Medical Physiology
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- ItemAbnormalities of bone and mineral metabolism in patients with eating disorders(Stellenbosch : Stellenbosch University, 2001) Conradie, Maria Martha; Hough, F. S.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division Medical Physiology.ENGLISH ABSTRACT: Osteopenia is a well documented complication of anorexia nervosa (AN). The pathogenesis of this bone loss is presently poorly defined in the literature. Pathogenetic mechanisms that have been implicated include certain nutritional factors, exercise abuse, hypogonadism, hypercortisolism and/or vitamin 0 deficiency. We studied, 59 Caucasian eating disorder patients aged 15-45yr. The eating disorder was classified by a single, qualified psychiatrist according to OSM IV R criteria as either anorexia nervosa (AN: n =25), bulimia nervosa (BN: n = 17) or eating disorder not otherwise specified (EONOS: n = 17). All patients were subjected to a detailed dietary and general history. We assessed the prevalence and severity (OEXA), the nature (osteocalcin, deoxypyridinoline) and site (vertebral versus hip) of osteopenla in these patients. he role of nutritional factors (energy intake, weight, height, BMI, plasma albumin, lipids), physical activity, hypercortisolemia (plasma and urinary free cortisol), vitamin 0 deficiency (plasma 250HD) and hypogonadism (amenorrhoea, E2, LH, FSH) in the pathogenesis of bone loss were also evaluated. Mild osteopenia (BMO decreased by more than 1SO below age-matched controls) was documented in 46% of the total study population, with more marked osteopenia (Z-Score < -2 SO) present in 15%. Both vertebral and hip osteopenia were documented. In the study population those patients with AN (Lumbar BMO (q/cm") = 0.869 ± 0.121) were most likely to develop osteoporosis, although a significant percentage of patients with BN (Lumbar BMO (q/crn") = 0.975 ± 0.16) and EONOS (Lumbar BMO (g/cm2) = 0.936 ± 0.10) were also osteopenic (29% and 35% respectively). Twenty four percent (24%) of the total patient population had a history of fragility fractures. These fractures were reported more commonly amongst patients with AN and EONOS (28% and29.4%). Fracture prevalence was however similar in patients with normal and low bone mass. Conventional risk factors were similar in patients with normal and low bone mass, except for a significantly longer duration of amenorrhoea (p = 0.009), a lower BMI (p = 0.0001) and greater alcohol consumption (p = 0.05) in the osteopenic patients. Nutritional parameters (S-albumin, protein, Ca, and P04 intakes), physical activity, as well as 25(OH) vitamin D levels were similar in AN and BN subjects, as well as in patients with a low versus normal BMD. Plasma and urine cortisol levels were also similar in these subgroups. With the exception of two patients with borderline osteopenia, significant bone loss was only documented in those patients with a past or current history of amenorrhoea. In the total patient population the duration of amenorrhoea was significantly (p<0.009) longer in patients with osteopenia versus those with a normal bone mass. A significant negative correlation between BMD (Z-Score) and duration of amenorrhoea was also documented in the total patient population (r = -0.4, P = 0.001) as well as in all three eating disorder groups (AN r - -0.4, P = 0.03; BN r = - 0.6, P = 0.008; EDNOS r = -0.6, P = 0.005). In the total patient population, those patients with amenorrhoea, had lower BMD and BMI values and lower estrogen levels compared to those with a normal menstrual cycle. We conclude that osteopenia commonly attends AN, as well as BN and EDNOS. Nutritional (with the exception of alcohol consumption) and mechanical factors as well as hypercortisolemia did not appear to contribute significantly to bone loss in this study population. Hypogonadism appeared to be the main cause of the bone loss observed in these patients.
- ItemAcrosome size and kinematics of human spermatozoa(Stellenbosch : University of Stellenbosch, 2007-03) Murray, George M.; Du Plessis, S. S.; Franken, D. R.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.For spermatozoa to gain access to the oocyte for fertilization, lytic enzymes need to be released during the acrosome reaction. These enzymes, which are stored and transported within an organelle termed the acrosome, make it possible for spermatozoa to collectively penetrate the layers of cells and glycoproteins that surround and protect an oocyte. Acrosomes may thus be viewed as essential for fertilization and their shape, size and volume were examined morphometrically by utilizing automated morphometric analysis equipment. In addition to the acrosome being necessary for normal unassisted fertilization, spermatozoa also need the ability to migrate to the oocyte. Following zona pellucida binding, sperm tail thrust movement initiates zona penetration into the space created by the digestive action of the acrosomal enzymes. Therefore the motion characteristics of spermatozoa were also quantified in terms of kinematic properties. In the treatment of male sub fertility, assisted reproductive techniques are applied. In the application of such techniques, a motile sub-population of spermatozoa was obtained by employing a procedure (swim-up selection) that selects cells on the basis of their kinematic ability. This study presents an analysis of the morphometric and kinematic qualities of spermatozoa populations that are subjected to swim-up selection and investigates the relationship of these morphometrical and kinematic qualities. Computer-assisted semen analysis, swim-up selection and automated sperm morphology analysis tests were all used to evaluate spermatozoa populations. Results indicated that, irrespective of acrosome size, higher kinematic parameter measurements were observed post-swim-up. A significant inverse relationship between the population’s average acrosome size and a number of kinematic parameters was observed. Our results indicated that for a post-swim-up population of spermatozoa an increase in the average acrosome size was significantly related to a decrease in the kinematic parameters VAP, VCL and the VSL within the same population.
- ItemThe anti-diabetic and insulin sensitizing potential of a watery extract of Agathosma tested in rat models of type 1 and type 2 diabetes(Stellenbosch : Stellenbosch University, 2015-03) Jansen, Vereneque; Huisamen, Barbara; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.ENGLISH SUMMARY: Introduction: Epidemiological data highlights that South Africa is currently facing a quadruple burden of disease of which non-communicable diseases (NCDs) are estimated to account for 29% of all deaths. These NCDs include, amongst others, cardiovascular disease, type 2 diabetes, hypertension, cancer, chronic lung disease and depression. Diabetes has become a major problem worldwide and in South Africa and is currently rated as the 5th largest cause of death by Statistics South Africa in 2011. This creates an enormous burden of disease on populations and the utilization of herbal remedies has escalated in popularity because of this. Buchu water is one of these herbal remedies advertised as having anti-diabetic properties. This water is a by-product of the extraction of the oil from the leaves of Agathosma and is already freely available to the public. The aim of this study was therefore to use animal models of type 1 diabetes, and obesity and insulin resistance to scientifically verify or refute these claims. Methods: We utilized male Wistar rats. Two different rat models were used: (i) a type 1 diabetic model; induced via a once-off intraperitoneal Streptozotocin (40mg/kg) injection ablating ~50% of pancreatic beta cells (T1D); (ii) A diet-induced obese model, rendering rats insulin resistant after receiving a high caloric diet for 16 weeks. Half of each experimental group was treated with diluted Buchu water for a period of 14 weeks and 16 weeks, respectively, while the rest consumed normal water. Water and food consumption were monitored, body weight and intraperitoneal fat measured, blood was collected to determine serum glucose and insulin levels, skeletal muscle was removed to test insulin sensitivity using radiolabelled deoxyglucose, pancreas and skeletal muscle harvested and stored in liquid nitrogen for further biochemical analysis. Results: One of the main findings of this study was that ingestion of Buchu water results in weight loss despite no decrease in food consumption. This occurred in both the pathological models and control animals. In the obese animals, this weight loss was due to a decrease in intra-peritoneal fat. A second important finding was that the ingestion of Buchu water in all instances, whether given as treatment (treated with Buchu water 3 weeks after the start of the experiment) or as prophylactic (treated with Buchu water from the start of the start of the experiment), resulted in normalization of glucose levels in a type-1 diabetic model with residual beta-cell mass. An insulin-sensitizing effect was not clearly established in skeletal muscle but this may be because of a large variation in values obtained, as well as the use of a slow-twitch muscle. A definite effect on pancreatic insulin secretion has been demonstrated by raised C-peptide levels in the diet-induced obese model. Conclusion: This study, with regard to the type 1 diabetic model, has confirmed the anti-diabetic effect of Buchu water by significantly lowering blood glucose levels of fasted and non-fasted blood and normalizing Intraperitoneal glucose tolerance test (IPGTT) curves. This was however not so evident in the obese model utilized. Despite this, animals lost weight which was mainly intra-peritoneal fat.
- ItemAntioxidant supplementation as protective strategy against stem cell impairment in Type 2 Diabetes(Stellenbosch : Stellenbosch University, 2021-03) Maartens, Michelle; Van de Vyver, Mari; Marais, Erna; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences.ENGLISH ABSTRACT: Type 2 Diabetes Mellitus (T2DM) is a global epidemic. It is a complex disorder that leads to cellular dysfunction and the development of co-morbidities. The underlying pathologic microenvironment in T2DM, associated with hyperglycaemia, include the accumulation of advanced glycation end products, excessive oxidative stress, and chronic inflammation. Bone marrow mesenchymal stem cells (MSC) are especially susceptible to this damaging microenvironment and as consequence the endogenous repair mechanisms within the body fail giving rise to secondary complications such as non-healing wounds, retinopathy, and neuropathy. There is thus a need for a holistic approach when it comes to disease management in which anti-diabetic drugs focussed on glucose control is complemented by supplementary treatment aimed at restoring homeostasis. Natural and synthetic antioxidants such as Ascorbic acid-2-phosphate (AAP) and N-acetyl-l-cysteine (NAC) are known to protect cells against oxidative stress-induced damage in vitro and have been shown to reduce inflammation in various models. The efficacy of these antioxidants to restore homeostasis and prevent cellular dysfunction in T2DM is however still unknown. The aim of this study was to investigate the protective effects of combined NAC and AAP supplementation against MSCs impairment using an animal model of obese diabetic (B6.C-Lepob/J) (ob/ob) (n=14) and wild type control (C57BL6/J) (n=20) mice. All mice received jelly cubes containing either antioxidants (NAC7.5mM+AAP0.6mM) or placebo (vehicle control) for a period of 6-weeks. Metabolic parameters (weight and blood glucose) were assessed on a weekly basis and the overall antioxidant status of animals assessed at the end of the 6-week period by analysing the total antioxidant capacity (TAC) and Malondialdehyde (MDA) levels in serum. Bone marrow MSCs were isolated from mice in each of the respective treatment groups and their ex vivo growth rate, viability, multi-lineage differentiation capacity and paracrine responsiveness upon stimulation with wound fluid assessed. Compared to the wild type (C) mice, excessive weight gain (weight: C 28.7±1.6 g; DM 44.3±3.5 g) (p<0.05) and hyperglycaemia (blood glucose: C 10.1±1.3 mmol/L; DM 23.6±5.7 mmol/L) (p<0.05) was evident in the DM animals validating the animal model as representative of T2DM. The antioxidant supplementation did not affect metabolic parameters indicating that differences observed between supplement and placebo treated mice were not due to weight loss or changes in glucose metabolism. NAC/AAP significantly (p<0.05) reduced lipid peroxidation in DM animals (DM:P 39.0±14.7 nmol/L; DM:S 21.5±11.6 nmol/L) to a level comparable to that of controls (C:P 22.9±11.4 nmol/L; C:S 30.5±3.3 nmol/L) and increased the overall TAC (C:P 3.7±1.3 U/mL; C:S 4.0±0.7 U/mL; DM:P 5.2±0.9 U/mL; DM:S 5.8±1.5 U/mL). The ex vivo growth rate of cells derived from DM animals were impaired (Time to confluence: C 8 days; DM 12 days). NAC/AAP supplementation did however improve the growth rate and viability of MSCs derived from both animal models. NAC/AAP furthermore reduced the adipogenic differentiation capacity of MSCs but could not restore osteogenesis in DM MSCs. Upon stimulation with wound fluid, slightly increased IL6 (DM:S+WF 1583.3±481.9 pg/mL) and IL10 (DM:S+WF 27.1±9.8 pg/mL) release was evident in MSCs derived from NAC/AAP supplemented animals. In conclusion, NAC/AAP supplementation was able to reduce oxidative stress in animals and improved MSCs viability.
- ItemATM Expression in peripheral blood mononuclear cells as a biomarker of insulin resistance(Stellenbosch : Stellenbosch University, 2019-04) Williams, Lois Esther; Huisamen, Barbara; Stellenbosch University. Faculty of Engineering. Dept. of Biomedical Sciences: Medical Physiology.Introduction: The Ataxia Telangiectasia Mutated (ATM) gene codes for the 350 kDa ATM protein kinase. ATM gene mutations cause inactivity/deficiency of the ATM protein, resulting in the autosomal recessive disease Ataxia Telangiectasia (AT). AT patients are predisposed to developing insulin resistance or further progress to type 2 diabetes and are at high risk of developing ischaemic heart disease. Due to the prevalence of insulin resistance in AT patients, we investigated the relationship between ATM protein levels and the degree of insulin resistance and proposed it as a possible early diagnostic technique for insulin resistance. Aim: To determine whether peripheral blood mononuclear cells (PBMCs) can be used to determine ATM levels in insulin resistant subjects and subsequently used as a biomarker of insulin resistance. Objectives: (i) To standardise a protocol for the isolation of PBMCs from rat blood. (ii) To isolate rat PBMCs and determine the ATM levels using Western blotting. (iii) To determine differences between ATM levels in obese rats and compare them to controls. (iv) To analyse PBMCs from a female Black Xhosa population with different degrees of insulin resistance and to determine a relationship with ATM levels. Methods: Male Wistar rats were fed an obesogenic diet (od) for 16 weeks to induce obesity and insulin resistance and compared to age-matched and young controls fed standard rat chow. Body weight and intraperitoneal (IP) fat mass were determined and oral glucose tolerance test (OGTT) was performed. PBMCs were isolated according to the standardised protocol and Western blotted for ATM and P22phox. The Western blotting protocol was repeated with samples collected from patients. Results from the animal model: 1. Effects of obesity/insulin resistance vs. age-matched controls: (i) larger IP fat mass; (ii) increased area under the curve of OGTT’s; (iii) elevated basal glucose levels. (iv) The phospho-(P)/total-(T) ATM ratio was decreased. 2.Effects of age: (i) As expected, older animals weighed more while T-ATM was decreased, P-ATMincreased and the P-/T-ATM ratio increased with age. In PBMC’s from patients, the following were observed: (i) Body mass index (BMI) was significantly higher in obese and pre-diabetic vs. control patients. (ii) The waist-to-hip ratio (WHR) of obese and pre-diabetic women was higher vs. controls. (iii) Trunk-to-limb fat mass (TF/LF) was increased in obese and pre-diabetics vs. controls but (iv) no differences in the lipid profiles were observed except for increased triglyceride levels between young pre-diabetic patients vs. their controls. (v) Fasting blood glucose of obese and pre-diabetic patients was significantly increased vs. controls. (vi) Significantly higher P-ATM levels were seen for obese and pre-diabetic vs. control patients. T-ATM levels increased with the state of insulin resistance. Effects of age: (i) BMI was significantly higher between young (Y) and middle aged (MA) control, obese and pre-diabetic groups vs. their respective controls while (ii) WHR of Y obese and Y pre-diabetic vs. Y controls also increased significantly. (iii) The TF/LF ratio was increased between Y and MA control and Y obese, Y pre-diabetic, MA obese and MA pre-diabetic women vs. their respective controls. Furthermore (iv) the blood glucose levels of Y pre-diabetics were increased vs. Y control. (v) The P-ATM levels was increased in Y pre-diabetic vs. Y control and therefore did not increase with age but the T-ATM levels significantly increased with age. Conclusion: ATM levels can be measured in PBMCs and are affected by the insulin resistant state and age. Unfortunately, due to the variation in ATM levels under different degrees of insulin resistance, it would be difficult to use ATM as a biomarker of insulin resistance.
- ItemBasic semen parameters assisted by Computer-Aided sperm analysis (CASA) and their correlations with advanced semen parameters in normozoospermic men with different abstinence periods(Stellenbosch : Stellenbosch University, 2018-03) Ayad, Bashir Mohamed; Du Plessis, Stefan S.; Van der Horst, Gerhard; Stellenbonsch university. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences : Medical PhysiologyIntroduction: Affordable basic semen analysis remains a fundamental procedure to be performed routinely during the diagnosis of male infertility. Advanced semen analyses, provide valuable clinical insights in treatment related decision-making, but these are highly expensive and lack universal standardization. The World Health Organization (WHO) guidelines for semen analysis have been adopted by most human andrology and fertility laboratories around the world for more than thirty years. According to the most recent prescribed guidelines of the WHO, subjects must remain abstinent for a minimum period of two days, but not longer than seven days before collecting a sample for a standard semen analysis. Several studies have sought to determine the optimal period for ejaculatory abstinence. However, the results are often found to be contradictory. The aims of this study are two-fold: Aim I: To investigate the effect of short (4 hours) and long (4 days) abstinence periods on sperm quality based on functional and biochemical parameters in a population of normozoospermic men, in addition to the prediction of various basic and advanced semen parameters of the second (4 hours) ejaculate from a set of basic parameters obtained from the first (4 days) ejaculate. Aim II: Establishing a correlation between basic semen parameters assisted by Computer-aided sperm analysis (CASA) and a set of advanced semen analysis tests. To determine cut-off values for advanced semen parameters from various basic parameters based on WHO defined reference values. Methods: Semen samples were collected from one hundred potentially fertile, normozoospermic men (20 to 30 years) who abstained for a period of exactly 4 days and 4 hours prior to collection of the first and second ejaculates respectively. Semen samples were analysed according to the WHO guidelines. Sperm concentration, total sperm count (T.S.C.), total and progressive motility and kinematic/velocity parameters were analysed by CASA. Sperm viability was performed by dye exclusion and morphology via SpermBlueTM staining techniques using Computer Aided Sperm Morphology Analysis (CASMA). Sperm acrosome status was evealuated by fluorescence microscopy. Sperm DNA fragmentation and intracellular superoxide (O2−•) levels were assessed by flow cytometry. Seminal antioxidant status [superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS)] were measured by means of spectrophotometry. Statistical comparisons between short and long abstinence periods were performed using paired Student’s t-tests on GraphPad Prism™ software, while the prediction of various basic and advanced semen parameters of the second ejaculate from a set of basic semen parameters of the first ejaculate was performed using linear regression models. Correlations were performed using Spearman rank correlation coefficients, while receiver operating characteristic (ROC) curves were used to determine cut-off values. Statistical significance was set at p<0.05. Results I: A significant increase in total and progressive motility as well as in the velocity parameters were observed after short (4 hours) abstinence compared to long (4 days) abstinence periods. DNA fragmentation and intracellular O2−• levels were not significantly different between short and long abstinence periods. Despite the observed decrease in semen volume, sperm concentration and T.S.C. after the short abstinence period, all mean values of the conventional semen parameters still remained above the lower reference limits as recommended by the WHO 5th edition. We were also able to make predictions of various basic (semen volume, sperm concentration, total motility, progressive motility, viability and normal morphology) and advanced (DNA fragmentation, seminal plasma CAT activity and TBARS) parameters of the second ejaculate from a set of basic semen parameters obtained from the first ejaculate with relative certainty. Results II: The proportions of total and progressively motile as well as rapid spermatozoa were positively correlated with CAT activity (p<0.05). A significant negative correlation was observed between VCL, VSL, VAP and both intracellular O2−• and TBARS levels. ALH was significantly and negatively correlated with intracellular O2−• levels and DNA fragmentation, while its correlation with SOD activity was positive (p < 0.05). A negative correlation was also found between the percentage of viable spermatozoa and both O2−• levels and DNA fragmentation, whereas the percentage of normal morphology was negatively correlated with O2−• levels and positively with CAT activity (p < 0.05). The optimal intracellular O2−• cut-off value to differentiate between asthenozoospermic and normozoospermic men was calculated to be 227 median DHE fluorescence intensity [MFI] (p < 0.01). At this cut-off value, the test was 80% sensitive and 86% specific. Sperm viability was associated with a seminal plasma TBARS cut-off value of 9.86 Umol/L (p = 0.02) with sensitivity and specificity of 81% and 80% respectively. Conclusion: Our data challenges the generally accepted guidelines regarding the prescribed prolonged abstinence periods since the results show that 4 hours of sexual abstinence yielded significantly better samples from a sperm functional point of view. The results obtained from this study further support the validity of some CASA parameters as sensitive indicators of changes in sperm oxidative status and DNA integrity. This study also enabled defining the cut-off values and prediction of certain advanced variables from the basic semen analysis.
- ItemCardio-metabolic risk profile of people living with HIV: Is retinal microvascular geometric morphology a marker of effect?(Stellenbosch : Stellenbosch University, 2022-04) Kgokane, Boipelo Mirriam Lepetjia; Strijdom, Hans; Everson, Frans; Kamau, Festus; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.Background and Aim Cardiovascular disease in people living with HIV has become of great concern. HI-viral factors, ART-toxicity and HIV/ART-associated cardiometabolic adverse effects have been implicated in the development of cardiovascular disease. Retinal microvascular geometric features may be potential useful markers of these effects. We aimed to investigate whether altered retinal microvascular geometric features are markers of HIV, ART and/or HIV/ART-associated cardiometabolic effects in a study population from the Western Cape Province. Methods The study followed a cross-sectional (HIV-free: n = 88 and HIV+ART: n = 122) and longitudinal (baseline vs. 18-month follow-up for HIV+ART only: n = 82) study design. Volunteering participants were recruited from health care clinics. Demographic, lifestyle, socioeconomic and anthropometric data were collected. Fasting blood and urine samples were collected and transported to the National Health Laboratory Services for biochemical analyses. Retinal images were obtained (Canon CR-2 camera) and vessel features quantified (MONA REVA 2.1.1 software). Linear stepwise regression (cross-sectional) and linear mixed model (longitudinal) analyses were applied to elucidate independent associations and statistical significance of p < 0.05. Results Population characteristics: The study population was relatively young (HIV-free:44.06±11.09 and HIV+ART:40.35±8.94years) and mostly women (HIV-free:80.7% and HIV+ART:63.1%). The baseline median/mean viral load (VL), CD4 cell count and ART-duration were 50 (10 to 675032) copies mRNA/mL, 539.92±237.16 cells/mm3 and 166 (1 to 707) weeks respectively. Cardiometabolic results: Body mass index (BMI) (24.50±6.65 vs. 28.25±7.68kg/m2, p < 0.001) was significantly lower in HIV+ART vs. HIV-free. ∆BMI in HIV+ART was significantly correlated with average arterial tree diameter (r = 0.323, p < 0.05), total length of skeletonised tree (r = 0.355, p < 0.01) and arteriolar branching angle (r = 0.234, p < 0.05). High density lipoprotein-cholesterol (1.59±0.74 vs. 1.39±0.45mmol/L, p = 0.019) and gamma-glutamyl transferase (GGT) (43.5 (14 to 494) vs. 27.0 (11 to 814)U/L, p < 0.001) were significantly higher in HIV+ART vs. HIV-free, but decreased in HIV+ART (Baseline vs. Follow-up HDL:1.62±0.77 vs. 1.44±0.64mmol/L, p = 0.017 and GGT:45 (14 to 494) vs. 41.50 (14 to 219)U/L, p = 0.004). HDL was significantly correlated with central retinal venular equivalent (CRVE) (r =-0.195, p < 0.01) and GGT with venular branching optimality (r = 0.180, p < 0.05). HIV and ART results: Cross-sectionally, HIV+ART status independently associated with CRVE (-0.146 (- 0.280 to -0.012), p = 0.033) and arteriolar and venular mother branch (D0), first daughter branch (D1) and second daughter branch (D2) (p < 0.05, respectively). VL (-0.198 (-0.025 to -0.001), p = 0.037) and ART- duration (0.188 (0.001 to 0.024), p = 0.047) were independently associated with arteriolar-venular ratio (AVR). Longitudinally, VL independently associated with CRVE (0.096 (0.017 to 0.175), p = 0.018) and AVR (-0.003 (-0.0006 to 0.000003), p = 0.046). CD4 cell count was independently associated with number of branchpoints 0.042 (-0.002 to 0.086), p=0.006) and endpoints (3.0 (0.750 to 5.250), p=0.010). HIV duration independently associated with lacunarity (-0.0080 (-0.0150 to -0.0010), p=0.036) and fractal analyses (0.011 (0.0001 to 0.021), p=0.045). 2nd-line ART was independently associated with CRVE (8.58 (0.35 to 16.81), p=0.041) and ART-duration with fractal analysis (-0.022 (-0.037 to -0.008), p=0.003). Discussion and conclusion HIV+ART appeared to have a more favourable cardiovascular risk profile vs. HIV-free. Various markers of HIV/ART and HIV-ART-associated cardiometabolic risk factors were associated with retinal vessel features and associations appeared mostly favourable/cardioprotective. These results indicate that retinal vessel geometric features may be potential markers of the effects of HIV/ART and/or associated cardiometabolic risk factors in the current study population.
- ItemChanges in hyo-laryngeal elevation post-pharyngeal electrical stimulation(Stellenbosch : Stellenbosch University, 2015-04) Basson, Tobias Johannes; Pillay, Mershen; Du Plessis, Stefan S.; Stellenbosch University. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.ENGLISH ABSTRACT: Swallowing disorders are prevalent in many elderly individuals and are common amongst individuals suffering from neurological diseases. These individuals are affected from slight swallowing difficulty to total swallowing inability. In severe cases this may cause aspiration pneumonia, dehydration, malnutrition and ultimately death. Swallowing disorders can be diagnosed and treated to increase quality of life. New treatment strategies to understand the pathophysiology and impaired swallowing response are needed. Neuromuscular electrical stimulation is used as rehabilitation method in various disciplines. This method of rehabilitation of physiological dysfunction is used in treating swallowing disorders and has become a focus for current research. To understand the effect of electrical stimulation to the swallowing centre it is proposed to study its mechanism on normal swallowing musculature. The outcome of the effect that electrical stimulation has on healthy individuals may possibly be used to extrapolate to clinical settings and its benefit for modern dysphagia rehabilitation. The purpose of this study was to report on the hyo-laryngeal movement pattern of young healthy, male and female, individuals and to measure the effect of a single neuromuscular electrical stimulation session on the hyo-laryngeal complex of 22 young healthy individuals. Lastly, the aim was to determine the detraining or lasting effect on the hyo-laryngeal swallowing complex of a single neuromuscular electrical stimulation session. The study reported on baseline hyo-laryngeal complex movement patterns by measuring the anterior movement and elevation of the hyo-laryngeal complex through the use of videofluoroscopy swallow study. Analysis of these measurements where done to report on the effect of electrical stimulation on the hyo-laryngeal complex movement pattern pre- and post- electrical stimulation. Significant changes were revealed with elevation of the hyo-laryngeal complex, however no significant effects could be found with anterior movement of the hyo-laryngeal complex pre- and post- electrical stimulation. It was found that elevation of the hyo-laryngeal complex lowered after a single electrical stimulation session. The hyo-laryngeal complex movement pattern remained similar between genders. Lastly it was found that a single electrical stimulation session showed significant reversibility towards baseline levels. This might be related to muscle fatigue and one would need to take into account muscle recovery for future research.
- ItemCharacterisation of high fat, high sugar diet-induced epigenetic changes in skeletal muscle of wistar rats and metabolic effects of an aspalathin-rich rooibos extract(Stellenbosch : Stellenbosch University, 2021-12) Myataza, Asive; Carmen, Pheiffer; Tarryn, Willmer; Shantal, Windvogel; Rabia, Johnson; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.Background Animal models are widely used to elucidate the pathophysiological mechanisms that underlie obesity and to test the efficacy of anti-obesity therapeutics. However, these models have been largely biased towards males, partly due to the complexity of hormonal fluctuations in females. The primary aim of this study was to elucidate DNA methylation profiles and gene regulatory networks that are altered in the skeletal muscle during the development of obesity in female and male Wistar rats, and to explore whether Afriplex-GRT™ could prevent aberrant DNA methylation patterns and the progression of metabolic disease. Methods Different animal models were employed where female and/or male Wistar rats were fed a standard or a high fat, high sugar (HFHS) diet for 12, 3 or 9 months. The effect of rooibos was investigated in the latter model, where 60 mg/kg of bodyweight Afriplex-GRTTM was co-administered with the diets. Parameters measured included food and water intake, bodyweight, and glucose, insulin, lipid and cytokine concentrations. Skeletal muscle was harvested for histology, gene expression measured using RT2 Profiler™ PCR arrays and Taqman® assays and global and gene-specific DNA methylation were quantified using pyrosequencing. To further explore the effects of DNA methylation, high glucose and fatty acids on skeletal muscle, C2C12 myocytes were differentiated with 7 μM 5-azacytidine, a global DNA methylation inhibitor, 33 mM glucose and 0.5 mM palmitate. Mitochondrial activity and oxidative stress were measured using appropriate assays. Myoblast differentiation and the expression of myoblast determination protein 1 (MyoD), myosin heavy chain 1 (Myh1), insulin growth factor 2 (Igf2), sterol regulatory element binding transcription factor 1 (Srebf1) and DNA methyltransferase 1 (Dnmt1) were assessed. Results The HFHS diet induced visceral adiposity and hypertriglyceridaemia in both male and female rats, while hyperinsulinaemia and significant bodyweight gain was observed in male rats, and systemic inflammation in females only. These changes were accompanied by increased expression of Igf2 and decreased expression of Srebfb1 and Dnmt1 in skeletal muscle of male, but not female rats. No differences in DNA methylation patterns were observed. Treatment with Afriplex-GRT™ did not ameliorate HFHS diet-induced metabolic dysregulation. In C2C12 cells, treatment with 5-azacytidine induced myoblast differentiation and MyoD and Myh1 expression, while palmitate inhibited differentiation and decreased the expression of MyoD, Myh1, Igf2 and Srebf1, which was restored by 5-azacytidine. High glucose increased Igf2 expression but did not affect Srebf1 expression, while a combination of high glucose and 5- azacytidine decreased Srebf1 and Dnmt1 expression. Conclusion The HFHS diet induced different metabolic responses and gene expression patterns in females and males. In general, females exhibited a dampened metabolic response, which we presume may be due to the intrinsic protective effects of female reproductive hormones. Afriplex-GRTTM did not prevent HFHS diet-induced weight gain, which contrasts previous findings where treatment with 60 mg/kg Afriplex-GRTTM decreased bodyweight in obese male rats, suggesting that Afriplex-GRTTM may be better targeted as a therapeutic than a preventative nutraceutical. This study provides novel information on how molecular differences in skeletal muscle may contribute to sex differences in response to HFHS feeding and may have important implications for the identification of therapeutic targets.
- ItemCharacterising pathways that contribute to post-TB lung disease(Stellenbosch : Stellenbosch University, 2024-02) Jacobs, Steve; Maarman, Gerald; Windvogel, Shantal; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.ENGLISH ABSTRACT: Tuberculosis (TB) is a major global health challenge, especially in low- and middle-income countries (LMICs). Post-tuberculosis lung disease (PTLD) is a common and debilitating sequela of TB, which can lead to chronic respiratory symptoms and impaired lung function. The pathogenesis of PTLD is not well understood, and much remains to be discovered, although some other authors have hypothesized that inflammation may be a key contribution, despite successful completion of TB treatment. Moreover, accumulating data suggests that PTLD might also include pulmonary hypertension (PH). The latter is a multi-organ disease and highly morbid clinical condition, with a broad range of clinical presentations and is caused by a large spectrum of underlying conditions. The relationship between PTLD and PH is complex and multifactorial, involving host, environmental, and pathogenic factors. Given the inflammatory nature of TB, it is likely that pro-inflammation may contribute to the development of PH post-TB, however, there is currently no data on this topic. This is concerning, as millions of people live with TB, close to 60 000 people die of TB in South Africa per annum, while every year almost 600 new cases of TB are reported. Given this context, it is worrying that TB patients (whether previous or current, or treated) are at risk of developing debilitating PTLD and fatal PH. This project, aimed to delineate the involvement of pathwaysthat may contribute to the development of PH in a post-TB context, and to explore the pathways that specifically contribute to PH in the same context. In our pursuit, we managed to highlight the instrumental roles of inflammatory pathways as part of PTLD pathogenesis. Our novel findings suggest that there is a pro-inflammatory state in active TB patients on treatment that persists post-TB, regardless of being successfully treated for TB. The unusual circulation pattern of inflammatory cytokines could be ascribed to mitochondrial dysfunction in immune cells. Considering the destructive nature of these cytokines, there is a need for further research to explore the implications of a persistent pro-inflammatory state, as it may predispose patients to PTLD. In terms of PH, we explored myriad pathways that may cause PH, now a new key feature of PTLD. Our review of the literature demonstrated a link between melatonin and PH as part of PTLD. Our findings demonstrate that melatonin is an important link between the gut microbiota and the development of PH (where suppressed melatonin-crosstalk between the gut and lungs could promote the development of PH). More studies are needed to investigate the link between the gut microbiota, melatonin and PH. Studies could also investigate whether microbiota genes play a role in the epigenetic aspects of PH. This is relevant because, e.g., dysbiosis (caused by epigenetic factors) could reduce melatonin signalling between the gut and lungs, reduce subcellular melatonin concentrations in the gut/lungs, or reduce melatonin serum levels secondary to epigenetic factors. PH is a fatal disease, and this essentially means that despite successful TB treatment thousands of patients are at risk of developing PH. Yet, there is no cure for PH and most developing countries do not have specialised PH drugs. Therefore, there is a need for research on better treatments for PH, particularly, in the post-TB context. We explored the potential of the repurposing of drugs for the treatment of PH especially in countries with resource limitations. Studies have demonstrated the benefits of medicinal plants against PH, most of which are of Indian or Asian descent. Africa is a rich source of multiple medicinal plants scientifically proven to counteract myriad pathologies. When perusing these studies one can notice that African medicinal plants afford biological effects that counteract the same molecular pathways (e.g., proliferation, vasoconstriction, inflammation, oxidative stress, and mitochondrial dysfunction) also involved in the pathogenesis of PH. Viable options include Aspalathus linearis, Allium sativium, Trifolium pratense L, Mimosa pigra L, and Aloe ferox. However, most of these plants have never been tested in an experimental PH model, and https://scholar.sun.ac.za 4 therefore, our proposition is hypothetical at the most. Regardless, we believe that future studies should investigate these and other African medicinal plants in appropriate models of PH, to test their efficacy and effectiveness. The relationship between PTLD and PH is complex and still requires several puzzles to be placed to fully understand it. The pathophysiology of PTLD that leads to PH, epidemiological factors and potential treatment options remains largely unexplored and are key areas for future research. Research into alternative and novel therapies is especially crucial as this has the potential to improve patient’s quality of life and clinical outcomes. Ultimately, further research will hopefully pave the way for the development of a comprehensive approach to PH prevention, detection and diagnosis, and treatment strategies.
- ItemCharacterization of a sonified peak flow monitor(Stellenbosch : University of Stellenbosch, 2000-03) Vermeulen, M. O.; Wessels, J. A.; Von Backstrom, T. W.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences.ENGLISH ABSTRACT: The Whistle Watch™, an innovative and commercialised peak flow monitor, inspired this study, with its abnormal and complex measuring behaviour. The Whistle Watch™ latter is an audible peak flow monitor with a threshold-activated whistle as the essential component. The whistle is calibrated for a certain flow, and then encased in a body with a variable exhaust valve to atmosphere. Using the Whistle Watch™, with the exhaust valve pre-set, executing a forced expiratory effort, the audible notification of the whistle would indicate a stable asthmatic condition at that setting. No audible notification would result in the use of medication as a preventative measure. Due to the absence of existing theories and literature on the mechanics of whistles, the Whistle Watch™ was empirically developed. This study therefore, focuses on the characterisation and consequent improved understanding ofthe mechanics ofa whistle, with the objective to monitor pulmonary function in a novel way. During this study, a novel technique was developed to determine the reed activation point, or onset of oscillation, in terms of pressure. This technique was then implemented throughout the study. The initial observation and experimentation underlined the whistle's activation sensitivity towards any irregularities of the reed surface. A statistical spread of reed activation pressures defined the reed's inherent non-linear properties. A high dependence of reed activation towards upstream geometry was noted, and a clarification hypothesis was formulated. The effect of reed dimensions on activation pressure was exposed as a complex unexplored field. Existing mathematical reed theories only accommodate steady state oscillations, whereas the completed study indicated a high sensitivity of the reed activation pressure towards different input envelopes. This sensitivity was encapsulated in a mathematical model, with initial support and proofprovided by a previous independent study. All the observed effects and phenomena had far reaching practical application towards the production and quality control ofthe Whistle Watch™.
- ItemChronic stress and semen parameters(Stellenbosch : Stellenbosch University, 2020-03) Van der Merwe, Esmari; Du Plessis, Stefan; Basson, E.; Stellenbosch University. Faculty of Health and Medical Sciences. Dept. of Biomedical Sciences: Medical Physiology.ENGLISH ABSTRACT: It has been well documented that stress has adverse effects on the body and can lead to various health issues. Stress has been investigated as a cause for unexplained infertility in both men and women. Semen quality is a key indicator of male reproductive health. Numerous studies have been done on the effect of stress on semen parameters and an association between chronic psychological stress and poor semen parameters have been reported. Managing psychological stress can help to improve the health of an individual. In order to address the problem it is therefore important to determine if an individual experience high levels of stress. This can be established through psychological questionnaires and various biomarkers, such as the screening test for time urgency perfectionism (TUP) and alpha-amylase in saliva. In general, more or less 84% of couples are estimated to conceive naturally within a year. The remaining 16% of couples are affected by infertility. Within this group, it is estimated that male reproductive factors are the sole cause of one-third of cases and a contributing factor in another 20% of cases. Management of chronic stress in female patients has shown improved IVF rate of 67% or higher. However, as of yet no study has been performed on males to correlate the levels of TUP-stress, alpha-amylase to semen parameters as well as other seminal stress markers such as DNA fragmentation and oxidative stress (ROS). This study compared TUP-categories (Low, Moderate, High) with respect to semen parameters, alpha-amylase levels, age and BMI and investigated if increased alpha-amylase levels correlate with semen parameters, age and BMI. The experiments were performed at Medfem Fertility Clinic in Bryanston Johannesburg and the Division of Medical Physiology in the Department of Biomedical Sciences at Stellenbosch University. A total of 62 male patients of Medfem Fertility Clinic adhering to the basic requirements enrolled in the study. Results showed no significant difference between age, semen parameters and alpha-amylase between TUP categories. Men in the High TUP category had a significant higher BMI compared to those in the Low and Moderate categories. No significant correlation was found between alpha-amylase, age, BMI and semen parameters. This study was unsuccessful in proving a significant relationship between the TUP categories, age and semen parameters. The High TUP category did show a significantly higher BMI compared to the Low and Moderate TUP groups. This finding confirms that there is a link between psychological stress and elevated BMI. Although there was no significant difference between the TUP categories with regards to sORP values, the Moderate and High categories were both higher than the normal value for sORP in semen. This implies that chronic stress leads to elevated levels of oxidative stress in semen. No relationship was found between TUP categories and alpha-amylase levels. Although both are used to detect chronic stress, the TUP questionnaire is used to detect personality types who are prone to chronic stress, whilst salivary alpha-amylase is a biomarker for chronic stress and functions in a completely different way. It is possible that whilst both can be used to detect chronic stress it is not advised to attempt to establish a relationship between the two as the mechanisms of both are very different.
- ItemA comparison of motility and head morphology of sperm using different semen processing methods and three different staining techniques(Stellenbosch : University of Stellenbosch, 2010-12) McAlister, Debra Ann; Du Plessis, Stefan; Van der Horst, Gerhard; Maree, Liana; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.ENGLISH ABSTRACT: Sperm morphology remains an important parameter in the prediction of fertility, both in vivo and in vitro. However, there remains a considerable level of concern surrounding the true potential of this parameter due to the lack of standardization of differential staining techniques used for the evaluation of sperm morphology. This study aimed at investigating two commonly used staining techniques, Rapidiff® (RD) and Papanicolaou (PAP), along with a new commercially available stain, SpermBlue® (SB), in the evaluation of sperm morphometry and morphology. Results indicated that significant differences in sperm morphometry exist due to the use of the staining techniques. Findings further indicated that RD causes sperm head swelling while PAP causes sperm head shrinkage. Results obtained using the SB staining technique have indicated measurements closest to that which would be obtained through the evaluation of fresh, unstained sperm. The lack of standardization and the different effects various stains have on sperm structure and overall sperm morphology evaluation should raise a level of concern, particularly when evaluating patients with borderline morphology. Based on this, the use of the SB staining technique is recommended over RD and PAP for effective and accurate morphology evaluation. In further support of this technique, SB was shown to be quick and simple in method, and allowed for the easy detection of sperm by computer aided sperm analysis (CASA) systems such as the Sperm Class Analyzer (SCA®). The second aim of this study was to examine the concentration, morphology and motility of the resultant sperm populations following semen preparation using the PureSperm® density gradient and swim-up techniques. Semen preparation is an essential step in any fertility treatment protocol, and it is important that the sperm obtained following semen preparation has sperm morphology and motility characteristics capable of improving assisted fertility success rates. Currently, the PureSperm® density gradient and sperm swim-up are the most widely employed techniques in fertility clinics. Although there is sufficient evidence to suggest they are each effective at extracting sperm with improved quality from neat semen, there remains insufficient evidence to suggest which of these two techniques is superior. The present investigation revealed that both sperm preparation methods were effective at improving sperm morphology and motility, however to varying degrees. The swimup method yielded a population of sperm with superior motility and morphology when assessed according to World Health Organisation (WHO) criteria, while the PureSperm® density gradient technique isolated a higher percentage of normal sperm, according to both WHO and Tygerberg strict criteria, with motility better than that of neat semen. Although results obtained via the swim-up method suggest it would be best for use in in vitro fertilization (IVF), the very low concentration of sperm isolated via this method remains a significant draw-back. The PureSperm® density gradient separation technique on the other hand is capable of isolating larger quantities of sperm, which is likely to be of more benefit with fertility treatments requiring larger quantities of sperm. Based on these findings, the use of PureSperm® density gradient technique is recommended, due to its ability to isolate large quantities of good quality sperm. However, a swim-up may still be of use when performing fertility treatment using a sperm sample which possesses a high concentration and motility.
- ItemCompounds specific to Aspalathus linearis protects the diabetic heart against oxidative stress: a mechanistic study(Stellenbosch : Stellenbosch University, 2016-12) Dludla, Phiwayinkosi Vusi; Johnson, Rabia; Huisamen, Barbara; Essop, M. Faadiel; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.ENGLISH ABSTRACT : In diabetics, hyperglycemia, hyperlipidemia and inflammation potentiates the development of cardiovascular diseases (CVDs). These conditions provoke excessive generation of oxidative stress that has been implicated in the pathogenesis of diabetic cardiomyopathy (DCM). In the diabetic state, excessive generation of free radicals can cause oxidative damage to DNA and alter protein and lipids, leading to the activation of various death-induced signaling pathways. Activation of these death pathways result in structural and functional modifications to the myocardium. Current diabetic drug therapies do not protect the diabetic heart at risk from developing cardiovascular complications. Thus, in search for new therapeutics, we aim to unravel the molecular mechanisms associated with the protective effect of two major bioactive compounds from Aspalathus linearis, phenyl pyruvic acid-2-O-β-D-glucoside (PPAG) and aspalathin against hyperglycemia-induced oxidative stress and apoptosis in H9c2 cardiomyocytes. The study showed that PPAG and aspalathin were able to decrease mitochondrial membrane depolarization and prevent hyperglycemia-induced myocardial apoptosis by increasing the Bcl2/Bax ratio. We revealed that while both compounds were able to reduce hyperglycemia-induced apoptosis, only aspalathin could ameliorate lipid toxicity and oxidative stress-associated with insulin resistance. An important feature of the failing heart is the observed shift in mitochondrial substrate preference that precedes the onset of oxidative damage. The current study revealed that aspalathin improved glucose metabolism by decreasing fatty acid uptake and subsequent β-oxidation. This was achieved through decreasing the expression of adenosine monophosphate-activated protein kinase threonine 172 (pAMPK (Thr172)) and carnitine palmitoyltransferase 1 (Cpt1), while increasing that of acetyl-CoA carboxylase (Acc) and glucose transporter 4 (Glut4). Additionally, it is known that cardiomyocytes have a very low antioxidant capacity and a shift in mitochondrial substrate preference can result in accelerated oxidative damage. In this study, we showed that aspalathin ameliorated oxidative stress by increasing the antioxidant capacity of the cells through activation of the antioxidant response pathway, nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) and its downstream target genes. Moreover, we showed that aspalathin was able to reverse lipid toxicity by increasing the expression of Adiponectin, C1Q and collagen domain containing (Adipoq) and concomitantly decreasing cluster of differentiation 36 (Cd36) and Cpt1 mRNA expression. We further observed that Adipoq negatively regulated sterol regulatory element binding transcription factor 1 (Srebf1), stearoyl-Coenzyme A desaturase 1 (Scd1) and solute carrier family 27 (fatty acid transporter), member 3/5 (Slc27a3/5). This led to reduced lipid accumulation in H9c2 cardiomyocytes, with an associated decrease in total cholesterol, triglycerides and low-density lipoprotein in leptin resistant db/db mice. This was accompanied by decreased mRNA expression of inflammation markers in H9c2 cells, including interleukin 3 and 6 (IL3 and IL6), tumor necrosis factor receptor superfamily, member 1b and 13 (Tnfrsf1b and Tnfsf13), Janus kinase 2 (Jak2) and mitogen-activated protein kinase 3 (Mapk3). Together our results infer that aspalathin can slow down the progression of DCM, and thus protect the myocardium against causal factors associated with the development and progression of CVD.
- ItemContribution of highly active anti-retroviral therapy to the development of non-alcoholic fatty liver disease with concomitant cardiovascular dysfunction in an obese rat model(Stellenbosch : Stellenbosch University, 2018-03) Kamau, Festus Maina; Ruduwaan, Salie; Waweru, Peter Maina; Strijdom, Hans; Stellenbosch University. Faculty of Medical and Health Sciences. Dept. of Biomedical Sciences : Medical Physiology.Introduction: HIV/AIDS mortality is declining due to successful highly active anti-retroviral therapy (HAART). However, obesity, non-alcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD) in treated HIV-infected populations are rising. Impaired peroxisome proliferator-activated receptor (PPARα/γ) activity is partly implicated. Aims: To assess the contribution of a protease inhibitor (Lopinavir/Ritonavir (LPV/r) and nucleoside reverse transcriptase inhibitor (Azidothymidine (AZT)/lamivudine (3TC)) HAART regimen in the development of NAFLD and CVD in rats with high caloric diet (HCD)-induced obesity, and to investigate dual PPARα/γ stimulation in limiting HAART-induced NAFLD and CVD. Methods: Wistar rats were randomised into rat chow and HCD groups (n=88/group; 16 weeks). From week 10, each group was further sub-divided into: vehicle/control, HAART, HAART+PPARα/γ agonist (Saroglitazar) and Saroglitazar only (n=22/group) administered via oral gavage. Endpoints: Daily food/water consumption, weekly total body mass (TBM), random+fasting blood glucose measurements (n=8/group); hearts exposed to either 20min global ischaemia/10min reperfusion for Western blot (WB) analysis (n=6/group) or 35min regional ischaemia/60min reperfusion (n=8/group) for haemodynamic and infarct size (IS) determinations. Liver samples were histologically assessed (n=12/group) and analysed by WB. Intraperitoneal (IP) fat was weighed. Fasting serum lipids, insulin and oxidative stress markers were measured (n=8/group). WB analyses of important signalling proteins in pre/post-cardiac ischaemia, liver and aorta tissues. Thoracic aorta (n=8/group) segments were subjected to isometric tension studies. Results: The HCD resulted in obesity (increased: TBM, %IP fat (HCD control 6.50±0.40% vs. lean control 3.60±0.3%; p<0.0001), liver mass, insulin and triglycerides (TGs). Additionally, HCD induced insulin resistance (IR). HAART+Saroglitazar led to reduced %IP fat in lean and HCD groups. HAART-induced IR, elevated cardiac mass and insulin in obese rats were limited by Saroglitazar co-treatment. HCD+HAART-induced oxidative stress (elevated conjugated dienes), was limited in combined HAART+Saroglitazar. Liver histology: HAART induced moderate hepatic steatosis in ~67% of obese rats and moderate inflammation in ~25% of cases. Combined HAART+Saroglitazar limited these changes and upregulated adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (PKB/Akt) activity, which were downregulated by HAART in HCD. Heart/aorta studies: Untreated obese rats had smaller %IS compared to lean control rats (19.1±1.6 vs. 26.1±1.6, respectively; p<0.05). IS for HCD+HAART animals were smaller (despite poor cardiac performance) compared to untreated obese rats. Post-ischaemia activity of extracellular-signal-regulated kinase (Erk1/2), PKB/Akt, AMPK and endothelial nitric oxide synthase (eNOS) was downregulated, whereas expression of p22-phox and caspase-3 was accentuated. HAART+Saroglitazar upregulated (1.5-fold) the expression of Erk1/2, PKB/Akt, AMPK and eNOS, and downregulated caspase-3 and p22-phox. Obese+HAART rats demonstrated poor aortic relaxation accompanied by downregulated eNOS and PKB/Akt, and upregulated p22-phox. However, Saroglitazar+HAART in obese animals improved aortic relaxation by ~30%, accompanied by upregulation of eNOS, and PKB/Akt, and downregulated p22-phox. Discussion and conclusion: HAART-induced NAFLD and CVD in obesity were limited by PPARα/γ agonist co-administration. HAART treatment for six weeks was not a cardiovascular risk factor per se, but it potentiated HCD-induced cardiovascular effects. Monitoring cardiovascular risk factors in obese+HAART patients is crucial, and our findings suggest that there is therapeutic potential in co-treatment with PPARα/γ agonists. The metabolic, functional and signalling disturbances in the liver, heart and aorta tissues in obese+HAART rats are interlinked, and partially limited by co-treatment with a dual PPARα/γ agonist.
- ItemA critical analysis of mitochondrial functioning and associated proteins in obesity-related cardiomyopathy(Stellenbosch : Stellenbosch University, 2013-03) George, Siddiqah; Huisamen, Barbara; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.ENGLISH ABSTRACT: INTRODUCTION: The mechanism behind obesity-related cardiomyopathies is at present not completely known, however, cardiac insulin resistance has been implicated as one of the main arbitrators of obesity-related cardiovascular disease. A few studies have associated perturbations in the insulin-mediated PI3K/PKB/Akt pathway in mediating this insulin resistance. Moreover, this pathway has been shown to regulate myocardial apoptosis, which in turn has been implicated in a number of cardiovascular diseases. Currently, few studies have compared the early onset and advanced effects of obesity on the heart. AIMS: To compare the early and advanced stages of obesity in terms of myocardial (i) PI3K/PKB/Akt signalling, (ii) apoptotic signalling and (iii) mitochondrial integrity. Furthermore, we aim to assess the cardiac mitochondrial (i) PI3K/PKB/Akt signalling, (ii) apoptotic signalling and (iii) integrity during the advanced stages of obesity. METHODS: Male Wistar rats were randomly assigned to either a control or diet-induced obesity (DIO) group. Controls were fed a standard rat chow diet and the DIO group fed a high caloric diet (standard rat chow supplemented with sucrose and condensed milk). The diets were implemented for either 8 or 20 weeks and thereafter, the body weight, intra-peritoneal fat mass, and fasting blood glucose and insulin levels (including intra-peritoneal glucose tolerance tests (IPGTTs)) were determined. Freeze-clamped hearts from both groups were subjected to cytosolic western blot analysis for PI3K p85 subunit, PKB/Akt, GSK-3α/β, Bad, Bax and Bcl-2. A fraction of each heart was also subjected to WB analysis of the mitochondrial electron transport chain (ETC) complexes (I-V). Thereafter, the above mentioned proteins were also probed for in mitochondria isolated from the 20 weeks group after administering insulin and exposing the hearts to ischemia. Oxidative phosphorylation (OXPHOS) capacity analysis was then conducted on mitochondria isolated from 20 weeks DIO and control groups and thereafter a citrate synthase (CS) activity assay was performed on these mitochondria. RESULTS: After the 8 and 20 weeks diet, the DIOs had significantly increased intra-peritoneal fat mass, fasting plasma glucose and insulin levels, compared to their controls. Cytosolic WB analysis: The tp85, pp85 and pPKB/Akt levels were significantly higher in the DIOs in comparison to the controls after 8 weeks of diet. Furthermore, pBad and Bax expression were significantly elevated in these animals. After 20 weeks of diet, the DIOs had significantly decreased pp85, tPKB/Akt and pPKB/Akt levels. The tBad was significantly elevated, while the Bad phosphorylated over total expression (P/T) ratio was significantly decreased, in these animals. CS activity assay: CS activity was significantly decreased in the DIOs, versus the controls, at 20 weeks. Mitochondrial ETC WB analysis: The subunit expression in complexes I-III and V did not differ significantly after 8 weeks however, the expression was significantly lower in complexes I and II after 20 weeks. Interestingly, the complexes III and V expression was significantly elevated. Mitochondrial OXPHOS analysis: The ADP/O ratio with (1) glutamate or (2) palmitoyl-L- carnitine as substrate, showed a significant decrease in the DIOs at 20 weeks. Mitochondrial WB analysis: The pp85 subunit was significantly elevated in the control and DIO groups, exposed to insulin and ischemia, in comparison to the untreated controls. The Bcl-2 levels were significantly decreased in the insulin and ischemia DIOs, when matched against the untreated DIOs. The tBad expression did not differ significantly between the insulin and untreated controls, while the tBad was significantly augmented in the ischemia controls versus untreated controls. All significant differences were taken as p<0.05. CONCLUSION: The results indicate that the initial stage of diet-induced obesity is associated with cardioprotection as there is augmented PI3K/PKB/Akt pathway signalling and a decrease in apoptotic markers. In contrast, during the advanced stages of obesity a decreased activity in PI3K/PKB/Akt pathway is associated with myocardial apoptosis and decreased mitochondrial function and integrity.
- ItemThe dietary ionic effects on sex ratios in animal models(Stellenbosch : Stellenbosch University, 2016-03) Linge, Augustine Peter Kavoo; Du Plessis, S. S.; Kimwele, C.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.ENGLISH ABSTRACT: X-linked disorders are more expressed in male offspring and prevention of these hereditary diseases is the only recourse to date. Influencing conception towards female offspring can circumvent this problem; however sex ratio adjustment remains highly contentious. Treatment of genetic disorders through sex ratio adjustments has been examined and adopted as acceptable, easier, cheaper, safer and legal. Historically, society has been rife with allegations that diet does influence sex ratios though this has not been proven fully. The dietary chemical compositions have been claimed to act as modulators that affect the electrical charges or potential of the membranes of the oocytes and cause allosteric modification or electrotactism through a process referred to as galvanotropism and cause selective attraction towards either of the male gametes and subsequently influence the gender of the conceptus. This study was performed in an attempt to address the various questions, allegations and speculations that have been rife in many societies concerning interplay between diet, fertilization and sex ratios so as to verify the validity of these social claims by taking them to the laboratory for experimental verification. Swiss Webster mice study In a double-blind fashion, nine double groups were set up comprising of 144 families of Swiss (Webster) mice, each receiving different ionic formulations in their drinking water: 1) water and 2) glucose as controls; high serum concentrations of single elements of 3) sodium, 4) potassium, 5) calcium and 6) magnesium; combined double elements 7) sodium + potassium and 8) calcium + magnesium; and finally a cocktail of the four elements 9) sodium + potassium + calcium + magnesium. Tests included the perinatal mortality rate; the relationship between high chemical composition diet and serum levels; the effects of the study chemicals on weight gains of the study models; the effects of birth order on sex ratios and the effects of seasonal variations on sex ratios. There were 1528 deliveries with 13,040 (6,348 females and 6,692 males) pups at 8.5 pups on average per litter. a) Glucose, sodium, potassium and sodium + potassium supplementation influenced the sex ratios towards male progeny (p<0.001). Calcium (p<0.014), magnesium (p<0.008) and calcium + magnesium (p<0.001) supplementation influenced sex ratios towards the female progeny. The water (p>0.61) and cocktail solutions (p>0.0609) had no influence. b) The perinatal mortality rate was 32/1000 and was female biased among the magnesium (p<0.005) and combined calcium + magnesium (p<0.044) groups only. c) Normal serum levels were observed in the control groups (p>0.165), while significant elevated serum levels were observed among the experimental groups (p<0.0001). d) The total mean weight gains were 11.12g and 10.55g among the females and males respectively. The weight trends were used to track the general wellbeing of the animal models. e) The mean litter size was 8.5 per delivery in all the groups and generations, while no influence due to birth order were detected. f) Seasons affect the litter size, in particular the rainy season, but not the gender ratios (p>0.061). Cat fish study Parallel double blind studies looking at the dietary chemical ionic effects on the oocyte membrane electrical potential were done utilising a cat fish model (n=108). The study sought to find out effects of the following solutions on the oocyte electrical charges: 1) plain electrolyte solution, 2) glucose solution 3) sodium solution 4) potassium solution 5) calcium solution 6) magnesium solution 7) sodium + potassium solution 8) calcium + magnesium solution and the 9) cocktail solution of the four elements combined. The results revealed that oocytes retrieved from the two control groups had baseline oval polar attraction significantly more towards the positive than the negative pole (p<0.0003). There was however more significant oocyte polar attraction towards the positive electrode among the oocytes retrieved from the sodium, potassium and the combined sodium + potassium solutions (p<0.0001). Oocytes retrieved from calcium, magnesium and combined calcium + magnesium solutions had significant affinity towards the negative electrode and minimal affinity towards the positive pole (p<0.0001). Oocytes harvested from the solutions constituted with all the salts demonstrated dual attraction with more attraction to the positive electrode than the negative electrode but of no statistical significance (p>0.0530). These study findings do confirm the social allegations that a positive relationship does exist between dietary components and sex ratios. The chemicals acted as the dietary modulators that ultimately influenced the electrical cellular gametal charges and subsequently the resulting progeny. Our platform of comfort is unlike artificial sex ratio adjustment methods; the natural sex ratio adjustment methods that include the dietary method under scrutiny in this study are practiced always at the comfort of many people’s homes and are difficult to quantify or have legislation on. However, this study shows that their long term effects conform to the Fishers principle of evolution towards 1:1 sex ratios and therefore do not have significant social gender skewing on a long term basis. The study clearly explains the molecular basis upon which ions of single valency attracts the Y-bearing sperm leading to a male conceptus and how cations of double valency attracts the X-bearing sperm leading to a female conceptus despite being positively charged. The study further reaffirms the natural feminine supremacy by demonstrating that it is the ova and by extension the woman who determines the sex of the conceptus. The study ultimately confirms that the dietary ionic effects on sex ratios can be used for prevention of X-linked disorders.
- ItemA diffusion tensor imaging study in HIV patients with and without apathy(Stellenbosch : University of Stellenbosch, 2010-12) Fouche, Jean-Paul; Strijdom, Hans; Carey, Paul Dermot; Spottiswoode, Bruce Shawn; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.ENGLISH ABSTRACT: HIV/AIDS is a global epidemic that accounts for a large percentage of the mortality in South Africa every year. Since the implementation of anti-retroviral treatment, HIV positive individuals have been living longer, and the cognitive impairment associated with the disease is becoming increasingly apparent. During the initial systemic infection of HIV, the virus migrates through the blood-brain barrier and inflicts axonal injury by causing upregulation of cytokines and neurotoxic proteins. HIV-associated dementia is a neuropsychological classification of cognitive impairment in HIV and a variety of symptoms have been classified as a part of the dementia complex. One of these is apathy, which is thought to be a precursor for dementia in HIV patients. Three groups of individuals have been recruited and scanned using magnetic resonance imaging (MRI) to examine changes in the brain. These are an HIV non-apathetic cohort, an HIV apathetic cohort and a healthy control cohort. Diffusion tensor imaging (DTI) is an MRI technique used to quantitatively assess white matter (WM) integrity using metrics such as fractional anisotropy (FA). Voxel-based analysis, tract-based spatial statistics (TBSS) and tractography are three established DTI analysis methods that have been applied in numerous studies. However, there are certain methodological strengths and limitations associated with each technique and therefore all three of these techniques were used to compare WM differences across groups. The frontal-subcortical pathways are known to be abnormal in apathy, and this has been demonstrated in a number of imaging studies. Most of these studies have examined apathy in the context of neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s. However, to our knowledge this is the first DTI study in HIV apathetic patients. With the tractography method, the anterior thalamic radiation and the corpus callosum were reconstructed for each individual to determine whether there were any global changes in these tracts. No significant changes were found. However, a variety of regions in the WM were significantly abnormal in the HIV cohorts when comparing the data at a voxel-based level and using TBSS. This included areas such as the genu and splenium of the corpus callosum, the internal capsule and corona radiata. Changes in frontal WM for the HIV apathy group are an indication of dysfunction in the frontal-striatal circuits, and previous literature has implicated these circuits in the neuropathology of apathy in a variety of central nervous system (CNS) disorders.
- ItemDoes sexual violence alter social behvaiour via a maladaptive HPA-axis?(Stellenbosch : Stellenbosch University, 2023-03) Fortuin, Chelsi Shante; Qulu, Lihle; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.ENGLISH ABSTRACT: Background and Aim: South Africa was identified as the rape capital of the world, recording one of the highest rates of sexual violence worldwide with 72.1 reported cases per 100 000 in the 2019/2020 year. The mental health and well-being of sexual assault survivors exhibit a dysregulated HPA-axis stress response, which may be a primary source of structural and functional alterations leading to PTSD symptoms, but they don’t always co-exist. Chronic stress and psychiatric diseases cause dysregulation of the HPA axis. A cortisol imbalance brought on by the dysregulation of the HPA-axis leads to a decrease in oxytocin secretion, which eliminates oxytocin's potential to attenuate HPA-axis activity. A lack of oxytocin has been linked to broken social and maternal bonds as well as losing relationship attachments as a result of trauma exposure such as sexual violence / rape. Oxytocin dysregulation has been associated in a large variety of dysregulated social behaviour and PTSD diagnosis. Additionally, when bound to cortisol, glucocorticoid receptor sensitivity modifies the stress response's equilibrium point, which in turn regulates the HPA axis's negative feedback loop. As a result, they have been linked to anticipating the effects of stress. Therefore, the aim of this study is to determine whether being subjected to sexual violence alters social behavior via a dysregulated HPA-axis. Methods: Data analysis was completed using PTSD, perceived stress and social support scores to investigate the effects of rape on mental health trajectory. Additionally, enzyme-linked immunosorbent assays were used, to explore the role of oxytocin and cortisol as indicators of PTSD in rape-exposed women. Both cortisol and oxytocin found within plasma samples were analysed over a one-year period at four time points (baseline [± twenty days], three months, six months and twelve months post rape) to assess whether these hormone levels, that are activated by sexual violence, were transient or long lasting, and whether these hormone levels may lead to the formation of PTSD. Mixed effect regression analysis were done to determine if cortisol and oxytocin concentrations predict PTSD outcomes. Results: PTSD symptoms significantly decreased overtime, p = 0.000. Similarly, perceived stress, p = 0.0023, and cortisol concentrations, p = 0.004, significantly decreased overtime. Whereas social support scores, p = 0.5851, and oxytocin concentrations, p = 0.995, had no significant changes. Additionally, cortisol concentrations, p = 0.1660, and social support, p = 0.827, were not able to predict PTSD outcome, however oxytocin concentrations, slope = 9.32, p = 0.002 and perceived stress scores, slope = -5.654, p = 0.033, could predict PTSD. Conclusion: These findings demonstrate a relationship between PTSD symptom severity and HPA-axis maladaptation, via augmented oxytocin responses in victims of rape. We conclude that these findings may have significant clinical ramifications for women who have been subjected to rape. Particularly, offering scientific based PTSD interventions to rape exposed victims may show promise in alleviating symptoms and "normalizing" HPA axis receptivity to stress related stimuli.
- ItemThe effect of 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and 5'-aminoimidazole-4-carboxamide-ribonucleoside-phosphate (ZMP) on myocardial glucose uptake(Stellenbosch : Stellenbosch University, 2005-03) Webster, Ingrid; Huisamen, Barbara; Lochner, Amanda; Stellenbosch University. Faculty of Medicine & Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.ENGLISH ABSTRACT: Introduction: Exercise increases skeletal muscle glucose uptake via AMP-activated protein kinase (AMPK) activation and GLUT4 translocation from cytosol to cell membrane. It also promotes glucose utilisation in type 2 diabetic patients via increased insulin sensitivity. Insulin stimulates GLUT4 translocation by activating P13- kinase and protein kinase B (PKB/Akt). We therefore postulated that a connection exists between these two pathways upstream of GLUT4 translocation. Understanding this connection is important in the development of treatment strategies for type 2 diabetes. This exercise-induced increase in AMP-activated protein kinase (AMPK) activation can be mimicked by a pharmacological agent, 5'-aminoimidazole-4- carboxamide ribonucleoside (AlGAR), which is converted intracellularly into 5'- aminoimidazole-4-carboxamide-ribonucleosidephosphate (ZMP), an AMP analogue. Aim: To investigate the effect of two pharmacological AMPK-activating compounds, ZMP and AlGAR, on the phosphorylation of AMPK, the phosphorylation of PKB/Akt as well as possible feedback on insulin-stimulated glucose uptake and GLUT4 translocation. Materials and Methods: Adult ventricular cardiomyocytes were isolated from male Wistar rats by collagenase perfusion and treated with 1 mM AlGAR or 1 mM ZMP in the presence or absence of 100 nM insulin or 100 nM wortmannin, an inhibitor of P13- kinase. Glucose uptake was measured via eH]-2-deoxyglucose (2DG) accumulation. PKB/Akt and AMPK phosphorylation and GLUT4 translocation was detected by Western blotting. Purinergic receptors were blocked with 8-cyclopentyl-1,3- dipropylxanthine (8CPT) and the effect on AMPK phosphorylation noted. Certain results were confinned or refuted by repeating experiments using the isolated rat heart model. Results: AICAR and ZMP promoted AMPK phosphorylation. Neither drug increased glucose uptake but in fact inhibited basal glucose uptake, although GLUT4 translocation from cytosol to membrane occurred. Both compounds also attenuated insulin stimulated glucose uptake. Wortmann in abolished glucose uptake and PKB/Akt phosphorylation elicited by insulin while, in the presence of wortmannin, AICAR and ZMP increased levels of PKB/Akt phosphorylation. Although AICAR and ZMP increased glucose uptake in skeletal muscle, this was not seen in cardiomyocytes. However both compounds increased GLUT4 translocation, clearly demonstrating that translocation and activation of GLUT4 are separate processes. 8CPT had no effect on the phosphorylation of AMPK by either AICAR or ZMP indicating that there was no involvement of the purinergic receptors. Conclusion: Although AICAR and ZMP increase glucose uptake in skeletal muscle, this was not seen in cardiomyocytes. Conversely, both compounds inhibited both basal and insulin stimulated glucose uptake despite increasing GLUT4 translocation. Inhibition of PI3-kinase in presence or absence of insulin unmasked hitherto unknown effects of AICAR and ZMP on PKB phosphorylation.