The primary aim of this dissertation was to examine the role of short-term immobilization on muscle function recovery, excitability of the muscle, evoked contractile properties of the muscle, and muscle proteins in the blood after contraction-induced muscle injury. In Studies I and II, the effects of four days of immobilization on recovery of muscle function and serum creatine kinase (CK) activity after eccentric exercise was examined in 26 males, who were placed into one of three groups: immobilization, control, or light exercise. When the damaged elbow flexor muscles were immobilized or exercised for four consecutive days, force recovery over eight days was significantly enhanced compared to a control. In Study II, during the four-day treatment period after eccentric exercise, immobilization resulted in a significant blunting of the CK response compared to the light exercise or control groups. However, increasing activity with light exercise did not have any effect on the CK response compared to control. The data from Study II suggested that reduced lymphatic transport with decreased muscular activity may have contributed to the lower CK response in the immobilized muscle. In Study III, mechanisms to explain the observations in Studies I and II with immobilization were undertaken. Muscle excitability and evoked contractile properties of the muscle were examined to determine whether immobilization altered the mechanical properties of the muscle to favor an enhanced force response. After eccentric exercise, there were immediate and prolonged reductions in the evoked contractile properties of the muscle. Immobilization, however, had no effect on these measures. CK and myoglobin were assessed during the four-day treatment period as well as during the five-day recovery period. There was a significant difference in the CK response between groups, with the immobilization demonstrating significant blunting of the CK response during the treatment period. Upon remobilization of the arm, CK activity increased but not as high as was anticipated. The myoglobin response, however, was not different between groups. Because their routes of entry into the blood differ, taken together, the myoglobin and CK response suggest that lymph transport likely contributed to the blunting of the CK response observed with immobilization.

At fertilization, the sperm induces a series of species-specific intracellular Ca2+ rises ([Ca2+]i) that are important for oocyte activation and embryonic development in all mammalian species studied to date. These have not been investigated in the horse, where in vitro assisted reproduction techniques generally provide suboptimal results. In this study, we characterize the activity of equine sperm extracts (sperm factor; eSF) in mouse oocytes, and demonstrate their ability to induce [Ca 2+]i transients and parthenogenetic activation in equine oocytes. However, sperm injected (ICSI) horse oocytes do not consistently mount [Ca2+]i responses, and this failure is not the result of inadequate sperm factor release into the oocyte. This research may explain suboptimal results reported for ICSI in the equine and suggests potential differences in fertilization induced-signaling mechanisms amongst different mammalian species.

Eccentric contractions promote short- and long-term adaptations in skeletal muscle proteins, but little is known of molecular alterations associated with these changes. The present work investigated adaptations particular to eccentrically-biased exercise by evaluating mRNA and protein expression of three heat shock proteins (HSP25, HSC70 and HSP70) in both a murine and human model. In the first model, untrained murine biceps brachii were examined following a single 15 minute bout of either uphill (+15°) or downhill (−15°) running. Uphill running elicited several mRNA changes but only one detected protein increase of HSP70 (3-fold) at 12 and 24 hours post-exercise (PX) and a significant decrease in HSP25 during exercise and at 6 hours PX. In contrast, downhill running also prompted significant mRNA changes as well as HSP70 protein accumulation (ranging from 2- to 5-fold) at 0.5, 1, 12, 48 hours and 1 week PX; HSP25 expression increased significantly at 24 hours and 1 week PX. HSC70, which is thought to be non-inducible, exhibited both short and long-term changes in abundance after downhill running, with significant increases in expression (also ranging from 2- to 5-fold) at 1, 24 and 72 hours as well as 1, 4 and 12 weeks PX. In the human model for eccentric exercise, untrained subjects performed 50 high-force eccentric contractions with their non-dominant biceps brachii (BB) and ran downhill (−10°) for 30 minutes. The 48-hour PX stress response was evaluated with immunoblotting and RT-PCR of material obtained in muscle biopsies. On the protein level, HSP27 and HSP70 abundance increased significantly PX in the BB (383% and 226% respectively; p < 0.01, but there were no significant HSP changes in the vastus lateralis (VL). The RT-PCR data supported these findings: BB HSP27 and HSP70C mRNA levels increased (135% and 128% respectively; p < 0.05); HSP70B increased in the VL only (206%; p < 0.05). In sum, a single bout of eccentrically-biased exercise elicits short- and long-term adaptations in the inducible expression of stress proteins HSP70 and HSP25 as well as constitutive proteins like HSC70. Further, these data indicate that HSP responses are exercise-specific and the consistently larger HSP response in the exercise with the most eccentric nature suggests that these molecules may be important to long-term skeletal muscle adaptations such as hypertrophy.

The mechanisms by which the sperm generates long lasting [Ca2+ ]i oscillations in mammalian oocytes is discussed in the first chapter. In Chapter 2, a study was undertaken to determine if injection of porcine sperm factor(s) (pSF) can be used to activate bovine oocytes during nuclear transfer. It was found that injection of 5 mg/ml pSF triggered [Ca 2+]i oscillations that resembled those associated with fertilization. This concentration of pSF supported in vitro and in vivo development up to 60–90 d of gestation. The effectiveness of pSF as an activating agent in bovine oocytes may have been compromised because it was unable to support oscillations pass 3 to 5 h post-injection. Likewise, a single injection of pSF failed to trigger down-regulation of the inositol 1,4,5-trisphosphate receptor-1 sub-type (IP3R-1). These results demonstrate that pSF can support early development in bovine nuclear transfer embryos; however, the efficacy may be limited due to the premature cessation of the induced oscillations. In chapter 3 we evaluated the temporal release of the Ca2+-active factor during mouse fertilization and its possible association with the perinuclear theca (PT) of mammalian sperm. Between 15–60 min post sperm entry a significant portion of the Ca2+ releasing activity became dissociated from the sperm head. The loss of the Ca2+ releasing activity coincided with exposure and solubilization of the sperm's PT, although disassembly of the PT did not appear to be required for the release of the Ca2+ factor. Lastly, the conditions in the egg that promote release of the sperm's Ca2+ factor do not appear to be cell cycle specific. Finally, in chapter 4 we evaluated the impact of the IP3R-1 on the generation of Ca2+ release in bovine oocytes. Bovine oocytes with fewer numbers of IP3R-1 were produced by injection of adenophostin A, a potent antagonist of the IP3R-1 that triggers degradation of the receptors. Western blot analysis revealed that >80% of the receptors were degraded. The ability of IP3R-1 deficient oocytes to trigger Ca2+ release was examined by injecting IP3 and pSF, two agonists of the IP3R-1. IP3-induced Ca2+ release was partially blocked in IP3R-1 deficient oocytes. Moreover, injection of pSF into IP3R-1 deficient oocytes failed to establish persistent Ca2+ responses. These results suggest that IP3R-1 number has significant impact on the generation of Ca2+ release in bovine oocytes.

The purpose of this work was to study the effectiveness of diet derived antioxidants from wheat based aqueous cereal extracts under simulated gastrointestinal pH conditions and to monitor their effectiveness in modulating iron mediated oxidation which has been suggested as a risk factor in chronic disease. Wheat based breakfast cereals of composition ranging from whole grain to a refined flour product were analyzed for potential antioxidant effects. The breakfast cereals were extracted under aqueous conditions and the resulting extracts were tested for their ability to inhibit phosphatidylcholine liposome oxidation. The extent of oxidation was monitored by measuring the formation of thiobarbituric acid reaction substances (TBARS) and lipid peroxides. The aqueous extracts were analyzed using solvent extraction, molecular weight fractionation, phytate analysis, soluble fiber analysis, and total phenolics assay to determine the types of compounds responsible for the antioxidant activity. The state and modulation of iron before and after simulated gastrointestinal pH changes was monitored using atomic absorption spectrometry and the bathophenanthroline test. The aqueous extracts from the whole grain wheat and wheat bran breakfast cereals displayed considerable inhibition to lipid oxidation, while the wheat flour product was less effective. Following molecular weight fractionation the high molecular weight fraction was found to retain most of the antioxidative properties. The aqueous extracts subjected to solvent extraction with chloroform resulted in an organic extract containing non-polar compounds found in the aqueous extract. The antioxidant activity of this organic extract was minimal suggesting that the majority of compounds responsible for inhibiting oxidation are polar. Phytate analysis along with the use of a non-metal catalyst revealed that the antioxidant mechanism is not solely due to metal chelation. Precipitation and isolation of soluble fiber from the aqueous extracts were found to have no effect on oxidation. The total phenolics assay indicated that high concentrations of phenolics are present in the aqueous cereal extracts and appear to contribute to the inhibition of lipid oxidation. Simulated gastrointestinal pH conditions resulted in a significant increase in antioxidant activity for all aqueous cereal extracts including the low molecular weight (molecular mass <3,000 Da) fraction following ultrafiltration. Simulated gastrointestinal pH conditions resulted in the solubilization of iron in the cereals fortified with elemental iron. This increase in soluble iron was minimal and was found to exist complexed and not in a free ionic state. The solubilized iron following gastrointestinal pH conditions did not significantly effect the oxidation rate of phophatidy1choline liposomes in the model system. The chemical state of iron in the cereal product which was fortified with ferric phosphate was uneffected by the simulated gastrointestinal pH conditions and remained insoluble.

Canids have long been considered to be conservative in their postcranial anatomy, so there are few studies examining individual canid taxa for locomotor adaptations. Canids are generally considered to be the most cursorial of the carnivorans. The limbs of large canids are generally adapted for rapid terrestrial locomotion, as these animals frequently rely on speed for prey capture. The prey animal is captured and killed using the jaws and teeth. Smaller canids, such as the red fox Vulpes and gray fox Urocyon, do not use their limbs primarily for fast running. The red fox appears to have many adaptations for running, including long slender legs, but these foxes do not run in their daily activities except when chased. The red fox uses its forelimbs to help in prey capture and its hind legs for leaping. The gray fox is an unusual canid since it regularly climbs trees. The limbs of the gray fox, especially the forelimb, are utilized in climbing. This dissertation contains a detailed description of the postcranial osteology and myology Vulpes and Urocyon cinercoargenteus and includes an analysis of these anatomical features in a functional framework. An examination of both the osteology and myology of the fore and hind limbs of these two foxes reveals that their behavior is reflected in a number of anatomical characters. Adaptations for leaping in the red fox include the presence of unusually long hind legs relative to the front legs, and an increase in the length of the distal bony limb elements relative to more proximal ones. In addition, the limb bones are very slender. Muscle bellies of tarsal and digital flexors and extensors are restricted to a proximal position on the limb, and muscles in general are emphasized that act along the long axis of the limbs. Adaptations of the gray fox for climbing include the presence of relatively short legs, a greater ability to rotate the radius on the ulna relative to other canids, and a relatively greater ability to abduct the hind limb. In addition, both red and gray foxes are able to retract their claws, an ability that is not generally associated with canids.

Most larval and permanently aquatic salamanders use undulatory swimming as their primary mode of steady aquatic locomotion. These swimming movements are powered by the segmented axial musculature. The hypaxial region of each segment consists of distinct muscle layers, which have a simple planar geometry and have varying architectural features. In an aquatic salamander Siren lacertina, the morphological features of the lateral hypaxial layers allow the shortening of muscle fibers to be amplified during contraction. The angled muscle fibers in these layers function to allow fiber shortening to be accompanied by substantial rotation of muscle fibers during contraction. The connective tissue sheets separating adjacent muscle segments (myosepta), allow the segment to bulge in a way that further amplifies muscle fiber rotation. The combined effect of architectural and moment arm variation ensures that muscle fibers from different layers undergo similar shortening patterns during swimming to allow for the generation of optimal tension during locomotion. In addition to steady swimming many salamanders respond to a predatory stimulus by performing a “C-start” aquatic escape response. This unsteady maneuver involves two kinematic stages, which function to propel the salamander away from the perceived threat. During metamorphosis, the tailfin of salamanders is resorbed and is thought to result in a substantial decrease in escape performance. However, in a stream salamander Eurycea bislineata , adults spend significant time in the water and behaviorally compensate for metamorphic changes in tail morphology by increasing the amplitude of escape responses. Aquatic locomotion in salamanders is not limited to axial swimming. Some salamanders also utilize their limbs to move along the substrate at slow speeds, while submerged. Structures used during aquatic walking face dramatically different mechanical loads compared to limbs used on land. The greater hydrodynamic resistance associated with water lowers the effective weight and can act to stabilize an organism throughout its gait. Therefore structures, such as the reduced limbs of S. lacertina, which would be considered ineffective on land, can be fully functional during aquatic walking.

The goal of this dissertation was to identify and characterize underlying mechanisms of exercise-induced muscle damage (EIMD) and muscle adaptation to damage. Study I examined contributions of central and peripheral factors to EIMD. Forty-six subjects performed voluntary and stimulated contractions before and immediately following eccentric exercise of the elbow flexors. Subjects demonstrating greater strength loss (a hallmark of EIMD) after eccentric exercise also had greater impairment of peripheral function, but similar central function compared with lower strength loss subjects, suggesting that the mechanism(s) driving variation in strength loss are localized mainly within the periphery. Study II further focused on peripheral factors, specifically molecular changes in gene expression within muscle tissue following eccentric exercise, to determine underlying molecular mechanisms of damage development. Three subjects performed an exercise in which one leg underwent concentric contractions, and the other leg performed both eccentric and concentric actions. Dependent variables included strength loss, soreness and serum creatine kinase activity. Muscle biopsy samples were taken 4-8h post-exercise. Microarray analysis of these samples identified upregulation of genes involved in inflammation, apoptosis (programmed cell death), structure and transcriptional regulation. These results provided the first global gene expression pattern of human muscle after eccentric exercise. EIMD is attenuated naturally via the repeated bout effect, where an initial bout of exercise confers a protective effect on muscle that results in less damage induced by a second bout of exercise. Study III aimed to identify mechanisms driving this adaptation. Seven subjects performed two bouts of eccentric exercise of the leg spaced 4wk apart. Muscle strength and soreness were evaluated and biopsies were collected at 6h post-exercise. Muscle samples were tested for expression of a subset of inflammatory genes identified in Study II. Study III showed upregulation of monocyte chemoattractant protein-1 and the transcription factors CEBPD and ZFP36 following the repeated bout. Monocyte chemoattractant protein 1 (MCP-1) was co-localized to macrophages and satellite cells, which are vital to muscle regeneration. These data suggest that specific alterations in the inflammation response may drive the repeated bout effect, possibly by enhancing communication between macrophages and satellite cells, which may strengthen muscle regeneration following EIMD.

Immunocytochemistry was used to study the distribution of gamma-aminobutyric acid (GABA) throughout the central visual nuclei and retina in Rana pipiens. In the diencephalon, intensely-labeled GABA immunoreactive neurons and nerve fibers were observed within the neuropil of Bellonci (nB) and corpus geniculatum (CG), while only immunoreactive puncta were found in the rostral visual nucleus (RVN). In the pretectal region, the posterior thalamic nucleus (nPT) contained the most intensely-labeled GABA immunoreactive perikarya and nerve fibers in the entire brain. Lightly immunoreactive perikarya were also found in the large-celled nucleus lentiformis mesencephali (nLM), as well as in the pretectal gray which contains neurons postsynaptic to the retinal terminal zones within nLM. In the optic tectum (OT), both immunoreactive perikarya and fibers were found within superficial layers 8 and 9; whereas only densely-packed immunoreactive perikarya were evident in the deep tectal layers (i.e. 2, 4, 6). The nucleus of the basal optic root (nBOR) contained a small number of lightly-labeled GABA immunoreactive perikarya mostly located in the dorsal half of the nucleus. A large number of perikarya within the nucleus isthmi (NI) were also lightly immunostained. In the retina, GABA immunoreactivity (both somata and fibers) was observed in all layers except the outer nuclear layer (ONL). Besides GABAergic putative horizontal and amacrine cells in the inner nuclear layer (INL), about 30% of total neurons within ganglion cell layer (GCL) expressed GABA immunoreactivity. Double-labeling studies indicated that about half of the GABA-containing perikarya in the GCL were retinal ganglion cells (RGCs). In addition, three GABAergic projection pathways existing in the visual system of Rana pipiens were demonstrated: (1) from RGCs to the contralateral OT; (2) from nBOR to the pretectal nLM; and (3) bilaterally from the NI to the OT. These results indicate that GABA is an important neurotransmitter in the frog visual system.

The purpose of this dissertation is to purify and characterize the carboxylesterase(s) associated with permethrin resistance in the permethrin-resistant (PE-R) strain of Colorado potato beetle (CPB), Leptinotarsa decemlineata, and to develop an immunoassay system for the detection of resistance in field populations of CPB. Most carboxylesterase (CbE) activity is found in the hemolymph and the soluble fraction of body tissue. Among a number of charged forms of CbE identified from hemolymph, the pI 4.5-4.9 CbEs are quantitatively elevated and are most responsible for permethrin resistance in the PE-R strain. Permethrin CbEs (i.e., pI 4.2-4.8 CbEs) have been purified from the hemolymph of the PE-R strain through several chromatographic procedures. The pI 4.8 CbE is a 46-48 kDa monomeric protein. The pi 4.5 CbE is likely a 57-59 kDa dimeric protein. All pI 4.5-4.8 forms are glycoproteins but the charge heterogeneity is not associated with N-glycan moieties. Biochemical properties of the pI 4.2-4.5 CbEs have been comparatively characterized through substrate kinetic analyses, specific inhibition studies, and pH-temperature experiments. The pI 4.8 and 4.5 CbEs share a number of similarities in their biochemical properties and functional role in resistance despite of their distinct molecular properties. The kinetics of inhibition of the pI 4.5-4.8 CbEs by permethrin and DDT are best described by a mixed-noncompetitive type and a noncompetitive type inhibition, respectively. The kinetic analyses indicate the presence of hydrophobic non-catalytic site(s) as well as hydrophobic catalytic site(s) that are available for the binding to hydrophobic insecticides. Along with a low level of permethrin hydrolysis, the hydrophobic binding nature of the pI 4.5-4.8 CbEs suggests that permethrin resistance is mainly conferred by sequestration rather than rapid hydrolysis of permethrin. The nonspecific sequestration by the pI 4.5-4.8 CbEs appears to be associated with the cross-resistance of the PE-R strain to other hydrophobic insecticides such as other pyrethroids, DDT, and abamectin. Polyclonal antisera have been generated against the 30, 48, and 60 kDa denatured CbE immunogens. A high degree of cross-reactivities of the antisera to different immunogens indicate that all CbE immunogens share a high level of structural similarity. An antibody capture immunoassay using denatured CPB hemolymph is shown to be effective in detecting the different levels of permethrin CbE in permethrin-resistant and -susceptible populations of CPB.