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<p>Please remove prior submission under the same title</p> / <p>Diabetes mellitus (DM) is associated with a significantly increased risk of microvascular complications, such as retinopathy, nephropathy, and neuropathy, as well as macrovascular disorders, including cerebro- and cardiovascular disease. Traditionally, the micro- and macro- vascular complications of DM have been considered distinct and independent disorders; however, data from several epidemiological and pathophysiological studies suggest they may be linked. It has been suggested that the <em>vasa vasorum</em>, a microvascular network which nourishes the walls of large muscular arteries, may play a role in macrovascular atherosclerosis. The effect of hyperglycemia on the microvessels of the vasa vasorum, and the potential impact of these effects on macrovascular atherosclerosis are not known.</p> <p>Here, we use a multiple-low-dose streptozotocin (STZ) injected apolipoprotein-E deficient mouse model to investigate the effects of hyperglycemia on the vasa vasorum, and to correlate such effects to atherosclerotic plaque progression. Hyperglycemia significantly increased plaque size and necrotic area (3- and 4-fold, respectively) relative to controls by 15 weeks of age. However, the density of vasa vasorum microvessels in the aortic wall of hyperglycemic mice was reduced at each time point examined. A similar vasa vasorum deficiency was also seen in STZ-induced hyperglycemic C57Bl/6J mice and hyperglycemic Ins2^Akita mice, and microvessel density could be corrected by insulin-mediated glucose normalization, suggesting a hyperglycema-specific effect. A localized deficiency in VEGF appears to be responsible for the reduced neovascularisation. Lastly, hyperglycemic mice fed standard chow supplemented with benfotiamine, a drug used to treat microvascular disorders in DM, appear to have reduced atherosclerosis.</p> <p>These findings provide the first indication that, in addition to retinal and glomerular capillary beds, hyperglycemia alters the microvessel structure of the vasa vasorum. Such microvascular changes directly correlate to the development and progression of atherosclerosis in hyperglycemic ApoE-deficient mice.</p> / Master of Science (MSc)

<p>Prenatal life events have been long observed to be able to influence disease into adulthood in both epidemiological and animal studies. Prenatal stress (maternal stress during gestation) is one of such factors that has been shown to impact cognition and behaviour of the offspring. However, the effects of prenatal stress on the immune system are not understood. This study has evaluated the effects of prenatal stress on a murine model. Prenatal stress increased allergic airway inflammation in male, but not female offspring following sensitization and challenge with cockroach extract. This corresponded with stress-induced changes in the immune environment of non-sensitized animals. These changes included a decrease in regulatory T cells at baseline in males compared to non-stressed controls and increased splenic dendritic cell percentage and cytokine, particularly IFN-γ, secretion compared to prenatally stressed females. In females, prenatal stress decreased allergic inflammation, which corresponded to a decreased percentage of dendritic cells in the lung and mesenteric lymph node. Prenatal stress did not affect dendritic cell antigen presentation in ether male or female offspring. There was no evidence to suggest a prenatal stress induced change in glucocorticoid sensitivity of dendritic cells. In order to explore the possibility of prenatal stress induced decrease of parasympathetic output, a vagotomy model was used as a proof of concept in naïve animals not exposed to prenatal stress. Vagal modulation of dendritic cell phenotype and function was assessed. While there was some evidence that vagotomy may indirectly modulate dendritic cell function, its effects on the immune system were different then the changes caused by prenatal stress and thus it is a role of reduced parasympathetic output was not supported. Overall this data indicates a role of prenatal stress on the immune system with clear sex differences, but the mechanism for how this occurs is currently unknown. Further research is needed to investigate the role of TLRs and IFN-γ in this model, as well as other possible mediators of prenatal stress such as the changes to the parasympathetic nervous system that may in turn mediate alterations to the immune system. Differences in when the effects of prenatal stress are expressed during postnatal life are discussed.</p> / Master of Science (MSc)

Fragile X syndrome (FXS) is identified by abnormal dendrite morphology and altered synaptic protein expression. Astrocyte secreted factors such as Tenascin C (TNC), may contribute to the synaptic changes, including maturation of the synapse. TNC is a known endogenous ligand of toll-like receptor 4 (TLR4) that has been shown to induce the expression of pro-inflammatory cytokines such as interleukin-6 (IL-6). At the molecular level, elevated IL-6 promotes excitatory synapse formation and increases dendrite spine length. With these molecular changes linked to the phenotype of FXS, we examined the expression and the mechanism of the endogenous TLR4 activator TNC, and its downstream target IL-6 in astrocytes from the FMR1 KO mouse model. Secreted TNC and IL-6 were significantly increased in FMR1 KO astrocytes. Exogenous TNC and lipopolysaccharide (LPS) stimulation of TLR4 induced secreted IL-6, whereas the antagonist of TLR4 (LPS-RS) had an opposing effect. Cortical protein expression of TNC and IL-6 were also significantly elevated in the postnatal FMR1 KO mouse. These results identify TNC as an endogenous ligand of TLR4, capable of effecting IL-6 secretion by astrocytes. In addition, there was an increase in the number of VGLUT1/PSD95 positive synaptic puncta of both WT and FMR1 KO neurons when plated with astrocyte conditioned media from FMR1 KO astrocytes, compared to those plated with media from wild type astrocytes. By assessing the cellular mechanisms involved, a novel therapeutic option could be made available to target abnormalities of synaptic function seen in FXS. / Thesis / Master of Science (MSc) / Autism spectrum disorders (ASDs) are neurodevelopmental disorders which arise from genetic and environmental factors. In the brain, a type of cell called the astrocyte is responsible for proper brain growth and development. Astrocytes release factors that promote inflammation, causing disruption of brain functions that control learning, memory and behaviour. Such factors released by astrocytes are capable of binding to their receptors, in turn impacting downstream targets, which have physiological effects. This research used various biological and genetic techniques to determine if the mechanism of an astrocyte-specific factor called Tenascin C (TNC) is impaired in the Fragile X mouse model. In a normal astrocyte, TNC with its binding partner is able to release molecules responsible for inflammation. Such molecules have been shown to increase the number synapses, where neurons and astrocytes exchange information, to control brain function. This proposed research would be the first to determine a role for TNC in ASDs. By assessing the cellular mechanisms involved between TNC and its binding partner, a novel therapeutic option could be made available in ASDs.

Fragile X Syndrome (FXS) is caused by the instability of a CGG-repeated tract at the 5’ end of the Fmr1 transcript. This instability causes silencing of the gene coding for FMRP. Higher levels of reactive oxygen species, lipid peroxidation, and protein oxidation within brain tissue have been found to be associated with the disease. These imbalances, along with altered levels of components of the glutathione system, provide evidence for increased oxidative stress. Astrocytes, glial cells within the brain, have many functions within neurodevelopment. Specifically, they regulate growth and synaptic contacts of neurons, regulate the level of excitability of synapses, and protect neurons at high levels of activity. To protect neurons from oxidative stress, astrocytes maintain oxidative homeostasis through their mitochondrial electron transport and antioxidant systems. This study examines the relationship between oxidative stress and FXS by assessing mitochondrial function and the antioxidant system of astrocytes. Using the Fmr1 knockout (KO) mouse model, mitochondrial respiration, and reactive oxygen species (ROS) production was analyzed in cultured cortical astrocytes. Astrocytes collected from male and female mice were analyzed under both normoxic and hypoxic conditions. In addition, western blots were conducted on both cortical tissue and cultured cortical astrocytes to determine potential differences in enzyme expression. Results indicate elevations of leak state respiration and ROS production in Fmr1 KO cultured cortical astrocytes alongside alterations in antioxidant and NADPH-oxidase expression. Characterization of mitochondrial function and the antioxidant system of astrocytes will be highly valuable to the understanding of glial roles during brain development and could provide future insight to direct clinically relevant studies of FXS and other neurodevelopment disorders. / Thesis / Master of Science (MSc) / Fragile X Syndrome (FXS) is the most common genetic cause of intellectual disability. It is characterized by the loss of FMRP, an important protein in brain development. Within the FXS brain there is evidence of oxidative stress. The cells that maintain oxidative homeostasis in the brain are astrocytes. Astrocytes are glial cells important for brain development. This thesis evaluated astrocytes' ability to maintain oxidative homeostasis in the FXS cortex. The findings of this thesis provide important insights into our understanding of FXS pathology and will help direct clinically relevant studies of FXS and other neurodevelopmental disorders.

BACKGROUND: Vibrio cholerae and Enterotoxigenic E. Coli (ETEC) are enteropathogens that are global causes of cholera and traveler’s diarrhea which are responsible for millions of diarrhea cases every year. ETEC and cholera are primarily found in Sub-Saharan Africa and Asia, particularly in nations with inadequate sanitation systems or little access to clean water. Infants and children are most vulnerable to these diseases, as severe infections can lead to stunting and death. The incidence of cholera and ETEC diarrhea have increased, due in part to changing weather patterns. At present, robust animal models for studies of ETEC colonization are lacking to study colonization and bottlenecks. The only licensed vaccines against cholera in endemic countries are killed whole cells, however, new live attenuated oral cholera vaccines (OCV) are in development and offer significant advantages. PanChol is a live attenuated OCV entering phase I trials. SPECIFIC AIMS: To propel studies of ETEC pathogenesis, I attempted to create a suckling mouse model of this globally important pathogen. To accomplish this goal, I constructed barcoded ETEC libraries that enabled me to determine founding population sizes along with intestinal ETEC burdens. To better understand PanChol, a new live attenuated OCV, I studied the shedding of the vaccine in the first 3 human volunteers to ingest this novel agent. METHODS: Triparental mating of donor strains MFDλpir pJMP1039 and MFDλpir pSM1 with recipient ETEC strains enabled construction of barcoded libraries. Neonatal CD-1 and C57BL/6 mice were infected with 104-107 CFU of wild-type ETEC to develop an infant mouse model. Founding population sizes of ETEC strains were compared via sequencing and STAMPR analysis while CFU burdens were determined via plating. Shedding of PanChol was done through enumeration of serial dilutions of fecal samples. Serotyping of shed PanChol was carried out using anti-Ogawa and anti-Inaba antisera. RESULTS: There were marked differences in ETEC small intestinal colonization in different mouse strains. Outbred CD-1 suckling mice only colonized with a 107 dose. In contrast, colonization of ETEC was approximately 106 CFU/small intestine at inocula sizes of 105 or greater in C57BL/6 mice. Laboratory studies using simulated bottlenecks made by serial dilutions established that the barcoded libraries accurately reflect founding population sizes up to 105 CFU. There was no difference in founding population sizes at the same inoculum size between WT ETEC and a hypervesiculation ∆mlaE mutant, though the founding population size increased with increasing input. PanChol retained the Hikojima serotype and shedding occurred in all volunteers with maximum colonization occurring 3 days post administration of 106 CFU. CONCLUSIONS: C57BL/6 P5 mice can serve as a new model to study ETEC intestinal colonization. Hypervesiculating ETEC did not produce a difference in founding population or colonization at the same input as WT ETEC strains. PanChol shows great promise as a viable OCV with shedding at 106 input and no serotype reversion.