One contentious issue regarding pancreatic acinar exocytosis concerns which SNAP25 isoform (SNAP23/29/47) mediates the various fusion events in this cell type. Based on dominant-negative over-expression studies, SNAP23 was hypothesized to be the putative isoform mediating apical exocytosis, basolateral exocytosis, and ZG-ZG fusion. Unfortunately, using a SNAP23 KD mouse model, 80% SNAP23 KD was insufficient to manifest any secretion phenotype. A novel syncollin-pHluorin exocytosis imaging technique initially meant to assess which fusion events are perturbed by SNAP23 KD was successfully developed and displayed improvements over previous imaging techniques. The syncollin-pHluorin imaging enabled visualization of apical and basolateral exocytosis as well as sequential ZG-ZG fusions. Combined with spinning disk microscopy, this assay allows 3D live exocytosis imaging with high temporal and spatial resolution. This novel imaging assay will be useful in visualizing apical, basolateral, sequential, and lateral fusion events for future acinar studies.

Our study aims to investigate the role of Syntaxin-3 in glucose stimulated insulin secretion (GSIS) and how it regulates the recruitment to plasma membrane and/or exocytotic fusion of insulin granules. We examined endogenous Syn-3 function by down-regulating its expression using siRNA/lenti-shRNA, which impaired GSIS. Although Syn-3 depleted cells showed no change in the number and fusion of docked granules, there was a reduction in newcomer granules and their subsequent exocytotic fusion. We then examined the effects of overexpressing Syn-3-WT, which enhanced biphasic GSIS. Since open conformation (OF) Syn-1A was reported to enhance exocytosis by promoting SNARE complex formation, we constructed OF Syn-3. Exogenous OF Syn-3 had no effect on secretion as it is unable to be trafficked to insulin granules. Taken together, we conclude that Syn-3 facilitates mobilization of newcomer insulin granules to the plasma membrane, to contribute to both first and second phase of GSIS in pancreatic beta cells.

Despite their undisputed importance to embryonic growth, the role of mRNA transport proteins in the developing visual system has been widely uncharacterized. Through RNA interference, this study aims to discover the function of Staufen 2 (Stau2), an mRNA transport protein, in chick eye development. When Stau2-miRNA was electroporated into the E1.5 primary optic vesicle, two days later they exhibited a reduction of eye size by 47%, whereas control miRNA did not significantly change eye size. TUNEL, β-III tubulin and BrdU staining were used to analyze the retinal apoptotic, differentiation and proliferative levels respectively, in response to Stau2 knockdown. These data suggest that the small eye is a result of a decrease in proliferation, and not cell death or pre-mature differentiation. Rescue experiments were done with each of the three Stau2 isoforms and confirmed both the direct effect of Stau2-miRNA and the involvement of these isoforms in eye development.

This thesis describes experiments to measure the ventilatory response to hypoxia at a constant (isocapnic) level of CO2 (HVR) in 18 subjects. So as to provide a complete picture of the autonomic responses, middle cerebral artery velocity, a surrogate for cerebral blood flow (CBF), as well as finger plethysmography blood pressure (BP) were also measured. Ventilatory responses have been previously described only in terms of an acute peak followed by a decline. However, rather than a single type of response, I found four types categorized as: Decline, Double, Plateau, or No response. The Double pattern, characterized by a second peak of response was the most common, yet is described here for the first time. These patterns are also characteristic of the CBF and BP responses. Furthermore the temporal correlations between these brainstem-controlled responses are also reported here for the first time.

The simultaneous application of interlaced innocuous warm and cool stimuli can elicit sensations of burning heat (the Thermal Grill Illusion, TGI). The TGI is thought to reflect changes in the central interactions between somatosensory sub-modalities (i.e. cold-inhibition of pain). Previous studies used multiple alternating warm and cool bars to elicit a TGI. The primary objective of this study was to evaluate the effects of altering the placements of warm and cool grill elements on the intensities of perceived pain and unpleasantness in 26 male subjects. Arranging the thermal grill elements in a warm flanking cool (WCW) pattern evoked significantly higher intensities of pain and unpleasantness than a cool flanking warm (CWC) pattern and a uniform warm stimulus. Conversely, CWC did not elicit significantly different intensities of perceived pain and unpleasantness than a uniform cool stimulus. This finding may reflect differences in the level of activation of central neurons.

The pathogenesis of diabetic nephropathy (DN) remains incompletely understood. In previous studies, we observed the activation of Tyr kinase Src by high glucose (HG) and showed that Src is required for MAPK activation and synthesis of collagen IV in cultured rat mesangial cells (MCs). Reactive oxygen species (ROS) are also important mediators of DN, and our present study aimed to investigate the role of Src in HG-induced ROS generation. In MCs, we found that HG led to ROS accumulation that was blocked by Src inhibitors or Src-specific siRNA. Downstream of Src, Vav2 was phosphorylated/activated leading to Rac1-dependent NADPH oxidase activation. Long-term HG exposure resulted in Src-dependent Nox4 protein induction. Nox2-specific siRNA abrogated ROS production only in short-term HG, while Nox4-specific siRNA blocked ROS production only in long-term HG. Taken together, our data indicate Src to be important in mediating ROS generation from both Nox2- and Nox4-containing NADPH oxidases.

Iatrogenic hypoglycemia is a limiting factor for managing diabetes mellitus and can have severe outcomes such as seizures and coma. Although several studies have investigated the central nervous system consequences of hypoglycemia, the effects of seizures, as well as possible treatment strategies, have yet to be elucidated in juvenile animals. The objective of this thesis was to establish an in vivo model of severe hypoglycemia and seizures in juvenile diabetic and non-diabetic rats. In both groups there existed a similar blood glucose threshold for seizures, and mortality only occurred following severe seizures, particularly with repeated seizures that were unresponsive to treatment. While the administration of anticonvulsants temporarily mitigated seizures, glucose administration was required to prevent mortality. Abnormalities in the hippocampal and brainstem electroencephalograms (EEG) were observed in hypoglycemic animals without a clear correlate to convulsive activity.

Master of Science / Department of Kinesiology / Timothy I. Musch / Skeletal muscle fatigue has a complex multifaceted etiology in which the ability to regulate intracellular calcium concentration ([Ca[superscript]2+][subscript]i) and the myofibrillar response to elevated [Ca[superscript]2+][subscript]i are key components. Pertinent to this issue, a calcium sensitizer compound has been shown to increase contractile function via altered myofibrillar Ca[superscript]2+sensitivity in in-vitro preparations. We tested the hypothesis that a calcium sensitizer compound would increase the endurance capacity and VO[subscript]2peak in young male Sprague-Dawley rats above saline control values. The exercise tolerance test consisted of a progressive exercise test in which each rat initially ran at a speed of 25 m/min up a 10% grade for 15 min. Thereafter the treadmill speed was increased by 5m/min every 15 min until fatigue (i.e., the rat could no longer maintain pace with the treadmill). VO[subscript]2peak was determined according to previously established methods used in our laboratory. Each rat initially ran at 25 m/min up a 10% grade for 2-3 minutes. The speed of the treadmill was the increased progressively in a ramp-like manner until fatigue. VO[subscript]2peak was defined either as the point at which O[subscript]2 uptake did not further increase despite increases in treadmill speed or the highest VO[subscript]2peak prior to fatigue. A calcium sensitizing compound or saline was administered via gastric gavage. There was a significant increase (P<0.05) in endurance capacity with 10 mg/kg of the calcium sensitizer compound, but not at lower (0.5-5 mg/kg) or higher (20-40mg/kg) doses. This improvement in endurance capacity occurred in the absence of any changes in VO[subscript]2peak. The highest dose (40 mg/kg) produced a significant decrease (P<0.05) in the endurance capacity as well as VO[subscript]2peak. These data demonstrate that the in-vitro observations of increased [Ca[superscript]2+][subscript]i sensitivity and improved muscle function with a calcium sensitizing compound can translate to improved whole body exercise performance. Further studies need to be conducted to explore the efficacy of calcium sensitizing agents in animal models of chronic disease (i.e. CHF and diabetes). It is possible that a calcium sensitizer compound could be used as a potential ergogenic aid for patients whom enhanced physical capacity could be of significant therapeutic value, and lead to increases in activities of daily living and quality of life.

Master of Science / Department of Mechanical and Nuclear Engineering / Steve Eckels / A new three dimensional transient human finger model was developed to predict the risk of frostbite in humans at different environmental conditions. The shape of the finger model was similar to that of a real human finger. Finite Element Techniques were used to build the finger model. Smith’s Model (1991) energy balance equations were used to calculate the temperatures in the current finger model. The current 3D finger model was validated against the experimental data of Wilson (1976) and Santee (1990). The model agreed well with the Wilson experiments and with the cold test in Santee experiments. The comparison indicates that the current finger model can be used to adequately predict the human finger responses in different environments. The current finger model was then tested in temperatures of 0, -10, -20, -25 and -30 oC and with different airspeeds 0, 3 and 6.8 m/s to assess the risk of frostbite in humans. Three resistances 0, 0.4 and 0.8 clo were used on the finger model to obtain responses in different environmental conditions. From the experimental results, an expression for safe glove resistance required to prevent frostbite in known temperatures was calculated. Also, the temperatures up to which a glove with known thermal resistance value can protect a human finger from frostbite was also computed.

Master of Science / Department of Kinesiology / Thomas J. Barstow / Dietary nitrate supplementation via beetroot juice (BR) has been shown to have positive effects on mitochondrial and muscle efficiency during large muscle mass exercise in humans, and more recently on locomotory muscle blood flow [Q-dot] in rats. To date, an integrated measure of these effects has not been performed in humans. Therefore, we assessed the influence of BR on [Q-dot] and muscle oxygenation characteristics during moderate and severe intensity handgrip exercise. Seven healthy men (age: 25 ± 3 yrs; height: 179 ± 4 cm; weight: 82 ± 9 kg) completed four constant-power exercise tests randomly assigned to condition (BR or placebo (PL)) and intensity (moderate (40% peak) or severe (85% peak)). Resting mean arterial pressure was significantly lower after BR compared to PL (79.3 ± 5.8 vs 86.8 ± 6.7 mmHg; p < 0.01). All subjects were able to sustain 10 min of exercise at moderate intensity in both conditions. BR had no significant effect on exercise tolerance during severe (342 ± 83 vs 382 ± 138 s, p = 0.382). Brachial artery [Q-dot] was not significantly different after BR at rest or any time during exercise in either intensity. Deoxygenated-[hemoglobin + myoglobin] was elevated at min 2 & 3 for moderate (p < 0.05) and throughout severe exercise (p = 0.03) after BR. The estimated metabolic cost ([V-dot]O₂) was not significantly different during either intensity after BR. These findings support the notion that an acute dose of BR may be valuable to reduce blood pressure in young adults, but revealed that it does not augment [Q-dot] or [V-dot]O₂ during small muscle mass handgrip exercise.