The process of autophagy is defined as the degradation of cellular cytoplasmic constituents via a lysosomal pathway. Herein I sought to examine the regulation of autophagy in the placental pathologies preeclampsia (PE) and intrauterine growth restriction (IUGR). I hypothesized that the Bcl-2 family proteins Mcl-1L and MtdL regulate placental autophagy and contribute towards dysregulated autophagy in PE. My results demonstrate that Mcl-1L acts to repress autophagy via a Beclin 1 interaction, while MtdL induces autophagy when it interacts with Mcl-1L. My data indicate that while autophagy is elevated in PE, a pathology characterized by oxidative stress, it is decreased in IUGR, a hypoxic pathology. Treatment with sodium nitroprusside to mimic PE caused a decrease in Mcl-1L and an increase in MtdL levels in response to oxidative stress, thereby inducing autophagy. Overall, my data provide insight into the molecular mechanisms contributing to the pathogenesis of preeclampsia.

Subarachnoid hemorrhage (SAH) comprises 7% of all stroke cases, and is associated with a disproportionately high morbidity and mortality with few therapeutic options available. The goal of this project was to understand the mechanism of neurological deterioration after experimental SAH, with a focus on cerebral vasospasm and brain injury after SAH. We tested the hypothesis that endothelial nitric oxide synthase (eNOS) is upregulated and uncoupled after SA, resulting in exacerbated neurological injury in a mouse model of SAH. The project entailed the investigation of eNOS-dimer uncoupling, its association with oxidative and nitrosative stress in the brain parenchyma and finally its association with secondary complications after SAH. In our studies we demonstrated the crucial role eNOS plays in anti-microthromboembolism, anti-apoptosis and maintenance of physiological superoxide (O2-)/NO balance. This study suggests that SAH up-regulates and disrupts eNOS, producing peroxynitrite (OONO-) and other radicals that further exacerbate the oxidative insult and neurological injury.

Type 1 and type 2 diabetes are characterized by an excessive loss of insulin producing β-cells. β-cells are particularly susceptible to increased oxidative stress induced apoptosis due to low expression of major antioxidants. Peroxiredoxin-2 (PRDX2) belongs to a group of antioxidants with antiapoptotic roles. Preliminary data indicate PRDX2 is expressed in the β-cells. Endogenous PRDX2 in the β-cell line MIN6 is found to decrease under oxidative stress conditions. I hypothesize that PRDX2 has a role in protecting β-cells against oxidative stress induced apoptosis. Overexpression or knockdown strategies were used to examine the role of PRDX2 in insulin-secreting MIN6 cells treated with various stimuli (cytokines, palmitate, streptozotocin) to induce apoptosis. Results showed that PRDX2 overexpression decreased oxidative stress induced apoptosis markers and cell death indicators, whereas knockdown of PRDX2 exaggerated oxidative stress induced toxicity. These findings suggest that PRDX2 plays a protective role in pancreatic β-cells under oxidative stress conditions.

Aerobic exercise such as cycling is known to elicit a post-exercise hypotensive (PEH) response. However, it is not known if swim exercise produces a similar effect in normotensive individuals. We tested the hypothesis that an acute bout of swimming would elicit a PEH response that is less compared to an equivalent bout of cycling. 10 trained and 11 untrained normotensive (SBP/DBP < 120/80 mmHg) individuals (23±1 years) underwent 30 min intensity-matched cycling and swimming sessions to assess changes in BP and cardiovascular responses. While PEH was similar between modalities within groups, the magnitude and temporality of change in BP following swimming was significantly different (p < 0.01) between groups, with untrained participants showing a significant PEH response. Attenuation of PEH in trained individuals was reflective of a significant increase in sympathetic outflow and slower vagal reactivation, suggesting training in an aquatic environment leads to alterations in post-exercise BP regulatory mechanisms.

The effective of additional muscle mass in a priming exercise on cardiac output (Q) and pulmonary oxygen uptake (VO2) kinetics (mean response time, s) were determined in cyclists. Outcomes were measured over four trials, each consisting of a 6-minute legs alone (UAL) or arms and legs (ULO) warm-up, 3 minute passive recovery, then 6 minutes leg cycling (PAL, PLO; respectively). Q was significantly higher preceding exercise onset with PAL compared to PLO or ULO (0.72 ± 0.13 vs. 0.58 ± 0.09, 0.43 ± 0.09 L∙min-1; respectively, P < 0.05). Q kinetics did not differ between unprimed (ULO: 38.9 ± 8.6) and primed exercise regardless of muscle mass (PLO: 38.6 ± 11.0; PAL: 40.7 ± 11.3). VO2 kinetics were faster (P < 0.05) with PAL (36.9 ± 6.0) compared to ULO (58.7 ± 10.5). Muscle mass employed during priming exercise had only slight effect on subsequent VO2 and Q responses.

Pumilio 2 (PUM2) is a member of the Puf family of mRNA binding proteins and translational regulators which are involved in various processes including embryonic patterning and memory formation. Nevertheless, its functions in the outgrowth of neuronal axons have not been studied. This study shows endogenous expression of PUM2 in neurites of dorsal root ganglia (DRG) neurons and transport of PUM2 along retinal ganglion cell (RGC) axons and their growth cones. Overexpression of PUM2 in DRG neurons resulted in shorter axons when compared to control neurons. Expression of either dominant negative mutation (dnPUM2) or PUM2W349G displayed a reduction in axonal length. PUM2 downregulation with microRNA (miRNA) also caused a reduction in neurite length compared to control neurons. Finally, PUM2 silencing did not alter eye size at E4, which allows investigation of axonal outgrowth in RGC in vivo. These results suggest a novel role for PUM2 in axonal outgrowth.

During pregnancy, 64-96% of women take at least one prescription drug. The placenta is the primary barrier between substrates in maternal and fetal circulation. The blood-brain barrier (BBB) acts as an additional barrier for the fetal brain, which is particularly susceptible to the effects of xenobiotics. Multidrug resistance phosphoglycoprotein (P-gp; encoded by Abcb1 mRNA) and breast cancer resistance protein (Bcrp1; encoded by Abcg2 mRNA) are efflux transporters localized on placental syncytiotrophoblast and capillary endothelial cells of the BBB. Placental Abcb1/P-gp and Abcg2/Bcrp1 limit maternal-fetal transfer of endogenous and exogenous substrates. Similarly, the neuroprotective roles of Abcb1/P-gp and Abcg2/Bcrp1 in the adult BBB have been demonstrated. However, developmental changes in expression and function and regulation of Abcb1/P-gp and Abcg2/Bcrp1 in these tissues are poorly understood. This thesis investigates gestational changes in expression and function of Abcb1/P-gp and Abcg2/Bcrp1 in the placenta and fetal brain, in addition to regulation by steroids, progesterone and glucocorticoids. The effects of glucocorticoids on Abcb1/P-gp and Abcg2/Bcrp1 in the placenta and fetal brain are of importance given that 10% of pregnant women are treated with synthetic glucocorticoids during the management of threatened preterm labour. These studies demonstrate that the decrease in placental Abcb1/P-gp mediated fetal protection near term is compensated by an increase in Abcb1/P-gp and Abcg2/Bcrp1 mediated neuroprotection in the fetal brain; likely in preparation for life ex-utero. The lack of effects of progesterone and the dose-, age- and sex- dependent regulatory effects of synthetic glucocorticoid have highlighted the complexity associated with regulation of these transporters. Further, these studies are the first to report sexually dimorphic glucocorticoid effects on Abcb1/P-gp and Abcg2/Bcrp1 expression and function, with the female fetus being particularly susceptible to glucocorticoid these effects. In this regard, Abcb1/P-gp and Abcg2/Bcrp1 transport capacity may be altered when synthetic glucocorticoid is administered as a co-therapy, and as such, recipient sex should be considered during pharmacotherapy. Understanding the regulation of Abcb1/P-gp and Abcg2/Bcrp1 expression and function in the placenta and fetal brain during normal development and under pathological conditions is critical for fetal health and development, particularly when therapeutic strategies are utilized in pregnancy.

Slow population activities (SPAs) are population activities in the brain with frequencies of less than 5 Hz. SPAs are prominent in many brain structures including the neocortex and the hippocampus. Examples of SPAs include the neocortical EEG δ waves and the hippocampal large amplitude irregular activities during NREM sleep. These in vivo SPAs are believed to play a fundamental role in brain plasticity. However, despite many experimental attempts to understand SPAs, their mechanisms are still not well understood. It is unclear how the individual neurons can sustain low frequency activities on the network as a whole. In this thesis, we demonstrate that a mathematical and computational perspective is indispensable in understanding slow population phenomena and generating testable hypotheses for future experiments. Our focus is on a hippocampal slice preparation exhibiting spontaneous, inhibitory-based SPAs (hippocampal SPAs). We develop a multi-pronged approach consisting of parameter extraction, simulation, and mathematical analysis to elucidate the mechanisms responsible for hippocampal SPAs. Our results suggest that hippocampal SPAs are an emergent phenomenon. In other words, the network “slowness” is not directly represented by any particular individual element within the network. Instead, the low frequency activities on the network are the result of interactions between synaptic and intrinsic characteristics of individual inhibitory interneurons. Our simulations quantify these characteristics which underlie hippocampal SPAs. Specifically, our simulations predict that individual interneurons should 1) be moderately fast-spiking above threshold before the increase in spike frequency slows down with increasing drive, and 2) be well connected with one another for SPAs to occur. We also predict that excitatory noise levels have a larger influence on hippocampal SPAs than mean excitatory drive. Subsequent mathematical analyses show that the synaptic and intrinsic conditions of individual interneurons as predicted by simulations promote network multi-stability. Hippocampal SPAs occur when the network switches from one network firing state to another. Since many of the parameters we use for simulations are extracted from experiments, our simulation model is likely a reasonable representation of actual biological mechanisms in hippocampal networks.

The vestibulo-ocular reflex (VOR) is a simple reflex that stabilizes gaze by moving the eyes in the opposite direction to the head. The gain of the VOR (ratio of head to eye velocity) can be increased or decreased during motor learning. It is thought that the memory for learned changes in the VOR gain is initially encoded within the cerebellar flocculus. Furthermore, these learned gain changes can be disrupted or consolidated into a long-term memory. In this thesis we describe novel results that show that consolidation of the VOR can take place rapidly, within 1 hour after learning has stopped. Furthermore, we demonstrated that unlike learning, which has been shown to have frequency selectivity, disruption and rapid consolidation generalize across the range of frequencies. We suggest that disruption and rapid consolidation in the VOR are local mechanisms within the cerebellar cortex, and do not require new learning. This thesis also provides additional evidence for the idea that learned gain increases and decreases are the result of separate mechanisms, most likely long-term depression and potentiation respectively, at the parallel fibre-Purkinje cell synapses. We demonstrate that learned gain increases, but not decreases, require the activation of type 1 metabotropic glutamate receptors (mGluR1) and B type γ-aminobutyric acid (GABAB) receptors. Blocking one or both of these receptors with an antagonist inverts gain-up learning, while the agonist augments gain-up learning. Furthermore, we provide novel evidence that these receptors are co-activated during gain-up learning.

Epidemiological studies in humans have revealed a relationship between altered development in utero and an increased incidence of pathophysiology during postnatal life. One of the mechanisms underlying this relationship is thought to be exposure to excess glucocorticoids during critical phases of brain development. The aim of the current set of studies was to determine the effects of prenatal stress during discrete developmental windows on behaviour and endocrine function in male and female guinea pig offspring. Guinea pigs were used as the model for these studies as they are a long-gestation species that give birth to neuroanatomically mature young and fetal brain development is well characterized. Pregnant guinea pigs were exposed to a high frequency strobe light during the period of rapid fetal brain growth or the period of rapid brain myelination. Pregnant guinea pigs were allowed to deliver normally and guinea pig offspring were tested for ambulatory activity, anxiety and hypothalamic-pituitary-adrenal (HPA) axis function. Male offspring whose mothers were exposed to stress during the period of rapid brain growth exhibited increased anxiety behaviour, increased basal plasma cortisol levels and decreased plasma testosterone levels. We found that replacing testosterone in these animals reversed the behavioural and endocrine differences. Male offspring whose mothers were exposed to stress during the period of rapid myelination exhibited an increased plasma cortisol response to activation of the HPA axis. Female offspring whose mothers were exposed to stress during the period of rapid brain myelination exhibited decreased ambulatory activity and a blunted salivary cortisol response to the stress of the strobe light, but only during the estrous phase of the reproductive cycle. Therefore, the current set of studies has demonstrated the effects of prenatal stress on behaviour and HPA axis activity are dependent on; 1) the timing of the prenatal stress and 2) the hypothalamic-pituitary-gonadal axis in both male and female offspring. These studies have begun to uncover the mechanisms underlying programming and provide the basis for continuing research in humans.