Cold-water immersion (CWI) is a common recovery modality used to facilitate restoration of pre-exercise muscle force generation and soreness following high-intensity exercise. Although it is commonly used by athletes and commonly studied in sport science, evidence is equivocal regarding its efficacy. We compared 4 CWI protocols (10 or 30 minutes at 10 or 20°C) of different durations and temperatures with passive rest for their effects on drop jump and squat jump height, inflammation (IL-6, IL-10, IL-8, MPO, IL-1β, TNFα, IFNγ, GM-CSF, IL-2), and ratings of soreness/impairment following high-intensity intermittent sprint-exercise. CWI for 10 minutes at 10°C promoted restoration of force generation, while CWI for 30 minutes at 10°C was associated with lower ratings of soreness/impairment, but higher plasma IL-8 and MPO at 2 hours post-exercise. Overall, minor functional benefits of CWI for 10 minutes at 10°C were observed, while longer duration CWI protocols may increase post-exercise inflammation.

An increasing number of women delay childbearing and consequently face infertility and pregnancy complications associated with age. The central contributor to compromised reproductive performance is poor oocyte quality. Despite advances in assisted reproductive technologies, a strategy to overcome the damage that oocytes receive with age is yet to be identified. This work focuses on the influence of TAp73, a protein that decreases in mouse and human eggs with age, on the developmental capacity of mouse oocytes. TAp73 deficient mice were found to have fewer active mitochondria and compromised clearance of damaged material in their oocytes, possibly due to reduced mTOR-TAp73 axis signaling. These qualities were shown to contribute to low oocyte maturation rates. Additionally, TAp73 likely mediates the action of coenzyme Q10, which restores oocyte TAp73 levels and mitochondrial quality in aged mice. Together these findings suggest that TAp73 is a promising therapeutic target for improving oocyte function.

An increasing number of women delay childbearing and consequently face infertility and pregnancy complications associated with age. The central contributor to compromised reproductive performance is poor oocyte quality. Despite advances in assisted reproductive technologies, a strategy to overcome the damage that oocytes receive with age is yet to be identified. This work focuses on the influence of TAp73, a protein that decreases in mouse and human eggs with age, on the developmental capacity of mouse oocytes. TAp73 deficient mice were found to have fewer active mitochondria and compromised clearance of damaged material in their oocytes, possibly due to reduced mTOR-TAp73 axis signaling. These qualities were shown to contribute to low oocyte maturation rates. Additionally, TAp73 likely mediates the action of coenzyme Q10, which restores oocyte TAp73 levels and mitochondrial quality in aged mice. Together these findings suggest that TAp73 is a promising therapeutic target for improving oocyte function.

Cold-water immersion (CWI) is a common recovery modality used to facilitate restoration of pre-exercise muscle force generation and soreness following high-intensity exercise. Although it is commonly used by athletes and commonly studied in sport science, evidence is equivocal regarding its efficacy. We compared 4 CWI protocols (10 or 30 minutes at 10 or 20°C) of different durations and temperatures with passive rest for their effects on drop jump and squat jump height, inflammation (IL-6, IL-10, IL-8, MPO, IL-1β, TNFα, IFNγ, GM-CSF, IL-2), and ratings of soreness/impairment following high-intensity intermittent sprint-exercise. CWI for 10 minutes at 10°C promoted restoration of force generation, while CWI for 30 minutes at 10°C was associated with lower ratings of soreness/impairment, but higher plasma IL-8 and MPO at 2 hours post-exercise. Overall, minor functional benefits of CWI for 10 minutes at 10°C were observed, while longer duration CWI protocols may increase post-exercise inflammation.

Calcium (Ca2+) influx through N-methyl-D-asparate receptors (NMDARs) is widely held to be the requisite step initiating delayed neuronal death following ischemic stroke. However, blocking NMDARs fails to prevent the accumulation of intracellular Ca2+ ([Ca2+]i) and subsequent neurotoxicity. This suggests that alternate, as yet uncharacterized Ca2+-influx pathways exist in neurons. Transient receptor melastatin 2 (TRPM2) is a Ca2+-permeable member of the transient receptor potential melastatin family of cation channels whose activation by reactive oxygen/nitrogen species (ROS/RNS) and ADP-ribose (ADPR) is linked to cell death. While these channels are broadly expressed in the central nervous system (CNS), the presence of TRPM2 in neurons remains controversial and more specifically, whether they are expressed in neurons of the hippocampus is an open question. Here, I employ a combination of molecular, biochemical and electrophysiological approaches to demonstrate that functional TRPM2 channels are expressed in pyramidal neurons of the hippocampus. Unlike in heterologous expression systems, the ADPR-dependent activation of TRPM2 in neurons required a concomitant rise in [Ca2+]i via either voltage-dependent Ca2+ channels or NMDARs. While short, repeated NMDA applications activated a TRPM2-like current in the absence of exogenous ADPR, sustained NMDA application to hippocampal neurons resulted in the activation of a pannexin1 (Px1) hemichannel. Px1 hemichannels are large conductance, nonjunctional gap junction channels that can be activated following periods of oxygen-glucose deprivation (OGD) in neurons. Activation of Px1 required the influx of Ca2+ through NMDARs. Supplementing the intracellular milieu with adenosine triphosphate (ATP) prevented Px1 activation, suggesting that hemichannels may be activated during periods of mitochondrial dysfunction and metabolic failure. Our findings have potential implications for the treatment of diseases such as cerebral ischemia and Alzheimer’s disease (AD) as they implicate two novel ion channels in the excitotoxic signaling cascade activated downstream of NMDARs.

Hypoglycemia is one of the most serious acute complications in intensively treated diabetes. Recurrent hypoglycemia predisposes individuals to subsequent hypoglycemia, and diminished counterregulatory hormone responses increase this threat. Elevated pancreatic and/or circulating somatostatin has been reported in diabetic humans and animals, and we postulated that excessive somatostatin contributes to the attenuation of counterregulatory hormone release during hypoglycemia in diabetes. It is known that somatostatin suppresses stimulated secretion of glucagon, epinephrine, and corticosterone. We hypothesized that selective somatostatin receptor type 2 (SSTR2) antagonism would: (Study 1) improve hormone counterregulation to hypoglycemia, and (Study 2) ameliorate hypoglycemia in recurrently hypoglycemic rats. Using both high (10 U/kg) and low (5 U/kg) dose insulin to induce hypoglycemia, we demonstrate that inhibiting the action of somatostatin on SSTR2 normalizes the severely attenuated glucagon and corticosterone responses to acute hypoglycemia in diabetic rats. These improvements were specific to diabetes since SSTR2 antagonism did not increase these hormones in non-diabetic rats in response to hypoglycemia. In the absence of hypoglycemia, SSTR2 antagonist neither markedly alters glycemia nor causes sustained elevations in counterregulatory hormones in diabetic animals. Diabetic rats exhibit up to 65% and 75% more pancreatic and plasma somatostatin than non-diabetic rats following hypoglycemia, respectively. Despite improvements of glucagon and corticosterone, expression of gluconeogenic enzymes PEPCK1 and G6Pase was unaltered. SSTR2 antagonism reduced the glucose requirement during a hypoglycemic clamp induced with a lower dose of insulin. In recurrently hypoglycemic diabetic rats, we demonstrate that SSTR2 antagonist treatment reduces the depth and duration of hypoglycemia and promotes the recovery to euglycemia, without affecting the glycemia-lowering effect of insulin. This amelioration of hypoglycemia by SSTR2 antagonism may be attributable in part to the observed modest improvements of glucagon, epinephrine, and corticosterone counterregulation following recurrent hypoglycemia. These results implicate an important role for increased pancreatic, and possibly circulating, somatostatin in defective hypoglycemic counterregulation in diabetes.

Bladder obstruction (neurogenic or anatomic) induces strain injury in detrusor smooth muscle cells. Signaling via strain injury in other systems has been highly studied, while in bladder obstruction, it has been quite limited to a small number of pathways. In our study we have examined the effects of strain injury using a combination of in vivo, ex vivo and in vitro models, with the aim of understanding disease pathogenesis in the bladder. Using a combination of literature searches, phospho-protein screens and pathway analysis, we uncovered three pathways activated by mechanical strain, ERK, STAT3 and mTOR, with potential for changing not only the way we understand but also the way we treat obstructive myopathies of the bladder. We found that not only were these pathways activated in response to strain and distension injury of BSMC, but they were also responsible for proliferation and sometimes de-differentiation. Included herein are three chapters, published in 2006 and 2010, on the role of ERK, STAT3 and mTOR pathways in bladder smooth muscle cell proliferation and differentiation, 8 Appendices containing the first pages of other papers and reviews published during the course of my studies.

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.

Master of Science / Department of Kinesiology / David C. Poole / Introduction: Dietary nitrate(NO[subscript]3[superscript]-) supplementation, via its reduction to nitrite (NO [subscript] 2 [superscript]-) and subsequent conversion to nitric oxide (NO) and other reactive nitrogen intermediates, reduces blood pressure and the O[subscript]2 cost of submaximal exercise in humans. Despite these observations, the effects of dietary NO [subscript]3 [superscript]- supplementation on skeletal muscle vascular control during locomotory exercise remain unknown. We tested the hypotheses that dietary NO [subscript]3 [superscript]- supplementation via beetroot juice (BR) would reduce mean arterial pressure (MAP) and increase hindlimb muscle blood flow in the exercising rat. Methods: Male Sprague-Dawley rats (3-6 months) were administered either NO [subscript]3 [superscript]- (via beetroot juice; 1 mmol · kg[superscript]-[superscript]1 · day[superscript]-[superscript]1, BR n=8) or untreated (control, n=11) tap water for 5 days. MAP and hindlimb skeletal muscle blood flow and vascular conductance (radiolabeled microsphere infusions) were measured during submaximal treadmill running (20 m · min[superscript]-[superscript]1, 5% grade). Results: BR resulted in significantly lower exercising MAP (control: 137 ± 3, BR: 127 ± 4 mmHg, P<0.05) and blood [lactate] (control: 2.6 ± 0.3, BR: 1.9 ± 0.2 mM, P<0.05) compared to control. Total exercising hindlimb skeletal muscle blood flow (control: 108 ± 8, BR: 150 ± 11 ml · min[superscript]-[superscript]1 · 100 g[superscript]-[superscript]1, P<0.05) and vascular conductance (control: 0.78 ± 0.05, BR: 1.16 ± 0.10 ml · min[superscript]-[superscript]1 · 100 g[superscript]-[superscript]1 · mmHg[superscript]-[superscript]1, P<0.05) were greater in rats that received beetroot juice compared to control. The relative differences in blood flow and vascular conductance for the 28 individual hindlimb muscles and muscle parts correlated positively with their percent type IIb + d/x muscle fibers (blood flow: r=0.74, vascular conductance: r=0.71, P<0.01 for both). Conclusion: These data support the hypothesis that NO [subscript]3 [superscript]- supplementation improves vascular control and elevates skeletal muscle O [subscript]2 delivery during exercise predominantly in fast-twitch type II muscles, and provide a potential mechanism by which NO [subscript]3 [superscript]- supplementation improves metabolic control.

Doctor of Philosophy / Department of Anatomy and Physiology / Timothy I. Musch / Nitric oxide (NO) is synthesized via distinct NO synthase (NOS) enzymes and constitutes an essential cardiovascular signaling molecule. Whereas important vasomotor contributions of endothelial NOS (eNOS) have been well-described, the specific vasomotor contributions of nNOS-derived NO in healthy subjects during exercise are unknown. The purpose of this dissertation is to test the global hypothesis that nNOS-derived NO is a critical regulator of exercising skeletal muscle vascular control. Specifically, we utilized the selective nNOS inhibitor S-methyl-L-thiocitrulline (SMTC) to investigate the effects of nNOS-derived NO on skeletal muscle vascular function within established rodent models of exercise performance. The first investigation (Chapter 2) identifies that nNOS inhibition with SMTC increases mean arterial pressure (MAP) and reduces rat hindlimb skeletal muscle blood flow at rest whereas there are no effects during low-speed (20 m/min) treadmill running. In Chapter 3 it is reported that nNOS inhibition with SMTC reduces blood flow during high-speed treadmill running (>50 m/min) with the greatest relative effects found in highly glycolytic fast-twitch muscles and muscle parts. Chapter 4 demonstrates that nNOS-derived NO modulates contracting skeletal muscle blood flow (increases), O2 consumption (VO2, increases), and force production (decreases) in the rat spinotrapezius muscle and thus impacts the microvascular O2 delivery-VO2 ratio (which sets the microvascular partial pressure of O2, PO2mv, and represents the pressure head that drives capillary-myocyte O2 diffusion). In Chapter 5 we report that systemic administration of the selective nNOS inhibitor SMTC does not impact lumbar sympathetic nerve discharge. This reveals that the SMTC-induced peripheral vascular effects described herein reflect peripheral nNOS-derived NO signaling as opposed to centrally-derived regulation. In conclusion, nNOS-derived NO exerts exercise-intensity and muscle fiber-type selective peripheral vascular effects during whole-body locomotor exercise. In addition, nNOS-derived NO modulates skeletal muscle contractile and metabolic function and, therefore, impacts the skeletal muscle PO2mv. These data identify novel integrated roles for nNOS-derived NO within healthy skeletal muscle and have important implications for populations associated with reduced NO bioavailability and/or impaired nNOS structure and/or function specifically (e.g., muscular dystrophy, chronic heart failure, advanced age, etc.).