Mitochondria serve as the major source of reactive oxygen species (ROS) production in cells resulting in antioxidant systems and cell signaling pathways that are unique to mitochondria. Thioredoxin-2 (Trx-2) is the mitochondrial member of the thioredoxin superfamily, and acts specifically to reduce the mitochondrial peroxidase, peroxiredoxin-3. It has been proposed that Trx-2 associates with cytochrome c, which functions in mitochondrial respiration and apoptosis. Homozygous Trx-2 deletion in mice is embryonic lethal and it is hypothesized here that Trx-2 lethality is caused by loss of mitochondrial function and oxidative stress. Results of experiments investigating mitochondrial integrity, cell viability, and ROS levels in Trx-2(-/-) mouse embryonic fibroblasts (MEFs), and results from Trx-2 siRNA MEFs, are similar to findings of knockouts in previously reported proteins that function in mitochondrial respiration and support the involvement of Trx-2 in this process. Mitochondrial ROS have also been implicated as major secondary messengers in cell signaling. Results reported here using cancer cells and cancer cells depleted of mitochondrial DNA, which consequently produce few ROS, have indicated that mitochondrial ROS produced in hypoxia are necessary for HRE and ARE activation, and are fundamental in the activation of SP-1 during reoxygenation. However, mitochondrial ROS are not required for HIF-1α protein expression in hypoxia, indicating a unique relationship between HIF-1α, hypoxia, and mitochondrial ROS.

Background: Hypertension is a pathological increase in blood pressure that affects nearly 30% of the U.S. population and is a primary modifiable risk factor for cardiovascular disease. Despite advancements in prevention and treatment, hypertension is still one of the most common conditions around the world, and for a majority of cases the causal mechanisms remain to be fully elucidated. A growing body of literature suggests that oxidative stress status may play an etiological role in many chronic conditions, including hypertension. Specifically, a systemic overabundance of reactive oxygen species may give rise to endothelial dysfunction, increased sodium and H2O retention, and alterations in sympathetic outflow, leading to an increase in blood pressure. Purpose: The main objective of this study is to investigate the prospective association between F2-isoprostanes, a validated biomarker of oxidative status, and development of hypertension in a large, multi-centered, multi-ethnic cohort of adults aged 40-69 at baseline. Methods: This is a secondary data analysis that utilized previously collected data from the Insulin Resistance Atherosclerosis Study. 844 participants were included in the analysis. Briefly, four urinary F2-isoprostane isomers (F2-IsoP1, F2-IsoP2, F2-IsoP3, and F2-IsoP4) were quantified using liquid chromatography/ tandem mass spectrometry and adjusted for urinary creatinine levels. Hypertension was assessed at baseline and follow-up visits and defined as systolic blood pressure > 140 mm Hg and/or diastolic blood pressure > 90 mm Hg and/or currently taking antihypertensive medications. Crude associations between study population characteristics and hypertensive status were analyzed with the chi-square and Wilcoxon-rank sum tests. Crude associations between study population characteristics and F2-isoprostane levels were analyzed with Wilcoxon-rank sum, Kruskal-Wallis, and Spearman’s rank correlation measures. Finally, the adjusted prospective associations between hypertensive status and F2-isoprostane concentrations were modeled using logistic regression. Results: Of the 844 participants who were included in the study, 258 (31%) were classified as hypertensive at baseline. Among the 586 participants who were normotensive at baseline, 123 (21%) developed hypertension over the five-year study period. Importantly, none of four F2-isoprostane isomers predicted a significant increase in the odds of developing hypertension, as indicated by their odds ratio 95% confidence intervals; F2-IsoP1: (0.85, 1.31), F2-IsoP2: (0.62, 1.13), F2-IsoP3: (0.80, 1.27), and F2-IsoP4: (0.84, 1.29). Conclusion: Previous studies have investigated the association between oxidative status and hypertension prevalence, however the cross sectional nature of the study designs have made it difficult to establish temporality between exposure and outcome. To our knowledge, this is the first study to model the odds of developing hypertension as a function of F2-isoprostane levels. The results of this study suggest that oxidative status is not involved in the development of hypertension.

The pathogenesis of diabetic nephropathy (DN) remains incompletely understood. In previous studies, we observed the activation of Tyr kinase Src by high glucose (HG) and showed that Src is required for MAPK activation and synthesis of collagen IV in cultured rat mesangial cells (MCs). Reactive oxygen species (ROS) are also important mediators of DN, and our present study aimed to investigate the role of Src in HG-induced ROS generation. In MCs, we found that HG led to ROS accumulation that was blocked by Src inhibitors or Src-specific siRNA. Downstream of Src, Vav2 was phosphorylated/activated leading to Rac1-dependent NADPH oxidase activation. Long-term HG exposure resulted in Src-dependent Nox4 protein induction. Nox2-specific siRNA abrogated ROS production only in short-term HG, while Nox4-specific siRNA blocked ROS production only in long-term HG. Taken together, our data indicate Src to be important in mediating ROS generation from both Nox2- and Nox4-containing NADPH oxidases.

The pathogenesis of diabetic nephropathy (DN) remains incompletely understood. In previous studies, we observed the activation of Tyr kinase Src by high glucose (HG) and showed that Src is required for MAPK activation and synthesis of collagen IV in cultured rat mesangial cells (MCs). Reactive oxygen species (ROS) are also important mediators of DN, and our present study aimed to investigate the role of Src in HG-induced ROS generation. In MCs, we found that HG led to ROS accumulation that was blocked by Src inhibitors or Src-specific siRNA. Downstream of Src, Vav2 was phosphorylated/activated leading to Rac1-dependent NADPH oxidase activation. Long-term HG exposure resulted in Src-dependent Nox4 protein induction. Nox2-specific siRNA abrogated ROS production only in short-term HG, while Nox4-specific siRNA blocked ROS production only in long-term HG. Taken together, our data indicate Src to be important in mediating ROS generation from both Nox2- and Nox4-containing NADPH oxidases.

Dravet syndrome (DS) is a severe, pediatric-onset epilepsy disorder linked to loss-of-function mutations in the sodium channel gene SCN1B. DS patients have a high risk of Sudden Unexpected Death in Epilepsy (SUDEP). Cardiac arrhythmias have been implicated as a potential cause underlying SUDEP. An exact pathway for how mutations in SCN1B leads to arrhythmia in DS is unclear. One cellular component linked to regulation of cardiac homeostasis are mitochondria, known as “the powerhouse of the cell” due to their ability to produce cellular energy (ATP) via the electron transport chain (ETC). The ETC is a major producer of reactive oxygen species (ROS). Typically, ROS are buffered by cellular antioxidants, to prevent oxidative stress, an imbalance of ROS that can lead to cell damage. Our previous work indicates that cardiac arrhythmias may result from mitochondrial instability and imbalances between ROS production and buffering. We analyzed whether Scn1b-/-mice are susceptible to arrhythmias due to altered mitochondrial ATP generation, ROS production, and compromised cellular antioxidant defenses. We isolated cardiac mitochondria from postnatal day (P) 15-20 KO and Scn1b+/+ (WT) mice. To assess mitochondrial ATP and ROS production, high-resolution respirometry (O2k, Oroboros) was used to measure mitochondrial O2 and H2O2 flux. We used a substrate-uncoupler inhibitor (SUIT) protocol to elucidate flux under different ETC pathways, including Complex I- and II-linked respiration. As a next step, we evaluated expression of superoxide dismutase (Sod) proteins associated with mitochondrial antioxidant defenses, including Cu/Zn-Sod (Sod1) and Mn-Sod (Sod2) in hearts from KO and WT mice pre- (P10) and post- (P17) seizure development. After addition of substrates supporting Complex-II linked respiration (succinate, ADP) there were no differences in O2 flux between mitochondria isolated from KO and WT hearts. Upon further addition of pyruvate to mitochondria to stimulate Complex I, O2 flux was significantly reduced (p < 0.0001) in mitochondria from KO mice, when compared to WT. Moreover, upon titration of rotenone (a Complex I inhibitor) its negative effect on O2 flux was not as substantial in KO mitochondria as in WT, suggesting that mitochondria from KO have deficits in Complex-I linked respiration. Furthermore, we detected significant differences in ROS production by mitochondria isolated from KO animals. Under conditions of reverse electron flow (succinate as substrate), a state where ROS production is highest, H2O2 flux was elevated significantly (p = 0.048) in mitochondria isolated from KO mice, compared to those isolated from WT. During our analysis of Sod expression, we found that Sod1 (p = 0.01) and Sod2 (p = 0.01) expression is significantly decreased at P17 in KO hearts compared to WT. Overall, our results suggest imbalances between mitochondrial activity and antioxidant defenses, which may underlie increased arrhythmia susceptibility in KO mice.

In the current work, the photophysics and photochemistry of the phototoxic red fluorescent protein (RFP) KillerRed was investigated. KillerRed's phototoxicity makes it useful for studying oxidative stress on cell physiology and for cell killing in photodynamic therapy. Spectroscopic probes were used to show that the phototoxicity of KillerRed stems primarily from a type I photosensitization mechanism producing radicals. The production of radicals was supported by electron paramagnetic resonance (EPR) studies, where a long-lived radical was observed in KillerRed and two other RFPs (mRFP and DsRed) following excitation. Transient absorption spectroscopy, various other spectroscopic techniques, and the published crystal structure of KillerRed indicate that the long-filled water channel is likely responsible for the increased phototoxicity of KillerRed. In the blue fluorescent protein (BFP) mKalama1, some of the same techniques were applied to understand the photophysics and photochemistry on the timescale ranging from femtoseconds to seconds. Transient absorption spectroscopy and previously published results demonstrate that two-photon excitation of mKalama1 likely results in the formation of a radical cation and solvated electrons. This may explain the blinking behavior which has been observed on the single molecule level for many fluorescent proteins, the identity of which has remained elusive. It was also shown that the chromophore, while neutral in the ground state, does not exhibit excited-state proton transfer (ESPT) during its nanosecond excited-state lifetime; however, the chromophore undergoes a deprotonation in the ground state after electronic relaxation. This work plays a key role in our understanding of fluorescent proteins and will help pave the way to developing new ones. The research on the BFPs was extended to improve them for cellular imaging. This was accomplished by identification of dark states in the BFPs which are longer in wavelength than the collected fluorescence. Using dual lasers, it was shown that these dark states could be optically depleted, thereby increasing the overall fluorescence without enhancing the background fluorescence. Rational site-directed mutagenesis was carried out on the BFPs and the mutants were screened for fluorescence enhancement. These proteins were then analyzed using transient absorption spectroscopy to elucidate the identity of the dark state(s) used for fluorescence enhancement.

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide (O2 • −) have been shown to serve as messengers in biological signal transduction, and many prokaryotic and eukaryotic proteins are now known to have their function controlled via ROS-mediated oxidation reactions occurring on critical cysteine residues. The tumour-suppressor protein p53 is involved in the regulation of a diverse range of cellular processes including apoptosis, differentiation, senescence, DNArepair, cell-cycle arrest, autophagy, glycolysis and oxidative stress. However, little is understood about the specific molecular mechanisms that allow p53 to discriminate between these various different functions. p53 is a multiple cysteine-containing protein and there is mounting evidence to suggest that redox-modification of p53 Cys residues participate in control of its biological activity. Furthermore, p53 activity has been linked to intracellular ROS levels. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) offers superior mass resolving power and mass measurement accuracy, which is beneficial for the study of intact proteins and the characterisation of their posttranslational modifications (PTMs). The primary goal of the work described in this thesis was to employ FT-ICR mass spectrometry to investigate the molecular details of p53 redox-regulation. The relative reactivity of each of the ten cysteine residues in the DNA-binding core domain of recombinant human p53 was characterised by treatment with the Cys-alkylating reagent N-ethylmaleimide (NEM) under various conditions. A combination of top-down and middle-down FT-ICR MS was used to unambiguously identify Cys182 and Cys277 as sites of preferential alkylation. These results were confirmed by site-directed mutagenesis. Interestingly, Cys182 and Cys277 have previously been implicated in p53 redox-regulation. Alkylation beyond these two residues was found to trigger rapid alkylation of the remaining Cys residues, presumably accompanied by protein unfolding. These observations have implications for the re-activation of mutant p53 with Cys-targeting compounds which result in the death of cancer-cells. Furthermore, the molecular interaction between p53 and the ROS hydrogen peroxide was investigated. p53 was found to form two disulfide bonds upon treatment with H2O2. An enrichment strategy was developed to purify oxidised p53 and top-down FT-ICR mass spectrometry revealed unambiguously that Cys176, 182, 238 and 242 were the oxidised residues. Interestingly, Cys176, 238 and 242 are Zn2+- binding residues suggesting that p53 contains a zinc-redox switch. The mechanism of H2O2 oxidation was investigated, and revealed that oxidation via an alternative pathway results in indiscriminate over-oxidation of p53. Moreover, Cys176, 238 or 242 was shown to act as a nucleophile, and the intracellular antioxidant glutathione (GSH) did not prevent oxidation of the Zn2+-binding Cys residues, providing further evidence for a role in p53 redox-regulation. This study has revealed hitherto unknown details regarding the chemistry of cysteine residues within the important tumour-suppressor protein p53. Furthermore, the analytical power of FT-ICR MS for the study of multiple Cys-containing proteins has been very clearly demonstrated.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand In fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2003 / Dietary iron (Fe) overload, originally referred to as Bantu Visceral Siderosis, is an Reloading condition that is still prevalent in rural populations of sub-Saharan Africa. The better known Fe loading disease, hereditary haemochromatosis (HFI) is frequently complicated by hepatocellular carcinoma (HCC) and, in rare instances this occurs in the absence of cirrhosis. The latter, together with recent evidence that dietary Fe overload in the Black African carries an increased risk for HCC, suggests that excessive hepatic iron may itself be carcinogenic. The aim of the study was to determine if Fe alone could induce HCC in experimental rat models and, if so, to investigate possible mechanisms of hepatocarcinogenesis. 360 Wistar albino rats (Rattus norvegicus) were divided into 6 groups. The first group, the control animals, was designated C group. Groups 2-6 were Fe-fed alone or in combination with other chemicals: group 2 Fe alone (Fe group), group 3 (Fe + V) vitamins A & E supplementation [50 mg all trans-retinol (vitamin A) and 500 mg a-tocopherol (vitamin E) per kg diet], group 4 (Fe - V) received a diet totally devoid of vitamins A & E, group 5 (Fe + ASA) received 20 mg aspirin (ASA) per day, group 6 (Fe + Cu) received 300 mg/kg diet of copper sulphate (CuS04) supplementation for 12 months followed by 3% copper hydroxide carbonate [CuC03»Cu(0H)2] / IT2018

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand In fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2003 / Dietary iron (Fe) overload, originally referred to as Bantu Visceral Siderosis, is an Fe- loading condition that is still prevalent in rural populations of sub-Saharan Africa. The better known Fe loading disease, hereditary haemochromatosis (HH) is frequently complicated by hepatocellular carcinoma (HCC) and, in rare instances this occurs in the absence of cirrhosis. The latter, together with recent evidence that dietary Fe overload in the Black African carries an increased risk for HCC, suggests that excessive hepatic iron may itself be carcinogenic. The aim of the study was to determine if Fe alone could induce HCC in experimental rat models and, if so, to investigate possible mechanisms of hepatocarcinogenesis. 360 Wistar albino rats (Rattus norvegicus) were divided into 6 groups. The first group, the control animals, was designated C group. Groups 2 - 6 were Fe-fed alone or in combination with other chemicals: group 2 Fe alone (Fe group), group 3 (Fe + V) vitamins A & E supplementation [50 mg all trans-retinol (vitamin A) and 500 mg a-tocopherol (vitamin E) per kg diet], group 4 (Fe - V) received a diet totally devoid of vitamins A & E, group 5 (Fe + ASA) received 20 mg aspirin (ASA) per day, group 6 (Fe + Cu) received 300 mg/kg diet of copper sulphate (CuS04) supplementation for 12 months / IT2018

Thesis advisor: Clare O'Connor / Within cells, reactive oxygen species (ROS) are synthesized naturally and in response to environmental stimuli. However, ROS have deleterious effects on a wide range of cellular molecules. Oxidative stress, caused by the ROS generated by the partial reduction of oxygen, is a major cause of cell damage linked to the initiation and progression of numerous diseases. Thioredoxin peroxidase (Tpx1) plays important roles in cellular defense against ROS. Although homologous genes and their functions have been identified in other eukaryotes, the level of activity as well as the necessity of this protective enzyme in S. pombe exposed to oxidative stress has yet to be fully elucidated. To explore the role of the Tpx1 protein in oxidative stress resistance, novel strains were constructed in which the tpx1 gene was overexpressed. The polymerase chain reaction was used to amplify txp1, and the amplified sequence was cloned into the yeast overexpression plasmid, pNMT41, which allows overexpression under the control of the powerful promoter. DNA sequencing was used to determine that the sequences had been properly inserted into the vector. The plasmids were transformed into two leu- yeast strains: FWP6 and TP108-3C. Production of the Tpx1 protein was ensured using Western Blot techniques. Experimentation to test the responses of the tpx1 strain to oxidative stress will employ a variety of reactive oxygen generators, including hydrogen n peroxide, menadione, tert-butyl hydroperoxide, and paraquat. The results generally supported the proposed role of Tpx1 to confer additional resistance against the oxidative stress. In a complementary line of investigation, knockout strains are being constructed to reduce the levels of the Tpx1 in S. pombe. Gene deletion cassettes were constructed for tpx1. Currently, the strains are being analyzed for the successful replacement of the endogenous tpx1 gene by homologous recombination. If the absence of the protein results in decreased cell viability, the role of Tpx1 indicated by the overexpression experiments could be supported. / Thesis (BS) — Boston College, 2006. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.