Les infections urinaires (UTI) sont la forme la plus courante des infections extra-intestinales causées dans 90 % des cas par la bactérie Escherichia coli. Plus de 100 000 personnes sont hospitalisées chaque année aux États-Unis en raison de complications rénales avec des risques de septicémies. La gestion des infections urinaires (UTIs) repose depuis longtemps sur le principe de la thérapie par les antibiotiques. Généralement ces derniers détruisent les bactéries en s'attaquant à leur structure cellulaire et en interférant profondément le cycle cellulaire normal. De ce fait, ces infections bactériennes deviennent une grande problématique au niveau mondial. La surconsommation et la mauvaise utilisation d'antibiotiques ont involontairement produit de nouvelles souches de bactéries résistantes qui ont développé des stratégies de défenses vis-à-vis de ces médicaments et qui peuvent donner naissance à des infections non traitables pouvant mener à la mort. Une nouvelle thérapie serait de mise pour l'éradication de ce phénomène en contre-carrant cette résistance par l'inhibition du premier contact entre la bactérie et les cellules et représenterait une alternative séduisante. L'adhésion bactérienne médiée par les interactions multivalentes protéines-glucides constitue le premier événement de l'infection à la surface urothéliale. La bactérie E. coli comportant des lectines Fim H comme protéines de surface sur leurs pili ne déroge pas à cette règle en se liant spécifiquement aux α-D-mannosydes des récepteurs d'uroplakines pour adhérer aux cellules urothéliales. Considérant l'importance de ce phénomène, notre travail s'est porté sur la synthèse de macromolécules glycosylées servant de mimétiques multivalents. Ces inhibiteurs synthétiques potentiels de l'adhésion bactérienne ont pu être obtenus de façon monodisperse, avec une valence controlée, sous la forme de «glycoclusters» ou «glycodendrimères». En effet, les dendrimères sont des macromolécules très bien définies avec une uniformité et une symétrie pouvant offrir une grande surface fonctionnelle multivalente. Concernant l'approche synthétique, la voie divergente a été empruntée, partant du coeur pour aboutir à des terminaisons fonctionnelles à la surface. Cette croissance dendritique a pu être développée et contrôlée avec l'utilisation d'éléments branchés de type aliphatique ou aromatique en utilisant des réactions efficaces de substitution nucléophile, de couplage peptidique, et de diazotransfert. La fonctionnalisation des dendrimères exhibant des épitopes a été effectuée avec la chimie « click » (ou CuAAc) sur trois dérivés mannosylés différents contenant les fonctions chimiques complémentaires requises. Ainsi, ces structures multivalentes mannosylées ont été synthétisées avec de hauts rendements, avec une variété architecturale controlée dont l'influence a été appréciée lors des investigations biologiques. Dans ce contexte, leur potentiel en tant que ligands synthétiques a été évalué par des tests préliminaires de turbidimétrie avec la lectine végétale Con A et des investigations plus avancées impliquant des tests d'inhibition sur des cellules cancéreuses U87 et des bactéries E. coli. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : E. coli uropathogénique, UTI, Anti-adhésines, Inhibition de l'adhésion bactérienne, Fim H, Mannosides, Multivalence, Glycoclusters, Glycodendrimères, Couplage pepridique, Diazotransfert, Chimie click, Tests d'inhibition d'hémagglutination, FACS.

Les souches uropathogéniques de Escherichia coli (UPEC) sont la cause de 80% des infections du tractus urinaire qui touchent une femme sur deux au moins une fois dans leur vie. Malgré l'utilisation d'antibiotiques, la récurrence du pathogène demeure un problème qui conduit à la cystite urinaire et la pyélonéphrite. La colonisation est favorisée par l'adhésion bactérienne à l'uroépithélium du tractus urinaire. Cette adhésion se fait par la reconnaissance moléculaire entre la partie glycosylée de l'uroplakine Ia et le domaine de reconnaissance aux carbohydrates (CRD) des pili bactériens (fimbriae de type 1). Récemment, la portion adhésine de 158 a.a. de la sous-unité FimH du pili de type 1 a été clonée (UPEC-JI6) et cristallisée en présence d'un ligand de haute affinité: le butyl a-D-mannopyranoside. La structure du complexe protéine-carbohydrate montre la proximité de quatre résidus d'acides aminés formant une couronne hydrophobe autour de j'aglycone (IIel3, Tyr48, Ile52, Tyrl37) dont deux résidus aromatiques électrodonneurs qui permettraient potentiellement d'optimiser l'interaction entre la lectine et des mannosides synthétiques en tirant profit des interactions hydrophobes et du recouvrement n-aromatique. Les dérivés O-aryl mannosides montrent des affinités de l'ordre du micromolaire pour la FimH. Nous avons donc réalisé la synthèse de dérivés a-D-mannosidiques possédant une extrémité aglyconique hydrophobe (espaceur aliphatique) munie de noyaux hétéro-aromatiques (triazole), en plus de quelques O-aryl mannosides. La synthèse de trois mini-clusters à terminaisons mannosidiques a aussi été effectuée par Click-Chemisty pour apprécier l'effet de multivalence en synergie avec les propriétés liantes de ces nouveaux analogues triazoliques. Les tests biologiques (voir en annexe) en résonance plasrnonique de surface (SPR) ont révélé des Kd allant de 651 à 27.7 nM pour ces nouveaux analogue monomérique, tandis que l'affinité par mannoside est augmentée jusqu'à 5.9 à 7.4 fois par l'effet de multivalence pour les clusters synthétisés (Kd de 14 et 8 nM). Finalement, une méthodologie de synthèse asymétrique de nouveaux synthons C-mannosidiques d'importance en chimie des sucres a été réalisée par allylboration diastéréosélective. Le développement de cette méthodologie, combinée à la métathèse de fermeture de cycle constitue un premier pas vers la synthèse de pseudodisaccharides C-liés d'un genre nouveau. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Chimie thérapeutique, UTI, E. coli, lectine, FimH, D-mannosides, affinités, Click-Chemistry, triazole, multivalence, cluster, C-glycosides, allylboration asymétrique, disaccharides C-liés, métathèse de fermeture de cycle, Grubbs, azidotranfert, modélisation moléculaire.

The current study used a probabilistic Quantitative Microbial Risk Assessment (QMRA) framework to describe the change of E. coli O157:H7 concentration in lettuce through a foodborne pathway, to develop a predictive model for risk estimation for E. coli O157:H7 infection associated with lettuce. The model consisted of a series of pathogen-associated events including initial contamination, growth during cooling, cold storage and distribution, disinfection (chlorine, gaseous chlorine dioxide and gamma irradiation), and dose response after consumption. A modified Baranyi growth model was proposed which described the initial physiological state of E. coli O157:H7 as a function of the initial temperature. The modified Baranyi growth model was used to predict E. coli O157:H7 growth under realistic time-temperature profiles, accounting for the time dynamics of temperature fluctuation. The risk assessment model was constructed in an Excel spreadsheet and Monte Carlo uncertainty analysis was simulated using Crystal Ball. The results in the current study showed that temperature control was the key measure for minimizing the risk of E. coli O157:H7 infection associated with lettuce. Disinfecting contaminated lettuce using the hypothetical methods examined in the study had limited effectiveness in risk reduction. Temperature abuse occurring before or after the hypothetical disinfections significantly diminished the disinfection effect and contributed to increased risk. Of all simulated scenarios, the lowest risk was associated with adequate temperature control and irradiation (44 infections per 1000 consumptions [95%: 94 infection per 1,000 consumption; 5%: 5 infections per 1,000 consumption]). The model can be used to explore the public health impact of other potential strategies that can be adopted to minimize the risk of E. coli O157:H7, while taking into account the possible amplification of pathogen through the food chain.

Research from the U.S. suggests that maternal HIV-infection negatively impacts children's psychosocial functioning and that resources (e.g., the parent-child relationship) positively influence their adjustment to maternal HIV-infection. Although HIV-infection in South Africa is most prevalent among Black South African women, there is limited research examining its impact on their children. In addition, as these children are exposed to numerous socio-cultural stressors beyond those associated with HIV-infection, they are at particular risk for psychosocial difficulties. This study had two aims: 1) to evaluate whether maternal HIV-infection confers risk for psychosocial difficulties (i.e., internalizing and externalizing behaviors) among Black South African children; and, 2) to examine potential protective resources for children of HIV-infected mothers that could ideally be addressed through appropriate community-level interventions. Three categories of resources were considered: material (familial economic stability); maternal (maternal psychological functioning; maternal social support); and, caregiving (the parent-child relationship; quality of the caregiver - co-caregiver relationship). Participants included women who self-identified as HIV-infected or non-infected and who were the biological mother of a child aged 11-16. Results indicated that there were no psychosocial adjustment differences between the two groups of children. The lack of differences suggests that in the context of the constellation of stressors Black South African children face, maternal HIV-infection may not serve as a unique stressor for psychosocial adjustment difficulties. However, the lack of differences should not be construed to mean that a child whose mother is HIV-infected is not affected his/her mother's diagnosis. Maternal HIV-infection is a complex phenomenon that warrants further study among Black South African children. The results did not illuminate any resources that were particularly salient to the children of HIV-infected mothers; rather, variables salient to all children were identified, notably economic stability, maternal depression, family social support, the parent-child relationship, and conflict in the mother- co-caregiver relationship. Given the overall risk present in the lives of Black South African children beyond maternal HIV-infection, it appears important to address the needs of all children. This study provides important information about individual and family-level variables that could be emphasized in family interventions with the population as a whole.

The purpose of this work was to investigate the adherence to basic hygiene routines among staff and to investigate if there is any difference between the adherences in diverse professions. The purpose was also to study how staff rates their own adherence to current guidelines. Data for the study were collected through observations and questionnaires. Altogether 35 people were observed and 15 answered the questionnaire. Maximum adherence was found in the category of "hand disinfection after patient contact" with a score of 96%. In the observation task “proper use of protective apron” a total adherence of 86 % was found. The observations in the category of "hand disinfection before patient contact" showed an adherence of 74 %. In the observation category "proper use of gloves" a total adherence of 52% was found. The greatest difference between professions was seen in the observation category of "proper use of gloves", where physicians’ observed adherence was 100% and the nurses observed adherence was 48%. In the observation category "proper use of apron” adherence varied between 76-91%. Adherence to “hand disinfection before patient contact” are lower in comparison with adherence to “hand disinfection after patient contact”. An awareness of this is seen in the responses also, with the majority indicating that their own routine deficiencies in this particular moment. Otherwise the compilation of the responses shows that staff rates their adherence lower than the observed adherence. We believe that our presence may have affected the staff during the observations with the consequence that adherence to the basic hygiene routines influenced in a positive direction. / Syftet med detta arbete var att undersöka följsamhet till basala hygienrutiner hos personal på en vårdavdelning samt undersöka eventuella skillnader i följsamhet mellan olika yrkeskategorier. Syftet med studien var också att undersöka hur personalen skattar sin egen följsamhet till gällande riktlinjer. Data till studien insamlades genom observationer och enkäter. Totalt observerades 35 personer och enkäten besvarades av 15 personer. Högst observerad följsamhet fanns i kategorin ”handdesinfektion efter patientkontakt” med ett resultat på 96 %. Observationsmomentet ”korrekt användning av skyddsförkläde” visade en total följsamhet på 86 %. Observationerna i kategorin ”handdesinfektion före patientkontakt” visade en följsamhet på 74 %. I observationsmomentet ”korrekt användning av handskar” fanns en total följsamhet på 52 %. Störst skillnad mellan personalkategorierna sågs i observationskategorin ”korrekt användning av handskar” där läkarnas observerade följsamhet var 100 % och undersköterskornas observerade följsamhet var 48 %.  I observationsmomentet ”korrekt användning av skyddsförkläde” varierade följsamheten mellan 76 – 91 %. Personalens observerade följsamhet till ”handdesinfektion före patientkontakt” är lägre i jämförelse med den observerade följsamheten till ”handdesinfektion efter patientkontakt”. En medvetenhet om detta syns i enkätsvaren där flertalet anger att deras egen rutin brister i just detta moment. I övrigt visar sammanställningen av enkätsvaren att personalen skattar sin följsamhet lägre än den observerade följsamheten. Vi anser att vår närvaro kan ha påverkat personalen vid observationerna med konsekvensen att följsamheten till de basala hygienrutinerna påverkats i positiv riktning.

The current study used a probabilistic Quantitative Microbial Risk Assessment (QMRA) framework to describe the change of E. coli O157:H7 concentration in lettuce through a foodborne pathway, to develop a predictive model for risk estimation for E. coli O157:H7 infection associated with lettuce. The model consisted of a series of pathogen-associated events including initial contamination, growth during cooling, cold storage and distribution, disinfection (chlorine, gaseous chlorine dioxide and gamma irradiation), and dose response after consumption. A modified Baranyi growth model was proposed which described the initial physiological state of E. coli O157:H7 as a function of the initial temperature. The modified Baranyi growth model was used to predict E. coli O157:H7 growth under realistic time-temperature profiles, accounting for the time dynamics of temperature fluctuation. The risk assessment model was constructed in an Excel spreadsheet and Monte Carlo uncertainty analysis was simulated using Crystal Ball. The results in the current study showed that temperature control was the key measure for minimizing the risk of E. coli O157:H7 infection associated with lettuce. Disinfecting contaminated lettuce using the hypothetical methods examined in the study had limited effectiveness in risk reduction. Temperature abuse occurring before or after the hypothetical disinfections significantly diminished the disinfection effect and contributed to increased risk. Of all simulated scenarios, the lowest risk was associated with adequate temperature control and irradiation (44 infections per 1000 consumptions [95%: 94 infection per 1,000 consumption; 5%: 5 infections per 1,000 consumption]). The model can be used to explore the public health impact of other potential strategies that can be adopted to minimize the risk of E. coli O157:H7, while taking into account the possible amplification of pathogen through the food chain.

Executive functions are important cognitive processes critical for survival. Damage to the prefrontal cortex impairs executive functions, such as working memory, decision making and set-shifting. Interestingly, patients diagnosed with different psychiatric disorders are also impaired in executive functions, especially in the set-shift domain, often measured by the Wisconsin Card Sorting Task (WCST). Set-shifting is an essential cognitive process, in that it allows the individual to suppress non-reinforcing strategies and engage in new rewarding strategies. To date, little is known about the etiology of executive dysfunction in psychiatric disorders. However, some epidemiological and serological experiments have shown strong correlations between prenatal infection and the increased risk to develop psychiatric disorders in the adult offspring. One study found that schizophrenic patients pre-exposed to a prenatal infection perseverated more during the WCST, than non-pre-exposed patients. Despite these findings, there are still numerous limitations (e.g., ethical concerns) when conducting these studies. Thus, animal models are important and can further elucidate the etiology of executive dysfunctions in psychiatric disorders. Prenatal infection animal models have consistently shown that inflammation during gestation in rodents induces behavioural, anatomical and cognitive changes in the adult offspring similar to psychiatric patients. However, no studies have investigated the effects of prenatal infection on set-shifting in the adult offspring. Therefore, the present thesis examined whether prenatal treatment with PolyI:C (a viral mimetic) during middle/late gestation of the rat would induce cognitive inflexibilities (i.e., set-shifting and reversal learning in an operant based task analogous to the WCST) in the adult male and female offspring. The results showed PolyI:C male offspring perseverated during the set-shift but had fewer regressive errors during the reversal learning day. PolyI:C treated female offspring were not impaired during any of the test days; however, females were slower to respond to the lever and required more training when compared the male rats. Taken together, these results give support for prenatal infection in inducing cognitive inflexibility, by potentially altering the PFC in the adult offspring. MS-based thesis: Zhang, Y., Cazakoff, B. N., Thai, C. A., & Howland, J. G. (2011). Prenatal exposure to a viral mimetic alters behavioural flexibility in male, but not female, rats. Neuropharmacology, [epub ahead of print]. doi:10.1016/j.neuropharm.2011.02.022

<p>Naturally occurring CD4+CD25+Foxp3+ regulatory T cells (TReg) are a cell lineage that develops in the thymus and exits to the periphery, where they represent 5-10% of the peripheral CD4+ T cell population. Phenotypically, they are characterized by the expression of the cell surface markers CD25, as known as the IL-2 receptor alpha chain, glucocorticoid-induced tumor necrosis factor receptor (GITR), and cytotoxic T-lymphocyte antigen-4 (CTLA-4), as well as forkhead box P3 (Foxp3), a transcription factor considered to be the most specific TReg marker. Functionally, TReg cells are defined by their ability to suppress the activation of multiple cell types including CD4+ and CD8+ T cells, B cells, natural killer (NK) cells, and dendritic cells (DCs). Suppression can be achieved by the production of immunosuppressive cytokines or direct cell-to-cell contact, with these mechanisms directly affecting suppressed cells or indirectly affecting them by modulating antigen presenting cells (APCs). The suppressive abilities of TReg cells are crucial in maintaining dominant tolerance--the active, trans-acting suppression of the immune system for the prevention of autoimmune diseases. In addition to preventing autoimmune diseases, studies have also demonstrated critical roles for TReg cells in down-modulating anti-tumor immunity, suppressing allergic diseases, such as asthma, and achieving transplant tolerance. Recent studies have also demonstrated roles for TReg cells during pathogen infection, which will be the focus of this thesis.</p><p>Studies examining TReg cells during infection have largely focused on chronic infection models. These studies have shown that TReg cells can affect responses to pathogens in various ways that can be beneficial or detrimental for either the host or the invading pathogen. In some infections, TReg cells downregulation effector responses, which can lead to pathogen persistence and, in some cases, concomitant immunity. TReg cell-mediated suppression can also reduce immunopathology at sites of infection, which can occur as a result of a vigorous anti-pathogen immune response. </p><p>In contrast to chronic infection, how TReg cells behave and function following acute infections remains largely unknown as, to date, very few studies have been conducted. Current work with acute infection models has indicated that TReg cells affect immune responses in some acute infection models, but not in all. Furthermore, the results of these studies have implicated that current approaches to examine TReg cells during acute infection by depleting the total TReg cell repertoire, as opposed to targeting pathogen-specific TReg cells, may not be ideal. Finally, it is unclear what happens to activated TReg cells following the resolution of infection. </p><p>Due to the lack of knowledge about the role of pathogen-specific TReg cells during acute infection, we sought to employ a different approach to address some of the outstanding questions in the field. Here, we utilized CD4+ non-TReg and TReg cells from T cell receptor (TCR) transgenic mice that recognize a pathogen-specific epitope found in three different models of acute viral infection: recombinant vaccinia virus, recombinant adenovirus, and influenza. Using this model system, we were able to track pathogen-specific TReg cells following acute viral infection to determine their kinetics during the course of infection, as well as their influence on CD4+ non-TReg cells during different times after infection. We also employed major histocompatibility complex (MHC) Class II tetramer technology to track the fate of endogenous pathogen-specific TReg cells following infection with influenza. </p><p>Using these models systems, we show that pathogen-specific TReg cells can be activated and expand upon acute viral infections in vivo. The activated TReg cells then contract to form a "memory" pool after resolution of the infection. These "memory" TReg cells expand rapidly upon a secondary challenge, secrete large amounts of IL-10, and suppress excessive immunopathology, which is elicited by the expansion of non-TReg cells, via an IL-10-dependent mechanism. The work presented in this thesis reveals a previously unknown "memory" TReg cell population that develops after acute viral infections and may help design effective strategies to circumvent excessive immunopathology.</p> / Dissertation

The overall goal of this research was to understand the role of inter-kingdom signaling in enterohemorrhagic Escherichia coli (EHEC) infections of the human gastro-intestinal (GI) tract from the perspective of both the invading pathogen and the human intestinal epithelial cells, which they colonize. Differential gene expression of EHEC was studied upon exposure to the human neuroendocrine hormones epinephrine and norepinephrine. We determined that these hormones increase EHEC chemotaxis, motility, biofilm formation, colonization of host cells, and virulence gene expression. We also studied the EHEC response to the GI tract commensal bacterial signaling molecules indole and autoinducer-2 (AI-2). We observed that indole decreases all the EHEC phenotypes that are increased by the human hormones and represses EHEC virulence. However, the effect of AI-2 was similar to that observed with hormones and opposite to that observed with indole, i.e. AI-2 increases EHEC virulence phenotypes. We studied changes in host cell transcriptome in the presence of the commensal bacterial signal indole. Indole increases expression of genes involved in tight junction and gap junction formation, and production of mucins and actin cytoskeleton genes. Indole also down-regulates genes encoding for pro-inflammatory cytokines, chemokines, and Toll-like receptors. The gene expression results were confirmed with phenotypic assays where we observed an increase in trans-epithelial resistance, increase in the anti-inflammatory cytokine IL-10, decrease in the pro-inflammatory cytokine IL-8, decrease in the activity of the pro-inflammatory transcription factor NF-κB, and decrease in colonization by EHEC of the indole-pre-treated HCT-8 cells. We established that factors secreted by epithelial cells are important determinants of EHEC virulence. Gene expression studies showed that 34 out of 41 LEE virulence genes were induced when EHEC was cultured in conditioned medium. In addition, the data showed increased expression of the shiga toxin-2 prophage 933W. These changes in gene expression were corroborated by a 5-fold increase in HCT-8 cell colonization and increased intracellular Stx2 phage titers. We determined that the HCT-8-secreted factor(s) was protein-based and that it was greater than 3 kDa in size. In conclusion, we have characterized the pathogen response to various eukaryotic and prokaryotic GI tract signals. We have established, for the first time, that the commensal bacterial signal indole is an inter-kingdom signal for the host epithelial cells. Overall, our studies provide a greater understanding of host-pathogen interactions.

Some pathogenic bacteria including Haemophilus influenzae and Neisseria meningitides produce a protease called IgA1 protease to impair a major antibody, immunoglobulin A1 (IgA1), on human mucosal surfaces. The iga mRNA is initially translated into a precursor containing four distinct domains: a 31-amino acids signal peptide which leads the precursor to the periplasmic space, an 105-kDa protease domain which cleaves host IgA1 molecule, a £]-domain responsible for autotransportation of the protease domain, and a short linker between the protease and the £]-domains. The autotransporter £]-domain can be further divided into three subdomains in Neisseria protease: an extracellular linking region £\-protein and a membrane-embedded £]-core, between which there is a distinguished sequence called surface region. The hydrolytic function of the protease and the transporter role of £]-core had been studied extensively, but the £\-protein and the surface regions were less defined, or had their role characterized. Thus this study is designed to reveal the possible pathogenic functions of the £\-protein and the surface region in bacterial adherence to human cell surfaces. To complete this project, recombinant £\-protein and the surface region were expressed in IgA1 protease-negative E. coli strain (UT5600) respectively and purified to homogeneity. These recombinant proteins were used in cellular assays for bacterial adhesion on human lung cancer cell (A549). Four different invasive strains of pathogenic bacteria (IgA1 protease-positive or negative), were recruited in adherence assays to determine the effect of the purified £\-protein and the surface region on bacterial adherence to A549 cells. Results showed that the both £\-protein and the surface region played a role in bacterial adherence in a species-dependent manner.