Philosophiae Doctor - PhD (Medical BioScience)

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Effect of Nicotine and Alcohol Exposure in utero on Vascular Intima Medial Thickness, Endothelial Function, and the Development of Cardiometabolic Risk Factors in Children in a Low-Income Setting.
(University of the western Cape, 2025) Ras (née Hartel) Tammy charlene; Juléy J.A. De Smidt
Foetal exposure to nicotine and alcohol during pregnancy may result in cardiovascular disease (CVD) later in life. In low socioeconomic regions, CVD risk factors have also presented in children and adolescents. The Developmental origins of Health and Disease (DoHaD) hypothesised that prenatal exposures will result in long-lasting, if not permanent adaptations in the structure, physiology and metabolism in the foetus, therefore predisposing children to CVD later in life. Several studies have reported associations between increased intima medial thickness (IMT) and CVD risk factors in offspring with in utero exposure to alcohol and nicotine. Studies have also proposed potential mechanisms of nicotine and alcohol such as oxidative stress, induced by chronic hypoxia in utero which affects nitric oxide (NO) bioavailability in the vascular endothelium. Aim: The aim of this study is to determine the effect of in utero exposure to nicotine and alcohol on vascular structure and function, and the development of cardiometabolic risk factors in adolescents in a low-income setting. Methods: This is a case-control study in 307 children aged 10-14 years. In Phase I, cardiometabolic parameters such as body mass index (BMI), waist circumference, subscapular and triceps skinfold thickness, blood pressure, lipid profiles and non-fasting blood glucose concentrations were measured. Mothers were interviewed to obtain data regarding maternal smoking and alcohol use, health status, and demographic characteristics. In phase II, subclinical signs of atherosclerosis were measured using non-invasive methods. Endothelial function was measured in the brachial artery (BA) using flow mediated dilation (FMD) to measure blood flow velocity and arterial diameter using Doppler and B-mode ultrasonography, respectively. Carotid artery IMT was measured using B-mode ultrasonography. All data was analysed using SPSS® software version 30.
Investigations on the antifungal and cancer modulating properties of extracts from selected species of Tulbaghia
(University of the Western Cape, 2012) Keyser, Zanephyn
Fusarium verticil/ioides (Sacc) Nirenberg a common phytopathogen of maize and maize-based products produces fumonisin B (FB) mycotoxins that have been related to several diseases such as equine leukoencephalomalacia (ELEM), porcine pulmonary edema (PPE), liver toxicity in several animals and esophageal and liver cancer in humans. In one of our studies we hypothesize that aqueous extracts of indigenous South African wild garlic species (Tulbaghia violacea, T. alliacea and T. simmleri) may enhance the efficacy of the fungicides, SporekilPu, Thiram, Itraconazole and Fluconazole against F. verticil/ioides (MRC 826). Data analysis from in vitro results indicates that for the 16 different mixtures of each plant extract and fungicide combination, several significantly (P
The relationship between "fertilizaiton environment" and structure and physiology of selected Anuran Spermatozoa
(University of the Western Cape, 1993) Wilson, Brian Adam
The propagation of sPecies and modes of fertilization of higher vertebrates are aspects which have been extensively researched' Studies on the spermatolory of lower vertebrates have not received the same attention. Anurans exhibit modes of fertilization which place them in a category between true aquatic and true internal fertilizers. They occupy fertilization environments ranging from aquatic to internal. This project was undertaken to establish baseline values for selected anuran sperrnatozoa and to test the hypothesis that fertilization environment plays a role in regulating sperm form and function. Anuran spermatozoon structure and ultrastructure were investigated and comparisons drawn between sperm from aquatic and terrestrial fertilizers. Measurements of sperm heads, acrosomes and tail complexes were used to discern whether significant differences exist between sperm from aquatic and terrestrial anuran fertilizers. Techniques were developed to determine baseline values for spermiograms of selected anuran species. These values were also compared to test the effect of selection pressures of the fertilization environment on sperm morphology and physiology. Sperm motion as a parameter to ascertain the viability of sperm was evaluated by using a Computer Assisted Sperm Motiliry Analysis (CASMA) system viz. the Sperm Motility Quantifier (SMO). The pattern and vigour of sperm motion were used as additional data to further elucidate the above mentioned hypothesis. spermatozoa were aspirated fuom Xenopus laeyir toads by developing methods to obtain sperm from amplectant males and via an electro ejaculation technique. The motility patterns were assessed and compared to determine similarities or differences between testicular and ejaculated spermatozoa' The research indicated that the fertilization environment plays a major role in modulating sPerm structure and function but that the effect of phylogeny is as important. The baseline values obtained during this project could possibly be utilised in programmes to protect endangered anuran species e.g. by using these values for the cryopreservation of sperm.
Investigation of the in vitro antidiabetic and neuroprotective effects of selected Helichrysum species against high monosaccharide-induced toxicity
(University of the Western Cape, 2022) Akinyede, Kolajo Adedamola
Chronic hyperglycaemia (glucotoxicity), a common complication in diabetes mellitus (DM), is known to cause cognitive decline in some of these patients, often affecting their overall quality of life. Some medicinal plants used in folk medicine for DM have also been reported effective in treating some of its co-morbidities, including cognitive decline. Plants of the Helichrysum genus (Asteraceae family) are well known in South African traditional medicine for their diverse health benefits, which make them potential sources of biologically active compounds. Thus, in the current study, the composition of bioactive compounds, as well as the antidiabetic and neuroprotective effects of selected Helichrysum plants viz: Helichrysum pandurifolium Schrank, Helichrysum foetidum, Helichrysum petiolare and Helichrysum cymocum, were investigated. Liquid chromatography-mass spectrometry (LC-MS) analysis was first used to characterise the aqueous acetone extracts of each plant, followed by bioactivity evaluation. LC-MS analysis and phytochemical screening revealed H. petiolare extract has the highest content of phenolics, saturated fatty acids, polyunsaturated fatty acids and total flavonoids. The best nitric oxide scavenging activity and total antioxidant capacity compared with other species were also demonstrated. Furthermore, the flavonoid composition varied in all extracts, with H. petiolare and H. pandurifolium Schrank extracts having the highest number of flavonoids. Thus, the aqueous acetone extract of H. petiolare (AAHPE) was further investigated for antidiabetic and neuroprotective potentials. The AutoDock Vina tool was used for molecular docking simulation. Treatment with AAHPE (25-75 µg/mL) improved the cell viability and increased the concentration-dependent percentage glucose uptake in the insulin-resistant HepG2 cell line significantly compared with the control. The highest AAHPE concentration (75 µg/mL) showed higher glucose uptake activity than the standard drug, metformin. Furthermore, AAHPE was found to inhibit both α-amylase and α-glucosidase enzymes in vitro, as corroborated by molecular docking results that showed strong binding (ΔG) of AAHPE flavonoids to α-amylase and αglucosidase compared with the drug, acarbose (ΔG for flavonoids = -7.2 to -9.6 Kcal/mol vs ΔG for acarbose = -6.1 Kcal/mol) for α-amylase; (ΔG for flavonoids = -7.3 to -9.0 Kcal/mol vs ΔG for acarbose = -6.3 Kcal/mol) for α-glucosidase. Analysis of in vitro neuroprotective effects of AHHPE against glucotoxicity induced on SH-SY5Y cells showed improved cell viability at all treatment concentrations (25-100 µg/ml). It also showed reduced reactive oxygen species production (ROS) and increased production of adenosine triphosphate (ATP) compared with cells treated with the standard acetylcholinesterase (AChE) inhibitor drug, donepezil (1 mM) or untreated cells (300 mM glucose). Molecular docking analysis showed that selected AAHPE flavonoids exhibited tight binding forces (better inhibitory profiles) with AChE compared with donepezil (-8.3 Kcal/mol). The flavonoids include 3, 5- dicaffeoylquinic acid (-9.9 Kcal/mol), isorhamnetin 3-galactoside (-8.8 Kcal/mol), 4,5- dicaffeoylquinic acid (-8.6 Kcal/mol), methyl 3, 5-di-O-caffeoyl quinate (-8.6 Kcal/mol), 3- caffeoylquinic acid (-8.4 Kcal/mol), quercetin-3-glucoside (-8.4 Kcal/mol) and sinocrassosideA1 (-8.4 Kcal/mol). Thus, the AAHPE and its bioactive phytocompounds, especially flavonoids, with their potential antioxidant effects, more effective glycaemic control than metformin, and better neuroprotective effects than donepezil against DM-associated disorders (e.g cognitive decline). Hence, they could be developed as commercially available dietary herbal supplements for managing postprandial hyperglycaemia.
Proteome profiling as tool to determine the effectiveness of traditional Africa therapies for Type 2 diabetes mellitus
(University of the Western Cape, 2022) Nouh, Ali Mohamed Balbout
Type 2 diabetes mellitus (T2DM) involves multiple organ dysfunction accompanied by a chronic inflammatory state and oxidative stress resulting from a combination of factors such as hyperglycaemia and dyslipidaemia. Although medication to treat T2DM is currently available, several limitations and harmful effects of anti-hyperglycaemic diabetes synthetic drugs make it necessary to investigate novel drug therapies that are safer and more efficient. The biggest challenges remain in the management of T2DM and its complications. Therefore, it is necessary to prevent the early onset of diabetes and the progression of the disease individually by using pharmacological and traditional therapies. Three traditional African herbs and a standard Western medicine for treating diabetes mellitus were selected for consideration in this study. The herbs selected were Trigonella foenum-graecum (T. foenum-graecum), Cinnamon verum (C. verum) and Artemisia afra (A. afra). The standard Western medicine chosen was metformin. These treatments were selected to assess potential effects on regulating the pro-inflammatory marker response and metabolism disorder bio-actives of mimic diabetic cells using in vitro assays. The murine macrophage cell line (RAW 264.7) cultures were selected to assess the effects of plant extracts and metformin on the inflammatory response and metabolic disorder biomarkers regulation. The effects on RAW 264.7 cells were monitored in the absence or presence of high glucose and lipopolysaccharide. The objectives of this study were to extract and analyse the chemical components of the plants T. foenum-graecum, C. verum and A. afra to verify their main bioactive compound concentrations; demonstrate cells expressing cytokines; assess the effects of plant extracts on pro-inflammatory (cytokines and chemokines) and biomarker activities in the presence or absence of high-glucose and lipopolysaccharide stimulation; evaluate the effects of herb extracts in comparison to metformin. In conclusion, this study presents parameters of the pro-inflammatory cytokines, chemokines, and growth factors of mimic diabetes cell expression. These protein molecules can be biomarkers and may be used for reducing diabetic complications because of low-grade/chronic inflammation, oxidation and lipid metabolism. They may also describe defects in the pathophysiology of T2DM and might offer an advantage in drug screening. Furthermore, evaluation of the effectiveness of traditional plants has revealed that the best herbal agent is A. afra ‒ it may have better anti-inflammatory action and the potential to regulate metabolism disorders, in the diabetic condition, than C. verum and T. foenum-graecum. These natural herbal extracts are more effective than metformin in inhibiting the pro-inflammatory response and regulation of oxidant activity in high glucose. This finding may be significant in contributing to increasing evidence that natural herbal therapies have potent anti-oxidant and anti-inflammatory properties that may result in the better regulation of dyslipidaemia. It may also open the way forward to novel therapeutic strategies for managing diabetic complications with minimal side effects. Patients with T2DM should be monitored and supplemented with antiinflammation and anti-oxidant therapies, combined with Western medicine, to avoid the progression of diabetes complications and achieve the most beneficial treatment.
The development of targeting nanosystems for the treatment of glioblastoma and neuroblastoma tumours
(University of the Western Cape, 2022) Boltman, Taahirah
Chlorotoxin (CTX) peptide selectively targets glioblastoma multiforme (GB) and neuroblastoma (NB) and has excellent blood-brain barrier permeability. Therefore, CTX is a promising targeting molecule for nanoparticle (NP) based diagnostic and therapeutic applications. Bimetallic gold-platinum nanoparticles (AuPtNPs) have recently attracted great attention for anti-cancer and catalysis applications due to the synergistic effects of combined metal atoms which enhance NP properties when compared to their monometallic NP counterparts. Therefore, the aim of this study was to develop gold NPs (AuNPs) and AuPtNPs, both conjugated to CTX for treatment in GB and NB cancer cells in vitro. This was achieved by synthesizing two novel CTX-NPs through the preparation of citrate capped AuNPs and AuPtNPs which was modified using two different types of polyethylene glycol (PEG) molecules, which allowed for conjugation of CTX onto the NPs. The physicochemical properties of the NPs were characterized, and ultraviolet-visible absorbance spectroscopy analysis demonstrated an increase in the absorption maxima (λ max) of all AuNPs, while the absence of an λ max for all AuPtNPs confirmed the formation of bimetallic NPs. Dynamic light scattering analysis for both citrate capped NPs showed a hydrodynamic size of approximately 7 nm, which doubled after surface functionalization with PEG and slightly increased following CTX surface functionalization. The zeta-potential revealed highly stable citrate NPs and decreased to more neutral charges following PEG and CTX surface functionalization confirming surface modification of NPs which was further supported by fourier transform infra-red spectroscopy analysis. Transmission electron microscopy (TEM) measurements revealed roughly spherical and monodispersed NPs with a core size of approximately 5 nm for all NPs. NPs were stable in biological media over 48-hours and CTXNPs demonstrated binding and uptake to U87 human GB and SH-SY5Y human NB cancer cell lines. NP-induced toxicity was investigated using the WST-1 cell viability assay at concentrations of 75-300 µg/ml for 48 hours in U87 and SH-SY5Y cells, while the non-cancerous KMST-6 human fibroblast cells served as a control cell line. The maximal inhibitory concentrations (IC₅₀ values) for all NPs in U87 and SH-SY-5Y cells was generally similar, however the most promising results was revealed in U87 cells with CTX-AuPtNPs treatment and was further investigated and showed the induction of apoptosis (using the APOPercentage™ apoptosis assay), the production of reactive oxygen species (using the CM-H2DCFDA probe), and decrease in mitochondrial activity (using the Tetramethylrhodamine ethyl ester, perchlorate probe) using flow cytometry. KMST-6 cell line highlighted selective toxicity towards the cancerous cell lines. CTX-NPs demonstrated significant decrease in cell survival through the inhibition of colony formation (using clonogenic assay) and inhibitory effects on cell migration (using wound healing assay) in U87 cell line. Gene expression analysis using real-time polymerase chain reaction (using the Human Molecular Toxicology Pathway RT² PCR Array) for investigating early cytotoxic effects of CTX-AuPtNPs, showed that genes involved in cellular stress responses were more significant, suggesting that U87 cells activated cytoprotective responses. In addition to the anti-cancer applications, AuPtNPs successfully reduced the 4-nitrophenol to 4-aminophenol at a catalytic rate constant (kcat) of 3.2 x 10-3/sec, demonstrating potential applications in catalysis.
Sperm function in different human sperm subpopulations
(University of the Western Cape, 2022) Keyser, Shannen
Human semen contains a heterogenous population of spermatozoa of which only a certain fraction is physiologically suitable for fertilization. Functional characteristics of these subpopulations and sperm separation techniques are not well understood. Furthermore, the questionable reliability of basic semen analyses has led to more emphasis being placed upon the assessment of detailed sperm functional parameters in determining male factor infertility as well as media supplements that could possibly enhance male fertility. Our study was designed to investigate and compare the functional characteristics of two sperm motility subpopulations and to determine which group of semen parameters provide predictive value into the presence of a high motility sperm subpopulation. In addition, the study aimed to investigate the effects of biological components present in follicular fluid on sperm functionality of different sperm motility subpopulations. Furthermore, it was aimed to determine whether the use of a new flagellar analysis and sperm tracking program (FAST) may provide more information on sperm motility functions as compared to computer-aided sperm analysis (CASA). Semen was separated into a high motile (HM) and low motile (LM) sperm subpopulation via double density gradient centrifugation. Subpopulations in human tubal fluid (HTF) were assessed for functional characteristics namely sperm viability, motility and kinematics, hyperactivation, acrosome reaction, reactive oxygen species and mitochondrial membrane potential, chromatin intactness and maturity. In the second phase, subpopulations were exposed to various media namely, HTF, capacitating medium (CAP), HD capacitating medium (HD-C), progesterone, myo-inositol, dopamine and prolactin, and subsequently subjected to the same functional tests as mentioned above. In the final phase of the study, subpopulations were exposed to selected concentrations of the media and individual spermatozoa further analysed with CASA and FAST for comparison of kinematic and flagellar characteristics. Two distinct sperm motility subpopulations were observed, with HM sperm subpopulations displaying significantly improved functionality compared to LM sperm subpopulations. Semen morphology abnormalities correlated with grouped motility parameters of the LM subpopulation. On the other hand, a combined group of semen total motility, progressive motility, viscosity and mucus penetration correlated with HM subpopulations’ grouped motility parameters. Media did improve sperm functionality, however progesterone and myo-inositol had the greatest effects on HM subpopulations, whereas dopamine and prolactin were more favourable to LM subpopulations. Furthermore, variable effects were observed for media on functional parameters. Collectively, total and progressive motility, viscosity and mucus penetration present a reliable group of semen characteristics for predicting the quality of a HM sperm subpopulation. Separating the same donor semen samples into two significantly varying motility sperm subpopulations can be a new approach to mimic the qualities of fertile and subfertile males’ sperm populations. Although motility subpopulations respond differently to various media, the study concludes that treatment of subfertile semen samples with biological substances present in follicular fluid could assist to develop new strategies for IVF treatment. Finally, FAST and SCA present with similar kinematic parameters, however flagellar parameters provide predictive value into rapid and medium progressive speed groups of individual spermatozoa.
The paracrine effect of normoxic and hypoxic cancer secretions on blood-brain barrier endothelial cells
(University of the Western Cape, 2022) Rado, Mariam Abobaker M
Cancer is the most common leading cause of death worldwide. Glioblastoma and breast cancer are the most aggressive solid tumour. The survival rate of these tumours depends on their ability to progress and spread. These cancers use their high proliferative capabilities for survival, increasing their malignancies. Glioblastoma is considered the most aggressive tumour initiated in the brain, whereas breast cancer is the most common metastatic cancer in the brain, both types of cancer are known as high infiltrated cancer and their invasiveness due to their capability to release factors that can alter the neighbouring cells to facilitate their progression. On the other hand, the brain remains a vital organ where the blood-brain barrier (BBB) plays a protective and homeostasis role, thus ensuring optimum brain function. The endothelial cells are the functional site of the BBB; these cells, with assistance from other brain cells such as astrocytes and pericytes, maintain the BBB protective function. The literature revealed perturbations and disruption in the BBB integrity in glioblastoma and metastatic breast cancer. The endothelial cells and other components of the BBB have been morphologically altered, resulting in impacting the BBB integrity. The interaction between cancer cells and brain endothelial cells is a complex scenario that is not entirely illustrated; however, a vital role in the crosstalk between cancer cells and endothelial cells is played by signalling mediated by soluble factors that can further promote cancer progression. This study aimed to investigate whether the secretions from glioblastoma and breast cancer cells could influence the brain endothelial cells. Brain endothelial cells (bEnd.3) were cocultured with glioblastoma (U-87) or breast cancer (MCF7) cells or subjected to their conditioned media. To mimic the heterogeneous physiological conditions of the solid tumour such as glioblastoma and breast cancer, U87 and MCF7 cells were cultured under normoxia (to reflect cancer cells exposed to oxygen levels in the perivascular regions of the tumour) or hypoxia (to reflect the hypoxic tumour area where invasive cancer cells are detached and invade the surrounding tissue forward to the blood vessels). Our findings underlined the involvement of paracrine secretion of cancer cells in modulating brain endothelial cells’ properties. Our data suggested that both cancer secretions derived from normoxia and hypoxia rendered changes in the brain endothelial cells at the level of mitochondrial activity. To investigate whether the exerted effects were associated with the acquisition of the brain endothelial resistance, we screened the transelectrical endothelial resistance (TEER) of brain endothelial cells after exposure to cancer secretions. Our findings revealed a decrease in TEER of the exposed brain endothelial cells. Moreover, gene expression of the tight junction (Claudin-5 and Occludin) were quantified in brain endothelial cells (bEnd.3) after exposure to normoxic and hypoxic cancer secretion using real-time qPCR; results showed upregulation of Claudin-5 after exposure to normoxic and hypoxic cancer secretion. Occludin gene expression was also upregulated after exposure to normoxic cancer secretion; however, the exposure to hypoxic cancer secretion decreased Occludin gene expression. In addition, the proliferation of endothelial cells (bEnd.3) exposed to cancer secretion was measured by cell counting, followed by analyzing the cell cycle; results showed that long term exposure to cancer secretion suppressed the proliferation of brain endothelial cells (bEnd.3), mostly after exposure to hypoxic cancer secretion. Cells were accumulated in the G1 phase. Overall, data suggest that factors secreted from normoxic and hypoxic cancer cells modulate brain endothelial cells, affecting their function.
A retrospective analysis of semen samples and reproductive hormones in Africa and the middle east
(University of the Western Cape, 2021) Moungala, Lionel Wildy
Semen analysis is the cornerstone for the investigation of male infertility. Semen quality can be influenced by geographical location, age, ejaculatory abstinence, and season. In 2010, the WHO published criteria for human semen characteristics that were markedly lower than those previously reported. Many reports have discussed the methodology used by the WHO to set the 2010 reference values. Some of the limitations of the WHO (2010) study included an undefined ejaculatory abstinence period, the limited representation of different age groups, and a limitation in geographical representation as the study did not include any data from Africa and Middle East. Therefore, the current cohort study was designed to provide retrospective data on semen quality (Africa and Middle East) and reproductive hormones (Middle East) in patients who underwent semen analysis and endocrine investigation at Andrology Laboratories in South Africa and Qatar. The effects of geographical location, age, ejaculatory abstinence and seasonal changes were evaluated. Furthermore, data from Africa (from the current cohort study) was compared to data from America, Asia, Australia and Europe obtained from global data from Cooper et al. (2010) and Campbell et al. (2021). Semen analysis reports (n = 70,765) for Africa and Middle East were obtained from Ampath Andrology Laboratory (n = 35,516), Lancet Andrology Laboratory (n = 24 967), Androcryos Andrology Laboratory (n = 7 450) and Hamad Medical Center (n = 2,832). Basic semen parameters such as semen volume, sperm concentration, total sperm count, progressive motility, total progressively motile count, sperm morphology, total normal sperm count, and functional sperm count such as DNA fragmentation, sperm viability and oxidation-reduction potential (ORP) were collected for the purpose of the study. Furthermore, reproductive hormones (estradiol, follicle stimulating hormone, luteinizing hormone, prolactin and testosterone), as well as seminal epithelial and red blood cells were investigated. All statistical analysis was done using the MedCalc® statistical software 19.5 with P-value of < 0.05 considered statistically significant. Men residing in Africa and Middle East had median ejaculate volume, sperm concentration, total sperm count, progressive motility and normal morphology within the normal thresholds recommended by WHO (2010). A prevalence of 20.3% for oligozoospermia and 3.6% for azoospermia were found in men residing in Africa and the Middle East. Men residing in the MENA region had sperm vitality below the recommended threshold and a median SDF higher than current recommended thresholds. Compared to Southern and Eastern Africa, the MENA region had generally worse semen parameters, most notably in the Middle East region. In Southern Africa, the highest semen parameters were found in men residing in Mozambique and Zimbabwe while the lowest were observed in patients residing in Zambia. In South Africa, the Free-State and Mpumalanga provinces had the lowest median sperm concentration. Age was found to negatively influence semen parameters in Africa and the Middle East. In the MENA region, an age-related decline in testosterone and prolactin, and increase in FSH was found, with no significant changes for LH and estradiol with age. The duration of abstinence had a statistically significant positive influence on semen volume, sperm concentration and progressive motility, while SDF worsened with the increased duration of abstinence. Furthermore, the results show a temporal decline in semen parameters between 2005 and 2019 among men from sub-Sahara Africa. A seasonal change in semen parameters of men residing in sub-Sahara Africa below the equator was found, with sperm concentration and total sperm count higher in winter compared to summer and autumn. The lowest sperm concentration was found in summer. Lastly, the results indicated a reduced semen quality in men residing in Africa compared to those living in America, Asia, Australia and Europe when comparing to global data from Cooper et al. (2010) and Campbell et al. (2021). The differences observed in semen quality and hormones in this study may indicate different environmental exposures and lifestyle changes in the investigated regions which requires further investigation.
The in vitro effects of heavy metals and nanoparticles on the immune system
(University of the Western Cape, 2017) Lategan, Kim Leigh
Heavy metals and nanoparticles may be released into the environment due to their use and applications. Sources of high, toxic metal concentrations may result from leachates from hazardous waste sites, discharge from industrial plants, and effluents from wastewater treatment plants being released into the environment. Nanoparticles may be found in a number of consumer products, and are used in medical applications such as drug delivery, bioimaging and biosensing. The release of heavy metals and nanoparticles to the environment may directly or indirectly impact abiotic and biotic systems. Three heavy metals and three nanoparticles were selected for this study. The heavy metals selected include cadmium (Cd), silver (Ag) and copper (Cu). The nanoparticles (NPs) chosen were silver nanoparicles (AgNPs), graphene oxide nanoparticles (GONPs) and carbon dots (CDs). These compounds were selected to evaluate the potential effects these compound may have on the immune system. The murine macrophage cell line RAW 264.7 and human whole blood cell cultures (WBCs) were selected as immune system representatives to assess the effects of heavy metals and nanoparticles on the immune system. The effects of heavy metals and NPs on RAW cells were monitored either in the absence or presence of the mitogen, lipopolysaccharide (LPS). The effects of heavy metals and NPs on WBCs were evaluated under basal conditions or in the presence of LPS or phytohaemmagglutinin (PHA). A number of parameters were monitored. These parameters included cytotoxicity, inflammatory biomarkers, cytokines of the acquired immune system and a proteome profile analysis. The first objective of this study was to assess the effects Cd, Ag and Cu on immune system biomarkers. No effects on were seen on cultures not stimulated by LPS. Cd was more cytotoxic than Ag, with Cu having no effect on cell viability of the RAW cells. The same trend was seen when evaluating the inflammatory biomarkers, nitric oxide (NO) and interleukin 6 (IL-6). Evaluating the effects of the metals on WBCs and the cytokines representing the innate (IL-6), humoral (IL-10) and cell mediated immunity (IFNγ) found that all the cytokines were reduced by the metals. The results show IL-6 was inhibited by Cu at lower concentrations than Cd, while Ag upregulated its synthesis. IL-10 was inhibited by Cd at lower concentrations than Cu, and Ag inhibited the production of this cytokine the least. IFNγ was reduced by higher concentrations of Ag, followed by Cu, and then Cd. The second objective of this study was to evaluate the effects AgNPs had on the immune system biomarkers. AgNP concentrations had no negative effect on RAW cell viability at concentrations used. However, AgNP cytotoxicity of WBCs was evident. Under basal conditions, AgNPs induced inflammation in RAW cells and WBCs respectively. Under a simulated inflammatory response, AgNPs inhibited the inflammatory response for both RAW and WBCs. The acquired immune cytokines IL-10 and IFNγ were both induced by AgNPs in the absence of PHA. IL-10 was partially inhibited by AgNPs when evaluated in the presence of PHA. Proteome profiles of RAW cell supernatants show that AgNPs do in fact modulate specific protein synthesis. Upregulated proteins due to AgNP exposure indicate induction of specific proteins indicative of inflammatory responses and wound healing. WBC supernatant proteome analysis indicates modulation of anti-inflammatory properties by AgNPs. The third objective was to monitor the effects of GONPs on the immune system biomarkers. GONPs were cytotoxic to both RAW and WBCs. In the absence of mitogens, GONPs elicited an inflammatory response from RAW and WBCs respectively. This activation was further corroborated by proteome profile analysis of both experimental cultures. GONPs inhibited LPS induced IL-6 synthesis and PHA induced IFNγ synthesis by WBCs in a dose dependent manner. In the absence of mitogens, GONPs stimulated IL-10 synthesis by WBCs. GONPs modulate immune system biomarkers and these may pose a health risk to individuals exposed to this type of nanoparticle. The last objective of this study was to evaluate the effects of CDs on the immune system. CDs were cytotoxic to RAW and WBCs respectively. Biomarkers associated with inflammation was induced by CDs under basal conditions for both RAW and WBCs respectively. The humoral immune system regulating cytokine IL-10 was increased by CDs under both basal and PHA activated WBCs. Proteome analysis supported the inflammatory data as proteins identified are associated with inflammation and provide potential biomarkers to be assessed upon CD exposure. The heavy metals and NPs assessed in this study can potentially be detrimental to human health as they are cytotoxic and induce immunomodulatory cytokines. This could potentially result in immunosuppression or immunostimulation in individuals exposed to these compounds.
Marine invertebrate sperm: assessment of sperm quality using computer-aided sperm analysis
(University of the Western Cape, 2019) Bennett, Monique
The Southern African marine ecosystems are dominated by a variety of marine broadcast spawners. In this study, five species of marine invertebrates, namely Parechinus angulosus (Echinoidea), Choromytilus meridionalis (Bivalvia), Crassostrea gigas (Bivalvia), Donax serra (Bivalvia) and Haliotis midae (Gastropoda) were investigated. The sperm morphology and base-line data were gathered concerning the sperm concentration, motility and sperm kinematic parameters using computer-aided-sperm analysis (CASA). Sperm morphology and sperm motility play an important role in determining sperm quality as they relate to fertilization success. More specifically, head length and total sperm length have been used to find associations with swimming speed for best rates of fertilization. The implementation of CASA allows detailed quantification of the nature of the sperm swimming track, percentage motility groupings and detailed kinematics for rapid-, medium-and slow swimming sperm subpopulations. The analysis of testicular sperm, taken directly from the gonads, and sperm activated by the introduction to sea water through the swim-up technique was determined. Using the CASA motility module, the behaviour of sperm was studied using Choromytilus meridionalis that was thermally induced to spawn and the activity of sperm in the presence of egg-water was determined using Parechinus angulosus as a model. A helical swimming pattern was a distinctive feature found in all species studied. However, species-specificity was found in the diameters and kinematic differences of the helical pattern, with Parechinus angulosus sperm creating the largest diameter tracks and Crassostrea gigas sperm the smallest diameter tracks, including a characteristic serrated helix. Parechinus angulosus and Haliotis midae maintained the progressive helical pattern, post-activation of 60 min, while mostly straight-line forward progressive sperm was evident in Choromytilus meridionalis and Crassostrea gigas sperm. The CASA quantitative sperm track features had a negative association with sperm morphology (head width, tail and total length), but has a strong positive association with sperm speed. It was evident from the Parechinus angulosus model that a decrease (thinner) in sperm head-width and shorter tail resulted in faster swimming sperm, creating a swimming track with a larger circumference and diameter which covered a bigger surface area. In conclusion, this finding shows that head shape played a significant role in sperm hydrodynamics and how it could relate within the fertilization environment. The different patterns of sperm motility could be related to a search strategy in quickly locating eggs and behaviour to reproductive strategies such as broadcast spawning and spermcasting. It is with great interest that this study elucidates the immense contribution CASA has made to being able to correlate so many of these sperm features and that these associations make biological sense.
Role of Rutin in 1-Mtthyl-4-Phenylpyridinium toxicity: Therapeutic implications for Parkinson's disease
(University of the Western Cape, 2018) Enogieru, Adaze Bijou
Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain. Although the etiology of PD is not completely known, it is believed to involve an association of various genetic, cellular, and environmental factors that individually or simultaneously advance neuronal degeneration. Neurotoxins such as 1-methyl-4-phenylpyridinium (MPP+) and 6-hydroxydopamine (6-OHDA) have been widely used to investigate distinct underlying mechanisms involved in the pathogenesis of PD. Presently, treatment options for PD are limited, as the available drugs are mainly focused on alleviating symptoms with limited ability to prevent disease progression. Accordingly, there is an increase in the use of natural compounds/products as potential neuroprotective agents. These neuroprotective treatments are believed to intervene in some stages in the pathogenesis of PD to suppress possible mechanisms of dopaminergic neuronal death such as apoptosis, mitochondrial dysfunction, oxidative stress, disturbances of calcium homeostasis, inflammation and autophagy. Thus, novel protective strategies for PD may be designed by targeting these mechanisms or intracellular signaling cascades that participate in PD pathogenesis. Plant-derived bioactive compounds used in traditional medicine have beneficial effects on some disorders including PD. For example, the bioflavonoid rutin, derives its common name from Ruta graveolens, a plant that contains high amounts of rutin. It is present in over 130 registered commercially available medicinal preparations and pharmacological studies have reported the beneficial effects of rutin in many disease conditions. Although rutin has been found to attenuate 6 OHDA toxicity in PC12 cells, its activity in MPP+-treated SH-SY5Y cells and fibroblasts have not been investigated. Consequently, for the first time, the protective activity of rutin in MPP+-treated SH-SY5Y cells and primary dermal fibroblasts was investigated, thus revealing possible molecular pathways and mechanisms of action. Findings from the cell viability studies show that rutin significantly protected SH-SY5Y cells and primary dermal fibroblasts from MPP+ toxicity. Additional findings further revealed that rutin pretreatment significantly attenuated MPP+ triggered increase in the production of nitric oxide (NO) and reactive oxygen species (ROS) in SH-SY5Y cells. The attenuation of increased ROS and NO production in SH-SY5Y cells is a crucial mechanism of action for its protection against MPP+ induced DNA damage and inflammation. This was demonstrated by a significant reduction in the expression levels of DNA damage (γH2AX) and inflammation (COX-2) markers following rutin pretreatment in SH-SY5Y cells. Also, rutin significantly suppressed MPP+ induced disruption of antioxidant enzymes and prevented MPP+ induced damage in nuclear morphology, clearly evidenced by fluorescence images from Hoechst staining showing shrinkage and fragmentation of SH-SY5Y cells. Meanwhile, the inhibition of p-Akt and p-NF-κB, as well as the activation of p-AMPK in MPP+ treated SH-SY5Y cells resulted in a cascade of apoptotic, autophagic and endoplasmic reticulum (ER) stress events leading to cell death. The ability of rutin to effectively regulate cell signaling pathways could be responsible for the protection of SH-SY5Y cells from the deleterious effects of apoptosis, autophagy and ER stress. This was demonstrated by a significant increase in the expression of full-length caspase 3 and GRP78/BiP, as well as a significant reduction in the expression levels of cleaved PARP, cytochrome C, LC3-II, p62 and CHOP proteins in pretreated SH-SY5Y cells. In confirmation of the western blot findings on autophagy, transmission electron images revealed abnormal presence/accumulation of numerous autophagosomes in our MPP+ treated SH-SY5Y cells while there was significantly reduced autophagic vacuoles in SH-SY5Y cells pretreated with rutin, perhaps due to the capacity of rutin to enhance efficient and speedy clearance of these vacuoles. Furthermore, increased levels of Ca2+ and significantly reduced mitochondrial membrane potential in SH-SY5Y cells, as well as significantly reduced maximal respiration and spare respiratory capacity in SH-SY5Y cells and fibroblasts, clearly highlights major characteristics of mitochondrial dysfunction in cells treated with MPP+. However, these effects were significantly attenuated following rutin pretreatment in SH-SY5Y cells and fibroblasts. Additionally, in SH-SY5Y cells, our findings show that rutin significantly improved basal and compensatory glycolysis as a compensatory response to an impaired oxidative phosphorylation system triggered by MPP+ which resulted in an insufficient ATP production. Taken together, proper regulation of the ROS-NO and cell signaling pathways, maintenance of Ca2+ homeostasis, mitochondrial protection and efficient autophagy clearance may account for the neuroprotective effects of rutin observed in our dopaminergic SH-SY5Y cells and fibroblasts. These findings further suggest that rutin may be a promising neuroprotective agent for the treatment of PD. Future studies will involve investigating its activity in animal models of PD.
Effects of Fucoidan and Chemotherapeutic agent combinations on Malignant and Non-Malignant breast cell lines
(University of the Western Cape, 2017) Abudabbus, Aisha Ibrahim
Background Breast cancer is currently one of the most common malignancies in women. Fucoidan (FUC) is a natural polysaccharide with anticancer properties. Despite a number of in vitro and in vivo studies reporting the efficacy of fucoidan in treating various cancers, few studies have measured the efficacy of fucoidan in combination with cancer drugs. Drugs like cisplatin, doxorubicin and taxol are important in breast cancer treatment. However, in recent years, supplements have gained importance in its treatment. Fucoidan, a sulfated polysaccharide mainly found in brown algae and seaweed, is a new candidate for breast cancer therapy because of its antitumour activity. This study was aimed at determining the cytotoxic, apoptotic and cell cycle distribution effects of fucoidan and its synergistic and/or antagonistic effects in combination with cisplatin, doxorubicin and taxol in the breast cancer cell line, MCF-7, relative to the normal MCF-12A non-malignant breast epithelial cell line. Methods The IC50 value of each agent was obtained against MCF-7 and MCF-12A cells using the MTTcytotoxicity assay. Apoptosis was determined with the Annexin VFITC/PI assay, Active Caspase-3/-7, and -9 and cell cycle assays, followed with Hoechst-33342 staining. MCF-12A non-cancerous epithelial breast cells was used as the control. Results Overall, fucoidan significantly increased the cytotoxic effect of the chemotherapeutic agents. Consistently, costimulation of MCF-7 cells with any chemotherapeutic agent in the presence of fucoidan further increased apoptosis induction, caspase-3/-7 and caspase-9 activation, particularly, in cisplatin- and taxol-challenged cells more than fucoidan-doxorubicin compared to untreated controls. Furthermore, fucoidan treatment resulted in G1 phase cell cycle arrest of MCF-7 cells and accumulation of the sub-G1 population as revealed by flow cytometry. Fucoidan-drug combinations strongly induced the accumulation of MCF-7 cells in the G2/M and sub-G1phase. In contrast, no significant differences for cytotoxicity and apoptosis or cell cycle profile were found between fucoidan treated and untreated MCF-12A cells. Conclusions Fucoidan is an effective antitumour agent either alone or in combination with cisplatin, doxorubicin and taxol in MCF-7 breast cancer cells. These findings suggest that fucoidan is a candidate natural product for breast cancer combination therapies. Further studies are required to evaluate cancer-specific and fraction-specific mechanisms of fucoidan for translation into in vivo tumour models.
An analysis of the morphological and biological properties of a novel human leukocyte- and platelet- rich concentrate
(University of the Western Cape, 2018) Peck, Mogammad Thabit
Wound healing is a complex process that involves several overlapping and interacting biological pathways. The consequences of delayed or abnormal wound healing may result in tissue formation that has impaired function or structural abnormalities. As a result, clinicians have sought ways to enhance this process. Recently, the use of autologous platelet concentrates have become popular in the management of wound healing sites. However, controversy exists as to how these biomaterials should be prepared and applied. We therefore sought to investigate whether a biologically viable and clinically effective platelet concentrate could be prepared using standard laboratory equipment. The findings are presented in a series of articles that have been published in peer-reviewed journals. The results suggest that the experimental platelet concentrate produced, has a morphological structure that consists of a dense fibrin network intermingled with platelets, has the ability to accelerate cellular growth in-vitro, has no adverse effects on cells in-vitro, can concentrate and release a systemically ingested antibiotic over a period of 24 hours in-vitro, can be stored for at least 60 minutes without showing signs of deterioration, and has shown clinical evidence of accelerating wound healing in sinus augmentation and alveolar ridge preservation procedures. The reduced cost of producing such a biomaterial allows it to be available to resource poor settings and to wider range of healthcare providers as compared to standard platelet concentration techniques. Further studies are required to investigate the clinical potential of this promising biomaterial.
Hyperglycinemia in captive-bred vervet monkeys with cataracts: genetic dynamics and associations
(University of the Western Cape, 2017) Magwebu, Zandisiwe Emilia
A small percentage (8%) of the captive-bred vervet monkeys (Chlorocebus aethiops) maintained at Primate Unit and Delft Animal Centre (PUDAC) of the South African Medical Research Council (SAMRC) were found to have high levels of glycine in their plasma (457795 µmol/L) and cerebrospinal fluid (CSF) (7.5-12.7 µmol/L). Additionally, these hyperglycinemic monkeys developed cataracts, a condition which has been previously characterized and reported in this specific colony of captive-bred vervet monkeys. This type of association has never been reported in literature before, therefore, this study will be the first of its kind to be investigated in non-human primates (NHPs). Nonketotic hyperglycinemia (NKH), also known as glycine encephalopathy, is well characterised in humans. The symptoms are exclusively neurological in nature, and clinically patients are diagnosed with abnormally high glycine levels in plasma (normal <350 µmol/L) and CSF (normal range 0-8). This neurological disorder is transmitted in an autosomal recessive form and is mainly instigated by a defective glycine cleavage system (GCS). In contrast to GCS, glycine transporter (GlyT1) which regulates glycine concentration at synapses and valproate administration have been associated with NKH. In this study, it was hypothesized that a correlation exists between hyperglycinemia and cataract in vervet monkeys. Since there is a close genetic relatedness between humans and NHPs, underlying genetic factors associated with cataract and hyperglycinemia in vervet monkeys are similar to those found in humans. Hence, genes that are implicated in cataract and NKH in humans were considered candidate genes in vervet monkeys. Two approaches were followed: (1) The animal intervention approach with valproate, sodium benzoate and dextromethorphan was conducted to compare the effectiveness of the current NKH treatment; (2) Molecular aspects of NKH were investigated using genotyping and gene expression techniques for valproate glucuronidation (UGT1A6 and UGT1A9), GlyT1 (SLC6A9), GCS (AMT and GLDC) and cataract (CRYAA and GCNT2) genes. Based on the findings from the animal intervention study, valproate induction in phase one elevated alkaline phosphatase, phosphate and platelet count. In phase two, the effect of valproate on the aforementioned biochemical parameters were reversed by sodium benzoate and dextromethorphan. The treatment was more effective in reducing glycine levels in plasma and CSF of the spontaneous group. Furthermore, the genotyping results for UGT1A6 revealed four missense variants, three silent variants in UGT1A9 and one silent variants in SLC6A9, and these sequence changes were not identified in the control group. Therefore, it is possible that these sequence variants played a role in valproate metabolism during the intervention study. A similar observation was made between mutated spontaneous individuals compared to the controls, and the results showed that both AMT and SLC6A9 genes were down-regulated in the spontaneous group. Therefore, AMT and SLC6A9 gene expression confirmed that there is a link between cataract formation and hyperglycinemia. The findings of the study conclusively suggest that a combination of drug therapy of sodium benzoate and dextromethorphan can be considered as treatment for normalizing glycine levels in plasma and CSF. Additionally, GCS and GlyT1 sequence variants may be responsible for the spontaneous hyperglycinemia in captive-bred vervet monkeys.
Aspects of the reproduction of male and female African penguins (Spheniscus demersus) with special reference to sperm biology and cryopreservation
(University of the Western Cape, 2018) Mafunda, Patrick Siyambulela
In the marine environment, penguins have been described as curators and serve a critical role in ecological balance. The African penguin (Spheniscus demersus) has undergone a rapid population decline, mainly due to disturbances in their natural habitat. The African penguin was up-listed from vulnerable to endangered on the IUCN Red List for Threatened Species in 2010 and thus urgent conservation action is required. Integral to long-term conservation action of any species is a basic knowledge of its reproductive biology, which is currently lacking for African penguins. The main aim of this investigation was to evaluate techniques for the collection of semen in African penguin and to determine sperm quality in order to cryopreserve sperm for in vitro fertilization (IVF) purposes of captive and wild populations. Semen was collected once a week during two breeding seasons from two captive African penguins. Ejaculates (n=51) were obtained over two breeding seasons (Jan-Feb and Jun-Oct) and evaluated for semen volume, sperm concentration, sperm vitality, sperm motility and sperm morphology. In addition twelve (six females and six males, n=4 were breeding pairs) captive African penguins were monitored for hormone (estradiol, testosterone, progesterone) levels prior to and after the egg-laying period. The testes were asymmetrical in adults, with the right testis on average shorter in length, width and volume respectively (16.80 ± 4.37 mm; 7.93 ± 2.63 mm; 0.75 mL) compared to the left testis (25.39 ± 5.85 mm; 11.48 ± 4.09 mm; 2.46 mL). The ovaries displayed variation in shape and size among the penguins, the adult ovary has a mean length of 25.72 ± 5.37 mm and a mean width of 9.02 ± 3.87 mm. The Follicles ranged from white small follicles with a diameter of <0.01 mm to mature, yellow large follicles which had a maximum diameter of 22 mm. The testis and ovary histological features such as structure, weight and size, can give a clear indication of breeding status in African penguin. Estradiol levels showed a biphasic pattern in three of the four breeding females, whereas no clear pattern could be followed in other hormones investigated. Semen volume ranged from 0.01 to 0.1 ml, sperm concentration from 802.6 to 7808.8 x106 /ml and total number of sperm per sample ranged from 3.42 to 740.18 x106. The percentage total motility was between 40.1 and 87.1%. The recorded velocities was for curvilinear velocity (VCL 81.5 ± 10.2 μm/s), straight-line velocity (VSL 42.72 ± 7.3 μm/s) and average path velocity (VAP 59.4 ± 8.2 μm/s), and kinematics at straightness of track (STR 71.4 ± 8.9 %), linearity of track (LIN 52.4 ± 8.1 %), amplitude of lateral head displacement (ALH 2.3 ± 0.2 μm) and beat cross frequency (BCF 16.8 ± 3.8 Hz). Sperm quality and semen parameters were similar across all samples collected over breeding seasons. In comparison to fresh semen, percentage total motility of thawed semen decreased to 16.8% after two hours in liquid nitrogen. Since spermatozoa differ notably in their morphology within species, phase-contrast microscopy, scanning electron microscopy and transmission electron microscopy were used to examine structural abnormalities. The sperm morphology is almost identical and largely resembles that of non-passerine birds in terms of the filiform head, small acrosome and mid-piece containing 13 spherical mitochondria, arranged around the proximal and distal centrioles in a single helix. The ultrastructure of the sperm principal piece revealed the typical 9+2 microtubular arrangement without any outer dense fibres. An unusual feature was the occurrence of multiple axonemes contained in one plasmalemma in 4% of spermatozoa. Multiple axonemes found in penguin flagella could be an apomorphism that distinguish them from other bird spermatozoa. This research represents a critical step in the conservation and long-term survival of the African penguin by evaluating techniques for the collection and determination of sperm quality in order to cryopreserve sperm for IVF.
The role of high-risk human papillomavirus in periocular cancers
(University of the Western Cape, 2018) Afrogheh, Amir H
Purpose: High risk human papillomavirus (HR-HPV) is well established as a causative agent of squamous cell carcinoma (SCC) of the orophaynx. HR-HPV has also been reported in periocular cancers and precancers, but controversy exists about its overall incidence and clinicopathologic profile. The purpose of this study is to evaluate the role of HR-HPV infection in periocular cancers and precancers, using multiple methods of detection. Design: Retrospective observational case series with laboratory investigations. Methods: Sequential surgical samples of 87 carcinomas (invasive SCC, SCC in situ and sebaceous carcinoma) from three different periocular sites (conjunctiva, lacrimal sac and the eyelid) diagnosed over a 15-year period (2000-2015) were selected for evaluation. Unstained paraffin sections of 87 cases of periocular carcinomas were analyzed with immunohistochemistry (IHC) for p16 as a screening test. p16 positive conjunctival- and lacrimal sac SCC were further evaluated for HR-HPV using DNA in situ hybridization (DNA ISH), and a subset was also analyzed by DNA Polymerase Chain Reaction (DNA PCR). p16 positive periocular sebaceous carcinomas (SC) were analyzed with PCR, and a subset of 18 cases was further studied with a novel method of mRNA ISH, an advanced technique with an enhanced sensitivity and specificity. Relevant patient clinical information was obtained from review of the electronic medical records. Results: Of 43 ocular surface squamous cell neoplasia (OSSN), 30% (n=13; 8 SCC in situ and 5 invasive SCC), were positive for HR-HPV. The HPV-positive OSSN occurred in 8 men and 5 women with a mean age of 60 years (range: 39 to 94 years). HPV type16 was detected in all conjunctival cases evaluated by DNA PCR. All 5 conjunctival invasive SCC (I-SCC) showed a non-keratinizing (n=4) or partially keratinizing (n=1) histomorphology, and were managed by simple excision. In contrast, the HPV-negative conjunctival I-SCC were predominantly keratinizing (11 keratinizing, 2 non-keratinizing). Of 9 lacrimal sac I-SCC (LSSCC), 66.7% (n=6/9) were positive for HR-HPV by p16 and DNA ISH; HPV subtypes were HPV-16 (n=5) and HPV-58 (n=1). In addition, two p16-positive cases with negative DNA ISH results were HR-HPV positive (HPV-16 and HPV-33) when evaluated by DNA PCR, suggesting that the rate of HRHPV positivity among the LSSCC may be as high as 89% (n=8/9). The combined group of HR-HPV positive LSSCC occurred in 4 men and 4 women with a mean age of 60 years (range: 34 to 71 years). Seven of the 8 HPV-positive LSSCC (87.5%) had a non-keratinizing or partially keratinizing histomorphology while 1 case (12.5%) was predominantly keratinizing. Strong immunohistochemical p16 positivity was present in 29 of 35 cases of periocular SC (82.9%). The selected 18 p16-positive cases tested were negative for HR-HPV using mRNA ISH. DNA PCR was unequivocal with adequate DNA isolated in 24 cases, 23 of which were negative for HR-HPV. One case was positive for HPV type 16, which was found to be a false positive as determined by mRNA ISH negativity. Conclusion: The presence of HR-HPV in 30% of OSSN and at least 66.7% of LSSCC confirms HR HPV as an important etiologic agent at these sites. No evidence was found for HR-HPV as an etiologic agent in the development of periocular SC using DNA PCR, and mRNA ISH to maximize sensitivity and specificity. p16 overexpression is common in periocular SC but unrelated to HR-HPV status. Immunohistochemical testing for p16, however, can be a valuable adjunct for identifying pagetoid intraepithelial spread of disease and small invasive cellular clusters. Although p16 may be used as a surrogate marker for HR-HPV status in conjuctival- and lacrimal sac carcinomas, this interpretation of p16 positivity is not applicable to periocular SC.
Identification and characterisation of cephalosporins and carbapenem-resistant Klebsiella pneumoniae isolates from Misrata, Libya
(University of the Western Cape, 2018) Shallouf, Mohamed Abdusalam
Background: Extended-spectrum beta-lactamase-producing (ESBL) and carbapenemaseproducing Gram-negative bacilli showing resistance to cephalosporins and carbapenems respectively, have been reported from several countries globally and recently among Libyan combatants who have been transferred to European countries for advanced medical care. However, there is a lack of data about their presence in Misrata and in Libya in general. This is the first documented study aimed at investigating the prevalence and resistance mechanisms of ESBL and carbapenemase-producing K. pneumoniae isolates from Misrata. Materials and Methods: Two hundred Gram-negative bacillus isolates were collected and identified from hospitals and pathology laboratories in Misrata. Following antimicrobial susceptibility screening, those showing resistance to cephalosporin and carbapenem were tested for ESBL activity using the Modified double disc synergy test, Sensititer ESBL confirmatory MIC plates and MAST AmpC detection sets D52C and D68C. Carbapenemase activity was detected using RAPIDEC CARBA NP test, Modified Hodge test (MHT), carbapenem inactivation methods (CIM), carbapenem combined test (CCT), and by MAST carba puls set. ESBL and carbapenemases genes were detected using multiplex PCR. Results: K. pneumoniae was the predominant species (85/200) of the 14 species identified, with 56 (65.8%) showing carbapenem resistance, 16 (18.8%) were cephalosporin-resistant carbapenem-susceptible and 13 (15.2%) were susceptible to all antibiotics except ampicillin. OXA-48 was the only carbapenemase detected, with SHV, TEM and CTX-M group 1 found in almost all carbapenem and cephalosporin resistant K. pneumoniae. Rep-PCR analysis revealed multiple clones and some K. pneumoniae strains were genetically related or indistinguishable despite differences in ESBL genes or carbapenemase activity. Conclusion: The findings of this study show that carbapenemase- and ESBL-producing K. pneumoniae are prevalent in Misrata and emphasize the urgent need for optimized infection control and antibiotic stewardship programmes in the Libyan hospitals to prevent further spread of these organisms.
Investigating the anti-cancer activity of novel phenothiazines in glioblastoma
(University of the Western Cape, 2018) Omoruyi, Sylvester Ifeanyi
Glioblastoma multiforme (GBM) remains the most malignant of all primary adult brain tumours. It is a highly invasive and vascularized neoplasm with limited treatment options and very low survival rate. GBM tumours are heterogeneous in nature with cellular hierarchy and at the apex of this hierarchy are the glioblastoma stem cells, known to promote tumourigenesis and resistance to chemotherapeutic agents and tumour recurrence. Currently, the standard care for GBM involves surgical resection, radiation, and chemotherapy treatment with temozolomide. Unfortunately, median survival after treatment is still daunting and tumour relapse is very frequent. Indeed, patients with recurrent glioblastoma have less than a year survival. To address this, novel therapies need to be developed with the early introduction of promising agents into clinical trials and subsequent approval for use. Importantly, for these novel therapies to be approved for GBM, they need to be safe, effective as well as being able to penetrate the bloodbrain barrier (BBB). Due to the high cost and process time for the development of new drugs, existing approved drugs are currently being repurposed for new indications and this is gaining significance in clinical pharmacology as it allows rapid delivery of useful drugs from bench to bedside. Drugs of the antipsychotic class are well known to cross the BBB due to their neuroleptic action. To this end, the aim of this study was to identify and characterize the anti-cancer activities of novel phenothiazine-derivatives belonging to the antipsychotic class of drugs in glioblastoma. To achieve this, several novel phenothiazine-derivatives were initially screened for possible anti-cancer activity in the U87 and U251 malignant GBM cells. Two lead compounds, DS00326 and DS00329, were identified and their anti-cancer activities were determined in U87 and U251 cells as well as in primary patient-derived xenograft (PDX) glioblastoma cultures. DS00326 and DS00329 significantly inhibited glioblastoma cell viability, with minimal effects observed in the non-cancerous FG0 fibroblasts. The IC50 values of DS00326 and DS00329 for U251, U87 and PDX cells ranged from 1.61 to 12.53μM. Flow cytometry analyses showed that DS00326 and DS00329 treatment led to an increase in the G1 population of cells. Additionally, DS00326 and DS00329 induced double-strand DNA breaks, which lead to activation of the canonical DNA damage response pathway. Furthermore, DS00326 and DS00329 induced apoptosis as shown by morphological markers, flow cytometry with annexin V-FITC/propidium iodide staining, as well as western blotting with an antibody to detect levels of cleaved PARP. Interestingly, treatment with DS00326 and DS00329 also induced autophagy as evident by the increase of acidic vesicular organelles in cells following staining with acridine orange as well as an increase in levels of the autophagy marker LC3-II. Autophagy was seen as a pro-death pathway in the U87 and U251 cells as inhibition of autophagy led to a reversal of cytotoxicity and consequently increased cell survival. Moreover, it was demonstrated that DS00326 and DS00329 inhibited the PI3/Akt pathway while modulating the mitogen-activated protein kinases p38, ERK1/2 and JNK signalling pathways. Importantly DS00326 and DS00329 displayed anti-cancer stem cell activities by the inhibition of neurosphere formation and regulation of stem cell markers SOX2 and GFAP in PDX cells. Together, the findings from this study suggest that DS00326 and DS00329 may be effective in the treatment of glioblastoma and provide a strong rationale for further clinical studies exploiting phenothiazines and their derivatives as treatments for glioblastoma.
The effects of green tea, green rooibos and their major flavonoids (EGCG and aspalathin) on testicular cell health in vitro
(University of the Western Cape, 2021) Booysen, Robin Alvin
The testes play a central role in the male reproductive system, as they represent the sites of male sex steroidogenesis and of spermatogenesis. Leydig cells, located at the testicular interstitium, produce predominantly testosterone upon stimulation with either chorionic gonadotropin (CG) or luteinising hormone by a series of enzymatic modifications of cholesterol. Sertoli cells respond to testosterone and follicle stimulating hormone to secrete inhibin and facilitate spermatogenesis by additional activities like maintaining Sertoli cell barrier (SCB) integrity and lactate secretion. Ultimately the Leydig cells, Sertoli cells etc. all work together to confer male fertility. However, infertility occurs globally; leading to the pursuit of treatment, including herbal medicines. Tea and rooibos are popular health drinks with global reach. Both have a green/unfermented form, which are said to possess potent health-beneficial properties. Polyphenols, and especially the flavonoids: epigallocatechin gallate (EGCG, from green tea) and aspalathin (from green rooibos), are held as primarily responsible for their health benefits. These flavonoids are known antioxidants that can interact with proteins, carbohydrates and fats, making them bioactive compounds of interest to health sciences. However, research on the effects of these teas and flavonoids on male reproduction are scarce and sometimes conflicting. Hence, this thesis aimed to determine some of the mechanisms by which green tea, green rooibos, EGCG and aspalathin affect model Leydig and Sertoli cells in vitro. The murine TM3 (Leydig) and TM4 (Sertoli) cell lines were cultured in vitro and exposed to varying concentrations of green tea, green rooibos, EGCG or aspalathin for 24 hours. Thereafter, the cells were assayed for oxidoreductase activity (by MTT: 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), morphological alterations (light microscopy), mitochondrial membrane potential (ΔΨm by tetramethylrhodamine ethyl ester) and reactive oxygen species (by chloromethyl 2',7'-dichlorodihydrofluorescein diacetate). TM3 cells, as well as boar testes ex vivo, were assayed for hCG-stimulated and unstimulated testosterone secretion. TM3 cells were assayed for mRNA expression of selected genes involved in steroidogenesis; namely Lhcgr, Star, Tspo, Cyp11a1, Cyp17a1 and Hsd17b3, as well as for Gapdh as a housekeeping gene. TM4 cells were also assayed for inhibin B secretion, lactate secretion and SCB integrity (by transepithelial electrical resistance). Within the tested concentration range of the teas and flavonoids used in this study, no cytotoxic effects were observed in Leydig or Sertoli cells. The experimental data further suggest that both green tea and green rooibos increase mitochondrial activity, resulting in lower cytosolic NADH, consequently decreasing oxidoreductase activity of Sertoli cells. Furthermore, lactate secretion was slightly reduced in Sertoli cells exposed to the teas. Contrary to this, their major flavonoids, EGCG and aspalathin did not exert the same responses. Sertoli cells exposed to EGCG or aspalathin showed a significant decrease in oxidoreductase activity without affecting lactate secretion or the SCB. In Leydig cells, both EGCG and aspalathin induced slight decreases in ΔΨm without affecting oxidoreductase activity. However, the flavonoids altered steroidogenic gene expression. EGCG increased both basal and hCG-stimulated Tspo expression, as well as stimulated Star expression. In contrast, aspalathin did not affect Star expression, but increased both basal and stimulated Tspo expression. All in all, these data suggest that both EGCG and aspalathin exerted pro-steroidogenic effects on Leydig cells. The murine TM3 (Leydig) and TM4 (Sertoli) cell lines are often used to investigate the effects of plant extracts on testicular functions. Several authors reported testosterone secretion in TM3 cells, and the TM3 cell population used here also proved to produce testosterone before the start of this study. However, in the interim, the cell physiology seems to have changed. As it turned out, both the TM3 and TM4 cell lines seemed to display a divergent physiology. The mRNA from some critical steroidogenic genes (i.e. Lhcgr, Cyp11a1 and Hsd17b3) could not be detected in the TM3 cells via PCR. That could explain why those cells were unresponsive to hCG. The TM4 cells did not secrete detectable levels of inhibin B either, indicating aberrant physiology for Sertoli cells.

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