Cutaneous fatty acid-binding protein (C-FABP) or FABP5, is a FABP family member that can bind to long chain fatty acids with high affinity. C-FABP was identified by our research group as a gene involved in malignant progression of prostate cancer and able to promote the growth of primary tumours and induce metastasis when transfected into rat benign Rama 37 model cells. It was demonstrated that C-FABP was a prognostic marker for patient outcome and a target of tumour-suppression for prostate cancer. As an initial step to understanding the complex molecular mechanisms involved in the cancer promoting activity of C-FABP, this study investigated the possible relationship between tumorigenicity-promoting activity of C-FABP and its fatty acid-binding capability. After single and double mutations were generated in the fatty acid binding motif of the C-FABP cDNA, wild type and mutant C-FABP cDNAs were excised from the mammalian vector pIRES2-EGFP and inserted into pBluescript cloning vector to generate a complimentary restriction sites at both ends of the cDNAs. The C-FABP cDNAs were excised from the pBluescript vector with KpnI and PstI and inserted into pQEs expression vector to form three constructs that express the wild type and two mutant C-FABPs (C-FABP-WT, C-FABP-R109A and C-FABP-R109/129A), respectively. SDS-PAGE and sequence analysis confirmed the correct insertion of C-FABPs into expression vector pQE. The pure recombinant proteins were subsequently produced and purified. The importance of fatty acid-binding activity to the cancer promoting function was assessed by comparing the cancer promoting abilities exerted by C-FABPs with different fatty acid-binding capabilities. To test whether the recombinant proteins produced were biological active, the fatty acid binding ability of wild type and mutant C-FABPs were tested. When fatty acid binding ability of the wild type C-FABP is set at 1, the average fatty acid binding ability the C-FABP-R109A and C-FABP-R109/129A were significantly reduced to 0.32% and 0.09%, respectively (Student t-test, P<0.005). These results suggested that fatty acid binding ability of C-FABP depends on the structured integrity of the binding motif. Thus, changing one amino acid in the motif significantly reduced the fatty acid binding ability by 68%, and changing two amino acids almost completely abolished the fatty acid binding ability of C-FABP. To access the importance of the structural integrity of the fatty acid binding motif to the tumorigenicity-promoting activity of C-FABP in prostate cancer, the effect of wild type and mutant C-FABPs on cell proliferation, invasion and colony formation as indication of tumourigencity were analysed. The average growth rate of cells stimulated with C-FABP-WT was significantly increased by 17% (Student t-test, P<0.05) when compared to control cells. Whereas, the average growth rate of cells stimulated with C-FABP-R109A and C-FABP-R109/129A were significantly reduced by 33% and 47%, respectively (Student t-test, P<0.005) when compared to control cells. The invasiveness of cells stimulated with C-FABP-WT, C-FABP-R109, C-FABP-R109/129A and the control cells were 256±40, 163±32, 80±26 and 96.6±15.2, respectively. The number of invaded cells stimulated with C-FABP-WT was the highest in all cell lines and more than 2.6-fold higher than the number of invaded cells from control (Student t-test, P<0.05). The average number of colonies produced in the soft agar by selected cells stimulated with C-FABP-WT, C-FABP-R109A, C-FABP-R109/129A and control were 1050±132.29, 283±76.38, 157±38.1 and 155±68.74, respectively. In comparison with the control, the average number of colonies produced by C-FABP-R109A stimulated cells was increased by 45% (Student t-test, P<0.01) whereas the average number of colonies produced by C-FABP-R109/129A stimulated cells was at same level as the control cells (Student t-test, P>0.5). The most significant change occurred in C-FABP-WT stimulated cells that produced more than a 6.7-fold (85%) increase in the number of colonies formed in soft agar when compared to controls (Student t-test, P<0.001). These results showed that the increased wild type C-FABP stimulation in prostate cell line significantly increased cell proliferation, invasiveness, and tumorigenicity. Whereas, the increased expression of both mutant forms of C-FABP did not significantly affect these characteristics. Overall, this study has confirmed that the biological potential of C-FABP to promote tumourigencity of prostate cancer cells depends on its ability of binding to fatty acids. Thus, C-FABP may facilitate cancer development through a mechanism involving transportation of intracellular fatty acids into cells. These results were supported by our recent data obtained from in-vivo studies performed in a nude mouse model.

Sustained, toxic elevations of pancreatic acinar cell cytosolic free calcium ion concentration ([Ca2+]C), such as those observed with supramaximal secretagogue stimulation (CCK) are implicated in acute pancreatitis. However, Cholecystokinin (CCK) has been thought to act only indirectly on human pancreatic acinar cells via vagal nerve stimulation, rather than by direct CCK receptor activation as observed in rodent pancreatic acinar cells. However, in the series of experiments presented here using human pancreatic acinar cells, CCK at physiological concentrations (1-20 pM) elicited rapid, robust, oscillatory rises of the cytosolic Ca2+ ion concentration ([Ca2+]C), showing apical to basal progression in acinar cells, in the presence of atropine and tetrodotoxin. The [Ca2+]C rises were followed by increases in mitochondrial ATP production and secretion, concluding that CCK acts directly on acinar cells in the human pancreas. The earliest pathological mechanisms, such as sustained, toxic elevations of the acinar cytosolic free calcium ion concentration ([Ca2+]C), incriminated in experimental pancreatitis have been previously demonstrated by non-oxidative metabolites of ethanol (FAEE’s), as well as bile salts, at supramaximal concentrations. However, in the clinical situation such hyperstimulation is unlikely to occur. To simulate a more clinically relevant stimulus, pancreatic acinar cells were stimulated with lower doses of FAEE’s and/or bile salts in combination with physiological doses of secretagogues - a process which may precipitate pancreatitis clinically. Illustrated here, the toxic transformation of secretagogue induced physiological Ca2+ signalling occurs with the perfusion of low doses of TLCS and POAEE resulting in cell injury. The intracellular second messengers implicated are IP3, cADPR and NAADP with the IP3 receptor channel pivotal with both toxins. However, as previously demonstrated with supramaximal concentrations of POAEE, if supplementary ATP is added to the intracellular milieu, cellular injury is avoided with continued extrusion of large quantities of Ca2+ from the cytosol indicating functional Ca2+ ATPase pumps. This is not observed in cells which do not receive supplementary ATP. The toxic sustained Ca2+ elevation is also be prevented by the removal of external Ca2+ or blockade of IP3 receptor using caffeine and cell injury is again avoided. Therefore, it may be concluded, that it is the large, sustained toxic [Ca2+]c load which impairs mitochondrial function and ATP production leading to Ca2+ATPase pump failure and ultimately cell death. Lowering sustained intracellular [Ca2+]c by blockade of IP3 receptor channels may reduce cell injury in clinical acute pancreatitis.

Chronic lymphocytic leukaemia (CLL) cells are derived from mature B lymphocytes and are distinctive with respect to overexpression of the classical protein kinase C isoform protein kinase CβII (PKCβII), which is encoded by PRKCB. Expression of PKCβII in CLL plays a vital role in the pathogenesis of the malignant cells in this disease, and within the microenvironment cells where it provides signals for the production of factors which support the survival of CLL cells. In CLL cells PRKCB transcription is stimulated by vascular endothelial growth factor (VEGF) through a mechanism involving activated PKCβII. However, at the beginning of this thesis the molecular regulatory mechanism(s) governing expression of the PKCβ gene were poorly described. Thus, to characterise the factors regulating PRKCB transcription in CLL cells I used different approaches including mithramycin treatment, a drug which intercalates into GC-rich areas of DNA to inhibit binding of specificity protein 1 (Sp1), specific Sp1 siRNA, promoter function assays and site directed mutagenesis and chromatin immunoprecipitation (ChIP). Experiments using these techniques showed that Sp1 has a direct role in driving expression of the gene coding for PKCβII in CLL cells. My results also show that Sp1 is highly associated with the PRKCB promoter in CLL cells compared to that in normal B cells, and suggest that this is likely because of the presence of histone marks permissive of gene activation. Examination of other transcription factors such as Sp3, MITF, RUNX1 and E2F1 that potentially bind the PRKCB promoter showed that they have static or indirect effects in regulating transcription of this gene. The exception to this is STAT3 which my data suggests plays a role in suppressing PKCβ gene expression in CLL cells. Exploration of the mechanism through which VEGF induces PRKCB transcription revealed that this growth factor stimulates increased association of Sp1 and decreased association of STAT3 with the PRKCB promoter. Thus, VEGF-stimulated activation of PKCβII may play a role in this process. Taken together, Sp1 is the major driver for overexpression of PKCβII in CLL cells, and because this transcription factor is also overexpressed in these cells, the mechanisms I describe controlling PRKCB transcription potentially provide a foundation for further study of other genes contributing to the phenotype of CLL cells that are regulated by this pleiotropic transcription factor.

Purpose: The objectives of this pilot study for women breast cancer survivors with lymphedema was 1) to evaluate recruitment rates, retention rates, adherence to Bowenwork (a noninvasive complementary therapy involving gentle muscle movements), home exercises, safety and comfort; 2) determine the effect of Bowenwork on quality of life (QOL), functional status, perceived pain, range of motion (ROM), arm/ankle circumference (to assess for localized and systemic changes). Methods: Participants received 4 Bowenwork sessions with home exercises. Initial and post assessments included QOL, functional status, and pain. ROM, arm/ankle circumference and pain measures were recorded before each session. Results: Twenty-one women enrolled in the study; 95% completion; adherence 100%; home exercises 95%; no adverse events. The intervention improved mental health (SF-36-MCS); breast cancer-related functional (FACT-B); increased ROM; reduced arm circumferences. P value set at <0.05. Conclusions: The Bowenwork intervention was safe and acceptable for women breast cancer survivors with lymphedema.

Cervical cancer is the third most commonly diagnosed cancer globally and it has also been identified as one of three AIDS defining malignancies. Highly active antiretroviral therapy (HAART) is a combination of three or more antiretroviral drugs which are classified as nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs). HAART has been shown to play a significant role in reducing the incidence of some AIDS defining malignancies, although its effect on cervical cancer is still unclear. It is hypothesized that HAART might reduce cancer risk by interacting with different signalling molecules and pathways that are involved in cancer in order to induce cell death and thus inhibit cell proliferation. The broader aim of this study was to understand the relationship between cervical cancer and HAART. This was achieved by studying the expression of key signalling molecules in cancer; MUC1 and NFkB (P65) and morphological features using scanning electron microscopy following 24 hour treatment of a cervical cancer cell line, HCS-2 with drugs which are commonly used as part of HAART; Emtricitabine (FTC), Tenofovir disoproxil fumarate (TDF), Efavirenz (EFV), Atripla combination (ATP) and Kaletra combination (LPV/r) at their clinical plasma concentrations. Quantitative real time polymerase chain reaction (qPCR) was used in order to study the gene expression of MUC1 and P65 and the data was analysed using the 2-ΔΔCT method to calculate fold change. The statistical analysis was conducted using JMP 11 software. MUC1 and P65 gene expression was reduced following drug treatment. Protein expression was studied by means of Immunofluorescence and MUC1 and P65 protein expression was reduced following drug treatment. Scanning electron microscopy revealed characteristic features of apoptotic cell death such as loss of cell contacts, reduced density and size of microvilli, increase in surface blebbing and budding and degradation of apoptotic bodies following treatment with all the drugs. In conclusion, the drugs used in this study

Thesis (MSc.Med.(Urology))--University of the Witwatersrand, Faculty of Health Sciences, 2014. / Bladder cancer is the second commonest urological malignancy and haematuria is the commonest symptom. Cystoscopy and urine cytology are integral for the investigation of haematuria, while the role of molecular markers such as the NMP22 BladderChek test is still being defined. The BladderChek is a qualitative point of care test developed for the detection of the elevated urinary levels of NMP22 associated with bladder cancer. No studies have been performed in South Africa using the BladderChek nor considered using this test to increase the efficiency of the workup of patients with gross haematuria. The primary aim was to establish the percentage of office cystoscopies done as part of a gross haematuria workup at Charlotte Maxeke Johannesburg Academic Hospital that are unnecessary and may be avoided if the BladderChek is positive under defined conditions. A cross-sectional study of the BladderChek test using prospective consecutive sampling, with special care to limit false positives and negatives, of 64 patients with a history of gross haematuria was conducted. The sensitivity, specificity, positive predictive value and negative predictive value for the BladderChek and the urine cytology were 78.9%, 84.4%, 68.2%, 90.5% and 36.8%, 93.0%, 70.0%, 76.9% respectively. The performance of the BladderChek was not affected by the history of gross haematuria, the stage nor grade of malignancy. Urine cytology detected only one malignancy missed by the BladderChek. Approximately 12.6% of office cystoscopies may be avoided and 78.9% of bladder tumours detected if the BladderChek is selectively applied as in this study. This may “fast-track” patients for transurethral resection of bladder tumour. The BladderChek may be a cost-effective alternative to urine cytology.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, February 2015 / Androgen Insensitivity Syndrome (AIS) is an X-linked disorder caused by mutations in the androgen receptor (AR) gene. The phenotype is variable and ranges from a complete feminine syndrome to simple gynecomastia. The phenotypes are described in terms of complete, partial and mild forms (CAIS, PAIS and MAIS). We describe novel and previously reported (recurrent) mutations in the AR gene for a family in which segregation of breast cancer (BC) and gynecomastia/MAIS is present. Methods: We studied a family of 16 members spanning four generations. Based on the presentation of symptoms, the family was divided into affected, unaffected, and control groups. Seven patients (six males diagnosed with MAIS and one female diagnosed with BC) formed the affected group, four genetically related individuals (two males and two females) formed the unaffected group and five genetically unrelated family members (one male and four females) served as controls. In each of these individuals, PCR amplification, cloning and the sequencing of exon 1 were carried out. Exons 2-8 were sequenced directly after PCR amplification. Exon 1 (CAG)n and (GGN)n repeats were classified according to their length: short (S) (n<23), long (L) (n>23) and wild type (WT) (n=23). Results: Part 1-The (CAG)n repeats varied among individuals and generations. In the 2nd generation, the unaffected male was S and the control female was WT. In the 3rd generation, three affected males were S, 2 of the controls were WT, one control was L and the other S. In the 4th generation, the 4 affected individuals were L, 1 of the unaffected was WT and the other 2 unaffected were L. Part 2- The (GGN)n variations also differed among individuals and generations. In the 2nd generation, the unaffected male and the control were S. In the 3rd generation, all three affected family members were S and among the controls, 1 was WT, 1 was L and 2 were S. In the 4th generation, 3 of the affected were S and one was WT and among the 3 unaffected, 2 were S and one was WT. Part 3- 30 unreported (novel) mutations as well as 13 recurrent (previously reported) mutations in exon 1 of the AR gene were identified. 17 novel and 5 reported mutations were identified in the affected group, 8 novel and 5 reported mutations, including one premature stop codon mutation, were identified in the related unaffected group and 7 novel and 4 reported mutations were found in the controls. Of the above-mentioned mutations, four mutations were identified in the activation function-1 (AF-1) domain of exon 1 in 4 members (3 affected: M-2, F-1 and 1 unaffected: F-1) of the family. All the point mutations identified were somatic in nature and were present in heterogeneous form i.e wild and mutant (mixture) as determined by cloning. The analysis of exons 2 through 8 revealed completely WT sequences. Conclusions: The (CAG)n and (GGN)n repeat analysis showed an indeterminate association with MAIS and BC in the family. Generation specific patterns of (CAG)n were detected and suggest generation specific modulation of the AR. Novel mutations including AF-1 region mutations were identified in exon 1. The disruption of the AF-1 domain may affect the transactivation activity of the AR.

A thesis submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy FACULTY OF HEALTH SCIENCES UNIVERSITY OF THE WITWATERSRAND, JOHANNESBURG 2014 / Breast tumour progression involves complex interactions between malignant cells and the tumour microenvironment. It is increasingly apparent that immunity is a critical determinant for tumour progression. T regulatory (TREG) lymphocytes, which dominate tumour infiltrating lymphocyte populations, are implicated in facilitating tumour immunoediting processes and suppressing Natural Killer (NK) cell anti-tumour function. To investigate such cellular interaction, experimentation traditionally involves using reductionist 2-dimensional culture systems that do not recapitulate the spatial dimensions of the in vivo microenvironment. Three-dimensional (3D) culture systems, conversely, recreate these dimensions, allowing tumour cells to assume a phenotype more representative of the tumour microenvironment. Given that immunity is a critical factor in determining tumour progression, a novel 3D culture system was established to investigate the interactions between TREG lymphocytes, NK cells and hormone-dependent (MCF-7) or hormone-independent (MDA-MB-231) breast cancer cells. Lymphocyte subpopulations were magnetically isolated, with the efficacy of the sorting procedure verified using flow cytometry. To generate 3D cultures, cell populations were resuspended in growth factor-reduced Matrigel and cultured for 72 hours. This culture system proved effective for RNA extraction for downstream applications; for immunolocalisation of selected tumour biomarkers (ER-α, TGF-β, MUC-1 and EGFR) for qualitative analysis; and for acquisition of cytokine data (IL-1β, IL-2, IL-6, TNF-α, IFN-, CCL2, CCL4 and CXCL8) for quantitative multivariate statistical analysis. Immune mediation was shown to induce the disruption of cell-cell associations, altering the expression of biomarkers and secreted cytokine profiles. Collectively, these results reflect tumour cell subversion of NK cell and/or TREG lymphocyte function to promote tumour progression by generating an inflammatory microenvironment. While hormone-dependent and hormone-independent breast cancer cells differed in their specific response to immune mediation, the mechanisms by which they elicited responses resulted in similar outcomes – that of enhanced evasive and invasive capacity. It is necessary to further elucidate the relationship between the investigated cytokines, biomarkers and immune cells, to understand their interactions and potentially provide more information for therapeutic intervention, given that these factors may contribute to tumours not responding favourably to combined modalities of therapy.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Medicine in the branch of Haematology. Johannesburg, March 2015 / The incidence of leukaemia in South Africa is 2.5 per 100 000 and has increased due to HIV. Accurate and timeous diagnosis of leukaemia directly impacts success of patient treatment and consequent survival. Usually the Full Blood count (FBC), white blood cell (WBC) differential count and review of the peripheral blood smear alerts the clinician to the possibility of leukaemia. However the number of qualified and skilled technologists in peripheral and central laboratories is on a continual decline making the performance of the critical function of peripheral blood review a challenge. The Advia 2120 haematology analyser performs a WBC and differential count using principles of flow cytometry and the cytograms generated can be used to classify haematological malignancies through the Peroxidase and nuclear density analysis (PANDA) classification system. The presence of myeloperoxidase (MPO) activity in 3% or more leukaemic blasts confirms acute myeloid leukaemia, and enzyme activity can be detected by immunophenotypic analysis or conventional cytochemistry . Research on the comparison of the Advia 2120 and manual morphologic assessment in the classification of leukaemias is limited in the South African setting, where leukaemia often coincides with infection. The aim of this study was to determine if the FBC, differential count and cytogram assessment by the Advia 2120 using the PANDA classification is as reliable as morphologic assessment in the initial classification of haematological malignancies from peripheral blood samples when using flow cytometry as the gold standard.. 150 cases of confirmed leukaemia were collected. The diagnosis obtained from either PANDA analysis and/or morphological assessment was compared to the diagnosis obtained by immunophenotypic analysis. Secondly, the MPO activity obtained by the Advia peroxidase cytogram was compared to the MPO obtained by conventional methods of immunophenotypic analysis and/or cytochemistry. Using the PANDA analysis system, only 48% (72/150) of cases overall were accurately classified. The inaccuracy was 9.3% (14/150) and 42.7% of cases could not be classified. The positive predictive value (PPV) was 88%. The most significant finding was all of the acute Page | iv promyelocytic leukaemia (APL) cases (8/8) had a distinct pattern and were accurately classified on cytogram analysis alone. Accurate sub-classification of other types of acute myeloid leukaemia using PANDA analysis alone was inconsistent. However, the accuracy in classifying leukaemia was improved when the Advia cytogram was used in conjunction with morphological analysis, as 90% (135/150) of cases were accurately classified. The sensitivity and specificity of the peroxidase cytogram in evaluating myeloperoxidase (MPO) activity was 85% and 88.6% respectively. The agreement between cytogram peroxidase activity and the reference methods was 89.1% and the Cohen’s kappa was 76.9%. To the best of our knowledge, there is no data comparing peroxidase activity on the cytogram to other methods. In conclusion, it was shown that the routine use of the Advia cytograms in conjunction with the morphology findings provides invaluable information in the initial screening of leukaemia. In cases with indistinct morphology, the cytograms have the potential to aid in a provisional classification. The peroxidase activity from the cytogram could be used as a surrogate marker for myeloperoxidase activity in leukaemia. Moreover, a tentative diagnosis of an APL is possible by simple analysis of the cytogram resulting in earlier diagnosis which could be life-saving.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfillment of the requirements for the degree of Master of Medicine in the branch of Internal Medicine / Medical Oncology. Johannesburg 1st September 2014 / Introduction: Tyrosine kinase inhibitors and EGFR mutations has changed the treatment approach to lung cancer globally. This retrospective study will look at factors associated with EGFR mutations and define the EGFR mutation rate in South Africa. Methods: Retrospective record review from NSCLC patients in South Africa who were tested for EGFR mutations at Lancet Laboratories during 1st September 2009 to 30th June 2012. Chi-squared test was used to determine association with categorical variables. Kaplan- Meier survival analysis was done for OS and PFS between EGFR mutation positive and negative patients. Cox proportional hazards were used for subgroup analysis. Treatment practices and response were described. Results: 170 lung cancer samples were evaluable for EGFR mutation and 37 were EGFR mutation positive (21.8%). There were 22 (59.5%) exon 19 deletions, 11 (29.7%) L858R mutations, two G719X mutations, one S768I mutation and one exon 20 insertion. The median age was 63 (range 27-85). There were more females (55.6%) than males (44.4%) sent for mutation testing. Most patients were whites (71%), followed by blacks (18.3%), and other race (10.7%). 85% of all NSCLC samples tested were adenocarcinoma. None of the squamous cell carcinoma tested was positive for EGFR mutation. Smoking status was inversely proportional to EGFR mutation status (p<0.001). Over 60% patients received chemotherapy first and second line and responses decreased with each line of chemotherapy. Median PFS and OS were not different between the EGFR mutation positive and negative groups (6.85 versus 6.8 months; HR 1.6; 95% CI 0.70-3.65; p=0.2543 and 11.5 versus 12.9 months; HR 0.70; 95% CI 0.28-1.75; p=0.44, respectively). On multivariate analysis, only non-white race was associated with decrease in OS (HR 6.66; 95% CI 2.31-19.19; p=0.0004). Conclusion: EGFR mutation rate in South African lung cancer patients was 21.8%. 89% of all EGFR mutations were either exon 19 deletions or L858R point mutations. Most EGFR mutations were associated with adenocarcinoma of the lung in non-smokers. These findings were consistent with current literature in western countries. Treatment practice remained chemotherapy based, with few patients receiving EGFR TKIs. Efforts should be made to prioritized targeted treatment approach in lung cancer in South Africa.