Browsing by Author "Kidson, Susan"

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    Open Access
    Birds of a white feather : a congenital hypopigmentary disorder in the chick
    (1999) Koubovec, Dominique Johanna; Kidson, Susan
    Dominant white, a mutation of the I gene, leads to amelanosis and is common to many commercial breeds of fowl, including Pile Games, White Plymouth Rocks and White Leghorns. Despite much investigation on the cellular mechanisms of Dominant white, its mode of action is still poorly understood. The aim of this study is to elucidate the molecular basis of amelanosis in WPR X PG chickens by addressing the following two questions: are melanocytes present in the skin regions and feather follicles in normal numbers of 8- to 13-day white chick embryos? If melanocytes are present in normal numbers, are they unable to synthesise pigment because of a defect in melanocyte differentiation? Two approaches were used to answer the first question. MelEM, a monoclonal antibody, shown to react specifically to quail melanocytes, was found to be unsuitable for localisation of chicken melanocytes. Secondly, in situ hybridisation with tyrosinase and tyrosinase related protein-2 (TYRP2) probes was carried out to quantitate the number of melanocytes at different stages of development. The results indicate that tyrosinase - and TYRP2 -expressing melanocytes are present in 10-day white chick skin and feather buds in normal, if not greater numbers than in the control (Black Australorp) breed. This suggests that amelanosis is not due the failure of migratory melanoblasts to reach the developing feathers, nor is it due to the selective elimination of melanocytes during migration. The results further showed that with increasing developmental age (12- and 13-days), there is a decline in the number of tyrosinase - and TYRP2-expressing melanocytes in the white chick breed in comparison to the black breed. This suggests that white skin melanocytes either downregulate tyrosinase and TYRP2 gene expression yet remain viable, or they undergo cell death. At 17-days, the results showed an absence of gene expression in both the black and white follicles due to the normal process of feather development. Thus, although WPR x PG melanocytes are present in normal numbers in 10-day skin and feather follicles, they never melanise. To address this issue, black and white neural crest cells were cultured in conditions resembling their respective skin environments. Firstly, black neural crest cells grown in defined medium with either black or white skin extract were able to synthesise melanin. This suggests that white skin contains the appropriate signals necessary to induce melanogenesis of black melanocytes. This in turn suggests that the white melanocyte itself is intrinsically defective. To test this, white chick neural crest cells were grown in defined medium in the presence of black or white skin extract. The results showed that white cells were able to respond to signals in extracts of skin from both breeds and became melanised, suggesting that white melanocytes are not intrinsically defective. Due to the intricate nature of this study and subsequent experimental limitations encountered, these contradictory results could not be completely resolved. However, a testable model in which the I gene is postulated to encode c-kit is presented.
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    The detection of occult metastatic disease in patients with cutaneous melanoma
    (1999) Hanekom Gideon S; Kidson, Susan
    The ability to identify melanoma patients with progressive disease is central to efficient management. The challenge therefore, is to develop prognostic markers and techniques which will allow the identification of those patients whom, at the time of primary tumor diagnosis, already have micrometastases (occult or clinically undetectable metastases). The use of the reverse transcription-PCR (RT-PCR) technique for the detection of circulating melanoma cells (CMCs) is potentially a powerful tool for identifying those patients at risk for developing metastases. The first aim of this study was to develop a more sensitive, reproducible, cost effective and clinically applicable assay and to eliminate the problem of false positives. A combined RT-PCR assay for tyrosinase mRNA, a marker specific for melanoma cells, was developed and tested. It was shown that the assay can reproducibly detect a single, viable melanoma cell in 10-15 ml of peripheral blood. Furthermore, a simple but effective procedure was developed to prevent carryover contamination. It was found that the chance of obtaining normal melanocyte contamination with the needle prick during blood sampling was only 2% and that illegitimate transcription does not contribute to sporadic false positives. The second aim of this study was to determine whether the early detection of CMCs is of any clinical value to monitor melanoma progression. Peripheral blood samples from 143 patients with primary melanoma (PM) were analysed by RT-PCR for the presence of tyrosinase mRNA. Seven percent (10/143) of the patients with PM had detectable CMCs. The percentage of PCR-positive patients was higher for stage II patients (9.0%) compared to stage I (5.3%) but the difference was not significant. A significantly higher percentage (P < 0.05) PCR-positive patients were found to have tumors greater than 1.5 mm thick and with ulceration present. Although this finding supports the notion that tumor thickness and ulceration are the two most significant prognostic factors, it was not possible at this stage, to link this directly to a poor prognosis since the majority of the PCR-positive patients have not yet (within four years) developed metastatic disease. However, the data does indicate that cells from tumors greater than 1.5 mm thick and with ulceration have a greater propensity to enter the circulation but that these cells do not necessarily have the ability to establish metastases. The results suggest that the detection rate of 9% for patients with stage II disease is much lower than would be expected, since 23.9% (16/67) of the stage II patients subsequently developed metastases. Of these 16 patients, only one was PCR-positive, one week before the metastases became clinically evident. Thus, the current technique fails to predict the likelihood of developing metastatic disease (P = 0.3485). The other nine PCR-positive patients had not yet developed metastases after a median follow-up period of four years. It is concluded that the current technique for the detection of CMCs is of limited clinical value to predict the likelihood of metastasis in patients with PM. It is suggested that other anatomic compartments, such as sentinel lymph nodes, should be explored for the identification of patients at risk for developing metastases. The third aim of this study was to determine whether high or low plasma levels and/or activity of plasminogen activator inhibitor type 1 (PAI1) correlate with the presence of metastatic disease in patients with melanoma. PAI1 is considered to be the main regulator of fibrinolytic activity in blood and has been identified as a key enzyme in the metastasis and vascularization of solid tumors. A unique enzyme-linked immunosorbant assay was developed to measure both the total amount of PAI1 in plasma as well as the active fraction of the inhibitor. This novel assay was then used to analyse and compare the plasmatic PAI1 levels and activity of a group of patients with advanced melanoma (AM) with a group of patients with primary disease and a control population. There was no statistical difference in the total plasmatic PAI1 levels between the controls and patients with PM and AM (P = 0.6199). In contrast, there was a significant difference in the active fraction of PAI1 between the controls and patients with PM or AM (P = 0.0076). A value of less than 44% active PAI1 was shown to be clinically meaningful by linear discriminant analysis. This means that a melanoma patient with a plasmatic PAI1 activity value less than 44% will have a 50% chance of harbouring metastases. Of the patients with PM, 19% had PAI1 activity values less than 44%, which strongly supports further investigations to determine whether plasmatic PAI1 activity levels might be predictive of metastatic disease. The false positive rate was 2.6%. It is speculated that this reduction in the active fraction of PAI1 for patients with AM might be attributed to tumor-derived tissue plasminogen activator and/or other melanoma-derived proteases or factors. The last section of this study describes several monoclonal antibodies (Mabs) that were developed against PAI1 in order to obtain useful reagents to study the regulatory functions of PAI1 in the metastasis and vascularization of solid tumors. The baculovirus expression system was used to express human PAI1 in insect cells and the crude infected cell population was used as the immunogen in mice. This approach was followed since the Escherichia coli-derived recombinant molecule elicited a poor immune response. A unique panel of anti-PAI1 Mabs was developed that were characterized with regard to their use for immunoblotting, immunofluorescence and immunocytochemistry. One of these antibodies blocked the binding of PAI1 to vitronectin and inhibited the activity of the inhibitor. Finally, two of these Mabs turned out to be extremely valuable and were used to develop a novel microtiter plate assay for measuring the active fraction of PAI1 in biological fluids by making use of Mabs against different epitopes of PAI1.
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    Generating a proteomic profile of neurogenesis, through the use of human foetal neural stem cells
    (2019) Garnett, Shaun; Blackburn, Jonathan; Kidson, Susan
    Introduction Neurogenesis, the development of new neurons, starts soon after the formation of the neural tube and is largely completed by birth. Development of the brain after birth is mainly reliant on the formation of new connections between surviving neurons. However, adult neurogenesis does continue in the subgranular zone of the hippocampus from quiescent adult neural stem cells. Traditionally neural stem cells were cultured as neurospheres, a heterogeneous agglomeration of neural cells at various stages of differentiation. This heterogeneity prevented accurate quantitative analysis. In 2008 Sun et al produced the first non-immortalised human foetal neural stem (NS) cell line from nine week old human foetal cortex. These cells are cultured as monolayers, have a radial glia like appearance, self renew and form all three neural cell types, neurons, astrocytes and oligodendrocytes upon differentiation. More recently human foetal neuroepithelial like (NES) stem cells have been produced from five week old human foetal hind-brain, they resemble neuroepithelial cells, with characteristic rosettes, upon differentiation they appear to form a pure population of neurons. These homogeneous monolayer cultures enable quantitative proteomic analysis, to increase our understanding of early brain development Methods Three NES and two NS cell lines were available for analysis. They proliferate by stimulation from FGF and EGF, removal of these growth factors results in spontaneous differentiation. Proliferating NES and NS cells were compared using SILAC labelling. In addition, each cell line was differentiated for 12 days, 6 timepoints were taken and compared using label free quantitation. Results 4677 proteins were quantitated with 473 differentially expressed, revealing fundamental differences between NES and NS cells. NES cells are less differentiated, expressing SOX2 and LIN28, have active cell cycle processes, DNA elongation, histone modification and miRNA mediated gene silencing. Whereas NS cells are more developmentally defined, express multiple membrane proteins, have activated focal adhesion, thereby increasing their binding and interaction with their environment. NS metabolism is more oxidative, utilises lipid metabolism, the pentose phosphate pathway and produces creatine phosphate. Upon differentiation the cell cycle processes are downregulated and neurogenic and gliogenic processes increased. Conclusion This work represent a detailed in vitro characterisation of non immortalised human foetal neural stem cells, it describes the regulatory, metabolic and structural changes occurring within neural stem cells in early brain development. The information herein points towards de-differentiation potentially through LIN28-let7, as a means to produce more neurogenic neural stem cells in vitro thus aiding regenerative therapies, as well as provides a wealth of information for better understanding neurological developmental disorders.
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    An in vitro investigation of the effect of Khellin and UVA (KUVA) on normal and transformed human melanocytes
    (1999) Carlie, Gadija; Kidson, Susan; Hulley, Phillipa
    The problematic and numerous side effects of PUVA (psoralen and UVA) and other treatments currently in use for vitiligo, have justified the search for an alternative treatment. In this study, the use of khellin, a naturally occurring furochromone, which is similar in structure to psoralens, is explored as an alternative treatment for vitiligo. When khellin is combined with UVA (KUVA), it is reported to repigment vitiligo skin as effectively as PUVA photochemotherapy, but without the adverse effects reported with PUVA. The exact mechanism for khellin-induced repigmentation is still to be determined and no cell biological studies have yet been done to elucidate its mechanism of action. The specific aim of this project was to set up an in vitro tissue culture system to determine the direct effects of khellin, UVA and KUVA on melanocyte proliferation and pigmentation. Studies were carried out on cultures of a human melanoma cell line (Mel-1), normal human melanocytes and 3T3 mouse fibroblasts. Cell proliferation assays revealed that the proliferation of melanoma cells and melanocytes were increased after exposure to khellin for four days at concentrations ranging from 1nM to 0.5mM. A peak of proliferation was obtained at 0.01mM khellin, which stimulated proliferation of melanoma cells and melanocytes by 2.3 5-fold and 2.1- fold, respectively. In contrast, khellin decreased proliferation of fibroblasts over the entire concentration range tested. At concentrations of 0.5mM and above, khellin was cytotoxic to both melanocytic cells and fibroblasts. Cytotoxic assays revealed that 1mM khellin was equally cytotoxic to both melanoma cells and fibroblasts. In addition, these assays revealed that the proliferative response observed with 0.01mM and 0.1mM khellin, did not mask an underlying cytotoxic effect. Exposure to single doses of UVA between 150-280mJ/cm², increased proliferation of melanoma cells with maximal proliferation at 250mJ/cm², while the proliferation of normal melanocytes and fibroblasts were unaffected by this UVA dose. More significantly than khellin or UVA alone, the treatment with the combination of khellin and UVA (KUVA) stimulated proliferation of the melanocytic cells. The combination of 0.01mM khellin plus a single dose of UVA at 250mJ/cm² was the most effective treatment. KUVA combination treatments were found to be more cytotoxic to the fibroblasts than khellin or UVA alone. To test the effect of khellin, UVA and KUVA on melanogenesis, standard radiometric assays were carried out. The combination of khellin and UVA enhanced melanogenesis of the melanocytic cells more significantly than khellin alone or UVA alone. The dose of 0.01mM khellin plus 250mJ/cm² UVA (maximal proliferative dose) increased melanogenesis of the melanocytes by 290% above the untreated control melanocytes. To determine whether khellin and KUVA act by increasing levels of melanogenic proteins, western blot analyses were carried out. The results revealed that there were no differences in the amounts of TRP-2 and tyrosinase in the melanocytic cells treated with khellin alone. In contrast, those treated with KUVA (or UVA alone) had increased levels of the glycosylated form of the enzymes and also possibly had increased levels of the de nova form of the enzymes. These results suggest that UVA might be enhancing glycosylation of melanogenic enzymes and provides a novel insight into the possible mechanism for UVA-induced melanogenesis. The results also revealed that 3T3 fibroblasts expressed the non-glycosylated form of TRP-2, as described by others. In conclusion, this study suggests a model in which khellin acts directly on melanocytic cells by acting as a mitogen and a melanogen at non-toxic concentrations. Khellin possibly increases melanogenesis by acting post-translationally or antagonizing or removing an inhibitor of the melanogenic pathway. The melanocyte-specific effect of khellin and even more so KUVA, seems to suggest that khellin acts along the signal transduction pathways which increases both proliferation and melanogenesis in melanocytes, possibly via endothelin-1 pathways.
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    Medical students' recognition of core knowledge in a supported problem-based learning curriculum
    (2008) Slater, Charles; Shay, Suellen; Kidson, Susan
    This study aims to achieve insight into how students identify core knowledge in a supported problem-based learning (PBL) medical curriculum. Self-directed learning and an emphasis on the clinical relevance of core knowledge are features of this curriculum.
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    Ontogenesis of the cornea and ciliary body : a morphological and molecular study
    (2005) Napier, Hugh Robert Lennox; Kidson, Susan
    The anterior segment of the eye includes the cornea, lens, iris, ciliary body and trabecular meshwork, with each of these elements playing a vital role in the maintenance of vision. The primary objective of this research is to contribute towards the understanding of how specific genes control tissue specification and structural morphogenesis of the developing anterior segment of the eye. Despite its extensive use as a model organIsm, very little is known about the structure and development of the ciliary body in the mouse eye. Using scanning electron microscopy (SEM) the ciliary processes in the adult mouse were shown to form an irregular pattern, crossing over and interweaving, rather than lying parallel to one another, as observed in other mammals. Histological and SEM studies from E14.5 to P7 revealed that the first morphological sign of differentiation in the ciliary body is the appearance of an annular bulge around the optic cup margin; this is then gradually moulded to form discrete ciliary processes. A striking similarity between the developing capillary network and the adult ciliary folds was observed and suggests that the patterning template for the ciliary processes could be the underlying capillary network. Cell proliferation measurements and cell height assessments indicated that one of the first events occurring during the morphogenesis of ciliary processes is a proliferative surge occurring at about PO in the outer ciliary epithelium. It is likely that this surge together with increasing cell heights leads to a bulging of this layer. After a slight delay, the inner ciliary epithelium responds by proliferating and extending inwards towards the lens. Final shaping of the ciliary processes is achieved through cell height reductions in the inner ciliary epithelium. Gene expression analyses revealed dynamic changes in Bmp4 and LEF] expression patterns over the period of ciliary folding, while TgffJ1i4 expression in the ciliary body did not change during morphogenesis. These differences suggest that these genes play different roles in directing the specification and morphogenesis of the ciliary body. The temporal correlation between mitotic and cell height changes during ciliary body morphogenesis suggests that these processes play an integral role in the shaping of ciliary processes.
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    Osseointegration potential for heat treated 3D Ti6Al4V scaffolds seeded with mysenchymal stem cells in vitro
    (2014) Van Heerden, Esther; Vicatos, George; Kidson, Susan
    Aseptic loosening of artificial joints occurs due to the loss of implant fixation. By implementing a 3D porous structure at the bone-implant interface, the ingrowth of bone will permit better and stronger interlocking of the implant to prevent loosening. In this study, it is hypothesized that the seeding of 3D scaffolding structures with mesenchymal stem cells (MSCs) will improve the potential for osseointegration of the implants, as the existing bone may be more inclined to unite with developing bone than with the implant substrate. Titanium-6 Aluminium-4 Vanadium (Ti64) is one of the most commonly used implant materials. Heat treatment of Ti64 was seen in tests done at the University of Cape Town to further improve against implant failure by vastly improving the materials strength and reducing debris formation. Thus the aim of this study was to investigate the effects the heat treatment of Ti64 would have on the capabilities of seeded MSCs in vitro.
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    Spinocerebellar ataxia type 7 in southern africa: an epidemiological, molecular and cellular study
    (2014) Smith, Danielle Claire; Greenberg, Jacquie; Kidson, Susan
    Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease caused by a pathogenic expansion of a CAG repeat within the ataxin 7 gene, resulting in an expanded polyglutamine tract in the ATXN7 protein. SCA7 patients suffer from selective degeneration of cerebellar Purkinje neurons and retinal photoreceptors, which leads to the development of various neurological symptoms, and blindness. SCA7 is considered to be a relatively rare disease, but South Africa has an increased prevalence of the SCA7 due to a founder effect within the black African population. In this study, three distinct but complementary approaches were taken to investigate SCA7 in South Africa, with the aim of estimating the prevalence of the disease, developing improved approaches for molecular diagnostic testing, and establishing a model for in vitro studies of pathogenesis.