Status epilepticus (SE) describes a state of persistent seizures which are unrelenting. First- line treatment for status epilepticus uses a group of drugs, the benzodiazepines, that promote the action of the major inhibitory neurotransmitter within the brain, gamma (γ)-aminobutyric acid (GABA). In a subset of patients however, benzodiazepines prove to be ineffective in terminating SE. Previous data from in vitro models has demonstrated that during single seizures, instead of being inhibitory, activation of the GABAA receptor can have an excitatory effect on neurons. To date, it is unknown whether this shift in GABAergic function contributes to SE, nor how it may modulate the anticonvulsant properties of benzodiazepines. In this thesis I explore the role of excitatory GABAergic signaling in an in vitro model of SE and how this may affect the anticonvulsant efficacy of the benzodiazepine, diazepam. Firstly, I confirm that benzodiazepine-resistant SE is prevalent in a South African paediatric population. Secondly, consistent with its established mechanism of action, I show that diazepam enhances GABAAR synaptic currents. Thirdly, using the in vitro 0 Mg²⁺ model of status epilepticus I show that whilst early application of diazepam has anticonvulsant properties, this is lost when the drug is applied during prolonged epileptiform activity. Fourthly, to investigate this phenomenon I use optogenetic activation of GABAergic interneurons to show that interneurons can drive epileptiform discharges during SE-like activity in vitro. Finally, I confirm that during seizure-like events there is a transient shift in GABAergic signaling that is caused by activity driven changes in the transmembrane Cl⁻ gradient. This thesis provides insight into how excitatory GABAergic signaling during prolonged seizures may contribute towards benzodiazepine resistance in SE. I believe that these results are relevant for understanding of the pathophysiology of SE and may help inform optimal treatment protocols for this condition.

The Karyopherin superfamily is a group of soluble transport proteins which are involved in nuclear-cytoplasmic trafficking. Studies have shown the involvement of Karyopherin proteins in nuclear pore assembly, nuclear membrane assembly and DNA replication. Since all these cell regulatory functions are critical for normal cell function, dysregulation of Karyopherin proteins may have an impact on cancer cell survival. Previous research in our laboratory and in that of others has shown that Karyopherin Beta 1 (KPNB1) is elevated in and necessary for the survival of cervical cancer cells as inhibiting its expression with siRNAs interfered with the proliferation of cancer cells. KPNB1 has thus been proposed as an anticancer target. In addition to inhibition by siRNA, an in silico screen for small molecules with potential to bind KPNB1 identified a number of compounds that are currently under investigation for their cancer cell killing effects. In this study, we investigated the ability of a novel small molecule 1-benzyl-4[(4-methoxy-1-naphyl) methylamino]-N-methyl pyrrolidine-2-carboxamide (Compound 53) to kill cancer cells and inhibit the activity of KPNB1 cargo proteins. In addition, the in vitro pharmacokinetic properties and in vivo toxicology of Compound 53 (C53) were investigated. Cervical (HeLa and CaSki) and oesophageal (WHCO6 and Kyse30) cancer cell lines were found to be more sensitive to C53 treatment compared to non-cancer cells (FG₀), with EC₅₀ values of ~20 μM for the cancer cell lines and ~30-40 μM for the non-cancer cells. C53 treatment significantly inhibited proliferation in cancer cell lines. The reduction in proliferation in cancer cells was associated with a block in the G1 phase of the cell cycle and a change in the expression of cell cycle related proteins such as CyclinD1 and CDK4. C53 treatment resulted in cell death via apoptosis as observed using Annexin V staining and PARP cleavage. To assess whether C53 interferes with KPNB1 associated nuclear import, we investigated the effect of C53 on the activity of KPNB1 cargo proteins, NFAT and NF-ĸB as well as investigate its effect on KPNB1 localisation. The results show that C53 has no effect on the localisation of KPNB1 but it does however block the nuclear activity of the KPNB1 cargoes, NFAT and NF-ĸB. In order to predict the behaviour of C53 in a living system, in vitro ADME pharmacokinetic studies showed that C53 has moderate solubility, permeability and protein binding however, rapid clearance was shown by liver microsome assay. In vivo repeated dose toxicology studies showed that C53 is tolerable in nude mice. Taken together, the data presented in this study shows that a novel small molecule, C53 has a negative effect on the proliferation of cancer cells, inhibits the nuclear import of KPNB1 cargoes, displays tolerable in vitro ADME pharmacokinetic properties and showed no toxic side effects in vivo. These results suggest that C53 targets KPNB1 and shows potential as an anticancer molecule.

The estimated global burden of STEC (Shiga toxin producing Escherichia coli) is 2,481,511 illnesses, 269 deaths, and 26,827 DALYs with 48% of these being foodborne. This thesis provides information on STEC diagnostic strategy, undetected STEC in a tertiary referral hospital in Cape Town, and the virulence and antimicrobial resistance properties of tellurite resistant diarrheic E. coli isolated on CHROMagar(TM)STEC (CHROMagar Microbiology, Paris, France). Deploying the One - Health surveillance approach to study selected diarrheic bacterial pathogens in an informal settlement setting, this study sheds light on the extent of bacterial foodborne pathogens in human and non-human sources.

BACKGROUND: Physician assistants are a profession that has adapted to the growing needs of the health care system. With an increasing shortage of doctors compared to patients, the flexibility of PAs has kept the profession in high demand and created a growing number of jobs in surgical specialties. Orthopedics is the third largest specialty to employ PAs but very little is known about the day to day life of these providers. LITERATURE REVIEW FINDINGS: This thesis contains a comprehensive literature review, composed largely of small cross-sectional studies, which summarizes the current research on job satisfaction and burnout in PAs. The literature review highlights that burnout is dependent on the medical specialty and is different from that of a physician. It also shows that there is greater burnout among oncology and emergency medicine PAs. There is presently a lack of literature on the burnout rates of orthopedic PAs, as well as the retention rates and the autonomy of these Pas within this specialty. PROPOSED PROJECT: This thesis proposes a cross-sectional study to identify the demographics, burnout, retention, and autonomy of orthopedic PAs compared to the general PA population. CONCLUSION: The study will match an orthopedic physician assistant and two general population PAs. The burnout, retention, and autonomy data will be compared and analyzed using a chi-square test to reveal correlations and differences. Significance: A provider who is burned out not only delivers sub-standard care but impacts the overall mental health of the provider and the patients are less likely to comply with their treatment plan if their provider suffers from professional burnout. In addition, high turnover of PAs is costly to the healthcare system.

Squamous cell carcinoma of the oesophagus is a formidable disease which poses a significant health risk in developing countries where the incidence is frequently high and access to health care facilities is often limited. The identification of genes involved in oesophageal tumourigenesis may provide new targets for therapy and improved diagnostics techniques, thereby improving the prognosis of this pernicious disease. In this study, real-time RT-PCR and immunohistochemistry described the overexpression of N-Myc Downstream-Regulated Gene 1 (NDRG1) in oesophageal squamous cell carcinoma (OSCC) tissue compared to normal tissue in a cohort of South African cancer patients. Despite more than ten years of research into the role of NDRG1 in cancer, the precise function of this protein remains enigmatic. Reports have been contentious, suggesting both tumour suppressor and tumour promoter functions for NDRG1, implicating it in tumourigenic processes such as metastasis and angiogenesis. Our immunohistochemical analysis if NDRG1 expression in OSCC tissue and matched normal epithelium (n=83) showed that NDRG1 expression is elevated by 2.6-fold in cancer tissue compared to normal tissue. Moreover, the expression and localisation of NDRG1 appeared to track with epithelial cell maturation where basal cells of normal oesophageal epithelium displayed plasma membrane-associated NDRG1 while maturing cells were mostly positive for NDRG1 in the cytoplasm and nucleus. Likewise, NDRG1 displayed interesting patterns of localisation in tumour tissue of the xiii oesophagus. Dysplastic tissue and poorly differentiated tumour tissue stained positively for NDRG1 in the plasma membrane, while moderately and well differentiated tumours displayed mixed staining for NDRG1 in the plasma membrane, cytoplasm and nucleus. Analysis of NDRG1 expression in cell lines cultured under anchorage-independent conditions revealed that NDRG1 expression is strongly induced when cells are prevented from adhering to the surface of culture dishes. Induced NDRG1 expression correlated inversely with mRNA expression of invasion genes, MMP-2 and MMP-9, as well as the mRNA expression of angiogenic factors Ang- 1, PDGF-B and VEGF-C but, in contrast, showed positive correlation with the angiogenesis cytokine, VEGF-A. Knock-down of NDRG1 expression with siRNA had no effect on anchorage-independent cell proliferation or apoptosis but did inhibit VEGF-A expression. Moreover, VEGF-A promoter activity, induced by culturing cells under anchorage-independent conditions was shown to be NDRG1-dependent. In order to identify factors that may drive NDRG1 transcription in cultured OSCC cell lines we cloned and partly characterised the NDRG1 promoter. Through the generation of promoter deletion constructs, site-directed mutagenesis and Chromatin Immunoprecipitation (ChIP) assays, we showed that both EGR-1 and cJun/AP-1 are capable of driving transcription of NDRG1 in response to 12-o-tetradecanoylphorbol- 13-acetate (TPA) through activation of PKC/MEK/ERK1/2 and JNK MAPK pathways. Taken together, we describe the regulation of NDRG1 expression by EGR-1 and AP-1 and we show that NDRG1 is overexpressed in squamous cell carcinoma of the oesophagus compared to normal oesophageal tissue. We associate NDRG1 with an xiv oncogenic function in OSCC through its potential role in angiogenesis via modulation of VEGF-A expression.

Oesophageal squamous cell carcinoma (OSCC) ranks as one of the deadliest tumours with a high incidence in developing countries in the areas of Southern Africa, Middle East and Far East. Moreover, its unfavourable prognosis is further complicated by the lack of knowledge about the molecular biology of this disease. In this thesis, we describe our work analysing the function of N-myc downstream regulated gene 1 (NDRG1, also known as Cap43 or Drg-1) in the neoplastic progression and maintenance of OSCC. Although NDRG1 has previously been implicated in breast, prostate, colon and liver carcinoma, the exact role of NDRG1 in OSCC still remains unclear. According to the immunohistochemical analysis of clinical OSCC tissue samples (n=52), NDRG1 expression was gradually increased in tumour tissue versus normal, indicating the potential involvement of NDRG1 in the neoplastic progression of OSCC. We next performed ectopic NDRG1 gain-of-function and loss-of-function studies using transfectants established from transduced OSCC cell lines (KYSE30 and KYSE150) by lentiviral vector mediated gene delivery. In KYSE30 cells, although no substantial effects on in vitro cell proliferation and differentiation were observed with altered NDRG1 expression, the ectopic overexpression of NDRG1 was found to be positively linked to metastasis, angiogenesis and apoptotic evasion as measured in cell culture. Accordingly, in the nude mouse xenograft model system, NDRG1 overexpression promoted the in vivo growth and metastasis of KYSE30 derived xenografts, which could be attributed to the reduced apoptotic and enhanced angiogenic activities promoted by this gene. Nevertheless, no significant phenotypic changes were observed in response to NDRG1 knock-down, suggesting that this gene was not essential for the neoplastic progression of OSCC. Moreover, null effect of either ectopic NDRG1 overexpression or knock-down were observed in KYSE150 cells, indicating ix that the function of NDRG1 may be largely dependent on the cellular context (Chapter 2). In addition to direct functional assays, evidence from analysing the regulation pattern of NDRG1 in OSCC cells was also presented to provide clues to indirectly predict the function of NDRG1 in OSCC. In Chapter 3, we demonstrated that NDRG1 could be actively regulated by various oncogenic stimuli such as cellular stress (genotoxicity and hypoxia) and mitogenic factors (EGF and IGF). Although these oncogenic regulatory effects on NDRG1 expression in OSCC cells may be dichotomous, the functional significance of NDRG1 upregulation, especially by hypoxia and EGF signalling, is highlighted. In our studies, the regulatory pattern of NDRG1 in OSCC is highly consistent with its oncogenic function revealed in ectopic studies (Chapter 2), further suggesting that phenotypic changes observed in the functional studies may not be artifactual, but may reflect the role of NDRG1 in the neoplastic progression of OSCC in physiological conditions. Taken together, our current data implicate NDRG1 as an effective but non-essential promoter in the neoplastic progression of oesophageal squamous cell carcinoma. Although the mechanism still needed to be further explored, our study suggests important clues regarding these mechanistic roles considering the impact of this gene on apoptosis, metastasis and angiogenesis.

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Includes bibliographical references (leaves 149-163). / Oesophageal cancer is the most common cause of cancer related deaths among black males in South Africa. Currently there are very limited treatment options, and patients have a very poor prognosis, due in part to the late stage at which this cancer is usually detected. In this thesis we describe the establishment of a screening assay using an oesophageal cancer cell line as a model. It was our hope that this screen would allow us to identify compounds which have activity against oesophageal cancer, that could be used as lead agents for further development of chemotherapeutic agents. Once our screen was established, we tested a wide range of extracts from southern African marine organisms, supplied by our collaborators from Rhodes University, South Africa. The marine environment represents a rich, untapped repository of novel and interesting compounds, and through our collaboration we had access to a wide range of marine-derived extracts and compounds. During the course of this project we provided screening data to assist in activity-directed fractionation from five active marine extracts, giving rise to 15 compounds of varying activity. These included several groups of novel active compounds such as the makaluvic acids from the sponge Strongylodesma aliwaliensis and the malonganenones from the octocoral Leptogorgia gi/christii. The identification of a number of novel, active compounds through our screening program highlights the potential of marine organisms from the southern African coast as a source of novel drug leads.