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.

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.

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.

The neuroanatomical distribution and the physiological role of the biogenic amine octopamine was investigated in the prosomal central nervous system (CNS) of the horseshoe crab, Limulus polyphemus. Perfusion of octopamine onto the isolated CNS elicited the rhythmic feeding motor pattern in the entocoxal motor nerves which innervate the feeding musculature. The specific effects of octopamine on the CNS and the pharmacology of its action are described. The stimulation of the feeding motor program at concentrations comparable to that of octopamine by the octopamine agonists NC-5, NC-7, and NC-13, the diterpene adenylate cyclase activator forskolin and the cyclic AMP analogue, 8-bromo cyclic AMP indicated that octopamine's effects may be mediated by a second messenger. The putative octopamine antagonists tested were ineffective or weakly blocked the effects of octopamine. The distribution and localization of octopamine in the prosomal CNS was determined by light and electron microscopic immunocytochemistry. Sixteen discrete clusters of octopamine-like immunoreactive (Oct-LIR) neurons were found in the circumesophageal ring (CER) of fused thoracic ganglia. The distribution of these immunoreactive cells are described. The immunoreactive somata are 40 to 100 $\mu$m in size and are found in clusters of 12-24 cells. There is extensive distribution of Oct-LIR nerve fibers in Limulus; the wide distribution of Oct-LIR provides an anatomical basis for the several effects of octopamine in Limulus. The subcellular localization of octopamine in Oct-LIR terminals in the CER was determined by postembedding immunoelectron microscopy with a 5 nm immunogold label. Labelled terminals are morphologically unique; they contain large, dense-core granules of a distinct shape, typically cylindrical with an indentation or depression in one end. These large granules are typically 100-150 nm in diameter and 150-400 nm in length. The dense labelling of these unusual granules with the gold particles indicates that octopamine is sequestered in or associated with these granules.

No description available.

No description available.

No description available.