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Mitochondria are crucial components of eukaryotic cells and exchange signalling molecules, metabolites, proteins and lipids with the rest of the cell. The organelle is key for energy metabolism as they provide most of the cellular ATP through oxidative phosphorylation and regulate intermediate metabolism. Mitochondria are also a major source of reactive oxygen species (ROS), which are by-products of aerobic respiration and recently recognised as important signalling molecules that control various cellular functions. To avoid the potential damaging effects of ROS, mitochondria contain protein antioxidant systems to help maintain thiol homeostasis. Mitochondria are emerging as an important redox signalling node and are involved in a myriad of signalling pathways, which have a redox component, either through a response to a particular ROS or the shift of the redox state of a responsive group. It is not surprising that mitochondria are therefore heavily regulated by retrograde signalling of the master regulator of cellular antioxidant defence, nuclear factor erythroid-derived 2-related factor 2 (Nrf2). Until now it has not been possible to disentangle the overlapping effects of mitochondrial ROS signalling compared to a redox signal stemming from disruption of mitochondrial thiol homeostasis. Furthermore, it is important to distinguish between disturbing the cytosolic and mitochondrial protein antioxidant systems. I characterised the effects of mitochondrial thiol homeostasis disruption on mitochondrial physiology with MitoCDNB, showing mitochondrial fission. I found that selective disruption of the mitochondrial glutathione pool and inhibition of its thioredoxin system led to Nrf2 activation, while using MitoPQ to enhance production of mitochondrial superoxide and hydrogen peroxide alone did not. To further our understanding of how mitochondrial redox homeostasis is sensed in the cytoplasm and signalled to the nucleus I used an RNAseq approach to investigate the intricacies of early mitochondrial retrograde signalling.

Reactive oxygen and nitrogen species (RONS) are continuously produced within the body during oxidative metabolism, and through normal immune function. When contained within a homeostatic balance, these molecules are part of a normal physiological function, and play integral signalling roles in cell growth, proliferation, and differentiation. This equilibrium is tightly regulated by a range of enzymatic and non-enzymatic antioxidants which seek to prevent detrimental reactions to biologically important macromolecules. The purpose of this thesis was twofold. Firstly, the use of various high-intensity exercise protocols was employed to investigate the effect on oxidative stress within circulating peripheral blood mononuclear cells and skeletal muscle tissue with a particular emphasis on DNA damage. Additionally, an environmental stressor was used via normobaric hypoxia to comprehensively elucidate the cell DNA damage-repair response, alongside systemic measures of oxidative stress following constant-load high-intensity exercise. Secondly, this thesis aimed to examine the efficacy of novel antioxidant supplementation on measures of exercise-induced oxidative stress; specifically, a plant-based combination of barley- and wheat-grass juice, and the mitochondrial-targeted antioxidant, Mitoquinone. Across all studies, it was confirmed that exercise of sufficient intensity (regardless of duration) provided a challenging physiological stress capable of inducing oxidative damage to DNA and lipids; likely through the generation of RONS, and concurrent reduction in antioxidant capacity. Furthermore, it was determined that peripheral blood mononuclear cells have an efficient and effective DNA damage-repair response, with maximal repair occurring within 24-hours for both single- and double-strand DNA damage; this response was marginally exacerbated in the hypoxic condition. The aforementioned modifications to DNA as a function of exercise, were concurrently aligned with systemic biomarkers of oxidative stress; including but not limited to, lipid peroxidation, lipid soluble antioxidants, and the ascorbyl free radical. With regards to plant-based antioxidant supplementation, although there was no meaningful prophylactic effect on a statistical level, a combination of barley- and wheat-grass juice did appear to provide a marginal protective effect against exercise-induced oxidative stress. On a more molecular level, mitochondrial-targeted supplementation for 21-days abrogated oxidative damage to the mitochondrial genome following high-intensity intermittent exercise; likely, through the scavenging of exercise-induced RONS, and/or secondary oxidation products of mitochondrial lipid peroxidation. The results of the studies demonstrate an increase in oxidative stress following high-intensity exercise as evidenced by an increase in DNA damage (single-strand breaks, double-strand breaks, base oxidation) across nuclear and mitochondrial genomes, and changes to other systemic measures, such as: lipid hydroperoxides, lipid soluble antioxidants, and the ascorbyl free radical. The DNA damage-repair response was exacerbated by normobaric hypoxia with single- and double-strand DNA damage returning to baseline by 48-hours and 24-hours respectively. Finally, the data also indicates a potential prophylactic effect by supplementing with a plant-based nutraceutical, and mitochondrial-targeted antioxidant; however, further research is warranted to elucidate the underlying biochemical processes.

OBJECTIVES: Recent studies have elucidated that diabetes mellitus (DM) patients exhibit an accelerated tumor progression, but the mechanism of its regulation is not yet fully understood. The following study seeks to examine the role of angiogenic factors in the growth of subcutaneously injected melanoma cancer using a diet-induced type II diabetic mouse model. METHODS: C57BL/6 mice were fed either a regular or high-fat, high-sucrose (HFHS) diet for 2 months (T2DM model; confirmed through a GTT) and subcutaneously injected with B16F0 melanoma cells. After a 1-week or 2-week incubation period, the tumor was extracted to examine its size, weight, vascularity, and gene/protein expression. In vitro studies were performed using endothelial cells to assess the effects of high-glucose on endothelial cell proliferation, migration, and tube formation. GLRX expression was examined in both tumor samples and endothelial cells. RESULTS: The results of the study showed that T2DM induced by a HFHS diet is able to promote tumor growth in both weight (2-week, p = 0.0070) and volume (1-week, p = 0.0351; 2-week, p = 0.0002). Tumors extracted from the HFHS diet group showed reduced expressions of angiogenic markers (ACTA2 (1-week, p = 0.0239; 2-week, p = 0.0123), KDR (1-week, p = 0.0091)) by western blot and a slightly reduced trend of angiogenesis (PECAM1) in histological analyses. GLRX expression was reduced in HFHS tumor samples (1-week, p = 0.0090) and, interestingly, lower amounts of GSH adducts (2-week, p = 0.0317) could be seen in 2-week tumors as well. In vitro studies of endothelial cells showed reduced trends of endothelial cell function (proliferation, migration, and tube formation) in high glucose medium. Also, it has been observed that high glucose may be able to stimulate GLRX expression in endothelial cells. CONCLUSION: The results of the following study have confirmed that B16F0 melanoma growth is, in fact, augmented in diet-induced diabetic mice; however, the vascularity and levels of angiogenic markers from the tumor tissues did not parallel the growth in its size. In vitro studies suggested that high glucose can impair EC function (i.e. proliferation, migration, and tube formation capabilities) as well as promote GLRX expression, which may be related to this discrepancy. Glutaredoxin-1 (GLRX), an enzyme which controls redox signaling, is upregulated in DM. Endothelial cell-specific GLRX overexpression in transgenic mice was found to stimulate subcutaneously injected melanoma (B16F0) growth, despite hindering limb revascularization after hind limb ischemia. The augmented tumor progression in DM may be associated with GLRX upregulation, alongside impaired ischemic limb revascularization and tumor angiogenesis; however, the mechanism of tumor growth in diabetes still lies inconclusive and further studies need to be examined to elucidate this phenomenon.

H2O2 acts as a second messenger that can modulate intracellular signal transduction via chemoselective oxidation of cysteine residues in signaling proteins, however, the protein targets of H2O2 as well as how oxidation influences protein activity has remained largely unknown. In the present study, we present the first direct demonstration of an essential role for sulfenic acid modification of the thiol peroxidase Gpx3 to communicate conditions of oxidative stress to the transcription factor Yap1 in Saccharomyces cerevisiae. We then describe the characterization of methods to profile ROS production and to monitor the redox status of the cellular glutathione pool, as well as the development of a method to profile global protein sulfenylation in Saccharomyces cerevisiae. Next we present the development and application of an alkyne-based probe for sulfenic acids, DYn-2. This new probe was used to reveal dynamic changes in global protein sulfenylation in response to epidermal growth factor stimulation of the human epidermoid carcinoma A431 cell line. This study identified three protein tyrosine phosphatases and the epidermal growth factor receptor as direct protein targets of hydrogen peroxide produced for epidermal growth factor signaling. Additionally, oxidation of the epidermal growth factor receptor was shown to enhance inherent kinase activity. Collectively, these results provide novel insight into how hydrogen peroxide can function as a second messenger to regulate eukaryotic signaling pathways and have broad implications for therapeutic development.

Oxidation of cysteine to sulfenic acid has emerged as a biologically relevant post-translational modification with particular importance in redox-mediated signal transduction; however, the identity of modified proteins remains largely unknown. In the present study we report the development of DAz-1, a cell-permeable chemical probe capable of detecting sulfenic acid- modified proteins directly in living cells. We then describe DAz-2, an analog of DAz-1 that exhibits significantly improved potency in vitro and in cells. Application of this new probe for global analysis of the sulfenome in a tumor cell line identifies most known sulfenic acid-modified proteins – 14 in total, plus more than 175 new candidates, with further testing confirming oxidation in several candidates. The newly identified proteins have roles in signal transduction, DNA repair, metabolism, protein synthesis, redox homeostasis, nuclear transport, vesicle trafficking, and ER quality control. Next we employ DAz-2 to discover two thioredoxin-related proteins that protect single cysteines from irreversible oxidation to sulfinic and sulfonic acid in Escherichia coli, which have functional homologues in eukaryotic cells. Finally we further refine these sulfenic acid probes to develop redox based probes of protein tyrosine phosphatases xxvii (PTPs) which show greatly increased sensitivity towards PTP sulfenic acid modification over previous probes. The combination of selective chemical enrichment and live-cell compatibility makes these sulfenic acid probes powerful new tools with the potential to reveal new regulatory mechanisms in signaling pathways, and identify new therapeutic targets.