Doctor of Philosophy / Department of Anatomy and Physiology / Thomas J. Barstow / The primary aim of this dissertation was to evaluate the factors that contribute to the cardiorespiratory and vascular responses following exercise conditioning and microgravity deconditioning. The first study of this dissertation (Chapter 2) revealed that exercise training in the head down tilt posture, which places increases central blood volume compared to upright, results in cardiorespiratory adaptations in both upright and head down tilt postures which are not completely expressed with exercise training in the upright posture. These findings suggest that augmentation of the ventricular volume load during exercise training may result in adaptations that transfer across multiple body positions. In the second and third studies measurements of blood velocity and flow were performed via Doppler ultrasound. In Chapter 3 we observed that in the brachial and femoral arteries blood moves with a slightly blunted parabolic velocity profile that is very stable across a range of mean arterial pressures and downstream limb resistances. We concluded that these findings support the current calculations of shear rate based on the assumptions of laminar flow. With these assumptions confirmed, the investigation in Chapter 4 could be performed. We observed that acute exposure to a sustained antegrade shear rate, via unilateral forearm heating, increased measurements of flow-mediated dilation and the overall rate of adjustment for forearm blood flow and vascular conductance during dynamic handgrip exercise. These findings suggest that one potential stimulus for improvements in vascular function and health following exercise conditioning may be exposure to elevations in antegrade shear. Lastly in Chapter 5 we changed focus to the cardiorespiratory deconditioning following long-duration microgravity exposure. We retrospectively reviewed and analyzed previous investigations of microgravity deconditioning and demonstrated that the decrease in maximal O2 consumption ( O2max) occurs as a function of duration of exposure and that both convective and diffusive O2 transport pathways substantially contribute to this decline. In addition we reviewed the current literature and highlighted potential mechanisms, across several organ systems, which may contribute to this decline in O2max. Collectively, these studies revealed the breath of plasticity for cardiorespiratory adaptations to a variety of stressors.

Master of Science / Department of Animal Sciences and Industry / Timothy G. Rozell / Since vascularization of the theca layer increases as follicles progress in size through preantral and antral stages, the principal angiogenic factor, vascular endothelial growth factor A (VEGFA), may influence follicle growth via regulation of angiogenesis. However, VEGFA may also influence follicular development through nonangiogenic mechanisms since its expression has been localized to nonvascular follicles and cells. Alternative mRNA splicing of 8 exons from the VEGFA gene results in the formation of different VEGFA isoforms. Each isoform has unique properties and is identified by the number of amino acids within the mature protein. Proangiogenic isoforms are encoded by exon 8a while a sister set of isoforms with antiangiogenic properties are encoded by exon 8b. The antiangiogenic isoforms comprise the majority of VEGFA expressed in most tissues while expression of the proangiogenic VEGFA isoforms is upregulated in tissues undergoing active angiogenesis. The Vegfa angiogenic isoforms (Vegfa_120, Vegfa_164, and Vegfa_188) were detected in developing rat ovaries, and quantitative RT-PCR determined that Vegfa_120 and Vegfa_164 mRNA was more abundant after birth, while Vegfa_188 mRNA was highest at embryonic day 16. The antiangiogenic isoforms, Vegfa_165b and Vegfa_189b, were amplified and sequenced from rat ovaries and quantitative RT-PCR determined that Vegfa_165b mRNA was more abundant around embryonic day 18, but Vegfa_189b lacked a distinct pattern of abundance. VEGFA and its receptors were localized to pregranulosa and granulosa cells of all follicle stages and to theca cells of advanced-stage follicles. Antiangiogenic VEGFA isoforms were localized to pregranulosa and granulosa cells of all follicle stages and to theca cells of advanced-stage follicles. To determine the role of VEGFA in developing ovaries, postnatal day 3/4 rat ovaries were cultured with VEGFR-TKI, a tyrosine kinase inhibitor that blocks signaling through the VEGFA receptors, FLT1 and KDR. Ovaries treated with VEGFR-TKI had vascular development reduced by 94%. In addition, treated ovaries had more primordial follicles, fewer early primary, transitional, and secondary follicles, and greater total follicle numbers compared with control ovaries. This suggests that VEGFA promotes follicle recruitment and early follicular development. These effects may be dependent upon increased ovarian vascularization or they may be mediated by nonvascular mechanisms.

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.

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.).

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.

Doctor of Philosophy / Department of Anatomy and Physiology / David C. Poole / The purpose of this dissertation is to expand our knowledge on the physiological effects of the ubiquitous signaling molecule nitric oxide (NO). Focus is given to the impacts of the nitrate (NO[subscript]3[superscript]-) nitrite (NO[subscript]2[superscript]-) NO pathway on skeletal muscle vascular and metabolic function during exercise. The NO[subscript]3[superscript]--NO[subscript]2[superscript]--NO pathway has garnered tremendous research interest due to its ability to upregulate NO bioavailability independently of NO synthase (NOS) function and thus impact the metabolic responses to exercise. Chapter 2 demonstrates that NO[subscript]3[superscript]- supplementation via beetroot juice (BR) augments the skeletal muscle vascular responses to exercise. Five days of BR supplementation resulted in a significantly higher skeletal muscle blood flow (BF) and vascular conductance (VC) during exercise when compared to control. The increases in BF and VC were preferentially directed to muscles and muscle portions comprised predominantly of fast twitch fibers. Furthermore, exercising blood [lactate] was reduced, suggesting improved metabolic control. In chapter 3, BR resulted in a slower fall in the microvascular PO[subscript]2 (PO[subscript]2[subscript]m[subscript]v, the soul driving force for blood myocyte O[subscript]2 flux) during the crucial rest-contraction transition thereby preserving the pressure head needed to move O[subscript]2 from the capillary into the myocyte. Chapter 4 examines the effects of BR on fast vs. slow twitch muscles in which BR raised the PO[subscript]2[subscript]m[subscript]v during the steady state of muscle contractions in fast but not slow twitch muscles, likely due to the lower PO[subscript]2[subscript]m[subscript]v at rest and throughout muscle contractions within these tissues. Chapter 5 investigates the effects of direct arterial NO[subscript]2[superscript]- infusion on skeletal muscle BF and VC during exercise in rats with NOS blockade via N[superscript]G-nitro-L arginine methyl ester. NO[subscript]2[superscript]- infusion restored MAP and VC to levels observed in healthy control animals (with intact NOS function) highlighting the potential for a NO[subscript]2[superscript]- based therapy to positively impact vascular function in those with compromised NOS function such that is evident in many prevalent diseases. These results provide crucial mechanistic insight into the improved exercise tolerance observed in humans following NO[subscript]3[superscript]- supplementation whilst also challenging our current understanding of NO’s role in physiology and pathophysiology.

Doctor of Philosophy / Department of Anatomy and Physiology / Thomas J. Barstow / The primary aim of this dissertation was to describe the changes in microvascular hematocrit, as total[hemoglobin+myoglobin] (T[Hb+Mb] measured with near-infrared spectroscopy (NIRS), during post-occlusive reactive hyperemia (PORH). Mechanisms of reactive hyperemia within skeletal muscle were also explored. The investigation detailed in Chapter 2 of this dissertation found that the differing time courses of the kinetic responses of both oxy- and deoxy[Hb+Mb], are related to changes in T[Hb+Mb]. We also determined that adipose tissue thickness had no effect on a purely temporal analysis of NIRS data. In Chapter 3 we observed that brachial artery reactive hyperemia preceded changes in T[Hb+Mb] during reactive hyperemia. Assuming that myoglobin remained constant, we posited that changes in T[Hb+Mb] must reflect alterations in red blood cell concentration in the microvasculature, i.e., microvascular hematocrit. In Chapter 4 comparisons were made between brachial artery blood flow, cutaneous and skeletal muscle flux and T[Hb+Mb]. The conduit artery response was faster than the microvascular responses in all tissues. Within skeletal muscle, time to peak and the time constant for the on-kinetics were faster in T[Hb+Mb] compoared with intramuscular flux as measured with intramuscular laser-Doppler. We observed no differences in temporal responses between cutaneous and intramuscular measures and suggested that in a purely temporal analysis the cutaneous microvasculature could serve as an analog for the skeletal muscle microvasculature. Finally, in Chapter 5 we found that prostaglandin inhibition with ibuprofen altered the initial T[Hb+Mb] response during PORH without impacting cutaneous flux or brachial artery blood flow. Chapter 5 also discussed that the addition of a wrist cuff to our standard instrumentation prevented the accumulation of T[Hb+Mb] during the occlusion period.