the role of rift-inheritance and segmentation for reactivation and to investigate the influence of asymmetric rifting on the syn-rift thermal evolution, using the Pyrenean-Cantabrian system as a natural laboratory. The study of the Pyrenean-Cantabrian junction discards the existence of a Pamplona transform fault between the two rift segments and argues for an accommodation zone where rift segments overlap north and south of the Basque massifs in relation with north-south direction of extension. During convergence, rift segmentation and the Triassic evaporite decoupling horizon controlled the reactivation and the local orogenic architecture. Finally, this study shows that asymmetric hyperextension is associated with asymmetric and diachronous thermal evolution, and highlights the importance of understanding the tectonic evolution to define the thermal architecture.

HIV infects over 33 million people worldwide with a new infection occurring every 9 seconds. Sex is the primary mode of transmission and the majority of new infections occur during unprotected sexual contact between an HIV-infected individual and an uninfected sexual partner(s) since HIV infected individuals tend to shed virus in their genital secretions. The infectiousness of an individual is closely tied to the amount of virus in blood, which is closely associated with HIV levels shed in semen or vaginal fluid or rectal secretions. Although, Highly Active Antiretroviral Therapy (HAART) is associated with complete suppression of HIV RNA in blood to undetectable levels, the impact of HAART on semen HIV RNA levels is less clear. I evaluated the correlation between systemic and mucosal HIV-specific CD8+ T cell immune responses and HIV RNA levels in blood and semen. Overall, there was a strong positive correlation between HIV RNA levels in blood and semen. Neither systemic nor mucosal (in semen) HIV-specific CD8+ responses were associated with HIV RNA levels in blood or semen, in fact CD8+ T cell immune responses in semen correlated with increased HIV RNA levels in semen. Furthermore, inflammatory cytokines (IL-6, and IL-8) CMV levels in semen were associated with increased semen HIV RNA shedding. HAART initiation was associated with complete suppression of HIV viremia, but a significant proportion of individuals on suppressive HAART continue to shed HIV RNA in semen even after 6 months, and this isolated virus was infectious and often present at high levels (> 5000 copies/mL). Nevertheless, long-term HAART was associated with complete immune reconstitution of CD4+ T cells in the sigmoid colon of HIV-infected individuals on long-term therapy. These findings demonstrate that neither systemic nor mucosal HIV-specific CD8+ responses, when assayed with IFN- production as an endpoint, were associated with reduced HIV RNA levels in blood or semen. Semen HIV RNA levels did correlate with local inflammatory cytokines and CMV reactivation. Furthermore, despite effective HAART a significant proportion of HIV-infected men continued to shed HIV RNA in semen. However, long-term completely suppressive HAART was associated with complete immune reconstitution of the sigmoid colon.

Epstein-Barr virus (EBV) latent infection is associated with lymphoid and epithelial tumours, including nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). Since EBNA1 protein is expressed in all EBV tumours, I explored whether EBNA1 alters the cellular environment in ways that would contribute to the development of these epithelial tumours. I have shown that EBNA1 disrupts nuclear bodies (NBs) formed by the PML tumor suppressor and degrades PML proteins in a proteasome dependent manner in NPC and GC cell lines. I have verified the role of EBNA1 in disrupting PML NBs through overexpression and silencing of EBNA1 and shown that EBNA1 alone is sufficient to mediate these effects. Using EBNA1 mutants I found that USP7 and protein kinase CK2 (two enzymes that negatively regulate PML NBs) are important for EBNA1-mediated disruption of PML NBs. Furthermore, I have shown that EBNA1 localizes to PML NBs, and interacts with PML IV, which mediates the enrichment of USP7 and CK2β with PML NBs and increases CK2 phosphorylation of PML proteins, a known prerequisite for PML degradation. Consequently, functions downstream of PML were impaired in the presence of EBNA1. In particular, cells expressing EBNA1 had decreased levels of p53acetylation, p21 and apoptosis in response to DNA damage. Furthermore, DNA repair was markedly impaired in these cells, despite the fact that they survived better after induction of DNA damage than cells lacking EBNA1. In keeping with these observations, immunohistochemistry staining of GC biopsies showed that EBV-positive GC biopsies had lower PML staining compared to EBV-negative samples. These results show that EBNA1 directly affects host cell processes that would be expected to promote malignant transformation. Additionally, I have shown that EBNA1's ability to disrupt PML NBs is important for reactivation of EBV from latency; hence, is required for efficient spread of EBV from host to host.

HIV infects over 33 million people worldwide with a new infection occurring every 9 seconds. Sex is the primary mode of transmission and the majority of new infections occur during unprotected sexual contact between an HIV-infected individual and an uninfected sexual partner(s) since HIV infected individuals tend to shed virus in their genital secretions. The infectiousness of an individual is closely tied to the amount of virus in blood, which is closely associated with HIV levels shed in semen or vaginal fluid or rectal secretions. Although, Highly Active Antiretroviral Therapy (HAART) is associated with complete suppression of HIV RNA in blood to undetectable levels, the impact of HAART on semen HIV RNA levels is less clear. I evaluated the correlation between systemic and mucosal HIV-specific CD8+ T cell immune responses and HIV RNA levels in blood and semen. Overall, there was a strong positive correlation between HIV RNA levels in blood and semen. Neither systemic nor mucosal (in semen) HIV-specific CD8+ responses were associated with HIV RNA levels in blood or semen, in fact CD8+ T cell immune responses in semen correlated with increased HIV RNA levels in semen. Furthermore, inflammatory cytokines (IL-6, and IL-8) CMV levels in semen were associated with increased semen HIV RNA shedding. HAART initiation was associated with complete suppression of HIV viremia, but a significant proportion of individuals on suppressive HAART continue to shed HIV RNA in semen even after 6 months, and this isolated virus was infectious and often present at high levels (> 5000 copies/mL). Nevertheless, long-term HAART was associated with complete immune reconstitution of CD4+ T cells in the sigmoid colon of HIV-infected individuals on long-term therapy. These findings demonstrate that neither systemic nor mucosal HIV-specific CD8+ responses, when assayed with IFN- production as an endpoint, were associated with reduced HIV RNA levels in blood or semen. Semen HIV RNA levels did correlate with local inflammatory cytokines and CMV reactivation. Furthermore, despite effective HAART a significant proportion of HIV-infected men continued to shed HIV RNA in semen. However, long-term completely suppressive HAART was associated with complete immune reconstitution of the sigmoid colon.

Epstein-Barr virus (EBV) latent infection is associated with lymphoid and epithelial tumours, including nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). Since EBNA1 protein is expressed in all EBV tumours, I explored whether EBNA1 alters the cellular environment in ways that would contribute to the development of these epithelial tumours. I have shown that EBNA1 disrupts nuclear bodies (NBs) formed by the PML tumor suppressor and degrades PML proteins in a proteasome dependent manner in NPC and GC cell lines. I have verified the role of EBNA1 in disrupting PML NBs through overexpression and silencing of EBNA1 and shown that EBNA1 alone is sufficient to mediate these effects. Using EBNA1 mutants I found that USP7 and protein kinase CK2 (two enzymes that negatively regulate PML NBs) are important for EBNA1-mediated disruption of PML NBs. Furthermore, I have shown that EBNA1 localizes to PML NBs, and interacts with PML IV, which mediates the enrichment of USP7 and CK2β with PML NBs and increases CK2 phosphorylation of PML proteins, a known prerequisite for PML degradation. Consequently, functions downstream of PML were impaired in the presence of EBNA1. In particular, cells expressing EBNA1 had decreased levels of p53acetylation, p21 and apoptosis in response to DNA damage. Furthermore, DNA repair was markedly impaired in these cells, despite the fact that they survived better after induction of DNA damage than cells lacking EBNA1. In keeping with these observations, immunohistochemistry staining of GC biopsies showed that EBV-positive GC biopsies had lower PML staining compared to EBV-negative samples. These results show that EBNA1 directly affects host cell processes that would be expected to promote malignant transformation. Additionally, I have shown that EBNA1's ability to disrupt PML NBs is important for reactivation of EBV from latency; hence, is required for efficient spread of EBV from host to host.