The misregulation of post-transcriptional mechanisms has been linked to the development and progression of numerous human diseases, in particular neurological disorders and cancer. Investigating these misregulated RNA pathways is essential to fully understand the disease mechanisms, identify novel biomarkers, and to develop effective therapies. In this thesis, I present three manuscripts that investigate the mechanisms behind the post-transcriptional misregulation of RNA in human disease, with a focus on pre-mRNA splicing. In the first manuscript (Bondy-Chorney et al., 2016a), we investigated the role of Staufen1 (Stau1) in splicing regulation in the neuromuscular disorder Myotonic Dystrophy Type 1 (DM1). Here we report the first insights into the mechanism that Stau1 uses to regulate the alternative splicing of INSR exon 11 through an interaction with Alu elements located in intron 10. Moreover, using a high-throughput RT-PCR screen, we uncovered a number of additional Stau1-regulated alternative splicing events in both wild-type and DM1 myoblast cell lines. As Stau1 is known to be aberrantly upregulated in DM1 skeletal muscle, our findings suggest that Stau1 acts as a disease modifier in this disorder. The second manuscript (Sanchez, Bondy-Chorney et al., 2015), describes a novel role of the protein methyltransferase Coactivator-Associated Methyltransferase-1 (CARM1), a protein found to be overexpressed in Spinal Muscular Atrophy (SMA). We found that CARM1 can act as a mediator in the nonsense-mediated decay pathway (NMD) and associated UPF1 to promoted its occupancy on PTC-containing transcripts. We identified a subset of natural non-PTC containing NMD targets that were dependent on CARM1, a number of which were misregulated in SMA. This work uncovered a novel role for CARM1 in the NMD pathway and revealed that defective targeting of PTC-containing mRNAs should be included in the complex array of molecular defects associated with SMA. Finally, the third manuscript (Bondy-Chorney et al., – in prep) examines the alternative iv splicing regulation of the Protein Arginine Methyltransferase PRMT1 exon 2, an event shown to alter the growth, survival, and invasion of breast cancer cells. Here, we used an RNA interference (RNAi) RT-PCR screen to uncover several splicing proteins that regulate the inclusion of exon 2, several of which we found to be misregulated in a panel of breast cancer cell lines and patient tumours. These findings confirmed that the inclusion of PRMT1 exon 2 was regulated by alternative splicing via splicing factors that are altered in breast cancer. Moreover, depletion of one of these splicing factors, RALY, resulted in a decrease in the motility and invasive potential of an aggressive breast cancer cell line. These three manuscripts represent a collection of work focused on elucidating the mechanisms involved in post-transcriptional misregulation of RNA in three diverse human diseases. Taken together, the data presented here highlight the broad impact that proteins, such as Stau1 and CARM1, can have in neuromuscular disorders. Moreover, we also uncovered novel misregulation of splicing proteins that alter alternative splicing patterns in breast cancer. Elucidating these mechanisms is of the highest importance in order to identify potential new and effective treatment avenues.

Breast cancers frequently metastasise to the skeleton where they cause osteolytic bone destruction. Effective treatment of bone metastasis remains a considerable clinical challenge. In the UK around 70% of the 12,000 patients that die from breast cancer annually have bone metastasis. Whilst existing therapies provide some pain relief, by limiting the tumour-mediated bone degradation, bone metastases are presently incurable. There is therefore an urgent need to develop therapies to prevent bone metastatic breast cancer. The transcription factor complex Runx2/CBFβ is a key regulator of bone development and is aberrantly expressed in breast cancer, leading to up-regulation of bone metastasis-associated genes. Previous work has demonstrated that Runx2/CBFβ determines the invasive phenotype of metastatic breast cancer cells and is required for the expression of metastatic genes. The Runx2/CBFβ complex also has a role in normal breast gene expression, activating expression of the milk protein β-casein in response to hormones. However, little is known about the normal role of Runx2/CBFβ in breast cells. The overarching aim of this project was to determine the role of Runx2/CBFβ in metastasis and identify the target genes that determine the metastatic phenotype. In order to understand the role of Runx2/CBFβ in breast cancer, initial experiments were performed to determine the role of Runx2/CBFβ in normal breast cells. A 3D culture system was established to examine the role of Runx2/CBFβ in regulating gene expression in non-cancerous differentiated epithelial breast cells. Attempts were also made to determine the Runx2/CBFβ target genes after lactogenic hormone stimulation. Unfortunately siRNA knockdown of Runx2 was incompatible with hormonal stimulation. However, 3D cell culture of normal mammary gland cell line HC11 showed Runx2 was expressed throughout the development of mammary acini structures. In addition the expression of CBFβ was confirmed in these cells. Having established the 3D culture system, experiments were subsequently performed to examine the role of CBFβ in the metastatic breast cancer cell line MDA-MB-231. These experiments demonstrated that depletion of CBF has a remarkable effect on the phenotype of the cells, leading to the development of mammary acini structures normally formed by non-cancerous breast cancer cell lines. Thus, depletion of CBF results in a reversion to an epithelial phenotype, suggesting that CBF is required to maintain the epithelial to mesenchymal transition (EMT). RT-PCR analysis also revealed changes in the expression of EMT marker genes. We also demonstrated that the EMT reversion could be rescued by re-expressing an inducible form of CBFβ. These data suggest that CBFβ is required to maintain the mesenchymal phenotype of metastatic breast cancer cells. Finally, a microarray analysis of MDA-MB-231 cells was performed to identify Runx2/CBFβ target genes that might contribute to the mesenchymal phenotype. Cells depleted of CBFβ and grown in 3D revealed reduced expression of IL11. This is known to be involved in bone remodelling. Inspection of the IL11 promoter revealed potential DNA binding sites which confirmed binding to Runx2 using EMSA.

Class of 2016 Abstract / Objectives: To determine the number of patients treated with eribulin who required an alternate dosing schedule other than “day 1/day 8” due to side effects. Methods: Chart reviews were conducted on all patients who met inclusion criteria. Data collected included patient demographics, history of surgery/radiation, number of past chemotherapy treatments, and lab values prior to each eribulin cycle. Results: A total of 37 patients met inclusion criteria for this study. Ten patients were initially started on the “day 1/day 8” schedule and 3 of those patients required a change to the extended “day 1/day 15” schedule. The remaining 27 patients were started on the extended schedule. Conclusions: The number of patients requiring a dosing schedule change due to side effects was not statistically significant. This finding was due to the fact that the majority of patients were started on an alternate dosing schedule in the beginning of treatment. More extensive studies would be required to determine if a majority of patients would require this alternate dosing schedule, and if this should be initiated in all patients starting on eribulin.

M.Tech. (Biomedical Technology) / The uncontrolled growth of cells in the body is often associated with cancer. It constitutes a major health problem and is one of the leading causes of death in the world. Cancers of the lung, breast, colon/rectum and prostate are no longer only associated with developed countries but are the most common occurring cancers worldwide. Breast cancer is the leading cancer faced by women in South Africa as well as in the world. Conventional cancer therapies often result in uncertain outcomes with numerous side effects and may be associated with limited therapeutic advantage. This has led to the development of safer and better treatment regimes with improved therapeutic outcomes. Photodynamic therapy (PDT) is a treatment used for a wide range of conditions, including cancer. This treatment utilises a photosensitiser (PS), a light activated chemotherapeutic agent, and light of a specific wavelength and power density. It is based on the selective tumour localisation of the PS and the ability to generate high levels of reactive oxygen species (ROS) in the presence of light. The generation of ROS causes permanent damage to the tumour cells resulting in cancer cell death. The distinctive criteria when comparing different PDT modalities is the choice of PS as the treatment outcomes are greatly influenced by the light dependent properties of the chemotherapeutic agent. Phthalocyanines are second generation PSs used in PDT. Effects of members of this PS family have been studied and they exhibited good photosensitising properties including lack of cytotoxicity in the absence of light, extended retention times in the tumour and high triplet lifetime of singlet oxygen species.

Recently developed potent and selective CDK4/6 inhibitors fall into two classes based on structure and toxicity profiles in clinical studies. One class, exemplified by palbociclib and ribociclib, exhibits neutropenia as a dose-limiting toxicity and requires discontinuous dosing. In contrast, the structurally distinct CDK4/6 inhibitor abemaciclib is dosed continuously, and has diarrhea and fatigue as dose-limiting toxicities. In preclinical models, palbociclib has been extensively studied and induces cell cycle inhibition in an RB-dependent manner. Thus far, abemaciclib has been less extensively evaluated. We found that abemaciclib cell cycle inhibitory activity is RB-dependent at clinically achievable concentrations. Abemaciclib elicited potent suppression of RB/E2F regulated genes associated with prognosis in ER-positive breast cancer. However, unlike palbociclib, at 250nM-1 mu M doses abemaciclib induced cell death in RB-deficient cell lines. This response was associated with a rapidlyinduced multi-vacuolar phenotype indicative of lysosomal membrane permeabilization that could be ameliorated with chloroquine. This event was not a reflection of inhibition of other CDK family members, but could be recapitulated with CBX4945 that inhibits casein and DYRK/HIPK kinases. To determine if these "off-target" features of abemaciclib were observed in vivo, mice harboring matched RB-positive and negative xenografts were treated with palbociclib and abemaciclib. In vivo, all of the apparent activity of abemaciclib was RB-dependent and strongly elicited suppression of cell cycle regulatory genes in a fashion markedly similar to palbociclib. Using gene expression data from cell lines and tumors treated with abemaciclib and palbociclib a composite signature of response to CDK4/6 inhibition was developed that included many genes that are individually required for tumor cell proliferation or viability. These data indicate that while abemaciclib and palbociclib can exert distinct biological and molecular effects, there are common gene expression features that could be broadly utilized in measuring the response to CDK4/6 inhibition.

Magister Artium - MA / This study explored the social support provided by family of women diagnosed with early stage breast cancer. The aim was to present an account of social support that is solicited by and given to women diagnosed with early stage breast cancer. There is a dearth amount of literature on support from families of women with breast cancer in South Africa. Thus, this study can potentially contribute to filling the gap of knowledge in this area in the country. Qualitative method was used and in-depth interviews were utilized to study the women's experience of family social support. The theoretical framework underpinning the study is the relational communicational perspective.

Philosophiae Doctor - PhD / The human epidermal growth factor receptor (HER)-family of receptor tyrosine kinases; human epidermal growth factor receptor 1, human epidermal growth factor receptor 2, human epidermal growth factor receptor 3 and human epidermal growth factor receptor 4 (EGFR/HER1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4) plays a major role in the pathogenesis of many solid tumours, in approximately 25 - 30% of breast cancers. Breast cancer is the second most common type of cancer and affects around 3000 women annually in South Africa alone. While the benefits of treatment and cancer progress to enhance therapeutic effectiveness for the patient are well documented, it is also important to employ or fabricate methods in which cancer can be screened at an early stage. A number of gene and protein based biomarkers have shown potential in the early screening of cancer. One specific biomarker that is over-expressed in 20 - 30% of human breast cancers is the human epidermal growth factor receptor 2 (Her2/neu). Several methods have been developed for detection of Her2/neu oncogene including immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), fluorescent in situ hybridisation (FISH) and polymerase chain reaction(PCR). However, these methods are subjected to interference problem. For these reasons an ultrasensitive, cheap and easy to use genosensor has been developed for early detection of the Her2/neu oncogene using electrochemical and spectroscopic methods. Due to their high surface-to-volume ratio, electro-catalytic activity as well as good biocompatibility and novel electron transport properties quantum dots are highly attractive materials for ultra-sensitive detection of biological macromolecules via bio-electronic or bio-optic devices. In this study a quantum dots (QDs)-based genosensor was developed in which Ga2Te3-based quantum dots were synthesised using a novel aqueous solution approach by mixing 3-mercaptopropionic acid (3MPA)-capped gallium metal precursor with reduced tellurium metal. The morphological, compositional and structural characterisation of the QDs was investigated prior to their utilization in DNA sensor construction.

Magister Scientiae - MSc / In the present study we report the synthesis and characterization of iron oxide magnetic nanoparticles doped with zinc in an attempt to enhance the magnetic properties. The nanoparticles were prepared via the co-precipitation route and capped with 3-phosphonopropionic acid (3-PPA). The amount of zinc dopant was varied to yield nanoparticles with the general formula ZnxFe3-xO4 (x=0, 0.1, 0.2, 0.3, 0.4). Characterization was carried out using high resolution transmission electron microscopy (HRTEM), X-ray diffraction spectroscopy (XRD), energy-dispersive X-ray spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and superconducting quantum interference device (SQUID) analysis. Results from HRTEM, XRD and SQUID confirm that doping took place and x=0.2 was found to be the doping limit for these nanoparticles with a maximum size of 10.73 nm and saturation magnetization of 73.37 emu/g. The EDS further confirmed successful doping with zinc, while FTIR and TGA confirmed successful capping with 3-PPA. Despite agglomeration at all doping levels, these nanoparticles show great potential for application in breast cancer diagnostic studies.

Philosophiae Doctor - PhD / The current diagnostic, prognostic, predictive and therapeutic monitoring methods used for breast cancer are limited. Thus, research into more specific, sensitive and effective strategies is required. Breast cancer is the most prevalent form of cancer in women worldwide and accounts for the most common cause of death in women every year. Cancer development is characterized by a wide spread of genetic abnormalities of gene sequences that can be used in detecting and monitoring treatment of the disease as a result of altered gene expression patterns which leave a trail of biomarkers. Seven candidate genes (Gene 1-7) were identified from a previous in silico study and their gene products (BRG 1-7) were annotated to be good candidate breast cancer biomarkers. Differential gene expression analysis using quantitative real-time PCR (qRT-PCR) validated the over-expression of Gene 3, Gene 4 and Gene 7 in a breast cancer cell line (MCF7), of which Gene 7, annotated as a Natural killer group 2, member D (NKG2D) ligand, was observed to be the most over-expressed gene. The innate immune system is the first line of the body's physiological defense against diseases and the natural killer (NK) cells, are central to mediating this type of immunity. NK cells are activated when a specific surface receptor such as the NKG2D receptor binds its ligands expressed by tumor cells. To evade being detected by the immune system, cancer cells are reported to shed off the NKG2D ligands and are expected to be present in the bodily fluids of cancer patients. Also, chemotherapeutics have been reported to suppress the natural anti-tumour immune response, thus should be taken into account when designing optimal therapy for cancer patients. The aim of this research was to validate these candidate genes as effective breast cancer biomarkers using several in silico methods as well as molecular techniques and study the effect of Gene 7 on modulating the effect of several pro-apoptotic compounds. The in silico part of the study investigated the functional, protein interaction, pathways, and tissue expression specificity of the candidate biomarkers using computational software such as DAVID, STRING, KEGG, Genecards and GEA. Also an in silico validation of the prognostic/predictive values of the genes was analysed using SurvExpress, KMplot, and GOBO. Protein expression of selected genes was analysed by Western blot, and immunofluorescence analysis. BRG 7 gene was cloned into pcDNA3.1 vector using recombinant DNA technology while commercial shRNA construct was used to 'knock-down' Gene 7 expression. The two constructs were used to transfect MCF-7 and MCF-12A cells. Over-expression and 'knock down' Gene 7 in transfected cells was confirmed using western blot analysis. Stably transfected cells were then treated with three pro-apoptotic compounds (Camptothecin, Doxorubicin and DMSO) for 24 hours. The apoptotic cells were stained with 3, 4, 5, 6-tetrachloro-2', 4', 5', 7' tetraiodofluorescein (TCTF) and then analysed using flow cytometry. Functional analysis linked Gene 1, Gene 2, Gene 4, Gene 6 and Gene 7 to different cancer related processes. The pathway analysis showed Gene 1, Gene 2, Gene 4 and Gene 7 were involved in pathways that can be linked to cancer modulation. The protein-protein interaction analysis showed only BRG 2 was directly linked to two major hallmarks of cancer (Apoptosis and Autophagy). Breast cancer associated Transcription factors were shown to regulate these genes. Gene 1 and Gene 5 as well as the three genes observed to be highly expressed in the qRT-PCR study were validated to differentially express in breast cancer. An additional protein (BRG 8) was identified and postulated to be a good biomarker candidate for breast cancer based on its direct interaction with BRG 7 and estrogen receptor protein (ESR). The prognostic value of the candidate genes were monitored in two datasets (DATA1 and DATA2) in SurvExpress. DATA1 showed that Gene 6 and Gene 8 while DATA2 showed that Gene 3, Gene 6 and Gene 7 were valuable candidate genes in breast cancer prognosis. The survival curves from the two datasets showed the combined genes could predict the outcome of breast cancer patients undergoing treatments. A plot box output from SurvExpress showed most of the genes were differentially expressed comparing two risk groups. The Kaplan Meier plotter confirmed, Gene 1, Gene 3, Gene 4 and Gene 7 have a significant P-value in predicting the survival outcome based on gene differential expression value. GOBO analysis showed the genes may accurately predict the survival outcome of estrogen positive subtype, ERBB2 subtype of estrogen receptor negative and lymph node negative subtype of ER- tumours, but not all subtype of ER- tumours. Western blot analysis showed BRG 7 may be highly expressed in MCF-7 as compared to MCF-12A, BRG 8 was found to be expressed in all cancer cell types analyzed except for MCF-7 and HT29. BRG 2 was found to be expressed in all cancer types analyzed. immunofluorescence analysis showed BRG 3, BRG 4 and BRG 7 are differentially expressed in breast cancer cell line and are more localized on the cell membrane when compared to the breast non-cancer cell line. Over-expression and gene knock down in cells were successfully confirmed with Western blot analysis. Stably transfected MCF-12A cell for over-expression of BRG7 protein, resulted in cell senescent and the cell stopped growing while stably transfected MCF-7 over-expressing BRG7 did not show any morphological changes. Apoptosis was enhanced in cells treated with camptothecin, doxorubicin and DMSO overexpressing BRG7. Apoptosis was reduced in camptothecin and DMSO treated gene 'knock-down' cells but not doxorucin treated. BRG7 gene 'knock down' in transfected cells showed varying response to all three pro-apoptotic compounds. From this study Gene 3, 5, 7 and 8 and their protein levels were confirmed to be differentially expressed in breast cancer cells and could serve as putative biomarkers for breast cancer. However the variance in the effectiveness of individual genes suggests that the set of genes would perform better than individual gene. The modulating role of BRG7 in drug induced apoptosis, suggest it could probably play an important role in personalised medicine and could serve as a biomarker to monitor the prognosis and/or therapeutic outcome of pro-apoptotic drugs in breast cancer patients. These findings will be further investigated in human breast tissues to validate these data.

Breast cancer is increasingly threatening the health of Chinese women. However, little is known about beliefs, attitudes or health practice related to breast cancer among Chinese women in the UK, which means that it is difficult for health professionals to provide evidence-based services to them. Using a triangulation research design, this study investigated the views on breast cancer among Chinese women in the UK. Moreover, factors influencing their views and the factors potentially threatening Chinese women's breast health were also examined. Chinese women who accessed the National Health Service (NHS) in the UK for primary care and were aged from 50 to 70 years were targeted. Twenty-two semi-structured interviews were conducted initially. Based on interview findings, a questionnaire was developed and a questionnaire-based survey was undertaken in a larger population. The findings showed that although negative views on breast cancer were held by the majority of the Chinese women, most women engaged in a healthy lifestyle and actively attended breast screening in order to promote health and prevent breast cancer. Breast cancer views were significantly influenced by the extent to which these Chinese women had been in contact with western culture. Chinese women who were more acculturated to western culture were more likely to hold positive views on breast cancer. Two factors potentially threatened Chinese women's breast health, which were lack of knowledge on breast and barriers to accessing health services. In order to promote early detection, practice nurses in communities need to develop culturally sensitive educational intervention to improve Chinese women's knowledge on breast cancer, in particular on warning signs and risk factors. In addition, barriers to accessing health services need to be tackled. In particular, interpretation services should be provided to those with limited English proficiency.