Philosophiae Doctor - PhD (Medical BioScience)

Permanent URI for this collectionhttps://hdl.handle.net/10566/17329

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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.
Semen characteristics of free-ranging African elephants (Loxodonta africana) and Southern white rhinoceros (Ceratotherium simum simum) using Computer-aided sperm analysis, Electron microscopy and Genomics as diagnostic tools
(University of the Western Cape, 2016) Luther, Ilse
The survival of free-ranging (in situ) African elephant and Southern white rhinoceros populations are currently being challenged on a daily basis in Africa. Reproductive health is considered a vital component of species conservation. Conservation of the last mega land mammals may ultimately require intervention by breeding management or combined with assisted reproductive technologies (ART). There is a strong case for gathering baseline information, both physiological and biological, of any species, as opportunities arise. During this study a total number of 21 ejaculates collected over two seasons from 12 free-ranging African elephant bulls were characterised, as well as 10 ejaculates collected from 10 free-ranging Southern white rhinoceros bulls from two populations. Ejaculates were collected from adult bulls by means of electroejaculation under anaesthesia. Routine semen analysis was combined with Computer-aided sperm analysis (CASA), Computer-aided sperm morphology analysis (CASMA), Transmission electron microscopy (TEM) and Genomics as diagnostic tools. Additionally, sperm functionality within different media was investigated and sperm subpopulation classification according to the motion pattern displayed. The results presented is based on the evaluation and classification of ≈ 45 000 individual African elephant spermatozoa and ≈ 18 000 individual Southern white rhinoceros spermatozoa. The average elephant ejaculate contained a total number of 47 x 109 spermatozoa (volume of 56 ± 38mL x concentration of 818 ± 750 x 106/mL) that recorded a total motility of 81 ± 29% of which 62 ± 26% were progressively motile. CASA recorded velocities for curvilinear velocity (VCL 241 ± 58µm/s), straight-line velocity (VSL 173 ± 181µm/s) and average path velocity (VAP 201 ± 54µm/s), and kinematics at straightness of track (STR 86 ± 85%), linearity of track (LIN 67 ± 16%), amplitude of lateral head displacement (ALH 4 ± 0.75µm) and beat cross frequency (BCF 21 ± 3Hz). Structural analysis revealed 68 ± 11% of the spermatozoa were viable (intact plasma membrane) and 77 ± 11% maintained acrosome integrity. Ejaculates contained 55 ± 14% morphologically normal spermatozoa, CASMA measured sperm head lengths at 6.83 ± 0.26µm and width 3.32 ± 0.18µm (total head area of 20.17 ± 1.96µm2) of which 38.95 ± 0.92% is covered by an acrosomal cap. The average rhinoceros ejaculate contained a total number of 1.1 x 109 spermatozoa (volume of 24 ± 24mL x concentration of 83 ± 96 x 106/mL) that recorded a total motility at 82 ± 8% of which 28 ± 23% were progressively motile. CASA recorded velocities for VCL (85 ± 29µm/s), VSL (44 ± 25µm/s) and VAP (69 ± 30µm/s, and kinematics at STR (63 ± 14%), LIN (51 ± 16%), ALH (2 ± 0.16µm) and BCF (16 ± 6Hz). Structural analysis revealed 73 ± 10% of the spermatozoa were viable (intact plasma membrane) and 76 ± 4% maintained acrosome integrity. Ejaculates contained 62 ± 14% morphologically normal spermatozoa, CASMA measured sperm head lengths at 5.5 ± 0.17µm and width 2.9 ± 0.19µm (total head area of 14.8 ± 1.43µm2) of which 36.3 ± 0.59% is covered by an acrosomal cap. Based on a Boolean argument and CASA data exploration it was possible to derive elephant and rhinoceros CASA cut-off criteria to sort between activated and hyperactivated motile spermatozoa. For the genomic component of this study, the CatSper1 (Loxodonta africana) gene was identified, sequenced and verified in a free-ranging (natural) African elephant population. Multivariate analysis (MVA) was applied to examine the associations between the semen and sperm parameters and the traits they accounted for in this study. Our understanding of wildlife reproductive sciences can substantially progress as the analytical techniques applied and the combination thereof is expanded. This investigation presents a new set of comprehensive semen and sperm threshold values for future investigations.
Optimizing embryo culture conditions and spent culture media analysis as predictors of embryo quality and pregnancy
(University of the Western Cape, 2021) Kaskar, Khalied
The aim of this thesis is first, to evaluate various culture conditions to improve embryo development, and secondly, to analyze spent culture media for any biomarkers that may be predictive of embryo health. Single-step and sequential culture media were compared in both Planer and EmbryoScope™ incubators. Single-step media resulted in better blastocyst development compared to sequential media and the EmbryoScope™ incubation system showed slight improvements in embryo development than the Planer system. The benefits of supplementing the culture medium with either insulin or insulin-like growth factor 1 (IGF-1) or culturing in a 2% O2 environment, using two different strains of mice (hybrid and C57), as well as the suitability of these strains for quality control were compared. In insulin, hybrid embryos were slower to blastulate and had a lower blastocyst rate, whereas C57 embryos were slower to the morula and faster to blastocyst stages, and lower blastocyst rate than the controls. IGF-1 showed no difference in time-lapse morphokinetics (TLM) or blastocyst rates compared to controls in both hybrid and C57 embryos. Under 2% O2, hybrid embryos showed no significant difference in TLM up to the 8-cell stage, but slowed down afterwards, resulting in blastocysts with significantly lower cell counts than the 6% O2 group. The C57 embryos were slower to reach morula and expanded blastocyst, and had lower blastocyst rates in 2%O2 vs 6%O2. The C57 strain had significant slower overall embryo development for all time points than hybrid embryos in insulin, IGF-1 and ultra-low O2, as well as lower blastocyst rates. Measurement of growth differentiation factor 9 (GDF-9) and oxidation-reduction potential (ORP) in spent media as markers for embryo health were evaluated. Day 5 human blastocysts yielded higher pregnancy rates and GDF-9 levels in spent media compared to Day 6 blastocysts, but TLM parameters showed no impact on pregnancy outcome. In Day 6 blastocysts, the non-pregnant group showed significantly faster embryo development compared to the clinically pregnant group up to the 8-cell stage and start of blastulation. GDF-9 did not show any significant differences between non-pregnant and pregnant groups of Day 5 or Day 6 embryo transfers. ORP in spent media from good quality Day 3 embryos that developed into blastocysts were significantly higher than from those that did not, with no difference in control medium ORP. Spent media from arrested embryos showed lower ORP than their corresponding controls. Arrested embryos had slower development at syngamy, morula, blastulation and blastocyst stages. The single step medium in the EmbryoScope™ is the preferred choice for embryo culture. Insulin or IGF-1 media supplementation or 2% O2 culture did not provide any benefit to embryo development. The C57 mouse strain is more sensitive and may be better to detect changes in culture conditions, and therefore better model for quality control assays. GDF-9 values decrease from Day 5 to Day 6 which gives new insight to understanding the role of GDF-9 during embryogenesis. ORP in spent media indicate that embryos that developed into blastocysts did not contribute to ROS, but maintained ORP balance.
Does Mondia whitei play a role in the treatment of prostate cancer and benign prostatic hyperplasia?
(University of the Western Cape, 2025) Koeras, Eurinah R.; Henkel, Ralf R.
Prostate cancer and benign prostatic hyperplasia are two of the most prevalent urological disorders affecting aging men worldwide. Conventional treatments including androgendeprivation therapy, 5α-reductase inhibitors, and surgery, are effective but often associated with adverse effects that compromise quality of life. Although these conventional treatments are effective, they are often associated with adverse effects such as sexual dysfunction, hormonal imbalance and lower urinary tract symptoms, resulting in a reduced quality of life. This has increased scientific interest in plant-derived therapeutics that are capable of modulating prostate physiology while preserving normal reproductive function. Mondia whitei is a traditionally used medicinal plant in sub-Saharan Africa, that reportedly possesses androgenic, antioxidant, anti-inflammatory and cytoprotective properties. However, the potential role of Mondia whitei in the prevention or treatment of prostate cancer and benign prostatic hyperplasia has not been scientifically established.
Cell culture biomarkers for monitoring of wastewater pollutants
(University of the Western Cape, 2021) Makene, Vedastus Wilfred
Wastewater is normally composed of a mixture of pollutants. The type and composition of pollutants in a particular wastewater depend on the source of origin. The source and characteristics of a particular wastewater determine the ideal method of sewage treatment. Specific treatment techniques are effective in the removal of certain types of pollutants and may have no impact on the levels of other types of pollutants. Therefore, a combination of treatments and assessment of the quality of effluent before release into the environment is normally recommended. The assessment of effluent can be achieved by various techniques including chemical analysis and biological assays. Chemical analyses are commonly employed; however, they often pose detection problems and are considered to be uneconomical. As a result increased efforts are being made to develop biological assays for pollutants that are easy to perform, have a broader pollutant detection range and are that are relatively cheap. The aim of this study was to use the mouse macrophage RAW264.7 cell line as a cell culture bioassay model for monitoring aquatic pollutants and quality of wastewater effluent. The RAW264.7 cells are mouse ascites leukaemia induced cells, which once stimulated secrete inflammatory mediators and pro-inflammatory cytokines. In this study the effects of selected common endocrine distrupting chemical (EDC) pollutants and effluent samples were evaluated using cytotoxicity and secretion of inflammatory mediators (NO) and pro-inflammatory cytokines (IL-6) in RAW264.7 cell culture. Cytotoxicity was evaluated by determination of cell viability. Inflammatory responses were evaluated by determination concentrations of NO and IL-6 in supernatant of RAW264.7 cell culture as biomarkers of inflammation. NO and IL-6 were determined in culture supernatant using Griess reaction assay and double-antibody sandwich enzymelinked immunoassay (DAS ELISA), respectively. The first specific objective of this study was to evaluate effects of endocrine disrupting chemicals on biomarkers of inflammation produced by lipopolysaccharide stimulated RAW264.7 macrophages. Endocrine disrupting chemicals (EDCs) are common pollutants in the environment and can induce disruption of the endocrine and immune systems. The EDCs investigated were Estradiol (E2), 5α-dihydrotestosterone (DHT) and Bisphenol A (BPA). To evaluate if the effects caused by EDCs were modulated by steroid hormone receptors, antagonists of estrogen and androgen receptors were used. The steroid receptor antagonists used were Tamoxifen, an estrogen receptor antagonist, and Flutamide an androgen receptor antagonist. The lipopolysaccharide (LPS) stimulated RAW264.7 cells were exposed to DHT, E2 and BPA alone or in combination with flutamide and tamoxifen. Secretion of biomarkers of inflammation, namely nitric oxide (NO) and interleukin 6 (IL-6), were monitored. RAW264.7 cell culture supernatants were collected for NO and IL-6 assays. The cells were used for cell viability assays. The results show that DHT, E2 and BPA at concentration of 5μg/ml had no cytotoxic effects to RAW264.7 cell culture. However, the same treatments significantly (P <0.001) induced suppression of both NO and IL-6 secretion in stimulated RAW264.7 cells. The suppression of NO and IL-6 indicates that DHT, E2 and BPA can induce anti-inflammatory activities in stimulated RAW264.7 cells. The anti-inflammatory effects were induced via their respective steroid receptors, because it was reversed by their respective antagonist compounds. DHT combined with flutamide reversed the anti-inflammatory effects of DHT, while a combination of an estrogenic E2 or BPA with tamoxifen reversed the effects of E2 and BPA respectively. The results show that stimulated RAW264.7 cells culture can be a useful bioassay model for monitoring androgenic and estrogenic pollutants. The second specific objective of this study was to assess toxicity and inflammatory activity of municipal wastewater samples using RAW264.7 cell culture model. The wastewater samples analysed were influent, post bio-filtration, post activated sludge treatment and final effluent collected from a wastewater treatment plant. RAW264.7 cell cultures were exposed to sterile filtered water samples from the treatment plant. RAW264.7 cell culture supernatants were collected for NO and IL-6 assays. The cells were used for cell viability assays. The results show that none of wastewater samples tested induced cell toxicity when compared to negative control. The results also show that all wastewater samples significantly (P<0.001) induced NO and IL-6 production in RAW264.7 cells. The highest inflammatory activities were induced by post bio-filtration wastewater sample. Final effluent sample induced the lowest inflammatory response. The lower inflammatory activity in final effluent indicates effective removal of pollutants upon sewage treatment. The findings of this study show that sewage samples can induce inflammatory responses in RAW264.7 cells. The results also give evidence that RAW264.7 cells can be used as a model for monitoring the quality of treated municipal sewage. The third specific objective of this study was to evaluate cytotoxicity and inflammatory activity of wastewater collected from a textile factory before and after treatment by coagulation-flocculation methods, using RAW264.7 cell culture model. RAW264.7 cell cultures were exposed to sterile filtered water samples from raw effluent and effluent treated with various coagulation-flocculation methods. RAW264.7 cell culture supernatants were collected for NO and IL-6 assays. The cells were used for cell viability assays. The results show that raw effluent induced cytotoxicity by reducing cell viability significantly (P<0.001) compared to the negative control. The results on inflammatory activities show that the raw effluent and effluent treated with 1.6g/L of Fe-Mn oxide induced significantly (P<0.001) higher NO production than the negative control. The inflammatory results further show that the raw effluent induced significantly (P<0.001) higher production of IL-6 than the negative control. Among the coagulants/flocculants evaluated Al2(SO4)3.14H2O at a dosage of 1.6 g/L was the most effective to remove both toxic and inflammatory pollutants from the textile effluent used in this study. Therefore, the inflammatory responses in RAW264.7 cells can be used as sensitive biomarkers for monitoring the effectiveness of coagulation/flocculation processes used for textile effluent treatment. The last specific objective of this study was to evaluate toxicity and inflammatory activities of a textile effluent treated with electrohydraulic discharge and coagulant/flocculants. A combination of coagulant/flocculants and electrohydraulic discharge (EHD) are tested for treatment efficiency of textile wastewater. Pre- and posttreatment samples were used to evaluate process efficiencies. Process efficiencies were evaluated using physicochemical characteristics, and cytotoxicity and inflammatory activities induced in macrophage RAW264.7 cell culture model. RAW264.7 cell cultures were exposed to sterile filtered water samples from raw textile industrial effluent and effluent after treatment with EHD in absence and presence of coagulant/flocculants. RAW264.7 cell culture supernatants were collected for NO and IL-6 assays. The cells were used for cell viability assays. The results show that raw effluent was characterized by high physicochemical parameters and intense colour. Treatment of textile wastewater with EHD alone successfully reduced the chemical oxygen demand (COD) and total organic carbon (TOC) values by 95.3 and 96.23 % respectively. The results show further that a single treatment approach is not effective in removing all pollutants. However, a combined treatment approach effectively removed complex organic pollutants, toxic and inflammatory pollutants based on the results of NO and IL-6 in RAW264.7 cell cultures. The study confirms that induction of NO and IL-6 secretions in macrophage RAW264.7 cells is a very sensitive model system to monitor the efficiency of textile effluent treated with electrohydraulic discharge and /or combined with other processes.
Differential toxicity of two murine endothelial cells to ROS duress: understanding oxidative stress-induced blood-brain barrier dysfunction
(University of the Western Cape, 2020) Alamu, Olufemi Akinyinka
The blood-brain barrier (BBB) is a critical interface between the blood circulation and brain tissue which performs critical selection of circulating molecules that gain access to the brain tissue. Its unique ability to adjust to changes in the constituents of the blood circulation confer in the BBB a dynamic nature enabling changes in its properties to suit the homeostatic needs of the brain. Dysfunction of the BBB has been established to be pivotal to the initiation and/or maintenance of an array of neurological disorders, most of which involve the production of excess reactive oxygen species (ROS) and oxidative stress in their pathophysiology. Thus, clinical trials of exogenous antioxidant agents have been proposed and initiated, with most results being inconclusive. Extensive studies of the impact, capacity and plasticity of endogenous antioxidants in the cells that constitute the blood-brain barrier, especially the brain endothelial cells, therefore, became necessary for the rational choice, timing, and the mode of application of antioxidants in the management of oxidative stress-mediated neurological diseases. In the present study, incremental hydrogen peroxide (H2O2) concentrations were dosed in cell culture medium to simulate OS and gauge the capacity of the brain endothelial cell (BECs) models of the BBB to resist ROS toxicity, the contribution of endogenous antioxidants to this resistance and to study the morphological changes in ROS-mediated BBB dysfunction. Two types of mammalian endothelial cells, b.End5 and bEnd.3, commonly used for in vitro studies of the blood-brain barrier, were selected for use in this study. A combination of spectrophotometry, fluorescent microscopy, trypan blue exclusion method and flow cytometry were used to assess cell viability, cellular glutathione (GSH) content, cell cycle changes, cellular death by apoptosis and necrosis in BECs exposed to incremental H2O2 concentrations for 24hr. Exogenous antioxidants were variably administered to study the effects of externally incident antioxidants when the cells were under H2O2 exposure. Results showed that b.End5 cell line significantly tolerated higher concentrations of H2O2 than the bEnd.3 cell line. GSH contents for both cell lines were fairly similar under physiological conditions but after exposure to H2O2, b.End5 cells demonstrated higher resistance to GSH depletion than the bEnd.3 cell line, although the two cell lines were obtained from the same animal species. Along incremental concentrations of H2O2, increased cell proliferation, cell necrosis and apoptosis and cell cycle arrest were observed concurrently. At H2O2 concentrations that defined OS, live cells were depleted in b.End5 cells used while there was significant increases in apoptotic and necrotic cells with apoptotic cells as the significant majority comparatively. Cell cycle studies showed arrest of cell division at the G2/M phase of the cell cycle at higher concentrations of H2O2. Application of exogenous antioxidants ameliorated the H2O2-induced cellular depletion as well as improved recovery in cellular viability following withdrawal of H2O2 after 24hr exposure. It was conclusive that apoptotic pathway of cellular death is a major pathway of BECs response to OS. Also, there was differential H2O2 toxicity and GSH de novo synthesis capacity between the b.End5 and bEnd.3 cell lines despite their common origin from the same animal species and their possession of similar contents of endogenous antioxidant GSH under physiological conditions. This finding calls for more caution for the choice of cellular models for specific studies of the BBB to ensure that results obtained are reproducible, reliable and sufficiently conclusive. Furthermore, our results tend to suggest that the processes responsible for the endothelial component of BBB dysfunction under conditions of oxidative stress occur concurrently and include increased proliferation, necrotic and apoptotic cell death as well as cell cycle arrest. Additionally, study suggests that the clinical administration of antioxidants could be an appropriate intervention for the alleviation of neurological diseases.
Protein expression and antifungal effect of fluconazole-resistant Candida species following effective in vitro treatment with K21, a novel antifungal agent
(University of the Western Cape, 2019) John, Cathy Nisha
Background: Oropharyngeal candidiasis, caused by the fungus Candida, is the most common opportunistic infection affecting the quality of life of immunocompromised patients. Fluconazole is widely used as the first line of treatment for fungal infections. However, the inappropriate and misguided use of the drug has led to the evolvement of fluconazole-resistant Candida organisms. This arising resistance resulted in the urgent need for the development of new antimicrobial drugs. The aim of the present study was to investigate the antifungal action of K21, a novel antimicrobial quarternary ammonium compound, on fluconazole-resistant Candida species. Materials and Methods: An in vitro study was conducted using a total of 143 Candida isolates obtained from HIV-positive patients. The ethical aspects of the study complied with the declaration of Helsinki (2013). The time-kill assay was used to evaluate the rate of action of K21 over time on Candida while the fungicidal effect of K21 against C. albicans (ATCC 90028) and C. glabrata (ATCC 26512) was observed at 2 hours. The minimum inhibitory concentration (MIC) of K21 was compared with the MICs of fluconazole. Synergy between K21 and fluconazole was evaluated by both the checkerboard microdilution method and time-kill assay. The modes of action of K21 and drug delivery were determined by performing postembedding immunogold labelling and for the sight-specific target and protein expression of Sap 1-3 and Sap 4-6 within the Candida cell. Results: Of the 143 isolates, 108 were fluconazole-resistant, 15 were fluconazole-intermediate and 20 were fluconazole-susceptible using the broth microdilution assay and breakpoint values recommended by the Clinical and Laboratory Standards Institute (CLSI). The MIC of K21 was 31.24 µg/mL for C. albicans when determined by the broth microdilution assay. About 103 Candida species were resistant and 13 were categorised as intermediate to FCZ with a MIC range between 64 µg/mL - 256 µg/mL and 16 µg/mL - 32 µg/mL respectively. However, the majority of the Candida species (n = 86) showed intermediate susceptibility to K21 with a MIC range between 62.48 µg/mL - 124.95 µg/mL and only 9 of the Candida species were resistant to K21 with a MIC value of ≥249.89 µg/mL. A statistically significant (p value = 0.000) was observed when the MIC values of K21 and FCZ were compared. No antagonism was observed in the study among the Candida strains. The time-kill and synergism assays showed significant differences over time with synergy between K21 and FCZ demonstrated for C. albicans (ATCC 90028 and NCPF 3281) C. dubliniensis (NCPF 3949a), C. tropicalis (ATCC 950) and C. lusitaniae (ATCC 34449). Scanning electron microscopy displayed major alterations in the morphology of Candida species between 2 hours and 24 hours, exhibiting cell lysis and cell death. Transmission micrographs of C. albicans (ATCC 90028) treated with K21 showed shrunken nuclei with disruption of cell walls and cell membranes. Immunogold labelling of C. albicans (ATCC 90028) and C. dubliniensis (NCPF 3949a) with Sap 1-3 antibodies exhibited the presence of gold particles confined to the cell wall and cell membrane, but, when exposed to Sap 4-6 antibodies there were a few non-specific gold particles found in C. albicans (ATCC 90028) and an absence of gold particles in C. dubliniensis (NCPF 3949a). C. tropicalis (ATCC 950) showed few gold particles along the cell membrane when treated with Sap 1-3 antibodies and no gold particels were found when treated with Sap 4-6 antibodies. Conclusion: The present study suggests that K21 acts as a potent antifungal agent and can be considered for development as an alternative treatment for fluconazole-resistant Candida species especially in immunocompromised patients.
The effects of various combinations of different classes of anticancer drugs and tyrosine kinase inhibitors on the human MCF-7 and triple-negative MDA-MB 231 breast carcinoma cell lines
(University of the Western Cape, 2020) Abrahams, Beynon
Globally, breast cancer is the most common cancer affecting women and it is predicted that in 2030 about 12 million deaths will occur with approximately 21.7 million new cases. Genetic risk factors as well as race and ethnicity, account for about 5-10% of all breast cancer occurrences. Triple negative breast cancer (TNBC), tumors that tested negative for oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), contribute to 10-20% of all breast carcinomas and is known to be a more aggressive type of cancer with varying degree of response to chemotherapeutic and radiation therapy. The epidermal growth factor receptor (EGFR) is abnormally activated in many cancers and its signal transduction pathways play an important role in regulating cell proliferation and survival. It is often overexpressed in TNBC. P-glycoprotein (P-gp) is a transmembrane glycoprotein responsible for active transport of substances across the cell membrane out of a cell. It functions as an efflux pump and is overexpressed in many cancer cells. The altered expression of both EGFR and P-gp parallels an aggressive clinical course and the development of resistance to anticancer and adjuvant endocrine therapies. Anti-EGFR compounds that specifically target the intracellular and extracellular domains of the EGFR include tyrosine kinase inhibitors (TKIs), e.g., gefitinib and erlotinib and monoclonal antibodies, e.g., cetuximab and trastuzamab, that are amongst the most effective agents that are currently used together with chemotherapeutic agents in clinical practice. However, the emergence of multidrug resistance (MDR), a mechanism that is not well understood, has, over time, eclipsed the progress that has been made in drug therapeutics. In this study we examined the effects of doxorubicin (DOX), cisplatin (CDDP) and one investigational TKI (EGFR inhibitor I, hereinafter referred to as EGFRi), individually and in combination, on human MCF-7 breast carcinoma cells and a triple negative breast cancer (TNBC) cell line (MDA-MB-231), with specific focus on P-gp and EGFR inhibition. Analyses of MCF-7 and MDA-MB-231 TNB carcinoma cells exposure to DOX, CDDP and EGFRi included growth and dose-response curves (cytotoxicity assays), caspase3/7 assays for apoptosis detection, P-glycoprotein function (intracellular Calcein-AM accumulation) and EGFR and P-glycoprotein (ABCB1 gene) mRNA expression, using quantitative Real-Time PCR. When used as an individual treatment, EGFRi demonstrated a higher growth inhibition in both MCF-7 and MDA-MB 231 cell lines, compared to DOX and CDDP. Combination treatment of DOX with CDDP at equimolar log dose concentrations, synergistically inhibited cell growth that was significantly different to the compounds used individually. The pairwise combinations of EGFRi with both DOX and CDDP also demonstrated synergistic and antagonistic drug interactions consistent with the Bliss independence and Loewe additivity synergy models. Apoptosis was confirmed in both MCF-7 and MDA-MB-231 TNBC cells after 24-hour drug treatments. Intracellular calcein accumulation that is indicative of P-glycoprotein inhibition, was determined using the Calcein-AM assay. Drug treatments significantly increased the intracellular accumulation of calcein inside the cells, but only at very high concentrations in MCF-7 cells. No significant levels of calcein accumulation were detected in MDA-MB-231 cells, following individual treatment with EGFRi, DOX and CDDP. The pairwise combination drug treatments of EGFRi + DOX demonstrated significant accumulation of calcein only in MCF-7 cells. RT-qPCR was utilized to quantify the gene expression levels of EGFR and ABCB1 in both MCF-7 and MDA-MB-231 TNBC cell lines following drug treatment. The expression levels of EGFR gene were significantly reduced in both cells following drug treatment, which correlates with the cellular growth inhibition results. The expression levels of ABCB1 gene could not be quantitated following optimization of expression due to undesirable CT values that was outside the normal acceptable ranges. This is a due to the undetectably low expression levels of ABCB1 gene in our samples. Our scientific findings confirmed our hypothesis of the EGFRi’s ability to successfully reduce the efflux function of P-glycoprotein as well as demonstrating its combinatorial potential with DOX and CDDP, as compounds from different classes are more effective in enhancing growth inhibition efficacy in the two human breast carcinoma cell lines.
Typha capensis - An electron rich resource for the synthesis of phytochemical-encapsulated gold nanoparticles through green nanotechnology
(University of the Western Cape, 2020) Pearce, Keenau Mark
Introduction Typha capensis (T. capensis), commonly known as bulrush, is a medicinal plant found growing in the wetland areas of South Africa. In traditional medicine, rhizome decoctions of T. capensis are used to treat a wide variety of ailments, including venereal disease, dysentery, diarrhoea and low libido in men. Previously, T. capensis rhizomes were shown to be a rich source of antioxidants, such as catechin and epicatechin, inhibiting both reactive oxygen species and reactive nitrogen species. The antioxidant capacity of such plant species serves as a reservoir of electrons to transport them into gold salt for the production of gold nanoparticles through green nanotechnology. Therefore, this study aimed to investigate the application of T. capensis in green nanotechnology and nano-medicine. Material and methods This study investigated the synthesis of gold nanoparticles (AuNP’s) from two aqueous T. capensis extracts, the S1 and S2 extracts, along with the bioactive compound naringenin. The following parameters were used for AuNP characterisation: spectrophotometry, dynamic light scattering, zeta potential, transmission electron microscopy (TEM), Folin-ciocalteu phenol assay, inductively coupled plasma mass spectrometry (ICP-MS). Hereafter, the effects of these AuNP’s were investigated toward the LNCaP and PC-3 prostate cancer, Panc1 pancreatic cancer, and HAEC human aortic endothelial cell lines over 24, 48 and 72 hours. Additionally, possible AuNP cell internalisation was investigated in the LNCaP and PC-3 cells by dark field hyperspectral microscopy, and definitive AuNP-cell internalisation in PC-3 cells by TEM imaging. Finally, selected AuNP’s, along with the S1 extract and naringenin, were investigated in vivo using SCID-mice bearing PC-3 prostate cancer tumour xenografts. Results Aqueous T. capensis rhizome extracts produced AuNP’s in a single step reaction, yielding the S1-AuNP’s, S1x2-AuNP’s, S2-AuNP’s and S2x2-AuNP’s. Similarly, the isolated bioactive compound naringenin also produced AuNP’s when the reaction was controlled at pH 7 and pH 8, yielding the Ng-AuNPs pH 7 and Ng-AuNP’s pH 8. These particles proved to be highly stable, showing no agglomeration over time. The extracts, naringenin, and their subsequent AuNPs yielded comparable levels of toxicity toward the LNCaP cells at each time point. Compared to the S1 extract, the S1-AuNP’s yielded significantly (P=0.0001, P=0.0004, P<0.0001) greater reduction in PC-3 cell viability at the highest concentration used, over each time point. Similarly, the highest concentration of the S2-AuNP’s produced significantly (P=0.0319, P=0.0006, P=0.0003) greater reductions in PC-3 cell viability at each time point in comparison to the S2 extract. The highest concentration of the Ng-AuNP’s pH 8 were also found to yield significantly (P=0.0009, P=0.0002, P=0.0125) greater reductions in PC-3 cell viability over each time point. The S1-AuNP’s, S1x2-AuNP’s, S2-AuNP’s, S2x2-AuNP’s, NgAuNP’s pH 7 and Ng-AuNP’s pH 8 yielded improved toxicity toward Panc1 cells in both a dose- and time-dependent manner. However, each AuNP formulation, along with their extract or bioactive compound counterpart, produced a degree of dose and time-dependent toxicity toward the non-cancerous HAEC cell line. Under in vivo conditions, the S1 extract and S1-AuNP’s were well tolerated by SCID-mice bearing PC-3 prostate cancer tumour xenografts, with 1.5 mg/kg of the S1-AuNPs significantly (P=0.0027) inhibiting tumour growth by the end of the study. Additionally, 1.5 mg/kg of the S1-AuNP’s yielded a significantly (P=0.0339) lowered neutrophil to lymphocyte ratio by the end of the study. Similarly, naringenin at a dose of 0.5 mg/kg, and the Ng-AuNPs pH 8 at 0.5 and 1.5 mg/kg significantly (P=0.0038, P=0.0038, P=0.006) inhibited tumour growth, and greatly improved bodyweight by the end of the study. Conclusion In summary, the collected data showthat highly stable gold nanoparticles, encapsulated with a plethora of phytochemicals from both Typha capensis and the bioactive compound naringenin, have been synthesized through a single step process. These nanoparticles exhibited robust stability under in vitro conditions and clear signs of cell internalisation, observed using darkfield microscopy and TEM. When compared to the effects of the extracts, similar levels of toxicity were observed in the case of the LNCaP and PC-3 cells. However, these nanoparticles were found to yield improved toxicity toward Panc1 cells in both a dose- and time-dependent manner. Furthermore, the present study demonstrated for the first time the positive therapeutic effects of T. capensis extract toward prostate cancer under in vivo conditions, adding a degree of validity to its clinical usage suggested by Ilfergane (2016). However, these positive effects were not found to be improved, nor maintained, by nano-encapsulation of the extract. The NgAuNP’s, however, were able to maintain the anti-cancer effects of free naringenin under in vivo conditions, having favourable outcomes toward tumour growth, body weight and blood parameters in tumour-bearing SCID-mice
Radiation-induced leukaemia in South Africa: response of lymphocytes and cd34+ cells to different radiation qualities
(University of the Western Cape, 2020) Engelbrecht, Monique
Epidemiological studies have highlighted that leukaemia can be considered as the most prominent malignancy after radiation exposure during childhood. The lifetime risk on radiation-induced leukaemia for a given dose is 3 – 5 times higher for children compared to adults. The high risk at a young age is related to the elevated sensitivity of the red bone marrow where haematopoietic stem and progenitor cells (HSPCs) are located. HSPCs self-renewal capacity and long-life span increase their susceptibility to DNA damage accumulation, making them a major target of radiation-induced carcinogenesis. Proton beam therapy (PBT) is increasingly used to treat paediatric brain tumours due to its dose sparing properties compared to conventional X-ray based radiotherapy. However, concerns regarding the carcinogenic potential of secondary neutrons produced during PBT, especially in terms of their effect on HSPCs harboured in the cranial bone marrow of paediatric patients, remain. In this study, the radiobiological differences between 60Co γ-rays and p(66)/Be(40) neutron exposure was investigated to resolve the underlying mechanisms for the high radiosensitivity of HSPCs (CD34+ cells) isolated from umbilical cord blood (UCB). For both radiation qualities, an apparent dose-dependent increase in the frequency of radiation-induced MN was observed in CD34+ cells. Furthermore, increased cytogenetic damage was observed with the CBMN assay after neutron irradiation, which highlights its leukaemogenic potential. In addition, no difference was observed in the nuclear division index of the CD34+ cells post-irradiation between both radiation qualities. The number of DNA DSBs was assessed by microscopic scoring of γ-H2AX foci, 2 and 18 hours after radiation exposure. A significant higher number of DNA DSBs were observed 2 hours after neutron irradiation with 0.5 Gy, but decreased to similar levels for both radiation qualities after 18 hours. Different stages of apoptosis in CD34+ cells were studied at 18 and 42 hours numerous time points post-irradiation by flow cytometry using the Annexin/PI assay. In contrast to the γ-H2AX foci results, a significant difference in late apoptosis was observed at 18 hours and 42 hours between the two radiation qualities. The results point towards a fast error-prone DNA repair in HSPCs after neutron irradiation, which might contribute to genomic instability and leukemogenesis. In the second phase of the PhD project, the impact of age on radiosensitivity was investigated by comparing newborn T-lymphocytes with adult peripheral blood (APB) T-lymphocytes. The major difference between UCB and APB T-lymphocytes, is their immunophenotypic profile. Since it is known that different T-lymphocyte subsets have a difference in radiosensitivity, the fraction of CD4+ , CD8+ , naïve (CD45RA+) and memory (CD45RO+) T-lymphocytes was determined via flow cytometry in the two groups. The cytokinesis-block micronucleus (CBMN) assay was used to determine the extent to which age influences the frequency of cytogenic damage in response to 60Co γ-rays radiation. For both APB and UCB, an outspoken dose-dependent increase in the frequency of radiation-induced MN was observed at 0.5, 1, 3 and 4 Gy. However, no significant difference was observed at 4 Gy when comparing MN yields of APB and UCB. An increased radiosensitivity of newborn to adult donors of 34%, 42%, 29%, 26% and 16% was observed based on the MN scoring at doses of 0.5, 1, 2, 3 and 4 Gy, respectively. The lowest radiosensitivity was identified at the highest dose, which might explain the non-significant difference at 4 Gy. In addition, there was a clear trend that females were more sensitive to 60Co γ-rays radiation than males in both adults and newborns, even though the difference was not significant. The immunophenotypic study revealed that that both the CD4+ and CD8+ T-lymphocytes of newborns are mainly naïve. This is illustrated by the coexpression of CD45RA+ on 90.70% (range: 80.80% – 98.40%) and 95.90%(range: 89.60% – 98.80%) of CD4+ and CD8+ cells respectively. The composition of adult T lymphocytes, in contrast, is clearly different with a more equal distribution between CD45RA+ and CD45RO+ subpopulations. This finding demonstrates that there are differences in the radiosensitivity between newborn and adult T-lymphocytes which might be linked to the immunophenotypic change of T-lymphocytes with age.
Advanced analysis of human sperm using computer-aided sperm analysis and flagellar applications
(University of the Western Cape, 2025) Webber, Tarryn Jean
Semen analysis provides insight into male fertility and overall reproductive health. While such analyses were traditionally subjective, technological advances have introduced a more objective approach, such as computer-aided sperm analysis (CASA). However, conventional CASA primarily tracks sperm head centroid movement and is not designed to track the sperm component responsible for sperm motility, namely the flagellum. As a result, the correlation among sperm head kinematics, flagellar dynamics and overall sperm quality remains poorly understood. Additionally, the optimization of technical factors (such as cut-off values for sperm parameters in CASA, frame rate and chamber depth for flagellar analysis) has not yet been established, forming an integral aspect of this study. This study aimed to investigate the effects of different frame rates and chamber depths for flagellar waveform analysis. It also sought to assess how biologically relevant environments influence the functionality of sperm subpopulations. The research intended to use established mathematical modeling software based on the physical principles of movement to explain sperm motility from a physiological perspective by comparing conventional CASA with flagellar tracking software and to provide insights into the structural features of the flagellum, such as the centrioles. Human semen samples were separated into high motile (HM) and low motile (LM) sperm subpopulations by the double density gradient centrifugation technique to model fertile and subfertile samples. In the first phase of the study, sperm subpopulations in human tubal fluid (HTF) were assessed using CASA to examine the impact of technical factors such as frame rate and chamber depth on sperm kinematics, thereby optimizing and standardizing procedures to identify the technical conditions for flagellar waveform analysis. In the second phase, the subpopulations were treated with a viscous 1% methylcellulose medium and HD capacitating (HD-C) medium to evaluate sperm functionality, including sperm kinematics, flagellar waveform features and hyperactivation in biologically relevant environments.
The characterisation of human coronavirus nl63 proteins
(University of the Western Cape, 2021) Gordon, Bianca
Human Coronavirus NL63 (HCoV-NL63) is one of seven coronaviruses (CoVs) that cause respiratory disease in the global population. The Membrane (M) and Nucleocapsid (N) proteins are part of the core CoV-structural proteins, crucial in viral replication and virion assembly. Here the expression of HCoVNL63 M and N was characterized across multiple in vitro systems including bacterial, insect and mammalian. To detect untagged proteins in viral structural studies, anti-peptide antibodies were generated in a mouse model. Polyclonal antisera and hybridoma-secreted antibodies exhibited specific binding to their respective full length protein antigens. Anti-peptide monoclonal antibodies were successfully generated against the HCoV-NL63 M and N proteins. During CoV infection, the interaction of CoV M and N is necessary for the production of infectious virions. For the first time, co-expressed, full length HCoV-NL63 M and N were assayed for protein-protein interaction in a mammalian cell system, allowing for native protein folding and modification. M protein formed higher order homomultimers in the presence and absence of co-expressed N. Complexed M and N were co-purified from mammalian cells and confirmed as interaction partners, even under denaturing conditions. HCoVNL63 M and N proteins formed stable interactions when co-expressed in vitro. The strong M-N association in the absence of other viral components highlights the importance of this interaction during virus replication in the host cell. The subcellular localization of HCoV-NL63 N was evaluated from early to late expression, and N exhibited no nucleocytoplasmic shuttling during protein synthesis and maturation. The identified pat4/pat7 nuclear localisation motifs within N may have been inactive; alternatively the localization of N expressed alone was modulated by multiple directive signals provided within the protein. A recombinant Baculovirus was used to express HCoV-NL63 M, towards examining CoV structural protein dynamics in an alternate eukaryotic insect system. Preliminary results indicated that M was expressed at 48-72 hours post-infection. CoV M and N each play multifunctional roles in the viral life cycle, and are implicated in the host-immune response. Investigating the relationships between these and other CoV structural components brings us closer to understanding CoV replication. Important viral protein-protein interactions could present new targets for exploitation in the hunt for an effective anti-coronaviral agent. HCoV-NL63 has been a human pathogen for over 500 years, and continues to adapt genetically, retaining viral fitness in the environment. Able to cause both upper and lower respiratory tract infections, HCoV-NL63 remains a clinically relevant and interesting HCoV species, that may hold the secrets to viral persistence and seasonal infection patterns of endemic HCoVs.
The effects of artificial and natural sweeteners on various physiological systems
(University of the Western Cape, 2011) Rahiman, Farzana
This study aimed to investigate the effects of commercially available natural (sugar cane molasses, white sugar and brown sugar) and artificial (Canderel™, Equal™, Natreen™, Sweetex™, Splenda™ and Swheet™) sweeteners on various physiological systems. The artificial sweeteners tested in this study may be categorised into their respective groups based on their primary ingredient. The brands Canderel™ and Equal™ contain aspartame, Natreen™ and Sweetex™ consist of saccharin and Splenda™ and Swheet™ are composed of sucralose. The inclusion of artificial or natural sweeteners in the human diet has been continually debated and their implication in the development of certain diseases has raised concern regarding their safe use. Therefore, it is necessary that these food products be subjected to a battery of tests to determine adverse effects on human health. Firstly, we investigated the effect of the popular sweetener, sugar cane molasses on the immune system. Whole blood cultures were used to assess the impact of molasses on cytokines regulating specific immune pathways. Lactate dehydrogenase activity was used to determine potential cytotoxicity of sugar cane molasses. Results showed that exposure of molasses to whole blood cultures caused no cell death. However, molasses stimulated interleukin-6 and interleukin-10 secretion, indicating effects on inflammation and humoral immunity respectively. The enhanced humoral response produced by molasses may be associated with increased antibody production that acts in defense against extracellular pathogens. Conversely, high IL-6 levels may be associated with the development of hypersensitivity reactions. Based on this outcome, we aimed to further investigate the comparative effect of molasses to other natural and artificial sweeteners using a similar model system. Results of this study showed that all artificial sweeteners had an inhibitory effect on the inflammatory response, while molasses stimulated the inflammatory process in vitro. The artificial, sucralose-containing sweeteners inhibited IL-10 under stimulatory conditions. Thus, exposure to sucralose induces a reduced humoral response that may be associated with adverse effects on the immune system. Secondly, we investigated the effects of sugar cane molasses on the male reproductive system. The effect of molasses on steroidogenesis was monitored using testicular cell cultures. Testes cultures were either stimulated or not stimulated with luteinizing hormone and exposed to molasses samples. The endpoints namely, lactate dehydrogenase activity, testosterone and estradiol synthesis were measured. Results show that molasses causes no cell death and has no impact on estradiol synthesis. However, molasses does exhibit a stimulatory effect on testosterone biosynthesis in vitro. Based on this outcome, we further investigated the comparative effect of molasses to various natural and artificial sweeteners. Results of these experiments indicate that in comparison to all tested sweeteners, molasses significantly enhanced testosterone secretion under stimulatory conditions. Perhaps the supplementation of molasses in diets of males suffering with low testosterone levels may be of beneficial use. The third objective of this study was to investigate the effects of natural and artificial sweeteners on the female reproductive system. Injury to the female reproductive tract was evaluated using frog, ovarian cell cultures. The biomarkers, testosterone and estradiol synthesis were measured to determine the impact of sweeteners on ovarian steroidogenesis. Cultured, oocyte fragments (Xenopus laevis) were exposed to either natural or artificial sweeteners under stimulated or unstimulated conditions. Results showed that the artificial sweetener, Natreen™ elevated testosterone levels while the natural sweetener, molasses enhanced estradiol levels. These results were supported by a correlating decrease on the E2/T ratio by Natreen™ and an increase in the E2/T ratio by molasses. Exposure to the aspartame and sucralose branded sweeteners reduced estradiol synthesis and this was confirmed by the decrease observed in the E2/T ratio. These results suggest that certain sweeteners have potential androgenic, estrogenic or anti-estrogenic characteristics that may beassociated with either harmful or beneficial effects on the female reproductive system. Sugar cane molasses demonstrated a significant effect on all investigated physiological systems in vitro. In order to validate these results, our final objective was to determine the impact of molasses on physiological systems using in vivo and in vitro methods. For this study, Balb/C, male mice were given an oral dosage of molasses for the exposure period of two months. Animals were allocated into either a molasses-treated or a control group. Parameters such as body weight, physiological changes and molasses intake were measured. Collected blood samples were assayed for potential toxicity using plasma biomarkers and liver enzyme activity. The effect of molasses on the immune system was investigated by monitoring antibody titre levels in immunised treated and untreated animals. Testes harvested from treated and untreated groups were cultured and assayed for testosterone synthesis. This was used to determine the impact of molasses on the process of testicular steroidogenesis. Results showed that molasses-treated groups consumed a significant amount of fluid and displayed symptoms of loose faeces. No significant change on body weight was observed in both treated and untreated groups. Plasma biomarkers and liver enzymes remained unaffected in molasses-exposed animals. However, a decrease in levels of IgG anti-antigen in treated groups indicated that molasses suppresses the humoral immune response. This may be associated with an increased risk to infection with extracellular pathogens. Molasses-treated animals significantly elevated levels of testosterone production in vitro. This result was consistent with previous findings and supports the use of molasses as a supplement to augment testosterone synthesis.
The Influence of maternal nicotine exposure on selected glycolytic and cytochrome P450 enzymes in developing neonatal rat lung
(University of the Western Cape, 2005) Gamieldien, Kareemah
The structural and functional integrity of a developing and maturing fetal and neonatal lung is critically dependent on carbohydrate metabolism. The energy derived from carbohydrate metabolism is utilized during the processes of cell growth and development. It is reported that maternal nicotine exposure during pregnancy and lactation results in the irreversible inhibition of glycolysis, for which no mechanism is currently proposed and a significant increase in glucose turnover. The principal objectives of this thesis are (1) to investigate the isoezyme patterns and transcript levels of selected glycolytic enzymes: Hexokinase (HK), Phosphofrutokinase (PFK), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and Lactate dehydrogenase (LDH) in control developing neonatal rat lung, (2) to investigate the transcript levels of selected cytochrome (CYP) P450 enzymes: CYP1A1, CYP2A3, and CYP2B1 in control developing neonatal rat lung and (3) to determine the influence of maternal nicotine exposure during gestation and lactation on the isoenzyme patterns and transcript levels of the selected enzymes in developing neonatal rat lung, in an attempt to elucidate the mechanism of inhibition of glycolysis observed. Tissue samples were obtained from the lungs of 1, 7, 14, 21, and 49-day old pups, from both control lung tissue and lung tissue exposed to nicotine during gestation and lactation (1mg/kg body weight/day). Isoenzyme separation is achieved using polyacrylamide gel electrophoresis (PAGE) techniques and was principally based on their differences in molecular weights. The PAGE gels are densitometrically analyzed and expressed as % Density/mg protein of lung tissue. Transcript levels are analyzed with the use of dot blots followed by densitometry of the blots. The results obtained for the mRNA subunits are expressed relative to ß-actin. The final data of both isoenzyme patterns and mRNA levels are analyzed statistically using the Wilcoxon unpaired T-test, in which P<0.05 is designated as significant. Results of this study show that: (1)All three isozymes, HKI, HKII and HKIII are detectable at the mRNA level however the method used is only sensitive enough to detect HKII from postnatal day 14. HKI is the dominant isoenzyme in the lung of control as well as rats exposed to nicotine at both the transcriptional and post translational levels during gestation and lactation. Furthermore, the developmental pattern of the isoenzymes as the lung matures follows the same trend in lung tissue of control and nicotine exposed offspring. (2) The three PFK sub-types are transcribed in control neonatal lung. PFK-M mRNA levels are expressed at significantly higher levels in control developing lung than PFK-L and PFK-C. However, maternal nicotine exposure during gestation and lactation results in over-expression of PFK-M and PFK-L, but has no influence on PFK-C mRNA levels. PFK-M was affected by maternal nicotine exposure during gestation and lactation, even at postnatal day 49. This implies that the long-term effect of maternal nicotine exposure on total PFK activity can be attributed to changes in the PFK-M isoenzyme activity. (3) GAPDH mRNA is over-expressed at postnatal days 14 and 21 in the lungs of rat pups exposed to maternal nicotine, however no difference is observed between control and experimental lung tissue at postnatal day 49. The expression of GAPDH mRNA of control lung increased gradually between postnatal days 1 and 49. On the other hand, GAPDH mRNA expression in lungs of nicotine exposed rat pups show a pronounced increase in expression after postnatal day 7 and reached a maximum at postnatal day 14. (4) Expression of all the LDH isoenzymes in control developing lung, except LD-1 between day 7 and day 14, decreased. LD-4 and LD-5 (the homozygous LD-M isoenzyme), the glycolytic associated isoenzymes displayed the greatest decrease. All LDH isoenzymes as well as sub-units at the mRNA level are over-expressed from day 7 onwards in the lungs of rats exposed to nicotine during pregnancy and lactation. LD-5 (the glycolytic sub-unit) and LD-M (mRNA homotetramer isoform) were particularly affected by maternal nicotine exposure. LD-1 is the dominantly expressed isoform at the transcriptional and post-transcriptional levels, in both control and nicotine-exposed lung at any age group analyzed. (5) The human orthologs of rat CYP1A1, CYP2A3 and CYP2B1 are transcribed in lung tissue of control neonatal rats and all display a significant increase in expression from postnatal day 1 to postnatal day 49. CYP2A3 is dominantly expressed at the mRNA level in control neonatal rat lung, followed by CYP2B1 and the lowest levels shown by CYP1A1. Maternal nicotine exposure results in the induction of CYP2A3 and CYP2B1, however has no influence on CYP1A1 expression. Moreover, CYP2B1 in control lung tissue remained significantly lower than CYP2A3, however in the experimental lung there are no differences between the two by day 49. It is concluded that the inhibition of glycolysis in lungs of rats exposed to nicotine during gestation and lactation is not due to changes in the HK or PFK isoenzyme levels in the lungs of the offspring. It is, however, evident that the maturing rat lung attempts to compensate for the glycolytic inhibition by over-expressing the isoforms at the mRNA level associated with glycolytic activity and gluconeogenic activity. The over-expression of GAPDH transcript levels in nicotine-exposed lung tissue takes place only when nicotine is present, and therefore maternal nicotine exposure has no long-term effects on GAPDH mRNA expression. Inhibition of the glycolytic pathway results in the over-expression of LDH at both the transcriptional and posttranscriptional levels and it is proposed that the over-expression is in an attempt to rectify the hindrance caused by maternal nicotine exposure. CYP1A1, CYP2A3 and CYP2B1 are transcribed in neonatal rat lung and induction of CYP2A3 and 2B1 in the lungs of the offspring by maternal nicotine exposure is irreversible and thus "programmed”. Furthermore, it is proposed that if the increased transcript levels reflect increases in activity of the CYP enzymes, this may explain the increase in glucose turnover observed in the lungs of offspring exposed to nicotine during gestation and lactation.
Fatty acids as cancer preventive tools in the dietary modulation of altered lipid profiles associated with hepatocarcinogenesis
(University of the Western Cape, 2005) Abel, Stèfan
Research into cell biology provid ed evidence that all living beings consist of individual cells whose activities, inheritance and ability to assemble into organisms can be understood in logical biochemical terms (1). This demonstrated that however miraculous the existence of life on earth might be, life's mechanisms can be understood. Cell biology can be divided into a number of branches which are becoming progressively intertwined as research advances are made (2). The study of biological membranes is the branch most responsible for elucidating cell function as cellular membranes are the very boundaries within which life exists. A membrane separates the vast array of biochemical reactions that define a living cell from the extracellular world. Within the cell, membranes also organize and separate these biochemical reactions from each other, generating compositionally and morphologically distinct compartments.
Potential anti-melanogenic effects of selected South African plants on b16 melanoma cells
(University of the Western Cape, 2019) Oyekunle, Olubunmi Simeon
Dyspigmentation is one of the commonest dermatological presenting complaints from patients, particularly hyperpigmentation. Hyperpigmentation can cause dangerous psychological and emotional impact on self-perception and health quality of lives of people affected. However, of all the diseases encountered globally, epidemiological data has shown that skin diseases account for almost 34% of all the diseases and these dermatological disorders have gotten worse over time. The gold standard for treatment of hyperpigmentation is hydroquinone. Despite its efficacy, hydroquinone and other current modalities of treatments are associated with some side effects. There are a number of natural products derived from medicinal plants that have proven to be an abundant source of biologically active compounds and a lot of these have served as the basis for the development of new lead chemicals for pharmaceutical. The present study focused on screening of selected South African plants (Maclura pomifera, Otholobium fruticans, Phyylica ericoides, Psoralea aphylla, Rhynchosia villosa, and Serruria furcellata) for their antimelanogenic potentials. Methanol and ethyl acetate were used for the extraction of plant materials. Standard methods were employed for evaluation of cytotoxicity of the methanolic leaf extracts (MLE), ethyl acetate leaf extracts (ELE) and melanin synthesis potentials on B16 melanoma cells. To elucidate mechanisms of melanin reduction action, intracellular tyrosinase activity was determined by measuring the rate of L-DOPA oxidation. Tyrosinase activity was assessed further with dihydroxyphenylalanine (DOPA) staining. The mode of action was further determined by evaluating reactive oxygen species (ROS) and expressions of melanogenesis gene using qPCR. The results showed that O. fruticans and S. furcellata reduced melanin synthesis without cytotoxicity. O. fruticans inhibited tyrosinase activity, increased ROS and suppressed the expression of TYR, TRP-1, TRP-2/Dopachrome tautomerase, MITF, MC1R but upregulated βCatenin. S. furcellata stimulated tyrosinase activity and did not increase ROS. It upregulated the expression of TYR, TRP-1, TRP-2, and MC1R while MITF and βCatenin were suppressed. The results showed that O. fruticans reduced melanin synthesis via cAMP pathway while S. furcellata reduced the synthesis via possibly degradation of melanin pigment. The present study on O. fruticans and S. furcellata has shown that leaves of these plants are candidate anti-melanogenic agents. However, more work still needs to be done to elucidate other possible mechanisms that are relevant to antimelanogenic effects of these two plants
The antifungal effect of Salvadora persica and Euclea natalensis on Candida isolates from Libyan patients with type 2 Diabetes mellitus
(University of the Western Cape, 2018) Esmaio, Mustafa Hasan Mustafa
Improved oral hygiene plays a vital role on quality of health and well-being of diabetic patients. Poor oral health leads to an increased incidence of oral diseases, particularly oral candidiasis. The emergence and global spread of azole- resistant Candida species has necessitated the need for novel, cost effective antifungals to stop further spread of resistant Candida infections. This project is the first documented investigation of Candida species prevalence in Libyan type 2 diabetes mellitus (T2DM) patients and investigated the antifungal effect of Salvadora persica and Euclea natalensis on azole-resistant Candida isolates. In this study, 182 Candida isolates from the oral mucosa of T2DM patients were identified using presumptive species identification by chromogenic media followed by confirmation using API ID 32 C, YST Vitek 2 and phenotype microarrays. Their drug susceptibility profiles were tested using the disc diffusion and the AST Vitek 2 compact system. High-pressure liquid chromatography and nuclear magnetic resonance were employed to separate, isolate and purify the bioactive compounds and fractions of the plant extracts which were then tested for their antifungal activity. The results showed that both Salvadora persica and Euclea natalensis promise to provide beneficial alternatives to conventional drugs in treating oral candidiasis in diabetic patients.
The assessment of Namibian water resources for endocrine disruptors
(University of the Western Cape, 2018) Faul, Andre Karel
Namibia is the driest sub-Saharan country in Africa and it is characterised by low and variable rainfall. As a result, potable water in this mostly semi-arid country is considered an extremely valuable resource. Given the variety of anthropogenic and natural chemicals released into the environment by a growing human population, many water resources worldwide present health risks to both man and wildlife. Many of these chemicals are classified as endocrine disruptors which are chemicals with the ability to adversely affect the physiological systems regulated by the endocrine systems of organisms. These include, among others, reproductive, neurological and immunological effects. Endocrine disrupting chemicals include: natural and synthetic hormones such as estrogen, estrone, estriol and testosterone; heavy metals such as tri-butyltin, lead and cadmium; pesticides such as organophosphates and organochlorides; and a number of compounds such as polycyclic aromatic hydrocarbons and polychlorinated biphenyls. Windhoek, the capital city of Namibia, has for long been at the forefront of water reclamation by being the first town in the world to reclaim sewage for direct potable re-use. Presently, reclaimed sewage contributes approximately a third of the potable water utilised in Windhoek, with the remaining water being sourced mainly from a three-dam system: the S von Bach, Swakoppoort and Omatako dams, as well as from boreholes tapping into the Windhoek aquifer. Prior to the research conducted for this thesis, no studies have been undertaken to determine the endocrine disrupting potential of the reclaimed sewage in Windhoek. Likewise, no such studies have been performed on any of the surface water sources in this country, including the three-dam system supplying Windhoek. During 2010 and 2011, raw sewage, treated sewage and reclaimed sewage samples from Windhoek were collected at different stages of the wet and dry season. These samples were analysed for cytotoxicity using a lactate dehydrogenase (LDH) assay, for neurotoxicity using an acetylcholinesterase (AChE) inhibition assay, for inflammatory activity using enzyme-linked immunosorbent-assays (ELISAs) to determine interleukin-6 (IL-6) and interleukin-10 (IL-10) concentrations, as well as for the presence and quantification of three selected steroid hormones: estradiol, estrone and testosterone using ELISAs. Simultaneously, surface water from nine dams in Namibia were collected and analysed for the same parameters. High estradiol, estrone and testosterone levels were detected in the raw sewage. The sewage treatment plant process significantly reduced the concentration of these hormones, but levels were still in the range where adverse effects can be expected in organisms exposed to this water. The reclamation process successfully removed these residual hormones. The AChE inhibition and inflammatory activity of the treated sewage was also significantly lower than in the raw sewage and were completely removed in the reclaimed water. Cytotoxicity was only present in the raw sewage. In all the dam waters, no samples showed cytotoxicity. Estrone was the only hormone detected at low levels, once in the Avis dam water sample and once in the Goreangab dam water sample. The highest acetylcholinesterase inhibition was noted in the Goreangab dam water. Water from all the dams induced high IL-6 production with the highest levels being in the Goreangab and Swakoppoort dam water. IL-10 was lower than IL-6 concentrations in all samples, but was also highest in the Goreangab and Swakoppoort as well as the Avis dam water samples. During 2017 the efficiency of the reclamation process of treated sewage in Windhoek was assessed using a range of immunotoxicological bioassays on the water samples. This again included LDH and AChE inhibition assays as well as IL-6 and IL-10 production. In addition interferon-γ (IFN-γ) and macrophage inflammatory protein (MIP) -1β production were also determined using ELISAs. As a broad screen for immunotoxicity, proteome profiling was performed to test for 36 different chemokines and cytokines. This is the first time that proteome profiling is used for determining the immunotoxicity of treated sewage reclaimed for direct potable water use. For the 2017 assays, no cytotoxicity was detected in treated sewage or reclaimed water. Based on the ELISAs, treated sewage induced IL-6, MIP-1β and IL-10 production, but not IFN-γ. The corresponding test results for the reclaimed water were negative. The proteome profile indicated the presence of interleukin-1ra (IL1ra), Monocyte Chemoattractant Protein-1 (MCP-1), MIP-1α/MIP-1β, IL-6 and interleukin-1β (IL-1β) in culture supernatants exposed to treated sewage, but not to the reclaimed water. In conclusion, the results of the studies indicated the usefulness of the in vitro bioassays to test the endocrine disrupting potential of water sources. Results indicated that intake water at the reclamation plant in Windhoek contains contaminants that can adversely affect human health. The reclamation process however successfully removed these. However, routine monitoring is required to ensure continued delivery of safe potable water. The study further indicated the usefulness of proteome profiling as a quick, cost-effective screen for the immunotoxicity of water sources. The proteome profile can be followed up with cytokine-specific ELISAs to better quantify the inflammatory potential of water sources.

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