Masters Degrees (Physiological Sciences)
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- ItemAgeing-associated oxidative stress and inflammation are alleviated by products from grapes(Hindawi, 2016) Petersen, K. S.; Smith, CarineAdvanced age is associated with increased incidence of a variety of chronic disease states which share oxidative stress and inflammation as causative role players. Furthermore, data point to a role for both cumulative oxidative stress and low grade inflammation in the normal ageing process, independently of disease. Therefore, arguably the best route with which to address premature ageing, aswell as age-associated diseases such as diabetes, cardiovascular disease, and dementia, is preventative medicine aimed at modulation of these two responses, which are intricately interlinked. In this review, we provide a detailed account of the literature on the communication of these systems in the context of ageing, but with inclusion of relevant data obtained in other models. In doing so, we attempted to more clearly elucidate or identify the most probable cellular or molecular targets for preventative intervention. In addition, given the absence of a clear pharmaceutical solution in this context, together with the everincreasing consumer bias for natural medicine, we provide an overview of the literature on grape (Vitis vinifera) derived products, for which beneficial effects are consistently reported in the context of both oxidative stress and inflammation.
- ItemAnthropometric characteristics and changes with HIV and ART in a randomly selected population in the Drakenstein region Western Cape Province(Stellenbosch : Stellenbosch University, 2014-12) Beukes, Dillan Charles; Nell, Theo A.; Essop, M. Faadiel; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Background - Highly active antiretroviral therapy (HAART) has extended life expectancy and enhanced the well-being of HIVpositive individuals. Since there are concerns regarding HAART-mediated onset of cardio-metabolic diseases in the long-term, we evaluated the anthropometric profile of HIV-infected individuals in the Drakenstein District (Western Cape, South Africa). - Objective of study - The primary objective of this study was to document the anthropometric characteristics within and HIV infected population in the Drakenstein region of the Western Cape Province of South Africa. - Methods - HIV-positive patients (n=44 males, n=102 females; 20-40 yrs.) were recruited for three groups: 1) control (HIVnaïve), 2) HIV-positive (HAART ≤ 0-36 months), and 3) HIV-positive (HAART ≥ 36 months). Participants underwent a) anthropometric (triceps skin fold [TSF], and b) bioelectrical impedance measurements (body cell mass [BCM], fat free mass [FFM], protein, muscle mass (MM), mineral, total body potassium (TBK) and calcium (TBCa), glycogen, and fat mass [FM]). - Results - Our data reveal that HIV-positive males on HAART ≤ 0-36 months displayed a trend for lower body cell mass (BCM), fat free mass (FFM), fat mass (FM), triceps skinfold (TSF) and protein content (vs. control). Females exhibited reduced BCM (p=0.001) and lower protein (p=0.003), muscle mass (p=0.001), glycogen (p=0.001), FM (p=0.0005) and FFM (p=0.002) content. However, with longer-term treatment (HAART ≥ 36 months), females displayed higher BCM (p=0.0001), protein (p=0.01), muscle mass (p=0.0003), glycogen (p=0.0001), FM (p=0.00003) and FFM (p=0.0002) vs. the 0-36 months treatment group. Their waist-to-hip ratio also increased vs. the naïve female group (p=0.02). By contrast, males on HAART ≥ 36 months did not show any significant increases vs. the HAART ≤ 0-36 month’s group. - Conclusions - This study demonstrates observed striking gender-based anthropometric differences in South African HIVpositive individuals on HAART. While both genders initially exhibit muscle wasting, HIV-positive females show a strong improvement with longer-term treatment vs. males. However, higher abdominal fat accumulation in females with longer-term treatment potentially increases their risk for the future onset of cardio-metabolic complications.
- ItemAre early proteomic and metabolic changes induced by long-term sugar-sweetened beverage consumption the key to unlocking the cardio-metabolic pandemic?(Stellenbosch : Stellenbosch University, 2017-03) Benade, Janina; Essop, M. Faadiel; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: INTRODUCTION: Cardio-metabolic diseases (e.g. type 2 diabetes mellitus) are a major cause of mortality worldwide. The incidence of cardio-metabolic diseases continues to increase, especially in low and middle income countries. This “pandemic” is possibly brought about by a fairly universal shift towards a more “Westernized” diet. High sugar consumption - a hallmark of the “Westernized” diet - may play a key role in the onset of cardio-metabolic diseases. Accordingly, our research focus moved towards sugar-sweetened beverages (SSBs) as it is a major source of added dietary sugars. The current study aimed to elucidate underlying mechanisms leading to the development of cardiometabolic diseases by exploiting a novel rat model of long-term SSB intake, and by focusing on the liver as a major metabolic organ. Here we evaluated well-known systemic markers together with hepatic proteome analysis and downstream consequences. METHODS: Male Wistar rats ( 200 g) were gavaged with 3-5.1 mL SSB daily for three and six months, respectively. The two control groups were gavaged with an iso-volumetric amount of water and iso-caloric amount of butter, respectively. Body weight and systemic blood markers were measured. A proteomic expression analysis was performed on the six-month liver samples. The rest of our experimental work was guided by the proteomic results. Four markers for oxidative stress were evaluated: malondialdehyde, conjugated dienes, reduced:oxidized glutathioneand oxygen radical absorbance capacity. The non-oxidative glucose pathways (NOGPs): polyol pathway, hexosamine biosynthetic pathway, advanced glycation end-products formation and protein kinase C activation; were measured as elevated activity could be indicative of impaired glycolytic flux. The liver histology was investigated with Hematoxylin and Eosin and Masson’s Trichrome stains, respectively. Finally, Western blotting techniques were used to evaluate markers of inflammation. RESULTS: SSB consumption had little effect on systemic markers of cardio-metabolic health. Our proteomic analysis revealed that the expression level of 140 proteins was significantly altered in the SSB group, with a major finding that SSB consumption induces hepatic endoplasmic reticulum (ER) stress. Initially the liver adapted to SSB-mediated nutrient overload by increasing oxidative phosphorylation, suppressing protein transcription, degrading misfolded proteins and improving protein folding capacity. However, due to prolonged stress liver cells entered an ‘’alarm phase’’ marked by a decrease in mitocholdrial metabolism. The proteomic results further revealed that SSB-induced effects are largely attributed to excess caloric intake versus SSBs per se. Surprisingly, oxidative stress did not precede ER stress as there were no significant changes in any of the oxidative stress markers here evaluated. The activity of the NOGPs did not increase significantly thus suggesting that moderate SSB intake did not suppress glucose metabolism and the glycolytic pathway in particular. Conversely, SSB intake increased hepatic lipid storage while limited changes were detected between the groups regarding inflammation and stress signaling. CONCLUSION: Frequent SSB consumption triggers metabolic changes in the liver, i.e. ER stress despite the lack of obvious manifestation of macroscopic “warning signs”. Thus the current study identifies hepatic ER stress as a relatively early result of long-term SSB consumption and it therefore emerges as a unique therapeutic target.
- ItemAn assessment of ischemia-reperfusion injury in rats exposed to chronic psychological stress(Stellenbosch : Stellenbosch University, 2019-12) Oliver, Lukas Van Zyl; Essop, M. Faadiel; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Cardiovascular disease remains the leading cause of death worldwide. Apart from known risk factors such as poor dietary intake, physical inactivity and smoking, chronic psychological stress is emerging as an important modifiable risk factor in the development of cardiovascular disease. The body relies on two physiological mechanisms to counter acute stressors and to achieve and/or maintain homeostasis, i.e. the hypothalamic-pituitary-adrenal axis and the autonomic nervous system. However, chronic activation of these systems can lead to the disruption of cellular and systemic processes that could potentially result in the development of neurological or psychosomatic diseases. Both chronic stress and ischemia-reperfusion injury are associated with a robust inflammatory response and the induction of oxidative stress. Does chronic psychological stress render the heart more susceptible to ischemia and reperfusion damage, and what are the role(s) of oxidative stress and inflammation in stress-related cardiac dysfunction? These questions will form the basis of this review. Following a comprehensive review, we established that chronic stress does render the heart more susceptible to damage following ischemia-reperfusion. After reviewing the mechanisms involved in both ischemia-reperfusion and chronic stress, we hypothesized that chronic stress induced inflammation and oxidative stress are major contributors in aggravated ischemia-reperfusion injury.
- ItemAssociation between antioxidant status and MnSOD Ala-9Val polymorphism in trained male athletes (rugby players) and sedentary male students controlled for antioxidant intake(Stellenbosch : Stellenbosch University, 2007-03) Seele, Maria; Senekal, M.; Steyn, N. P.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: The human body has developed an integrated antioxidant defence system to protect against free radical damage. Acute exercise may result in the increased generation of free radicals, including reactive oxygen species, and this may overwhelm antioxidant defence systems resulting in oxidative stress. However, it has been shown that individuals who undergo regular exercise training may have improved antioxidant capacity when compared to sedentary controls. Results from research regarding the association between antioxidant capacity and exercise training are however not conclusive and further investigation is required. Therefore, the aim of this study was to investigate the association between the total plasma antioxidant status and selected plasma indicators of antioxidant status and the MnSOD Ala-9Val (-28C®T) polymorphism in trained male athletes (rugby players) and sedentary male students while controlling for dietary intake of the major antioxidants using a validated dietary assessment method. In order to address the potential confounding effect of dietary antioxidant intake on antioxidant status in the main study, a FFQ that measures vitamin C, vitamin E, carotenoid and flavonoid intake was developed. The reproducibility was assessed by the repeat administration of the FFQ (n = 38), while the va lidity was assessed using a 28-day closeended dietary record and repeated plasma vitamin C values (n = 18). Several statistical tests were conducted to compare the values obtained from the FFQ with values obtained from the various reference methods. While results from Bland-Altman plots suggested that the reproducibility and validity of FFQ was not completely satisfactory, similar mean values, moderate to strong correlation coefficients, and a high percentage of individuals classified correctly according to quartiles of intake indicated satisfactory reproducibility and validity of the FFQ in assessing antioxidant intake. Furthermore, moderate to strong validity coefficients obtained from the method of triads also indicated satisfactory validity for the FFQ. The main study involved a cross-sectional study that compared plasma vitamin C and carotenoid levels as well as total plasma antioxidant status in trained rugby players (n = 76) and sedentary male subjects (n = 39) with different MnSOD genotypes, while controlling for dietary antioxidant intake. Rugby players had significantly higher plasma vitamin C and carotenoid levels compared to sedentary students, which indicated more satisfactory plasma antioxidant status. This was also reflected in the tendency for total plasma antioxidant status (ORAC assay) to be higher in rugby players than sedentary students. MnSOD genotype did not influence plasma vitamin C and carotenoid levels or plasma total antioxidant status, with or without control for dietary antioxidant intake. Dietary vitamin C, vitamin E, carotenoid an flavonoid intake (from foods + supplements) was similar for rugby players and sedentary students and was adequate for both groups. Thus the association between antioxidant status and MnSOD genotype in rugby players and sedentary students seemed not to be influenced by dietary antioxidant intake. In conclusion therefore, rugby players undergoing regular exercise training had a more satisfactory antioxidant status compared to sedentary students. Based on this conclusion, the widespread use of antioxidant supplements by athletes is questioned.
- ItemAssociation between cancer, adipose tissue and selected systemic markers : a possible classification according to body shape(Stellenbosch : Stellenbosch University, 2016-03) Mentoor, Ilze Lauren; Nell, Theo A.; Kruger, Maritza J.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Background: The metabolic syndrome (MetS) is a cluster of risk factors associated with an increased risk of developing chronic diseases of lifestyle, and has more recently been associated with cancer risk. Currently, the pathophysiology of the MetS and cancer risk is still unknown; however it is proposed to involve several factors. These include the effects of body composition (android and gynoid shapes), and insulin resistance on the bioavailability of growth factors, inflammatory markers and sex hormone profiles. Various anthropometrical measurements have been used to investigate body composition, however, due to their limitations, a new metric namely a body shape index (ABSI) has been proposed to be a better measure of fat distribution and body shape. Aims: To determine the prevalence of the MetS, and the possible risks of developing cancer in relation to metabolic status, body composition, growth factors as well as inflammatory and sex hormone parameters. Methods: Female participants between the ages of 20-60 years were classified according to the International Diabetes Federation’s (IDF) definition of the MetS and according to body shape (android/gynoid) by photoscopic somatotyping. A series of tests and assessments were conducted; such as blood pressure assessments, anthropometric measurements, bioelectrical impedance analyses (BIA) and blood analyses. Blood analysis included fasting glucose, fasting insulin, lipid profile, insulin-like growth factor-1 (IGF-1), inflammatory marker (C-reactive protein (CRP)); and sex hormone parameters (oestrogen, female testosterone, sex hormone binding globulin; and free androgen index). Results: The prevalence of the MetS was found to be 57.5 %; with abdominal obesity (73.8 %), elevated blood pressure (BP, 68.8 %) and low high density lipoprotein-cholesterol (HDL-c) levels (68.8 %) being the more prevalent risk factors. Both metabolic status; and body shape alone were found to be predictors influencing anthropometric, BIA, physiological and biochemical blood parameters. Metabolic status was found to have an effect on several parameters in the gynoid body shape groups, i.e. body mass (BM) (p<0.001), hip circumference (HC) (p<0.01), body mass index (BMI) (p<0.001), fat mass (FM) (%) (p<0.01), fat free mass (FFM) (%) (p<0.01), waist circumference (WC) (p<0.001), HDL-c (p<0.001), triglycerides (TG) (p<0.05), systolic blood pressure (SBP) (p<0.05) and diastolic blood pressure (DBP) (p<0.01), while metabolic status showed an effect on BM (p<0.001), BMI (p<0.01), TG (p<0.05), SBP (p<0.01) and DBP (p<0.01) in the android body shape groups. Both metabolic status and body shape did not show any effect on ABSI, total cholesterol (TC), low density lipoprotein-cholesterol (LDL-c), fasting insulin, CRP and all sex hormone parameters. Correlation analyses revealed significant correlations for several anthropometric, BIA and blood parameters. Conclusion: This study showed that metabolic status, body shape and/or both could predict changes in various body composition, physiological and biochemical parameters in women. However, no effects were evident for any parameters linking the MetS to cancer risk. Thus, no accurate conclusion could be drawn regarding the pathophysiology. Our findings on ABSI, still warrants future investigation to substantiate the use of this metric in relation to the MetS, body shape and cancer risk.
- ItemThe association between the metabolic syndrome and bone mineral density in pre- and post-menopausal farm workers(Stellenbosch : Stellenbosch University, 2016-12) Marais, Sumine; Nell, Theo A.; Kruger, Maritza J.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Introduction and aims: The prevalence of the metabolic syndrome (MetS) is increasing, both globally and in South Africa. Albeit so, limited data have been published in the South African context. One of the factors that appear to influence the prevalence of the MetS is menopausal status, with both the MetS, and menopausal status influencing bone mineral density (BMD); however, the reported results are inconsistent. Therefore, the aim of this study was to determine the prevalence of the metabolic syndrome, investigating bone health as well as the interactions between the MetS, menopausal status and bone health, in a farm working female population in the Western Cape. Methods: A total of n=80 females were recruited and classified with the MetS, using the International Diabetes Federations’ definition. The data collected included basic anthropometric measurements, blood pressure, BMD, and several questionnaires to obtain information regarding physical activity, demographic information, menstrual-, diet- and family health- history. The blood parameters that were measured included alkaline phosphatase (ALP), vitamin D, parathyroid hormone (PTH), oestradiol (E2), fasting insulin (FI), fasting glucose (FG) and a lipid profile. Results: A relatively high prevalence of the MetS (55.0%) was reported in the current study. When investigating the separate MetS risk factors, most of the study participants had three risk factors (32.5%), with increased BP being the most prevalent MetS risk factor (72.5%). Factors that differed between MetS and Non-MetS sub-groups (according to menopausal status and age) included waist circumference (WC), high-density lipoprotein-cholesterol (HDL-c), systolic blood pressure (SBP) and diastolic blood pressure (DBP). Significant associations between body mass (BM) and E2, and body mass index (BMI) and E2, were limited to the PreM (20-39 years) age group with the MetS (r=0.58, p=0.03, and r=0.60, p=0.02). A total of 78.8% of the study participant had normal BMD. When correlating BM and speed of sound (SOS), significant associations were limited to the PreM (≥40 years) group (MetS: r=0.56, p=0.04, Non-MetS: r=0.76, p=0.00), and significant associations between BMI and SOS were noted in both PreM groups (MetS PreM 20-39 years: r=0.53, p=0.05, Non-MetS PreM ≥40 years: r=0.73, p=0.00). The significant correlations between FI and ALP (r=0.72, p=0.00), FG and ALP (r=0.89, p=0.00), and triglycerides with ALP (r=0.82, p=0.00) were limited to the PreM (≥40 years) group. Conclusion: The prevalence of the MetS was higher than that reported by previous South African studies. Irrespective of metabolic and menopausal status, most of the participants of the current study population had normal BMD. Key words: Metabolic syndrome, bone mineral density, menopause
- ItemAssociation between the metabolic syndrome and cancer risk : the potential role of fatty acids on body composition(Stellenbosch : Stellenbosch University, 2016-12) Johnson, Olga; Nell, Theo A.; Kruger, Maritza; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Background: Sub-Sahara Africa is experiencing an epidemiological transition with an increasing burden of non-communicable diseases (NCDs) including obesity, hypertension, insulin resistance (IR) and dyslipidaemia. These NCD are collectively labelled the metabolic syndrome (MetS). The MetS is characterised by dyslipidaemia, and in particular, distorted fatty acid (FA) metabolism. Additionally, the MetS and its components are also associated with different FA classes. Furthermore, several lifestyle-cancers have also been associated with the MetS and its components. Aim: To determine the association and interaction between the MetS and cancer risk and the likely influence of FAs on body composition in a female population residing in the Western Cape, South Africa. Methods: Female farm workers in the Cape Winelands region (n=80) aged 20-60 years were randomly selected and categorised as having the MetS (n=34 MetS and n=46 non-MetS) using the International Diabetes Federation (IDF) criteria. All participants were additionally classified according to their body mass index (BMI). Blood pressure was measured, followed by blood sampling to determine blood glucose and insulin levels, as well as a full lipid profile. Selected red blood cell (RBC) membrane FAs and FA ratios were analysed, and enzyme-linked immunosorbent assays (ELISAs) were used to quantify serum insulin-like growth factor-1 (IGF-1) and leptin concentrations. Anthropometric measurements and bioelectric impedance analyses (BIA) were also performed. Results: The prevalence of the MetS was 42.5 % with abdominal obesity (100.0 % for the MetS, and 39.1 % for the non-MetS), hypertension (82.4 % for the MetS, and 47.8 % for the non-MetS), and low high-density lipoprotein cholesterol (HDL-c) (76.5 % for the MetS, and 34.8 % for the non-MetS) being the most prevalent MetS risk factors. Several statistically significant differences were observed between the MetS and non-MetS groups for blood parameters, including insulin and HDL-c levels (p<0.001), and glucose, IGF-1, and leptin levels (p<0.05). The MetS group also presented with significantly higher anthropometric measurements, including BMI (p<0.05), waist circumference (WC), waist-to-hip ratio (W:H), and the sagittal abdominal diameter (SAD) (all p<0.001). Furthermore, BIA (including visceral adipose tissue (VAT) area, percentage VAT (VAT %) and -subcutaneous adipose tissue (SAT %), and VAT to SAT ratio (VAT:SAT) (p<0.001 for all) also differed between the MetS and non-MetS groups. No significant differences were noted for any of the individual FAs or FA ratios. Categorisation according to metabolic status and BMI was shown to influence several metabolic-associated blood parameters, anthropometric measurements, BIA. However, metabolic status and BMI did not influence individual FA levels or FA ratios. The obese MetS group presented with significantly higher IGF-1 levels compared to their normal weight non-MetS counterparts. Correlation analyses indicated several significant associations between anthropometric measurements, BIA, FAs and metabolic-associated blood parameters. Conclusion: The results from this study suggest that metabolic status alone, and the combined effect of metabolic status and BMI, may predict alterations in metabolic-associated blood parameters, anthropometric measurements, and BIA in women, possibly linking obesity and the MetS to an increased risk of developing lifestyle-associated cancer. Keywords Metabolic syndrome, fatty acid profile, body composition, leptin, cancer risk
- ItemCan the Sutherlandia herb or resistance exercise reverse the stress inducing effects of a mild-intermittent stress procedure(Stellenbosch : University of Stellenbosch, 2006-03) Neethling, Ian Garth; Myburgh, Kathryn H.; Smith, Carine; University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences.This study aimed to assess the effect of mild psychological stress in male Wistar rats using incremental, intermittent stress on parameters of atrophy, including body mass, soleus and extensor digitorum longus (EDL) muscle mass, and mechanisms possibly contributing to atrophy. Serum corticosterone concentrations, 20s proteasome activity, glutamine synthetase (GS) and tyrosine amino-transferase (TAT) activities were determined. I also assessed whether Sutherlandia (Su) or resistance exercise was able to reverse the effects of stress on any of these parameters.
- ItemCardio-metabolic effects of anti-retroviral treatment in the Cape Winelands region of South Africa(Stellenbosch : Stellenbosch University, 2016-03) Abaid, Faten E. Bashir; Essop, M. Faadiel; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Background: Although highly active antiretroviral therapy (HAART) has significantly improved the survival of human immunodeficiency virus (HIV)-infected patients there are increased concerns regarding the onset of co-morbidities (e.g. cardio-metabolic complications) and mortalities. Although South Africa is burdened with the highest number of HIV-infected individuals globally, there is a relative paucity of data regarding potential links between HIV infection, HAART and cardio-metabolic risk/onset. Methods: This cross-sectional study therefore investigated the prevalence of cardio-metabolic risk factors in HIV-infected individuals within the Cape Winelands region of South Africa. Here we collected anthropometric, biochemical and lifestyle-related data for HIV-positive HAART naive (n =25) and HIV-positive individuals on HAART (n = 50) patients (20–55 years old) at the Worcester Community Day Centre (CDC) (Worcester, Western Cape, South Africa) during 2014 and 2015. Subjects on HAART were further divided into two sub-groupings, i.e. first line (n = 25) and second line treatments (n = 25). Results: Our data reveal the relatively high prevalence of traditional, cardio-metabolic lifestyle risk factors in HIV-infected individuals. There was a relatively high prevalence of smoking, i.e. 88% for the HIV-positive HAART naive group and 27% for the HIV-positive group on HAART (P = 0.001), while more than half of the HIV-positive individuals exhibited a positive history of familial cardiovascular diseases (CVD). There were no significant differences for fasting blood glucose (FBG) and insulin levels between HIV-positive HAART naive and HIV-positive on HAART. Lipid metabolite analyses (Total cholesterol [TC], low-density lipoprotein [LDL], high density lipoprotein [HDL] and triglyceride [TG]) also did not reveal significant changes when comparing HIV-positive on HAART versus HIV-positive HAART naive groups. However, additional analyses (using established cut-off values for HDL, LDL) showed a significant difference in the proportion of individuals categorized with ‘’low HDL’’ status, i.e. 68% for the HIV-positive HAART naive compared to 40% for the HIV-positive on HAART group (P =0.022). HAART also enhanced anthropometric measures of obesity, with significant differences for weight gain, triceps skin fold (TSF), biceps skin fold (BSF), waist circumference (WC) and mid-upper arm circumference (MUAC) between the naive and HAART groups. This applied similarly for first and second line treatments. Conclusion: The study established the prevalence of several traditional lifestyle CVD risk factors in both HIV-positive naive and HIV-positive on HAART in the Cape Winelands region of South Africa. HAART enhanced several measure of weight gain and lipid profile, suggesting a restoration to health and well-being. However, there was a relatively high prevalence of obesity in the HIV-positive on HAART group (especially females) thus placing them at a greater risk for the onset of future cardio-metabolic complications. We are unable to distinguish whether this risk is due to HAART or lifestyle-related risk factors, and this question requires further investigation. The findings of this study indicate that clinicians should be attentive of lifestyle-related CVD risk factors in HIV-positive persons and make an effort to counsel patients to adopt improved lifestyle choices.
- ItemThe cellular response of triple-negative breast cancer to short-term starvation: implications for chemosensitivity(Stellenbosch : Stellenbosch University, 2021-03) Prangley, Charne; Engelbrecht, Anna-Mart; Davis, Tanja; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Introduction: Breast cancer is currently the most common cancer among women globally. Triplenegative breast cancer (TNBC) is an aggressive and often drug-resistant sub-type of breast cancer that is correlated with poor patient outcomes. As a result, adjuvant therapies that may improve drug sensitivity are currently being sought. Due to the unique metabolic hallmarks of cancer, metabolic adjuvant therapies have become an area of increasing interest. We therefore set out to investigate the effect of short-term starvation (STS) on the growth, viability, and metabolism of TNBC cells and a benign breast epithelial cell line. We also investigated the effect of STS on chemotherapyinduced cytotoxicity in these cells to determine whether STS may enhance the effect of doxorubicin in TNBC. Methods: Three cell lines were utilised for this study: a benign breast epithelial cell line (MCF- 12A), and two triple-negative breast cancer cell lines (BT-549 and MDA-MB-231). Western blotting was employed to determine the effect of starvation over time on growth and proliferation signalling pathways (PI3K/Akt) and markers of autophagy (Atg5, p62 and LC3-II). Immunocytochemistry was utilised to quantify autophagic puncta. Cell cycle progression and viability were assessed using flow cytometry and a WST1 assay, respectively. The effect of STS on chemosensitivity was then established by incubating cells in standard or starvation-mimicking media for 24 hours, whereafter they received doxorubicin at a concentration of 2.5 μM. Chemosensitivity was then established in terms of live cell number, cell death and viability, and cell cycle progression. Results and Discussion: In response to STS, the MCF-12A cells downregulated pro-growth signalling pathways, while the MDA-MB-231 cells showed significant upregulation. A 24-hour starvation period had no significant effects on these pathways or on autophagic flux in the BT-549 cells. Both the MCF-12A and MDA-MB-231 cell lines significantly upregulated autophagic flux in response to STS, with the latter achieving the most significant effect at 24-hours. This may have offered protection to these cells, as a period of starvation prior to drug administration reduced doxorubicin-induced G2/M arrest. Additionally, STS had no other significant effects on chemosensitivity in these cells. In the BT-549 cells, however, starvation was able to significantly increase the percentage of dead cells in the group that received STS prior to doxorubicin treatment. As autophagy was not significantly increased in this cell line during starvation, this suggests that autophagy may indeed play a role in drug resistance. Conclusion: In summary, the cell lines which displayed an upregulation of autophagy at 24 hours of starvation were not sensitised to doxorubicin in terms of cell death, and also experienced amelioration of doxorubicin-induced G2/M arrest. This supports the notion that autophagic upregulation may protect cancer cells from doxorubicin-induced cytotoxicity and contribute to drug resistance. However, to gain a more thorough understanding of this phenomenon, future studies investigating the mechanisms by which autophagy promotes chemoprotection are recommended.
- ItemChanges in acetylcholine receptor expression : neuromuscular junction morphology and associated myonuclei in BALB/C mice following muscle contusion injury(Stellenbosch : Stellenbosch University, 2015-03) Louw, Elizabeth Adrienne; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Contusion injuries cause significant muscle damage, that effects skeletal muscle in its entirety, including the innervating motorneuron and the myofibre’s neuromuscular junction (NMJ). Upon injury, the acetylcholine receptors (AChR) scatter and disintegrate yet reaggregate over time to re-create an optimally functioning motor end-plate. This process involves the upregulation of the receptor subunits’ – α, β, γ, δ, ε – expression to varying degrees. Satellite cells are key role players in muscle regeneration, but studies linking the regenerative roles of satellite cells to the rehabilitating NMJ are limited. Moreover, the majority of studies on acetylcholine receptors investigate the effects of a denervation event rather than an injury that affects the muscle tissues in their entirety. Bromodeoxyuridine (BrdU) is a useful tool in labelling and tracking proliferated satellite cells. Two experimental groups (referred to as PCR and BrdU group) of male BALB/C mice were subjected to a hind limb contusion injury induced with the mass-drop technique. Alzet® mini-osmotic pumps delivering BrdU (50 mM, 1.0 μl.h−1 release rate) were inserted into the BrdU group prior to injury. Animals were sacrificed at days 1, 3, 5, 7, 10 and 14 post injury. Both injured and contralateral, non-injured gastrocnemius muscles were collected. qPCR was performed for AChR-γ and AChR-ε mRNA expression on the muscles of the PCR mice. Muscles from the BrdU group were cryo-sectioned in longitudinal orientation and stained with 1) H&E and 2) immunohistochemically with α-bungarotoxin to visualise AChRs; and also with antibodies against laminin and BrdU. Images were obtained by light microscopy (1) to detect and describe contusion injury in longitudinal section and confocal microscopy (2) to observe the form, prevalence and arrangement of NMJs along both the injured and non-injured muscle. AChRs position themselves into junctional folds that adopted a coral-like appearance – identifiable as a NMJ. A 3D z-stack image at 40x magnification revealed myonuclei residing beneath the NMJ in intimate connection. These NMJ were arranged along the muscle in central band; however contusion injury resulted in a disintegration of part of or the entire junctional complex. Super-resolution microscopy revealed in depth structural arrangement in the intact NMJ. This became jagged and dispersed following contusion injury, by 7 days. Robust, regenerating NMJs were detected in muscle sections at 14 days post injury. Surface area and volume were measured and revealed a trend towards a decrease in NMJ size at 7 days post injury, followed by an exaggerated increase in NMJ size by day 14 post injury. A two-step staining procedure exposed BrdU+ cells residing beneath the neuromuscular junction at 14 days post injury. The results of this study show that NMJ morphology is indeed affected by muscle contusion injury, and repairs itself by increasing its AChR subunit production. We explored novel techniques for analysis of neuromuscular morphology and its changes after injury and during regeneration. We have also ascertained the migration of satellite cells to beneath the NMJ following contusion injury. These findings lay the foundation for future research to better understand the role players involved in neuromuscular regeneration.
- ItemCircadian rhythms as novel chemotherapeutic strategies for breast cancer(Stellenbosch : Stellenbosch University, 2014-12) Mitchell, Megan Irvette; Engelbrecht, Anna-Mart; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Introduction: Mammalian circadian rhythms form an integral physiological system allowing for the synchronisation of all metabolic processes to daily light/dark cycles, thereby optimising their efficacy. Circadian disruptions have been implicated in the onset and progression of different types of cancers, including those arising in the breast. Several links between the circadian protein Per2 and DNA damage responses exist. Aberrant Per2 expression results in potent downstream effects to both cell cycle and apoptotic targets, suggestive of a tumour suppressive role for Per2. Due to the severe dose limiting side effects associated with current chemotherapeutic strategies, including the use of doxorubicin, a need for more effective adjuvant therapies to increase cancer cell susceptibility has arisen. We therefore hypothesize, that the manipulation of the circadian Per2 protein in conjunction with doxorubicin may provide a more effective chemotherapeutic strategy for the treatment of breast cancer. The aims of this project were thus to: (i) Characterize the role of Per2 in normal breast epithelial cells as well as in ER+ and ER- breast cancer cells; (ii) to determine the role of Per2 in doxorubicin-induced cell death, (iii) to determine the role of Per2 in autophagy and finally (iv) to assess whether the pharmacological inhibition of Per2 with metformin, can sensitize chemo-resistant MDA-MB-231 breast cancer cells to doxorubicin-induced cell death. Methods: An in vitro model of breast cancer was employed using the normal MCF-12A breast epithelial, estrogen receptor positive (ER+) MCF-7 and estrogen receptor negative (ER-) MDA-MB-231 breast adenocarcinoma cell lines. Circadian rhythmicity of Per2 protein expression was determined using western blotting, and Per2 cellular localization was assessed using fluorescent confocal microscopy. Per2 was then silenced by means of an endoribonuclease-prepared siRNA, and silencing efficiency was determined with the use of western blotting. The roles of Per2 in doxorubicin-induced cell death and autophagy were assessed by treating MDA-MB-231 breast cancer cells under the following conditions (1) Control, (2) 2.5 μM doxorubicin or 10 nM bafilomycin A1 (3) 30 nM esiPer2 and (4) 30 nM esiPer2 in combination with 2.5 μM doxorubicin or 10 nM bafilomycin A1. Following treatments cell viability was assessed using the MTT assay, western blotting for markers of apoptosis including p-MDM2 (Ser166), p-p53 (Ser15), cleaved caspase-3 and –PARP as well as markers of autophagy (AMPKα, mTOR and LC3). Furthermore, cell cycle analysis, G2/M transition and cell death (Hoechst 33342 and propidium iodide staining) were assessed by means of flow cytometry. The pharmacological inhibition of Per2 was achieved by treating MDA-MB-231 cells with 40 mM metformin as well as in combination with 2.5 μM doxorubicin. MTT cell viability assays, cell cycle analysis (flow cytometry) and western blotting for apoptosis (Per2, p-AMPKα (Thr172), p53, caspase-3 and PARP) were assessed. Results and discussion: A circadian pattern of Per2 protein expression was observed in the normal MCF-12A and MDA-MB-231 cancer cells with protein levels peaking at ±700% and ±500% of baseline was observed. However, no rhythmic expression was observed in the MCF-7 cancer cells. Immunostaining for Per2 showed localization OF Per2 in the cytoplasm as well as in the nucleus of both the MCF-12A and MDA-MB-231 cells. Concentration curves showed a significant reduction in cell viability following 2.5 μM doxorubicin treatment for 24 hours. Per2 protein expression was significantly reduced with both esiPer2 and metformin treatment. Silencing of Per2 in combination with doxorubicin treatment resulted in cell cycle arrest with a significant increase in apoptosis, indicating that Per2 silencing effectively sensitized the MDA-MB-231 cancer cells to the anti-carcinogenic properties of doxorubicin. Modulation of Per2 protein expression was effectively achieved with the use metformin although this decrease occurred independently of AMPKα phosphorylation. A significant increase in apoptosis was observed following treatment with metformin in combination with doxorubicin treatment. However, no changes in cell cycle regulation were observed. Per2 appears to be involved in the regulation of autophagy as a significant increase in autophagy flux was observed when Per2 was silenced. Additionally, this increase in autophagic flux resulted in a significant increase in MDA-MB-231 cancer cell death which was enhanced further when autophagy was inhibited with bafilomycin A1 subsequent to Per2 silencing. Conclusions: Per2 protein expression was shown to display a 24 hour circadian rhythm in the MCF-12A cells, and to a lesser extent in the MDA-MB-231 cells. However, the MCF-7 cells failed to show rhythmic changes in Per2 protein expression. Per2 was shown to be located predominantly in the cytoplasm, with nuclear localization observed when cytoplasmic fluorescent intensity was lower. Per2 silencing effectively sensitized the chemo-resistant MDA-MB-231 breast cancer cells to both doxorubicin-induced cell death and autophagic inhibition.
- ItemThe comparison between two high-intensity interval training protocols on skeletal muscle and satellite cell dynamics(Stellenbosch : Stellenbosch University, 2019-03) Sugden, Cameron; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Introduction: High intensity interval training (HIIT) interventions are popularly used by endurance athletes to increase muscle strength, peak speed and aerobic capacity. Running involves the use of both eccentric and concentric contractions, with the level of the running surface determining the ratio between the two. Downhill running is considered eccentric- biased exercise. Conversely uphill running is considered concentric-biased exercise. Uphill running and downhill running are therefore two different role players in muscle adaptation, although potentially both act through regulating satellite cell (SC) dynamics. Hypothesis: The different modes of HIIT will result in differing skeletal muscle damage, satellite cell activity and morphological adaptation, resulting in differing muscle adaptation, aerobic capacity, muscle strength and running performance. Methods: 12 healthy active males were randomized into either a downhill running (DHG) or an uphill running group (UHG). Subjects underwent baseline and post training performance testing which consisted of a flat VO2max treadmill test, maximal isometric strength test and a 5km road time trial. Training consisted of 10 HIIT sessions over a period of 4 weeks. Each session consisted of 6 intervals at either +5% gradient and 80% peak treadmill speed or -10% gradient and 90% peak treadmill speed. Muscle biopsies and blood draws were taken at baseline, as well as 6 hours after the first and the last session. Results: Performance testing: The UHG, but not the DHG, improved VO2max from baseline (59.48 ± 1.73 ml.kg.min-1 – 61.86 ± 1.28 ml.kg.min-1). The DHG, but not the UHG, improved maximal isometric after the 4 weeks of HIIT (734 ± 133 N - 893 ± 55 N). Both groups improved their 5km TT performance by 3.5 ± 1%. The DHG but not the UHG had a significant increase in CK levels 6 hours after running (p < 0.05). Muscular response to 4 weeks HIIT for the DHG included an increased CSA (p < 0.05), increased SC pool size (0.1 ± 0.001 SC/fibre - 0.3 ± 0.02 SC/fibre), and an increase in myoD after the first bout of exercise (p < 0.05). Muscular adaptations in the UHG included increased capillary to fibre ratio (1.76 ± 0.18 – 2.55 ± 0.20) and capillary density (249 ± 39 mm2 – 304 ± 57 mm2) with training. Conclusion: Four weeks of uphill or downhill HIIT resulted in physiological adaptation by different mechanisms, one by enhanced SC activity and a more forceful contraction and the other involving muscle perfusion and oxygen utilization. The mechanisms of adaptation are training specific, yet they both result in a similar improvement in 5km race performance.
- ItemA comparison of compounded-bioidentical hormone formulations versus FDA-approved hormone formulations in breast cancer progression(Stellenbosch : Stellenbosch University, 2023-03) Mochoele, Kamano Angela; Engelbrecht, Anna-Mart; Africander, Donita; Du Plessis, Manisha; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Introduction: Oestrogen and oestrogen receptor-induced signalling plays an important role in breast cancer development and progression. Studies have shown that certain menopausal hormone therapies {MHTs} containing oestrogens and oestrogens in combination with progestogens, increase the risk of invasive breast cancer. Compounded-bioidentical hormone therapies {cBHTs}, not FDA-approved or regulated by the Medicines Control Council of South Africa, have become a popular MHT and are advertised as safer efficient alternatives. Oestrogen alone and in combination with progestogens such as medroxyprogesterone acetate {MPA} and norethindrone {NET} enhance breast cell proliferation, migration and invasion. lt is therefore important to determine the effects of compounded oestrogen formulations in the development and progression of breast cancer. This study aims to provide a comparative profile of the effects of traditional menopausal therapies {estrone + MPA and estrone + NETA}, an FDA-approved bioidentical formulation {oestradiol + progesterone {bE2+bP4}} with the compounded bioidentical biest hormone formulation E2 + estriol {bE2+bE3} on the progression of breast cancer. Methods: Human ER+ mammary adenocarcinoma cells {MCF7} were used. Proliferation was assessed by determining the cell viability through water-soluble tetrazolium salt {WST-1} assays. The cell cycle was analysed with flow cytometry. Western blot analyses were performed to assess the proliferation marker MCM2, the Pl3K/Akt signalling pathway and epithelial-to-mesenchymal transition {EMT} markers; E-cadherin, N-cadherin, Snail and β-catenin. Migration was measured through a wound healing assay. Results and discussion: All treatment combinations significantly increased cancer cell viability. The cell cycle analysis shows that FDA-approved estrone + MPA and estrone + NETA treatments induced the accumulation of MCF7 cells in the GO/Gl phase of the cell cycle. Western blot analysis revealed that all hormone treatments did not activate the Pl3K/Akt pathway. Furthermore, treatment of BE2 + BP4 indicated mesenchymal characteristics of EMT. The wound closure assay showed that the hormone treatments did not induce migration. Conclusion: According to our findings, there are both similarities and differences among the compounded biest combinations and FDA-approved hormone formulations. Concerningly, cBHT increases cell viability in a manner consistent with the FDA-approved formulations. Similar to FDA- approved therapies, they did not cause migration or activate the Akt pathway for cell proliferation. In contrast, when compared to their FDA-approved counterparts, cBHT formulations exhibited different effects on EMT and the cell cycle. All together these results demonstrate that cBHT treatments did not stimulate the pathways associated with breast cancer progression that was stimulated by the FDA-approved formulations. Future recommendations include investigating the effects of cBHT preparations on other pathways involved in breast cancer initiation and progression in comparison to the FDA-approved formulations.
- ItemA comparison of the wound healing dynamics in an acute and newly developed chronic wound model using Neutral endopeptidase, an inhibitor of Substance P.(Stellenbosch : Stellenbosch University, 2020-03) Boodhoo, Kiara; Van de Vyver, Mari; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Chronic wounds affect millions of people with diabetes world-wide. Various diabetic wound healing animal models exist, none of which currently replicate chronic wounds in diabetic patients. Animal models that do not closely resemble human conditions delay application of promising therapeutic strategies in clinical settings. Substance P is a neuropeptide that promotes wound healing by binding to neurokinin-1 receptor initiating the inflammatory process. Neutral endopeptidase (NEP) is a zinc metalloprotease that degrades substance P, thereby hindering the healing process. Increased NEP enzymatic activity is evident in chronic wounds of diabetic patients unlike in current animal models. This study aimed to determine the optimum dose of NEP for injection into wound edges in obese pre-diabetic mice (B6.Cg- Lepob/J, ob/ob) in order to create a chronic non-healing wound. Thereafter, the study investigated wound healing dynamics of this chronic non-healing wound compared to an acute wound treated with saline. In addition to a saline control, three concentrations of NEP [low NEP (0.33 mg/μL), medium NEP (0.68 mg/μL) or high NEP (1.02 mg/μL)] were used to obtain a dose-response curve. NEP activities did not differ between treatment groups however, substance P was lower in the wound areas for high NEP compared to low NEP groups. Therefore, less substance P was present to initiate wound healing with high NEP. MMP-9 was higher with high NEP treatment on day 2 compared to saline, low and medium NEP groups. Similarly, the cytokine profile within pooled samples of day 2 wound fluid was lowest with high NEP treatment. Therefore, high NEP application did mimic some characteristics of a chronic wound during the early stages post wounding, including delayed onset of inflammation. When comparing the healing dynamics of this chronic non-healing wound, induced by high NEP, to an acute wound [wild-type (C57BL/6J) saline-treated mice] that follows the normal progression of healing, the chronic wound displayed delayed wound closure (day 7), significantly greater MMP-9 expression (day 0, 2 and 7) with no formation of granulation tissue, re-epithelization and angiogenesis (day 7). Proteomic analysis of the acute saline-treated and chronic high NEP wounds at day 2 indicated significant differences in expression of proteins such as Stefin-1, Stefin-3, Microtubule-associated protein 1B, Band 4.1-like protein 2, Caveolae-associated protein 1 and Gamma-synuclein. These proteins have not been previously described as involved in wound healing and may be directly involved or have downstream interactions in the wound healing processes. This study identified two proteins previously described as playing a role in wound healing, via their involvement in inflammation (Alpha-1- acid glycoprotein 1) and proliferation and remodelling (Nidogen-1). In conclusion, high NEP administration in the wound boarders of obese pre-diabetic mice resulted in a chronic wound model that better mimics human diabetic chronic wounds, which could be used to test various treatment strategies. High NEP administration resulted in less wound closure that could be explained by higher MMP-9, lower cytokine content in wound fluid and differences in several proteins identified by proteomic analysis.
- ItemThe detection of stress-related diseases: establishment of two unique methods to discover circulatory phospho- and glycoproteins(Stellenbosch : Stellenbosch University, 2023-12) Smith, Logan Jason; Essop, M. Faadiel ; Joseph, Danzil; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Introduction: Psychosocial stress has strong links to numerous chronic diseases related to the dysregulated activation of the physiological stress system. This heightens the burden of mortality as there is a robust relationship between chronic psychosocial stress and non- communicable diseases. Hence there is a robust impetus for the identification of novel, circulating biomarkers to earlier detect stress-related chronic diseases. Although protein post-translational modifications such as glycosylation and phosphorylation can act as putative markers of pathophysiology, their relatively low abundance complicates extraction and identification from samples with a high dynamic range. The main aim of this study was therefore to establish two unique enrichment methods for circulatory glycoprotein and phosphoprotein extraction that would then be applied in a preclinical model of chronic psychosocial stress. Methods: Phosphoprotein enrichment was performed using functionalized magnetic particles while glycoprotein enrichment occurred using lectin-bound magnetic particles. Both these methods were tested using a known purified phosphorylated and glycosylated protein and compared to bottom-up proteomics methodology using rat serum. The latter was obtained from a rat model of chronic stress that is well-established in our laboratory (n = 16 controls versus n = 16 stressed rats). These were randomly selected for proteomics analysis to assess the efficiency of retrieval in enriched versus unenriched samples. Fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and proteins visualized using Coomassie and specific fluorescent staining. Here, the relevant Pro-Q stains were employed for the identification of glycosylation and phosphorylation, respectively. The coupling of such enrichment methods to LC – tandem mass spectrometry (MS/MS) was enabled by employing various preparation steps such as deglycosylation and digestion. An exogenous protein was also included as part of the sample preparation to ensure quality control analysis of the LC-MS/MS experiment. Results: SDS-PAGE analyses and staining methods revealed non-specific enrichment with regards to intact protein retrieval. In addition, LC-MS/MS data demonstrated that enrichment using the current set of affinity materials was inadequate for glycopeptide and phosphopeptide retrieval in serum. Conclusion: A lack of enrichment indicates that stringent sample preparation is needed for biological materials with a high dynamic range. This may also be due to the porous nature of both materials employed for phospho- and glycoprotein/peptide enrichment respectively. A combination of enrichment and/or depletion methods may therefore be beneficial for deeper analysis of the blood proteome. These enrichment techniques and the subsequent sample preparation still require further optimization to derive more definitive conclusions in the chronic stress context.
- ItemDeubiquitination : does the answer to cardiotoxicity lie within the ubiquitin proteasome pathway(Stellenbosch : Stellenbosch University, 2017-03) Ogundipe, Temitope R.; Sishi, Balindiwe J. N.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Introduction: Cardiotoxicity, a complication that arises from anthracycline use is one that has confounded scientists for decades. Attempts have been made to attenuate the development of this condition through the use of anti-oxidants with little success and this has led to calls for new adjuvant therapies. One area that has been identified as a potential intervention involves the ubiquitin proteasome system (UPS) and its regulation and degradation of proteins that control mitochondrial morphology, apoptosis and cellular anti-oxidants. This process can be reversed through the use of de-ubiquitinating enzymes (DUBs); however their role in this context is relatively unknown. Therefore, this study aimed to investigate the role of specific DUBs relevant in this context and whether the manipulation of their protein expression levels will be beneficial. Methods: Chronic doxorubicin (DOX)-induced toxicity was induced in H9C2 cardiomyoblasts and male Sprague-Dawley rats for 120 hrs (0.2 μM) and eight weeks (2.5 mg/kg/week) respectively. Baseline protein expression of DUBs as well as their down-stream factors was determined by western blotting on both models. Immunocytochemistry was undertaken only for in vitro studies. DUBs were down-regulated using SiRNA, and the subsequent effect on downstream proteins was determined through western blotting. Mitochondrial morphology was evaluated by fluorescence microscopy, while cellular toxicity and ATP production were assessed using a mitochondrial toxicity assay. Results and Discussion: DOX increased the expression of USP9x (103.7 ± 4.7%, p<0.01), which regulates MCL1 (long-fragment), an anti-apoptotic protein which was down-regulated in this scenario. Interestingly, the pro-apoptotic short-fragment of MCL1 was up-regulated, suggesting a mechanism by which DOX uses USP9x to promote apoptosis. DOX treatment also reduced USP30 expression (27.5 ± 3.7%, p<0.01), as well as its downstream target, the mitofusin proteins (22.7 ± 5.9%, p<0.001) which regulate mitochondrial fusion during mitochondrial dynamics. USP36 showed little variation between the two groups however, DOX reduced SOD2 expression (250.9 ± 6.8%, p<0.001). While both models utilised produced similar results, there was minor variation in the results. When DUB (SiRNA) was initiated in the presence of DOX, mitochondrial morphology appeared to improve. Interestingly, while the known-down of some DUBs (USP30 and USP36) did not modify mitochondrial toxicity except when USP9x was abolished, ATP synthesis was significantly upregulated in all intervention groups when compared to DOX treatment alone. Although more research into this topic is urgently needed, it is clear from the positive results obtained above that de-ubiquitination may be a mechanism that can be exploited as a potential treatment strategy in this context.
- ItemDevelopment of a transendothelial shuttle by macrophage modification(Stellenbosch : Stellenbosch University, 2016-12) Visser, Johan Georg; Smith, Carine; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Background: Targeted stem cell delivery via macrophage modification is a novel and relatively non-invasive therapeutic intervention. Monocytes circulate through the vasculature and infiltrate damaged tissue in response to chemotactic signalling. Here they differentiate into functionally different macrophage phenotypes - the classically activated pro-inflammatory (M1) or alternatively activated anti-inflammatory (M2) phenotypes. The M1 macrophages are able to cross endothelial barriers, while the M2 anti-inflammatory macrophages are unable to transverse endothelium and instead remain tissue associated. The focus of our research was to produce M1 macrophages in vitro that could transverse endothelium while carrying engulfed stem cells, in order to deliver more stem cells in a relatively short time, to any injured tissue to facilitate recovery. Methods: Primary isolated monocytes were cultured with Granulocyte Monocyte Colony-Stimulating Factor, Lipopolysaccharide and Interferon gamma for 6 days to pre-differentiate them into M1 macrophages. Cells were treated with a Wortmannin-Concanamycin A-Chloroquine cocktail to achieve phagosome maturation arrest and thus preserve ingested cells in a viable state. Preservation of engulfed stem cells (simulated with fluorescent latex beads covalently labelled with IgG antibody) was qualitatively and quantitatively determined by flow cytometry and live cell imaging, respectively. Bead-containing macrophages were co-cultured with HUVEC cells in a Transwell system, and exposed to Monocyte Chemoattractant Protein 1 (added to bottom well) to determine migration capacity. Results: Monocytes were differentiated into M1 classically activated macrophages. The majority of these cells (68.67±3.51%) were able to engulf opsonised beads after successful induction of phagosome maturation arrest – a capacity similar to that of untreated cells (61.19±4.68%). Ingested beads were preserved within macrophages for the duration of our protocol (2 hours), determined by retained red antibody signal on beads and perturbed phagosome acidification. 72.86±16.0 phagosome maturation arrested macrophages were able to transverse a HUVEC coated membrane with 8 μm diameter pores (simulated endothelial layer), while only 70.14±12.6 cells per well migrated when carrying a bead cargo. Conclusion: A delivery system capable of engulfing, preserving and delivering cargo was successfully induced. Further optimisation of this technique could lead to translation into a novel method for delivery of stem cells with regard to regenerative medicine and may even be used as a drug delivery system for the treatment of various malignancies.
- ItemDissection of autophagy machinery and protein cargo flux(Stellenbosch : Stellenbosch University, 2020-04) De Wet, Sholto; Loos, Ben; Stellenbosch University. Faculty Science. Dept. of Physiology.ENGLISH ABSTRACT: Introduction: Neurodegenerative diseases, such as Alzheimer’s disease, are characterised by the increased histological expression of insoluble protein aggregates. Evidence has demonstrated that the soluble protein oligomers which constitute these insoluble protein aggregates, are a major source of neurotoxicity. Autophagy is known to be a major protein degradative pathway and has been shown to be active during neurodegenerative diseases. In the past, macroautophagy has been described as a non-specific means of degrading long-lived cytoplasmic proteins. However, recent evidence has demonstrated that various subtypes of macroautophagy (hereafter autophagy) exist, distinguished from one another by their preferred cargo. Of particular interest in the context of neurodegenerative diseases is regulating and controlling protein degradation through autophagy. p62 has been shown to be the preferred autophagy cargo receptor during protein degradation. In addition to p62, NBR1 has been shown to be another autophagy cargo receptor of proteins and has been shown to compensate for a loss of p62 expression levels. How these two receptors behave with respect to one another to support effective degradation over a 24 hour period of autophagy induction is unknown. Additionally, it is unknown to what extent these receptors interact with autophagosomes to contribute toward effective autophagy flux. Aims and methods: The aim of this study was to investigate the changes that occur to components of the autophagy system within an autophagy model system established in mouse embryonic fibroblasts (MEFs) not expressing any disease symptoms. MEFs were micropatterned as a means of standardising cell shape and size. Autophagy changes were induced using Rapamycin and Spermidine, respectively at relatively high and low concentrations. Studies were conducted over 24 hours to understand what impact time had on autophagy and its components. Western blotting was used to measure the abundance changes of LC3-II, p62, NBR1 and LAMP2a. Additionally, fluorescence microscopy was used to observe GFP-LC3, p62 and NBR1 puncta counts. Furthermore, studies were done in the presence and absence of Bafilomycin A1, an inhibitor of autophagosome/ lysosome fusion, to better understand the clearance activities of each protein. Results and discussion: Initial investigations using western blotting techniques demonstrated that Rapamycin caused an increase in LC3-II abundance levels but does not change receptor levels. Additionally, Spermidine treatment caused an increase in autophagosome clearance and receptor abundance but does not change receptor clearance levels. Fluorescence microscopy imaging revealed that autophagy induction with 1 μM Rapamycin caused an increase in GFP-LC3 and receptor puncta count 2 hours after incubation. However, no change was seen in the receptor clearance as was shown by the lack of co-localised puncta clearance. 10 nM Rapamycin on the other hand demonstrated fewer autophagosomes, however; effective receptor turnover, was demonstrated, especially of p62. Spermidine results demonstrated different behaviours, as 20 nM Spermidine showed a slower increase in GFP-LC3 than 1 μM Rapamycin, but demonstrated highly effective p62 clearance at 2 hours, followed by effective NBR1 clearance at the same time and at 8 hours, where p62 turnover was found to be at its lowest. 5 nM Spermidine did not induce the system in the same way as 20 nM Spermidine as was seen by less effective GFP-LC3 turnover. However, 5 nM Spermidine did demonstrate effective p62 clearance at 8 hours as well as effective co-localised puncta clearance at 2 hours and 8 hours of treatment. Conclusion: The means by which autophagy is induced, either by mTOR-dependent or –independent inducers, has an impact on autophagy components expression levels. Furthermore, treatment with higher concentrations of drugs demonstrated a more robust and immediate response of the autophagy components measured as well as their clearance. Conversely, lower drug treatment concentrations demonstrate different times of effectiveness. Taken together this study has shown that the effectiveness of autophagy flux is multifactorial and should be adjusted according to the autophagosome as well as receptor involvement for future research to be successful.