Doctoral Degrees (Medical Physiology)

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Browsing Doctoral Degrees (Medical Physiology) by browse.metadata.advisor "Du Plessis, Stefan"

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    Gamete proteomic profile of male patients suffering from sexually transmitted infections
    (Stellenbosch : Stellenbosch University, 2016-03) Flint, Margot Gwyneth; Du Plessis, Stefan; Van der Horst, Gerhard; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Medical Physiology.
    ENGLISH ABSTRACT: The aim of this study was to provide better insight into the effects of Neisseria gonorrhoea, Chlamydia trachomatis, and Trichomonas vaginalis on semen characteristics. In addition, a crucial focus was also to determine the protein profile of spermatozoa isolated from these infected semen samples. Identification of semen samples positive for bacterial colonisation of N. gonorrhoea and T. vaginalis were done through the use of differential isolation mediums. For the detection of samples positive for C. trachomatis, the ImmunoComb® was employed, which is a quantitative indirect enzyme immunoassay. Macro- and microscopic semen and spermatozoa parameters were assessed, including: volume, pH, viscosity, concentration, motility, morphology, viability, acrosome reaction, leukocyte count, and PMN-elastase concentration. To assess prostate and seminal vesicle functioning, the seminal concentrations of citric acid and fructose were photometrically quantified. The quantification of the level of reactive oxygen species (ROS) production was determined by means of flow cytometry and the DNA fragmentation was detected using a commercially available assay. For proteomic analysis, samples were iTRAQ (isobaric tags for relative and absolute quantification) labelled and underwent liquid chromatography-mass spectrometry, followed by data analysis, protein identification, and quantification. Results of the study showed that amongst the three microorganisms, the most prevalent occurrence rate in the population tested was that of T. vaginalis. The effects of the studied sexually transmitted infections (STIs) on the spermatozoa parameters demonstrate the negative impact of the microorganisms on the fertility outcome of the male partner. The study has shown the relationship between leukocyte-derived ROS and DNA fragmentation, which can significantly impair the fertility outcome. Additionally, the findings of decreased concentrations of fructose in the ejaculates positive for N. gonorrhoea, demonstrates the effect of an STI on the glandular functioning of the secretory activity of the seminal vesicles. The observed negative correlation between ROS and DNA fragmentation in samples positive for N. gonorrhoea demonstrates the impact of a pro-oxidant overload on spermatozoa DNA integrity. A considerable number of 178 differentially expressed proteins (DEPs) were identified in the STI positive sample groups. The study subsequently focused on specific proteins according to their role in male fertility. Insight into the role DEPs may play in spermatozoa metabolism and the impact on the motility was provided. Amongst structural related proteins, the down-regulation of outer dense fibre 2, in samples positive for C. trachomatis, was evaluated. Oxidative stress related proteins included the up-regulated superoxide dismutase 1 in the ejaculates positive for N. gonorrhoea and down-regulated peroxiredoxin 5 in the C. trachomatis positive group. From the proteins involved in the response to physiological stress, heat shock proteins was discussed, with the down-regulation of the 70-kDa heat shock protein and the up-regulated HSP 90-kDa-beta member 1, both identified in samples positive for N. gonorrhoea. Among immune response proteins, prolactin-inducible protein (C. trachomatis) was found to be down-regulated, while azurocidin (N. gonorrhoea) and filamin-B (T. vaginalis) were up-regulated. Proteins involved in DNA condensation included down-regulated protamine-2 (C. trachomatis). The identification of proteins, which are differentially expressed between spermatozoa from samples positive for STIs, can provide crucial insight into their possible influence on male fertility and role as potential biomarkers for further research. Research on a proteomic and molecular level could allow for the prevention of the long-term obstacles facing partners experiencing compromised fertility. Despite the ongoing research focusing on the sperm proteome, it can be stated with reasonable certainty, that this is the first study into the proteomic profile and corresponding parameters of spermatozoa isolated from semen samples positive for STIs.
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    Ischaemic preconditioning : an investigation of the patterns of kinase activation and protein expression profiles during reperfusion in the rat heart
    (Stellenbosch : Stellenbosch University, 2013-12) Hattingh, Susanna Maria (Suzel); Du Plessis, Stefan; Engelbrecht, Anna-Mart; Salie, Ruduwaan; Stellenbosch University. Faculty of Medical Sciences. Dept. of Biomedical Sciences. Division of Medical Physiology.
    ENGLISH ABSTRACT: Introduction: Coronary heart disease (CHD) is the leading cause of death worldwide with 3.8 million men and 3.4 million women dying globally each year. Although existing myocardial reperfusion strategies such as thrombolysis and percutaneous coronary intervention (PCI), if applied in a timely manner, limit myocardial infarct size, the mortality and morbidity remains significantly high. Ischaemic preconditioning (IPC) may offer the potential to attenuate myocardial ischaemia/reperfusion injury through cardioprotective signaling pathways which is recruited at the time of myocardial reperfusion, thereby improving clinical outcomes in patients with coronary artery disease. Ischaemic preconditioning is a phenomenon whereby short intermittent episodes of coronary occlusion followed by reperfusion protect the myocardium against a subsequent period of sustained ischaemia. This protection is reflected in the limitation of infarct size and improved functional recovery of the ischaemic heart during reperfusion. Despite intensive research efforts, the promise of an effective cardioprotective strategy using the endogenous protective mechanisms of the heart which underlies IPC, has not yet been materialized. Although progress has been made in terms of signaling mechanisms in the preconditioned heart, the identification of the myocardial reperfusion phase as the critical “window” for cardioprotection, requires the elucidation of the signal transduction pathways during the reperfusion phase after IPC. In view of the above, the aims of the present study were to investigate: i. the involvement of the RISK pathway and p38 MAP kinase pathway in IPC during early and late reperfusion ii. the involvement of heat shock protein-27 (HSP-27), heat shock protein-70 (HSP-70), GSK-3β, CAMKII, AMPK and the transcription factor CREB in the context of IPC during early reperfusion iii. the involvement of autophagy and apoptosis during early and late reperfusion after IPC iv. the correlation of the protein kinases with the hemodynamic parameters of the heart v. the mechanism of IPC by means of two-dimensional (2D) proteomics Methods: The isolated perfused working rat heart model was used with functional recovery as end-point. Hearts were preconditioned (IPC) for 3x5 min global ischaemia, alternated with 5 min reperfusion. Hearts were subjected to 25 min sustained global ischaemia, followed by 5, 10, 15 or 30 min reperfusion when hearts were snap-frozen for western blotting analysis. Alternatively, hearts were reperfused for 30 min to record hemodynamic parameters and measure functional recovery. Non-preconditioned (Non-IPC) hearts were stabilized for 30 min and subjected to 25 min sustained global ischaemia followed by 5, 10, 15 or 30 min reperfusion when hearts were snap-frozen. Alternatively Non-IPC hearts were reperfused for 30 min to serve as control for the 30 min reperfused IPC group. Activation of the protein kinases was determined by western blotting analysis. For the proteomic study mitochondrial and cytosolic proteins were isolated from heart tissue and separated in the first dimension by isoelectric focusing, followed by separation in the second dimension by two dimensional gel electrophoresis. The PD Quest software programme was used to identify significantly expressed protein spots. Protein spots of interest were excised and subjected to in-gel digestion and the resulting peptides were analysed by mass spectrometry. Proteins were identified by Mascot and the Swiss Prot database. Results: Western blotting analysis demonstrated that the RISK pathway and p38 MAPK are activated very early in reperfusion, but the activation is not sustained during the reperfusion period. Autophagy is also upregulated during this early reperfusion phase; it is attenuated in the middle reperfusion phase and increase for a second peak of upregulation in the late reperfusion phase. In addition, we identified CAMKII as a novel marker of functional recovery in IPC after reperfusion. The proteomic analysis identified twenty differentially expressed mitochondrial and thirty six differentially expressed cytosolic proteins between Non-IPC and IPC hearts. Functions ascribed to the majority of these individual proteins were directly related to cardiac metabolism. Conclusion: Activation of the majority of the protein kinases investigated in the present study is associated with the hemodynamic parameters of the heart instead of functional recovery. Results indicated that the variable signaling patterns could be attributed to differences in heart rate and the effect thereof (ejection fraction, minimum and maximum rate of contraction), as a result of sympathetic stimulation due to psychological stress in the animals before slaughtering. Proteomics results demonstrated that IPC hearts which failed after ischaemia /reperfusion are metabolically compromised and “worse off” compared to non-IPC hearts.
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    The Possible Ameliorating effects of Rooibos, Honeybush and Sutherlandia on diabetes-induced reproductive impairment in adult male Wistar rats
    (Stellenbosch : Stellenbosch University, 2020-03) Omolaoye, Temidayo Siyanbola; Du Plessis, Stefan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.
    ENGLISH ABSTRACT: The global prevalence of infertility is on the rise, and so is male factor infertility. Out of the approximately 72.4 million infertile couples worldwide, male factor is responsible for 50%. It has been reported that sperm quality is on the decline, even in healthy men. Several aetiologies have been identified, and this includes disease-related male infertility, such as diabetes mellitus (DM). DM is a chronic non-communicable disease, marked by increased hyperglycaemia which occurs as a result of lack in insulin secretion or due to the insensitivity of the target tissue to the metabolic effect of insulin. It has been reported that 422 million people are affected with DM globally, of which ≤10% is type 1 diabetic and ≤90% is type 2. Studies have shown that although most autoimmune diseases usually affect a higher percentage of women, type 1 diabetes is more prevalent in boys and men. The consequential detrimental effects of DM on male fertility have been reported in both experimental animals and diabetic men. This includes, decreased sperm motility, reduced normal sperm morphology, increased fragmented spermatozoa and many more defects. In the course of unravelling the pathways through which DM affect male fertility, studies have elucidated the role of endocrine dysfunction, formation of reactive oxygen species (ROS) and the subsequent development of oxidative stress (OS). However, other possible related pathways such as the involvement of glycated proteins, are yet to be explored. Since there are evidences that DM is detrimental to male fertility, it is important to firstly understand the underlying pathophysiology and then develop and explore strategies to combat this disease and its associated male reproductive complications. Many natural and artificial agents have been proposed to have antioxidant properties due to their potential in reducing oxidation. Antioxidants work either by scavenging ROS or by preventing its formation. Rooibos (Aspalathus linearis), honeybush (Cyclopia intermedia) and sutherlandia (Lessertia frutescens) are plants endemic to Southern Africa. Herbal teas are derived from the leaves and stems of these plants. These infusions have been shown to be caffeine-free. Studies have also reported that the infusions from these plants contain bioactive chemical compounds with therapeutic benefits. Additionally, studies have shown their beneficial role in preventing the development of diseases and some have shown their ameliorative effects. However, their role in male reproduction is little understood as this represents the first study to report the effects of honeybush and sutherlandia on male reproductive function. Based on the expressed gaps in knowledge, the study was divided into two main parts, with each having several objectives. For the first part, thirty adult male rats were randomly divided into three groups of ten, which included a vehicle and two streptozotocin (STZ) groups receiving either 30mg/kg (STZ30) or 60mg/kg (STZ60) intraperitoneally. For the second part, ninety animals were divided into nine groups of ten without bias. The groups included a control (water only), vehicle (STZ control, citrate buffer + water), rooibos (2% fermented rooibos), honeybush (4% fermented honeybush), sutherlandia (0.2% unfermented sutherlandia), diabetic control (STZ45mg/kg + water), diabetic + rooibos (STZ45mg/kg + 2% rooibos), diabetic + honeybush (STZ45mg/kg + 4% honeybush), and diabetic + sutherlandia (STZ45mg/kg + 0.2% sutherlandia) group. Animals in the diabetic infusion treated groups were pre-treated with the respective infusion one week before DM induction. Animals were monitored closely throughout the treatment period and were sacrificed seven weeks after DM induction. Blood, testes and epididymides were collected for further analysis.The results of the first part of this study showed the negative effects of DM on male reproductive function, as diabetic animals presented with a decrease in spermatozoa with normal morphology and an increased number of spermatozoa with a higher deformity index. Histological and histomorphometrical analysis of the testis showed alteration in the seminiferous tubules’ cellular association in diabetic animals. Additionally, the first section further sheds some light on the involvement/expression of advanced glycation end products (AGE), its receptor (RAGE), mitogen-activated protein kinases and the activation of apoptosis in diabetes, which may contribute to the impairment seen in male fertility. Briefly, in DM, there is altered antioxidant enzyme activity (↓Catalase) which may result in OS. The development of OS can on the one hand instigate the breakdown of polyunsaturated fatty acids (PUFA), which can be measured by the levels of malondialdehyde (MDA) and also indirectly increase AGE formation. The peroxidation of PUFAs attracts more ROS formation, which can activate apoptotic induction, resulting in impaired sperm function. AGEs work by binding to their receptor (RAGE). The AGE-RAGE complex initiates the activation of the MAPKs, which may induce apoptosis by increasing the cleavage of PARP, hence resulting in nDNA damage. All these effects collectively result in reduced male fertility. To properly understand the role of the infusions (rooibos, honeybush and sutherlandia) on male reproduction of both healthy and diabetic animals, the second part of the study was divided into four sub-studies. Subsection one of the second part of the study investigated whether treatment with rooibos, honeybush and sutherlandia will impact sperm functional parameters positively or otherwise, in healthy rats. Animals treated with the respective infusions presented with a percentage increase in SOD activity but showed reduced sperm motility and decreased normal The results of the first part of this study showed the negative effects of DM on male reproductive function, as diabetic animals presented with a decrease in spermatozoa with normal morphology and an increased number of spermatozoa with a higher deformity index. Histological and histomorphometrical analysis of the testis showed alteration in the seminiferous tubules’ cellular association in diabetic animals. Additionally, the first section further sheds some light on the involvement/expression of advanced glycation end products (AGE), its receptor (RAGE), mitogen-activated protein kinases and the activation of apoptosis in diabetes, which may contribute to the impairment seen in male fertility. Briefly, in DM, there is altered antioxidant enzyme activity (↓Catalase) which may result in OS. The development of OS can on the one hand instigate the breakdown of polyunsaturated fatty acids (PUFA), which can be measured by the levels of malondialdehyde (MDA) and also indirectly increase AGE formation. The peroxidation of PUFAs attracts more ROS formation, which can activate apoptotic induction, resulting in impaired sperm function. AGEs work by binding to their receptor (RAGE). The AGE-RAGE complex initiates the activation of the MAPKs, which may induce apoptosis by increasing the cleavage of PARP, hence resulting in nDNA damage. All these effects collectively result in reduced male fertility. To properly understand the role of the infusions (rooibos, honeybush and sutherlandia) on male reproduction of both healthy and diabetic animals, the second part of the study was divided into four sub-studies. Subsection one of the second part of the study investigated whether treatment with rooibos, honeybush and sutherlandia will impact sperm functional parameters positively or otherwise, in healthy rats. Animals treated with the respective infusions presented with a percentage increase in SOD activity but showed reduced sperm motility and decreased normal The results of the first part of this study showed the negative effects of DM on male reproductive function, as diabetic animals presented with a decrease in spermatozoa with normal morphology and an increased number of spermatozoa with a higher deformity index. Histological and histomorphometrical analysis of the testis showed alteration in the seminiferous tubules’ cellular association in diabetic animals. Additionally, the first section further sheds some light on the involvement/expression of advanced glycation end products (AGE), its receptor (RAGE), mitogen-activated protein kinases and the activation of apoptosis in diabetes, which may contribute to the impairment seen in male fertility. Briefly, in DM, there is altered antioxidant enzyme activity (↓Catalase) which may result in OS. The development of OS can on the one hand instigate the breakdown of polyunsaturated fatty acids (PUFA), which can be measured by the levels of malondialdehyde (MDA) and also indirectly increase AGE formation. The peroxidation of PUFAs attracts more ROS formation, which can activate apoptotic induction, resulting in impaired sperm function. AGEs work by binding to their receptor (RAGE). The AGE-RAGE complex initiates the activation of the MAPKs, which may induce apoptosis by increasing the cleavage of PARP, hence resulting in nDNA damage. All these effects collectively result in reduced male fertility. To properly understand the role of the infusions (rooibos, honeybush and sutherlandia) on male reproduction of both healthy and diabetic animals, the second part of the study was divided into four sub-studies. Subsection one of the second part of the study investigated whether treatment with rooibos, honeybush and sutherlandia will impact sperm functional parameters positively or otherwise, in healthy rats. Animals treated with the respective infusions presented with a percentage increase in SOD activity but showed reduced sperm motility and decreased normal morphology. Paradoxically, they presented with increased sperm concentrations. Hence, rooibos, honeybush and sutherlandia may enhance sperm concentration, which represents the sperm quantity. However, they may impair sperm quality (sperm motility and morphology), when consumed by healthy animals. Subsection two of the second part of the study investigated the testicular oxidative stress and apoptosis status in diabetic rats following treatment with rooibos, honeybush and sutherlandia infusions. This section highlighted the negative impact of DM on sperm functional parameters through increased lipid peroxidation and reduced antioxidant activity. The infusion treatment groups displayed increased antioxidant enzyme activity, which may be partly responsible for the observed improvement in sperm motility and morphology of diabetic animals receiving rooibos (DRF) and diabetic animals receiving sutherlandia (DSL). Additionally, the current study have shown the increased expression of apoptotic biomarkers in the diabetic control (DC) animals, which were not alleviated by the infusions. This suggests that these infusions play a role in alleviating diabetes-induced sperm function impairment through suppression of OS, but their role in apoptosis is still unclear. Subsection three of the second part of the study investigated the role of rooibos, honeybush and sutherlandia on insulin signalling in the testes of diabetic rats. All diabetic groups presented with a significant increase in blood glucose levels after diabetes induction. However, the diabetic animals treated with the infusions showed only a mild decrease in fasting blood glucose. The diabetic control animals showed a decrease in testis protein expression of IRS-1, PkB/Akt and GLUT4. Diabetic animals treated with rooibos (DRF) and honeybush (DHB) displayed an upregulation in IRS-1, while diabetic animals treated with sutherlandia (DSL) showed improvement indecrease in PkB/Akt, DHB and DSL animals displayed upregulation compared to control. All diabetic animals showed increased phosphorylated ERK1/2 and reduced total ERK1/2 when compared to control and vehicle. Additionally, diabetic control animals presented with a non significant decrease in plasma testosterone concentration compared to control, while DRF and DSL showed a significant decrease and DHB showed upregulation. This suggests that (i) the activation of IRS, PkB/Akt, ERK and GLUT translocation is important in testicular insulin signalling (ii) rooibos, honeybush and sutherlandia may play a role in testicular insulin signalling, however, through different pathways (iii) honeybush may mildly enhance testosterone production in diabetes. Lastly, the fourth subsection of the second part of the study investigated the descriptive histological evaluation of the testis and cauda epididymis after treating with rooibos, honeybush and sutherlandia in both healthy and diabetic rats. The infusion control groups (RF, HB and SL) showed normal seminiferous tubule cellular association, presence of spermatozoa in the epididymal lumen and normal overall architecture. Both the testicular and epididymal morphology were altered in DM, but these disruptions were mildly alleviated by rooibos, honeybush and sutherlandia. It appears, from appreciating the results of the entire study, that diabetes does have detrimental effects on male reproduction, both at the tissue and cellular levels. The cellular impact may be exerted through protein glycation, development of OS and apoptosis induction. The use of rooibos, honeybush and sutherlandia, when healthy, should be taken moderately and with caution, as too much may be detrimental and may impair sperm functional parameters. However, in disease conditions such as diabetes, these infusions may be beneficial, as: (i) Rooibos and sutherlandia may mildly improve sperm quality (motility and morphology), (ii) Rooibos, honeybush and sutherlandia may mildly reduce hyperglycaemia by enhancing insulin signalling, (iii) Honeybush may enhance testosterone production, (iv) Rooibos, honeybush and sutherlandia may mildly improve the histomorphological architecture of the seminiferous tubule and cauda epididymal tubule.