Behavioural phonotaxis (oriented movement in response to sound) is an effective means to quantify auditory perception in acoustically communicating insects. Previous phonotaxis studies on the acoustic parasitoid fly Ormia ochracea (Diptera: Tachinidae) have described stereotyped, reflex-like responses towards auditory stimuli modeled after their preferred cricket hosts, yet their ability to demonstrate plasticity of responses in the context of dynamically changing auditory cues has not previously been described. Using a behavioural sensitization protocol, I compared phonotaxis towards behaviourally irrelevant (non-attractive) test stimuli presented alone, and when preceded with the natural, response-evoking cricket song (attractive). Results demonstrate the cricket song as a sensitizing stimulus mediating phonotaxis towards otherwise non-attractive sounds, and differential walking patterns depending on temporal delay between song offset and test stimulus onset. My findings suggest an ecological purpose of sensitization, allowing flies to maintain orientation towards a cricket host amidst conditions of signal disruption in the environment.

Behavioural phonotaxis (oriented movement in response to sound) is an effective means to quantify auditory perception in acoustically communicating insects. Previous phonotaxis studies on the acoustic parasitoid fly Ormia ochracea (Diptera: Tachinidae) have described stereotyped, reflex-like responses towards auditory stimuli modeled after their preferred cricket hosts, yet their ability to demonstrate plasticity of responses in the context of dynamically changing auditory cues has not previously been described. Using a behavioural sensitization protocol, I compared phonotaxis towards behaviourally irrelevant (non-attractive) test stimuli presented alone, and when preceded with the natural, response-evoking cricket song (attractive). Results demonstrate the cricket song as a sensitizing stimulus mediating phonotaxis towards otherwise non-attractive sounds, and differential walking patterns depending on temporal delay between song offset and test stimulus onset. My findings suggest an ecological purpose of sensitization, allowing flies to maintain orientation towards a cricket host amidst conditions of signal disruption in the environment.

In this thesis I investigated the neural circuits that trigger cataplexy in mice. Specifically, I first addressed the theory that cataplexy is a REM sleep disorder. I then investigated a role for the noradrenergic and dopaminergic systems in murine cataplexy. Finally, I addressed the role of the amygdala in triggering cataplexy. From this work several specific conclusions can be drawn: 1. Cataplexy does not share a common executive mechanism with REM sleep, although the two may share a common mechanism that generates muscle atonia. Muscle tone during REM sleep and cataplexy is similar, however increasing REM sleep pressure does not increase cataplexy and positive affective stimuli that can increase cataplexy tend to decrease REM sleep. 2. Systemic manipulation of dopamine receptors can modulate cataplexy without affecting behavioral state. Specifically, manipulation of D2-like dopamine receptors at specific doses can modulate cataplexy while having no affect on sleep-wake state or sleep attacks, and manipulation of D1-like receptors potently affects sleep-wake state and sleep attacks without affecting cataplexy. 3. Systemic modulation of noradrenergic activity in orexin KO mice is sufficient to modulate cataplexy. Specifically, activation of excitatory α1 receptors reduces the occurrence of cataplexy while blockade of these receptors exacerbates it. 4. Withdrawal of an endogenous α1-mediated noradrenergic drive from motor neurons during wakefulness contributed to the loss of muscle tone during cataplexy. Re-establishing this excitatory drive exogenously alleviated cataplexy-dependant muscle atonia. 5. The amygdala is a critical part of the neural mechanism that triggers cataplexy in orexin KO mice. Ablation of the amygdala resulted in significant decreases in both baseline cataplexy and emotionally-induced cataplexy. The amygdala may trigger cataplexy through direct projections to brainstem areas that regulate muscle atonia.

Irx5 is a homeodomain transcription factor that negatively regulates cardiac fast transient outward K+ currents (Ito,f) via the KV4.2 gene and is thereby a major determinant of the transmural repolarization gradient. While Ito,f is invariably reduced in heart disease and changes in Ito,f can modulate both cardiac contractility and hypertrophy, less is known about a functional role of Irx5, and its relationship with Ito,f, in the normal and diseased heart. Here I show that Irx5 plays crucial roles in the regulation of cardiac contractility and proper adaptive hypertrophy. Specifically, Irx5-deficient (Irx5-/-) hearts had reduced cardiac contractility and lacked the normal regional difference in excitation-contraction with decreased action potential duration, Ca2+ transients and myocyte shortening in sub-endocardial, but not sub-epicardial, myocytes. In addition, Irx5-/- mice showed less cardiac hypertrophy, but increased interstitial fibrosis and greater contractility impairment following pressure overload. A defect in hypertrophic responses in Irx5-/- myocardium was confirmed in cultured neonatal mouse ventricular myocytes, exposed to norepinephrine while being restored with Irx5 replacement. Interestingly, studies using mice virtually lacking Ito,f (i.e. KV4.2-deficient) showed that reduced contractility in Irx5-/- mice was completely restored by loss of KV4.2, whereas hypertrophic responses to pressure-overload in hearts remained impaired when both Irx5 and Ito,f were absent. These findings suggest that Irx5 regulates cardiac contractility in an Ito,f-dependent manner while affecting hypertrophy independent of Ito,f. On the other hand, Irx5-ablation attenuated calcineurin (Cn)-induced hypertrophy in hearts and cultured cardiomyocytes, suggesting that the effect of Irx5 on hypertrophy involves the Cn-NFAT signalling cascade. Biochemical assessments further revealed that Irx5 can positively mediate Cn-NFAT activities as well as Nfatc3 and Gata4 expression, and interacts with Nfatc3 and Gata4, suggesting the formation of a transcription complex for hypertrophic gene regulation. Taken together, these studies have identified Irx5 as a vital cardiac transcription factor, important for contractile function of the heart by regulating Ito,f, and compensatory hypertrophic response to biomechanical stress in the heart by affecting the Cn-NFAT (and Gata4) signaling pathway.

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.

Master of Science / Department of Diagnostic Medicine and Pathobiology / Melinda J. Wilkerson / Failure of passive transfer (FPT) is one of the main reasons for increased mortality rate in newborn calves and diagnosis is dependent on determination of serum IgG concentrations (diagnosis is based on < 1 g/dL of total IgG). Several qualitative assays are available, but the reference method, single radial immunodiffusion assay (SRID), albeit quantitative measures only one subclass at a time. We set out to develop a competitive multiplex microsphere flow cytometry assay to measure bovine IgG1 and IgG2 concentrations in 30 serum samples acquired from newborn Holstein calves prior to and 24 hours after ingestion of colostrum and to compare the values with SRID. A triplex bead assay was created by mixing three distinct sets of Quantum plex carboxylated fluorescent microspheres that were coated with purified bovine IgG1, IgG2 or albumin using a two step chemical reaction. The triplex protein coated beads were reacted with a cocktail of sheep anti-bovine IgG1 and IgG2. Evaluation of analytical specificity demonstrated cross reactivity between anti-bovine IgG2 and IgG1 coated beads that precluded determination of IgG2 > 0.5 g/dL. Cross reactivity between anti-IgG1 and IgG2 coated beads was minimal and did not affect IgG1 concentrations between 0.15 to 1.2 g/dL. A competitive linear decrease in the fluorescence intensity was observed in the triplex assay when 2-fold dilutions spanning a concentration range of 12 mg/dL – 100 mg/dL of either purified bovine IgG1 or IgG2 were included as a competitive inhibitor of the reaction. Precolostral serum samples from 29 calves were determined to be < 0.4 g/dL by SRID. Standard calibrants for the flow assay were prepared from two fold serial dilutions of purified bovine IgG (stock concentration 10 g/dL) using a precolostral calf serum pool as the diluent. The standard calibrants (IgG1 was 1.0- 0.16 g/dL and IgG2 was 3.4 – 0.22 g/dL) were used as the inhibitors in a triplex assay to develop a standard curve for unknown samples. Dilutions of bovine reference serum containing known amounts of IgG1 (1.2 – 0.15 g/dL) and IgG2 (1.6 – 0.2 g/dL) was used as positive control. The intra Intra-assay and inter-assay precision of the mutiplex assay was good (coefficient of variation < 10%). Since the IgG2 concentrations of post colostral samples were below detection limit, only IgG1 values were compared to the SRID. The agreement between triplex microsphere assay and SRID for IgG1 was poor with a mean bias of 0.743 g/dL towards triplex microsphere assay (95% confidence interval of 0.382 to 1.105 g/dL). Method comparison studies between total IgG determined by SRID and the gamma-globulin fraction determined by serum electrophoresis indicated that the SRID calculated higher values than the protein method (mean bias of -1.4 g/dL, 95% confidence interval was -1.8 to -1.05 g/dL). We hypothesized that the positive bias for the microsphere assay was explained in part by the use of dilution factors, use of standards that had a low analytical range, and erroneously high standards used in the SRID method.

Master of Science / Department of Kinesiology / Craig A. Harms / Diaphragmatic fatigue is known to limit endurance performance during heavy exercise in humans. Previous reports have shown that diaphragmatic fatigue is reduced in rats with N-acetylcysteine (NAC; a nonspecific antioxidant) infusion, suggesting that oxidative stress contributes to this fatigue. However, it is not known if oral supplementation of NAC will reduce respiratory muscle fatigue during heavy exercise in humans. Therefore, the purpose of this study was to determine the effect of an acute oral dose of NAC on respiratory muscle fatigue during whole body heavy exercise. Eight healthy, non-smoking men (22+/-2 yrs), with no history of cardiovascular or lung disease, completed baseline pulmonary function tests followed by an incremental cycle VO[subscript 2peak] test. A randomized, double blind crossover design was then used where subjects were given either placebo (PLA) or NAC (1800 mg) 45 min prior to a 30 minute constant load (85% VO[subscript 2peak]) discontinuous (six-five minute stages) or continuous (cycle until volitional exhaustion) exercise test. Tests were separated by approximately one week. Maximum pressures (inspiratory, PImax; expiratory, PEmax) and venous blood samples (plasma lactate and total plasma glutathione) were made prior to- and following each 5-min of exercise in discontinuous tests and pre- and post-exercise in continuous tests. Subject's VO[subscript 2peak] was 43+/-5 ml/kg/min. There was no difference (p>0.05) in PImax between NAC (127.9+/-34.1 cmH[subscript2]O) or PLA (134.1+/-28.1 cmH2O) at rest. During exercise, PImax was significantly lower ([similar to]14%) in 6 of 8 subjects with PLA compared to NAC at minutes 25 and 30 of the discontinuous test indicating respiratory muscle fatigue. With NAC, PImax did not change (p>0.05) from rest throughout exercise indicating no respiratory muscle fatigue. There was no difference (p>0.05) in PEmax, plasma glutathione, lactate, oxygen uptake (VO[subscript 2]), ventilation (VE), heart rate (HR), or rating of perceived exertion between PLA and NAC at rest or during exercise. Time to exhaustion was not different (p>0.05) during the continuous tests (PLA: 1263 + 334 sec; NAC: 1047 + 136 sec). These results suggest that an acute dose of NAC reduces respiratory muscle fatigue during high intensity exercise but does not alter other ventilatory or metabolic indices. The significance of this reduced respiratory muscle fatigue with NAC on whole body exercise performance remains to be determined.

Master of Science / Department of Kinesiology / Thomas J. Barstow / Background and Aim: Long-term microgravity exposure, via spaceflight or -6° head-down tilt bedrest, has been shown to produce significant cardiovascular deconditioning and decreases in exercise performance. However, there is little known about how acute microgravity exposure influences the cardiovascular system’s ability to adjust to increases in physical work. Therefore, the aim of this study was to compare cardiovascular and exercise performance during acute upright, supine and -6° head-down tilt positions. Methods: Seven healthy inactive men performed maximal cycle exercise (VO2peak) tests in the upright, supine, and -6° head-down tilt on separate days. Oxygen consumption and heart rate were measured continuously throughout the testing procedures. Cardiac output (acetylene exhalation technique) was measured periodically and interpolated to the 100-watt work rate. Stroke volume was calculated from cardiac output and heart rate data. Results: Peak oxygen uptake and heart rate were significantly decreased in the supine and -6° head-down tilt positions compared to the upright (VO2peak 2.01±0.46, 2.01±0.51 versus 2.32±0.61 L/min respectively; peak heart rate 161±13, 160±14 versus 172±11 bmp). However, cardiac output at 100-watts was similar in all three-exercise positions. Calculated stroke volume at 100-watts was significantly higher in the -6° head-down tilt position compared to the upright position (76.6±4.7 versus 71.2±4.5, ml). Conclusion: These results suggest that exercise capacity is immediately decreased upon exposure to a microgravity environment, prior to any cardiovascular deconditioning. Therefore, an astronaut’s exercise performance should be evaluated with exercise tests in the -6° head-down tilt position prior to space flight in order to establish a baseline response.

Master of Science / Department of Animal Sciences and Industry / Duane L. Davis / The common choice of medium for culturing pig umbilical cord matrix stem cells (PUCs) is high glucose Dulbecco’s Minimum Essential Medium (HG-DMEM) supplemented with fetal bovine serum (FBS). FBS is a chemically undefined supplement that encourages attachment of explants and cells and is useful for long-term proliferation in an undifferentiated state. Removing FBS from the culture medium would decrease the possibility of microbial contamination and might produce more consistent results. A defined medium would facilitate experiments to determine requirements for specific growth factors and nutrients. Starting PUCs in a FBS-free environment proved to be a challenge. The results of 15 experiments testing various media, supplements, and culture conditions indicate that PUCs initially plated in an FBS-free environment do not attach as readily as those in HG-DMEM supplemented with FBS. PUCs were collected using enzyme digestion of the whole cord or by plating explants from the cord in culture medium. In the final experiment PUCs were seeded in 24-well plates (5.0 * 10[superscript]4 viable cells per well) with a collagen coating and cultured in Knock-out DMEM (KO-DMEM) with basic fibroblast growth factor (5ng/mL) and platelet derived growth factor (5ng/mL) in a low oxygen atmosphere (5% O[subscript]2/ 5% CO[subscript]2/ 90% N[subscript]2). The total non-adherent cell count at passage 1 was 1.78 * 10[superscript]5 +or- 3.68 * 10[superscript]4 and the total adherent cells were 2.58 * 10[superscript]5 +or- 9.29 * 10[superscript]4. The well confluence during initial cell proliferation appeared similar to cells cultured in the control media with 20% FBS (total adherent cells = 6.40 * 10[superscript]5 +or- S.E. 1.61 * 10[superscript]5 and total non-adherent cells = 2.88 * 10[superscript]5 + 7.60 * 10[superscript]4). However the number of adherent cells recovered for passage 2 was considerably less for cultures in FBS-free media than for the control group. Serum may affect attachment by providing attachment factors or it could change expression of integrins or other attachment molecules on the PUCs that enhance attachment to plastic or other substrates. In future studies the requirements for attachment of PUCs should be further evaluated.

Master of Science / Department of Kinesiology / Timothy I. Musch / During exercise aged individuals exhibit endothelial dysfunction and decreased levels of whole-limb blood flow (BF), both of which may be linked mechanistically to age-related increases in reactive oxygen species (ROS). Ascorbic acid (AA) reduces levels of ROS and has been shown to alleviate vascular and hyperemic dysfunction at rest (Jablonski et al., 2007) and during small muscle mass exercise in humans (Kirby et al., 2009). However, the effect of AA on vascular function and BF to individual muscles during whole-body exercise is not known. PURPOSE: To test the hypothesis that a single high-dose infusion of AA would increase BF to the hindlimb musculature of old rats at rest and during treadmill running. METHODS: 18 old (~28 months) Fischer 344 x Brown Norway rats were randomized into rest (n=9) and exercise (n=9) groups. BF to the total hindlimb and individual muscles (28 individual muscles and muscle parts) was evaluated via radiolabeled microspheres before and after intra-arterial AA administration (76 mg/kg in 3 ml heparinized saline, 30 minute infusion) at rest and during submaximal treadmill running (20m/min, 5% grade). Total antioxidant capacity (TAC) and thiobarbituric acid reactive species (TBARS) were measured before and after AA to determine the ability of this specific dose of AA to increase levels of plasma antioxidants and decrease levels of ROS, respectively. RESULTS: At rest: AA increased TAC (~37%, P<0.05) but did not change TBARS (Pre: 6.8±0.7 vs Post: 7.0±1.0 µM, P>0.05). AA decreased total hindlimb BF (Pre: 25±3 vs Post: 16±2 ml/min/100g, P<0.05) and BF to 8 of the 28 muscles that were evaluated. During exercise: TAC was increased (~35%, P<0.05) and TBARS were decreased (Pre: 9.8±2.0 vs Post: 7.0±1.0 µM, P<0.05). However, there was no effect on either total hindlimb BF (Pre: 154±14 vs Post: 162±13, P>0.05) or BF to any of the individual muscles evaluated. CONCLUSIONS: Increased TAC via AA infusion reduces hindlimb muscle BF at rest but had no effect on BF during whole-body dynamic exercise. Thus, even though TBARS decreased, there was no evidence that AA supplementation increases blood flow to the locomotor muscles of old rats during whole-body exercise.