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The time-dependence of molecular evolution, specifically over short timescales, has been shown to be a major confounding factor in the analysis of nucleotide changes between closely related strains or species. The assumption that selection works extremely quickly to purge all of the deleterious changes is at odds with the Nearly Neutral model of evolution, whereby the majority of changes are only mildly deleterious and therefore impose only a minor fitness cost so they are relatively rapidly purged only in populations with large effective population sizes. The aim of this project was to explore the patterns of nucleotide changes evident between the core genomes of nine E. coli and Shigella strains, with the latter having adopted a specific ecological niche in the recent evolutionary past. The Shigellae and E. coli show little difference in their extant genome compositions, in terms of nucleotide composition and genome size, however there are a markedly higher number of pseudogenes and insertion sequences present in the Shigella genomes. The polymorphism profiles of the core genomes reveal a time-dependency of dN/dS, Ti/Tv, +AT/+GC and the Metabolic cost of Amino acid changes, the nucleotide data showing a clear separation of the E. coli from the Shigellae, with the latter showing trends indicative of weaker purifying selection. Additionally these differences are evident when examining the nucleotide ratios (+AT/+GC & Ti/Tv) along the core genome, also revealing patterns of evolution associated with genome position. A simulation based approach reveals different projected nucleotide contents for the E. coli and Shigellae genomes further highlighting their different evolutionary paths as evident from the polymorphism profiles. The methods employed and developed in this study provide a useful and effective toolset for examining the evolution of bacterial genomes over short timescales, especially in light of the availability of multiple whole genome sequences for a given 'species'.

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La Sclérose en Plaques (SEP) est une maladie d'origine multifactorielle qui se développe chez des individus génétiquement susceptibles en présence de facteurs environnementaux inducteurs. Ma thèse avait pour objectif d'analyser les effets d'un facteur génétique, le variant R63W du gène Vav1 et d'un facteur environnemental, la souche "Escherichia coli Nissle 1917", sur le développement de l'encéphalomyélite auto-immune expérimentale (EAE), un modèle animal de la SEP. Une région de 1cM comportant un polymorphisme dans le gène Vav1 a en effet été identifiée au laboratoire comme étant responsable de la résistance des rats Brown-Norway à l'EAE. Afin d'établir formellement le rôle de ce polymorphisme dans ce modèle, une souris Knock-In Vav1R63W a été générée. Nous avons montré que les souris Vav1R63W développent une EAE moins sévère. Ceci est associé à un défaut de production de cytokines inflammatoires intrinsèque aux lymphocytes T (LT) CD4 qui n'est pas lié à une augmentation de la fréquence de LT régulateurs. Sur le plan moléculaire, Vav1R63W présente une activité adaptatrice défectueuse conduisant à la diminution de la phosphorylation de ERK, AKT et p38 mais à une activité enzymatique normale. Nos résultats montrent un rôle de la fonction adaptatrice de Vav1 dans les fonctions des LT CD4 et son implication dans la susceptibilité à l'inflammation du système nerveux central (SNC). L'analyse de l'effet d'un traitement oral par le probiotique E. coli Nissle 1917 (ECN) montre un effet bénéfique sur le développement de l'EAE. Ceci est associé à un défaut de la sécrétion de cytokines par les LT CD4, ainsi qu'à une diminution de l'infiltration de LT CD4 auto-réactifs dans le SNC. De plus, la barrière intestinale est moins altérée chez les souris traitées par ECN au cours du développement de l'EAE. L'effet bénéfique de ECN semble être dû à la production d'une génotoxine, la colibactine. Par contre, la colonisation néonatale des souris C57BL/6 par ECN ne reproduit pas le même effet observé à l'âge adulte. Dans l'ensemble, nos résultats montrent un effet bénéfique du changement de la fonction de Vav1 ainsi que du traitement par ECN sur le développement de l'EAE. L'analyse approfondie des mécanismes mis en jeu, permettra une meilleure compréhension de la pathogenèse de la SEP et pourrait contribuer à l'identification de nouvelles options thérapeutiques. / Multiple sclerosis (MS) is a demyelinating disease of the central nervous system. It develops in genetically susceptible individuals when they encounter specific environmental factors. The aim of my thesis was to analyze the role of a genetic factor (Vav1R63W variant) and an environmental factor (Escherichia coli Nissle 1917) in the development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Previous genetic studies of my team suggest the implication of a polymorphism in the Vav1 gene in the resistance of Brown-Norway rats to EAE. In order to analyze the role of the identified polymorphism in the susceptibility to EAE, we generated a Knock-In mouse bearing the same polymorphism (Vav1R63W). Using this model, we showed that Vav1R63W mice develop less severe EAE due to a defect in cytokine production by CD4 T cells. This defect is intrinsic to CD4 T cells and is not linked to the increased proportion of regulatory T cells observed in Vav1R63W mice. We also showed that Vav1R63W present an altered adaptor function as shown by reduced ERK and AKT phosphorylation and decreased calcium flux after TCR stimulation, with no effect on Vav1 enzymatic activity. Thus, our results highlight the role of Vav1 adaptor function in CD4 T cell functions and susceptibility to central nervous system inflammation. Next, I analyzed the impact of the treatment with the probiotic E. coli Nissle 1917 (ECN) on EAE development. Our results showed that the daily oral treatment of adult C57BL/6 mice with ECN ameliorates the course of the disease. In addition to their defect in cytokines production, MOG specific CD4 T cells from ECN treated mice were increased in the periphery. Consequently, ECN treated mice exhibited reduced CD4 T cell infiltration in their central nervous system. Furthermore, analysis of intestinal permeability revealed that its alteration after MOG immunization was partially reversed after ECN treatment. The reduced EAE seems to be due to the secretion of a genotoxin by ECN, the colibactin. In contrast, neonatal colonization of C57BL/6 mice did not protect against EAE. Together, our data showed a beneficial role of the imbalance of Vav1 function and ECN treatment in EAE development. Further analysis of the involved mechanisms will help us to better understand the pathogenesis of MS and to develop new therapeutic strategies for MS.

Aim of this study was to select environmental E. coli isolates that produced colicins against the swine pathogen E. coli K88+. In initial evaluation using a modified plate method with 18 colicinogenic E. coli constructs, colicins E3, E4, E5, E9, Ia, K and N were found to possess inhibitory activity against 12 ETEC K88+ strains. A total of 463 environmental isolates from cattle rumen, cattle feces, pig feces and hog manure-amended soil were screened for colicin production by a modified plate test. Further, colicinogenic isolates were screened for five toxin genes LT, STa, STb, VT1 and VT2 as well as K88 (F4) fimbriae using PCR reactions. Fourteen non-pathogenic isolates were subjected to characterization of colicin genes by PCR using 9 new primer sequences, antibiotic susceptibilities and substrate utilization. Two potential probiotic strains of E. coli, UM-2 and UM-7 which produced colicins that could utilize potato starch and inulin were selected for in-vitro competition with E. coli K88+ strain 2-12. In vitro competition between the synbiotics and E. coli K88+ revealed inhibition of E. coli K88+. Based on the present in vitro studies it could be concluded that carefully selected potential synbiotics should be further studied for their role in protecting piglets from post-weaning diarrhea without antibiotics. / October 2007

Bacteria are exposed to evolutionary pressure to an extent unlike other organisms. This leads to a high degree of adaptation to its surrounding. This thesis investigates the degree of adaptation towards higher accuracy in the transcription of DNA to RNA in coding sequences in the DNA of the E. coli bacteria. Two coding sequences with different nucleotide contents were investigated. Goodness-of-fit tests were performed but no significant adaptation towards higher transcription accuracy was found in the investigated sequences.

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Beneficial management practices for controlling and managing pollutant loadings from entering source waters are essential to preserving the natural habitat of lakes and rivers. Artificial Floating Wetlands (AFWs) are a promising technology, when applied with other beneficial management practices, for the removal of water borne contaminants in urban and agricultural runoff. To evaluate AFWs for their application for nutrient-rich water remediation, a pilot scale study was assessed at the Centre for Alternative Wastewater Treatment in Lindsay Ontario. Fifteen ponds, averaging in size from 7.2 m width to 9.7 m length to 3.8 m depth were divided into two controls and three treatments, each with three replicates. The controls were not covered with AFWs and were used for comparative purposes while the treatments had AFW coverage averaging 67% of the total surface area of each pond. The treatments and controls received varying inputs of simulated stormwater runoff during the study. This study was used to determine the effects of high surface area coverage of AFWs on temperature, pH, DO and ammonia. A lab scale study was designed to investigate the results obtained from the pilot study and to assess the effectiveness of 100% AFW coverage for nutrient-rich water treatment. The study consisted of twelve 150 L containers, with four of the containers covered with the AFW, four without coverage and the final four with coverage without vegetation. Water quality parameters including phosphorus, nitrogen, E.coli, pH and temperature were tested on a weekly basis for two five week retention periods. Results from these studies suggest that AFWs can provide a valuable method of treatment when combined with other beneficial management practices. The pilot scale study demonstrated that AFWs inhibit algae growth and create a cooler environment below the wetland surface. Results from the second study indicate AFWs have a much better and quicker removal of phosphorus and ammonia when compared to the treatment with coverage without vegetation. E.coli levels were completely reduced within the water column for all treatments during the testing period. / Thesis (Master, Civil Engineering) -- Queen's University, 2014-04-30 12:57:03.197