There is increasing evidence that hydrogen peroxide (H2O2) can act as a signalling molecule capable of modulating a variety of biochemical and genetic systems. Using Jurkat T-lymphocytes, this study initially investigated the involvement of H2O2 in the activation of a specific signalling protein extracellular signal-regulated protein kinase (ERK). It was found that as a result of H2O2 treatment, mitochondrial complex activities decreased which led to subsequent increase of mitochondrial reactive oxygen species (ROS) production. The increase of ROS resulted in higher cellular H2O2 as well as increased ERK activation. This study demonstrated that in an oxidative stress setting, H2O2 production from the mitochondria was an essential component in maintaining the activation of a signalling protein. One way in which H2O2 could influence protein function is by the oxidation of susceptible thiol groups of cysteine residues. To further understand the variety of signalling pathways that H2O2 may be involved in, an improved proteomics technique was developed to globally identify proteins with susceptible thiol groups. The

Thesis (M.Sc.)--York University, 2004. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 100-121). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL:http://gateway.proquest.com/openurl?url%5Fver=Z39.88-2004&res%5Fdat=xri:pqdiss&rft%5Fval%5Ffmt=info:ofi/fmt:kev:mtx:dissertation&rft%5Fdat=xri:pqdiss:MQ99388

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 76-93).

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 May 24

Several studies have found a positive correlation between hypertension and hyperhomocysteinemia. Increasing evidence implicates oxidative stress as one of the initiating events closely linked to the homocysteines ability to damage endothelium, subsequently causing vascular dysfunction. We previously found that heparin protects cultured endothelial cells from free radical injury and oral heparin at 1 mg/kg/48h prevents venous thrombosis in a rat model in vivo. Our objective was to study the protective effects of oral heparin in a rat model with elevated plasma homocysteine (Hcy) concentrations, and begin to elucidate whether the pathophysiological effects of Hcy are mediated through an oxidative mechanism causing endothelial dysfunction.<p> Elevated plasma Hcy levels were induced by feeding male Wistar Kyoto rats a diet containing an additional 1.7% methionine for 8 weeks. Groups included rats fed additional methionine, methionine plus oral heparin (1 mg/kg/48h by gastric feeding tube), and age-matched controls fed normal rat chow. At the end of 8 weeks of treatment, rats were anesthetized using 1.5% isoflurane in 100% oxygen. Hemodynamics parameters were assessed by inserting a Millar Mikro Tip pressure transducer into the left ventricular chamber and the thoracic aorta. Fasting plasma total Hcy levels were measured using a Hcy immunoassay kit with an Abbott IMx instrument. Malondialdehyde (MDA) concentrations, a lipid peroxidation product and marker for oxidative stress, was measured by a spectrophotometric method in serum and tissue samples. Glutathione (GSH) concentrations, an important antioxidant for low-level oxidative stress was measured by HPLC in plasma and tissues samples. Lastly, tissue samples from each experimental group were stained with the TUNEL method to assess their respective percentage of apoptotic endothelial cells. Results were expressed as mean ± S.E. Unpaired Students two-tailed t-test was employed to assess the difference between groups with p < 0.05 considered significant.<p> Plasma Hcy was significantly elevated after 8 weeks in the methionine (7.17 ± 0.46 umol/L) and methionine plus heparin treated rats (7.02 ± 0.40 umol/L) compared to control (5.46 ± 0.36 umol/L). All measures of arterial pressure, systolic (SP) and diastolic pressure (DP) and mean arterial pressure (MAP), were significantly elevated in rats fed the methionine diet without heparin (119.9 ± 3.9 mmHg; 90.3 ± 3.5 mmHg; 97.7 ± 2.9 mmHg, respectively) compared to controls (107.8 ± 2.5 mmHg; 79.2 ± 2.1 mmHg; 88.8 ± 2.2 mmHg, respectively) but not compared to heparin (114.7 ± 3.3 mmHg; 83.4 ± 2.4 mmHg; 93.8 ± 2.7 mmHg, respectively). Left ventricular end diastolic pressure (LVEDP) was significantly elevated with the methionine diet without heparin (14.2 ± 2.5 mmHg) but not with heparin treatment (8.4 ± 1.9 mmHg) versus controls (7.1 ± 1.1 mmHg). Also, left ventricular systolic pressure (LVSP) was significantly elevated in the methionine fed rats after 8 weeks (122.6 ± 3.2 mmHg) compared to controls (112.3. ± 2.9 mmHg). Heparin treatment had no effect on LVSP (119.9 ± 3.2 mmHg). <p> Additionally, the results of this study showed that oral heparin treatment significantly decreased liver MDA concentrations (2.42 ± 0.28 nmol/mg protein) compared to the methionine treated group (5.10 ± 0.96 nmol/mg protein) and methionine treatment alone significantly reduced MDA concentrations in kidney tissue (1.59 ± 0.12 nmol/mg protein) compared with controls (3.26 ± 0.66 nmol/mg protein). Methionine diet significantly decreased GSH concentrations in plasma (0.59 ± 0.59 µmol/L) compared with controls (4.24 ± 0.94 µmol/L) and oral heparin treatment significantly attenuated the decrease in GSH concentrations in left ventricle tissue samples (0.0229 ± 0.0023 µmol/mg protein) compared with methionine treatment alone (0.0135 ± 0.0016 µmol/mg protein). <p> Elevated plasma homocysteine levels, induced by methionine diet feeding significantly increased the percent of apoptotic endothelial cells in the aortas (17.04 ± 3.74%) and superior mesenteric arteries (17.99 ± 1.90%) of WKY rats compared with control aortas and mesenteric arteries (6.08 ± 3.24%; 7.43 ±1.62%, respectively) and compared to oral heparin treated mesenteric arteries (7.31 ± 1.18%). <p> The results of this study showed that elevated plasma levels of Hcy correlate with the development of hypertension, defined as significantly increased arterial pressure. Oral heparin treatment prevented the significant increase in arterial pressures and LVEDP, decreased MDA concentrations and therefore the oxidative stress on the liver, attenuated the decrease caused by elevated plasma Hcy in left ventricle GSH concentrations, and significantly reduced the number of apoptotic endothelial cells in the superior mesenteric artery of high methionine fed rats. We conclude that elevated levels of plasma Hcy contributes to the development of hypertension and furthermore towards the onset of heart failure likely through an oxidative mechanism and that oral heparin reduces the overall oxidative stress in specific physiological environments, preventing Hcy mediated endothelial cell apoptosis.

Friedreich Ataxia (FRDA) is an autosomal recessive degenerative disorder. It is caused by an abnormal expansion of GAA trinucleotide repeats in the first intron of the gene encoding frataxin. Since rates of cell division have been linked to oxidative stress, we have examined several parameters of oxidative stress in a FRDA primary fibroblast cell line that had a dramatically different growth rate. In the FRDA fibroblasts, the high level of reactive oxygen species (ROS) indicated elevated oxidative stress. The elevated glutathione peroxidase (Gpx) activity in the ROS defense system may represent an adaptive response to the high oxidative stress. The increased mitochondrial membrane potential (MMP) likely contributed to increased oxidant production, which could be contributed by elevated ROS. This increased oxidant production might be responsible for increased rate of progression through the cell cycle.<p> Furthermore, the elevated oxidative stress is also associated with progressive neural pathology of FRDA. In FRDA, pathology is first seen in the dorsal root ganglia and the dorsal columns of the spinal cord. Due to the abnormal metal distribution seen in the FRDA spinal cord and medulla, we hypothesized that metal binding proteins were abnormally distributed in FRDA. In our FRDA samples, we observed the well established histopathology of FRDA and examined the distribution of some metal binding proteins (frataxin, ferritin and metallothionein) through immunohistochemistry. Our results showed demyelination and loss of axons in the degeneration areas of the two FRDA cases. In addition, we found that the metal binding proteins were abnormally distributed in the FRDA spinal cord and the medulla. The abnormal distributions of the metal binding proteins were characterized by low expressions of iron binding proteins, especially frataxin and cytosolic ferritin, and undetectable expression of the copper and zinc binding protein, metallothionein. In summary, the rapid cell growth is a feature of FRDA fibroblast cell lines. We also tested Gpx activity, measured oxidant levels and determined the MMP in a FRDA primary fibroblast cell line that had a dramatically fast growth rate. The FRDA histopathology studies showed the metal binding proteins including frataxin, ferritin and metallothionein were abnormally distributed in the spinal cord and the medulla.

Restenosis is one of the major adverse outcomes of Percutaneous Coronary Intervention (PCI). Previous studies have shown conflicting reports for homocysteine as a predictor of restenosis following PCI. The conflicting reports may be due to oxidative factors (stimulation of polymorphonuclear leukocyte [PMNL]-induced reactive oxygen species generation, xanthine- xanthine oxidase, and arachidonic acid metabolism) other than homocysteine which could cause endothelial cell dysfunction leading to restenosis. Malondialdehyde (MDA), a lipid peroxidation product, is a marker for oxidative stress and is related to all oxidative factors. Therefore, it is possible that serum MDA may be a better predictor of restenosis than plasma homocysteine. The purpose of this study is to determine whether or not the pre-procedural serum MDA and plasma homocysteine levels are elevated in patients who develop restenosis post PCI. <p>The study included fifty-one patients undergoing elective PCI who consented to participate in a protocol that was approved by the Ethics Committee of the University of Saskatchewan. Homocysteine and malondialdehyde were measured in the plasma and serum respectively. Blood samples were collected pre-procedural, 0 time, 8 hours, 24 hours, and 6 months post-procedure. Exercise tolerance tests were performed at two weeks, and six months post-procedure to determine if there was any evidence of restenosis. <p>The results of the study showed that pre-procedural values of plasma homocysteine in the restenosis and non-restenosis groups were 10.37 ± 0.46 and 10.73 ± 0.49 respectively. These values were not significantly different (p=0.60) between the groups. The pre-procedural levels of plasma homocysteine were not significantly different (p=0.08) from the post-PCI values of those patients who did not develop restenosis at the 6-month time interval. However, the pre-procedural levels of plasma homocysteine were significantly different from the post-PCI values of those patients in the restenosis group at the 24hr (p=0.04) and 6-month (p=0.002) time intervals. In the restenosis group there was a significant increase (24%) after six months in the values of homocysteine from the pre-procedural levels. Thus, this indicates that restenosis is associated with higher post-PCI levels of homocysteine. <p>The pre-procedural levels of serum MDA in the restenosis and non-restenosis groups were 0.124± 0.16 and 0.147± 0.02 respectively. There was no significant difference (p=0.60) between the two groups. There was also no significant difference (p=0.053) between the pre-procedural values and the 6-month post-PCI values in those patients who did not develop restenosis. However, there was a significant difference (p=0.001) between the pre-procedural values and the 6-month post-PCI values in those patients who developed restenosis. The levels of serum MDA in patients with restenosis at 6-months increased by 109% and were significantly different (p=0.001) in the restenosis group. <p>The results suggest that pre-procedural levels of plasma homocysteine and serum MDA were not predictors of restenosis following PCI. However, the post-PCI six-month levels of both homocysteine and MDA are predictors of restenosis. Moreover, the post-PCI levels of MDA were better predictors of restenosis than the post-PCI levels of homocysteine because the increase in MDA levels were greater at six months than the rise in homocysteine levels at the same time interval.

Damage to cardiac contractile proteins such as myosin light chain 1 (MLC1), during oxidative stress is mediated by reactive oxygen species such as peroxynitrite (ONOO-), resulting in impairment of cardiac systolic function. The purpose of this study is to investigate the effects of the increased level of ONOO- on MLC1 degradation by the proteolytic enzyme matrix metalloproteinase-2 (MMP-2) during oxidative stress which ultimately decreases cardiac function.<p> In the present study two distinct models were utilized to demonstrate the mechanism by which MLC1 is modified by ONOO- and how these post-translational modifications lead to its increased degradation by MMP-2. In a model of newborn hypoxia-reoxygenation in piglets we demonstrated that ONOO--induced nitration and nitrosylation of tyrosine and cysteine residues of MLC1 increase its degradation by MMP-2. Furthermore, we found nitration of a tyrosine residue located adjacent to the cleavage site for MMP-2. We verified these results by using a model of isolated rat heart myocytes to determine that the same mechanism responsible for cardiac dysfunction in newborn piglets occurs in isolated myocytes and that the MMP-2 involved in degradation of MLC1 is located within the myocytes. Moreover, we were able to determine that this mechanism occurs during ischemia itself before the onset of reperfusion. Furthermore, we have found that pharmacological intervention aimed at inhibition of MLC1 nitration/nitrosylation during ischemia by the ONOO- scavenger FeTPPS (5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III]), or inhition of MMP-2 activity with phenanthroline, provides an effective protection of cardiomyocyte contractility. The work presented here provides new evidence on the mechanisms of regulation of contractile proteins during the development of contractile dysfunction.

Oxidative stress and mutations in DJ-1, a redox sensitive protein, are linked to Parkinson's Disease. The protective mechanism of DJ-1 is unclear. I hypothesized that: 1) DJ-1 mediates protection by translocating to mitochondria after oxidative stress and, 2) when DJ-1 is downregulated, apoptotic pathways regulated by calcineurin are also downregulated. In PC12 cells and rat cortical neurons, oxidative stress resulted in the upregulation of DJ-1 and increased DJ-1 in the nucleus, but did not increase mitochondrial translocation of DJ-1. In cortical neurons and wildtype mouse embryonic fibroblasts, H2O2 induced cleavage of CnA into an inactive fragment. DJ-1 knockout fibroblasts had less nuclear localization of the transcription factor NFATc4, a substrate of calcineurin involved in apoptosis. H2O2 increased CnA cleavage in DJ-1 knockout fibroblasts, but NFATc4 localization was unchanged. These results suggest that the downregulation of apoptotic pathways regulated by calcineurin may be a compensatory response to the downregulation of DJ-1.