Over 270 million people play soccer worldwide, and its popularity grows every day. In team sport exercise, fatigue may result from numerous factors including limited fuel, depleted energy stores and production of compounds that promote an inflammatory response. While inflammation is an essential mechanism for repairing damaged muscle tissue with exercise, prolonged inflammation leads to increased production of reactive oxygen species that can damage cell membranes, muscle, and signaling proteins. To prevent this response and improve performance, athletes are increasingly looking to nutritional interventions. Carbohydrate and antioxidant supplementation have both shown evidence of producing an ergogenic effect and attenuating inflammation and oxidative stress with prolonged endurance exercise. Less is known about how these interventions may influence intermittent, high-intensity exercise characteristic of soccer. In particular, this exercise presents a unique challenge in that opportunities for nutrient intake are limited to pre-game and half-time. In our first study, we had 10 male collegiate soccer players perform a 90-min. soccer-simulation test, that we developed, which was followed by a progressive shuttle run (PSR) test to exhaustion. They consumed a honey-sweetened beverage (H), a sports drink (S), or a placebo (P) before and half-way through the protocol. Both H and S provided 1.0 g·kg⁻¹ carbohydrate and ~17.6 mL·kg⁻¹ total volume for each trial. Overall, the test resulted in increased fatigue and production of inflammatory markers and antioxidant capacity. There was no significant difference between treatments for any performance measure. Mean times for a high intensity run and rating of perceived exertion increased with time, and there was an overall decrease in PSR time compared to baseline (-22.9%). There was a rise in glucose (15.6%), IL-6 (548%), IL-1ra, IL-10 (514%) and ORAC (15%) post-test but no change in cortisol. Insulin was significantly lower by 1 h-post. IL-1ra levels increased post-test for H (25.8%), S (65.5%), and P (63.9%), but the change for H was less than the other treatments. No treatment effects for the other blood measures were observed. The lack of an ergogenic effect of carbohydrate on soccer performance calls into question the benefit of supplementation at a frequency typical of a regulation soccer match in highly trained athletes with adequate energy stores. Since acute carbohydrate ingestion in the first study did not attenuate some markers of inflammation (e.g. IL-6), we chose to focus on an alternative theory for the rise in inflammatory markers with strenuous exercise in our second study. One aspect of soccer, repeated sprinting, results in increased ROS production partially through the activation of the enzyme xanthine oxidase (XO). Quercetin, a flavonol in plants that has shown some ergogenic effects with endurance exercise, inhibits XO in vitro. The effect of quercetin on team sport exercise had not been studied. We gave recreationally active males a commercial sports drink (S) or S + 500 mg of quercetin (Q) 2x/d for 1 wk prior to a repeated sprint test (RST). Sprint times increased (5.9%) for both treatments as did plasma XO activity (47%), IL-6 (77%), and uric acid (25%) from pre-test to post-test. Q supplementation did not attenuate plasma XO activity or IL-6 and actually increased one calculated index of fatigue, percent fatigue decrement (5.1%- Q and 3.8%- P). These findings add to the growing body of literature that quercetin supplementation does not attenuate exercise-induced inflammation and oxidative stress in vivo. Collectively, this research has practical implications for sports drink companies who are exploring the use of flavonoid compounds in product formulation. Specifically, they should reconsider adding quercetin to their beverages if they are marketing to team sport athletes. Also, soccer players should be made aware that, at ingestion frequencies typical of a soccer match, they may not expect a significant performance benefit from acute carbohydrate supplementation. / Ph. D.

The entire epidermis of patients with vitiligo exhibits accumulation of up to 10-3M concentrations of hydrogen peroxide (H2O2) (Schallreuter, Moore et al. 1999). Over the last decade our group and others have focused on the effect of H2O2-mediated oxidative stress on the function of many proteins and peptides due to oxidation of target amino acid residues in their structure including L-methionine, L-tryptophan, L-cysteine and seleno cysteine (Rokos, Beazley et al. 2002; Gillbro, Marles et al. 2004; Hasse, Kothari et al. 2005; Schallreuter, Chavan et al. 2005; Spencer, Chavan et al. 2005; Chavan, Gillbro et al. 2006; Elwary, Chavan et al. 2006; Gibbons, Wood et al. 2006; Schallreuter, Bahadoran et al. 2008; Shalbaf, Gibbons et al. 2008; Wood, Decker et al. 2009). Moreover, it was shown that patients with vitiligo possess up regulated wild type functioning p53 protein in their skin (Schallreuter, Behrens- Williams et al. 2003). The reason behind this up regulation has remained unclear (Schallreuter, Behrens-Williams et al. 2003). Therefore the aim of this thesis was to get a better understanding of these puzzling data. Along this project different techniques have been used including Western blot, dot blot, immuno precipitation, immuno fluorescence, EMSA and computer modelling. In this thesis we confirmed the previous result on up regulation of p53 in vitiligo and we showed that p90MDM2, the master regulator for p53 protein is not different in patients and healthy controls. Therefore we decided to test for expression of p76MDM2 which mediates the inhibition of p90MDM2-p53 binding. Our results show for the first time the presence and over expression of p76MDM2 protein in vitiligo compared to 3 healthy individuals. This result could provide an explanation, why up regulated p53 is not degraded in this disease. Since epidermal H2O2 accumulation has been extensively documented in vitiligo, we wanted to know whether other ROS could also contribute to the overall oxidative stress in this scenario. Therefore we turned our interest to nitric oxide (NO) and its possible effects on p53 protein. In order to elucidate this role in more detail, the expression levels of epidermal nitric oxide synthesase (iNOS) and the oxidation product of NO and O2 - i.e peroxynitrite (ONOO-) were investigated. Our data revealed over expression of iNOS and nitrated tyrosine residues, the foot print for ONOO-. Moreover, we show for the first time the presence of abundant nitration of p53 protein in vitiligo. In addition using purified p53 from E. coli strain (BL21/DE3) and mutant p53 protein from HT-29 cells (colon cancer cells), we show that nitration takes place in a dose and time dependent manner. On this basis we investigated the effect of both H2O2 and ONOO- on p53-DNA binding capacity employing EMSA, since this is the most acceptable technique to follow the binding between proteins and DNA. Our results revealed that ONOO- abrogated p53-DNA binding capacity at concentrations >300 ¿M, meanwhile oxidation of p53 protein with H2O2 at the same concentrations does not affect binding capacity. Importantly, a much higher p53- DNA binding capacity was observed after exposure to both ONOO- and H2O2. Taken together, p53 is regulated by both ROS (H2O2) and RNS (ONOO-). Next we identified the presence of phosphorylated and acetylated p53 in vitiligo. Phosphorylation of ser 9 and ser 15 residues of the protein are associated with over expressed ATM protein kinase, while acetylation of lys 373, 382 residues correlates with increased PCAF expression. We show that up regulated p53 is associated with over expressed p21 (cyclin dependent kinase inhibitor 1) and induced PCNA 4 expression. Hence, we can conclude that p53 in patients with vitiligo is up regulated, activated and functional. Finally we show up regulated BCL-2 supporting the long voiced absence of increased apoptosis in vitiligo. Given that patients with vitiligo have no increased risk for solar induced skin cancer and increased photo damage (Calanchini-Postizzi and Frenk 1987; Westerhof and Schallreuter 1997; Schallreuter, Tobin et al. 2002), despite the presence of increased DNA damage as evidenced by increased 8-oxoG levels in the skin and in the plasma, our findings suggest that both p53 and PCNA provide a powerful machinery to mediate DNA repair via hOgg1, APE1 and DNA polymerase ß (Shalbaf 2009). On this basis it is tempting to conclude that DNArepair is the overriding mechanism to combat oxidative stress in this disease. / Egyptian government; Institute for Pigmentary Disorders in association with the EM Arndt University of Greifswald, Germany.

Equine athletes are subjected to environmental and physical stressors resulting in oxidative stress that can negatively impact performance. Oxidative stress can result in lipid peroxidation, cell damage, and DNA degradation leading to physiological dysfunction and increased instance of disease. It has been established that humans are able to adapt to oxidative stress when exposed to extended periods of high-intensity exercise, however, this has yet to be established in the equine model. In the present study, we sought to establish patterns of oxidative stress expression immediately following exercise and adaption to prolonged exposure to exercise training in the equine model. Results indicate horses express changes in oxidative stress biomarkers at the onset of exercise training but adapt with prolonged exercise regimes. Future research should focus on mitigation techniques and therapeutics for oxidative stress in equine athletes.

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Dilated cardiomyopathy (DCM) or round heart disease is a muscle disease of the heart characterized by left ventricular dilatation and abnormal systolic and diastolic ventricular function. In animals, including turkeys and humans, DCM is a major cause of morbidity and mortality that results in heart failure. In the turkey, DCM can be idiopathic or induced. Since idiopathic or spontaneous DCM occurs in about 2-4 % of normal turkeys, it is of significant concern to the poultry industry. This dissertation was designed to increase our understanding of the pathophysiology of DCM in commercial turkeys. Specific objectives included: evaluating the influence of dietary selenium (Se) and vitamin E on poults fed toxic levels of furazolidone (Fz). Evaluating differences among reciprocal crosses of turkey varieties in susceptibility to a toxic level of Fz that induces DCM were used to assess the role of genetics in DCM. Using glutathione (GSH), glutathione peroxidase (GPx), malondialdehyde (MDA), and plasma uric acid (PUA) as biomarkers, oxidative stress (OS) levels were evaluated. Oxidative stress was also evaluated in poults fed diets containing varying concentration and combinations of vitamin E (0, 50 and 100 IU/kg) and Se (0.0, 0.3 and 0.5 mg/kg). Results from echocardiography measurements at four weeks of age, for poults fed toxic levels of Fz, showed the Narragansett x Bourbon Red reciprocal cross had the lowest internal-diastolic (LVIDd) and systolic dimensions (LVISd), while the Bourbon Red x Narragansett reciprocal cross had the largest LVIDd and LVISd. Left ventricular internal-diastolic and systolic dimension were lower for cross bred than parental poults. In treatment poults, heterosis for ventricular dilation was most significant for Bourbon Red x Narragansett cross. The data suggest that reciprocal crosses respond differently to toxin that induces DCM and genetics may influence a turkey's response to toxic levels of Fz that causes DCM. Results from OS measurements in poults fed normal and those fed normal diets with Fz at two weeks of age, showed no significant differences in MDA and GPx levels. PUA and GSH levels were however significantly increased for poults fed Fz-containing diets. At four weeks of age, no differences were observed for MDA and GPx measurements between poults fed normal and Fz-containing diets. PUA levels increased for poults fed normal diets with Fz, while GSH levels increased only for those fed normal diets. Differences between poults fed normal and Fz-containing diets were significant for GPx measurements. Results of this study showed that, feeding diets with Fz does not increase OS. Measure of the influence of feeding diets supplemented with different concentrations and combinations of Se and vitamin E to poults fed either normal or normal diets with Fz at two and four wks of age, showed higher MDA levels for poults fed Fz-containing diets supplemented with 0.3 mg/kg Se and 100 IU/kg vitamin E. For antioxidant biomarkers, GPx activity were increased for poults fed normal diets with Fz supplemented with 0.5 mg/kg Se and those fed 100 IU/kg vitamin E. Poults fed normal diets supplemented with 100 IU/kg vitamin E had the highest GPx. PUA levels were higher for poults fed normal diets with Fz supplemented with 0.5 mg/kg Se at two wks of age. At four wks of age, PUA concentrations were higher for poults fed Fz-containing diets supplemented with 100 IU/kg vitamin E. Increased PUA were also observed for poults fed diets supplemented with 0.5 mg/kg Se and 50 IU/kg vitamin E and 0.5 mg/kg and 100 IU/kg vitamin E. Poults fed diets supplemented with 50 and 100 IU/kg vitamin E had the highest GSH at two wks of age. Measurements taken at 2 wks of age, for poults fed normal diets supplemented with different concentrations and combinations of Se and vitamin E had increased GSH levels when compared with those fed diets with Fz at four wks of age. In this study, we showed that supplementation of poults fed normal diets with Fz with different concentrations and combinations of Se and vitamin E did not reduce DCM at 2 wks of age. However, at 4 wks of age, though DCM was not decreased by feeding diets supplemented with different concentrations and combinations of Se and vitamin E, reduced oxidant and antioxidant biomarkers were observed. / Ph. D.