Pathogenic Yersinia cause a manifold of diseases in humans ranging from mild gastroenteritis (Y. pseudotuberculosis and Y. enterocolitica) to pneumonic and bubonic plague (Y. pestis), while all three have a common virulence strategy that relies on a well-studied type III secretion system and its effector proteins to colonize the host and evade immune responses. However, the role of other protein secretion and/or translocation systems in virulence of Yersinia species is not well known. In this thesis, we sought to investigate the contribution of twin-arginine translocation (Tat) pathway and its secreted substrates to the physiology and virulence of Y. pseudotuberculosis. Tat pathway uniquely exports folded proteins including virulence factors across the cytoplasmic membranes of bacteria. The proteins exported by Tat pathway contain a highly conserved twin-arginine motif in the N-terminal signal peptide. We found that the loss of Tat pathway causes a drastic change of the transcriptome of Y. pseudotuberculosis in stationary phase at environmental temperature with differential regulation of genes involved in virulence, carbon metabolism and stress responses. Phenotypic analysis revealed novel phenotypes of the Tat-deficient strain with defects in iron acquisition, acid resistance, copper oxidation and envelope integrity, which we were partly able to associate with the related Tat substrates. Moreover, increased glucose consumption and accumulation of intracellular fumarate were observed in response to inactivation of Tat pathway implicating a generic effect in cellular physiology. We evaluated the direct role of 22 in silico predicted Tat substrate mutants in the mouse infection model and found only one strain, ΔsufI, exhibited a similar degree of attenuation as Tat-deficient strain. Comparative in vivo characterization studies demonstrated a minor defect for ΔsufI in colonization of intestinal tissues compared to the Tat-deficient strain during early infection, whereas both SufI and TatC were required for dissemination from mesenteric lymph nodes and further systemic spread during late infection. This verifies that SufI has a major role in attenuation seen for the Tat deficient strain both during late infection and initial colonization. It is possible that other Tat substrates such as those involved in iron acquisition and copper resistance also has a role in establishing infection. Further phenotypic analysis indicated that SufI function is required for cell division and stress-survival. Transcriptomic analysis revealed that the highest number of differentially regulated genes in response to loss of Tat and SufI were involved in metabolism and transport. Taken together, this thesis presents a thorough analysis of the involvement of Tat pathway in the overall physiology and virulence strategies of Y. pseudotuberculosis. Finally, we propose that strong effects in virulence render TatC and SufI as potential targets for development of novel antimicrobial compounds

La protéine du VIH-1 est une protéine essentielle à la transcription et à la réplication du virus. Elle a donc un rôle crucial dans la cellule infectée. On sait qu'une partie de la Tat cellulaire peut être sécrétée, malgré l'absence de séquence signal. En effet, les 2/3 de la Tat cellulaire sont exportés par la cellule T-primaire infectée. Le mécanisme de sécrétion de Tat est non conventionnel et se produit directement à travers la membrane plasmique où Tat est recrutée grâce à sa très forte affinité pour le phosphatidylinositol(4,5)-biphosphate ou PI(4,5)P2 qui est exclusivement localisé à ce niveau. La Tat extracellulaire a un effet délétère sur de nombreux types cellulaires et agit donc comme une toxine virale. Elle constitue un facteur déterminant de l'évolution vers le SIDA. En accord avec cette efficacité de sécrétion par les cellules T primaires infectées, Tat est essentiellement localisée sur la membrane plasmique des T primaires infectées par le VIH-1. Une fraction importante de Tat est donc résidente à la membrane et nous avons recherché un mécanisme pouvant expliquer cette rétention et mis en évidence la palmitoylation. Nos travaux montrent que Tat est palmitoylée, dans des cellules T ainsi que dans les ‘cibles' comme les neurones et les macrophages. Cette palmitoylation, qui inhibe la sécrétion de Tat, est réalisée sur la cystéine 31 de Tat (qui possède 7 cystéines) par l'enzyme DHHC20 avec comme cofacteurs nécessaires les immunophilines (prolyl-isomérases), Cyclophiline A et FKBP12, qui interagissent avec Tat au niveau de la membrane. Nos résultats indiquent aussi qu'en présence de Gag, la palmitoylation de Tat est inhibée. Nous pensons que l'export de la CypA dû à son encapsidation diminuerait la quantité de CypA disponible pour Tat, inhibant la palmitoylation de Tat et permettant sa sécrétion efficace par les cellules infectées. En effet, le VIH-1 encapside 250 copies de CypA/virion, le taux de CypA régulant la virulence des virions produits. Dans les cellules cibles, Tat serait fortement palmitoylée ce qui induirait sa fixation quasi irréversible au PI(4,5)P2, l'empêchant de ressortir de la cellule et permet ainsi un effet cumulatif des doses reçues par la cellules. Cette accumulation de Tat perturbe des processus membranaires dépendant du PI(4,5)P2 tels que la phagocytose et la neurosécrétion. La palmitoylation de Tat est nécessaire pour ces effets. Ces actions de la Tat extracellulaire pourraient participer au développement des infections opportunistes et des troubles neurologiques observé lors du SIDA. / HIV-1 Tat is a small protein that is required for viral transcription and multiplication. It thus has a crucial role in the infected cell. It was known that Tat can be secreted despite its lack of signal sequence. In fact 2/3 of cellular Tat are exported by infected primary T-cells. The unconventional secretion of Tat relies on its interaction with phosphatidylinositol(4,5)-biphosphate or PI(4,5)P2, a lipid that is concentrated within the inner leaflet of the plasma membrane and is strictly required for Tat secretion. Exogenous Tat has deleterious effects on several cell types, indicating that extracellular Tat is involved in evolution to AIDS. Consistent with this secretion efficiency, Tat is mainly localized at the plasma membrane of primary T-cells infected by HIV-1. A large fraction of Tat is resident at the membrane and we looked for a mechanism that could explain this retention and discovered that Tat is palmitoylated. Our studies show that Tat is palmitoylated, both in T-cells and also in ‘target' cells such as neurons and macrophages. Tat palmitoylation inhibits its secretion and is performed on Tat cysteine 31 (Tat has seven cyteines) by the enzyme DHHC20 using immunophilins (prolyl ismerases), Cyclophilin A (CypA) and FKPB12, as cofactors. Our results also indicate that the presence of Gag inhibits Tat palmitoylation. We believe that the export of CypA due to its encapsidation will make less CypA available for Tat, thereby inhibiting Tat palmitoylation. Indeed, HIV-1 encapsidates 250 copies of CypA/virion and the amount of CypA regulates the virulence of produced virions. In target cells, Tat is strongly palmitoylated and this modification induces its almost irreversible binding to PI(4,5)P2, preventing its secretion and allowing cumulative effect of minute Tat doses.Tat palmitoylation enables Tat to severely inhibit various PI(4,5)P2-dependent processes such as phagocytosis and neurosecretion. These effects of extracellular Tat likely contribute to the development of opportunistic infections and neurological disorders observed during AIDS.

La transcription est une étape fondamentale dans l'expression des gènes. Cependant, elle reste incomplètement caractérisée dans les cellules vivantes. Pour mieux comprendre la dynamique de la transcription, notre laboratoire a amélioré le système de marquage d'ARN en utilisant la séquence codante pour MS2, facilement fusionnée avec le promoteur d'intérêt et inséré copie unique dans deux lignées cellulaires HeLa cellules. Cette construction permet une vue quantitative de la transcription, a l’échelle de la molécule unique, en temps réel. Nous avons trouvé que le VIH-1 est transcrit par des groupes de polymérases nominés convois. La transcription oscille de manière aléatoire avec des périodes actives (ON) et inactives (OFF) et est contrôlée indépendamment.Sur la base de cette découverte, nous avons étudié: (i) comment l'architecture de différents promoteurs de mammifères contrôle la cinétique transcriptionnelle; et (ii) le rôle du transactivateur transcriptionnel (Tat), le régulateur principal de la transcription du VIH-1, dans les cellules vivantes. Pour traiter ces questions, une nouvelle méthode de modélisation a été établi, combinant l'information des fluctuations transcriptionnelles avec différentes résolutions temporelles. Cela a donné une vue complète et précise du processus stochastique, décrit par le modèle de Markov. Cinq des six promoteurs de mammifères pourraient être définis par trois états, probablement contrôlés par des mécanismes différents. Le passage entre ces états est défini par les constantes de vitesse et l'écart entre eux pourrait potentiellement expliquer la différence dans la quantité d'ARN produit. De manière intéressante, nous avons constaté que les taux de passage entre les états inactifs et profondément silencieux sont la marque distinctive de différents promoteurs, suggérant que les événements cruciaux définissant les profils transcriptionnels sont en fait des événements pré-transcriptionnels.Pour étudier le rôle de Tat, des lignées cellulaires contenant un rapporteur du VIH-1 et une quantité différente de Tat ont été produites. Avec cette approche décrite ci-dessus, nous avons montré que Tat, précédemment caractérisé en tant qu'acteur dominant dans la libération de la polymérase en pause, agit longtemps avant que la transcription soit initiée. Ces résultats frappants apportent de nouvelles perspectives concernant la dynamique transcriptionnelle du VIH-1 contrôlée par Tat. / Transcription is a fundamental step in gene expression. However, it is incompletely characterized in single living cells. To address this question, our laboratory developed the improved RNA tagging system using MS2-binding protein that could easily be fused with the promoter of interest inserted in a single copy in HeLa cell lines. This construct allows quantitative, single molecule view of the transcription in a real time. We have found that HIV-1 is transcribed by groups of closely spaced polymerases referred as convoys. The transcription oscillates randomly between active (ON) and inactive (OFF) periods that are controlled independently.On the basis of this discovery, we further investigated: (i) how architecture of different mammalian promoters controls the transcriptional kinetics; and (ii) the role of transcriptional transactivator (Tat), the master regulator of in HIV-1 transcription in living cells. To address this, new pipeline for the quantification was established, combining the information of transcriptional fluctuations with different temporal resolutions. This gave the full and precise view of the stochastic switching, described by the Markov model. Five of six mammalian promoters could be defined by three states, probably controlled by different mechanisms. Switching between them is defined by the rate constants and the discrepancy among them could potentially explain the difference in the amount mRNA produced. Interestingly, we found that switching rates between inactive, deeply silent states are the hallmark of different promoters, suggesting that the crucial events defining the transcriptional profiles are in fact pre-transcriptional events.To address the role of Tat, cell lines containing HIV-1 reporter and different amounts of Tat were produced. With the above described approach, we found that Tat, previously characterized as dominant player in the release of the paused polymerase, actually acts long before the transcription is initiated. These striking results bring new insights of HIV-1 transcriptional dynamics controlled by Tat.

Thesis (Ph. D.)--Cornell University, August, 2007. / Vita. Includes bibliographical references (leaves 165-176).

Les symptômes de conversion, que Freud a jusqu’à la fin de sa vie qualifiés de « mystérieux », continuent aujourd’hui de susciter de nombreux questionnements. L’un d’entre eux concerne le rapport qu’ils entretiennent avec la douleur : si les douleurs sans étiologie médicale retrouvée sont parfois considérées comme des symptômes de conversion au même titre que les symptômes sensori-moteurs, elles semblent pouvoir correspondre à des pathogénies variées (névrose d’angoisse, hypocondrie, manifestations psychosomatiques…). À l’appui de ce constat, nous faisons l’hypothèse que là où les symptômes sensori-moteurs représenteraient le résultat d’un mécanisme de conversion pouvant être sous-tendu par des registres conflictuels divers mais impliquant nécessairement une dimension d’expression symbolique de ces conflits, les symptômes douloureux pourraient quant à eux s’inscrire le long d’un continuum allant d’un pôle conversif, marqué par cette valeur symbolique, à un pôle non conversif, porteur d’une valeur moins symbolique qu’économique – étant cependant entendu qu’il existerait des possibilités de passage d’un pôle à l’autre. Au pôle conversif, les symptômes seraient sous-tendus par de bonnes capacités d’élaboration psychique de l’excitation, des enveloppes psychiques relativement solides et souples et de bonnes capacités à s’appuyer sur un objet psychique interne ; au pôle non conversif, ils seraient sous-tendus par des difficultés d’élaboration psychique de l’excitation, des enveloppes psychiques marquées par la fragilité et des difficultés à s’appuyer sur un objet psychique interne.Afin de mettre nos hypothèses à l’épreuve, nous avons, dans le cadre d’une vaste recherche mise en place par le Dr Lisa Ouss à l’hôpital Necker-Enfants malades, rencontré 27 enfants et adolescents âgés de 8 à 15 ans et présentant un trouble de conversion ou un trouble douloureux (diagnostics DSM-IV-TR) ; 8 présentaient des symptômes exclusivement sensori-moteurs, 11 des symptômes à la fois sensori-moteurs et douloureux, et 8 des symptômes exclusivement douloureux. Nous avons recueilli et analysé les données issues des entretiens et des épreuves projectives (Rorschach et TAT) de chaque patient dans une perspective psychodynamique, puis avons procédé à des comparaisons inter-groupes.Les résultats montrent que les patients présentant des symptômes sensori-moteurs (accompagnés ou non de symptômes douloureux) disposent de capacités d’élaboration psychique et de capacités à s’appuyer sur un objet psychique interne significativement meilleures que les patients présentant des symptômes exclusivement douloureux, ce qui tend à confirmer notre hypothèse de départ ; il n’existe en revanche pas de différence significative entre les groupes en matière de qualité des enveloppes psychiques. Les résultats montrent en outre que les (pré)adolescents (11-15 ans) disposent de capacités d’élaboration psychique et de capacités à s’appuyer sur un objet psychique interne significativement meilleures que les enfants en période de latence (8-10 ans), et que les filles disposent de capacités d’élaboration psychique, de capacités à s’appuyer sur un objet psychique interne et d’une solidité des enveloppes psychiques significativement meilleures que les garçons. / Conversion symptoms have been considered “mysterious” by Freud until the end of his life. They still raise numerous questions. One of them deals with their relation to pain: whereas medically unexplained pain symptoms are sometimes considered to be conversion symptoms in the same way as sensorimotor symptoms, they can correspond to various pathogeneses (anxiety neurosis, hypocondria, psychosomatic symptoms...).We thus hypothesize that whereas sensorimotor symptoms result from a conversion mechanism (which might be underlied by various conflicts but necessarily imply a symbolical expression of these conflicts), pain symptoms, as for them, may range along a continuum extending from a conversive end characterized by this symbolical dimension to a non conversive end characterized by a more economical than symbolical dimension (including possibilities of moving from an end to another). On the conversive end, symptoms would be underlied by good abilities in psychical elaboration of excitation, strong and supple psychical envelopes and good abilities to rely on a psychical internal object. On the non conversive end, symptoms would be underlied by difficulties in psychical elaboration of excitation, fragile psychical envelopes and difficulties to rely on a psychical internal object.In order to test our hypotheses, we have met 27 children and adolescents (8 to 15 years old) diagnosed as having a conversion disorder or a pain disorder (DSM-IV-TR diagnoses), in the framework of a wide research set up by Dr Lisa Ouss at the Necker-Enfants malades hospital (Paris). Among these patients, 8 had exclusively sensorimotor symptoms, 11 had both sensorimotor and pain symptoms, and 8 had exclusively pain symptoms. We have gathered and analysed the data of clinical encounters and projective tests (Rorschach and TAT) of each patient in a psychodynamic way. We then have made intergroup comparisons.The results show that patients with sensorimotor symptoms (accompanied or not by pain symptoms) have significantly better abilities in psychical elaboration of excitation and significantly better abilities to rely on a psychical internal object than patients with exclusively pain symptoms, which tends to confirm our initial hypothesis. Yet there is no significant difference between the groups as far as psychical envelopes’ quality is concerned. The results also show that (pre)adolescents (11 to 15 years old) have significantly better abilities in psychical elaboration of excitation and significantly better abilities to rely on a psychical internal object than latent children (8 to 10 years old), and that girls have significantly better abilities in psychical elaboration of excitation, significantly better abilities to rely on a psychical internal object and significantly stronger psychical envelopes than boys.

This study attempted to determine the relationship between fantasy aggression and behavioral aggression, and whether fantasy aggression measured by the Thematic Apperception Test is related to behavioral aggression. Participant TAT protocols from psychology clinic files were scored for fantasy aggression, and these scores were correlated with self-reported presence or absence of behavioral aggression. The scoring system used was a blend of popular aggression scales used in the 1960s and newer theory. Other variables that were examined were story length and gender in relation to the measured amount of fantasy and behavioral aggression.

Myelin pallor is frequently reported in HIV patients, and can occur in the CNS prior to other evidence of disease process. Our exploratory studies showed that oligodendrocytes (OLs) are direct targets of HIV-1 Tat (transactivator of transcription). Tat induces a dose-dependent increase of intracellular Ca2+ level ([Ca2+]i) in cultured murine OLs, which can be attenuated by ionotropic glutamate receptor (iGluR) antagonists MK801 and CNQX. The Tat-induced [Ca2+]i increase leads to increased death in immature (O4+, MBP-), but not mature (O4+, MBP+) OLs, over 96 h. In addition, Tat-induced [Ca2+]i increase also reduced myelin-like membrane production by mature OLs. Calcium/Calmodulin dependent kinase IIβ (CaMKIIβ) and glycogen synthase kinase 3β (GSK3β) have been known to regulate differentiation, myelination, and apoptosis in OLs. Since both CaMKIIβ and GSK3β are important downstream modulators of [Ca2+]i change, we hypothesized that the detrimental effects of Tat on immature/mature OL viability and function are mediated via CaMKIIβ and GSK3β activation. Our results showed that Tat activates both CaMKIIβ and GSK3β in immature OLs, but only activates CaMKIIβ in mature OLs. MK801 completely blocks Tat-induced CaMKIIβ and GSK3β activation in both immature and mature OLs, while CNQX blocks GSK3β activation, but has only a partial effect on CaMKIIβ activity. Blocking iGluRs or inhibiting GSK3β both rescue Tat-induced immature OL death, but only MK801 reverses the membrane injury in mature OLs. Together, these data strongly suggest that 1) activity of CaMKIIβ and GSK3β in OLs can be regulated by Tat-induced iGluRs activation and 2) OLs at different developmental stages show different responses to Tat, possibly due to activation of different signaling pathways.

The Twin arginine translocation pathway translocates fully folded proteins across cellular membranes and is only utilised by proteins that fold before translocation. It is a unique process that is found in many bacteria, archaea and also in plant chloroplasts. Investigation of the bacterial and thylakoidal systems has revealed much of the substrates and the components involved in their translocation. Unfortunately, there are still many unanswered questions such as how substrates are directed to the membrane and the actual mechanism of translocation. This thesis specifically investigates the Tat pathway of halophilic and thermophilic archaea. To date, there has been a lack of research into the archaeal Tat pathway and it is possible that there are unique adaptations because of the extreme environments that these organisms inhabit. Chapter 3 specifically investigates the thermophiles Sulfolobus solfataricus and Sulfolobus tokodaii and attempts to purify their Tat complexes. By doing so it was hoped to learn more about the Tat components and their interactions. Further experiments were also performed to determine if the two S. solfataricus Tat operons provide specificity to the Tat substrates that translocate. Four separate areas of the Tat pathway of halophilic archaea (haloarchaea) were investigated in Chapters 4-7. Firstly, site-directed mutagenesis was used to analyse the signal peptides of haloarchaeal Tat substrates in more detail. Consequently, the resulting data led to the use of bioinformatics to analyse the Haloarchaeal signal peptide. The bioenergetics of the Tat system was then determined by analysing the effect of a variety of ionophores on translocation of the Tat substrates AmyH and SptA. Finally, a series of folding and stability assays were used to increase our understanding of AmyH, which could provide further information on why this protein, like many other haloarchaeal proteins, requires the Tat pathway for translocation.

Studies on repeated exposure to opioids have been carried out for decades yet the mechanisms for certain phenomena such as tolerance are still not fully understood. Furthermore, different medications, such as frequently prescribed benzodiazepines, or different disease states, such as HIV, have their own effects and interactions with chronic opioid exposure that are not fully understood. The overall objective of this dissertation was to investigate the complexities of chronic opioid exposure and how different disease states and medications may modulate the effects of chronic opioids. Our findings demonstrate that the administration of diazepam, at doses that are not antinociceptive or have any motor effects, reverse both antinociceptive and locomotor tolerance to orally active opioids. These doses of diazepam did not potentiate the acute effects of these prescription opioids. We also found that HIV-1 Tat expression significantly attenuated the antinociceptive potency of acute morphine in non-tolerant mice while not significantly altering the antinociceptive tolerance to morphine. Consistent with this, Tat attenuated withdrawal symptoms among morphine-tolerant mice. Pretreatment with maraviroc, a CCR5 antagonist blocked the effects of Tat, reinstating morphine potency in non-tolerant mice and restoring withdrawal symptomology in morphine-tolerant mice. Protein array analyses revealed only minor changes to cytokine profiles whether morphine was administered acutely or repeatedly; however, 24 h post repeated morphine administration, the expression of several cytokines was greatly increased. Tat further elevated levels of several cytokines and maraviroc pretreatment attenuated these effects. With the understanding that gap junctions may be involved in both HIV-Tat effects on opioid antinociception as well as tolerance, we investigated the role of gap junctions in opioid antinociceptive tolerance. We observed that carbenoxolone, a gap junction inhibitor, administered systemically attenuated the development of opioid antinociceptive tolerance. Furthermore, we observed a small percentage of carbenoxolone in brain tissue compared to the amount found in blood, suggesting a peripheral site of action. Finally, we show preliminary evidence that in vivo administration of carbenoxolone is able to attenuate tolerance to morphine in DRG neurons.

Les protéines solubles sont généralement faiblement immunogènes, ce qui constitue une limite pour le développement de vaccins sous unitaires à base de protéines. Mes travaux de thèse ont eu pour objectif de décrypter certains mécanismes moléculaires et cellulaires qui contribuent à l'immunogénicité et d'en tirer partie pour développer des approches originales permettant d'améliorer la capacité des protéines à déclencher la réponse immunitaire. Pour cela, j'ai principalement utilisé le transactivateur transcriptionnel (Tat) du VIH-1. J'ai montré que l'oligomérisation de Tat permet à un mécanisme de collaboration B-TH-2 d'induire la réponse immunitaire en absence d'adjuvant. J'ai identifié le déterminant minimal responsable de l'effet et montré qu'il confère la propriété adjuvante à d'autres antigènes. J'ai ensuite montré que la présentation aux cellules T restreinte aux CMH I et CMH II est accrue lorsque les protéines sont dotées de la capacité à lier des sucres sulfatés d'expression ubiquitaire: les héparanes sulfate. Ces travaux ont permis de définir de nouvelles approches pour améliorer l'immunogénicité de protéines susceptibles d'être intégrées dans des préparations vaccinales.