Thesis (DMed (Obstetrics and Gynaecology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Chapter 1 is a literature review investigating the incidence of cancer in children and young adults. It describes the most important treatment options including chemotherapy, radiotherapy and surgery and the effect of treatment on future endocrine development and fertility. Different primary cancer sites are discussed in more detail. Chapter 2 is a literature review on the effects of cancer surgery in women and the options for fertility sparing. Cervical cancer and pre-cancer are discussed in detail with options for more conservative surgery in selected patients. A summary of the available published cases of trachelectomy with pregnancy outcomes is included. Other gynaecological cancers requiring surgery are also discussed with reference to conservative options. Chapter 3 is a literature review about the medical (pharmacological) options for protection of ovarian function in patients undergoing oncotherapy. The role of gonadotrophin releasing hormone analogues and hormonal contraceptives in ovarian suppression is discussed in detail. Chapter 4 This chapter examines germ cell physiology with reference to cryopreservation. It includes two major parts. Part 1 is the description of germ cell- and follicle physiology, the principles of cryobiology followed by a review of oocyte cryopreservation and ovarian tissue preservation. Both slow freezing and vitrification techniques are described. The second part of chapter 4 is a report on a randomised controlled evaluation of two different slow freezing cryopreservation protocols. This experimental study compared ultrastructural changes in fresh and previously cryopreserved ovarian cortical tissue after equilibration and thawing using two different cryoprotectants. This is the first randomised investigation into DMSO and PROH as cryoprotectants. Chapter 5 is an investigation into cryopreservation of ovarian tissue as a strategy to protect hormonal function and fertility against gonadotoxic treatment. This chapter consists of two parts. The first part is a thorough literature review of all the published work about grafting of previously cryopreserved ovarian tissue. The largest case series found from a single institution was five patients. Another report of six patients included patients from various sites in Denmark. Part 2 is a description of a cohort of patients followed up after re-implantation of previously cryopreserved ovarian cortical tissue. Follow-up hormone levels of 13 individual cases are described in detail. This is the largest case series ever reported. The experimental study described in Chapter 4 and the clinical study described in Chapter 5 was approved by the ethical research committee of the Faculty of Health Sciences, Stellenbosch University, project number N05/10/182. Chapter 6 provides an integrated overview of the incidence and treatment of cancer in young women and how its negative effects may be prevented or mitigated. Aspects of chemotherapy, radiotherapy and surgery are evaluated where it may affect future reproductive health. The role of oocyte and ovarian tissue cryopreservation is discussed. Guidelines are provided for clinicians. / AFRIKAANSE OPSOMMING: Hoofstuk 1 Hierdie is ‘n literatuuroorsig wat die insidensie van kanker in kinders en jong volwassenes ondersoek. Dit sluit die mees belangrike behandelingsopsies in, naamlik chemoterapie, radioterapie en chirurgie en die effek wat behandeling mag hê op toekomstige endokriene ontwikkeling en fertiliteit. ‘n Verskeidenheid kanker tipes word in meer detail beskryf. Hoofstuk 2 Hoofstuk 2 is ‘n literatuuroorsig oor die effekte van kankerchirurgie in vroue en die geleenthede tot beskerming van fertiliteit. Servikale kanker en voorlopers van servikale kanker word bespreek en die opsies vir konserwatiewe chirurgie in uitgesoekte pasiënte word gegee. ‘n Opsomming van die inligting wat beskikbaar is oor tragelektomie en swangerskap uitkomste word ingesluit. Ander ginekologiese kankers wat chirurgie mag benodig, word ook bespreek met verwysing na konserwatiewe hantering. Hoofstuk 3 ‘n Literatuuroorsig oor die mediese (farmakologiese) opsies vir die beskerming van ovariële funksie in pasiënte wat behandeling ontvang vir kanker. Die rol van gonadotropien-vrystellingshormoon-analoë en hormonale kontrasepsie vir ovariële onderdrukking word in detail bespreek. Hoofstuk 4 Hierdie hoofstuk ondersoek kiemselfisiologie met verwysing na vriesbewaring. Dit is verdeel in twee dele. Deel 1 is ‘n beskrywing van kiemsel- en follikelfisiologie en die beginsels van vriesbiologie. Dit word gevolg deur ‘n oorsig van oösiet vriesbewaring en ovariële weefselbewaring. Stadige bevriesing en vitrifikasie- metodes word bespreek. Die tweede deel van hoofstuk 4 is ‘n verslag oor ‘n gerandomiseerde, gekontroleerde evaluasie van twee stadige bevriesingsmetodes. Hierdie eksperimentele studie het die ultrastrukturele veranderinge vergelyk in vars en voorheen bevrore ovariële kortikale weefsel na ekwilibrasie en ontdooiing met twee verskillende vriesbeskermers. Dit is die eerste gerandomiseerde studie oor DMSO en PROH as vriesbeskermers. Hoofstuk 5 Hierdie hoofstuk handel oor ‘n ondersoek na vriesbewaring van ovariële weefsel as ‘n benadering tot beskerming van hormonale funksie en fertiliteit teen gonadotoksiese behandeling. Die hoofstuk bestaan uit twee dele. Die eerste deel is ‘n deeglike oorsig van die literatuur oor al die beskikbare werk wat handel oor terugplasing van voorheen bevrore ovariële weefsel. Die grootste pasiëntreeks van ‘n enkel instelling was slegs vyf pasiënte. ‘n Ander beskrywing van ses pasiënte het pasiënte van verskeie eenhede in Denemarke ingesluit. Deel 2 is ‘n beskrywing van ‘n groep pasiënte wat opgevolg is na oorplanting van voorheen bevrore ovariële kortikale weefsel. Opvolg hormoonvlakke van 13 gevalle word in detail bespreek. Hierdie is die grootste pasiëntreeks wat tot nog toe beskryf is. Die eksperimentele studie wat in hoofstuk 4 beskryf word en die kliniese studie wat in hoofstuk 5 beskryf word, is goedgekeur deur die etiese navorsingskomitee van die Fakulteit Gesondheidswetenskappe van die Universiteit Stellenbosch met die projeknommer N05/10/182 Hoofstuk 6 Hierdie is ‘n geïntegreerde oorsig van die voorkoms en behandeling van kanker in jong vroue en hoe die negatiewe effekte daarvan voorkom of verminder kan word. Aspekte van chemoterapie, radioterapie en chirurgie word geëvalueer ten opsigte van die effek op toekomstige reproduktiewe gesondheid. Die rol van oösiet- en ovariële weefselvriesbewaring word bespreek. Riglyne vir klinici word gegee.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

No description available.

Acute Myeloid Leukemia (AML), the most common form of acute leukemia in adults, is only curable in approximately 30% of all cases. Despite prognostic risk stratification using sub-typing and cytogenetic analysis to direct therapy, the mortality and relapse rate remains high. AML patients with normal karyotypes are defined as intermediate risk and are the most challenging to treat. Somatic mutations may be the key in refining prognostic stratification and providing useful therapeutic targets. The FMS-like tyrosine kinase 3 (FLT3) and Nucleophosmin (NPM1) genes have common mutated forms that are associated with overall survival and response to therapy. We assessed mutations in the FLT3 and NPM1 genes and their levels of expression in twenty eight AML patients in the presence and absence of HIV and their response to induction therapy. Furthermore, we used a novel technique, High Resolution Melting (HRM) Analysis to detect FLT3 Internal Tandem Duplications (ITD) and NPM1 exon 12 mutations. Five of the patients in this study were HIV positive, three of whom did not survive post-induction therapy. Of the AML patients, 17.9% were positive for the NPM1 mutation and 21% had mutated FLT3. Interestingly, the presence of the FLT3 and NPM1 mutations were coupled with an increase in expression levels of FLT3 and NPM1 from presentation to post-induction respectively and the loss of these mutations were coupled with a decrease in levels of expression from presentation to post-induction. However, an increase/decrease from presentation to post-induction did not necessarily denote the presence/absence of a mutation. Therefore, while mutational status of genes may generally confer mRNA levels, our results showed that there existed no definitive trend between mRNA levels of NPM1 and FLT3 expression and mutational status. We found that the HRM method was definitive for the simpler NPM1 mutation however detection of the FLT3-ITD mutation was challenging. There isn’t a clear distinction between mutated and non-mutated FLT3 due to the formation of hetero-duplexes during analysis, making detection highly subjective and error-prone. Sequencing allowed confirmation of mutated FLT3 and non-mutated FLT3 which were not in all instances in concordance with HRM analysis. The prognostic value in terms of overall survival of NPM1 and FLT3 mutations in this study is indefinite. Furthermore, the analysis of the HIV positive AML patients revealed no clear correlation between NPM1 and FLT3 levels of mRNA expression and mutational status. Also, the small number of HIV positive AML patients did not allow for conclusions to be made regarding HIV status and survival when affected with AML. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Extracts of the African potato have long been believed to have anti-cancer properties. The aim of the current research was to isolate hypoxoside (HYP) from Hypoxis hemerocallidea (African potato) and synthesize the dimethyl (DMH) and decaacetyl (DAH) derivatives and to test their selective cytotoxicity on a model consisting of a normal (MCF10A) and premalignant, invasive breast epithelial cells (MCF10A-NeoT). Hypoxoside was extracted from the H. hemerocallidea corms using ethanol, purified using a C-18 reverse phase column and the compound examined by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry and found to be of high purity. This was also the case for the synthesized compounds. To assess possible selective effects (cytotoxicity) of derivatized and underivatized hypoxoside, effects on the metabolism of premalignant and normal cells were assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Effects on cell number (total counts) and cell death [trypan blue and propidium iodide (PI) staining for dead cells versus a lack of staining for live cells] were, thereafter, assessed. Imaging of live adherent cells was also carried out using acridine orange (AO) and PI for live and dead cells (respectively). Propidium iodide staining of detached cells was carried out for flow cytometric determination of cell death (PI indicating early apoptotic or late apoptotic/necrotic cells). After treatment of normal (MCF10A) breast epithelial cells and premalignant cHa-rastransfected (MCF10A-NeoT) derivative breast epithelial cells with HYP, DMH and the DAH derivative, the MTS assay and the Duncan‟s multiple range, analysis of variance (ANOVA) post hoc analysis of the MTS results revealed that only the 150 and 300 µM DAH derivative had a statistically significant effect on the metabolic activity of the abnormal cell line relative to the dimethyl sulfoxide (DMSO) and revealed no significant effect on the normal MCF- 10A cell line after treatment with any of the test compounds. Supravital PI staining of adherent cells seemed to indicate a far higher rate of induction of cell death in abnormal cells than evident in the MTS assay and the PI-based flow cytometry or the trypan blue exclusion assays and need re-investigating, though result trends were similar. Total cell counts, show that HYP and its derivatives appear to increase both cancer and normal cell proliferation significantly, except in the case of DAH at 150 and 300 μM in the MCF10A-NeoT, without affecting the MCF-10A cell line. The trypan blue method for detection of cell death, together with total cell counts, the Duncan‟s analysis of MTS results and a 24 hour exposure to test compounds, seems to constitute an optimal system for drug screening and indicates the statistically significant selective toxicity of the DAH compound at 150 and 300 μM in the MCF10A-NeoT, suggesting that the DAH derivative at 150 and 300 µM would have significant, selective therapeutic potential on Ras-related malignancies. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Three plant species (Phylica paniculata Willd., Pergularia daemia Forssk. and Monsonia / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

Lateral diffusion of membrane components (lipids and proteins) is an important membrane property to measure since the essential process of absorption of anti-cancer and other drugs -some of which are not soluble in lipids and therefore would not be able to penetrate the cell membrane through passive diffusion- lies on it. In particular, the procedure of diffusion into the cell cytoplasm is reliant on free volumes in the membrane (passive diffusion) as well as carrier proteins (facilitated diffusion). By enhancing the mobility of lipids and/or proteins, the possibility of the carrier protein to "encapsulate" pharmacological components maxim- izes, as a "scanning" of the proteins gets performed due to the fluid phase of a biological membrane. At the same time, the increased mobility of the lipids facilitates the passage of lipid-soluble molecules into the cell. Thus, given that the success of anticancer treatments heavily depends on their absorption by the cell, a significant enhancement of the cell mem- brane permeability (permeabilisation) is rendered vital to the applicability of the technique. For this reason, there is augmented interest in combined methods such as Nanotechnology based drug delivery that is focused on the development of optimally designed therapeutic agents along with the application of shock waves to enhance the membrane permeability to the agents. This study examines the impact of shock waves on a numerical model of a biological membrane. Cont/d.

Prodigiosins and tambjamines are natural compounds from bacterial and marine sources belonging to a family containing a common 4-methoxy-2,2'-bipyrrole core. These compounds have received a lot of interest due to their promising biological activities. Studies have suggested DNA as a potential therapeutic target for the natural prodigiosin and tambjamine due to their ability to facilitate oxidative DNA cleavage in the presence of Cu2+. Based on this we sought to study the metal binding activity of new prodigiosin and tambjamine analogues. A new prodigiosin analogue was synthesized and complexed with Cu2+. This revealed 1:1 complex formation between the tripyrrole and Cu2+ that was confirmed by mass spectra and NMR spectra analysis. In addition in situ studies also revealed that our new analogues of prodigiosin cannot bind Zn2+ when the methoxy group on ring B is replaced by an alkyl group or when one of the ring nitrogens is methylated. Our UV-Vis experiments with calf thymus DNA showed that prodigiosins and tambjamines bind DNA mainly through an external mode, suggesting that hydrogen bonding between the pyrrole ring nitrogens and the bases of DNA takes precedence over stacking interactions. For the new Cu2+ complex synthesized however, we observed spectral changes that suggest intercalation into DNA. DNA cleavage experiments revealed that the prodigiosin-Cu complex is able to convert supercoiled DNA into its linear form. The data from the gel shift assays fit well to a first-order consecutive reaction model. In addition to preformed metal complexes, we showed DNA cleavage by in situ complexation of the ligands in the presence of Cu2+. However, although we showed Zn2+ complex formation with prodigiosin analogues, in situ studies did not show induction of DNA cleavage by Zn2+ complexes under our experimental conditions.

Submitted in fulfillment of the requirements for the degree of Doctor of Philosophy: Biotechnology, Durban University of Technology, Durban, South Africa, 2015. / Photodynamic therapy (PDT) is emerging as a viable alternative to invasive anti-cancer treatment regimens such as surgery, chemotherapy or radiotherapy. A series of metal – based phthalocyanine complexes have been discovered that may be used as a drug or photosensitizer in photodynamic therapy for the treatment of cancers. During photodynamic therapy the photosensitizer is administrated intravenously or topically to the patient before laser treatment at an appropriate wavelength is delivered to the cancerous site to activate the photosensitizer. The activated photosensitizer will react with oxygen typically present in the cancerous tissue to produce reactive oxygen species for the eradication of the cancerous tissue. This is the first study where gallium (GaPcCl), indium (InPcCl) and iron (FePcCl) Pc chloride complexes were used for photodynamic research. These metal – based phthalocyanine complexes were investigated using different cancer cell lines (Caco-2, MCF-7, melanoma and A549). Also, the baseline cellular uptake and photodynamic effect of these complexes were established on healthy normal cells (human fibroblast cells). Fluorescent spectrophotometry showed that all three photosensitizers accumulated in a time-dependent manner in Caco-2, MCF-7, melanoma and A549 cancer cells, as well as in healthy normal fibroblast cell in amounts which increased over a period of 24 hours, with emission peaking at 24 hours for all cell lines. Dark toxicity effects and photodynamic therapy efficacy were established with a MTT assay. High concentrations of inactive GaPcCl, InPcCl and FePcCl was toxic to Caco-2, melanoma, A549 and fibroblast cells. However, all three photosensitizers were in its inactive state at low and high photosensitizing concentrations were highly toxic to MCF-7 cancer cells. On the other hand, in vitro photodynamic therapy treatment with both low and high concentrations of GaPcCl, InPcCl and FePcCl were observed to be potently cytotoxic towards all four cancer cell lines upon exposure to laser light for 22 seconds (2.5 J/cm2), 39 seconds (4.5 J/cm2) and 74 seconds (8.5 J/cm2). These results revealed that all three photosensitizers reacts to photodynamic therapy in a concentration-dependent (photosensitizer) and dose-dependent (light dose/time) manner. At 24 hours after photodynamic therapy, the most effective treatment parameters were laser treatment for 74 seconds with FePcCl concentrations from 60 µg/ml - 100 µg/ml which resulted in 0% cell survival of Caco-2 cancer cells. A short laser treatment time of 74 seconds for activation of FePcCl (20 µg/ml) resulted in 0% cell survival of MCF-7 cancer cells. Similarly, FePcCl (40 µg/ml - 100 µg/ml) activated for 22 seconds, 39 seconds and 74 seconds resulted in 100% cell death of A549 cancer cells. Photodynamic therapy treatment with GaPcCl and InPcCl were very effective in reducing the cell viability of melanoma cancer cells. Healthy normal fibroblast cells survived in vitro photodynamic therapy treatment with all three photosensitizers much better than the cancer (Caco-2, MCF-7, melanoma and A549) cells. This confirms the previously reported results that photosensitizers such as phthalocyanines and its metal-based complexes preferentially accumulate in cancer cells than normal healthy cells. All three photosensitizers localized in mitochondria and lysosomes of the Caco-2, MCF-7 and A549 cancer cells. In melanoma cancer cells InPcCl also localized in the mitochondria and lysosome, but GaPcCl and FePcCl localized in mitochondria only. Apoptosis was identified via microscopical and flow cytometric investigations, as the dominant mode of cell death induced by GaPcCl, InPcCl and FePcCl mediated photodynamic therapy in cancer cell lines tested. Therefore, this study concludes that GaPcCl, InPcCl and FePcCl are effective photosensitizers for the in vitro PDT treatment of cancer cells. The effective in vitro PDT treatment for each cell line was dependent on the photosensitizer concentration and illumination period for each of the different photosensitizers. / D