Philosophiae Doctor - PhD (Medical BioScience)
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Item Antibody production with synthetic peptides against human Coronavirus-NL63 ORF3 protein(University of the Western Cape, 2025) Mnyamana, Yanga EddieHuman Coronavirus (HCoV) NL63 was detected in an infant in the Netherlands in 2004 and will hereafter be referred to as CoV-NL63. Four years later, it was classified into Group 1 coronaviruses by the International Committee for Taxonomy of Viruses (ICTV), which was modified into alphacoronavirus in 2009. It appears that CoV‐NL63 originates from bats and its hosts are bats and palm civets. A 27,553‐bp CoV‐NL63 genome structure and the spike glycoprotein (S1 domain) use angiotensin‐converting enzyme 2 (ACE2) for infectious entry. Furthermore, CoV‐NL63 simplifies binding to ACE2 by attaching to heparan sulphate proteoglycans on the cell surface. ACE2 has a significant expression in airway differentiated epithelial cells and CoV‐NL63 enter these cells from their apical surface. It is imperative to note that CoV‐NL63, CoV‐229E, CoVHKU1 and CoV‐OC43 infect polarized epithelial cells, and the entry and release of these viruses are related to apical cells in addition to respiratory signs, there are two non-respiratory symptoms including gastroenteritis and Kawasaki disease, both of which are associated with CoV‐NL63. Moreover, neurological manifestations of CoV-NL63 have been reported, especially in children. The main aim of this research study was to generate monoclonal antibodies (mAbs) against the human coronavirus NL63 Open Reading Frame 3 (ORF3) protein. Firstly, bioinformatic tools were used to assist in identifying antigenic determinants and antigenic epitopes were used to generate peptides of ORF3 protein. Thereafter, the ORF3 protein was characterized using various molecular techniques across the bacterial and mammalian expression systems. A CoV-NL63 full-length ORF3 protein (Protein Identity Number: ABE97138.1) was utilized for the synthesis of the peptide from the N-terminus and peptides conjugated with a MAP; KLH and MAP+KLH linkage on the carboxyl end. The mAbs were generated in vivo by immunizing BALB/c mice. Antigen-specific ELISA assessed the antibody specificity and sensitivity. The mAbs that exhibited the highest specificity and sensitivity for antigenic peptide (s) were used to characterize the ORF3 protein by Western blot and immunofluorescence analysis (IFA).Item Assessment of the biological quality of raw and treated effluents from three sewage treatment plants in the Western Cape, South Africa(University of the Western Cape, 2012-03-06) Hendricks, Rahzia; Pool, Edmund J.The aim of this study was to compare the water quality of raw wastewater and treated sewage effluents from three different sewage treatment plants in the Western Cape, South Africa. The treatment plants investigated are on the same river system. Sewage treatment plant 1 and 2 use older technologies, while sewage treatment plant 3 has been upgraded and new technologies (membrane bioreactor) were incorporated in the treatment processes. The first objective was to determine the occurrence of total coliforms, Escherichia coli (E. coli) and fluoroquinolone and sulfamethoxazole antibiotic residues in raw wastewater and treated sewage effluents. Bacteria in treated sewage effluents can result in diseases such as dysentery, gastroenteritis, and typhoid upon exposure. A chromogenic test was used to screen for coliforms and E. coli. Enzyme linked Immunosorbent Assays (ELISA) were used to quantitate antibiotic residues (fluoroquinolones and sulfamethoxazole) in raw wastewater and treated sewage effluents. This study showed that bacteria are present in raw wastewater and residual bacteria are released with treated sewage effluents from sewage treatment plants.Item Apoptotic markers in ejaculated human spermatozoa(University of the Western Cape, 2005-12-01) Brooks, Nicole Lisa; Van der Horst, Gerhard; Dyer, SilkeThe role of male germ cell death in spermatogenesis is an important one as it removes dysfunctional or genetically damaged germ cells and is necessary to maintain an optimal germ cell to Sertoli cell ratio. The formation of the bloodtestis barrier requires the elimination of excessive germ cells and a surge of germ cell apoptosis occurs prior to puberty regulating the ratio of germ cells to Sertoli cells. The aim of this study was to evaluate the presence of four apoptotic markers on sperm from patients with various grades of fertility using flow cytometry. Furthermore, any correlations between the apoptotic marker assays and the standard semen analysis results were identified. This study compares early and late parameters of apoptosis with morphological features in spermatozoa in the same samples. The three sample groups were identified as: teratozoospermic [G-pattern] (n=26), teratozoospermic [P-pattern] (n=98) and oligoteratozoospermic [Ppattern] (n=36). Standard semen analysis was conducted on the semen samples according to the WHO guidelines. Four apoptotic marker assays using flow cytometry was applied in this study to examine the apoptotic alterations in ejaculate sperm. These assays included the Annexin-V staining for the determination of phosphatidylserine exposure, APO-Direct to identify DNA fragmentation, caspase-3 to detect expression of this active protease during early apoptosis and Fas expression. For the Annexin-V and caspase-3 assays, statistically significant differences (P<0.05) were evident between the three groups. No significant differences (P>0.05) were found between the groups with respect to the APO-Direct assay. A significant difference (P<0.05) was found when comparing the teratozoospermic [G-pattern] group and the oligoteratozoospermic [P-pattern] group for the Fas assay. A strong positive correlation was evident between the Fas and the caspase-3 assays in the teratozoospermic [G-pattern] group. For the teratozoospermic [P-pattern group] the following positive correlations existed between the APO-Direct and the Fas assays, APO-Direct and caspase-3 assays and between caspase-3 and Fas assays. The only strong positive correlation was between the caspase-3 and APO-Direct assays in the oligoteratozoospermic [P-pattern] group. The presence of spermatozoa showing microscopic features resembling apoptosis has been identified in ten human ejaculate samples per sample group. Electron microscopy was used to identify morphological features of apoptosis in these human sperm samples. Classical apoptosis as observed in diploid cells could be identified in sperm and these included: loose fibrillarmicrogranular chromatin network, presence of vacuoles in the nuclear chromatin, membranous bodies within the vacuoles of the chromatin, partially disrupted nuclear membranes, plasma membrane protuberances and apoptotic bodies containing cytoplasmic vacuoles and dense masses. This study has confirmed that semen samples with abnormal semen parameters exhibit the presence of apoptotic markers in sperm. The identification of apoptotic markers on the sperm suggests that abnormalities occur during their developmental process, however, the exact mechanism thereof remains unclear. These findings may suggest that certain apoptotic markers may be an indicator of abnormal sperm function and possibly indicative of male infertility.