Thesis (MSCMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2006. / In this study, we presented data on the role of free radicals in human spermatozoa, particularly in the context of centrifugation and the potential development of defective sperm function. In order to achieve this, methods were developed to directly measure intracellular free radicals in human sperm and the effects of exogenously applied free radicals on sperm function were established. The role of brief and prolonged centrifugation and the associated generation of free radicals was also investigated.

Lysophosphatidic acid (LPA) belongs to a class of extracellular lipid signaling molecules. In the vasculature, LPA may regulate platelet activation and modulate endothelial and smooth muscle cell function. LPA has therefore been proposed as a mediator of cardiovascular disease. The bulk of circulating LPA is produced from plasma lysophosphatidylcholine (LPC) by autotaxin (ATX), a secreted lysophospholipase D (lysoPLD). Early studies suggest that some of the production of circulating LPA is platelet-dependent. ATX possesses an N-terminal somatomedin B-like domain suggesting the hypothesis that ATX interacts with platelet integrins which may localize ATX to substrate in the membrane and/or alter the catalytic activity of ATX. Using static adhesion and soluble binding assays we found that ATX does indeed bind to platelets and cultured mammalian cells in an integrin-dependent manner which is blocked by integrin function-blocking peptides and antibodies. This binding increases both the activity of ATX and localization of its product, LPA, to the platelet/cell membrane. LPA is generally stimulatory to human platelets although platelets from a small population of donors are refractory to LPA stimulation. Likewise LPA is inhibitory to murine platelets. We previously found that LPA receptor pan-antagonists reduce agonist-induced platelet activation, and partial stimulation of LPA5 specifically increases platelet activation in humans. Since both LPA5 and LPA4 are present at significant levels in human platelets, we hypothesized that LPA4 is responsible for an inhibitory pathway and LPA5 is responsible for an inhibitory pathway. We used mice deficient in LPA4 to test this model. Isolated platelet function tests revealed no major difference between lpa4-/- mice compared with WT mice although lpa4-/- mice were more prone to FeCl3-induced thrombosis. Paradoxically, chimeric mice reconstituted with lpa4-/- deficient bone marrow derived cells were protected from thrombosis. These discrepancies may be explained by involvement of endothelial cells and the relative scarcity of LPA receptors in murine platelets compared with human platelets. Taken together, these results demonstrate two critical regulators of LPA signaling and open up new avenues to further our understanding of atherothrombosis.

It is well documented that both quantitative and qualitative changes in the murine hematopoietic stem cell (HSC) population occur with age. In mice, the effect of aging on stem cells is highly strain-specific, thus suggesting genetic regulation plays a role in HSC aging. In C57BL/6 (B6) mice, the HSC population steadily increases with age, whereas in DBA/2 (D2) mice, this population declines. Our lab has previously mapped a quantitative trait locus (QTL) to murine chromosome 2 that is associated with the variation in frequency of HSCs between aged B6 and D2 mice. In these dissertation studies, I first aim to characterize the congenic mouse model which was generated by introgressing D2 alleles in the QTL onto a B6 background. Using a surrogate assay to mimic aging, I analyzed the cell cycle, apoptotic and self-renewal capabilities of congenic and B6 HSCs and show that D2 alleles in the QTL affect the apoptotic and selfrenewal capabilities of HSCs. In the second aim of these studies, I used oligonucleotide arrays to compare the differential expression of B6 and congenic cells using a population enriched for primitive stem and progenitor cells. Extensive analysis of the expression arrays pointed to two strong candidates, the genes encoding Retinoblastoma like protein 1 (p107) and Sorting nexin 5 (Snx5). B6 alleles were associated with increased p107 and Snx5 expression in old HSCs therefore both genes were hypothesized to be positive regulators of stem cell number in aged mice. Finally, in the third aim of these studies, I show that the individual overexpression of p107 and Snx5 in congeic HSCs increases day35 cobblestone area forming cell (CAFC) numbers, therefore confirming their roles as positive regulators of HSC number in vitro. These studies uncover novel roles for p107 and Snx5 in the regulation of HSC numbers and provide additional clues in the complex regulation of HSC aging.

Olfactory sensory decline has been associated with normal aging as well as neurodegenerative disorders, yet the underlying mechanisms are unclear. The overall aim of this dissertation was to investigate the fundamental molecular and cellular mechanisms associated with olfactory neurodegeneration. This investigation uses an integrative approach, combining proteomics and gene expression analyses with cellular and tissuelevel characterization. Using these approaches, two model systems were investigated: 1) normally aging C57BL/6 mice of ages 1.5-, 6- and 20-months; and 2) a mouse model of elevated endogenous oxidative stress-associated neurodegeneration, namely, the Harlequin mutant mouse. The first specific aim was to test the hypothesis that oxidative stress is associated with aging of the olfactory system. Using proteomics, I demonstrated that olfactory aging was accompanied primarily by increased oxidative stress-, mitochondrial metabolism- and synaptic/transport-associated changes. The second specific aim was to test the hypothesis that the olfactory system accumulates oxidative stress-mediated macromolecular damage over time, predisposing it to neurodegeneration. Two types of protein oxidation, namely, carbonylation and nitration, accumulated with aging in the olfactory system. Protein and cellular targets of oxidative stress-associated damage were identified using redox proteomics coupled with immunohistochemical localization. The third specific aim was to test the hypothesis that elevated oxidative stress in the olfactory system results in apoptosis/neurodegeneration. The Harlequin mutant mouse was critically selected and validated as a model for studies of oxidative stress-associated olfactory neurodegeneration at both the cellular and molecular levels. The Harlequin mouse had decreased levels and altered distribution of apoptosis inducing factor protein in mature olfactory sensory neurons, increased oxidative DNA damage and apoptosis in the olfactory epithelium, and pronounced cytoskeletal disorganization. The molecular studies confirmed and extended our cellular data and identified several significantly regulated genes associated with elevated oxidative stress and apoptosis. This novel study, by combining contemporary proteomics and genomics with cellular and tissue-level analyses, has provided a road map for understanding fundamental molecular mechanisms of olfactory degeneration.

Parkinson's disease is characterized by progressive degeneration of substantia nigra (SN) and subsequently loss of the nigrostriatal circuit. Many strategies have attempted to reconstruct this circuit but failed to satisfy clinical trials. The inhibitory environment of the adult CNS and the long distance between the SN and the striatum make true reconstruction difficult. To reconstruct this circuit, we used a transplant-pathway targeting model. Several putative pathway targeting molecules were examined for their ability to direct the growth of axons from a dopaminergic transplant. For a proof-of-principle study, adenoviral and lentiviral encoded glial cell line-derived neurotrophic factor (GDNF), GDNF-receptor alpha1 (GFRa1 ), or netrin-1 were injected along the corpus callosum individually or in combination. Treatment with individual factors leads to modest growth with few axons extending the entire length of the pathway. Combined treatment with either GDNF/GFRa1 or GDNF/netrin-1 induced the most robust growth towards the contralateral striatum. GDNF/netrin-1 showed the most consistent growth, with about 80% of the axons growing to the farthest injection site on the contralateral side. To determine if this combination of guidance molecules could be used to reconstruct the nigrostriatal pathway, we examined axon outgrowth from transplants placed within the SN in the 6-0HDA-Iesioned hemiparkinsonian animal model. A pathway from the SN to the striatum was made by injecting lentivirus encoding either GDNF and netrin-1 or GDNF and GFRa1, along the internal capsule, from the SN to the striatum. In another cohort of animals lentivirus encoding GFP was used as a control. A piece of embryonic VM tissue was transplanted into the SN two weeks after lentivirus injections. Compare to the GFP control group, a significantly greater number of dopaminergic axons grew from the transplants towards the striatum ten weeks after transplantation. Retrograde tract tracing showed the dopaminergic axons were from A9 cells in the transplant. Behavioral studies showed a significant reduction in number of amphetamine-induced rotations in GDNF/netrin-1 treated animals. Functional recovery strongly correlated with the number of dopaminergic fibers growing out from the transplant. This study shows that a functional nigrostriatal pathway can be reconstructed by guiding axonal growth from the dopaminergic neurons transplanted in the SN along a preformed growth-supportive pathway extending into the striatum. Refinement of this technique could be beneficial for PD patients in the future.

Dietary saturated fat intake contributes to diabetes and cardiovascular disease, as shown in numerous animal and human studies. However, the hypothesis that stearic acid, a saturated fat, has beneficial effects on these conditions has not been adequately tested. Leptin receptor deficient mice (db/db) and wild-type mice were fed either chow or a high fat diet enriched in either stearic acid or oleic acid for ten weeks. The progression of diabetes was evaluated with blood glucose, insulin, and metabolic parameter measurements. At the conclusion of the study, pancreatic islet organization was examined, and blood, liver and feces were assayed for fatty acid content. The stearic acid enriched diet prevented increases in blood glucose levels independently of weight loss in db/db mice compared to an oleic acid or chow diet. Diabetic mice fed stearic acid maintained insulin responsiveness and pancreatic islet organization compared to the db/db mice fed chow and oleic diets. The islet organization of the stearic acid fed mice did not change over the course of the study and was similar to that of wild-type mice fed the same diet. Conversely, diabetic mice fed oleic acid and chow diets had decreased insulin responsiveness and disorganized islets. Stearic acid fed db/db mice had high fecal fat content and caloric intake calculations indicated low absorption of this fat. Switching to stearic acid after prolonged hyperglycemia had a rescue effect on blood glucose levels. After feeding diabetic and wild-type mice standard chow diets for 6, 8, and 10 weeks to establish hyperglycemia, mice switched to a high fat diet enriched in stearic acid, but not one enriched in oleic acid diet, had significant reductions in blood glucose levels. The ability of a stearic acid enriched high fat diet to slow the progression of diabetes and reverse hyperglycemia in db/db mice argues that risks and benefits of fats in the diet depend on the chemical structure, rather than the chemical class, of fats ingested. The beneficial effect of stearic acid appears to be associated with a decreased absorption of dietary fat.

The sense of smell relies upon the detection of odorants by neurons located in the nasal cavity. These neurons, referred to as olfactory sensory neurons (OSNs), line the olfactory epithelium and extend axons that make synaptic connections with mitral/tufted cells in the olfactory bulb. The mechanisms by which these synaptic connections form remain largely unknown. The development of these synaptic connections relies on the axons of immature OSNs innervating the olfactory bulb. The primary goal of this dissertation was to identify components of the mechanisms used by immature OSN axons to innervate the olfactory bulb. To accomplish this goal, a knockout mouse model was used. OSN axons, of Emx2 knockout mice fail to innervate the olfactory bulb. As EMX2 is a transcription factor, this model was used investigate the possible causes of the defective OSN axon growth. To gain a better understanding of OSN axon growth, differences in expression of axon growth and guidance genes in immature and mature OSNs was investigated. This analysis revealed that many axon growth and guidance genes are differential expressed, and helped to identify immature OSN specific genes. The data also revealed a previously unrecognized developmental stage, termed nascent OSNs, identified by the expression of Cxcr4. Analysis of Emx2-/- mice revealed that EMX2 is necessary for OSN survival, odorant receptor expression and expression of the axonogenesis related gene Ablim1. EMX2 is necessary for the expression of many odorant receptor genes; however the loss of odorant receptor expression does not explain the axon growth defects. Apoptosis is increased in Emx2-/- mice, an outcome that may be due to the failed axon growth. Analysis of axon guidance gene expression identified a large reduction in Ablim1 expression in Emx2-/- mice. Ablim1 is expressed by immature OSNs, placing it in the proper cell type to regulate OSN axon growth. The loss of Ablim1 expression in Emx2-/- mice indicates defective signaling in the axon growth cone and a possible mechanism regulating OSN axon growth into the olfactory bulb. The data presented in this dissertation provide new insight into the regulation of odorant receptor gene expression and OSN axon growth.

Acute myocardial infarction (AMI) is the leading cause of death worldwide. Currently, the best method of treating cardiac ischemia is early reperfusion which, itself, induces myocardial damage. The mTOR complex is a key regulator of cardioprotection against cell stressors. We hypothesized that reperfusion therapy with Rapamycin, a potent mTOR inhibitor, would reduce infarct size in adult mouse hearts. Rapamycin was administered at the onset of reperfusion following 30 min in situ LAD ligation. After 24 hours of reperfusion, myocardial infarct size and apoptosis were significantly reduced in rapamycin-treated mice compared to control. Rapamycin inhibited pro-apoptotic protein Bax and phosphorylation of ribosomal protein S6 (target of mTORC1), while it induced phosphorylation of AKT (target of mTORC2). Rapamycin also induced phosphorylation of ERK, while significantly reduced phosphorylation of p38. Thus, our study shows that reperfusion therapy with Rapamycin provides cardioprotection through induction of the phosphorylation of Akt and ERK.

The hamster estrous cycle lasts four days and is considered to be a physiological model for angiogenesis. Angiogenesis is the formation of new capillaries from preexisting vessels, and it occurs extensively during corpus luteum formation in the estrous cycle. Von Willebrand Factor (vWF) is a glycoprotein that is secreted uniquely in endothelial cells and megakaryocytes. It is frequently used as an endothelial cell marker and it is able to detect vessels within tissues when it is used in immunohistochemical staining techniques. This study explores von Willebrand Factor expression within Golden Hamster ovarian tissue. In particular, this study uses cage control and antiorthostatic cage suspension tissue. Antiorthostatic cage suspension is a model developed to mimic and study the physiological effects caused by microgravity, such as that experienced in space flight. It is hypothesized that simulated microgravity caused by antiorthostatic cage suspension would result in lower levels of vasculature and expression of vWF within ovarian tissue. Due to financial considerations, conclusive data was not obtained due to a lack of statistics. However, our study indicates that vasculature and vWF expression may be increased in antiorthostatic cage suspension tissue.

Electrical potentials exist across the membranes of nearly every cell type in the body. In addition, excitable cells, such as neurons, myocytes and even some endocrine cells elicit electrochemical fluctuations, action potentials (AP), in the cell membrane to initiate cell-to-cell communication or intracellular processes. The basis for the electrical potential is rooted within an array of complex interactions between monovalent ions and their associated membrane channels and transporters that regulate the flux of these charged species across the hydrophobic bilayer. Here, an expansion of our recently published work [1] will serve to explore the modern concepts regarding the origin of the AP as well as to examine the mechanisms by which intracellular calcium ([Ca2+]i) is regulated within the HL-1 mouse cardiac myocyte.