Dissertations / Theses on the topic 'Medical Biochemistry'
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1
Woodhouse, Jennifer Ann. "Plutonium pharmacokinetics and blood biochemistry." Thesis, University of Central Lancashire, 1997. http://clok.uclan.ac.uk/20148/.
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Since its discovery in the early 1940s the element plutonium has been seen by mankind as both an opportunity and a threat. As a radioactive nuclide plutonium presents health hazards in its handling and if mankind is to make the most of this element's potential benefits it is essential that these hazards be understood. Both overestimation and underestimation of these hazards are damaging to its proper utilisation. Many studies have been carried out to determine the effects of plutonium exposure and a broad picture of the biological behaviour of plutonium has been built up. Radiological protection standards are based on such broad understanding and a "Central Dogma" has arisen viz, plutonium is bound avidly in liver and bone; clearance half-lives from these organs differ (by a factor of 2.5) but are very long - a minimum of 50 years for bone; this is why plutonium urinary excretion levels are very low. Despite all the research work that has been carried out there are many important areas of plutonium behaviour which are not well understood or in which the central ideas adopted for radiological protection purposes are questionable. One such questionable area is extended half-life in the body. Two rather different areas relate to the molecular binding interactions which plutonium enters into in body tissues and transfer mechanisms from blood into cellular organelles. Very little is known about these processes and the speciation that plutonium demonstrates within the body. This thesis explores understanding of plutonium behaviour by application of pharmacokinetic theory to observed human behaviour, both following occupational exposure and experimental injection. Occupational exposure data demonstrated behaviour consistent with pharmacokinetic expectations over periods of 25 years or more. Long-term half-lives were 10 to 30 years rather than 50 to 100 years or more. There was no evidence of differing half-lives between liver and bone. Very low renal clearance was seen in intravenous injection studies suggesting either very extensive plutonium binding to the protein transferrin in blood or pointing to reabsorption in the kidney tubule after glomerular filtration. This latter possibility might lead to a "Plutonium blood pressure" which effectively forces activity into tissues irrespective of the strength of binding forces. Experimental work indicated species differences in transferrin binding which may have relevance for extrapolation from animals to humans.
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Zhong, Sheng-Ping. "Biodegradation of medical polymers." Thesis, University of Liverpool, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.333769.
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Schrift, Greta Lynn. "Energetic consequences of structural features and dynamics changes upon nucleotide binding to ribonuclease SA molecular basis for nucleotide binding specificity /." Diss., University of Iowa, 2004. http://ir.uiowa.edu/etd/120.
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Driver, Cathryn Helena Stanford. "The development of a radiolabelled macromolecule as a therapeutic agent for the treatment of cancer." Doctoral thesis, University of Cape Town, 2015. http://hdl.handle.net/11427/15540.
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One of the major focus areas of anticancer therapy is the design of new radiotherapeutic agents that are able to specifically target and destroy cancer cells with minimal side effects and damage to healthy, normal cells. This thesis describes studies towards the synthesis of a macromolecular bioconjugate that was designed to: i) co-ordinate a radioisotope through a tetra-amine macrocycle (cyclam), ii) lead to passive tumour targeting via the EPR effect and a suitably large carrier such as human serum albumin and iii) induce active targeting through a glucose moiety recognised by the over-expressed glucose transporters on the surface of highly metabolically active cancer cells. The various cyclam functionalisation strategies explored were relatively unsuccessful, but eventually a bis-aminal cyclam was successfully converted, through nucleophilic substitution, into a precursor pro-conjugate: a di-functionalised cyclam containing a β-glycoside tether and a long chain primary alkylamine. The glycoside tether was synthesised via glycosylation of a glycosyl iodide with decandiol followed by oxidation of the terminal hydroxyl group to an acid chloride for cyclam acylation. The second linker attached to cyclam was synthesised by conversion of decanediol to a brominated alkyl amine. This amine would then be converted into a maleimide functionality suitable for Michael addition with a free thiol group contained within the proposed bio-carrier to form the desired bioconjugate. Further studies described towards the synthetic construction of the bioconjugate include: 1) The construction of a maleimide group 2) The attachment of an imaging radioisotope, ⠹⠹m Tc, or therapeutic isotope, ¹⠰³ Pd, to the pro- conjugate and other glucose-cyclam precursors 3) The determination of the potential uptake of the bioconjugate through glucose transporters by using a fluorescent dansyl-glucose compound as a model and monitoring its uptake into WHCO1 oesophageal cancer cells. 4) The HPLC analysis of the coupling of a glucose-maleimide model compound to bovine serum albumin to investigate the Michael addition of the free thiol in HSA to a maleimide 5) The development of a potentially alternative nanoparticle carrier by synthesis of palladium and magnetic nanoparticles with commercially available thioglucose or glucuronic acid moieties as the surface targeting and stabilising agent. In summary, this thesis outlines a number of synthetic, radiological and biological aspects towards the development of a fully functioning radiolabelled macromolecular bioconjugate that could be tested for improved targeted cancer radiotherapy.
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Viljoen, Katie S. "Integrative genomic analyses of bacterially-associated colorectal cancer." Doctoral thesis, University of Cape Town, 2015. http://hdl.handle.net/11427/15761.
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Sporadic colorectal cancer (CRC) has been linked to various lifestyle factors, including the consumption of alcohol and red meat, smoking, and obesity. CRC is one of most extensively characterised cancers, both at a molecular and 'omic' level; nevertheless, the precise mechanism driving CRC initiation remains unknown. To date, numerous studies have identified changes in the microbial profiles of CRCs compared to adjacent normal mucosa and compared to healthy controls; however, CRC-associated bacteria have not been concurrently quantified across a single cohort; nor have the relationships between CRC-associated bacteria, clinicopathological features of CRC and genomic subtypes of CRC been investigated. The main aim of this thesis was therefore to gain insight into the potential contribution of CRC-associated bacteria in the aetiopathogenesis of CRC by leveraging both host genomic and clinicopathological data as well as to investigate patterns of tissue colonisation between different CRC-associated bacteria. The objectives were 1) to quantify, using quantitative-PCR, CRC-associated bacteria in a cohort of 55 paired tumour and adjacent histologically normal samples collected during surgical resection as well as in an additional 18 formalin-fixed paraffin-embedded (FFPE) samples; 2) to determine their relationships to patient age, gender, ethnicity, stage of disease, site of disease and MSI status (Chapter 4); 3) to evaluate the relationship between each bacterium and host gene expression (Chapter 8) and methylation changes (Chapter 6); and 4) to determine genomic subtypes of CRC using unsupervised clustering of gene expression data in the context of patient clinicopathological features and bacterial quantitation data; and 5) to gain a deeper biological understanding of the results from the objectives 1–4 using pathway analyses of the genomic subtypes obtained (Chapter 7). The main finding of this thesis is that a transcriptomic subtype of colorectal cancer, characterised by an increase in CpG island methylation, displays an increased frequency of colonisation by Enterococcus faecalis and by high levels of Fusobacterium. At the pathway-level, this subtype is enriched for pathways related to damage response, infection, inflammation and cellular proliferation; notably, these findings were confirmed in a well-defined publically available CRC gene expression dataset of colorectal adenocarcinomas (N=155). These findings suggest that specific bacterial colonisation underlie s a distinct genomic subtype of colorectal cancer that is characterise d by inflammatory-related gene expression changes ; these findings however require validation in a larger cohort. In addition, novel associations between colonisation by specific bacteria and host clinicopathological, transcriptomic and DNA methylation features were identified.
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Esau, Luke Emmanuel. "Proliferative and survival pathways in oesophageal cancer." Doctoral thesis, University of Cape Town, 2011. http://hdl.handle.net/11427/12279.
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Includes abstract.Includes bibliographical references.
Oesophageal squamous cell carcinoma (OSCC) is the 8th most common cancer worldwide with high incidence in areas that include China, Iran and South Africa. The current treatment available for OSCC does not significantly enhance patient survival. A better understanding of proliferative and survival pathways activated in OSCC could allow identification of more specific therapeutic targets, potentially improving management of OSCC. Cell surface receptors are known to play important roles in relaying signals from the extracellular environment...The aim of this study was to determine the role of EGFR, IGF-1R and CXCR2 in proliferation and survival of OSCC cells.
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Gordon, Kerry. "Protein-protein interactions of human somatic angiotensin-converting enzyme." Doctoral thesis, University of Cape Town, 2011. http://hdl.handle.net/11427/10535.
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In this study, novel disulphide bridges were engineered into the linker region of ACE [Angiotensin-converting enzyme] in an attempt to limit inter-domain movement, thereby producing a candidate for crystallisation and to determine the effect of these bridges on inter-domain movement.
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Matejcic, Marco. "Identification of genetic polymorphisms associated with oesophageal squamous cell carcinoma risk in South Africa." Doctoral thesis, University of Cape Town, 2013. http://hdl.handle.net/11427/3139.
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Includes abstract.Includes bibliographical references.
Oesophageal squamous cell carcinoma (OSCC) is a complex disease, determined by the interaction of genetic factors with environmental risk factors. In South Africa, OSCC is a major malignancy occurring with high incidence in the Black and Mixed Ancestry populations. Previous studies by our research group have reported that genetic polymorphism of xenobiotic metabolizing enzymes influence greatly the detoxification of tobacco-related carcinogens in vivo, and may therefore have an important role in determining susceptibility to oesophageal cancer.
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Parker, Ayesha. "The characterisation of the ectodomain shedding of the low density lipoprotein receptor." Doctoral thesis, University of Cape Town, 2009. http://hdl.handle.net/11427/3145.
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Ronacher, Katharina. "Internalisation of the type II gonadotropin-releasing hormone receptor of marmoset monkey." Doctoral thesis, University of Cape Town, 2003. http://hdl.handle.net/11427/8599.
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Bibliography: leaves 102-124.The mammalian type II GnRH receptor has a C-terminal tail unlike the mammalian type I GnRH receptor, which uniquely lacks the cytoptasmic C- terminal domain. lnternalisation of a mammalian type ll GnRH receptor has never been investigated, therefore this thesis studies the internalisation pathway of the type ll GnRH receptor. As the C-terminal tail mediates rapid internalisation of many G protein-coupled receptors this research investigates the functional role of the C-terminal tail and intracellular loop in receptor internalisation. The internalisation pathway of the type ll GnRH receptor in COS-1 cells was investigated by co expressing dominant negative mutants and wild- type constructs of G protein-coupled receptor kinases (GRKs), dynamin-1 and β-arrestin 1 and 2 with the type II GnRH receptor. The results show that internatisation of the receptor requires GRK 2 and dynamin but does not require β-arrestin 1 and 2. Furthermore, inhibitors to both the caveolae pathway as well as the clathrin coated vesicle endocytosis abolished receptor internalisation indicating that both structures are involved in internalisation of the receptor. Even though in COS-1 cells the type ll GnRH receptor internatises in a β-arrestin independent manner, internalisation of this receptor can be enhanced by over-expression of wild type β-arrestin. This indicates that the type ll GnRH receptor is able to utilise a β-arrestin mediated internaltsation pathway if high levels of β-arrestin are present in the cell. The mammalian type ll GnRH receptor internalises with enhanced rate and extent compared to the tail-less human type I GHRH receptor. The role of the C-terminal tail of the type ll GnRH receptor in internalisation was investigated by measuring internalisation of C-terminally truncated mutants. It was found that the region between Gly 343 and Ser 335 within the C-terminal domain is important for receptor internalisation. Substitution of putative phosphorylation sites within this region revealed that Ser 338 and Ser 339 are critical for rapid receptor internalisation. Furthermore a serine residue in intracellular loop three (Ser 251) was shown to play a role in signalling as well as in internalisation. Since dominant negative GRK 2 could not inhibit internalisation of a mutant lacking all three serine residues, but could reduce internalisation of the wild-type receptor, we suggest that Ser 251, 338 and 339 are target of phosphorylation by GRK. However these phosphorylation sites as well as the C-terminal tail are not necessary for β-arrestin dependent internalisation. Taken together this thesis elucidates the internalisation pathway of a mammalian type lI GnRH receptor and identified residues within the C-terminal tail and intracellular loop three that are critical for rapid internalisation.
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Sutherland, Jason Robert. "The role of seminal plasma in cervical carcinoma." Doctoral thesis, University of Cape Town, 2010. http://hdl.handle.net/11427/10617.
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Includes bibliographical references (leaves 211-264).Cervical cancer is a worldwide public health problem with in excess of 370,000 cases being reported each year. It is the leading cause of death from cancer among women in the developing world where 80% of cases occur. Human papilloma virus (HPV) has been identified as the main causative factor linked with the development and progression of cancer of the cervix, although other factors are known to exist and include genital warts, consenting to sex at an early age, smoking, long term use of contraceptive pills and multiple sexual partners.
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Coetsee, Marla Catherine. "Ligand-induced selective signalling at the gonadotrophin releasing hormone receptor." Doctoral thesis, University of Cape Town, 2008. http://hdl.handle.net/11427/3123.
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Includes abstract.Includes bibliographical references (p. 179-202).
The pituitary gonadotrophin releasing hormone (GnRH) receptor regulates reproduction by activation of Gq/11 proteins. In contrast, GnRH receptors at extrapituitary sites induce anti-proliferative effects that do not correlate with Gq/11 activation. We propose that the two endogenous ligands, GnRH I and GnRH II, and certain antagonists selectively activate distinct signalling pathways by stabilisation of distinct active conformations of the GnRH receptor, a concept termed ligand-induced selective signalling (LiSS). This dissertation has investigated LiSS at the GnRH receptor using several approaches. The sequences of GnRH I and II differ in positions 5, 7 and 8. I investigated the interaction of position 5 of GnRH I and GnRH II with Tyr6.58 of the receptor. Compared with the Leu and Ala mutants, the Tyr6.58Phe mutant had higher affinity for native GnRHs, but not Ala5-substituted GnRHs, suggesting that Tyr5 of GnRH I and His5 of GnRH II interact with Tyr6.58 by aromatic interactions. Our molecular models show that GnRHs interact with distinct rotamer conformations of Tyr6.58. This is supported by the Tyr6.58Leu receptor, which makes compensatory interactions that improve binding affinity and receptor activation for GnRH II, but not GnRH I, compared with the Tyr6.58Ala receptor. Together these results suggest that GnRHs stabilise distinct receptor active conformations. To identify the most proximal signalling proteins that mediate GnRH receptordependent anti-proliferative effects, I established a range of [35S]GTPS binding assays. I confirmed that the GnRH receptor activates Gq/11, but in contrast to previous proposals, my results show that the GnRH receptor cannot directly activate Gi. I subsequently identified a novel GnRH receptor signalling partner, the SH2 domaincontaining phosphatase 2 (SHP-2). I propose that SHP-2 mediates the antiproliferative effects of the receptor. I show that the SHP-2 pathway is activated independently of Gq/11 and suggest that signalling occurs by a direct interaction of SHP-2 and src with the GnRH receptor. Furthermore, this pathway is activated by a classical Gq/11 antagonist or by Gq/11-uncoupled GnRH receptor mutants. My results provide convincing evidence supporting LiSS at the GnRH receptor and may facilitate development of therapeutics with increased signalling specificity at this receptor.
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Abera, Aron Berhanie. "Characterization of signalling cross-talk between the EP2 and FP receptors in endometrial epithelial cells." Doctoral thesis, University of Cape Town, 2009. http://hdl.handle.net/11427/3159.
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Uterine fibroids are benign tumors that arise from the smooth-muscle uterine cells (myometrium) and are the most common uterine disorder occurring in as many as 30% of women over 35 years of age. Despite their frequent occurrence, the etiology of uterine fibroids is not well elucidated. Several studies have shown that numerous tumors can be regulated by cyclooxygenase (COX) enzyme products but their role in uterine fibroids is not well established. The initial aim of the study was to determine the expression level of COX enzymes and prostaglandin receptors in fibroids and autologous myometrium samples from women with fibroids. Real-Time reverse-transcriptase polymerase chain reaction (RT-PCR) revealed that the expression of COX enzymes, EP1, EP2 and EP4 prostanoid receptors and prolactin were not significantly altered while the EP3 subtype receptor was significantly down-regulated in fibroids compared to adjacent myometrium samples. The EP3 receptor has a protective role in tumor development suggesting the role for down-regulation of the receptor in uterine fibroids pathology. In addition, the expression of COX enzymes, prostaglandin receptors and prostaglandin-mediated genes were assessed in endometrium samples from women with and without uterine fibroids in different stages of the menstrual cycle. COX-2 and interleukin-8 (IL-8) mRNA expressions were significantly higher in both proliferative stage and early-mid secretory, EP2 receptor and IL-11 were elevated in the proliferative stage, vascular endothelial growth factor (VEGF) was highly expressed in the early-mid secretory phase while FP receptor was up-regulated in all stages of the menstrual cycle in endometrium samples from women with fibroids. These data suggest that up-regulation of COX-2 and prostaglandin receptors (EP2 and FP) in endometrium can induce expression of angiogenic and mitogenic factors such as VEGF, IL-8 and IL-11 which might act in a paracrine manner on neighboring myometrial/fibroid tissue to promote angiogenesis and facilitate tumor growth. XVII Furthermore, since EP2 and FP receptors were up-regulated in the proliferative phase of endometrium from uterine fibroid patients and the receptors are co-expressed in endometrial adenocarcinoma (Ishikawa) cells, this study investigated a possible cross-talk that influences intracellular signalling by using Ishikawa cells stably expressing the EP2 and FP receptors (FPEP2 cells) as a model cell line. Real-Time RT-PCR, Western blot analysis and immunofluorescence microscopy confirmed stable expression of the EP2 and FP receptors in FPEP2 cells localized to the perinuclear and plasma membrane. Using FPEP2 cells, the integrated effect of Butaprost (EP2 receptor ligand) and PGF (FP receptor ligand) co-administration on inositol phosphate (IP3) and adenosine 3-,5-cyclic monophosphate (cAMP) release was assessed to study a possible heterologous-interaction or cross-talk between the EP2 and FP receptors. The study showed that in FPEP2 cells, PGF alone does not alter cAMP production, but in combination with Butaprost augments EP2 receptor-mediated cAMP release. PGF-mediated potentiation of cAMP release was abolished by antagonism of the FP receptor, inhibition of phospholipase C (PLC) and IP3-receptor whereas inhibition of protein kinase C (PKC) had no effect suggesting the cross-talk is mediated by FP receptor activation of IP3 release. Moreover, inhibition of calcium effectors using calmodulin antagonist (W7) or Ca2+/calmodulin-dependent kinase II (CaMK-II) inhibitor (KN-93) abolished PGF potentiation of Butaprost-mediated cAMP release. Using short interfering RNA (siRNA) molecules targeted against the adenylyl cyclase 3 (AC3) isoform, the study showed the isoform to be responsible for the cross-talk between the FP and EP2 receptors. In order to determine the integrative effects of the EP2 and FP receptors co-activation on gene expression, a whole genome array profiling in FPEP2 cells in response to Butaprost and/or PGF was performed. The gene array revealed 228 genes that are regulated by co-activation of the EP2 and FP receptors that are involved in cell morphology, proliferation and differentiation. XVIII In addition, co-activation of EP2 and FP receptors with their respective ligands enhanced or repressed a set of EP2 receptor-regulated genes. One of the genes identified, SAT1 (Spermidine/ N1-acetyltransferase), was regulated by the EP2 and FP receptors cross-talk via the calcium sensitive AC3 isoform. SAT1, with known role in regulation of tumorigenesis was also up-regulated in the proliferative stage of endometrium samples from women with uterine fibroids suggesting the EP2 and FP receptor cross-talk characterized in vitro can also happen in vivo. In conclusion, this study reports that COX-2, EP2 and FP receptors, VEGF, IL-8, IL-11 and SAT1 are up-regulated in endometrium from women with uterine fibroids. These genes play a major role in development of fibroids by facilitating angiogenesis and cell growth and by inhibiting apoptosis via autocrine/paracrine mechanisms. In addition, this study demonstrates that co-activation of the EP2 and FP receptors results in enhanced release of cAMP via the FP receptor-G +-q-Ca2+-calmodulin pathway by activating the calcium-sensitive AC3 isoform and modulates a molecular switch which alters the trans-activation of a subset single-receptor induced genes that have important functions in the pathogenesis of reproductive pathologies.
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Maranyane, Hapiloe 'Mabaruti. "Phosphoglucomutase 1 (PGM1) expression and regulation in cancer cells." Doctoral thesis, University of Cape Town, 2015. http://hdl.handle.net/11427/16694.
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Includes bibliographical referencesCancer cells undergo metabolism that is significantly different to normal cells, with an increased dependence on glucose metabolism as a hallmark of most cancers. Changes in global gene expression patterns are the major driving forces behind cancer progression. These changes trigger events that result in the dysregulation of key enzymes associated with metabolic processes. Gene expression profiling studies done previously in our laboratory identified a group of genes involved in glucose metabolism to be differentially expressed in cervical cancer patient material. Of these, Phosphoglucomutase 1 (PGM1) was identified to have elevated expression in the cancer group. PGM1 is a phosphotransferase that catalyses the reversible conversion of the glycogen breakdown product, glucose-1-phosphate into glucose-6-phosphate, a substrate for glycolysis and the pentose phosphate pathway. This places PGM1 at a critical traffic point of glucose metabolism. In this study we investigated the expression, regulation and biological significance of PGM1 in cancer cells. Our results showed that PGM1 expression was elevated in cervical cancer tissue compared to normal. Its expression was also high in cervical, oesophageal and breast cancer cell lines. Elevated PGM1 expression associated with high promoter activity as well as with E2F and HIF1α activities in cancer cells. PGM1 expression at the level of mRNA, protein and promoter activation was significantly stimulated in hypoxia mimicking conditions. Our data showed that PGM1 expression in cancer cells was required mainly for glycogen accumulation with marginal changes on glycolysis and the pentose phosphate pathway. While PGM1 expression did not appear necessary for cancer cell proliferation in normoxia and nutrient sufficiency, our data shows that it is required for proliferation under conditions of glucose deprivation combined with hypoxia. Together these findings suggest that PGM1 expression is altered in cancer cells, that it is required for aberrant glycogen expression in cancer cells and that it has a role in cancer biology during severe stress conditions.
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Teng, Huajian. "Identification of signalling pathways regulating TBX2 gene expression and its target genes." Doctoral thesis, University of Cape Town, 2008. http://hdl.handle.net/11427/3151.
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Includes abstract.Includes bibliographical references (leaves 83-97).
Members of the T-box family of transcription factors provide an important link between development and cancer. T-box factors play critical roles in embryonic development and results from recent studies suggest that they function in controlling cell cycle progression and also in the genesis of cancer. Importantly, the T-box factors Tbx2 and Tbx3 are overexpressed in several cancers including melanoma, small cell lung carcinoma, breast, pancreatic, liver and bladder cancers and can suppress senescence, a cellular process which serves as a barrier to cancer development. However, the precise role of most T-box factors is poorly defined, in part, because their target genes are still poorly characterised and very little is known of the signalling pathways that regulate their expression and activity. The broad aim of this study was therefore to contribute towards the identification of Tbx2 target genes as well as to identify signalling pathways that regulate TBX2 expression. The specific aims were thus to (1) investigate the regulation of type1 collagen gene expression by Tbx2; (2) clone the human TBX2 regulatory region and to identify cis-acting elements involved in the basal transcription of the TBX2 gene and (3) investigate the regulation of TBX2 gene expression by signalling pathways.
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Peters, Julian S. "Comprehensive proteomic profiling of clinically relevant strains of Mycobacterium tuberculosis." Doctoral thesis, University of Cape Town, 2014. http://hdl.handle.net/11427/12959.
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Includes bibliographical references.Tuberculosis is an airborne infectious disease caused by the bacillus known as Mycobacterium tuberculosis. Despite limited genetic variability, Mycobacterium tuberculosis strains exhibit vast discrepancies in phenotypic presentation in terms of virulence, elicited immune response and transmissibility. This study aims to use Mass Spectrometry (MS) tools to quantitatively and qualitatively investigate the total proteome expressed by various epidemiologically significant strains within the Mycobacterium tuberculosis complex (MTBC) as well as a clinically relevant non-tuberculous Mycobacteria (NTM) strain when cultured in vitro. We aim to use the experimental data obtained using discovery mass spectrometry to identify candidate proteins to use in the design of multiple reaction monitoring (MRM) MS experiments for targeted biomarker validation in patient derived biological samples such as sputum. Liquid chromatography mass spectrometry (LC MS/MS) and data capture were carried out using the LTQ Orbitrap Velos. 1D LC was carried out on gel fractionated samples to increase proteome coverage. This allowed a significant increase in the number of protein identifications of up to 80% proteome coverage per strain. Comparative analysis of the datasets was carried out to identify and define the core-proteome expressed across all strains as well as to identify differentially expressed proteins amongst the strains.
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Chang, Cheng-Fu. "Structure-function and regulation studies of angiotensin-converting enzyme 2." Doctoral thesis, University of Cape Town, 2009. http://hdl.handle.net/11427/3122.
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Dzobo, Kevin. "Matrix-mediated regulation of type 1 collagen synthesis and degradation in cultured fibroblasts." Doctoral thesis, University of Cape Town, 2009. http://hdl.handle.net/11427/3125.
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Includes abstract.Includes bibliographical references (leaves 122-157).
Stromal cells and the extracellular matrix (ECM) components provide the microenvironment that is pivotal for cell growth, motility, attachment and differentiation. Fibroblasts are some of the cells responsible for the synthesis of most of the extracellular matrix proteins. Type I collagen is the most abundant extracellular matrix protein in the human body and is found in tissues requiring high tensile strength. In this study we investigated the effect of a pre-formed fibroblast-derived extracellular matrix on the expression of type I collagen and associated matrix metalloproteinases in fibroblasts.
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Kröger, Wendy Lee. "A molecular basis for the C-domain selectivity of angiotensin-converting enzyme." Doctoral thesis, University of Cape Town, 2009. http://hdl.handle.net/11427/3134.
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Mwapagha, Lamech Malagho. "The role of viral sequences in genetic aberrations and malignant transformation." Doctoral thesis, University of Cape Town, 2014. http://hdl.handle.net/11427/12870.
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Includes bibliographical references.Cancer is a leading cause of death worldwide and viral infections such as HBV/HCV and HPV have been known to be responsible for up to 20% of cancers in low- and middle-income countries. Approximately 500,000 of these deaths are due to oesophageal squamous cell carcinoma (OSSC) alone, one of the major cancers in Eastern and Southern Africa, Latin America and Asia. Previous studies have shown HPV DNA to be integrated in nearly 40% of oesophageal tumours whereas it was present in only 3% of normal healthy asymptomatic individuals, implicating it as a possible risk factor. The aim of this study was to compare the roles and effects of the E6 gene from the low risk HPV11 and high risk HPV18 on the cellular gene expression profile in order to identify genes required for the initiation of cellular transformation and also to identify genomic alterations associated with oesophageal squamous cell carcinoma. Cancer is a leading cause of death worldwide and viral infections such as HBV/HCV and HPV have been known to be responsible for up to 20% of cancers in low- and middle-income countries. Approximately 500,000 of these deaths are due to oesophageal squamous cell carcinoma (OSSC) alone, one of the major cancers in Eastern and Southern Africa, Latin America and Asia. Previous studies have shown HPV DNA to be integrated in nearly 40% of oesophageal tumours whereas it was present in only 3% of normal healthy asymptomatic individuals, implicating it as a possible risk factor. The aim of this study was to compare the roles and effects of the E6 gene from the low risk HPV11 and high risk HPV18 on the cellular gene expression profile in order to identify genes required for the initiation of cellular transformation and also to identify genomic alterations associated with oesophageal squamous cell carcinoma.
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Larmuth, Kate Morgan. "Angiotensin-converting enzyme cleavage of the Alzheimer's beta-amyloid peptide." Doctoral thesis, University of Cape Town, 2015. http://hdl.handle.net/11427/16561.
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Includes bibliographical referencesAngiotensin-1 converting enzyme (ACE) is a zinc metallopeptidase that consists of two homologous catalytic domains (N and C) with different substrate specificities. ACE is a central component of the intrinsic brain renin angiotensin-aldosterone system (BRAAS), well renowned as the regulator of blood pressure. The BRAAS has alternate functions that extend beyond fluid and blood pressure homeostasis into areas such as neurological function. As a result, it is implicated in many neurodegenerative diseases including Alzheimer's disease (AD). ACE's specific mechanistic role in AD is not entirely clear and is somewhat controversial. However, it has been shown that ACE hydrolyses the amyloid beta (Aβ) peptide, the putative causative agent of AD. This study aimed to investigate the molecular basis of ACE hydrolysis of Aβ by determining : 1) the kinetic parameters of five different forms of human ACE with various N-terminal amyloid beta (Aβ) substrates; 2) the specific active site determinants of Aβ-domain selectivity; and 3) the high-resolution crystal structures of the N-domain of ACE in complex with Aβ(1-16), Aβ(10-16), Aβ(4-10), the FRET Aβ(4-10)Y and Aβ(35-42) peptides. For the physiological Aβ(1-16) peptide, a novel ACE cleavage site was found at His14/Gln15. Furthermore, Aβ(1-16 ) was preferentially cleaved by the truncated N-domain; however, the presence of an inactive C-domain in full-length ACE greatly reduced enzyme activity and affected domain-selectivity. Two fluorogenic substrates, designed specifically to assess ACE's mechanism of Aβ hydrolysis Aβ(4-10)Q and Aβ(4-10)Y, underwent endoproteolytic cleavage at the Asp7/Ser8 bond. The Aβ(4-10)Q peptide was a poor substrate of ACE but was N-selective, with a selectivity driven largely by interactions with the domain-specific residues of the S2 and S2' pockets. The selectivity of the S2' residues were confirmed with a similar, more physiological, fluorogenic Aβ(4-10)Y peptide. This work provides further understanding towards the substrate determinants of N-selectivity, highlighting the importance of the S2' Ser357. ACE C-domain hydrolysed Aβ(4-10)Y with modest efficiency compared to the other substrates, where hydrolysis under the same conditions did not occur. Moreover, Aβ(4-10)Y also displayed N-domain selectivity. In contrast to Aβ(1-16) and Aβ(410)Q, both sACE and the double C-domain (CC-sACE) construct showed positive domain cooperativity towards Aβ(4-10)Y. The high-resolution crystal structures of the N-domain in complex with five Aβ peptide fragments provided an overlapping, conserved, molecular mechanism of peptide binding and evidence of the enzyme's broad exoprotease activity. In addition to the kinetic and structural studies, ACE's signalling response to the N-selective Aβ(1-16) and Aβ(1-42) was investigated using immunodetection and mass spectrometry. Similar to the ACE inhibitor lisinopril, the Aβ peptides elicited ACE signalling by phosphorylation of the cytoplasmic Ser1270 residue and JNK activation. The signalling response of ACE was coupled to increased ACE activity an d expression on treatment with Aβ(1-42). These studies allowed us to rationalise the increased ACE activity and expression found in AD, may arise through direct interactions with Aβ. This work provides a kinetic, structural and mechanistic understanding of the selective cleavage of Aβ by the N and C catalytic sites of ACE. Due to the broad substrate specificity of the two domains of ACE, and the overarching N- selectivity of Aβ hydrolysis, these findings provide rationale for further in vivo pharmacological studies on the mechanism of action C- domain-selective inhibitors, in the context of AD.
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Chi, Ru-pin Alicia. "Investigating a novel small molecule inhibitor of nuclear import as an anti-cancer approach." Doctoral thesis, University of Cape Town, 2016. http://hdl.handle.net/11427/22896.
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The identification of novel cancer-associated biomarkers against which drugs can be developed is anticipated to be beneficial in multiple ways; including their use as monotherapies and in combination with current chemotherapeutic agents for improved anti-cancer treatment outcome. Recently, research in our own laboratory and others have reported elevated expression of the nuclear transporter Kpnβ1 in multiple cancers. Using the cervical cancer model, we showed that its inhibition using small-interfering RNA (siRNA) resulted in cancer cell death via apoptosis while sparing normal cells, suggesting it has potential as a target for anti-cancer therapy. An in silico screen for Kpnβ1 inhibitors identified several small molecules that showed inhibitory effects on nuclear import as well as cancer killing activity. In this study, we aimed to examine the potential of one such small molecule, the Inhibitor of Nuclear Import-43 (INI-43) as a lead compound with anti-cancer activities using multiple cancer models. Through culture-based in vitro assays, we demonstrated that INI-43 inhibited the proliferation of cancer cells grown anchorage-dependently and independently. These effects were similarly observed in Kpnβ1 knock-down cells, and Kpnβ1 over-expression was able to partially reverse these effects, suggesting that the anti-cancer effects of INI-43 is mediated through interference of the Kpnβ1 function. Toxicology studies and liver microsomal assay showed that INI-43 has an acceptable toxicity profile in nude mice and is metabolically stable, allowing its use in in vivo testing. Intraperitoneal administration of INI-43 significantly reduced the growth of subcutaneously xenografted cervical and oesophageal tumour cells in nude mice, supporting its anti-cancer activity in vivo. To examine the potential of using INI-43 in combination therapy, we examined the effects of the combined treatment of INI-43 and Cisplatin (CDDP), a first-line chemotherapeutic agent used in the treatment of many cancers. INI-43 treatment at sub-lethal concentrations enhanced cancer cells' sensitivity to CDDP, which was similarly observed in Kpnβ1 knock-down cells. Using an ovarian cancer model, we demonstrated that CDDP treatment led to elevated expression and nuclear localization of Kpnβ1, suggesting that Kpnβ1 is involved in CDDP-induced stress response. INI-43 treatment impeded the CDDP-induced nuclear accumulation of Kpnβ1 which correlated with increased cell death, suggesting that nuclear localization of Kpnβ1 may be important for ovarian cancer cell survival when challenged with genotoxins such as CDDP. Using the cervical cancer model, we demonstrated that INI-43 enhanced CDDP-induced cell death synergistically, and that the enhanced cell death is mediated through stabilizing p53 protein. This associated with decreased levels of Myeloid Cell Leukemia 1 (Mcl-1), an anti-apoptotic factor negatively regulated by p53. Furthermore, INI-43 treatment reduced the nuclear import of NFκB, a stress-regulated response known to promote cancer cell survival. Decreased levels of various downstream pro-survival and DNA-repair targets of NFκB were observed, including cyclinD1, c-Myc and X-Linked Inhibitor of Apoptosis Protein (XIAP), which correlated with increased DNA damage and apoptosis. Taken together, we show that nuclear import inhibition using small molecules could have therapeutic benefits in the treatment of cancer, and that INI-43 is a promising candidate for further development to be used in anti-cancer monotherapy or combination chemotherapy.
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Woodman, Zenda. "Characterisation of the ectodomain shedding of angiotensin-converting enzyme." Doctoral thesis, University of Cape Town, 2003. http://hdl.handle.net/11427/3156.
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Zemanay, Widaad. "Altered protein expression patterns in oesophageal cancer." Doctoral thesis, University of Cape Town, 2009. http://hdl.handle.net/11427/3157.
Full textAbstract:
Includes abstract.Includes bibliographical references (leaves 125-143).
Oesophageal squamous cell carcinoma presents a significant health burden in South Africa. It is one of the most common causes of cancer-related mortality of South African black males, as a result of its asymptomatic progression leading to late diagnosis and poor prognosis. The aim of this study was to identify membrane or membrane-associated proteins that are expressed at different levels in oesophageal tumour tissue when compared to normal tissue. The identification of such proteins would be an important step towards the development of better diagnostic and therapeutic strategies for this disease. Two proteomic approaches, were employed to identify differentially expressed proteins.
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Douglas, Ross Gavin. "Significance of active site residues in the n-domain selectivity of angiotensin-converting enzyme." Doctoral thesis, University of Cape Town, 2011. http://hdl.handle.net/11427/11786.
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Angiotensin-converting enzyme (ACE) is a zinc metallopeptidase that plays an important role in vascular function; with ACE inhibitors being clinically utilised in the treatment of cardiovascular disease and diabetic nephropathy. Somatic ACE consists of two homologous catalytically active domains (designated N- and C-domains) that share high overall sequence identity and structural topology. Despite the high degree of similarity between domains, each domain displays differences in substrate processing and inhibitor binding abilities. This suggests that active site residues differing between the two domains could provide unique interactions within the N-domain that allow for N-selective binding and processing. Literature reports of ACE crystal structures and studies with substrate and inhibitor analogues have implicated unique residues present in the S2 and S2' subsites in providing important interactions for N-selectivity.
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Duarte, Jessica Da Gama. "Proteomic studies on patient responses to chemotherapy, radiotherapy and immunotherapy in cancers." Doctoral thesis, University of Cape Town, 2015. http://hdl.handle.net/11427/20261.
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There is increasing evidence that the aberrant expression of cancer-testis (CT) antigens - a family of ca. 150 proteins that are both autoimmunogenic and mainly restricted to tumours in various types of human cancers - makes them attractive immunotherapy targets, as well as possible cancer diagnostic markers. We carried out a retrospective serological study of primary and secondary autoimmune responses of various cohorts of cancer patients prior to and/or following a variety of distinct treatments (chemotherapy, radiotherapy and immunotherapy), using a large number of archived human serum samples. Our goals were to develop and validate a novel cancer-testis and -associated antigen microarray platform and to then explore its utility and general applicability in the cancer immunology field. In addition, we sought to cross-correlate our protein microarray data from specific cohorts with in vitro T-cell re-stimulation assays for a selected subset of patients. Furthermore, as a means of determining the biological significance of our protein microarray data, we also collected clinical patient data where possible. The underlying hypothesis of our study was that there were measurable differences in autoantibody repertoires towards tumour-specific and -associated antigens between pre- and post-treated cancer patient samples (using various trial therapies), potentially augmented by prior chemo- or radiotherapy, which would correlate with likelihood of response of individual patients to a given therapeutic treatment - including those treatments that aim to generate T-cell responses - and which would also correlate with the nature and extent of individual patient responses to treatment.
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Ebrahim, Roshan. "Biogenesis of lysosomes in macrophages : intracellular pathway of lysosomal membrane protein to lysosomes." Doctoral thesis, University of Cape Town, 2008. http://hdl.handle.net/11427/3126.
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Van, der Watt Pauline Janet. "Expression and regulation of the nuclear transport proteins, Crm1 and Kpnß1, in cervical cancer and transformed cells." Doctoral thesis, University of Cape Town, 2009. http://hdl.handle.net/11427/3152.
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Faurholm, Bjarne. "Gene structure, transcripts and transcriptional regulation of primate type II gonadotropin-releasing hormone receptors." Doctoral thesis, University of Cape Town, 2004. http://hdl.handle.net/11427/3127.
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Rose, Beverley Ann. "The regulation of type I collagen gene expression in stromal fibroblast by breast tumour cells." Doctoral thesis, University of Cape Town, 2011. http://hdl.handle.net/11427/10861.
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Includes abstract.Includes bibliographical references (leaves 110-135).
Recent studies have revealed that interactions between tumour cells and the surrounding stroma play an important role in facilitating tumour growth and invasion. Stromal fibroblasts produce most of the extracellular matrix (ECM) components found in the stroma, including type I collagen. Previous in vivo studies in our laboratory have shown that type I collagen mRNA levels are decreased in stage II and III breast tumour tissue compared to adjacent normal tissue.
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Anthony, Colin Scott. "The importance of N-linked glycosylation on the N-domain of angiotensin-I converting enzyme." Doctoral thesis, University of Cape Town, 2011. http://hdl.handle.net/11427/10051.
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Angiotensin-I converting enzyme (ACE) is an important drug target in the treatment of heart disease due to its role in the regulation of blood pressure. ACE contains two domains, the N- and C-domains, both of which are catalytically active and heavily glycosylated. Glycosylation is one of the most important forms of post-translational modification, having a wide range of functions including protein folding, modulation of the immune response, and providing targeting signals. Glycosylation is required for the expression of active ACE and structural studies of ACE have been fraught with severe difficulties because of surface N-glycosylation of the protein. This problem has been addressed to a large extent with respect to the C-domain, where the role of glycosylation has been extensively characterised and a minimally glycosylated form was able to crystallise reproducibly. As yet, little is known about the degree and importance of N-linked glycosylation on the N-domain. The generation of minimally glycosylated N-domain, however, requires a greater understanding of the relative importance of the individual N-linked glycosylation sites.
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Lokanga, Rachel Adihe. "Somatic expansion of premutation alleles and the role of the mismatch repair and base excision repair proteins on repeat expansion in a mouse model of the fragile X-related disorders." Doctoral thesis, University of Cape Town, 2016. http://hdl.handle.net/11427/20853.
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The Fragile X-related disorders arise from an unusual mutation in the X-linked FMR1 gene. The mutation involves expansion, or an increase in the number of repeats, in a CGG•CCG repeat tract located in its 5' untranslated region. FMR1 alleles carrying 55-200 repeats are called Premutation (PM) alleles, and cause Fragile X associated tremor/ataxia syndrome (FXTAS) and Fragile X-associated primary ovarian insufficiency (FXPOI). FMR1 alleles having more than 200 repeats are referred to as full mutation (FM) alleles and cause Fragile X syndrome (FXS). These different alleles arise by intergenerational expansion of the repeat tract from smaller unstable alleles by a mechanism that is unknown. We have shown that in addition to germ line expansion, somatic expansion also occurs in a human cell line in vivo and in a FX PM mouse model. In the mouse model, we found that the extent of somatic instability is dependent on age, gender and tissue. Specifically, organs such as brain, liver and gonads are susceptible to expand more than heart and kidney and expansion is much more frequent in males than in females. No differences were found between male and female mice in the levels of the DNA repair proteins that had already been implicated in repeat expansion in model systems of other disorders thought to arise via a similar mechanism. Neither were there any differences between males and females in the amounts of proteins produced from X-linked DNA repair genes. We also showed that estrogen did not protect against expansion. However, we found that PM alleles expanded exclusively when they were located on the active X chromosome. Thus some of the differences between males and xii females in the level of somatic expansion might be due to the fact that females undergo X inactivation and thus have the PM allele on the inactive X chromosome in half (~50%) of their cells. It also indicates that transcription and/or an open chromatin configuration is required for expansion in the FX PM mouse.
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Watermeyer, Jean Margaret. "Structural determinants of the domain-selectivity of novel inhibitors of human testis angiotensin-converting enzyme." Doctoral thesis, University of Cape Town, 2008. http://hdl.handle.net/11427/3154.
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Sales, Kurt Jason. "Expression and functional role of cyclooxygenase enzymes in cervical carcinoma." Doctoral thesis, University of Cape Town, 2001. http://hdl.handle.net/11427/3149.
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Bibliography: leaves 133-156.Cervical cancer is considered an important clinical problem in sub-Saharan Africa. Recent studies have suggested that epithelial tumors may be regulated by cyclooxygenase enzyme products. The purpose of this thesis was to determine the expression, localisation and possible functional role of cyclooxygenase enzymes in cervical carcinomas. The initial aim of the study was to determine whether cyclooxygenase-1 and cyclooxygenase-2 expession and prostglandin E₂ synthesis are up-regulated in cervical cancers. Real-time quantitative reverse-transcriptase polymerase chain reaction and Western blot analysis confirmed cyclooxygenase-1 and cyclooxygenase-2 ribonucleic acid and protein expression in all cases of squamous cell carcinoma and adenocarcinoma investigated. In contrast, minimal expression of cyclooxygenase-1 or cyclooxygenase-2 was detected in histologically normal cervix. Immunohistochemical analyses localised the site of cyclooxygenase-1 and cyclooxygenase-2 expression and prostaglandin E₂ synthesis to neoplastic epithelial cells of all squamous cell carcinomas and adenocarcinomas studied.
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Siyo, Vuyolwethu Penelope. "Molecular mechanisms involved in the anticancer activity of BISPMB in oesophageal cancer cells." Doctoral thesis, University of Cape Town, 2016. http://hdl.handle.net/11427/20422.
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BisPMB (E, Z)-1,8-(Bis-p-methoxyphenyl)-2,3,7-trithiaocta-4-ene 7-oxide) is a synthetic analogue of the garlic compound ajoene. It is 12 times more active at inhibiting the growth of oesophageal squamous cell carcinoma WHCO1 cells and displays selectivity for cancer cells over normal cells. BisPMB is therefore attractive as a potential cancer therapeutic. In this study, bisPMB was found to inhibit WHCO1 cancer cell proliferation in a time and concentration dependent manner with 24 hour IC50's between 6.7 - 8.1 μM against a range of oesophageal cancer cell lines including WHCO1, KYSE30 and WHCO6. The normal oesophageal epithelial cell line, HET1A was found to be five times less responsive to bisPMB. Furthermore, bisPMB was found to induce apoptosis and G2/M cell cycle arrest in WHCO1 cells. Gene expression data obtained from the microarray analysis showed that bisPMB primarily targets the unfolded protein response (UPR) in WHCO1 cells. We also found that bisPMB deregulated the ER stress genes involved in protein processing in the endoplasmic reticulum and also deregulated MAPK pathways in WHCO1 cells. At a protein level, bisPMB was found to induce an increase in protein ubiquitination and in the expression of ER stress and UPR genes ATF4, Grp78 and CHOP in WHCO1 cells. We also observed a decrease in ATF6 90 kDa protein and transient XBP-1 mRNA splicing. The activation of p38, JNK and ERK MAPK pathways in bisPMB treated WHCO1 cells was also observed. Furthermore siRNA mediated knock-down of CHOP abolished the anti-proliferative effect of bisPMB in WHCO1 cells. However, inhibition of JNK and p38 MAPK by chemical inhibitors, SP600125 and SB 203580 respectively, had no effect on bisPMB antiproliferative activity against WHCO1 cells. On the other hand, inhibition of ERK1/2 MAPK by U0126 enhanced the anti-proliferative effect of bisPMB in WHCO1 cells. These results support the hypothesis that ER stress and MAPK signalling pathways are essential for bisPMB induced cytotoxicity in oesophageal cancer cells.
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Li, Dong-Ping. "Genetic polymorphisms in the drug metabolizing genes and their roles in the development of oesophageal cancer." Doctoral thesis, University of Cape Town, 2008. http://hdl.handle.net/11427/3137.
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Includes bibliographical references (leaves 109-130).Although the incidence and mortality due to the oesophageal squamous cell carcinoma (OSCC) in Black South Africans is extremely high, very little is known about the aetiology and molecular biology of the disease. In order to make a contribution to the understanding to the causes of this disease we investigated the role of the polymorphisms in the genes coding for the cytochrome P450 (CYP1A1, CYP1A2, CYP1B1, CYP2E1), sulphotransferase 1A1 (SULT1A1), glutathione S-transferases (GSTT1. GSTM1 and GSTP1) alcohol dehydrogenase (ADH2 and ADH3) and aldehyde dehydrogenase (ALDH2) because the products of these genes are involved in the metabolism or biotransformation of harmful compounds.
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Shunmoogam-Gounden, Nelusha. "An investigation into the molecular mechanisms induced by derivatives of natural products in oesophageal cancer." Doctoral thesis, University of Cape Town, 2014. http://hdl.handle.net/11427/13237.
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Includes bibliographical references.Current chemotherapies for oesophageal cancer display poor efficacy and tolerability, highlighting an unmet need for novel chemotherapeutic agents. Artemisinin derivatives, currently used to treat malaria, were recently shown to possess potent anticancer activity. This study investigated the potential of two first generation artemisinin derivatives (artesunate and dihydroartemisinin), together with novel artemisinin hybrid compounds, as cancer chemotherapeutic agents and explored the mechanism of action in oesophageal cancer. Artesunate and dihydroartemisinin including seventeen other artemisinin derivatives were screened against oesophageal cancer cells using the 3 - [4,5-dimethylthiazol-2 -yl]-2,5 - diphenyltetrazolium bromide (MTT) assay and GraphPad Prism Software to calculate IC 50 (50% inhibitory concentration) values. Novel halogenated artemisinin - isatin hybrid compounds displayed the best activity against oesophageal cancer cells, and were more potent than artesunate and dihydroartemisinin in a small panel of oesophageal, breast and cervical cancer cell lines tested. The novel derivatives induced a G0/ G1 cell cycle arrest whilst the parental compounds induced a G2/ M block of the cell cycle, using flow cytometry. This suggested a different mechanism of action for the novel compounds. Dihydroartemisinin and the most active novel hybrid, EXP57EA, were investigated to understand their molecular mechanisms of action in oesophageal cancer.
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Moorad, Razia. "Computer-aided drug design and the biological evaluation of anti-cancer drugs." Doctoral thesis, University of Cape Town, 2016. http://hdl.handle.net/11427/20715.
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Computer-aided drug design has become a promising alternative to high-throughput screening by identifying potential hits in silico for in vitro evaluation. In this study a combination of ligand-based and structure-based virtual screening was performed to identify in silico hits. This was based on finding similar inhibitors to 6-amino-4-(4-phenoxyphenylethylamino) quinazoline, a potent inhibitor of the Nuclear Factor kappa B (NF-κB), a transcription factor that has a pivotal role in cancer survival and Pentamidine, an anti-parasitic drug that has recently been demonstrated to possess tumour-killing activity. A hierarchical methodology consisting of a similarity search followed by structure-based virtual screening of the ZINC database was performed. In order to perform the docking studies, binding sites for 6-amino-4-(4-phenoxyphenylethylamino) quinazoline on the NF-κB/IκBα complex were identified through blind docking. In addition, the National Cancer Institute (NCI) database was screened, utilising existing structure-activity relationship data from literature. A pharmacophore search was designed to test the hypothesis of the structural features necessary for activity as seen with quinazoline inhibitors of NF-κB. No virtual hits from the ZINC database were confirmed with in vitro activity. On the other hand, three compounds identified from the pharmacophore search were confirmed to inhibit cancer cell proliferation in vitro, with compound NSC727152 demonstrating the most potent activity. In order to determine if NSC727152 acted similarly to 6-amino-4-(4-phenoxyphenylethylamino) quinazoline by inhibiting NF-κB, the effects of NSC727152 on the expression of NF-κB targeted genes, including the Growth Arrest and DNA Damage 45 (GADD45) α and γ and the Interleukin 6 (IL-6) genes were evaluated. GADD45 α and γ have been shown to be regulated by NF-κB during cancer progression and aberrant IL-6 gene expression has been implicated in cancer progression and mortality and its expression is at least partially mediated via constitutive activation of NF-κB. In this study, it has been demonstrated that GADD45 α and γ are upregulated after treatment with NSC727152. A down-regulation of the IL-6 promoter activity and mRNA expression in cancer cells treated with NSC727152 has also been demonstrated in this study. However, no hits similar to Pentamidine were confirmed with in vitro activity. In conclusion, the compound NSC727152 has been shown to inhibit NF-κB and further analysis is necessary to determine its full potential as an NF-κB inhibitor.
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Strydom, Erin. "Investigating Karyopherin B1: small molecule interactions for cancer therapy." Doctoral thesis, University of Cape Town, 2016. http://hdl.handle.net/11427/22897.
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The advent of gene expression profiling studies has allowed for the identification of genes with potential as disease markers and therapeutic targets. Our laboratory identified the eukaryotic nuclear importer protein Karyopherin B1 (KpnB1), to be up-regulated in different cancer cell lines, including cervical and oesophageal as well as transformed cells. Inhibition of KpnB1 in these cells using small interfering RNA (siRNA) resulted in significant cancer cell death via apoptosis, suggesting KpnB1 is essential for cancer cell survival. Within our laboratory, we established that candidate small molecules targeted against KpnB1 identified using a rational drug design approach. The outcome of this research is for examine inhibitors of KpnB1 for potential as future anti-cancer agents using. Based on the long-term goal of this research, this particular project was aimed at investigating a small molecule inhibitor identified in our laboratory, known as Inhibitor of Nuclear Import-43 (INI-43) for its potential to bind to the nuclear importer, KpnB1. Using conventional assays as well as cutting edged techniques including circular dichrosim (CD) and isothermal titration calorimeter (ITC), an examination of INI-43 and its interactions with KpnB1 was made. In vitro analysis showed that INI-43 exhibits cytotoxic effects on cervical cancer cells with an IC₅₀ of ≈10μM and induces apoptotic cell death. The NFAT dual luciferase assay measured nuclear import of KpnB1 associated proteins, showing that INI-43 inhibits nuclear import/activity of NFAT in a dose dependent manner. Confocal microscopy of exogenous FRFP-KpnB1 as well as endogenous KpnB1 in the presence of INI-43 showed a change in the localisation of KpnB1 upon drug treatment. Both FRFP-KpnB1 and endogenous KpnB1 appear to be prevented from entering the nucleus, and is retained in the peri-nuclear space and the cytoplasm suggesting that INI-43 inhibits KpnB1 movement into the nucleus. To investigate KpnB1-INI-43 interactions, purified KpnB1 was prepared and used in biophysical techniques. Purified KpnB1 protein was prepared using GST-tagged purification methods and the tagged protein confirmed by mass spectrometry. Purified GST-KpnB1 was used in drug binding studies including circular dichrosim (CD) isothermal titration calorimetry (ITC). CD showed a drug concentration dependant shift in the spectra at around 233nm, indicative of drug protein interaction possibly occurring in a region of KpnB1 containing aromatic amino acids. The purified GST-KpnB1 was used in ITC, which confirmed an interaction between KpnB1 and INI-43, a relatively weak interaction. In conclusion, our data shows that the small molecule, INI-43 kills cancer cells, likely by interfering with KpnB1 associated nuclear import pathways. We show that INI-43 interferes with the nuclear localisation of KpnB1 itself and biophysical assays provide evidence for possible KpnB1-INI-34 interactions. Small molecules such as INI-43 present as promising tools to studying the potential of KpnB1 as an anticancer target.
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Fromme, Bernhard Johannes. "The role of extracellular loop three of the human gonadotropin releasing hormone receptor in ligand selectivity." Doctoral thesis, University of Cape Town, 2001. http://hdl.handle.net/11427/3129.
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Bibliography: leaves 123-146.The hypothalamic neuropeptide, gonadotropin releasing hormone (GnRH) perferentially inteacts with GnRH type I receptors on the gonadotropes in the anterior pituitary. Activation of the GnRH receptor is required for the biosynthesis and release of luteinizing hormone (LH) and follicle stimulating hormone (FSH), which regulate reproductive function. Multiple forms of GnRH are present in most vertebrate species and are thought to have physiological functions in addition to regulating pituitary hormone release. The mammalian type I GnRH receptor is proposed to discriminate between endogenous forms of GnRH (King and Millar, 1995). In this thesis the mechanism of the GnRH selectivity by a mammalian type I GnRH receptor is examined at molecular level and previous hypotheses are re-evaluated.
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Folefoc, Asongna Theresia Forkem. "Functional consequences of South African mutations of the HIV-1 co-receptor, CCR5." Doctoral thesis, University of Cape Town, 2007. http://hdl.handle.net/11427/3128.
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Includes bibliographical references (p. 100-119).Four mutations of the CCR5 receptor have been identified in the South African population, but the effects of these mutations on CCR5 function and HIV infection are unknown. We have used in vitro methods to assess the ffect of the mutations, Asp2Val, Leu107Phe, Arg225Gln and Arg225stop, on CCR5 interactions with chemokine ligands and HIV.
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Matsha, Tandi Edith. "Human Papillomaviruses in oesophageal cancer." Doctoral thesis, University of Cape Town, 2003. http://hdl.handle.net/11427/3140.
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Bracher, Jacqueline Claire. "Expression and regulation of N-Myc Downstream- Regulated gene 1 in squamous cell carcinoma of the oesphagus." Doctoral thesis, University of Cape Town, 2009. http://hdl.handle.net/11427/3120.
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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.
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Wei, Wei. "Functional analysis of N-MYC downstream regulated gene 1 (NDRG1) in Oesophageal squamous cell carcinoma." Doctoral thesis, University of Cape Town, 2009. http://hdl.handle.net/11427/3155.
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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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Whibley, Catherine Evelyn. "South African marine compounds as anticancer agents." Doctoral thesis, University of Cape Town, 2006. http://hdl.handle.net/11427/8393.
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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.
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Phillips, Pumza Samantha. "The role of Gai in the Gonadotropin-releasing hormone (GnRH) receptor inhibition of cell proliferation." Master's thesis, University of Cape Town, 2011. http://hdl.handle.net/11427/11787.
Full textAbstract:
Includes abstract.Includes bibliographical references (leaves 72-78).
The activation of Gonadotropin-releasing hormone receptor (GnRHR) by the GnRH ligand has been shown to mediate antiproliferative effects in extra-pituitary cells and in reproductive cancer cell lines. The GnRHR couples to Gαq in pituitary gonadotropes. However, the GnRHR expressed in reproductive cancer cell lines is thought to couple to Gαi. Recent evidence also suggests that the antiproliferative effects may be mediated via Gαq in these cells. Therefore our study involved determining the role of Gαi in the antiproliferative effects mediated by the GnRHR. The results suggest that the Gαi pathway could play a role in mediating the antiproliferative effects of GnRH.
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Nair, Omesan. "Functional effects of cytochrome P450 variants on drug metabolism and adverse drug reactions: developing and extending high throughput P450 protein technology platforms." Master's thesis, University of Cape Town, 2014. http://hdl.handle.net/11427/13238.
Full textAbstract:
Includes bibliographical references.Cytochrome P450 (CYPs) are a superfamily of heme containing enzymes that catalyse a diverse range of biological reactions. They are responsible for over 80% of primary metabolism of currently available drugs and are therefore central to its medical importance. Investigating the effects of these enzymes on drugs by metabolite detection and kinetic studies is a step forward to the vision of personalised medicine. The enzyme family is known to be associated with the development of adverse drug reactions which are usually only discovered in late stages of drug development, therefore screening for potential adverse drug reactions earlier on would aid minimising such adverse events occurring. There is therefore a need to analyse the interaction profile of new drugs with CYPs in a cost effective and high throughput manner for early stage screening, since drug discovery efforts tend to utilise large compound libraries. Recently, a novel functional CYP microarray has been developed in the Blackburn laboratory at UCT to enable label-dependent analysis of metabolism of substrates by the major CYP3A4 isoform in a high throughput manner. This thesis describes efforts involved in expanding the functional CYP microarray format to the other major CYP isoforms namely, CYP2C9 and CYP2D6 and developing a new immobilisation-free technology with label-free mass spectrometric identification and quantitation of metabolites formed. The goals of expansion of functional CYP microarrays were achieved by using microarray or confocal fluorescence scanning in conjunction with atomic force microscopy to more accurately quantitate active CYP3A4, CYP2C9 and CYP2D6 protein levels for catalytic substrate-dependent turnover rates. Finally the label- and immobilisation-free CYP technology was evaluated using probe substrates and a complex drug, rifampicin. These two platforms are primed to be a useful tool in pre-clinical drug screening for use in the drug discovery field by the academic, pharmaceutical and biotechnology industries.
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Ujma, Sylvia. "The role of surfaceant protein A in immunity to HPV16 pseudovirus infection." Master's thesis, University of Cape Town, 2018. http://hdl.handle.net/11427/29526.
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Infection by oncogenic human papillomavirus (HPV) is known to be the causative agent for the development of various anogenital cancers, including cervical cancer. Worldwide, the majority of cervical cancer cases occur in less developed regions, and while prophylactic vaccines exist to combat HPV infection, they are largely unattainable in these areas. Therefore, alternative preventative measures against HPV infection are needed to help eradicate cervical cancer over time. Since HPV employs multiple mechanisms to evade the host immune response, a proposed method for preventing infection may be by enhancing HPV recognition by the immune system. Surfactant proteins A and D (SP-A and SP-D) are innate immune proteins with a variety of functions including recognition and opsonisation of pathogens. They are primarily found in the lung, but have also been shown to be expressed at other sites of the body, including the female reproductive tract. It was hypothesised that SP-A and/or SP-D may enhance immune recognition of HPV, thereby preventing infection. To assess this hypothesis, co-immunoprecipitation and flow cytometry experiments were performed to determine whether SP-A and/or SP-D bind to HPV16 pseudovirions (HPV16- PsVs). SP-A was shown to bind to HPV16-PsVs as well as enhance viral uptake by RAW264.7 murine macrophages, while SP-D bound HPV16-PsVs weakly and had no effect on viral uptake. To confirm these observations and to assess whether SP-A had an effect on HPV16- PsVs infection in vivo, a well-established, but not yet available murine HPV16-PsVs cervicovaginal challenge model system was set up at UCT. It was determined that neither naïve nor C57BL/6 mice challenged with HPV16-PsVs expressed SP-A in the female genital tract. However, under the experimental conditions established herein, pre-incubation of HPV16-PsVs with purified SP-A at a 1:10 weight per weight ratio resulted in a reduction in infection. This study is the first to describe a biochemical and functional association of HPV16 virions with the innate immune molecule SP-A. In the long term, these observations may contribute to the development of topical microbicides incorporating recombinant fragments of SP-A to reduce the burden of new HPV infections.
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Mayevu, Ntateko Merriam Immogen. "The contribution of His7.36(305) of the GnRH receptor to ligand binding and receptor activation." Master's thesis, University of Cape Town, 2004. http://hdl.handle.net/11427/3141.
Full textAbstract:
Bibliography: leaves 88-104.The current study was perfonned to give insights into the role of individual amino acid residues of both the GnRH receptor and the GnRH ligand in receptor function. A His7.36(3o5) residue on transmembrane helix seven of the GnRH receptor was investigated for its contribution to the overall function of the receptor.
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Wagiet, Mateen. "Modulating ADAM-10 activity and expression in cervical and oesophageal cancer cells." Master's thesis, University of Cape Town, 2016. http://hdl.handle.net/11427/22732.
Full textAbstract:
The ADAMs (A Disintegrin And Metalloproteinase) is a family of transmembrane and secreted proteins essential in cellular fate determination, wound healing, cell migration, proliferation and angiogenesis. Previous studies have linked a range of ADAMs, which include ADAM10 to cancer development and progression. Research in our laboratory found endogenous ADAM10 levels to be higher in both oesophageal and cervical cancer cell lines. Reports in the literature have highlighted a correlation between high levels of ADAM10 expression with that of cancer cell biology; hence ADAM10 shows promise as an anti-cancer target. The aim of this study was to modulate ADAM10 activity in oesophageal and cervical cancer cell lines using the small molecule inhibitor GI254023X as well as previously undescribed two molecules generated en route to synthesizing GI254023X, namely SN-254 and SN-311. A CX₃CL1 ELISA functional assay as an indicator of ADAM10 activity showed a decrease in CX₃CL1 cleavage after treatment with GI254023X, SN-311 and SN-254 suggesting that all three compounds substantially inhibited ADAM10 activity. The effects of these compounds on the cell biology of WHCO5 oesophageal and HeLa cervical cancer cells were monitored. Our data shows that GI254023X, SN-254 and SN-311 inhibit oesophageal and cervical cancer cell proliferation, and cause cell death via apoptosis as observed by PARP cleavage, and elevated Caspase 3/7 activity. Drug treatment also resulted in an increase in cellular adhesion as well as a significant decrease in the invasion and migration of WHC05 and HeLa cells. The effect of ADAM10 inhibition on typical markers of the epithelial to mesenchymal transition state was also examined. An increase in epithelial cell markers (E-Cadherin, B-Catenin) and a decrease in mesenchymal marker expression (Vimentin) post treatment with the compounds tested strongly suggested that ADAM10 plays a role in mesenchymal cell transition. These results suggest that ADAM10 activity is necessary for the biological phenotypes that associate with cervical and oesophageal cancer cells and that targeting ADAM10 with inhibitors have potential as anticancer therapies.