[Truncated abstract] Current understanding of atherosclerosis suggests that it is a chronic inflammatory disease, and that increased oxidative stress may be an important pathological event contributing to the disease process. There has been interest in the ability of dietary derived nutrients such as vitamin E, to act as antioxidants and protect against atherosclerosis. Despite promising epidemiological data which suggested benefits from a higher intake of &alpha-tocopherol (&alphaT), one of the major forms of dietary vitamin E, for protection against atherosclerosis, large scale, randomised controlled trials have generally shown no protective effect of high dose &alphaT supplementation. Recent studies suggest that the other major dietary tocopherol isomer, &gamma-tocopherol (&gammaT), may possess biological activities not shared by &alphaT. Supplementation with &gammaT, or mixtures of tocopherols rich in &gammaT, have shown biological activities that may help protect against atherosclerosis. The aim of this PhD project is to further characterise the biological relevance of ?T for protection against CVD... Both ?- and mixed tocopherol supplementation resulted in reduced plasma F2-isoprostanes (P < 0.001 and P = 0.001, respectively) but did not affect 24 hour urinary F2-isoprostanes and erythrocyte antioxidant enzyme activities. Neither &alphaT nor mixed tocopherol supplementation affected any measured plasma markers of inflammation. The tocopherol supplementation also did not affect COX-2 activity as assessed by 14 stimulated whole blood prostaglandin E2 synthesis, and urinary prostacyclin metabolite output. Compared to the placebo group, stimulated neutrophil leukotriene B4 (LTB4) production decreased significantly in the mixed tocopherol group (P=0.02) but not in the &alphaT group (P=0.15). The ability of both pure &alphaT and mixed tocopherol supplementation to reduce systemic lipid peroxidation in patients with type 2 diabetes, suggests potential benefits of vitamin E supplementation in this population. However, despite decreasing oxidative stress, our results also suggests that in populations with well controlled type 2 diabetes, supplementation with either &alphaT, or mixed tocopherol rich in &gammaT, is unlikely to confer further benefits in reducing systemic inflammation. Future research into the possible unique biological activity of different tocopherol isomers other than &alphaT, for example, their ability to affect the 5-LO pathway and production of inflammatory mediators such as LTB4, is warranted.

Thesis (Ph. D.)--University of Texas Health Science Center at Houston, School of Public Health, 2007. / Source: Dissertation Abstracts International, Volume: 68-12, Section: B, page: 7975. Adviser: Mary Ann Smith. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2010. / Title from document title page. Document formatted into pages; contains x, 178 p. : ill. Includes abstract. Includes bibliographical references.

Sturgeon are an ancient family of fish which have remained essentially unchanged for 200 million years, rendering them physiologically distinct from the more modern teleosts. Of the 26 known species of sturgeons all are likely endangered. North American populations have been declining steadily since the 1800s due to factors such as overharvesting, habitat alterations and increasing pollution. White sturgeon (Acipenser transmontanus), endemic to Western North America, are the largest freshwater fish on the continent. Protecting white sturgeon is of interest because nearly all Canadian populations are endangered and they are culturally and economically important. Factors such as great size, longevity, position in the food chain and benthic life style render white sturgeon particularly susceptible to bioaccumulation of toxicants. They are known to be among the most sensitive species to pollutants such as metal ions, dioxin-like compounds and endocrine disrupters. However, little is known about their susceptibility to other priority contaminants such as selenium (Se). Selenium, in its organic form selenomethionine (SeMet) has become a contaminant of particular concern as it is a known toxicant that efficiently bioaccumulates and biomagnifies in the food chain. It is also of interest as Se is an essential micronutrient that becomes toxic at only marginally greater than optimal doses. Current elevated concentrations of SeMet in white sturgeon prey, with predicted increases in anthropogenic releases, have made it a contaminant of concern for this species. It is hypothesized that increased releases of Se to aquatic environments have contributed in part to sturgeon declines; however, to date little is known about its specific effects on this species. Therefore, the purpose of the present study was to investigate the sensitivity of three year old white sturgeon to dietary SeMet and to link physiological effects to key molecular events of toxicity and to elucidate the mechanism of toxicity. Specifically, this thesis focused on oxidative stress in liver tissue as a hypothesized primary mechanism of toxicity. For 72 days sturgeon were given either a control diet of 1.4 µg Se/g feed or a diet spiked with SeMet (5.6, 22.4 or 104.4 µg Se/g feed dry mass). These doses corresponded to an uptake necessary for proper health, two environmentally relevant exposures, and a worst-case scenario for industrial Se release, respectively. A subsample of fish was taken at day 10 to investigate molecular endpoints. Within 10 days of exposure, pathological effects were observed in fish given the high dose. Occurrence of severe edema causing exophthalmos developed within 15, 23 and 52 days in high, medium and low dose group fish, respectively. There was a 54% and 22% occurrence of lethal effects in the high and medium dose groups, respectively. Se accumulated in a dose dependent manner and reached equilibrium in high dose fish after approximately 40 days. Growth, liver weight and hepatosomatic index were all significantly lower in the high dose group. Histology of 72 day liver samples showed a significant and dose dependent increase in melanomacrophage aggregates and decrease of energy stores and cell size. Food avoidance was also observed in sturgeon exposed to the high dose. To investigate oxidative stress, 10 day liver samples were tested for changes in gene expression coding for glutathione peroxidase (GPx), superoxide dismutase, catalase, glutathione S-transferase, apoptosis inducing factor and caspase 3, using real-time PCR. Only GPx was significantly induced. Day 72 liver samples were tested for the presence of lipid hydroperoxides but there were no significant differences between dose groups and controls, which shed doubt on oxidative stress being the main driver of toxicity. Taken together the data makes a strong case for the sensitivity of white sturgeon to Se accumulation and indicates a general suppression of health due to toxic levels of exposure. However, in contrast to other fish species exposed to Se, oxidative stress is not likely the main mechanism of toxicity in white sturgeon. Findings from the present study could be used for the risk assessment of sturgeon to anthropogenic Se in aquatic ecosystems.

This study examines three possible methods for improving the digestibility and bioavailability of zinc to rainbow trout (Oncorhynchus mikiss). The first method was to examine the availability of the zinc utilisation from commonly used protein sources; the second was to assess the efficacy of the upstream use of an enzyme treatment of the raw materials; the third was to assess the use of organically complexed mineral supplements as opposed to the inorganic salts widely used at present. The first section indicated that the zinc from the soyabean meal was the most available (49.4%). The zinc digestibilities of the animal based protein used in this current study were 15.1% for LT94 fish meal, 26.6% for the Provimi 66 white fishmeal and 15.8% for poultry meat meal. The zinc in the maize/corn gluten meal was 31.9% digestible and from the NuPro 26.1%. Gram for gram maize gluten meal supplied the least amount of zinc to the fish (3.66 mg per kg). Based on these results the diets for the subsequent supplementation trials were formulated. The liver, eye and caudal fin were identified biomarkers of a severe zinc deficiency. The second part of the study revealed a soybean product, treated by exogenous enzymes, had a higher phosphorus digestibility, (49.0%, vs. 36.6%) and zinc digestibility (30.7% vs. 7.9%) The treatment did not improve the protein digestibility (85%). The third part of the study showed the organic source proved more digestible than the inorganic, 37.4% and 26.9% respectively. The fish fed the organic source maintained a higher level of zinc in both the eye and caudal fin. The liver zinc levels were unaffected by both dietary level and zinc source. Analysis of the liver for a zinc dependant protein showed that under stress conditions only the organic supplemented fish were able to synthesis this protein. The analysis of the mRNA levels coding for this protein indicate the fish on both zinc forms up regulated the production of the mRNA to the same extent when stressed. Finally this study also examined the viability of using a stable isotope to identify different ‘preferences’ for one form of supplementation over the other in different tissues. This method illustrated a tissue dependant difference to how the fish attempted physiologically to compensate for zinc deficiency. The rate of turnover was fastest in the liver, then the caudal fin and then the eye, and also showed that when the diet was more deficient there was an increased ability for the tissues to take up the organic form.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Tuberculosis (TB) has become a global health epidemic affecting millions of people worldwide with a high incidence in third-world countries. The emergence of multi-drug and extremely-drug resistant M. tuberculosis strains together with the HIV/AIDS pandemic warrants the need for new drugs or new drug combinations. The folic acid synthesis pathway is one of the key pathways that are essential for the survival of bacteria in general. Sulfonamides are a group of compounds that target folic acid synthesis, particularly dihydropteroate synthetase, the first enzyme in the folate pathway. Some of these sulfonamides were used during the introduction of chemotherapy for the treatment of TB in the 1930s, but had toxic side effects. Newer derivatives became safer, but were not employed again for TB treatment. In a recent case study it was reported that the combination of trimethoprim-sulfamethoxazole (Bactrim), which is used to treat various bacterial infections, such as urinary tract infections, had activity against M. tuberculosis. In light of this and the fact that trimethoprim-sulfamethoxazole is well tolerated by humans, we have investigated their antimycobacterial activity with particular interest in the combinational effect of sulfamethoxazole and trimethoprim with the first-line anti-TB drugs, Isoniazid, Rifampicin and Ethambutol against M. tuberculosis. Since sulfonamides are known to produce oxidative stress, we also investigated the contribution of this factor to the efficacy of sulfamethoxazole using a mycothiol deficient strain of M. tuberculosis, ΔmshA. Though trimethoprim-sulfamethoxazole targets the folic acid pathway, we also investigated the possibility that trimethoprim-sulfamethoxazole may have other cellular targets and applied proteomic analysis. We have found that Trimethoprim-Sulfamethoxazole has activity against M. tuberculosis and that Sulfamethoxazole is the active compound. However, our observation was that not all sulfonamides are active against M. tuberculosis. In addition we observed that sulfamethoxazole enhances the activity of Rifampicin against M. tuberculosis in a synergistic way. We also observed that a mycothiol deletion mutant was more susceptible to Sulfamethoxazole compared to the wild type strain CDC 1551. Through global protein expression profiling (Proteomics) we were also able to show that sulfamethoxazole could also kill M. tuberculosis by oxidative stress production as we identified oxidative stress responsive proteins that were differentially regulated upon exposure to sulfamethoxazole. As trimethoprim-sulfamethoxazole is a registered drug combination, inexpensive and widely available, we propose that this regimen could be used in our fight against M. tuberculosis infection. / AFRIKAANSE OPSOMMING: Tuberkulose (TB) is ‘n globale gesondheidsprobleem wat miljoene mense wêreldwyd affekteer met ‘n besoderse hoë voorkoms in die derdewêreld lande. Die voorkoms van multi-middel weerstandige en uitersweerstandige M. tuberculosis stamme, tesame met die HIV/VIGS pandemie, steun die erns vir die ontwikkeling van nuwe middels teen M.tuberculosis . Die foliensuur sintesepad is essensieël tot die oorlewing van bakterieë in die algemeen. Vir daardie rede is daar vele middels ontwerp om hierdie metaboliese pad te teiken. Die sulfonamiedes is ‘n groep antibiotika wat foliensuursintese, spesifiek dihidropteroaatsintese, die eerste ensiem in die foliensuursintese pad, teiken. Van hierdie sulfonamiedes is voorheen in die 1930’s gebruik vir die behandeling van tuberkulose, maar het toksiese newe-effekte getoon. Nuwe, minder toksiese derivate, is later ontwikkel maar is nooit vir TB behandeling weer aangewend nie. In ‘n onlangse gevallestudie is daar gerapporteer dat die kombinasie trimethoprim-sulfamethoxazole (TMP/SMX. Handelsnaam: Bactrim), wat normaalweg gebruik word vir die behandeling van algemene bakteriële infeksies soos blaasinfeksies, aktiwiteit teen M. tuberculosis getoon het. Na aanleiding hiervan en dat Bactrim veilig in mense gebruik kan word, het ons die aktiwiteit van Bactrim komponente teen M. tuberculosis bepaal en in die besonder die aktiwiteite van SMX en TMP in kombinasie met die eerstelinie anti-tuberkulose middels Isoniasied, Rifampisien en Ethambutol. Aangesien sulfonamiedes ook oksidatiewe stres intrasellulêr genereer, het ons ook die bydrae van hiervan tot die doeltreffendheid van SMX bepaal deur gebruik te maak van ‘n mycothiol-gemuteerde M. tuberculosis stam ( mshA). Omdat TMP/SMX die foliensuur-pad hoofsaaklik teiken het ons ook die moontlikheid ondersoek dat SMX ander sellulêre teikens het en het ons proteomiese (Proteomics) tegnieke hiervoor aangewend. Ons het gevind dat TMP/SMX aktiwiteit teen M. tuberculosis toon en dat SMX die aktiewe komponent van Bactrim is teen M. tuberculosis . Ons wys ook dat sulfonamiedes in die algemeen nie noodwendig ook aktiwiteit teen M. tuberculosis toon nie. Ons het ook waargeneem dat SMX die aktiwiteit van rifampisien bevorder en dat die twee middels saamwerk op ‘n sinergistiese wyse. Ons het ook getoon dat oksidatiewe stres ‘n rol speel deurdat‘n mycothiol delesie-mutant meer vatbaar was vir SMX in vergelyking met die wilde-tipe stam van M. tuberculosis (CDC1551). Met globale proteïenkartering (Proteomics) het ons ook getoon dat SMX M. tuberculosis kan doodmaak deur oksidatiewe stres te genereer omdat ons oksidatiewe stres reaktiewe proteïne geïdentifiseer het wat differensieël gereguleer is gedurende blootstelling aan SMX. Aangesien Bactrim ‘n reeds geregistreerde middel is, goedkoop is en geredelik beskikbaar is, stel ons voor dat Bactrim moontlik geïnkorporeer kan word in die huidige behandeling van Tuberkulose.

Francisella tularensis is capable of infecting numerous cell types, including professional phagocytes. Upon phagocytosis, F. tularensis resides within the phagosome before escaping into the cytosol to replicate. Phagocytes constitute a hostile environment rich in ROS, which are employed as a means of killing pathogens. ROS interact with and disrupt the function of vital molecules such as DNA, proteins and bacterial structures. Iron potentiates the danger of ROS through the Fenton reaction where ferrous iron reduces H2O2 causing the formation of highly reactive hydroxyl radicals and anions. Low levels of ROS are formed during normal aerobic metabolism and pathogens thus have a need for defense mechanisms to handle the ever present levels of ROS but even more so to combat the onslaught of ROS experienced within a host. This thesis was focused on the investigation of the iron status and oxidative stress response of F. tularensis; thereby identifying key players controlling the bacterial iron content, its adaptation to oxygen-rich environments and defense against ROS. We identified subspecies-specific differences in iron content, where F. tularensis subsp. tularensis was found to contain significantly less iron than strains of subsp. holarctica. The reduced iron content resulted in an increased tolerance to H2O2, despite simultaneously causing a decrease in the activity of catalase - the iron-dependent enzyme responsible for degrading H2O2 in F. tularensis. This strongly suggests that the restricted iron uptake and storage by subsp. tularensis strains is beneficial by rendering the bacteria less susceptible to H2O2, thereby evading the toxic effects of the iron-driven Fenton reaction. This evasion is likely to be an important part of the higher virulence displayed by subsp. tularensis as compared to subsp. holarctica. We further identified that the global regulator, MglA, is important for the adaptation of LVS to oxygen-rich environments. Deletion of mglA from LVS resulted in a mutant, ΔmglA, with impaired defense to oxidative stress, as manifested by an inability to grow to wild-type levels under aerobic conditions, an accumulation of proteins with oxidative damage, a suppressed expression of iron-uptake related genes, an increased catalase activity, and an increased tolerance to H2O2. This phenotype was reversed in a microaerobic environment. We therefore conclude that MglA is an important factor for the defense of LVS to oxidative damage under aerobic conditions and speculate that MglA is of greatest importance in oxygen-rich foci. We also studied the role of OxyR in LVS by creating a ΔoxyR mutant as well as a double mutant, ΔoxyR/ΔkatG. The in vitro response of these mutants, as well as of ΔkatG, to defined ROS was assessed using H2O2, the O2- generating agent paraquat, and the ONOO- generator SIN-1. ΔoxyR was more susceptible to all ROS than LVS as was ΔkatG, with the exception of O2- Strikingly, ΔoxyR/ΔkatG was significantly more susceptible to all ROS tested compared to either single deletion mutant. LVS, ΔoxyR and ΔkatG replicated efficiently in bone marrow-derived macrophages whereas ΔoxyR/ΔkatG showed no replication. In mice, the ΔoxyR mutant displayed impaired replication in liver, but intact replication vs. LVS in spleen. Collectively, our results demonstrate an important role of OxyR in the oxidative stress response and virulence of F. tularensis, and further reveal overlapping roles of OxyR and catalase in the defense against ROS. The results thus shed new light on the complexity of ROS defense in F. tularensis.

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 μM, 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.

published_or_final_version / abstract / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biological Sciences / Doctoral / Doctor of Philosophy