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.

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.

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.

Precise regulation of heterotrimeric G-protein signaling is important for maintaining proper cardiovascular system function. Indeed, G-protein signaling is frequently upregulated during cardiovascular disease suggesting that identifying mechanisms for inhibiting G-protein signaling may be an effective therapeutic strategy for the treatment and prevention of disease. The work presented in this thesis is directed at two RGS proteins, RGS2 and RGS5, the two highest expressing RGS proteins in VSMCs. Despite the large number of studies published on them, there is still much to be learned about the specific G-protein pathways that each RGS protein controls. Using genetic and molecular models, we set out to identify novel regulatory pathways controlling RGS2 and RGS5 function. We hypothesize that characterizing the determinants and regulation of RGS protein function will provide a better understanding of the signaling that occurs within VSMCs under both physiologic and pathophysiologic conditions. Our work presented in the first three studies of this thesis, describes novel regulatory pathways that are involved in regulating RGS2 protein function. We describe the production of RGS2 protein isoforms that are the result of alternative translational start site usage. Interestingly, the expression pattern of these proteins is controlled by the signaling status of the cell. In the second two studies, we identify a functional consequence of RGS2-interaction with the plasma membrane. We show that this is dependent on the interaction between the amphipathic α-helix and anionic phospholipids present in the plasma membrane. We further show that disruptions in this interaction, as occurs in the human population, can lead to reduced RGS2 function and thus potentially hypertension. Finally, our last study focuses on the function and regulation of RGS5, the single highest expressing RGS protein in VSMCs. We show that the regulation of RGS5 is dependent, similar to other VSMC-specific genes, on the activity of SRF and myocardin. However, interestingly, RGS5 expression is further controlled by the extent of DNA methylation that occurs in its proximal promoter. We show that this is an important regulator of RGS5 expression both in development as well as during disease, specifically in-stent restenosis.

In this thesis, I examined the inhibitory control of REM sleep motor activity using both a pharmacological rat model and a genetic mouse model. I characterized the role for GABA and glycine in mediating the REM-specific suppression of muscle activity as well as their involvement in regulating the phasic muscle twitches that punctuate this atonia. Based on four specific research objectives, the following conclusions were drawn: 1. REM atonia is not directly mediated by glycinergic or GABAA-mediated inhibition. These data refute the prevailing hypothesis that REM atonia is caused by glycinergic inhibition. These receptors are, however, important in the regulation of phasic muscle twitch activity. 2. GABAB receptors can modulate REM atonia but only when acting in concert with GABAA and glycine receptors. Blockade of all three receptor types results in a partial reversal of REM atonia, suggesting a functional interaction is occurring between these receptors during REM sleep. 3. The phasic glycinergic/GABAA-mediated inhibitory drive present in REM sleep regulates the temporal pattern of phasic twitch activity that is seen across this state. I hypothesize that this progressively decreasing inhibitory input counteracts a gradually increasing excitatory input to shape the temporal distribution of muscle twitches across REM sleep. 4. A loss of normal inhibitory function may play a causal role in the pathology of REM sleep behaviour disorder (RBD), the sleep disorder characterized by excessive motor activity in REM sleep.

The hypothalamus is a central regulator of energy homeostasis. Recently, proglucagon-derived peptides have emerged as potential appetite regulators. The proglucagon gene is expressed in the periphery and also in selective hypothalamic neurons. The regulation of hypothalamic proglucagon by two key regulators of energy balance, insulin and leptin, remains unstudied. Central glucagon-like peptide (GLP)-1 receptor (GLP-1R) activation by exendin-4, a long-acting GLP-1R agonist, induces anorexia; however, the specific hypothalamic neuronal populations activated by exendin-4 remain largely unknown. The role of GLP-2 as a central appetite regulator is poorly understood. In this thesis, using murine hypothalamic cell lines and mice as experimental models, mechanisms involved in the direct regulation of proglucagon gene by insulin and leptin were studied, and the actions of exendin-4 and GLP-2 on hypothalamic neuropeptides were determined. It was found that insulin and leptin regulate hypothalamic proglucagon mRNA by activating Akt and signal transducer and activator of transcription 3, respectively. Insulin and leptin did not regulate human proglucagon promoter regions, but affected proglucagon mRNA stability. In mice, intracerebroventricular exendin-4 and GLP-2 induced anorexia, activated proopiomelanocortin- and neuropeptide Y-expressing neurons in the arcuate nucleus and neurotensin- and ghrelin-expressing neurons in major hypothalamic appetite-regulating regions. In the hypothalamic neuronal models, exendin-4 and GLP-2 activated cAMP-response element-binding protein/activating transcription factor-1, and regulated neurotensin and ghrelin mRNA levels via a protein kinase A-dependent mechanism. Overall, the in vivo and in vitro findings suggest that these neuropeptides may serve as potential downstream mediators of exendin-4 and GLP-2 action. This research demonstrates direct regulation of hypothalamic proglucagon by insulin and leptin in vitro, and reports a previously unrecognized link between central GLP-1R and GLP-2R activation and regulation of hypothalamic neuropeptides. A better understanding of the regulation of hypothalamic proglucagon and central GLP-1R and GLP-2R activation is important to further expand our knowledge of feeding circuits.

An acute increase in hypothalamic glucose and its downstream metabolite lactate lower glucose production (GP) and plasma glucose (PG) levels in normal rodents. However, the effectiveness of this nutrient-sensing mechanism in metabolic disease is unknown. We assessed the effects of intracerebroventricular (i.c.v.) or intra-hypothalamic glucose and lactate on in vivo glucose kinetics in conscious rats. Study I revealed that i.c.v. lactate lowered PG via a suppression of GP in rodents with uncontrolled diabetes and diet-induced insulin resistance. Study II demonstrated that i.c.v. glucose was ineffective at suppressing GP in uncontrolled diabetic rodents or rodents with a prior 24 h whole-body or hypothalamic hyperglycemic insult. When PG levels per se were normalized in diabetic rodents hypothalamic glucose sensing to lower GP was rescued. As such, sustained hyperglycemia per se impairs hypothalamic glucose effectiveness in diabetes. Further studies are necessary to determine defective mechanisms upstream of lactate metabolism hindering CNS glucose sensing.

Little is known of the temporal cerebral blood flow response to a chemical stimulus consisting of increased PCO2 measured over time. Currently, there is only one study suggesting multiple phases in the CBF-CO2 response. Time constants of middle cerebral artery blood velocity (MCAV) response to a change in PETCO2 have been reported to be between 3 and 99.4 s. We studied the MCAV response in 28 subjects (10 females) to a sustained +10 mmHg above baseline (10 min) acute increase of PETCO2. We found that there were three distinct MCAV response patterns among subjects. Additionally, the responses of males and females differed. These studies suggest that there are multiple overlapping mechanisms controlling the chemoresponse of cerebral blood vessels and that these mechanisms may differ between men and women.