Doctoral Degrees (Physiological Sciences)

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Anthracycline-induced cardiotoxicity : the role of proteolytic pathways
(Stellenbosch : Stellenbosch University, 2012-03) Sishi, Balindiwe J. N. (Balindiwe Jennifer Nonkosazana); Engelbrecht, Anna-Mart; Loos, Benjamin; Van Rooyen, Jacques; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Introduction: The anthracyclines (ACs), daunorubicin (DNR) and doxorubicin (DXR) are two of the most effective drugs known for the treatment of systemic neoplasms and solid tumours. However, their clinical use is often hampered by their dosedependent cumulative cardiotoxicity, which leads to irreversible and fatal druginduced congestive heart failure. The mechanism by which ACs induces heart damage is not fully understood. Recent reports have indicated that DXR activates autophagy and ubiquitin proteasome-mediated degradation of specific transcription factors, however, no reports exists on the effect of ACs on the E3 ubiquitin ligases, MuRF-1 and MAFbx. The aim of the first part of the study was therefore to investigate the effect of DNR treatment on the protein and organelle degradation systems in the heart and to elucidate the signalling mechanisms involved. Although this model was ideal in allowing the investigation of the signalling pathways which are affected by DNR, it did not allow for further exploration or manipulation of signalling pathways that may be of potential benefit in this context. The in vitro model was therefore used to validate the hypothesis that increased autophagy alleviates AC-induced cardiotoxicity and delays the onset of cardiomyocyte death. The aims for the second part of the study were (i) to characterize the effect of DXR in H9C2 cells, (ii) to determine whether the induction/inhibition of autophagy in combination with DXR alleviates cytotoxicity and (iii) to investigate the influence of increased/decreased autophagy in combination with DXR on reactive oxygen species (ROS) production, mitochondrial function, endoplasmic reticulum (ER) stress and the ubiquitin proteasome pathway. In the final part of this study, an in vivo model was used to assess the potential benefit of autophagy in a novel GFP-LC-3 tumour bearing mouse model of acute DXR-induced cardiotoxicity. Material and Methods: Adult rats were divided into two groups where one group received six intraperitoneal injections of 2 mg/kg DNR on alternate days and the other group received saline injections as control. Hearts were excised and perfused on a working heart system the day after the last injection and freeze clamped for biochemical analysis. H9C2s were cultured and treated with Bafilomycin A1 (10 nM, inhibitor of autophagy) for 6 hrs, Rapamycin (50 μM, inducer of autophagy) for 24 hrs, DXR (3 μM) for 24 hrs or a combination of these drugs. Following treatment, cells were harvested and assessed for cell death, proteolytic activity and oxidative stress using western blotting, fluorescence microscopy and flow cytometry. In the final phase of the study, twenty-four female mice were injected at 8 weeks with a mouse breast cancer cell line (EO771) and after observation of tumour growth, animals were either treated with one injection (i.p.) of Rapamycin (4 mg/kg), two injections (i.p.) of DXR (10 mg/kg) or a combination of the two drugs. After the experimental protocol, mice were terminated and their hearts were rapidly excised. The hearts were divided cross-sectionally and utilized for biochemical and histological analyses. Results and Discussion: DNR treatment significantly attenuated myocardial function and increased apoptosis in the ex vivo heart model. DNR-induced cardiac cytotoxicity was associated with the upregulation of two E3 ubiquitin ligases, MuRF-1 and MAFbx as well as a significant increase in two markers of autophagy, beclin-1 and LC-3. These changes observed in the heart were also associated with attenuation of the PI3-kinase/Akt signalling pathway. The augmentation of autophagy with rapamycin before DXR treatment significantly reduced cell death in the in vitro model. Indeed, rapamycin treatment demonstrated to be a vital survival mechanism for acute DXR-induced cardiotoxicity as it decreased cellular ROS production, improved mitochondrial function and prevented nuclear translocation of DXR. Moreover, these changes in cardiomyocytes were also associated with a reduction in the ubiquitin-proteasome pathway (UPP). In the final part of this study, a novel tumour bearing GFP-LC3 mouse model was developed to confirm the results obtained in the in vitro study. It was demonstrated that acute DXR-induced cardiotoxicity resulted in increased apoptosis, the inhibition of autophagy and increased proteolysis via the UPP. These findings were associated with a reduction in body weight and cardiomyocyte cross-sectional area. The cardiotoxic effects of DXR were substantially reduced when autophagy was induced with rapamycin. Taken together, our data strongly indicates that it is possible to attenuate the cardiotoxic effects of doxorubicin in cancer patients by carefully controlling the levels of autophagy using rapamycin as adjuvant therapy.
Assessment of Metabolic Therapy for Acute Heart Failure
(Stellenbosch : Stellenbosch University, 2017-03) Kimar, Charlene Patricia; Essop, M. Faadiel; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Introduction Acute heart failure (AHF) is the most common primary diagnosis for hospitalized heart disease cases in Africa. Increased fatty acid oxidation (FAO) with heart failure (HF) triggers detrimental effects on the myocardium, we hypothesized that diabetic rat hearts subjected to AHF display lower cardiac function vs. controls and that Trimetazidine (TMZ) (a partial FAO inhibitor) counters this effect. Aims 1)To establish an ex vivo AHF model for diabetic hearts; 2) Assess whether TMZ treatmentoffers cardioprotection to diabetic rat hearts subjected to an AHF protocol; and 3) Delineate underlying mechanisms by evaluating markers for oxidative stress, mitochondrial uncoupling, apoptosis and metabolic dysregulation. Methods Vehicle control male Wistar rats were injected with citrate buffer. To induce diabetes rats were administered streptozotocin (60 mg/kg) for one week vs. non-diabetic controls. Hearts were perfused on the Langendorff retrograde perfusion system for three phases: Stabilization - (11 mM glucose- non-diabetic, and 30 mM glucose- diabetic hearts) at 100 cm H2O (30 min); AHF – (1.5 mM palmitic acid, 2.5 mM glucose) at 20 cm H2O (35 min); and Recovery– (1.5 mM palmitic acid, 11 mM glucose or 30 mM glucose) at 100 cm H2O (30 min). 1 μM TMZ was administered at the start of recovery. In addition, we evaluated necrosis and infarct size by tetrazolium (TTC) staining at the end of the AHF phase. Western blotting was performed for markers of apoptosis (pBAD/BAD), oxidative stress (superoxide dismutase 2 [SOD2], conjugated dienes [CDs], thiobarbituric acid reactive substances (TBARS), reduced/oxidized glutathione [GSH/GSSG] analysis, oxygen radical absorbance capacity [ORAC]), mitochondrial uncoupling (uncoupling protein 2 [UCP2]) and metabolic dysregulation (advanced glycation end product [AGE] and polyol pathway analyses). We investigated direct effects of TMZ (1 μM) in H9c2 cardiomyoblasts exposed to 500 μM palmitate for 21 hours and assessed the effects of TMZ treatment on fatty acid-induced oxidative stress and apoptosis. Results Reduced function was seen for all groups in recovery vs. controls, while AHF-diabetic showed worse outcomes vs. AHF alone. TMZ treatment resulted in a robust increase in left ventricular developed pressure (LVDP) for diabetic hearts vs. controls. Infarct size assessment showed no differences. TMZ treated diabetic hearts also displayed lower AGE and higher polyol pathway activation vs. respective controls. However, several markers of the AGE pathway did not show any significant differences for any groups. Non-diabetic and diabetic hearts displayed increased oxidative stress (TBARS) compared to their counterparts. TMZ treatment resulted in anti-apoptotic effects in hearts subjected to AHF. TMZ exhibited antioxidant effects by lowering fatty acid-induced mitochondrial oxidative stress in cells. Conclusion This study successfully established a novel ex vivo model of AHF for the diabetic rat heart, and TMZ treatment resulted in cardioprotection for diabetic hearts. Our data suggest that TMZ may mediate some of its cardioprotective effects by acting as an anti-oxidant to lower myocardial oxidative stress triggered during AHF. The findings also indicate that TMZ treatment may lower the formation of damaging AGEs in the diabetic heart. TMZ therefore, emerges as a putative therapeutic target to be considered as sole and/or combined treatment (with more conventional drugs) for AHF patients.
The association between genotype and BMI, health and lifestyle indicators as well as weight loss outcomes in overweight/obese Caucasian adults
(Stellenbosch : University of Stellenbosch, 2011-03) Harbron, Janetta; Senekal, Marjanne; Zaahl, Monique; Kotze, Maritha J.; University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Genetic screening to improve obesity treatment outcomes is available despite the lack of conclusive evidence, specifically for Caucasian South Africans, in this regard. The aim of this study was to investigate the association between genotype (seven polymorphisms) and body mass index (BMI), health and lifestyle indicators in a cross-sectional sample of overweight/obese Caucasian adults (n=133), as well as the association between genotype and weight loss outcomes following an intervention (n=88) using a quasi experimental study design (time-series). The intervention consisted of a 24-week conservative weight loss programme that included dietary, physical activity and behavioural components. The primary null hypothesis for the cross-sectional sample, namely that there is no association between genotype and BMI, has not been rejected. A number of the secondary/exploratory hypotheses were rejected of which the most plausible associations (based on support by the literature and a physiological basis for the findng) are: 1) the mutant TT homozygotes of the GNB3 C825T polymorphism may have a higher risk to develop the metabolic syndrome (MetS) as they had significantly higher fasting triglyceride and glucose levels, a higher number of traits that met the diagnostic cut-off criteria for MetS and higher number of these subjects was diagnosed with MetS compared to the wild-type C-allele carriers; and 2) subjects with mutant alleles of either the FTO rs1421085 or rs17817449 polymorphisms may have poorer eating behaviours (a higher rigid control, habitual and emotional disinhibition, perceived hunger and internal locus for hunger) and higher intake of high-fat foods. The primary null hypothesis for the intervention sample, namely that there is no association between genotype and weight loss outcome, was not rejected for the FABP2 Ala54Thr, INSIG2 rs7566605, FTO rs1421085, ADRB3 Trp64Arg and GNB3 C825T polymorphisms. However, it was rejected in some instances indicating the following associations: 1) The wild-type TT homozygotes of the FTO rs17817449 polymorphism lost significantly more weight during the first two months of the program compared to the mutant allele carriers (this is a novel finding); 2) The wild-type Arg16Arg homozygotes of the ADRB2 Arg16Gly polymorphism lost significantly more weight during the first month of the program compared to the mutant allele carriers (this finding is supported by one other intervention study); 3) Subjects with a mutant C-allele of the INSIG2 rs7566605 polymorphism and a mutant Gly16-allele of the ADRB2 Arg16Gly polymorphism lost significantly less weight over the six month intervention period (this is a novel genegene interaction finding). A number of secondary/exploratory hypotheses were rejected, of which the most plausible finding include that the improvement in emotional disinhibition in the wild-type TT subjects of the FTO rs1421085 polymorphism was associated with a more pronounced decrease in BMI over the six month weight loss period. The integration of the results from this study with the literature indicates that there is insufficient evidence at this stage for genetic screening of the polymorphisms investigated in this study and the provision of evidence-based personalized recommendations for weight loss in obese individuals. It is recommended that these associations should be viewed as priority in future research.
Cell death in hyppxic injury : signaling mechanisms and dynamics in the decision making process
(Stellenbosch : University of Stellenbosch, 2009-12) Loos, Benjamin; Engelbrecht, Anna-Mart; University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Three main morphologies of cell death have been described in the diseased myocardium, type I, better known as apoptotic cell death, which is characterized by cell shrinkage and chromatin condensation, type II, or cell death with autophagy, presents a morphology with intracellular accumulation of autophagic vacuoles and type III, better known as necrosis, is characterized by cellular swelling and rapid loss in cellular membrane integrity. However, recent literature strongly argues against rigid classifications in the context of cell death mechanisms but rather suggests to adopt a view of cell death as a dynamic and integrative cellular response. Furthermore, the contribution of autophagy in cell death or cell survival is still poorly understood. Therefore the aims of this study were twofold: (i) to characterize the contribution of each cell death type in context of the severity and duration of an ischaemic insult and (ii) to determine whether manipulation of the autophagic pathway affects the contribution of cell death and translates into protection of the heart. Rodent derived cardiac myoblast cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and incubated under 5% CO2 conditions. Cells were submitted to protocols of 2, 4 and 8 hrs of simulated ischaemia (SI) under hypoxic conditions in a humidified environment containing 0.1% O2, 5% CO2 and the balance N2, followed by 1 hr of reperfusion respectively. We employed a modified ischaemic buffer containing either 2-deoxy- D-glucose, sodium dithionate or both, with the aim to create an ischaemic insult of mild (mild SI), moderate (moderate SI) and severe (severe SI) character respectively. We evaluated the contribution of each cell death mode using a combination of viability- and ATP assays. Molecular markers for each cell death process such as LC3, PARP and HMGB1 were evaluated using 3-dimensional fluorescence techniques as well as western blot analysis and flow cytometry. Next, autophagy was induced or inhibited prior to the ischaemic insult, using rapamycin and 3MA respectively, and similar parameters were evaluated after 2 hours of mild or moderate SI. Propidium Iodide exclusion and Fluorescence Resonance Energy Transfer (FRET) in combination with mitochondrial inner membrane depolarization were employed to assess the onset of cell death dynamically. Flow cytometry was employed to evaluate the degree of protection. In addition, the ATP levels and reactive oxygen species (ROS) were evaluated. Our results strongly indicate a differential induction of cell death, which is dependent on the severity and duration of the ischaemic insult. Mild SI led to the induction of autophagy and apoptosis, whilst moderate or severe SI induced both apoptotic and necrotic cell death without an indication of autophagy. Only mild SI, but not moderate and severe SI, resulted in an ATP surge. Moreover, our data provide direct evidence that increased autophagy delays the loss of cellular membrane integrity and delays caspase-3 activation as well as mitochondrial depolarization in ischaemic cardiomyocytes. Our results show a profound effect of increased autophagy on the onset of apoptosis as well as necrosis under simulated ischaemic conditions, providing cellular protection. This ATP surge observed during mild SI was abolished with increased autophagy. Furthermore, our results indicate a profound effect of autophagy on ROS generation. Under normoxic conditions, increased autophagy induced a significant decrease in ROS while the inhibition of autophagy significantly increased ROS generation. However, when increasing or decreasing autophagy prior to the ischaemic insult, ROS increased significantly in both scenarios. The results suggest that the severity of ischaemia determines the mode of cell death differentially. An increase in autophagic responsiveness and flux, as induced through rapamycin treatment, provides a selective advantage for tissue against injury, possibly by maintaining intracellular ATP levels through the provision of metabolic substrates. Autophagy is described as an inherent cellular mechanism v which affects the onset of cell death and exhibits protective effects in the ischaemic myocardium when upregulated prior to the ischaemic insult. The protective effect of increased autophagy was mirrored in the isolated perfused rat heart model, reflected by improved functional recovery during ischaemia/reperfusion.
Chemoresistance in a breast cancer animal model: the role of obesity and inflammation
(Stellenbosch : Stellenbosch University, 2019-12) Mentoor, Ilze; Nell, Theo A.; Engelbrecht, Anna-Mart; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Background: Globally an increase in obesity incidence represent a major health concern amongst a rising incidence of impaired treatment outcomes of breast cancer patients. Adipose tissue and/or adipocytes, in the tumour microenvironment serve as an exogenous energy source for the survival of breast cancer cells, especially since adipose tissue is abundant in breast tissue. Breast cancer cells modulate lipid metabolism (de novo fatty acid synthesis and lipolysis), by altering the secretion of adipokines through adipocytes, resulting in the release of free fatty acids to provide energy substrates for breast cancer cells to sustain its high proliferation demand for survival. Evidence on the role of obesity and lipid metabolism especially fatty acids in breast cancer treatment resistance is lacking. This motivates investigation to identify and understand the underlying physiological and molecular mechanisms by which chemotherapeutic treatment resistance is achieved. We therefore hypothesise that obesity-induced inflammation alters lipid metabolism in adipose tissue/adipocytes and contribute to the development of doxorubicin chemotherapeutic treatment resistance in breast cancer cells. Methods: A diet induced obesity animal model was established by feeding female C57BL6 mice a high fat diet for 12 weeks. After developing the diet induced obesity phenotype, breast tumour xenographs were induced by subcutaneous inoculation in the fourth mammary gland with E0771 triple negative breast cancer cells.Once tumours became palpable, mice received either vehicle treatment (Hanks balance salt solution) or doxorubicin treatment (cumulative dose of 12 mg/kg). Plasma inflammatory markers, fatty acid profiles and protein expression of lipid metabolism markers (de novo fatty acid synthesis and lipolysis) was determined in mammary adipose and tumour tissue. To validate the in vivo model findings, we developed an in vitro model using a conditioned media approach. A human adipose tissue derived stem cell line was used for the differentiation of mature adipocytes after which conditioned media was collected to assess the paracrine effect between adipocytes and doxorubicin treated MDA-MB-231 triple negative breast cancer cells. Cell viability was assessed with WST-1 assays. Western blots were used to determined alterations in protein expression of apoptotic and lipid metabolism markers (de novo fatty acid synthesis and lipolysis). An Inflammatory marker as well as free fatty acid profile was also analysed in treatment conditioned media. Results: Diet induced obesity significantly increased tumour growth and decreased doxorubicin treatment efficacy in E0771 triple negative breast tumours (p<0.0001), resulting in treatment resistance. Our findings also showed that diet induced obesity supressed de novo fatty acid synthesis (decreased SCD-1) and lipolysis (decreased HSL) in mammary adipose tissue of doxorubicin treated mice. Conversely an increase in de novo fatty acid synthesis (increased SCD-1) and lipolysis (increased ATGL) was found in tumour tissue, leading to significant changes in FAs composition of both tissues. Diet induced obesity also significantly increased plasma leptin (p=0.025) and resistin levels (p=0.046) and increased NFĸB protein expression in mammary fat of doxorubicin treated mice, thereby inducing systemic and local inflammation. Furthermore, we also report that adipocytes promoted acquired breast cancer treatment resistance by significantly increasing the cell viability of doxorubicin treated MDA-MB-231 triple negative breast cancer cells (Dox+CM vs Dox, p=<0.0001). This was achieved by attenuating doxorubicin’s efficacy to induce apoptosis (decreased cleaved-caspase-3, p<0.05), in a paracrine manner. Adipocytes also induced inflammation (increased leptin and MCP-1) as well as lipolysis (increased HSL) in doxorubicin treated breast cancer cells (Dox vs Dox+CM p=0.03), thereby altering the free fatty acid profile of breast cancer cells. Conclusion: Our data suggest that adipose tissue/adipocytes significantly contribute to treatment resistance in triple negative breast cancer cells. We have demonstrated in both in vivo and in vitro models that adipose tissue/adipocytes secretory factors induce inflammation in the breast tumour microenvironment, which leads to the induction of lipolysis in triple negative breast cancer cells. This resulted in altered metabolic behaviour i.e. increased free fatty acid utilization, which can be utilized as energy substrates or induce lipid saturation in order confer to acquired treatment resistance by evading apoptosis We propose that this could be a novel mechanism by which adipose tissue/adipocytes within the tumour microenvironment can contribute to the development of breast cancer treatment resistance under obesogenic conditions. This study also significantly contributed to the identification and understanding of molecular mechanisms underlying breast cancer treatment resistance in obese patients.
Chronic stress-associated accelerated ageing: inflammation and oxidative stress treatment
(Stellenbosch : Stellenbosch University, 2020-12) Petersen-Ross, Kelly Shirley; Smith, Carine; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: In recent years, the incidence of non-communicable diseases (NCD) normally associated with advanced age has begun presenting in younger populations. This has resulted in a growing burden on global healthcare systems and decreasing quality of life in individuals. Cardiovascular diseases, cancers, chronic respiratory diseases, chronic inflammatory diseases and diabetes are some of the many NCD’s and all these have two maladaptive characteristics in common, namely chronic low-grade inflammation and increased oxidative stress. The aim of this research was to identify a threshold prior to maladaptation in both redox and inflammatory status which can be targeted with preventative medicine strategies; in this way, we may identify suitable models which are sensitive enough to identify this threshold as well as show small effect sizes so that they can be used for drug screening of preventative medicine treatments. In order to elucidate this threshold, two rodent models were employed to simulate a pre-onset and an early onset state. The pre-onset state was simulated by chronic D-galactose injections to mimic cumulative oxidative stress as is associated with chronological ageing. The early onset state was simulated with a collagen induced rheumatoid arthritis (RA) model. A grape seed polyphenol supplementation was employed to assess the sensitivity of the models. Comprehensive end-point analysis of the oxidative and inflammatory state of various compartments were performed. Analysis of parameters associated with ageing were also included as measure of relative ageing status in models. The results of both studies indicated that the threshold or point of onset of accelerated ageing was indeed identified. In the D-galactose model, a novel finding was the compromised antioxidant capacity in plasma, even in the absence of experimentally elevated oxidative damage, observed as decreases in plasma FRAP. However, oxidative damage was observed in tissue specific investigations, such a morphological changes in the mesenteric lymph nodes. In the RA model, decreases in antioxidant capacity was noted along with oxidative damage in plasma, but not in all tissue types investigated - particularly the brain. This novel finding of pre-damage oxidative changes in the brain was indicated by decreases in MDA and increases in FRAP. This combined with a switch to a pro-inflammatory state within the circulation, confirms the early disease state within the RA model. This investigation has elucidated the importance of monitoring the oxidative state within multiple compartments to identify the threshold at which disturbances to homeostasis turns into maladaptation and FRAP may be the most sensitive parameter to display this. The effect changes noted after supplementation with an antioxidant treatment also enhanced our knowledge of which parameters and tissue are susceptible to oxidative and inflammatory modulation to prevent maladaptations which may result in pathology.
Comparative assessment of neurological vs metabolic allostasis as reflected in human skin fibroblasts
(Stellenbosch : Stellenbosch University, 2022-12) Benecke, Rohan Meerholz; Smith, Carine; Van de Vyver, Mari; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Incidence of mental health disorders are rising in modernity. Many mental health disorders share molecular and genetic overlap as well as having high incidence of comorbidities. Stress and the compounded effect of multiple low-grade stressors may be contributing to a relative increase in the pro-inflammatory and oxidative state seen in mental health disorders and other complex diseases. This leads to allostatic changes that potentially contribute to disease aetiology and progression. Allostasis is the process of homeostatic equilibrium under stress. Inflammation, which is often used to measure allostatic load, is potentially the incorrect measure as inflammation is transient and the damage ascribed to chronic inflammation is due to increases in reactive oxygen species (ROS) and decreases in antioxidant capacity. Post-traumatic stress disorder (PTSD) is a mental health disorder that is characterised by severe stressors and a maladaptive response to these stressors. Although the role of inflammation and oxidative stress have been implicated in the disease aetiology it is still a relatively neglected aspect of PTSD research. Furthermore, despite the high rate of comorbidities associated with PTSD there is still a lack of understanding in terms of the peripheral effects of PTSD. PTSD and potentially comorbid obesity, present ideal health paradigms to assess this relative neglect of allostatic changes, in particular those in the periphery, that may be contributing to disease outcome in PTSD. A novel therapeutic target, namely the trace amine system, is investigated as a potential anxiolytic in zebrafish larvae that could address allostatic changes of chronic diseases such as PTSD and obesity. Patient derived fibroblasts are used as model cell type to investigate potential functional changes in the periphery of PTSD patients as result of allostatic load. Finally, the potential for peripheral signalling to influence central function is explored in astrocytes, that represent the ideal candidate cell to investigate allostatic load in the context of mental health. Changes in peripheral calcium function and central redox function indicate the allostatic load of PTSD can modulate the chemiosmotic potential of cells through longitudinal shifts in the homeostatic set point. As a result, low grade cumulative stressors may be damaging to cellular function without activating endogenous defence mechanisms.
The contribution of inflammatory mediators to delayed secondary muscle damage
(Stellenbosch : Stellenbosch University, 2013-03) Van de Vyver, Mari; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Background: Understanding the contribution of divergent individual response patterns remains a key objective in identifying mechanisms of inflammation and potential factors limiting the resolution of inflammation. The purpose of this research project was to investigate downstream effects of inflammation following exercise-induced muscle damage in human subjects. Methods: For three different studies, a total of 53 untrained healthy male participants were recruited and divided into a non-exercising control (n=13) and exercise-induced muscle damage groups (n=40). The study design for the three studies was the same (with few exceptions): Downhill running (DHR) (12 x 5min bouts, 10% decline, 15 km.h-1) with blood samples taken pre, post, after 2 and 4 hours post-exercise (2h, 4h) and on days 1, 2, 3, 4 and 7 (d1-d7). Serum was analysed for creatine kinase activity (CK), myoglobin (Mb), cortisol, cytokine (TNFα, IL-1ra, IL-1β, IL-4, IL-6, IL-8, IL-10, sIL-6R), chemokine (G-CSF, MIP-1β) and adhesion factor (sICAM-1, sP-selectin) concentrations. Tissue degradation was assessed by serum matrix metalloprotease (MMP-9) and myeloperoxidase (MPO) content. White blood cell differential count was determined and the surface expression of various cluster of differentiation factors (CD11b, CD163, CD68, CD88, CD34) as well as intracellular MPO were assessed in whole bood using flow cytometry. Nuclear localization of the inflammatory mediator NFĸB in isolated perhipheral blood mononuclear cells (PBMCs) was determined using immunofluorescence microscopy. Muscle biopsies (vastus lateralis) taken at baseline, 4h, d1 and d2 were analysed for fibre type, inflammatory and stress-induced pathways (STAT3, IĸBα, p38MAPK), myogenic factors (MyoD, myogenin), neutrophil activity (MPO) and satellite cell number (Pax7). Results: Participants in the DHR group were subdivided into those with a normal recovery (DHR1) and those who developed secondary damage (DHR2). CK peaked on d1 in both subgroups (DHR1: 1512 ± 413 u.L-1, DHR2: 1434 ± 202 u.L-1) and again on d4 only in the DHR2 group (1110 ± 184 u.L-1). A similar IL-6 and IL-10 response was evident immediately post DHR in all individuals. Additional IL-6 was released in the DHR2 subgroup peaking at 4h (10.3 ± 4.2 pg.mL-1) whereas IL-10 had returned to baseline. IL-1ra (23.6 ± 8.8 pg.mL-1), CD68+ (5%) and CD163+ (3%) monocytes were significantly higher in the DHR2 subgroup. Neutrophil count at 2h (DHR1: 8.6 ± 0.8 x109 cells.L-1, DHR2: 11.4 ± 1.8 x109 cells.L-1) was significantly (p<0.02) correlated to CK activity on d4. PBMC NFĸB p65 nuclear localization was slightly less at 2h in the DHR2 compared to the DHR1 and control groups. Intramuscular STAT3 signalling and MPO were significantly higher in the DHR2 compared to the DHR1 subgroup at 4h and d2 respectively. The progenitor cell response was similar for all DHR individuals with an increase in Pax7+ SC observed at 4h (0.06 ± 0.01 Pax+ SCs/fibre) and d1 (0.07 ± 0.02 Pax+ SCs/fibre). Conclusion: Healthy young men can be divided into those with a adequate and those with a less efficient capacity to control the post damage inflammatory response. The early cytokine response, especially IL-6, seems to be a key role player in the cascade of events leading to late secondary skeletal muscle damage.
Correlations between stress-associated anxiety and physiological determinants of health in adolescents
(Stellenbosch : Stellenbosch University, 2016-03) Viljoen, Monet; Smith, Carine; Seedat, Soraya; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Anxiety disorders are among the most prevalent of psychiatric disorders across age groups, with onset typically in childhood or early adolescence, and risk for developing an anxiety disorder increasing with trauma/childhood maltreatment. Little is known about biomarkers of resilience/vulnerability in relation to subclinical anxiety, especially when trauma-exposed adolescents are implicated. Therefore, better elucidation of the neuro-endocrine and -immunological underpinnings relative to anxiety and trauma, may highlight specific avenues to target with more effective diagnosis, monitoring and/or treatment strategies in the context of youth at risk for later development of anxiety disorders. Thus, our aims were to elucidate the central and peripheral neuroendocrine and immunological profiles in association with anxiety proneness, in comparison to childhood trauma, in older adolescents, and to assess potential outcome modulators. A total of 43 participants, aged 15-18, were selected from an initial cohort of 1149 adolescents. Participants were delineated into four groups based on levels of anxiety proneness and trauma exposure, using questionnaires and a structured diagnostic interview. Blood obtained from each participant was analysed for an HPA-axis hormone profile (cortisol, prolactin, testosterone and dehydroepiandrosterone-sulphate (DHEAs) and immune status (total white blood cell count, leukocyte glucocorticoid receptor (GR) expression and serum cytokine and myeloperoxidase (MPO) levels). Resilience (coping capacity), self-esteem and handedness were assessed via questionnaires. Verbal- and visuospatial working memory, as well as executive neurocognitive function, were assessed by means of the administration of neurocognitive tests. A structural Magnetic Resonance Imaging (MRI) was performed to determine left versus right grey matter volumes of the thalamus, amygdala, hippocampus, and Prefrontal cortex (PFC). Finally, HPA-axis responsivity and concurrent state anxiety to an in vivo Bexamethasone suppression test, in conjunction with as a psychosocial stress test (TSST), were assessed. In terms of neurophysiological maladaptations, main findings included a relatively larger association with anxiety proneness, compared to childhood maltreatment. Specifically, anxiety proneness was associated with poorer neurocognitive function, increased right amygdala volume, lower serum DHEAs levels, lower peripheral leukocyte counts, and increased GR expression. In terms of potential outcome modifying factors (OMFs), resilience and self-esteem were affected by trauma, but not anxiety proneness, while a higher degree of right handedness was associated with poorer neurophysiological outcomes. Furthermore, increased serum IL-12p70 and MPO (suggesting relatively more pro-inflammatory state) were associated with anxiety scales and emotional/physical abuse. Also, better PFC neurocognitive function and larger left PFC volumes were associated with better physiological outcome as indicated by levels of GR expression and DHEAs. In conclusion, this is the first study to have investigated neurophysiological adaptations, as well as psycho-physiological responses to HPA-axis suppression and a psychosocial stress test, in association with anxiety proneness and trauma exposure, in adolescents of low socio-demographic background. Results suggest for the study population, a) chronic hypo-activity and acute hypo-reactivity of the lower HPA-axis, b) neurophysiological perturbations associated relatively closely with anxiety proneness, when compared to trauma exposure, c) central correlates associated with physiological outcome, and d) a higher degree of consistent right handedness to be a potential marker of vulnerability in terms of neurophysiology and anxiety.
The Damara sheep : an appraisal of its reproductive performance and potential
(Stellenbosch : University of Stellenbosch, 1998-03) Schoombee, Cornelius Johan Albertus; Coetzer, W. A.; Barry, D. M.; University of Stellenbosch. Faculty of Agricultural Sciences. Human and Animal Physiology.
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Dietary red palm oil-supplementation offers cardioprotection against Ischaemia/Reperfusion injury : possible cellular mechanisms involved
(Stellenbosch : University of Stellenbosch, 2005-12) Esterhuyse, Adriaan Johannes; Van Rooyen, Jacques; Du Toit, Eugene F.; University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Activation of the NO-cGMP pathway is associated with myocardial protection against ischaemia/reperfusion injury. However, high-cholesterol diets alter function of this pathway and these alterations have been implicated in both ischaemic/reperfusion injury and the development of ischaemic heart disease. Little is known about the effects of supplements such as Red Palm Oil (RPO) on the myocardial NO-cGMP-signalling pathway. RPO consists of saturated, mono-unsaturated and poly-unsaturated fatty acids and is rich in antioxidants such as β-carotene and Vitamin E (tocopherols and tocotrienols). The aims of this study were: 1) to determine whether dietary RPO-supplemention protects against ischaemia/reperfusion injury in rats fed a standard rat chow (control) and cholesterol-enriched diets and 2) if so, to investigate possible mechanisms for this protection. Male Long-Evans rats were fed a standard rat chow or a standard rat chow plus cholesterol and/or RPO-supplementation for 6 weeks. Myocardial functional recovery was measured and hearts were freeze-clamped for determination of myocardial phospholipid, cAMP/cGMP concentrations, total myocardial nitric oxide concentrations, lipid hydroperoxide production and superoxide dismutase- and nitric oxide synthase activity in isolated rat hearts subjected to 25 minutes of normothermic total global ischaemia. In addition, the degree of phosphorylation of extracellular signal-regulated kinase (ERK), p38, c-Jun N-terminal protein kinase (JNK) and protein kinase B (PKB/Akt) was investigated. Furthermore, the effect of RPO-supplementation on caspase-3 activation and poly (ADP-ribose) polymerase (PARP)-cleavage in hearts subjected to ischaemia and reperfusion was also investigated. Our data show that dietary RPO-supplementation protects the hearts of rats on a standard rat chow (control) and hypercholesterolaemic diet against ischaemia/reperfusion injury as reflected by improved aortic output recovery. Increased intracellular cardiomyocyte NO concentrations as observed in control hearts supplemented with RPO after 120 minutes hypoxia may contribute to the elevated cGMP concentration and may confer some of the cardioprotection to the ischaemic/reperfused heart. Although improved functional recovery with RPO-supplementation of a high-cholesterol diet was also associated with an increase in intracellular cardiomyocyte NO production after hypoxia compared to the non-hypoxic conditions, it could not be linked to increased NO-cGMP signalling. These data are in agreement with other studies, which showed that high-cholesterol diet impairs NO-cGMP signalling and confirms our hypothesis that elevated cGMP concentrations may not be the only mechanism of protection. We have also shown that RPOsupplementation caused increased phosphorylation of p38 and PKB, reduced phosphorylation of JNK and attenuation of PARP cleavage, which may contribute to the protection of the cell against apoptosis. Based on our results we propose that the myocardial protection offered by RPO-supplementation of rats on a normal and hypercholesterolaemic diet may be associated with either its antioxidant characteristics and/or changes in the fatty acid composition of the myocardium during ischaemia/reperfusion. Furthermore, we demonstrated for the first time that RPO-supplementation protects the isolated perfused working rat heart during reperfusion from ischaemia/reperfusion-induced injury through a MAPK-dependent pathway.
Differential effects of TNfα on satellite cell differentiation
(Stellenbosch : Stellenbosch University, 2007-03) Fouche, Celeste; Smith, Robert M.; Niesler, Carola U.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Tumour necrosis factor alpha (TNFα) is a pleiotropic cytokine and has a wide variety of dose dependent cellular effects ranging from cell growth and differentiation, to inducing apoptosis. It has long been implicated in muscle and non-muscle inflammatory disorders, such as muscle wasting in chronic disease states, and rheumatoid arthritis. However, a physiological role for TNFα in muscle regeneration has been proposed as elevated levels of the cytokine are present when muscle regeneration processes are initiated: TNFα is secreted by infiltrating inflammatory cells, and by injured muscle fibres. Adult skeletal muscle contains a population of resident stem cell-like cells called satellite cells, which become activated, proliferate and differentiate following muscle injury to bring about repair of damaged muscle. Much research on the effects of TNFα on satellite cell differentiation has been conducted in recent years. It is however difficult to get a complete characterisation of the cytokine’s action as all models used slightly differ. We aimed therefore at providing comprehensive assessment of the effects of increasing doses of chronically supplemented TNFα on differentiating C2C12 cells. Cells were allowed to differentiate with or without TNFα supplementation for 7 days. Differentiation was induced at day 0. The effect on differentiation was assessed at days 1, 3, 5, and 7 by western blot analysis, and supplementary immunohistochemical analysis at days 1, 4, and 7 of markers of differentiation - muscle regulatory factors: MyoD and myogenin, markers of the cell cycle p21, PCNA, and the integral signalling molecule, p38MAPK. TNFα supplementation at day 1 tended to positively regulate early markers of differentiation. With continued supplementation however, markers of differentiation decreased dose dependently in treated cultures as the initial effect appeared to be reversed: A trend towards a dose dependent decrease in MyoD, myogenin and p21 protein existed in treated cultures at days 3, 5, and 7. These findings were significant at day 5 (p21, p<0.05), and day 7 (myogenin, p<0.05). A significant dose dependent decrease in p38 phosphorylation was evident at day 3 (p<0.05), while phospho-p38 was dose dependently increased at day 7 (p<0.05). Taken together, these data show that TNFα supplementation for 24 hours following the induction of differentiation in vitro, tends to increase levels of early markers of differentiation, and with continued TNFα supplementation decrease markers of differentiation in a dose dependent fashion. This study provides a comprehensive characterisation of the dose and time dependent effects of TNFα on satellite cell differentiaton in vitro. The model system used in the current study, allows us to make conclusions on more chronic disease states.
Differential tolerance of a cancer and a non-cancer cell line to amino acid deprivation : mechanistic insight and clinical potential
(Stellenbosch : Stellenbosch University, 2012-03) Thomas, Mark Peter; Engelbrecht, Anna-Mart; Strijdom, Hans; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Introduction – Due to spatial separation from the native vascular bed, solid tumours develop regions with limited access to nutrients essential for growth and survival. The promotion of a process known as macroautophagy may facilitate in the maintenance of intracellular amino acid levels, through breakdown of cytoplasmic proteins, so that they remain available for macromolecular biosynthesis and ATP production. Several studies point to the potential ability of some cancers to temporarily increase autophagy and thereby prolong cell survival during metabolic stress. The validity of these claims is assessed when a commonly used breast cancer cell line and an epithelial breast cell line are starved of amino acids in this study. Furthermore, we go on to hypothesize that acute amino acid deprivation during treatment will result in an elevated sensitivity of MDAMB231 cells to doxorubicin toxicity but limit its cytotoxic side-effects in MCF12A cells. Methods and study design- Human breast cancer cells (MDAMB231) and breast epithelial cells (MCF12A) cultured in complete growth medium were compared to those incubated in medium containing no amino acids. Steady state autophagy levels were monitored using classical protein markers of autophagy (LC3-II and beclin-1) and the acidic compartmentalization in cells (Lysotracker™ red dye) in conjunction with autophagy inhibition (bafilomycin A1 and ATG5 siRNA). Cell viability was monitored using several techniques, including caspase 3/7 activity. ATP levels were assessed using a bioluminescent assay, while mass spectrometry based proteomics was used to quantify cellular amino acid levels. Similar techniques were used to monitor autophagy during doxorubicin treatment, while cellular doxorubicin localization was monitored using immunofluorescence microscopy. Finally, a completely novel GFP-LC3 mouse tumour model was designed to assess autophagy and caspase activity within tumours in vivo, during protein limitation and doxorubicin treatment. Results - Amino acid deprivation resulted in a transient increase in autophagy at approximately 6 hours of amino acid starvation in MDAMB231 cells. The amino acid content was preserved within these cells in an autophagy-dependent manner, a phenomenon that correlated with the maintenance of ATP levels. Inhibition of autophagy during these conditions resulted in decreased amino acid and ATP levels and increased signs of cell death. MCF12A cells displayed a greater tolerance to amino acid starvation during 24 hours of amino acid starvation. Evidence indicated that autophagy was important for the maintenance of amino acid and ATP levels in these cells and helped prevent starvation-induced cell death. Furthermore, data showed that concomitant amino acid withdrawal resulted in decreased cellular acidity in MDAMB231 cells, and increased acidity in MCF12A cells, during doxorubicin treatment. These changes correlated with evidence of increased cell death in MDAMB231 cells, but a relative protection in MCF12A cells. A novel model was used to apply these techniques in vivo, and although mice fed on a low protein diet during high dose doxorubicin treatment had increased mean survival and smaller tumour sizes, evidence suggested that autophagy is protecting a population of cells within these tumours. Conclusions - This novel approach to tumour sensitization could have several implications in the context of cancer therapy, and given the delicate relationship that autophagy has with the cancer microenvironment, efforts to determine the mechanisms involved in autophagy and sensitization could lead to new and innovative treatment opportunities for cancer management.
The effect of altered trace aminergic signalling and estrogen on intestinal inflammation, within an IBS context
(Stellenbosch : Stellenbosch University, 2022-12) Pretorius, Lesha; Smith, Carine; Van Staden, Anton du Preez; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Irritable bowel syndrome (IBS) is a widespread (≈10% global prevalence) female predominant functional gastrointestinal (GI) disorder. While it is known that IBS is underpinned by relative microbial dysbiosis and chronic microinflammation, current therapeutic strategies often only provide transient symptomatic relief (with relative neglect of inflammation) and are thus unsatisfactory in many cases. As such, the development of targeted therapeutics to alleviate GI inflammation and consequential symptomologies are required. We suggest that the trace aminergic system, which connects several IBS risk factors (sex, dysbiosis, diet, inflammation and anxiety), may be a pretermitted regulatory system that could be manipulated as a therapeutic target. In addition, existing data supports an interpretation of sex dependence in trace aminergic signalling. As such, fluctuations of female reproductive hormones, such as 17β-estradiol (E2), may alter subsequent signalling cascades. Therefore, this thesis aimed to investigate the GI modulatory effects of selected trace amines (TAs), with consideration of the context of female predominance. To elucidate mechanisms at play, a multidisciplinary approach was necessitated. As such, multiple model systems were utilised, including both in vitro (microbial cultures and human tissue cultures) and in vivo (zebrafish larval) models. In this regard, microbial (probiotic and commensal strains) culturing techniques, coupled with the development of a novel multianalyte mass spectrometry methodology, allowed for the accurate assessment of microbial TA generation. Indeed, data generated in these studies highlighted firstly, the dependence of probiotic secretome profile on host hormonal status, and secondly, that specific rooibos supplementation strategies may be able to negate E2-induced alterations in secretome TA profiles, both of which have important implications in TA-associated symptom management in females with GI disorders. Data generated in vitro in HT-29 colon adenocarcinoma cells and in vivo in zebrafish larvae, in which the effects of increased TA load were assessed, demonstrated potential differences in the mechanisms of actions between TYR and AGM in particular. In this regard, extensive occludin redistribution was observed following TYR-exposure, which was associated with increased reactive oxygen species and pro-inflammatory cytokine levels, as well as tight junction disruption – an outcome prevented by E2 treatment. In contrast, AGM administration promoted the colocalization of ZO-1 and occludin to promote tight junction integrity but was also associated with risk of pro-oxidant damage when AGM metabolism was insufficient. In conclusion, this dissertation contributes significantly to our understanding of the role of TAs in GI physiology, consistently illustrating (across in vitro and in vivo models), that while some TAs may promote disease symptomology, others may have therapeutic benefit when responsibly administered. From a therapeutics standpoint, data presented here crucially highlights the importance of dosage and administration optimisation to achieve benefit and minimize adverse side effects when targeting TA signalling in the context of functional GI disease. In addition, potential mechanistic insights by which E2 - or rather the transient cyclic lack thereof - is associated with trace aminergic signalling, was elucidated.
The effect of melatonin treatment on doxorubicin-induced skeletal muscle atrophy within a cancer model
(Stellenbosch : Stellenbosch University, 2018-12) Isaacs, Ashwin Wayne; Engelbrecht, Anna-Mart; Loos, Ben; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Background and Aim: Skeletal muscle atrophy is a major concern in patients suffering with malignancy. Chemotherapeutic agents, such as doxorubicin (DOX), can further exacerbate this loss of skeletal muscle. Although many cancer patients on chemotherapeutic agents suffer from this condition, there are no therapies routinely used to moderate muscle atrophy. The aim of the study was to investigate whether melatonin (MLT) can attenuate doxorubicin‐induced skeletal muscle and myotube atrophy in an in vivo rodent model of breast cancer as well as in an in vitro model of DOXinduced myotoxicity respectively. The safe and cost‐effective role of melatonin as a possible therapy to limit the burden of doxorubicin‐induced muscle toxicity in cancer patients serves as rationale for the in vivo study and the in vitro study allows for the exploration of more invasive mechanistic aspects using the cell lines, which would not be possible when viewing excised tissue. Methods: Female Sprague‐Dawley rats were inoculated with LA7 cancer cells and were randomly assigned to six groups: Control, Tumour control (TCON), Vehicle control (VEH), MLT, DOX and DOX + MLT (DM). Prophylactic treatment of MLT (6 mg/kg) was administered in drinking water daily and rats received three intraperitoneal injections of DOX (4 mg/kg, 3 times at 3‐day intervals). Following sacrifice blood samples (whole blood counts) and skeletal muscle tissue were collected for histological, immunoblot, antioxidant capacity and immunofluorescence analyses. Furthermore, C2C12 myoblasts grown to confluency and differentiated into myotubes were pretreated with MLT (50 nM) for 48h followed by DOX treatment (0.8 μM) for 24h. The effect of MLT treatment on C2C12 myotube diameter, mitochondrial reactive oxygen species (mtROS) production, sirtuin levels and autophagy activity was then assessed. Results: DOX treatment significantly reduced animal weight (279.1 ± 21.34 g vs. 222.2 ± 20.40 g, p˂0.0001) compared to DM weight (281.5 ± 7.11 g vs. 284.0 ± 6.53 g) and gastrocnemius muscle weight (1.4 ± 0.13 g vs. 0.99 ± 0.076 g, p˂0.0001) and cross sectional area (CSA), while increasing markers of muscle degradation compared to MLT treated groups. Serum myoglobin levels were significantly elevated in the DOX group compared to the DM group (572.6 ± 444.19 ng/mL vs. 218.2 ± 83.66 ng/mL, p˂0.0001); while, white & red blood cell counts (WBC & RBC) were significantly decreased in the DOX group compared to the MLT treated groups respectively (2.06 ± 1.59 x 109L‐1 vs. 4.13 ± 1.56 x 109L‐1 & 4.00 ± 1.52 x 1012L‐1 vs. 5.66 ± 1.03 x 1012L1, p˂0.0001). Furthermore, MLT treatment significantly increased intramuscular antioxidant capacity, mitochondrial biogenesis and satellite cell number. In vitro DOX treatment resulted in increased myotube atrophy, mitochondrial ROS levels and these effects were significantly reduced with MLT pre‐treatment. Discussion: The improvement in animal weight, muscle to body weight ratio, muscle CSA as well as the reduction in myoglobin levels in the treatment groups compared to the DOX group indicate that MLT protects against DOX‐induced atrophy. Moreover, MLT pre‐treatment improved circulating levels of WBC & RBC compared to the DOX only group and attenuated skeletal muscle atrophy by reducing cell apoptosis and increasing satellite cell number suggesting that MLT assists with muscle repair. The in vitro study indicated that DOX‐induced myotube atrophy was preceded by increases in mitochondrial ROS. Conclusion: Results indicate that pre‐treatment with exogenous MLT protects against skeletal muscle wasting induced by DOX in a pre‐cachectic tumour‐bearing rat model.
The effect of the TGF-β isoforms on progenitor cell recruitment and differentiation into cardiac and skeletal muscle
(Stellenbosch : University of Stellenbosch, 2007-12) Schabort, Elske Jeanne; Niesler, Carola U.; University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences.
Definition: Stem cells are unspecialised cells with the capacity for long-term self-renewal and the ability to differentiate into multiple cell-lineages. The potential for the application of stem cells in clinical settings has had a profound effect on the future of regenerative medicine. However, to be of greater therapeutic use, selection of the most appropriate cell type, as well as optimisation of stem cell incorporation into the damaged tissue is required. In adult skeletal muscle, satellite cells are the primary stem cell population which mediate postnatal muscle growth. Following injury or in diseased conditions, these cells are activated and recruited for new muscle formation. In contrast, the potential of resident adult stem cell incorporation into the myocardium has been challenged and the response of cardiac tissue, especially to ischaemic injury, is scar formation. Following muscle damage, various growth factors and cytokines are released in the afflicted area which influences the recruitment and incorporation of stem cells into the injured tissue. Transforming Growth Factor-β (TGF-β) is a member of the TGF-β-superfamily of cytokines and has at least three isoforms, TGF-β1, -β2, and -β3, which play essential roles in the regulation of cell growth and regeneration following activation and stimulation of receptor-signalling pathways. By improving the understanding of how TGF-β affects these processes, it is possible to gain insight into how the intercellular environment can be manipulated to improve stem cell-mediated repair following muscle injury. Therefore, the main aims of this thesis were to determine the effect of the three TGF-β isoforms on proliferation, differentiation, migration and fusion of muscle progenitor cells (skeletal and cardiac) and relate this to possible improved mechanisms for muscle repair. The effect of short- and long-term treatment with all three TGF-β isoforms were investigated on muscle progenitor cell proliferation and differentiation using the C2C12 skeletal muscle satellite and P19 multipotent embryonal carcinoma cell-lineages as in vitro model systems. Cells were treated with 5 ng/mℓ TGF-β isoforms unless where stated otherwise. In C2C12 cells, proliferating cell nuclear antigen (PCNA) expression and localisation were analysed, and together with total nuclear counts, used to assess the effect of TGF-β on myoblast proliferation (Chapter 5). The myogenic regulatory factors MyoD and myogenin, and structural protein myosin heavy chain (MHC) were used as protein markers to assess early and terminal differentiation, respectively. To establish possible mechanisms by which TGF-β isoforms regulate differentiation, further analysis included determination of MyoD localisation and the rate of MyoD degradation in C2C12 cells. To assess the effect of TGF-β isoforms on P19 cell differentiation, protein expression levels of connexin-43 and MHC were analysed, together with the determination of embryoid body numbers in differentiating P19 cells (Chapter 6). Furthermore, assays were developed to analyse the effect of TGF-β isoforms on both C2C12 and P19 cell migration (Chapter 7), as well as fusion of C2C12 cells (Chapter 8). Whereas all three isoforms of TGF-β significantly increased proliferation of C2C12 cells, differentiation results, however, indicated that especially following long-term incubation, TGF-β isoforms delayed both early and terminal differentiation of C2C12 cells into myotubes. Similarly, myocyte migration and fusion were also negatively regulated following TGF-β treatment. In the P19 cell-lineage, results demonstrated that isoform-specific treatment with TGF-β1 could potentially enhance differentiation. Further research is however required in this area, especially since migration was greatly reduced in these cells. Taken together, results demonstrated variable effects following TGF-β treatment depending on the cell type and the duration of TGF-β application. Circulating and/or treatment concentrations of this growth factor could therefore be manipulated depending on the area of injury to improve regenerative processes. Alternatively, when selecting appropriate stem or progenitor cells for therapeutic application, the effect of the immediate environment and subsequent interaction between the two should be taken into consideration for optimal beneficial results.
The effects of nutrient deprivation on macroautophagic flux and chaperone-mediated autophagy in a model of alzheimer's disease
(Stellenbosch : Stellenbosch University, 2018-12) Ntsapi, Matlakala Claudia; Loos, Benjamin; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Introduction: Alzheimer’s disease (AD) is a devastating neurodegenerative disease characterized by progressive cognitive impairment, particularly in brain regions crucial for learning and memory. These symptoms are caused by neuronal death resulting from two pathological features: extracellular senile plaques composed of aggregated amyloid-beta (Aβ) peptides, and intracellular neurofibrillary tangles generated by the hyperphosphorylation of tau protein. Although AD is a multifactorial disease, much of the AD research continues to be guided by the amyloid cascade hypothesis, which posits that Aβ aggregation is the key initiate in AD pathogenesis. Aβ is generated from the proteolytic cleavage of the amyloid precursor protein (APP) by β - and γ-secretase. Accordingly, research efforts to modulate APP processing, and better clarify the mechanisms that regulate intracellular Aβ metabolism and clearance during AD progression have been explored in the treatment of AD. Autophagy, a lysosome-based proteolytic pathway that plays a crucial role in intracellular protein quality control, has been implicated in both the production and clearance of Aβ peptide. Cumulative evidence shows that AD-related autophagic dysfunction coincides with the detection of Aβ within autophagic vacuoles (AVs) that accumulate within dystrophic neurites with the initial increase in Aβ neurotoxicity. Therefore, autophagy dysfunction may exacerbate Aβ pathology and further augment disease progression; however, when in this context autophagy becomes dysfunctional remains unclear. Moreover, although it is known that Aβ levels themselves may induce autophagy, how long autophagy remains upregulated and functional in this process is unclear. It also remains unclear whether autophagy plays a causative, or protective role in Aβ neurotoxicity; or whether autophagy dysfunction is a consequence of the disease process itself. Therefore, the aims of this study were (i) to characterize the expression profile of key amyloidogenic pathway proteins, both macroautophagy and chaperone-mediated autophagy (CMA) proteins as well as the extent of neuronal toxicity using a unique APP overexpression model, (ii) to dissect the interplay between proteolytic pathways and cell death markers in the context of APP overexpression using a proteomics approach, (iii) to assess macroautophagic flux in the context of APP overexpression and to unravel the extent of autophagy dysfunction, (iv) to assess the contribution of macroautophagy and CMA in Aβ clearance and neuronal toxicity by modulating each pathway, and (v) to assess the effects of prolonged intermittent fasting (IF) on the modulation of macroautophagy and CMA in a paraquat (PQ)-induced in vivo brain injury model. Methods: A unique AD overexpression model, the N2a mouse neuroblastoma cell line stably overexpressing the human Swedish double mutation was utilized. APP overexpression was characterized, and the induction of macroautophagy, CMA, and apoptosis was assessed over time using a combination of cell viability assays, western blot analysis, fluorescence microscopy, transmission electron microscopy (TEM), and correlative light and electron microscopy techniques. Moreover, the effect of APP overexpression on a global proteome level was quantified using high resolution liquid-chromatography coupled to tandem mass spectrometry. Finally, a PQ – induced brain injury model was established and utilized to assess the effects of prolonged IF on macroautophagy and CMA using GFP–LC3 transgenic mice. Mice were injected twice weekly with 10 mg/kg PQ for a duration of 3 weeks. A prolonged IF protocol of 48 hrs fasting, followed by 24 hrs refeeding was implemented for a duration of 3 weeks. Modulation of macroautophagy and CMA following chronic oxidative stress exposure, and prolonged IF was evaluated in selected brain regions by western blot analysis, fluorescence microscopy, comparative haematoxylin and eosin staining, and TEM analysis. Results: The results indicate that APP overexpression leads to prominent apoptosis induction after 48 hrs and activates the autophagy machinery in a time-dependent manner. To our surprise, macroautophagic flux analysis reveals that autophagy is upregulated upon APP overexpression but remains elevated in the presence of apoptosis induction. Our CMA analysis indicates that APP overexpression activates the CMA machinery, particularly during the 48 hrs time point. However, induction of apoptosis proceeded despite elevated levels of CMA activity. Next, our proteome analysis reveals a time-dependent increase in APP proteinprotein interaction partners over time. Cumulatively, the in vitro results suggest that the modulation of macroautophagy and CMA augments Aβ clearance and mitigates neuronal toxicity. In vivo, a significant decrease in cytochrome c, and 4HNE expression were observed with prolonged IF intervention in selective brain regions. These changes were associated with elevated levels of macroautophagy and CMA induction, as evidenced by the significant increase in LC3II and LAMP2A protein expression. Therefore, suggesting that protection was brought about by the prolonged IF intervention through the modulation of macroautophagy and CMA. Conclusion: Our findings indicate that autophagy is upregulated in the presence of high levels of APP and Aβ, and to our surprise, remains upregulated even in the presence of apoptosis induction, suggesting an insufficient autophagy response in the mitigation of Aβ neurotoxicity. However, enhanced Aβ clearance was observed with a sufficiently high autophagy response even during 48 hrs APP overexpression, suggesting that autophagy modulation may be a viable treatment approach long into disease progression. These findings were also confirmed with prolonged IF intervention, where markers of apoptosis, and lipid peroxidation were notably decreased in brain regions associated with neurodegeneration. Further studies, specifically using in vivo APP overexpression models are warranted to further verify the clinical use of autophagy control.
An evaluation of the hepatic proteomic signature in identifying cancer tolerance and resistance mechanisms in a mouse allograft system
(Stellenbosch : Stellenbosch University, 2017-03) Van Niekerk, Gustav; Engelbrecht, Anna-Mart; Loos, Benjamin; Nell, Theo A.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Background The unfavourable therapeutic index of most treatment modalities has greatly impeded progress in the development of effective cancer therapy. Therefore a need exists for treatment modalities that are less taxing on a patient’s health status (i.e. maintain a patients reserve capacity and thus prolong survival) while additionally not invoking counter evolutionary strategies from tumour cells. Plant biologist have long distinguished between the host’s ability to accommodate pathogen burden, as oppose to its ability to antagonise pathogen load. Hence, the disease trajectory is not only dependent on the hosts’ ability to resist an infection, but also on the capacity to tolerate pathogen burden. This distinction have only recently been applied to animals. A number of observations suggest that this distinction may be of great immunological relevance, including the prevalence of asymptomatic carriers and natural variation in the population with regards to disease progression. Thus, the tolerance/resistance (T/R) paradigm represents a novel approach for understanding disease progression. We hypothesise that similar mechanisms might underlie host-tumour dynamics. Study aims and experimental design The current study aimed to evaluate the application of the T/R framework within an oncological context. A syngeneic mice model system was used to compare tolerance and resistance between two cancer cell lines. C57BL/6 mice were inoculated with either mammary carcinoma cells (EO771) or melanoma cells (B16). In a clinical setting, health status would not only be influenced by tumour load, but also by therapeutic interventions such as cytotoxic therapies, which must also be tolerated. Thus, a second study was performed using chemotherapeutic regimes as a variable to explore the effect of high (5 mg/kg) and low (2 mg/kg) dose doxorubicin (DXR) treatment on tolerance and resistance in mice. In addition, attempts were made to identify mechanisms underlying differences between groups with regards to variation in tolerance and resistance. To this end, a combination of immunoblotting and proteomic analyses were performed. Methodology: quantifying tolerance and resistance Resistance was quantified as the slope of a regression line, with tumour volume as response variable, and time as independent variable. Tolerance was measured similarly, but with body weight as response variable and tumour load as independent variable. Differences in regression slopes was used to compare tolerance and resistance. To confirm tolerance, differences in gastrocnemius muscle cross-sectional area (MCA) were compared between groups. Results Mice inoculated with melanoma (B16) cells showed a significantly lower resistance compared to mice inoculated with breast cancer EO771 cells. With regards to tolerance, B16 cells also exhibited lower tolerance, though tests for homogeneity of regression slopes demonstrated that these differences did not reach significance (p = 0.0856). Similarly, B16 and EO771 groups did not exhibit any difference in MCA. Comparing the effect of high and low dose DXR on mice bearing EO771 revealed that DXR decreases resistance: both low dose and higher dose DXR increased tumour growth as demonstrated by significantly steeper slopes in DXR groups compared to the tumour control group. In order to explain the increase in EO771 tumour growth in mice receiving DXR, the activation of a panel of signalling proteins associated with cell growth and survival (cRaf, ERK, p38 MAPK, JNK, PTEN, PI3Kp85, PDK1, Akt, mTOR, Bcl-2) as well as apoptotic markers (Caspase 3, 8 and 9) in tumour samples were evaluated by western blot analyses. However, the only significant finding include elevated ERK activation in mice receiving DXR, suggesting that extracellular signalling molecules might drive tumour growth. Since the liver plays a critical role in energy homeostasis, as well as in the production and clearance of circulating factors, western blot analyses were performed on liver samples. Markers of autophagy (p62 and LC3B-II) as well as growth signalling proteins (Akt and mTOR) and apoptosis (Caspase 3) were evaluated by western blot analyses. Mice inoculated with B16 demonstrated a marked increase in both p62 and LC3B-II, signifying an increase in autophagosome pool size, most likely due to dysfunctional lysosomal fusion. Surprisingly, other makers in both EO771 and B16 did not significantly differ from control liver samples. Subsequently, liver proteomics were performed making use of a Gene Ontology approach in order to describe biological, functional, structural and other processes that are uniquely altered between groups. Interestingly, a comparison between livers of mice inoculated with B16 melanoma cells and EO771 breast cancer cells also suggested that autophagic activity was not upregulated compared to the control group. DXR groups also did not exhibit differences in autophagic processes, though proteins involved in the proteasomal pathway were upregulated in mice receiving high doses of DXR. An increase expression of enzymes associated with retinoic acid metabolism was observed in the B16 group, which might explain decrease tolerance and resistance in this group. An increase in steroid metabolism was also observed in mice receiving DXR. Since cholesterol form a key component of cell membranes, it is possible that cholesterol synthesis might enable rapidly growing tumours of mice receiving DXR. Finally, concurrent up- and downregulation of certain proteins involved in radical scavenging in DXR mice might suggest a differential free radical scavenging response, thus explaining why anti-oxidant therapies have not proven successful in clinical settings in response to DXR. Collectively, these observations highlight alteration in hepatic activities through which tolerance and resistance mechanism might manifest. In summary, this study have demonstrated the implementation of the T/R framework within an oncological setting. Evidence suggest that defects in hepatic autophagy might contribute to lower tolerance, and possibly also resistance. Autophagy was not significantly upregulated in response to DXR which was associated with lower tolerance. Similar, mice inoculated with B16 tumours exhibited lower tolerance as well as evidence for suppressed lysosomal fusion with autophagosome. These observations suggest that a compromised autophagic apparatus might contribute towards the lower tolerance. Proteomic results are also suggestive of a potential role played by altered liver metabolism, including retinoic acid and steroid metabolism. Future studies evaluating the role of this pathways might identify novel tolerance-promoting pathways.
Exercise, stress and immune system functional responses
(Stellenbosch : University of Stellenbosch, 2004-12) Smith, Carine; Myburgh, Kathryn H.; University of Stellenbosch. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Stress related to chronic exercise affects both the immune and endocrine systems, but there are still many issues that are poorly understood, particularly effects of stress on the functional capacity of immune cells. This thesis probed some of these issues using physiological models of physical and psychological stress. Both exercise training stress and chronic psychological stress in human subjects were shown to result in an up-regulation of spontaneous reactivity of white blood cells in vitro, using two different assays, namely a) a peripheral blood mononuclear cell (PBMC) culture assay measuring immune cell responsiveness and b) a relatively new flow cytometry technique for assessing activation status of cells by their expression of the surface marker CD69, in a lymphocyte subpopulation-specific manner. An up-regulation of immune cell activation in the absence of an additional stressor was associated with a decreased capacity to mount a response to a subsequent mitogen stimulus in vitro after chronic psychological stress and acute, extreme exercise stress. Another novel finding was that cortisol high-responders to chronic psychological stress exhibited a higher spontaneous reactivity of both CD4+ and CD8+ lymphocytes when compared to cortisol low-responders. This result indicates that chronic exposure to cortisol may decrease its usual inhibitory effect on spontaneous T lymphocyte responsiveness. After optimisation of an animal model of mild, psychological stress, we demonstrated (using an IL-6 antibody) that IL-6 is necessary for a full-blown cortisol response to chronic, intermittent mild stress. Results also suggest that IL-6 plays a role in regulation of its own secretion by PBMCs in response to a stressor, by maintaining the production of IL-1β in the face of stress. Basal serum corticosterone concentration was shown to be the main determinant of the magnitude of mitogen-stimulated PBMC secretion of IL-6 in vitro in the stress-free controls. However, after blocking of IL-6 in vivo, IL-1β was identified as a major regulator of IL-6 secretion by mitogen-stimulated PBMCs in vitro, independently of the presence or absence of stress. The implications of these novel findings are that proinflammatory cytokines are sensitively regulated during mild stress.Mean serum cortisol concentration at rest was not a useful tool to assess chronic exercise stress after training intervention. However, classification of athletes at baseline into two groups according to their resting serum cortisol concentration illustrated two distinct patterns for the responses of both cortisol and the cortisol:testosterone ratio to chronic stress. These studies on the effects of chronic stress on parameters of the endocrine stress-axis and the immune system led to the following main conclusions: a) chronic exposure to cortisol results in a decreased inhibition of spontaneous immune cell activity at rest, b) this increased spontaneous activation of immune cells at rest in the absence of a stressor, is associated with a suppression of immune capacity to respond to a subsequent challenge, c) the latter finding is not evident under stress-free conditions where cortisol promoted immune cell IL-6 secretion, and d) IL- 1β and IL-6 are involved in the regulation of each others’ secretion.
Faktore wat die prestasie en gesondheid van vroue-atlete kan beinvloed
(Stellenbosch : Stellenbosch University, 2003-12) Strauss, Johannes Albertus de Wet; Myburgh, Kathryn H.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
ENGLISH ABSTRACT: Although it is common knowledge that regular exercise has many beneficial effects on the human body, it is also true that many highly competitive athletes neglect their health for the sake of performance. With this as a general objective for the study, women athletes of the Matie Athletics Club were recruited as subjects and were monitored and tested for several health-related parameters. Current results indicate that, although the average total cholesterol (TC) concentrations of the group were within normal ranges, quite a number of the sprint and field athletes had TC values regarded as a cardiovascular risk (> 5.2 mmol.l"). Serum testosterone levels of the sprint and field athletes were also higher than those of the distance athletes, but a correlation between TC and testosterone was not established. In general, cholesterol intake of women athletes was within the recommended daily allowance (RDA) prescriptions. The high-density lipoprotein fraction was also within the norm, but a better chemical pathological range had been expected. All haematological parameters were within the normal ranges of distribution, but the red blood cell count, haemoglobin concentration and hematocrit were on average lower than the standard average for females. Athletes, quite often, have higher plasma volumes than average and this can disguise normal haematological values and is described as sport anaemia. The current study has also indicated an iron deficiency (83% RDA) in the diet of female athletes in general. Thus the relatively low observed red blood cell count could not necessarily be attributed to sport anaemia. The energy intake was also poor and did not comply with the energy needs of the athletes. Bone mineral density (BMD) and plasma electrolytes were normal. Distance athletes had a higher BMD of the hip compared to the lumbar spine area. This is probably related to the stress to the hip associated with running. A correlation was observed between TC and BMD of the hip of eumenorrheal and amenorrheal athletes, which had not been observed before. The influence of the phase of the menstrual cycle on the immune system is controversial, and the results of the thesis confirm those of other studies that indicated no influence. In addition, it has been shown that the exogenous ingestion of glutamine, before the onset of exercise, can increase the plasma concentration thereof, and that the formerly observed decline (also seen in the current study) after intense exercise can be totally neutralized. This had not been reported before. The physiological significance of this has not been established, but the assumption is that a continuous adequate supply of glutamine will benefit the immune cells with regard to its reaction to pathogens. As reported by others, it has been shown that the ingestion of 5% glucose during long duration exercise eases the stress on the immune system, as both leucocytes and cortisol levels were attenuated compared to intake of a placebo. A new discovery, however, was that the ad libitum ingestion of glucose was not enough to produce desired significant results. The importance of this finding may have practical implications with regard to desirable amounts of glucose supplementation during races. In conclusion: Female athletes of club performance level are on general in a healthy condition, but are not excluded from the risk with regard to cholesterol. The screening of TC alone is insufficient with regard to competitive athletes, unless the sub-fractions are screened as well during routine medical examinations. Adjustments with regard to the energy and iron content of the diet are suggested. Supplementation of glutamine and glucose before and during exercise could be beneficial to the immune system. More studies with regard to the association of cholesterol with BMD are recommended.

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