Master of Science / Department of Kinesiology / Craig A. Harms / Subjects with prehypertension are at risk for developing hypertension (HTN). Hypertension is associated with low-grade systemic inflammation (LGSI). Aerobic exercise training (ET) is a proven means to reduce both blood pressure and LGSI in healthy and diseased subjects. Recently, high intensity interval training (HIIT) has been show to elicit similar cardiovascular and metabolic adaptations as ET in healthy and at-risk populations in a more time efficient manner. Therefore, we hypothesized that HIIT would elicit greater reductions in blood pressure and LGSI than ET. Twelve pre-hypertensive subjects (systolic blood pressure 127.0 ± 8.5 mmHg; diastolic blood pressure 86.2 ± 4.1 mmHg) were randomly assigned to an ET group (n=5) and a HIIT group (n=7). All subjects performed an incremental test to exhaustion (VO2max) on a cycle ergometer prior to, after 4 weeks, and after 8 weeks of training. Resting heart rate and blood pressure were measured prior to and three times a week during training. LGSI was measured via high-sensitivity C-reactive protein (hs-CRP) prior to, after 4 weeks and after 8 weeks of training. ET subjects performed an eight week exercise training program at 40% VO2 reserve determined from the VO2max test, while HIIT subjects performed exercise at 60% peak power determined from the VO2max test. ET group trained four days/week while HIIT trained three days/week. ET exercised for 30 minutes continuously at a constant workload and cadence of 60 rpm while HIIT performed a protocol on a 1:1 work-to-rest ratio at a constant workload and cadence of 100 rpm. Both groups showed similar (p<0.05) decreases in mean arterial (ET = -7.3%, HIIT = -4.5%), systolic (ET = -6.6%, HIIT = -8.8%), and diastolic (ET= -9.7, HIIT= -8.2%) blood pressure. HIIT decreased in LGSI (-33.7%) while ET did not change LGSI (p>0.05). VO2max increased ~25% with both HIIT and ET with no differences (p>0.05) between groups. These data suggest both HIIT and ET similarly decreased resting blood pressure and increased VO2max while HIIT was effective in decreasing LGSI in subjects who were pre-hypertensive.

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.

Distinct membrane fusion events in the polarized pancreatic acinar cell involve highly specific interactions between distinct sets of SNARE proteins forming exocytotic complexes, whose assembly is modulated by distinct Munc18 proteins. The Munc18 isoform responsible for these exocytotic events in the acinar cell is unknown. Here, I postulate Munc18b to regulate apical exocytosis in the acinar cell. Current dogma for the pathogenesis of acute pancreatitis, including alcoholic pancreatitis, is mis-targeting and deregulated fusion of zymogen granules with lysosomal bodies in the acinar cells. This derangement results in premature activation of proteolytic zymogens and autophagic digestion of cellular contents. I have hypothesized an alternate mechanism, which is pathologic exocytosis occurring at the basolateral plasma membrane, and further propose Munc18c to mediate this process in alcoholic pancreatitis. The aims of this thesis are to demonstrate the roles of Munc18b and Munc18c in regulated apical exocytosis and pathologic basolateral exocytosis underling alcoholic pancreatitis, respectively. In Chapter Three, using both real-time and static imaging techniques and biochemical tools, I demonstrated that Munc18c is dissociated from the acinar basal plasma membrane (BPM) when stimulated with postprandial CCK8 preceding preincubation of acini with postprandial 20-50mM ethanol concentrations. This activated Syntaxin (Syn)-4 and SNAP-23 on the BPM to complex with VAMP proteins on the granule to form the exocytotic SNARE complex that triggered basolateral exocytosis. This molecular mechanism of pathologic basolateral exocytosis was recapitulated in an Ethanol-diet rat model of pancreatitis. In Chapter Four, I determined Munc18b to be in the apical pole of the acinar cell to appropriately bind cognate Syn-2 and Syn-3 in the apical PM and ZGs. Here, I examined the structure-function of Munc18b on amylase secretion by employing Munc18b mutants with distinct affinities to Syn-2 and Syn-3. In Chapter Five, I discovered a novel EF-hand Ca2+-binding protein called Cab45b, which binds Munc18b to regulate its membrane targeting and interactions with Syntaxins in the acinar cell in a manner that influenced Ca2+-induced amylase release. Taken together, these studies clarify our understanding of the role of Munc18 proteins involved in regulated and pathologic membrane fusion events underlying physiologic digestive enzyme secretion and clinical alcoholic pancreatitis.

The inter-relation between inflammation, the immune system and leukocytes is multifaceted, with communication between stroma and immune cells mediated by cytokines, growth factors, chemokines, integrins and other molecules. Proteolysis plays an important role in regulating these molecules. Proteolytic cleavage can not only destroy some molecules but can activate or shed others, converting local juxtacrine signalling proteins into effectors that act at a distance. Shedding can also convert membrane-bound receptors into soluble ligand-binding inhibitors. Finally, cleavage can convert agonist molecules into antagonists. As a wide-ranging inhibitor of metalloproteinases, tissue inhibitor of metalloproteinase 3 (TIMP3) has the potential to down-regulate many of these activities. We explore the role of TIMP3 in the regulation of inflammation, revealing that loss of TIMP3 leads to a more rapid increase of soluble TNF, higher levels of soluble TNF receptors and ultimately to increased TNF signalling in systemic inflammation. We also demonstrate TIMP3 loss impacts local inflammation. In addition we investigate the importance of TIMP3 in the expansion of hematopoietic cells.

Zinc levels in pancreatic islets are amongst the highest in the body and reduction in its levels in the pancreas has been associated with diabetes. The link between zinc, diabetes and islet dysfunction has recently been reiterated by genome-wide association studies that identified an islet cell membrane zinc transporter, SLC30A8 (ZnT8), as one of the risk loci for type 2 diabetes. Here we begin to elucidate the molecular mechanisms linking ZnT8 and type 2 diabetes by characterizing global and beta cell specific ZnT8 knockout mice. Our results associate absence of ZnT8 with a reduction in zinc sequestration into insulin vesicles, abnormal insulin granule morphology, down regulation of insulin processing enzymes, abnormal insulin secretion, elevated plasma proinsulin levels and diet-induced obesity and insulin resistance. Furthermore, we observed differential zinc uptake properties by two human ZnT8 variants. We report here that the W325 variant of ZnT8 is more efficient in mediating zinc transport than the at risk variant, R325. Cumulatively, these results suggest that ZnT8 is crucially important for zinc transport and zinc-insulin crystallization in insulin granules of the pancreatic beta cell.

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.

This thesis consists of four chapters. In this thesis we explored associative plasticity of human motor cortex with the use of noninvasive transcranial magnetic stimulation (TMS). Paired Associative Stimulation (PAS) has grown in popularity because of its potential clinical applications. We used TMS techniques in combination with electromyographic (EMG) measurements to study cortical excitability and kinematic features of arm movement. This work has focused in a cohesive approach to answer certain fundamental questions about a) the rules of cortical plasticity and mechanism of PAS, b) the interaction between the state of neuronal excitability at the targeted cortical network and the effects of PAS, and c) translation of these effects into obvious measurable kinematic changes starting from network level changes and ending up with the behavioral modulation of arm movement. First we explored the role of GABAergic intracortical networks and intracortical facilitation on modulation of cortical excitability by showing for the first time that PAS can be conditioned by these inhibitory and facilitatory intracortical networks. Next, using standard indirect approaches utilizing peripheral EMG measures, we showed a graded excitability response for the PAS technique and showed that interactions of PAS with motor learning depends on the degree as well as the state of cortical excitability. Rules governing the interactions of brain stimulation techniques and motor learning are important because brain stimulation techniques can be used to modify, improve or disrupt motor adaptation and skill learning with great potential for clinical applications such as facilitation of recovery after stroke. TMS provide us with a unique opportunity to study the rules of plasticity at a systems level, which is a combination of synaptic and nonsynaptic (metaplastic) changes. These changes can occur either in the direction to limit the physiological range of neuronal functioning (homeostatic) or against the direction established state of neurons.

This thesis tested the hypothesis that ethanol suppresses respiratory muscle activity by effects at the central motor pool and/or by state-dependent regulation of motor activity via influences on sleep/arousal processes. Ten rats were implanted with electroencephalogram and neck electrodes to record sleep-wake states, and genioglossus and diaphragm electrodes for respiratory recordings. Studies were performed following intraperitoneal injection of ethanol (1.25g/kg) or vehicle. The effects on genioglossus activity of ethanol (0.025-1M) or vehicle applied directly to the hypoglossal motor nucleus were also determined in sixteen isoflurane-anaesthetized rats. The results of these studies suggest that ethanol at physiologically relevant concentrations promoted sleep, and altered electroencephalogram and postural motor activities indicative of a sedating effect. The lack of effect on genioglossus activity with ethanol applied directly to the hypoglossal motor pool suggests that the suppression observed with systemic administration may be mediated via effects on state-dependent processes rather than direct effects at the motor pool per se.

Transforming growth factor-beta 1 (TGF-β1) is a multifunctional cytokine that orchestrates key events of development, disease and repair in the central nervous system (CNS). To investigate the effects of chronically producing TGF-β1 on synaptic structure and synaptic transmission, I performed immunohistochemistry and immunoblot of brain tissues from transgenic mice (TGF-β1 mice) that over-express active form of TGF-β1 from astrocytes in the CNS. Immunohistochemical assays showed that synaptophysin increased in the CA3 subfield whereas calbindin-D28K decreased in the mossy fibres. Immunoblot analysis revealed that several α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor subunit proteins were up-regulated in the hippocampus of TGF-β1 mice. To examine the direct effect of TGF-β1 alone on glutamatergic synaptic activity, cultured hippocampal neurons were treated with or without TGF-β1. Electrophysiological recordings displayed that TGF-β1 significantly increased the amplitude of glutamate-evoked current (p<0.05). Taken together, these data suggest that TGF-β1 modulates hippocampal glutamatergic synaptic protein expression and regulates synaptic transmission.

Rationale: Vascular remodeling and right-ventricular (RV) dysfunction are features of refractory pulmonary hypertension (PHT) in human neonates. These features are replicated in rats chronically exposed to hypoxia (13% O2), in which increased pulmonary vascular resistance (PVR) was acutely normalized by Y-27632, a Rho-kinase (ROCK) inhibitor, but not by inhaled nitric oxide. Objective: To examine the reversing effects of sustained ROCK inhibition on haemodynamic (RV dysfunction and increased PVR) and structural (RV hypertrophy and arterial wall remodeling) changes of chronic hypoxic PHT. Methods: Rat pups were exposed to air or hypoxia from birth for up to 21 days and received Y-27632 (15 mg/kg/b.i.d.) or vehicle from day 14. Results: Y-27632 normalised RV dysfunction and reversed remodeling secondary to chronic hypoxia. These changes were accompanied by increased apoptosis of smooth muscle and attenuated endothelin-1 expression in pulmonary arteries. Conclusion: ROCK inhibitors hold promise as a rescue therapy for refractory PHT in neonates.