The aim of this body of work was to characterise a novel cisplatin drug delivery system and to develop new tools based on biophotonic imaging that could be used to enhance studies of drug delivery in vivo. Cucurbiturils (CB) are macrocycles which are formed by acid catalysed condensation of glycoluril and formaldehyde. The internal cavity of CB[7] encapsulates a single molecule of cisplatin and the hypothesis was that encapsulation would reduce thiol degradation of the drug. Drug sensitivity studies in vitro with the cisplatin-sensitive human ovarian cancer cell line, A2780, and a cisplatin-resistant derivative, A2780/cp70, showed that the CB[7] encapsulated cisplatin retained activity but that this encapsulation drug delivery system was not able to overcome resistance to platinum. However, when these cell lines were grown as subcutaneous xenografts in nu/nu mice, the encapsulated cisplatin was able to reduce the growth of A2780/cp70 tumours which are resistant to the maximum tolerated dose of cisplatin in vivo. One possible explanation of this observation is that encapsulation might alter the pharmacokinetics of cisplatin and a method for the detection of platinum in biological samples by ICP-MS was established and validated. This assay was sufficiently sensitive to detect the low levels of platinum present in mouse plasma 24 hours after administration of either free or encapsulated cisplatin. Plasma and tissue pharmacokinetics show that encapsulation had no effect on the peak plasma concentration of cisplatin but did reduce the rate at which cisplatin was cleared from the plasma. The increased plasma AUC of cisplatin resulted in a non-selective increase in the delivery of cisplatin to both tumour and normal tissues. However, there was no apparent increase in toxicity which could be explained by the fact that encapsulation, unlike an increase in the dose of free cisplatin, had no effect on the peak plasma concentration. Subcutaneous xenografts lack critical features of human tumours. The development of more complex models for use in drug development has been limited due to lack of a method for monitoring tumour growth. Biophotonic imaging was, therefore, investigated to determine whether it is sufficiently sensitive and reproducible to be able to evaluate growth of disseminated tumours in mice. The bioluminescent signal is dependent on the metabolism of luciferin by luciferase. Subcutaneous injection of luciferin was shown to produce a consistent signal in all injected mice. The bioluminescent signal was transient but reached a maximum intensity 6 minutes after injection and remained stable for about 4 minutes which defined the window during which measurements were taken. Sensitivity was shown to be dependent on the level of expression of luciferase by the cells. Injection of commercially available HCT116Luc cells, where the luciferase gene was inserted by a lentiviral system, was shown to allow detection of 10,000 cells in the lungs of mice. This sensitivity was about 10 fold greater than was obtained by lipofectamine based gene transfection. When HCT116Luc cells were grown as subcutaneous xenografts in mice, an exponential growth pattern was easily detected by bioluminescence imaging and the reproducibility between mice was comparable to that routinely obtained by calliper measurements. Activity of encapsulated cisplatin was determined in a model of disseminated ovarian cancer. Rab25, a member of the RAS oncoprotein superfamily, is up-regulated in around 80% of ovarian cancer samples compared to normal ovarian epithelium. Rab25 contributes to tumour progression by enabling the tumour cells to invade the extracellular matrix by altering the trafficking of integrin. Transfection of Rab25 into A2780 cells results in cells that can grow in the peritoneal cavity of mice. A2780-Rab25 cells were 4 fold resistant to cisplatin in vitro which confirms a previous observation that Rab25 expression in A2780 makes them less sensitive to the induction of apoptosis in response to stress. A2780-Rab25 cells that express the luciferase gene (A2780-Rab25Luc) were injected into the peritoneal cavity of mice and growth was measured by biophotonic imaging. Exponential growth was clearly apparent at a stage at which no obvious abdominal distension was apparent. The disseminated A2780-Rab25Luc tumour xenografts were less sensitive to cisplatin than are subcutaneous xenografts of A2780. This is the first study that suggests that Rab25 over-expression results in reduced drug sensitivity in vivo. In contrast, a very significant growth inhibition was observed when mice were treated with an equivalent dose of encapsulated cisplatin regardless of whether it was administered by the intraperitoneal or subcutaneous route. These results are very encouraging since they confirm the enhanced activity of encapsulated cisplatin and also demonstrate the value of biophotonic imaging for measurement of tumour growth in vivo. Pharmacodynamic measures of drug activity in vivo in animal models are often based either on measures of surrogate tissue response or on single measures on tumour tissue removed at the end of the experiment. Biophotonic imaging in vivo allows the translation of reporter assays used in cell lines in vitro to studies of tumour response in vivo. A plasmid was prepared that links the p53 transcriptional response element to the luciferase gene and it was then transfected in to A2780 cells which express wild type p53. Stable transfectants of A2780p53Luc were treated with cisplatin, doxorubicin and paclitaxel and induction of p53 determined by bioluminescence and confirmed by Western blotting. A very low bioluminescent signal was present in untreated cells and a clear dose dependent increase in bioluminescence was seen in response to all three drugs. When A2780p53Luc cells were grown as subcutaneous xenografts the bioluminescent signal was significant in untreated tumours but was markedly increased 24 hours after treatment of the mice with cisplatin. Induction of p53 in the tumours was confirmed by immunohistochemistry and this also confirmed significant expression of p53 in untreated tumours. The possible implications of these findings for the improved delivery of cisplatin are discussed.

Protein tyrosine phosphatases (PTPs) have been identified that mediate a range of physiological and pathological processes, such as proliferation and tumour metastasis. PTPRK and PTPRM belong to the same subfamily of PTPs. This study aims to investigate the role of PTPRK and PTPRM in cancer development and progression. Knockdown of PTPRK expression was performed in PC-3 and DU-145 cells. Functional assays were then carried out on these cells in order to determine any changes in their biological properties. Knockdown of PTPRK significantly reduced the growth and adhesion of both PC-3 and DU-145 cells. The experimental results suggested that reduction of cell growth is potential involvement of p53 and/or caspase-3 and -8 and its up-stream molecule JNK. The decreased expression of PTPRK and PTPRM are associated with poor prognosis and reduced survival. Knockdown of PTPRK resulted in increased adhesive and invasive abilities, and promoted cell proliferation and motility of breast cancer cells. Moreover, PTPRM knockdown resulted in elevated adhesion, invasion, and proliferation of breast cancer cells. Activation of ERK and JNK by tyrosine phosphorylation and consequent elevated MMP9 activity is involved in increased cell migration and invasion by PTPRM knockdown. These results suggested that PTPRK and PTPRM are involved in the disease progression of prostate and breast cancer by regulating a complex network of pathways and molecules. This provides further proof of the importance of the R2B subfamily, a subgroup of PTP superfamily, in cancer. In addition, it sheds some light on the use of PTPs as prognostic indicators of disease, aiding in diagnosis and treatment. The major effect is the promotion of motility and invasiveness of cancer cells via ERK and JNK pathways. However it can also impair the apoptosis mediated by JNK pathways in certain cancer cells, such as prostate cancer cells. Such contrasting effects on survival and motility require further investigation, and should also be considered when treating cancers by targeting these molecules.

CLL is an incurable B-cell malignancy associated with profound tumour cell-mediated immune dysfunction. It therefore represents a challenging disease for the successful application of immunotherapeutic strategies aimed at promoting anti-tumour T-cell responses. In this study, extensive immunophenotypic analysis of T-cells from the blood of CLL patients was performed, in order to better characterise their dysfunctional status within the disease. Analysis of CLL patient blood samples revealed a skewing of T-cells towards a highly differentiated effector memory phenotype as well as the expression of markers associated with exhaustion/senescence (CD28- and CD57+) and immunosuppressive molecules (PD-1 and CD200). In addition this study revealed the expansion of CD8+ T-cells in a subset of CLL patients leading to an inversion in the normal CD4:CD8 ratio. The presence of an inverted CD4:CD8 ratio was subsequently shown to be associated with a shorter time to first treatment and reduced progression-free survival. Characterisation of T-cells identified several molecules that could be targeted therapeutically in order to break T-cell tolerance in CLL patients and potentially restore normal immune responses. Investigation of the immunosuppressive molecules PD-1 and CD200 showed that they are over expressed in CLL patients, suggesting that they may be involved in maintaining T-cell tolerance in the disease. However, blockade of PD-1-PDL-1 and CD200-CD200R signalling pathways failed to enhance T-cell responses from CLL patients in vitro. Investigation of an alternative approach to enhance T-cell responses in CLL involved the use of a bi-specific antibody targeting CD19 and CD3 called blinatumomab. In vitro testing showed that blinatumomab can induce T-cell activation, promoting the release of pro-inflammatory cytokines and granzyme B secretion from both CD4+ and CD8+ T-cells. In addition, blinatumomab was shown to mediate T-cell dependent killing of CLL cells requiring the formation of T-cell:CLL cell conjugates. Finally this study provided clear evidence that blinatumomab can break T-cell tolerance in CLL and strongly advocates the progression of blinatumomab into clinical trials as a novel therapeutic agent in CLL.

CD8+ T-cells recognise pathogens and cancer through a specific interaction between the T-cell receptor (TCR) and a 8-14 amino-acid residue peptide presented by class I major histocompatibility complex (pMHCI) molecules expressed on the target cell surface. The first structures of murine and human TCR/pMHC complexes, published in 1996, revealed a number of important features of the TCR/pMHC interface. Currently, <25 unique human TCR/pMHC complexes are reported in the literature. This is a relatively low number compared with the number of antibody or unligated pMHC structures. The lack of structural information regarding human TCR/pMHC complexes has compromised the determination of a comprehensive and accepted set of rules that govern T-cell antigen recognition. Difficulties in generating TCR/pMHC complex crystals partly explain the low number of these structures. The first part of this thesis reports the development of a new crystallization screen (TOPS) designed specifically for the generation of such protein crystals. I also had access to MART-1-specific TCRs, the MART-1 protein being expressed by virtually all fresh melanoma tumour specimens. Different human leukocyte antigen (HLA)- A*0201-restricted peptides from this protein are presented at the melanoma cell surface. As TCRs are known to bind to cancer-derived “self” peptides with weak affinity, there is considerable interest in designing enhanced affinity TCRs for the recognition of HLA-A*0201-MART-1. My work concentrated on the MART-1- specific TCR MEL5 and its affinity-enhanced variant selected by phage display, α24β17. I analysed the biophysical properties of α24β17 and determined that it bound HLA-A*0201-MART-1 with >30,000-fold enhanced affinity and distinct thermodynamics. Comparison of TCR/HLA-A*0201-MART-1 complex structures solved with TOPS and binding biophysics showed that: (i) TCR affinity can be enhanced by increasing interactions between the TCR and the MHC surface; (ii) soluble α24β17 retains the peptide specificity by a novel mechanism involving interactions with solvent molecules; and, (iii) MEL5 interaction with the physiologically relevant MART-127-35 nonameric antigen led to a peptide anchor residue switch, a TCR-induced modification that has never been observed before. I also initiated a preliminary study on the generation of genetically modified Jurkat cells and CD8+ T-cells expressing a range of affinity-enhanced TCRs directed against melanoma for adoptive cell therapy. These results suggested that melanoma specificity is retained after MEL5 transduction and that there is no need to optimize beyond a TCR affinity threshold to obtain optimal T-cell activation. Collectively, these data shed light on the complex and unpredictable nature of T-cell antigen recognition.

<p> The purpose of the study was to examine the impact of cancer caregiving on primary caregivers, exploring their personal narratives looking back on the entire experience from diagnosis, through treatment, and beyond. Caregiving is associated with exacerbation of stress-related disorders such as hypertension and heart disease and may also be associated with increased mortality rates. Transitions theory served as the conceptual framework for the study. Eleven adult caregivers, pre-retirement age, each participated in two semi-structured interviews. Caregivers were recruited from a community cancer resource center and were purposively selected to achieve maximum variation in terms of outcome of cancer treatment. The sample included 8 females and 3 males; there were 3 husbands, 6 wives, and 2 daughters. Caregivers provided care for patients with a variety of cancer types and a variety of treatment outcomes, from cancer free with sequelae to deceased. Each caregiver interview recording was transcribed, and preliminary examination of each transcript helped guide subsequent interviews. NVivo9 software was used to assist with data management. Data saturation was achieved. Narrative within-case analyses as well as thematic analysis were used to address research questions. Thematic analysis resulted in seven themes: Burden: The Load that Never Ends; Disconnectedness and Isolation: The Invisible Person; Helplessness and Loss of Control: Tied to This Ride; Dealing with the Healthcare System; Role Disruption: Spinning the Plates; Loss, Change, and Grief: Reaction to the Whole; and Carrying Forward with Scars: New Priorities and Permanent Change. All of the caregivers changed their employment or social responsibilities due to the demands of caregiving. Themes were present in different parts of the cancer trajectory and in differing intensities in all interviews. Findings included disconnectedness and isolation as a central feature of cancer caregiving, plus significant grief present through the cancer trajectory, especially in the post-treatment phase. Furthermore, the experience of cancer caregiving remained one of significant impact years after treatment had ended. Successful transitioning requires connectedness and mastery, but participants in this study identified that their caregiving trajectories were full of isolation, grief, burden, and helplessness. Many suggested the need for support, even though they tended to deny their own physical and emotional needs while caregiving. Healthcare professionals can help by providing information, support, listening, and grief counselling. Research is needed on interventions that may reduce isolation, helplessness, and burden for caregivers.</p>

The effect of CD40, CD54/ICAM-1 and CD95 (Fas/APO-1) ligation in murine B lymphomas was investigated. Crosslinking ligation of CD40 induced p53, the p53-regulated CDKI p21Cip1/Waf1, and apoptosis in 3 lymphoma cell lines of a mature phenotype (A20, M12 and TA3), but not in 2 lymphomas of an immature phenotype (WEHI-279 and WEHI-231). Association of Mdm2 with p53 was reduced in A20. Expression of Bax and Bcl-2 was unaffected by CD40 ligation in all lines. Ligation of CD95 induced apoptosis in A20, not in M12, TA3 or WEHI-279. Crosslinking ICAM-1 on A20 had no effect on growth, but induced tyrosine phosphorylation of a 90--100KDa band in ICAM-1 immunoprecipitates, consistent with ICAM-1 itself. My results demonstrate that CD40 and CD95 differentially affect B cell lymphomas according to their developmental stage and show the potential for tumor suppression by signals that promote growth of non-transformed B cells.

Gap junctional intercellular communication (GJIC) allows for the transport of small signaling molecules between adjacent cells. Gap junctions and their constituents, connexins, are often impaired in cancer. Restoration of connexins and GJIC in cancer cells leads to reversal of the transformed phenotype, reduction of the rate of cell growth, and inhibition of in vivo tumorigenicity. In addition, restoration of connexin43 (Cx43), one of the most abundant connexins that is often reduced in cancer, allows for the transfer of chemotherapeutic agents among neighboring cells, a phenomenon known as the "Bystander Effect". In chapter 2, I'm reporting evidence that induction of Cx43 and GJIC with cyclic-AMP (cAMP) can enhance the cytotoxic effect of the prodrug ganciclovir following infection of breast cancer cells with an adenovirus expressing the ganciclovir-activating enzyme viral thymidine kinase (vTK). This induction may provide a therapeutic advantage in suicide gene therapy due to the bystander effect when a small proportion of the cell population is expressing vTK. In chapter 3, I'm reporting that breast tumor tissue samples from patients had low to undetectable Cx43 levels, in contrast to their matched normal tissues. In addition, Cx43 protein and RNA levels were undetectable in a panel of human breast cancer lines and in rat breast tumors induced by the carcinogen dimethylbenz[a]anthracene. Together, these observations indicate that the loss of Cx43 is a common marker of cancer cells. In chapter 4, I examined the hypothesis that Cx43 downregulation occurs primarily at the transcriptional level. To determine the mechanisms behind the transcriptional regulation of Cx43, the human Cx43 promoter was investigated. Overexpression of the H-Ras oncogene in NIH3T3 cells leads to the induction of the human Cx43 promoter activity, as well as Cx43 RNA and protein levels. This stimulation involves the MEK-ERK pathway downstream of H-Ras. The promoter sequence responsible for

Objective : An audit of the use and outcomes of Radioactive Iodine (RAI) therapy in the definitive management of thyrotoxicosis at Inkosi Albert Luthuli Central Hospital (IALCH), KwaZulu-Natal, South Africa. Methods : The clinical records of all new patients with thyrotoxicosis, referred in a 4 year period between 01/01/2003 and 31/12/2006, were analysed. Response to RAI was monitored using biochemical parameters (namely, Thyroid Stimulating Hormone and Free T4 levels). Rates of euthyroidism (cure), hypothyroidism and hyperthyroidism (treatment failure) were correlated to dose of RAI. Patients were followed-up for at least 2 years or until the onset of hypothyroidism. The follow-up period was until 31/12/2007. Results : One hundred and fourteen patients (37.7%), of a cohort of 302 new thyrotoxic patients treated with RAI, met the inclusion criteria. Ninety-six patients (84.2%) had Graves Disease (GD) whilst 18 had Toxic Nodular Disease (TND). At 2 year follow-up, 91 patients (79.8%) were hypothyroid, 10 (8.8%) were euthyroid and 13 (11.4%) were hyperthyroid. The average dose of RAI to achieve euthyroidism was 10mCi and hypothyroidism, 9.7mCi. The average time to achieve euthyroidism was 5.9 months and 10.1 months to become hypothyroid. Thirty-one patients (27.2%) remained persistently hyperthyroid after one dose of RAI. Patients with GD (88.5%) were more likely to become hypothyroid (p < 0.001) whilst 38.9% of TND patients remained hyperthyroid (p = .001). Baseline TFT values were significant in terms of outcomes correlated with the prescribed RAI dose i.e Low Dose (<8mCi) vs. Intermediate Dose (8-9mCi) vs. High Dose (>9mCi)(TSH p = 0.05; FT4 p = 0.003; FT3 p = 0.001). Conclusion : The majority of patients became hypothyroid over time, in keeping with reported data. In the public health sector, where early access to RAI (in terms of waiting times for appointments for RAI) and follow-up are major problems, early cure is essential to minimize the morbidity of thyrotoxicosis and this may be achieved with an initial high dose of RAI. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2011.

Introduction Prostate cancer (PCa) is the most commonly diagnosed male malignancy and the second leading cause of male cancer death in the Western world. In the United States of America (USA), African American men (AAM) have among the highest rates of PCa in the world. They develop the disease 1.5 times more frequently than European American men (EAM) of the same age .The mortality rate is approximately two to three times higher for AAM compared to EAM. There is a dearth of literature exploring the incidence and treatment outcomes of this disease based on racial profiling in a South African population. This study aims to evaluate racial disparities with a focus on patients with PCa managed in the public health care sector in the province of Kwazulu Natal (KZN). Patients and methods The study was a retrospective analysis of patients with PCa treated at Inkosi Albert Luthuli Central Hospital and Addington Hospital, which are both based in the Durban Metropolitan area in the province of KZN. Data extracted from the folders of patients with PCa who presented between March 2003 and December 2007 were collated onto a data capture form and analysed. Patient data were analysed according to the following categories: „h Patient demographics; „h Patient follow-up period; „h Disease risk profile; „h Response to treatment; „h Compliance on treatment. SPSS version 15.0 was used to analyse the data. Within each disease category, the response variables were analysed by race group using non-parametric Kruskal-Wallis tests. Multiple comparisons were made using pairwise Mann-Whitney tests and Bonferroni adjusted significance levels according to the number of multiple comparisons made. In order to control for other confounding factors such as age, serum PSA levels and compliance, Cox proportional hazards models were used. Hazard ratios and 95% confidence intervals were also reported. Results In KZN, the majority of the population is classified as blacks (82.9%). The Indian population group makes up 9.0% of the provincial population while white and coloured people make up 6.1% and 2.0% of the provincial population respectively. In this study population, Blacks made up 57.7% and whites made up 27.5%, followed by 11.4% of Indians and 3.4% of coloureds. The racial frequency distribution of the study population demonstrated that whites had a higher incidence of PCa when analysing their demographic profile in the province. Blacks had the highest median total serum prostate specific antigen (PSA) levels on presentation. When compared to that of the white study population, this was found to be statistically significant (p < 0.001). There was a significant association between stage of disease and race (p = 0.001). In the black group, a greater proportion had metastatic rather than localised or locally advanced disease, and in the white group the converse was seen, whereas in the Indian and coloured groups an almost equal proportion had localised or locally advanced disease versus metastatic disease. A crude analysis of progression free survival (PFS) data in patients with metastatic disease demonstrated that PFS was significantly (p = 0.003) longer for whites compared to blacks. Cox regression analysis did not confirm the influence of race on disease progression but this was confounded by incomplete data. Discussion The high incidence of whites in our study population relative to their racial distribution in the province may be explained by better educational and awareness levels of PCa and better access to healthcare facilities in this race group as compared to blacks. The data demonstrating a more advanced stage of disease presentation and higher median PSA levels in the black population may be reflective of an informational void on screening and awareness of PCa and/or a more aggressive disease course in this population group. The hypothesis that the black population may have a more aggressive disease course is given further credence by the crude analysis data suggesting a longer PFS for whites when compared to blacks. Conclusions This study invites further exploration of racial trends in PCa incidence, risk profile and outcomes amongst the diverse population groups of SA. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2009.

The Bcr-abl translocation arises from a reciprocal translocation between chromosomes 9 and 22 and results in the augmentation of the tyrosine kinase activity of c-Abl. Chronic myelogenous leukemia (CML) is one of several hematological malignancies associated with Bcr-abl expression. The pathogenesis of CML, associated with P210Bcr-abl, is bi-phasic consisting of an initial chronic phase followed by a severe terminal phase referred to as acute blast crisis. The chronic phase of the disease is characterized by granulocytic hyperplasia but in which normal hematologic maturation is still intact. Patients ultimately enter the terminal blast crisis where hematologic maturation is lost, resulting in the accumulation of immature blast cells and a severe immuno-compromised state. Progression to the blast terminal phase is associated with genomic instability demonstrated by the accumulation of genetic and cytogenetic abnormalities. Results from in vitro cell line systems expressing Bcr-abl have suggested that the loss of cell-cycle arrest and induction of apoptosis, as a result of genotoxic stress, might be responsible for this phenotype. In this study, I utilized a transgenic mouse model which expresses P190Bcr-abl to extend those observations to an in vivo model for leukemia. I observed normal cell-cycle arrest and induction of apoptosis following the induction of DNA damage. However, using the Big Blue in vivo mutagenesis mouse assay system, I evaluated genomic instability in P190Bcr-abl mice by measuring mutation frequencies in vivo. I observed an increase in mutation frequencies in spleens and kidneys from P190Bcr-abl mice. This Bcr-abl-induced mutator phenotype may explain the inherent genomic instability associated with the progression of CML and other diseases associated with the expression of activated tyrosine kinases.