Nitric oxide (NO), a free radical released by macrophages (a subset of white blood cells) as a response to infection, is noxious to organisms due to its ability to disable crucial biomolecules such as lipids, proteins and DNA. Although normally effective at eradicating invading bacteria, several pathogens have developed mechanisms to detoxify NO and its toxic by-products, reactive nitrogen species (RNS). While some of these detoxification processes have been characterized, very little is known about the metabolic changes that enable microbes to survive this deleterious environment. Investigations into the effects of RNS on microbial physiology have shown that these harmful radicals inactivate the citric acid cycle and oxidative phosphorylation, the series of reactions responsible for making energy aerobically. The central aim of this thesis was to determine how the organism counteracts the detrimental effects of RNS, while bypassing the ineffective central metabolic pathways. The findings presented herein show that P. fluorescens engineers an elaborate metabolic network to generate ATP whilst withstanding the injurious effects of nitrosative stress. Crucial to this adaptation is the ability to produce energy via substrate level phosphorylation, a necessity that arises out of the cells’ inability to produce a substantial amount of ATP using the electron transport chain (ETC). The up-regulation of the enzymes citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate, phosphate dikinase (PPDK) helps the organism accomplish this feat. Blue native polyacrylamide gel electrophoresis (BN-PAGE), high performance liquid chromatography (HPLC) as well as co-immunoprecipitation (CO-IP) studies were applied to demonstrate that these proteins form a metabolon, a transient complex of enzymes that ensures citrate is converted into its desired end products, pyruvate and ATP. In order to gauge the individual contributions iv of phosphoenolpyruvate-dependent kinases, a novel in-gel activity assay was developed to probe these enzymes under disparate conditions. These results suggest that the organism switches from an ATP-dependent metabolism to one based on the utilization of pyrophosphate (PPi). The rationale for this appears to be energy efficiency, as pyrophosphate-dependent glycolysis can theoretically produce five ATP rather than the two yielded by Embden-Meyerhof glycolysis. Additionally, the up-regulation in activity of the enzymes adenylate kinase, nucleoside diphosphate kinase and acetate kinase seem to ensure that ATP generated by PPDK is properly shuttled and stored when aerobic metabolism is defective. The lower activity of inorganic pyrophosphatase likely ensures an adequate supply of pyrophosphate for the activity of PPDK. Taken together, this research reveals the critical role metabolism plays in the survival of microbes under the onslaught of NO and RNS. As several of these enzymes are absent in mammalian systems, they present themselves as novel targets for the development of new antibacterial agents. A comprehensive awareness of bacterial defense systems in response to NO may lay the groundwork to developing more effective treatments to impede microbial infections.

Salmonella enterica serovar Typhimurium is responsible for a variety of diseases in domestic animals and humans. The infection of mice causes similar disease progression to human typhoid fever, thus representing a model for this systemic disease. The ability of S. Typhimurium to reside in a macrophage phagosome is important for their survival and spread to different organs. The antimicrobial mechanisms in this compartment include reactive oxygen species, reactive nitrogen species and elevated copper levels. S. Typhimurium possesses two copper-exporting P1B-type ATPases, CopA and GolT, both of which contribute to copper resistance. A previous study has shown that copper export by CopA and GolT confers a survival advantage in resting macrophage phagosomes. In this study the role of copper resistance systems has been examined further. The reduced survival of ΔcopA/ΔgolT in macrophages is detected beyond 8 hours post infection and coincides with increased nitrite production by macrophages. We have established that ΔcopA/ΔgolT display some increased sensitivity to reactive nitrogen species. However, whilst treatment of macrophages with the iNOS inhibitor L-NMMA reduced macrophage bactericidal activity against wildtype S. Typhimurium, this was not the case for ΔcopA/ΔgolT. In contrast, survival of ΔcopA/ΔgolT was not impaired in macrophages treated with the copper-chelator BCS. Furthermore real-time PCR confirmed the expression of copA and golT is elevated during infection of macrophages treated with IFN-γ or L-NMMA, but is reduced during infection of BCS treated macrophages. This indicates that bactericidal activity in macrophages is associated with copper availability and this is unaffected by reactive nitrogen species released due to iNOS activity. In contrast to Escherichia coli Salmonella lacks a cus system associated with export across the outer membrane and hence the mechanism of copper export from the periplasm is not known. TolC was investigated as a potential outer membrane copper exporter based on clustering of TolC dependent systems to genes with sequence similarity to the S. typhimurium periplasmic copper chaperone CueP, across several bacteria. Mutation of tolC gave reduced copper tolerance and over-accumulation of copper at non-lethal concentrations under aerobic conditions. However TolC does not provide a role in copper tolerance or homeostasis under anaerobic conditions. TolC also does not provide tolerance or homeostasis to other divalent cations: Zn, Ni and Co. TolC therefore provides specific transport of copper under aerobic conditions in S. Typhimurium.

No description available.

Tumor necrosis factor (TNF) is upregulated in a number of cardiomyopathies. This thesis investigates TNF in triggering the expression and activation of matrix metalloproteinases (MMPs) in pressure overload cardiac disease, and explores the role of superoxide. Cardiac pressure overload was generated in adult wild-type and TNF-/- mice by transverse aortic constriction. Isolated cardiomyocytes and cardiofibroblasts from neonatal mice ventricles were treated with recombinant TNF (rTNF), and MMP induction and activation were assessed, with and without apocynin (a NADPH-oxidase inhibitor). TNF-/- mice showed less superoxide production and MMP activation, compared to wild-type mice, following pressure overload. rTNF upregulated the production of NADPH-dependent superoxide in cardiomyocytes as early as 1 hour (24 hours in cardiofibroblasts). rTNF also increased the expression of MMP-9 and MMP-12 in cardiomyocytes more than in cardiofibroblasts, and MMP-8 and MMP-13 more in cardiofibroblasts. This induction in both cardiac cell types was concomitant with superoxide production.

No description available.

Oxidative stress, caused by increased levels of reactive oxygen species (ROS)and reactive nitrogen species (RNS) and/or a decrease in antioxidant capacity, can result in the oxidation of various bio-molecules, such as proteins, lipids and deoxyribonucleic acid (DNA). These oxidized bio-molecules may contribute to pathologies such as cardiovascular diseases, neurodegenerative disorders and cancer. The Sympathetic Activity and Ambulatory Blood Pressure in Africans (SABPA) study was initiated in 2008 to investigate the coping styles and catecholamine metabolic markers of Africans, contributing to their higher sympathetic output and poorer psychosocial wellbeing. This study forms part of the SABPA study, but with a specific aim to investigated lipid and protein oxidation markers in hypertensive Africans versus their normotensive counterparts. Analytical methods for the quantification of specific lipid and protein oxidation markers were optimized and validated. Urine samples from 172 urbanized black South Africans were collected and 3-nitrotyrosine (3NT) and thiobarbituric acid reactive substances (TBARS) were quantified in these samples, using the optimized spectrophotometric and LC-MS/MS methods. Statistical analyses showed that in both males and females, TBARS and 3NTcorrelated with each other. In males, 3NT also correlated with physical activity level (PAL) and C-reactive protein (CRP), while TBARS also correlated with body mass index (BMI). In females 3NT correlated with BMI, while TBARS correlates with PAL. These correlations meant that they could influence the calculations of the true effect of 3NT and TBARS levels between normotensive and hypertensive subjects. After analyses of covariance (ANCOVA) analyses it was determined that the hypertensive male subjects had higher TBARS values than the normotensive male subjects did (p-value = 0.03) and the normotensive female subjects had higher 3NT levels compared to the hypertensive female subjects (p-value = 0.04). These results partially supported the hypothesis that that elevated concentrations of specific urinary lipid and protein oxidation markers will be observed in the hypertensive test subjects compared to their normotensive counterparts. The results also indicated that there were indeed a difference in lipid and protein oxidation between hypertensive and normotensive subject. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013

Oxidative stress, caused by increased levels of reactive oxygen species (ROS)and reactive nitrogen species (RNS) and/or a decrease in antioxidant capacity, can result in the oxidation of various bio-molecules, such as proteins, lipids and deoxyribonucleic acid (DNA). These oxidized bio-molecules may contribute to pathologies such as cardiovascular diseases, neurodegenerative disorders and cancer. The Sympathetic Activity and Ambulatory Blood Pressure in Africans (SABPA) study was initiated in 2008 to investigate the coping styles and catecholamine metabolic markers of Africans, contributing to their higher sympathetic output and poorer psychosocial wellbeing. This study forms part of the SABPA study, but with a specific aim to investigated lipid and protein oxidation markers in hypertensive Africans versus their normotensive counterparts. Analytical methods for the quantification of specific lipid and protein oxidation markers were optimized and validated. Urine samples from 172 urbanized black South Africans were collected and 3-nitrotyrosine (3NT) and thiobarbituric acid reactive substances (TBARS) were quantified in these samples, using the optimized spectrophotometric and LC-MS/MS methods. Statistical analyses showed that in both males and females, TBARS and 3NTcorrelated with each other. In males, 3NT also correlated with physical activity level (PAL) and C-reactive protein (CRP), while TBARS also correlated with body mass index (BMI). In females 3NT correlated with BMI, while TBARS correlates with PAL. These correlations meant that they could influence the calculations of the true effect of 3NT and TBARS levels between normotensive and hypertensive subjects. After analyses of covariance (ANCOVA) analyses it was determined that the hypertensive male subjects had higher TBARS values than the normotensive male subjects did (p-value = 0.03) and the normotensive female subjects had higher 3NT levels compared to the hypertensive female subjects (p-value = 0.04). These results partially supported the hypothesis that that elevated concentrations of specific urinary lipid and protein oxidation markers will be observed in the hypertensive test subjects compared to their normotensive counterparts. The results also indicated that there were indeed a difference in lipid and protein oxidation between hypertensive and normotensive subject. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013

Non-thermal plasma has a lot of ways for using in nowadays medicine. It presents many useful actions like charged particles, UV light, electric field, radicals, excited atoms and molecules. That complicated chemistry directs to uncountable synergistic interaction between cold plasma and biological systems, involve cells and tissues. This thesis is about effects of cold plasma to wound healing. Two different microwave plasma systems were used for the presented study. The first one was argon plasma torch generated by surface wave using the quartz capillary, the second one was plasma torch with reverse vortex argon flow. Diagnostics of plasma jet by optical emission spectroscopy shown the presence of active particles, which are responsible for a lot of impact of plasma treatment. Concentrations of active particles generated by plasma are dependent on conditions of plasma generation like power of generator and gas flow. For visual evidence of effects on skin caused by active particles was created simulation of skin tissue. Interaction between plasma jet and artificial skin tissue shown that UV light and temperature are not responsible for all observed effects which are noticed after plasma treatment. Some part of experiments was realized in collaboration with Medical University of Sofia in Bulgaria. The theory of positive effect to wound healing was supported by experiments based on treating artificially created wounds on laboratory mice by cold plasma. It was proved, that process of wound healing is significantly shorter after using plasma treatment in comparison with normal wound healing. Plasma treating of wound for 10 seconds in two consequent days seems like more effectively than application of plasma only one day. This Thesis was carried out as a part of international project PLASMABORDER that was supported by European commission under cohesion funds; programme INTEREG SK-CZ under contract No. 304011P709.

No description available.

No description available.