<p>Crohn’s Disease is a chronic inflammatory bowel disease characterized by an overzealous immune response to a microbial trigger in genetically susceptible individuals. Although this microbial trigger is unknown, <em>Escherichia coli</em> with adherent and invasive properties (Adherent-Invasive <em>Escherichia coli</em>, AIEC) is preferentially enriched in a proportion of Crohn’s Disease patients. AIEC can adhere to and invade intestinal epithelial cells and replicate intracellularly within epithelial cells and macrophages <em>in vitro</em>. One important colonization factor expressed by AIEC is the type 1 fimbrial adhesin protein FimH. FimH mediates colonization of CEABAC10 transgenic mice and can bind several host cell receptors including the macrophage receptor CD48 <em>in vitro</em> indicating a potential role for FimH in macrophage interaction. However, it was not known whether FimH contributed to phagocytosis of AIEC or colonization of wild-type mice. Here we show that FimH enhances early intracellular AIEC levels <em>in vitro</em> and colonization <em>in vivo</em>. We found that deletion of <em>fimH</em> may reduce intracellular AIEC burden at 2 hours post-infection and that this effect was modulated by bacteria opsonisation. Using a competitive index assay, we show that a Δ<em>fimH</em> mutant is unable to chronically colonize CD-1 mice at the same levels as the parental strain. Our results demonstrate that FimH is an important AIEC colonization factor and may increase interaction with macrophages. Identifying factors such as FimH which contribute to colonization and persistence will further our understanding of AIEC survival strategies within the host. Development of therapeutics targeting FimH may provide a means to reduce harmful bacteria overgrowth particularly after surgical intervention.</p> / Master of Science (MSc)

The tachykinin NK-3 receptor is a G-protein coupled receptor activated by mammalian tachykinin neuropeptides, which can modulate dopaminergic neurotransmission, and alter dopamine-mediated behaviors. The NK-3 receptor is currently under investigation as a novel therapeutic target for cocaine addiction. Our studies, as outlined in this dissertation, sought to determine if NK-3 receptors have a functional role in the acute as well as long-term behavioral effects of cocaine. Administration of NK-3 receptor agonists or antagonists potentiates or attenuates dopamine-mediated behaviors, respectively. Based on these findings, we hypothesized that blockade of neurokinin-3 receptors would alter acute and long-term behavioral responses to cocaine. We investigated whether acute and repeated administration of the NK-3 receptor antagonist SB 222200 altered hyperactivity induced by cocaine, and determined a possible mechanism involving dopamine D1 receptors in the striatum. We also determined whether NK-3 receptor blockade altered the development and expression of behavioral sensitization after repeated cocaine administration. Lastly, we investigated whether modulation of behavioral effects of acute and repeated cocaine by NK-3 receptors involved GSK3 phosphorylation in the nucleus accumbens. As described in this dissertation, we show that acute administration of the NK-3 receptor antagonist SB 222200 before a cocaine injection attenuated stereotypic responses produced by cocaine. Repeated administration of SB 222200 enhanced stereotypic activity produced by either cocaine or a low dose of SKF 82958 (0.125 mg/kg, i.p.) when administered seven days later. Dopamine receptor binding studies were performed to determine the mechanism of enhanced stereotypic responses. Binding studies showed a 19.7% increase in dopamine D1 receptor density in the striatum seven days later after repeated SB 222200 administration. These findings demonstrate that acute blockade of NK-3 receptors attenuated cocaine-induced behaviors in agreement with previous studies. Furthermore, these studies also show novel effects of repeated blockade of NK-3 receptors, which causes subsequent enhancement of cocaine and dopamine D1 receptor-mediated behaviors, possibly resulting from dopamine D1 receptor up-regulation in the striatum. In order to determine a role of NK-3 receptors in the development of cocaine-induced behavioral sensitization, the NK-3 receptor antagonist SB 222200 (2.5 or 5 mg/kg, s.c.) was administered prior to daily cocaine injections for 5 days. After a 7-day drug-free period, behavioral responses to a cocaine challenge were measured. Repeated administration of cocaine for 5 days induced a sensitized response upon a cocaine challenge 7 days later. Administration of SB 222200 prior to daily cocaine attenuated the development of behavioral sensitization. Moreover, administration of SB 222200 prior to the cocaine challenge blocked the expression of behavioral sensitization. These findings demonstrate that NK-3 receptor activity is involved in the development and expression of behavioral sensitization to cocaine. Lastly, we examined GSK3 phosphorylation in the nucleus accumbens induced by acute and repeated cocaine administration and determined if phosphorylation was altered by NK-3 receptor blockade. Similar to the drug administration regimens used in the behavioral studies, the NK-3 receptor antagonist SB 222200 was administered 30 mins prior to an acute cocaine injection. The nucleus accumbens was examined for changes in GSK3 phosphorylation by Western blot analysis. Increases in phosphorylation of the isoforms, GSK3α and GSK3β in the nucleus accumbens were detected 20 mins after an acute injection of cocaine. NK-3 receptor blockade prior to cocaine administration did not alter the cocaine-induced increase in GSK3 phosphorylation. Similar to the behavioral sensitization studies, SB 222200 was administered prior to repeated cocaine for 5 days, and 7 days later GSK3 phosphorylation was measured after a subsequent cocaine challenge. In contrast to the increases in GSK3α and GSK3β in the nucleus accumbens after an acute cocaine injection, no regulation of GSK3 phosphorylation was found after prior repeated cocaine administration and cocaine challenge. Administration of SB 222200 prior to repeated cocaine produced an increase in GSK3α and GSK3β phosphorylation after a cocaine challenge. Collectively, these data point to involvement of NK-3 receptor activity in changes in the phosphorylation of GSK3 in the nucleus accumbens produced by cocaine. In summary, functional involvement of NK-3 receptors in acute and long-term behavioral effects of cocaine was investigated. In agreement with previous findings, studies in this dissertation demonstrate that acute blockade of NK-3 receptors attenuates cocaine-induced behaviors. In addition, we found novel effects of repeated blockade of NK-3 receptors on cocaine-induced hyperactivity. There is enhancement of subsequent cocaine and dopamine D1 receptor-mediated behaviors possibly due to dopamine D1 receptor up-regulation in the striatum. NK-3 receptor activity was shown to be involved in long-term behavioral effects of cocaine and molecular changes in GSK3 phosphorylation in the nucleus accumbens. Blockade of NK-3 receptors prevented the development and expression of behavioral sensitization to cocaine and also blocked the changes in the phosphorylation of GSK3 in the nucleus accumbens. This dissertation has demonstrated a role of NK-3 receptors in modulating acute as well long-term cocaine-induced behavioral hyperactivity. Therefore, there is potential clinical relevance of NK-3 receptors in cocaine abuse and dependence as a therapeutic target for treatment, which warrants further characterization in future preclinical and clinical investigations. / Pharmacology

Vitamin D is important for bone development with immunomodulatory effects. This study investigated whether feeding CD-1 and interleukin 10 (IL-10) knockout (KO) dams low (25 IU/kg diet) or high (5,000 IU/kg diet) vitamin D affected bone health of dams as well as their offspring. Offspring were weaned to 1 of the 2 diets and followed to young adulthood. Unlike CD-1 dams, IL-10 KO dams experienced greater femur strength with high vitamin D. CD-1 male offspring had reduced femur neck strength and female offspring had smaller, weaker femurs, and weaker lumbar vertebra 2 (LV2) with high maternal vitamin D. IL-10 KO male offspring had larger femurs and female offspring had stronger femurs when weaned to high vitamin D. Low vitamin D did not adversely impact bone health but the optimal level of dietary vitamin D seems to differ between healthy and inflammatory states.

Vitamin D is important for bone development with immunomodulatory effects. This study investigated whether feeding CD-1 and interleukin 10 (IL-10) knockout (KO) dams low (25 IU/kg diet) or high (5,000 IU/kg diet) vitamin D affected bone health of dams as well as their offspring. Offspring were weaned to 1 of the 2 diets and followed to young adulthood. Unlike CD-1 dams, IL-10 KO dams experienced greater femur strength with high vitamin D. CD-1 male offspring had reduced femur neck strength and female offspring had smaller, weaker femurs, and weaker lumbar vertebra 2 (LV2) with high maternal vitamin D. IL-10 KO male offspring had larger femurs and female offspring had stronger femurs when weaned to high vitamin D. Low vitamin D did not adversely impact bone health but the optimal level of dietary vitamin D seems to differ between healthy and inflammatory states.

Puberty is a critical period for sexual maturation during which the sex-specific reorganization and remodelling of the pubertal brain facilitate sex biases in stress sensitivity. Pubertal (i.e., six-week-old) CD-1 mice treated with the bacterial endotoxin lipopolysaccharide (LPS; 1.5 mg/kg body weight, ip) show several sex-specific changes to the neuroendocrine and behavioural systems of several reproductive and non-reproductive functions. One promising explanation for the elusive mechanisms driving the sex-specific outcomes of pubertal immune challenge may lie in the cascade of neuroimmune events induced by this systemic immune stressor. This doctoral thesis tested the hypothesis that sex-specific responses of the pubertal neuroimmune network contribute to sex differences in the enduring outcomes of pubertal immune challenge on hippocampus-dependent cognitive processes. Male and female CD-1 mice are equally vulnerable to enduring impairments in spatial memory following pubertal LPS exposure. Across brain regions for cognition and stress regulation, pubertal LPS treatment alters baseline sex differences in microglial expression and morphology in a sex-dependent manner. The temporary female-specific increase in whole-brain blood-brain barrier permeability during LPS-induced sickness may have facilitated the apparent female bias in LPS-induced changes to pubertal microglia. In the context of sex- and region-specific residual effects of pubertal LPS-induced sickness on microglial expression and morphology, pubertal LPS treatment may accelerate certain neurodevelopmental processes in males but not females. The innate sex differences in the pubertal neuroimmune network highlighted by these studies underscore how a systemic immune challenge precipitates sex biases in immune-mediated disorders of brain and behaviour during adulthood.