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This thesis is concerned with the chemical control of breathing during exercise in humans. Chapter 1 reviews some of the relevant studies in animals and humans. Chapter 2 describes the experimental apparatus and the technique of dynamic end-tidal forcing performed using a computer-controlled gas-mixing system. Chapter 3 describes a study of the effects of sustained hypoxia on ventilation during steady exercise. The acute ventilatory response to hypoxia (AHR) was increased during exercise as compared with rest, but the magnitude of the subsequent decline in ventilation (HVD), expressed as a fraction of the AHR, was reduced. A simple model of the hypoxic peripheral chemoreflex is proposed, in which the mechanisms underlying AHR and HVD are functionally separate and can be independently modulated by external factors. Chapter 4 assesses changes in peripheral chemoreflex sensitivity to hypoxia in terms of the degree of decline in AHR measured in the resting periods shortly after prior conditioning periods of hypoxia and/or exercise. At rest, a second AHR measured 6 min after a period of sustained hypoxia had declined by 30% as compared with the initial AHR. In contrast, the AHR measured in the resting period after a period of sustained hypoxic exercise was only 11% smaller in magnitude than the AHR measured after a period of euoxic exercise. The results suggest that the degree to which hypoxic sensitivity declines during sustained hypoxia is genuinely attenuated, rather than masked, by exercise. Chapter 5 describes the changes in respiration during prolonged exercise breathing air with and without added CO₂. During prolonged poikilocapnic exercise, ventilation remained constant, but metabolic CO₂ production, respiratory quotient and end-tidal P_CO2 declined; a result which suggests that in man, ventilation can be dissociated from the CO₂ flux. During hypercapnic exercise, ventilation progressively increased; this was interpreted as being due to a correction by end-tidal forcing of the natural tendency for end-tidal CO₂ to decline, together with an independent effect of CO₂ per se on the ventilation. Chapter 6. Electrical muscle stimulation was used as means of inducing non-volitional exercise. Electrically-induced exercise increased the AHR as compared with rest, and with voluntary exercise at matched external work rate. The AHRs during electrical stimulation and voluntary exercise matched to the internal work rate were similar. Chapter 7. Electrical muscle stimulation was used in paraplegic subjects in whom there would be no neural control of exercise. Electrically-induced exercise increased the AHR as compared with rest. When compared with the data from Chapter 6, the results suggest that the observed increase in AHR during normal voluntary exercise can be wholly accounted for by the increase in metabolic CO₂ production, or closely related factors. Chapter 8 presents a brief summary of the findings in this thesis.

Mitochondria perform functions which are central to the life of most eukaryotic cells. These organelles can be considered the ultimate energy power house of a living cell. The role of mitochondria in cancer phenotype remains a fertile area of research. Several carcinogens are known to enter the mitochondria, resulting in impaired functioning and altered structure. Aflatoxin BI (AFB1) a primary type I mycotoxin elaborated by Aspergillus flavus and Aspergillus parasiticus, is carcinogenic for a wide species range. The epoxide is capable of binding to nucleic acids and proteins, resulting in induced mutations, cellular toxicity, and eventually carcinogenesis. Approximately 250 000 deaths occur annually in both China and Africa due to patients presenting with Hepatocellular Carcinoma (HCC). The causative agents being AFB1-ingestion via contaminated foods and feeds, and the Hepatitis B Virus infection. The toxin has a multifaceted mode of attack, capable of being activated to a highly reactive and carcinogenic derivative, the AFB1-8,9-epoxide, via the cytochrome P450 enzyme system of the microsomes, endoplasmic reticulum and also the mitochondria. The epoxide is capable of binding to nucleic acids and proteins, resulting in the formation of covalent adducts. The repeated occurrence of gold labelled toxin within mitochondria from hepatomas of patients presenting with HCC suggested that these organelles were direct sites of toxin binding. Despite observations that mitochondria appear as direct and perhaps preferential targets for attack by AFB1, the actual in vivo immunolocalisation and characterisation of bound AFB1 within liver mitochondria has not been reported previously. In addition the role of AFB1-protein binding within mitochondria was investigated to determine the mode of action of the toxin, within the mitochondrial system. Liver sections from rats treated with a single lethal dose of AFB1, showed distinct ultrastructural abnormalities viz. large nuclei, increased heterochromatin, and swollen mitochondria. Immunocytochemistry revealed for the first time, the selective localisation of conjugated gold labelled toxin within the mitochondria. Toxin was found in the intracristal and peripheral spaces and frequently within the mitochondrial matrix. The mitochondria isolated from treated rats revealed significant alterations and damage to the mitochondrial membranes. The cristae were also markedly swollen with the associated clearing of the mitochondrial matrix. Western blot immunoassays revealed the presence of five AFB1-bound proteins (150kDa, 50kDa, 25kDa, 18kDa, 14kDa) in the inner mitochondrial fraction of isolated mitochondria. High pressure liquid chromatography also revealed that a significant proportion (84%) of an initial dose of toxin, was absorbed by mitochondrial protein. This study is the first to show the presence of specific mitochondrial proteins involved in toxin binding. In addition, the presence of toxin within the mitochondria and the specific binding to inner mitochondrial proteins suggest that the toxin specifically targets the electron transport chain and hence effects ATP production. This study conclusively indicates that mitochondria are direct targets for attack by AFB1 during experimental carcinogenesis. Mitochondria therefore play an important role in AFB1-mediated carcinogenesis. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1998.

Pre-eclampsia, often described as toxaemia of pregnancy, historically represents one of the most widely investigated conditions relating to human reproduction. To date no firm cure has been found and a clear, well defined mechanism has not been ascribed to the pathogenesis of the disease. Researchers seem to focus on single pathways in isolation of others. The disease rather represents a multitude of possible underlying pathologies nvolving genetics, immune dysregulation, vascular maladaptation, and sociobiological factors thus complicating the approach to treatment. However, a central theme is the presence of reduced placental perfusion resulting in a hypoxic and/or ischaemic placenta and the subsequent secretion of various factors that initiate the maternal syndrome. It is within this context that we examine how an intervention such as increasing placental perfusion may represent a promising treatment strategy for this disease. We sought to manipulate the vasodilatory mechanisms of the uterine vasculature using sildenafil citrate and a flavonoid extracted from Eriosema kraussianum (Kr2), in Sprague-Dawley rats that exhibited preeclampsia-like manifestations. Both treatment regimens improved fetal outcomes and reduced blood pressure amplification and proteinuria. They also reduced the plasma concentrations of the two anti-angiogenic factors; sFlt1 and sEng. Only sildenafil citrate improved nitric oxide levels which was expected, suggesting that Kr2 causes vasodilation by some other mechanism. Nevertheless, both compounds improved both pup and placental weights, suggesting that they also improve utero-placental perfusion. These findings that selective uterine vascular dilation improves placental perfusion may be promising in averting possible death to mothers and their babies from pre-eclampsia especially in low resource environments. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.

Oxidative stress resulting from an imbalance between free radicals and antioxidants is considered to be an important etiological factor in the development and maintenance of hypertension. Angiotensin II (Ang II) has been shown to be an important regulator of blood pressure and acts to elevate blood pressure by its pressor effects. The pressor effects of Ang II are well documented but recent evidence has suggested another possible role of Ang II in elevating blood pressure, whereby it acts via an independent mechanism that is directly linked with oxidative stress. The spontaneously hypertensive rat (SHR) is a widely used model in the investigation of the pathophysiological mechanisms involved in hypertension. This study was therefore undertaken to determine whether Ang II acts as a causative factor via oxidative stress in the development and maintenance of hypertension in the SHR. This was elucidated by evaluating the role of both the endogenous in vivo levels of Ang II as well as an exogenous sub-pressor dose of Ang II, on oxidative stress and its associated parameters. The parameters evaluated included, the antioxidant status of the model on the basis of the levels of the major antioxidant enzymes viz. SOD, GPx and catalase; the free radical generating capacity, by assessing the activity of the membrane bound enzyme NADPH oxidase and the levels of H2O2. The study also evaluated the levels of the endogenous vasodilator nitric oxide (NO), remodelling of the vasculature and the level of tissue oxidative stress in the kidney. The results show that the SHR has an elevated plasma Ang II level and an elevated level of oxidative stress, thus showing that in this model there is an intimate link between oxidative stress and Ang II. The SHR also shows depleted levels of NO and thus a decreased vasodilatory capacity and increased remodelling of the vasculature. The kidney showed an increased level of lipid peroxidation, which was due to the elevated levels of oxidative stress. All of these pathophysiological changes contribute to the elevation in blood pressure in this model. The long term infusion of the sub-pressor dose of Ang II affected the SHR to a greater extent than the Wistar. Although the dose of Ang II elevated the blood pressure in both models, the degree of the pathophysiological changes associated with the elevation in blood pressure was greater in the SHR. The Ang II infusion in both these models demonstrated that in the SHR which is genetically predisposed to hypertension, adjustments are made to the antioxidant system, that result in an elevated level of protection against oxidative stress. These results show that Ang II acts as a causative factor in the pathogenesis of hypertension in the SHR via its well documented pressor effects, as well as via a multitude of independent mechanisms that are linked to oxidative stress. This is substantiated by the significant decrease in NO that is caused by the elevated oxidative stress, as well as the previously described pathophysiological changes. This study has therefore shown that Ang II has an intimate causative link with oxidative stress that results in parallel mechanisms that work concomitantly with each other in hypertension in this model. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.

Recent clinical studies have highlighted the effectiveness of immunotherapy for Gram-negative bacteraemia in humans. Studies in America, undertaken on patients with Gram-negative bacteraemia, have shown that mortality was reduced by virtually 50% in patients who received specific antiendotoxin antiserum. In India, mortality from pseudomonas septicaemia was significantly reduced by the administration of small quantities of a anti-pseudomonas immunoglobulin. The antibodies in those studies were raised by vaccination of healthy volunteers with heat-killed Gram-negative bacteria or vaccines containing endotoxin. Adverse side effects in volunteers as well as logistic and legal problems make it difficult to produce antiserum on a large scale, in this manner. In Israel, S.L. Gaffin and coworkers found that approximately 7% of plasma units in a blood bank had antiendotoxin antibody concentrations of 40 ).1g/m1 or greater. This high titre human plasma significantly protected cats from lethal endotoxic shock secondary to haemorrhage. The immunoprecipitin technique used by them to measure antiendotoxin antibody concentrations was unsuitable for screening large numbers of blood samples. To overcome this problem we have devised an enzyme-linked imounosorbent assay (ELISA) for determining the level of antiendotoxin immunoglobulin G in human plasma. The assay, which is suitable for large scale use, was found to be specific for antiendotoxin antibodies. It was calibrated using a serum sample of specific antibody concentration as determined by an ilununoprecipitin assay. Serum samples found to be high in antiendotoxin titres (> 40_ug/m1) were tested for their specificity towards endotoxins from 12 bacterial iv strains and species. While each sample was found to have its own characteristic specificities, most were found to react strongly with Sh. flexneri, S. typhimurium and S. enteritidis. The Natal Blood Transfusion Service has found that in Natal, blood units containing high concentrations of specific antibodies occur with a frequency of 3,6% among all White donors and 10,35% among all African donors. They found that African females, in turn, had almost twice the frequency of high titre serum as African males. In this study, Indian female hospital patients did not have a statistically higher frequency of high-titre serum than Indian male patients. Blood units donated to the Natal Blood Transfusion Service are now routinely screened by ELISA for antiendotoxin antibodies and those units with high concentrations (> 40 ug/ml) of antibody were pooled and fractionated to obtain a gamma globulin, Lot LG-l. The binding capacity of the LG-1 antibodies towards 12 endotoxins was examined. Binding was found to be highest with endotoxin from Sh. flexneri, S. abortus equi and S. typhimurium and intermediate with S. enteritidis and E.coli 026:B6. Binding with the other endotoxins tested was relatively low. Differential absorption experiments showed that LG-1 was made up of a mixture of cross-reacting as well as specific antibodies For example, the antibodies binding Sh. flexneri endotoxin were mainly specific. Those binding E. coli 026:B6 endotoxin were specific and cross-reacting in almost equal proportions. Antibodies to the endotoxins from the salmonella strains tested were mainly cross-reacting. The specificities of the LG-1 antibodies towards endotoxins from the various Gram-negative bacteria did not in most cases reflect the incidence of these organisms in blood cultures taken from hospital patients. V The activity of LG-1 antibodies was compared to that of normal human immunoglobulin preparations obtained from the National Blood Fractionation Centre, Pinetown and to an anti-pseudomonas immunoglobulin prepared by Wellcome Laboratories, England. The binding capacity of the antibodies in the standard globulin preparations towards most of the endotoxins tested was less than 15% of that of the LG-1 antibodies. The anti-pseudomonas immunoglobulin was shown to bind poorly to most of the endotoxins tested in comparison with binding by LG-1 antibodies. / Thesis (MMedSc.)-University of Natal, Durban, 1984.

Folic acid is a water-soluble, B-complex vitamin influencing a number of biological processes in humans and particularly important in the prevention of neural tube defects (associated with spinal bifida) in unborn children. Reliable analytical methods are therefore needed for quantisation of the amount of total folic acid (FA) in biological materials of quality assurance and regulatory purposes. What is particularly needed are rapid and reliable methods for ensuring that the correct amount of FA is consumed and the degradation rates of these compound is kept at minimum during the extraction process. Analytical methods for determination of folic acid in biological materials have been around for decades and the most common procedures include microbiological assay; biomolecular interaction analysis (BIA); immunoassay; conventional chromatographic procedure such as thin-layer column chromatography (TLC) and high performance liquid chromatography (HPLC). These procedures were replaced by HPLC, which is more rapid and in many instances yields a better resolution. Current HPLC methods uses C-18 column and reverse phase conditions in combination with ion-pair or ion suppression techniques; fluorescence or electrochemical detector, unfortunately, excitation and emission of folic acid is found not sufficiently to allow physiological levels of the form of the vitamin to be detected. In addition, ion-pair reagent nullifies the mobile phase and interferes with the absorption! fluoresce spectrum resulting in poor separation. Therefore this study was carried out to address and improve the problems that are in the existing HPLC methods. Currently scarce information is available on the determination of folic acid in biological materials by HPLC with UV detection. Serum samples were spiked with folic acid standard to check the efficiency of the method. Other wavelengths from 200 nm to 300 nm were attempted for detection of folic acid, in which the wavelength 250 nm was found to have better absorbance compared to other wavelengths. Folic acid was detected at 250 nm wavelength under isocratic elution using a mobile phase consisting of citrate phosphate buffer: acetic acid: methanol. Folic acid in maize meal was detected at 290 nm using mobile phase containing potassium phosphate containing ascorbic acid/sodium ascorbate mixture and 2-mercaptoethanol under gradient elution. The mobile phase used for gradient and isocratic elution was suitable for separation of folic acid from other compounds with flow rate of 3 ml/min modified to Iml/mim to avoid overloading of the column under isocratic elution. For good separation of folic acid under gradient elution the flow rate was set at 0.8ml/min with pH of mobile phase modified from pH 2.2 to pH 2.5. The recovery of folic acid added to human serum was 91% -100% and recovery of folic acid added in unfermented maize meal and fermented maize meal ranged from 55% - 73%. Folic acid level from unfermented maize meal and fermented maize meal ranged between 1.29 - 1.3 [!g/g and 1 - 2.1 [!g/g respectively. In conclusion the optimised method in this study gives better analytical results when compared with earlier HPLC method in terms of efficiency, reproducibility and sensitivity for folic acid in human serum and maize meal. However, there is a need to minimise the loss of folic acid during the sample treatment. The outcome of this work indicated that more work has to be done to improve extraction procedure for specific foods with minimum time preparation to sample analysis. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2007.

This study was aimed at investigating the potential of lactic acid fermentation in reducing myco toxin concentration in maize meal products. Maize meal was spiked separately with aflatoxin Bi, fumonism Bi, and zearalenone, and fermented for four days. During this period the concentration of each toxin and the pH of the fermented maize meal were monitored. There was a significant (p= 0.000) decrease in the concentration of all the mycotoxins, with a percentage reduction of 55-69 by the third day and 68-75 by the fourth day, respectively. Commercial amahewu samples were also screened for the presence of these three mycotoxins, and the results indicated that the samples were not contaminated with detectable levels of these toxins. An attempt was made to characterise the metabolic derivatives (by-products) of each mycotoxin following lactic acid maize meal fermentation. To achieve this maize meal samples were separately spiked with each of mycotoxin, fermented for four days and screened for specific mycotoxin derivatives (by-products) using GC/MS, HPLC and relevant standards (i.e. partially hydrolysed fumonisin Bi, aflatoxin B2a, a- and Pzearalenol). None of the targeted derivatives could be detected in the fermented maize meal samples. The potential cytotoxicity of the mycotoxin-spiked fermented samples was investigated using an SNO cell line. The fermented toxin-spiked maize meal samples with a starter culture were comparatively less toxic (29 - 36%) to SNO oesophageal cells than samples spiked with toxin without a starter culture (24 - 30%). However, this observed difference was not statistically significant (p = 0.295 - 0.681). Furthermore, cells that were only inoculated with the cell culture medium had significantly (p = 0.000) high percentage cell viability. This study indicates that it is possible to significantly reduce the concentration of mycotoxins using lactic acid maize fermentation to trace levels. However, such a reduction will not significantly alter the possible chronic toxic effects of such toxins in the diet, particularly a maize based diet containing poor quality protein. The trace amounts of these toxins in fermented and unfermented maize meal should continue to be a cause for concern. / Thesis (M.Med.Sc.)-University of Natal, Durban, 2003.

Aflatoxin Bi (AFBi), a highly toxic fungal metabolite (mycotoxin) of certain strains of Aspergillus, has long been known to be carcinogenic in animal species. Accumulation of epidemiological evidence led to its classification, in 1993, by the International Agency for Research on Cancer as a Group I human carcinogen. Aflatoxin Bi contaminates the food supply in most tropical and sub-tropical countries, where it is associated with increased incidence of hepatocellular carcinoma (HCC). In these countries, AFBi is also linked to kwashiorkor, jaundice, and Rey's syndrome. The biological action of AFBi is through its oxidation to AFBi-8,9 epoxide (AFBiO). This epoxide binds to macromolecules like DNA, RNA and proteins as well as amino acids to form AFBi-macromolecular adducts. Quantitation of these adducts is thought to be the most promising approach in the development of methods to measure levels of exposure to aflatoxins. Aflatoxin Bi was produced, isolated and purified using preparative thin layer chromatography (TLC). The toxin was oxidised to AFBiO using dimethyldioxirane and the UV spectra of both the AFBi and AFBiO were determined. Reaction of selected Na-acetyl amino acids (AA) with AFBiO was studied and UV spectrophotometry, TLC, high performance liquid chromatography (FfPLC) and high performance capillary electrophoresis (CE) were used to characterise the reaction products. The epoxide was also reacted with albumin and DNA. Aflatoxin Bi-albumin reaction mixture was hydrolysed and characterised by TLC. Spectrum measurement of the oxidative product of AFBi gave peaks at 266 and 367nm. Qualitative TLC and the epoxide spray reagents confirmed that epoxidation was successful. The in vivo reaction of selected Na-acetyl AA with the epoxide gave peaks between 300 and 400 nm. Naacetyl-arginine, Na-acetyl-lysine and Na-acetyl-histidine showed reaction with AFBiO with maximum wavelengths at 392, 397 and 391 nm respectively. These results strongly suggest that AFBiO is able to covalently bind to lysine, histidine and arginine in albumin. A total of twenty nine blood samples were analysed by HPLC for the presence of AFBilysyl adduct. Of the twenty nine samples, ten were from HCC patients, ten from control patients and nine from kwashiorkor patients. The results show that AFBi-lysine does occur in patients at King Edward VIII Hospital (KEH) and the highest level was detected in HCC patients followed by kwashiorkor patients. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1998.

The main purpose of this study was to evaluate the amount of time which was spent in giving preventive pressure area care in both a sample of hospital patients and a sample of community patients. A pilot study was carried out to test the methodology, which was subsequently used with only minor modifications, for the main study. Bedfast or chairfast patients were studied from admission to the selected hospital wards or community nursing areas for a period of six weeks or until they were discharged from care, developed pressure sores, died, or became mobile. Data was collected by means of interviews and observations made of patients, nurses and relatives. A diary sheet was designed for use by nurses in hospital and by nurses and relatives in the community, on which they were asked to record pressure area care as it was given. Information collected by this means included the time spent in care, the method used and observation of the skin areas. The researcher also collected data about the patient's appetite, Norton Score, age, sex and diagnosis. The outcome measure used was whether or not the patient developed a pressure sore which was defined for this study as a break in the skin due to pressure. Due to geographical dispersion of patients within the community in the health district used for that part of the study, fewer community patients (n = 30) were included in the study than the number of hospital patients studied (n = 88). Discriminant analysis was used on the results to distinguish between groups of patients. Results of this study showed that a higher percentage (29%) of the hospital patients developed pressure sores than among the community patients studied (20%). The average total time spent on pressure area care daily was higher for the community patients than for the hospital patients. Interestingly, of the six community patients who developed pressure sores, five were dependent entirely upon the nursing service for pressure area care, whilst the usual pattern at home was that relatives and nurses shared the care. Frequency of pressure area care given showed a significant relationship with outcome for both hospital and community patients. It should be noted that whilst the number of patients who developed sores is reported here, and this is related to the total number of patients studied, this study is not an incidence or a prevalence study, and should not be considered as such. The study appears to show that nursing care devoted to the prevention of pressure sores in terms of time and frequency is significantly related to outcome and thus to effectiveness.