Recently, viral hemorrhagic septicemia virus (VHSV IVb) was associated with several walleye (Sander vitreus) mortality events in the Great Lakes. To examine the effects of route, strain-variation and temperature, walleye were experimentally infected with VHSV IVb using intraperitoneal (i.p.)-injection (102-108 pfu/fish) and immersion (w.; 1.4 x 107 virions mL-1). Walleye were relatively resistant to experimental infection with VHSV IVb, regardless of route or water temperature. High cumulative mortality (64-100%) and severe gross lesions associated with VHSV-IVb infection were only evident in fish i.p.-injected with 108 pfu at 12°C, which had mild to moderate, multifocal necrosis of several tissues including the gill and heart. There were significant differences in mortality between four walleye strains following i.p.-infection. Viral antigen was found in both i.p. and w.-exposed walleye using immunohistochemistry, mostly within the gill and skin epithelium of w.-exposed fish. VHSV IVb was detected in walleye tissues from 6-21 d post-infection using RT-qPCR. / Great Lakes Fisheries Commission and NSERC

Bacteria perform biological nitrogen fixation (BNF) which leads to conversion of N2 to ammonia. One of the best studied models of BNF is the symbiotic association of Sinorhizobium meliloti - Medicago sativa (alfalfa). Since alfalfa is a major source of animal feed and the fourth largest crop grown in the USA, enhanced understanding of this symbiosis can have implications for increasing crop yields, reducing environmental contamination and food costs. Studies discussed here focus on two symbiotically important bacterial traits, type IVb pili and chemotaxis. Chapter 2 characterizes S. meliloti type IVb pili encoded by flp-1 and establishes their role in nodulation. Bundle-forming pili were visualized in wild-type cells, while cells lacking pilA1, the pilin-encoding gene, showed an absence of pili. Competitive nodulation assays with alfalfa concluded that cells lacking pili had a significant nodulation defect. Regulation of pilA1 expression via a quorum sensing regulator, ExpR, was confirmed. Chapter 3 describes the role of the flp-2 cluster in establishing symbiosis. PilA2 is a pilin subunit encoded from flp-2. The pilA2 deletion strain was defective in nodulation by 31% as compared to the wild type. A non-significant change in nodulation was seen in pilA1pilA2 strain. Thus, both flp-1 and flp-2 have a significant role in establishing symbiosis. Chapter 4 focuses on the deviations of S. meliloti chemotaxis from the enterobacterial paradigm. Transcriptional fusions showed that S. meliloti chemoreceptors (MCPs) are class III genes and regulated by FlbT. Quantitative immunoblots determined the cellular amounts of chemoreceptors. Chemoreceptors were grouped in three classes; high, low, and extremely-low abundance, similar to the high and low abundance chemoreceptors of Escherichia coli. Importantly, the MCP:CheA ratio in an S. meliloti cell was observed to be 37:1, similar to that in Bacillus subtilis of 24:1, but quite different from that in E. coli of 3.4:1. In conclusion, our data indicates that soil bacteria may have optimized their chemotaxis system based on their milieu, which is different from enteric bacteria. These studies have enhanced our understanding of two symbiotically important processes in S. meliloti, and pave the way for future manipulations of the system to increase symbiosis and reduce our dependence on synthetic fertilizers. / Ph. D.

La souche de P. aeruginosa PA14 est un isolat humain hautement virulent. PA14 possède deux îlots de pathogénicité. L'îlot de pathogénicité PAPI-1 de 108 kb est un élément intégratif et conjugatif (ICE), capable de s'auto-transférer à des souches de Pseudomonas par un mécanisme de conjugaison. Le mécanisme de transfert fait intervenir un pilus de Type IVb, encodé dans l'îlot PAPI-1. Mon travail de doctorat a eu pour but de caractériser à un niveau moléculaire le pilus de Type IVb (Pil-PAPI-1). J'ai d'abord, inséré un promoteur constitutif en place du promoteur endogène pour activer l'expression du locus pil2. J'ai démontré que 9 des 10 gènes sont requis pour le transfert d'ADN. J'ai ensuite initié la caractérisation de composants formant la machine de conjugaison. J'ai caractérisé le produit des gènes pilL2 et pilN2. J'ai démontré que PilL2 est une protéine de membrane externe (ME) et exposée dans le périplasme. Cette protéine est essentielle pour la fonctionnalité (transfert d'ADN) de la machinerie de conjugaison. Malgré ses caractéristiques prédites de lipoprotéine, aucune des mutations réalisées n'a pu modifier la localisation de ME de PilL2. J'ai aussi démontré que PilL2 forme un sous complexe de ME avec PilN2, la sécrétine du système. PilN2 forme des multimères stable, et présente les caractéristiques d'une liposécretine, capable d'auto-insertion et d'auto-multimérisation dans la ME. J'ai démontré que le N-ter de PilN2 mature est critique pour la formation d'un pore fonctionnel, mais n'est pas impliqué dans l'interaction avec PilL2. Ces résultats suggèrent que PilL2 et PilN2 forment un nouveau type de sous complexe de ME dans la famille des TFPb. / The P. aeruginosa PA14 strain is a highly virulent human isolate. PA14 possesses two pathogenicity islands. The 108-kb pathogenicity island PAPI-1 is an integrative and conjugative element (ICE), capable of self-transferring to any recipient Pseudomonas strain, by a conjugative mechanism. The transfer mechanism is mediated by a Type IVb pilus, encoded within the PAPI-1 Island. My PhD work aimed to characterize this Type IVb pilus (Pil-PAPI-1) at a molecular level. The pil2 locus is poorly expressed under laboratory condition. I, first, introduced a constitutive promoter to turn on expression of the pil2 locus. I demonstrated that 9 of the 10 genes are required for DNA transfer. Then, I initiated the characterization of components forming the conjugation machinery. I characterized the products of pilL2 and pilN2 genes. I demonstrated that PilL2 is an OM protein and protruding in the periplasm. This protein is essential for the functionality (DNA transfer) of the conjugative TFPb machinery. Despite its predicted lipoprotein-hallmarks, none of the mutations engineered were able to abrogate the OM-localization of PilL2. We also demonstrated that PilL2 forms an OM sub-complex with PilN2, the secretin of the system. We provide evidence that PilN2 forms stable multimers, which presents the features of a liposecretin, capable of self-insertion and self-multimerization in the OM. We demonstrated that while the N-terminus of the mature PilN2 is required for the formation of a functional pore, it is not involved in interaction with PilL2. These results suggest that PilL2 and PilN2 could form new type of OM sub-complex in the TFPb family.

Philosophiae Doctor - PhD / The domesticated apple (Malus x domestica Borkh.) has been distributed into diverse climatic conditions worldwide for commercial production of fruit. Apple trees need exposure to cold temperatures, referred to as chill unit (CU) accumulation during winter, in order for budbreak to occur promptly and uniformly after winter. In warmer production areas the application of dormancy breaking chemicals has enabled successful production of high chilling requiring apple cultivars in suboptimal environmental conditions. In the Western Cape region of South Africa it is common orchard practice to apply dormancy breaking chemicals after winter in order to stimulate vegetative growth. If this is not done prolonged dormancy symptoms (PDS) are experienced which include extended rest, less synchronised breaking of buds and reduced branching. An increasing awareness of both global warming and the negative effects associated with the use of chemical sprays (for both pest and disease resistance and growth regulation) has resulted in the need to breed cultivars better adapted to current and future environmental conditions. The breeding of new cultivars using conventional breeding methods is a time consuming process, especially in perennial tree species with a long juvenile phase such as apple. The implementation of marker-assistedbreeding (MAB) and selection (MAS) will enable the selection of favourable genotypes at a very early seedling stage. Although markers linked to genes involved in disease resistance for a variety of known apple pathogens have been identified and are already in use in breeding programs, the genetic determinants of dormancy related characteristics residing within the bud itself iii (endodormancy) are poorly understood. This hampers the genetic improvement of such characters. Although this study focused on time of initial vegetative budbreak IVB, there are various other characteristics that can be associated with dormancy, such as position and number of budbreak and budbreak duration.

Mucopolysaccharidoses are a group of diseases that belong to lysosomal storage disorders. A common sign of these monogenic multisystem diseases is a gene mutation leading to a deficiency of the lysosomal enzyme participating in glycosaminoglycan degradation. It results to their accumulation in the tissues and organs, where they cause a progressive damage. There is no efficient treatment available for most mucopolysaccharidoses. Moreover, the research is complicated because of the low prevalence and type of affected tissues. Animal models of these human diseases are used for an evaluation of newly developed therapeutic approaches. However, they also have many limitations due to the different pathogenesis and catabolic pathways of the accumulated substrates between humans and animals. Therefore, animal models are replaced by human cell models. In this thesis, the development of four mucopolysaccharidoses human cell models is reported (MPS IIID, MPS IVA, MPS IVB, MPS VI). Corresponding genes (GNS, GALNS, GLB1, ARSB) were inactivated using CRISPR/Cas9 technology, where plasmids containing specific inserts are delivered to the target human induced pluripotent stem cells (iPSC), using electroporation. Isolated clones, which represent iPSC disease models, were characterized by Sanger sequencing, enzyme...

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