With little recent improvement in osteosarcoma (OS) outcomes, identification of therapeutic targets is critical. RNA interference (RNAi) and drug screens using OS tumour cell lines (TCL) were used to identify novel genetic dependencies and validate tractable therapeutic targets. Cell viability data from RNAi screens in 18 OS TCL was integrated with whole-exome sequencing and protein expression data. Comparison with non-osteosarcoma TCL, demonstrated OS to be more reliant on skeletal morphogenesis pathways and FGFR1/2, with increased sensitivity to FGFR1 inhibitors. OS TCL positive for FGFR1 amplification and polysomy were significantly more sensitive to FGFR1 inhibitors than unknown or non-amplified OS TCL, providing further evidence for a clinical trial in an enriched population. Correlation of RNAi results with the presence of recurrent driver gene alterations revealed that sensitivity to selective silencing of DYRK1A was associated with deficiency of RB1. This finding was validated using RNAi in the OS TCL, an additional 34 breast TCL, and a DYRK1A kinase inactive model. Harmine, a DYRK1A inhibitor, resulted in greater apoptosis in an RB1 deficient OS TCL than in a RB1 wildtype model. DYRK1A has been identified as a protein interaction partner of RB1 and is pharmacologically tractable. Further work is necessary to mechanistically understand this synthetic lethality. The model system was also used as a tool to validate the potential role of BRCAness in OS, recently identified as a potential target in genomic studies. This determined the majority of OS TCL not to be profoundly sensitive to PARP inhibition. However, LM7 (an OS TCL) created by repeated pulmonary murine passage of SAOS2, demonstrated this acquired phenotype. Absence of RAD51 foci in LM7 in contrast to SAOS2, identifies this as a suitable, mechanistically relevant, tool for studying ‘BRCAness’ in OS. Integrated screens provided a framework for pre-clinical identification and validation of tractable therapeutic targets to facilitate translation into development of clinical trials.

Inhibitor of apoptosis (IAP) proteins are negatively regulated by the mitochondrial pro-apoptotic protein, SMAC, through a tetrapeptide binding-motif. A class of drugs mimicking this motif have been developed as anti-cancer agents and have further highlighted IAP signalling in regulation of cell death. These SMAC mimetics (SMs) activate the non-canonical NF-κB pathway, induce TNFα secretion and selectively promote cell death in tumour cells; an effect potentiated in a TNFα-rich microenvironment. However, due to this mechanism of action, SMs have the potential to induce toxicity in non-tumour cells residing in this environment. The aim of this thesis was two-fold: to determine the effect of SMs in non-tumour derived cells both in vitro and in vivo. I determined a robust methodology in sensitive and resistant tumour cells with SMs as single agents. I found that MDA-MB-231 and MDA-MB-468 cells were sensitive and resistant to SMs as single agents, respectively. Furthermore, the pan caspase inhibitor, zVAD, actually inhibited TNFα secretion in combination with the bivalent SM, BV6. In hepatocyte-like and epithelial non-tumour cells in vitro, I found that SMs did not induce cell death, even when co-treated with TNFα. These data suggest that SMs under clinical development do not have significant toxicity in non-tumour derived cells suggesting a large therapeutic window in the clinic. As liver regeneration is also TNFα-dependent, SMs have the potential to exacerbate liver injury. To determine whether the SM, TL-32711, could induce NF- κB activation in vivo, I used mice expressing an NF-κB reporter gene (miceluc) allowing for quantification of NF-κB activity via in situ live imaging. Miceluc were dosed with TL-32711, either alone, or in combination with two hepatotoxins. Strikingly, TL-32711 alone induced significant liver-specific NF-κB activation and protected against liver injury. These data show that SMs could have real clinical relevance for use as a treatment to ameliorate hepatoxicity.

Previous reports indicate resveratrol can modulate many processes and targets in colorectal cancer (CRC) cells and rodent models but it is uncertain whether these effects translate to humans in vivo. The lack of relevant experimental models, particularly premalignant colorectal cells, the major targets for prevention, is a significant obstacle to translation of effective preventive agents to the clinic. Development of improved 3-dimensional organoid models with discrete mutational drivers (Apc, Apc+Kras and Braf) representing different subtypes of early CRC is an aim of this thesis. A further aim is to evaluate resveratrol across these models and examine in greater detail the ability of resveratrol to interfere with the development of cancers driven by mutant BrafV600E. Organoid cultures using intestinal cells isolated from genetically engineered mice and human cancer/adenoma tissue were successfully established and conditions optimised to allow long-term maintenance. Notably, the organoids derived from adenomas arising in Villin-Cre/BrafV600E mice constitute a novel preclinical model of CRC. After first demonstrating that resveratrol had anti-proliferative activity across a panel of human CRC cell lines with different mutation profiles it was tested in the organoids at clinically-achievable concentrations for effects on autophagy, senescence, apoptosis and stem cells. Administration of a high-fat diet to Villin-Cre/BrafV600E mice enhanced Braf-induced cryptal hyperplasia in a short-term study and this was significantly reduced by resveratrol. In a follow-up survival study, high dose resveratrol greatly prolonged the lifespan of Villin-Cre/BrafV600E mice on high-fat diet. However, resveratrol had no effect on the survival of mice given standard diet. Overall, these findings suggest a specific interaction between mutant Braf expressed in mouse intestine and high-fat, and that the effects are blocked by high dose resveratrol. Future studies are needed to investigate underlying mechanisms. Results suggest resveratrol may have value in the prevention of colorectal adenomas/cancers with different genetic alterations, including mutant BrafV600E.

Multiple myeloma (MM) is a life-threatening haematological malignancy, which is rarely curable by conventional therapies. Immunotherapy, using autologous antigen specific cytotoxic T-lymphocytes (ASCTL), may represent a useful adjunct therapy for MM. In this study, I assessed the ability of previously described hybrid cell lines, generated by chemical fusion of myeloma tumour cells and the EBV B-lymphoblastoid cell line (EBV B-LCL) HMy2, to induce ASCTL in vitro from peripheral blood lymphocytes from patients with MM (and from healthy individuals). The tumour associated antigens (TAAs) hTERT, MUC1, MAGE-C1 and CS1 were selected as potential inducers of ASCTL, based on their prevalence of expression in MM patients. Expression of these TAAs was assessed in four B-LCL/myeloma hybrid cell lines, using real time PCR and flow cytometry, and two of the hybrid cell lines were selected as in vitro stimulator cell lines in long-term activated T-cell cultures, using PBMCs from HLA-A2+ healthy donors and multiple myeloma patients. Induction of ASCTLs was assessed by HLA-A2-peptide pentamer staining and flow cytometry, Europium release cytotoxicity assays, and interferon-gamma and perforin ELIspot assays, using known HLA-A2 restricted peptide epitopes of the TAAs. The hybrid cell lines induced ASCTLs to the 4 selected TAAs, after 4 rounds of in vitro stimulation with the hybrid cell lines, in PBMCs from both (HLA-A2+) healthy donors and MM patients. In contrast, the (HLA-A2+) myeloma cell line, U266, failed to activate ASCTL in vitro. Hybrid cell lines, generated by fusion of EBV B-LCL and myeloma tumour cells, can induce ASCTL in PBMCs from healthy individuals and multiple myeloma patients in vitro, and may represent a novel strategy for use in immunotherapy of MM.

Literature review: A systematic review of the existing literature was conducted, eliciting ten studies which met the inclusion criteria examining psychosocial predictors of prophylactic mastectomy in women with a confirmed BRCA gene alteration. Narrative synthesis identified that results coalesced around temporal, familial and other factors including conceptualisation of cancer and perceived risk. The relative scarcity of published research, and an accompanying dominant biomedical focus, highlight that further exploration of psychosocially predictive factors, particularly those which are modifiable, is needed. Research report: Interpretative Phenomenological Analysis (IPA) was utilised to explore five women’s experience and sense-making of their decision to opt for prophylactic mastectomy, and how they experienced the period between opting for preventative surgery and waiting for this to occur. Four superordinate themes were identified: ‘It’s a no-brainer’ illuminated how women approached and made sense of their decision; ‘good breast/bad breast’ reflected women’s experience of simultaneously holding conflicting views towards their breasts; ‘big B on my shoulder’ highlighted worry held in relation to geneticised identity; and ‘the preciousness of life’ illustrated the impact of familial and existential experience. Findings emphasised the importance of clinicians remaining mindful to experiential, emotional and systemic motivations for surgery and to recognise and support women with the potential tension they may still hold as they debate and navigate prophylactic mastectomy. Critical appraisal: A reflective account is presented to support the consolidation of personal and professional learning points and reflections made during the research process.

Conventional B-mode ultrasound provides information on the anatomical features using acoustic impedance differences in the tissues. Ultrasound elastography uses a variety of techniques to map soft tissue elasticity. Tissue stiffness is a novel indicator of the tissue health, as many pathologies can alter the tissue stiffness such as cancer and fibrosis. Accurate and early detection of tissue elasticity can guide towards reliable diagnosis, and prognosis of diseases. The objectives of the research reported in this thesis are to implement strain and shear wave elastography techniques on the locally developed ultrasound systems, along with identifying current challenges in elastography and proposing solutions to develop ultrasound elastography as an accurate, and reliable clinical tool. In the first study, strain elastography was implemented and novel strain estimation quality assessment approach was proposed to discard noisy strain images. The second study proposed a shear wave generation method, called Dual Push Beam (DPB) to address challenges of the current shear wave elastography techniques, such as to reduce data acquisition events and to improve imaging depth. Further, the thesis includes the study which introduced a new angle-aligned shear wave tracking method, which improved displacement estimation quality for shear compounding. Final study designed seven different elastography schemes and investigated variations in elasticity estimation across the image by changing shear waves generation beam parameters such as aperture size and focal depth and its implication for liver fibrosis and breast cancer diagnosis.

Tuberculosis, caused by Mycobacterium tuberculosis (M.tb) is one of the world's most prevalent infectious diseases. The quest for an effective vaccine and an immune correlate of protection are on going. Previous literature reports links between the iron status of the host and disease severity, but the underlying mechanisms are not clear. Here, a murine model of M.tb infection was used to determine the effect of M.tb infection on the expression of hepcidin and other genes involved in the regulation of iron homeostasis. Female BALB/c and C57BL/6 mice were infected with M.tb Erdman via aerosol. Change in expression of iron-responsive genes was measured by liver qRT- PCR. Bacterial burden was determined in organ homogenates. The effect of iron deficiency on disease outcome was also investigated, where mice fed an iron-deficient or control diet were infected with M.tb. To determine whether hepcidin has a role in disease outcome, Hamp1-/- mice were also infected with M.tb. In the time course experiments, changes in hepcidin kinetics post-M.tb infection were observed, which were likely regulated by BMP/SMAD and not IL-6/JAK-STAT signalling. In the iron- restricted diet experiments, iron deficiency did not reproducibly affect bacterial burden. Moreover, deletion of Hamp1 did not affect outcome of in vivo M.tb infection. The data in this thesis indicate that iron homeostasis is altered during M.tb infection, but that altering host iron homeostasis by inducing iron deficiency or deleting the hepcidin gene does not compromise the host's ability to control infection.

This thesis is concerned with the synthesis of small molecules that mimic tetramate- and pyrrolidinone-containing natural products, and the evaluation of their antibacterial and anticancer properties. Chapter 1 exposes the need for new antibiotics due to the emergence of resistance to current drugs, and it discusses the value of the natural product inspired approach for drug discovery. It also gives an overview of the structure, synthesis and biological properties of the natural products pramanicin and oxazolomycin, both of which contain a pyrrolidinone core. Chapter 2 describes an efficient synthetic route which allowed the preparation of a library of acyltetramates via Grignard addition to a Weinreb amide. This route was also used to prepare mimics of the acyl chain of pramanicin, comprising dienones and epoxy-enones of different lengths, and of the left-hand segment of oxazolomycin. In Chapter 3, reduction of the tetramate core of different closely-related systems was performed, and it was found that the diastereochemical outcome could be controlled by appropriate choice of substituents in the bicyclic system. Preparation of reduced acyltetramates was only successful via hydrogenation of the Weinreb amide and subsequent Grignard substitution. Chapter 4 describes the introduction of β-lactones to pyrrolidinones. Conditions for the selective formation of either spiro- or fused-β-lactones were found, which relied on the activation of the precursor β-hydroxyacid, accessed via N,O-acetal deprotection and ester saponification of the bicyclic systems. Further functionalisation of these analogues to mimic oxazolomycin was thwarted by the instability of some of the intermediates. Finally, the biological properties of the analogues are discussed in Chapter 5. The antibacterial activity against Gram-negative E. coli and Gram-positive S. aureus was examined using the holeplate method, which showed that the aliphatic lipophilic tetramates were the most potent inhibitors, with selective Gram-positive activity. An anticancer high-content screen was also designed, but only weak activities of the synthesised compounds were observed against four cancer cell lines.

A variety of epidemiological studies have provided support for an increased leukaemia incidence following exposure to both high and low dose radiation. There is a close histopathological similarity between human and murine acute myeloid leukaemia with mouse models of the disease being most often used. We report here, for the first time, that allogeneic haematopoietic stem cells can compete for niches in the nonmyeloablated NSG bone marrow compartment and that the nonmyeloablated NSG bone marrow microenvironment is capable of supporting allogeneic long-term HSC engraftment and differentiation. Using this NSG transplantation model, we then provided evidence of a reduced contribution of low dose irradiated HSCs towards longterm haematopoiesis. We further report on the generation and characterisation of a novel Chr2MDRmCh mouse model where a construct positioned in the minimal deleted region following radiation exposure carries the fluorescent protein mCherry, which is expressed under control of the ubiquitous Rosa26 promoter. Crossing these mice with Sfpi1GFP mice allowed the early detection of an expanding haematopoietic clone which had lost mCherry fluorescence following radiation exposure (and so also the chromosome 2 homologue carrying the fluorescent construct and the Sfpi1 gene). Finally, we report on a gender-dependent leukaemic progression and characterisation where 3 Gy irradiated male mice only present with acute myeloid leukemia and irradiated female mice mainly develop leukaemia with a lymphoid phenotype. In conclusion, the work in this thesis postulates data obtained from two novel mouse models and which could help to further identify key molecular mechanisms involved in leukaemia initiation and development.

Genomic instability is a hallmark of cancer. The tumour suppressors BRCA1 and BRCA2 play key roles in genome integrity by promoting homologous recombination DNA repair and replication fork stability. Deficiency in these functions has been described as the Achilles heel of BRCA-defective tumours. This provides an important rationale for further exploring the roles of BRCA1 and BRCA2 in DNA replication and genome stability. G-quadruplexes, alternative DNA structures formed by guanine-rich single- stranded DNA, represent natural replication fork barriers. This study demonstrates that treatment with the G-quadruplex-stabilising compound pyridostatin selectively decreases viability of BRCA1- and BRCA2-deficient cells by inducing replication stress and DNA damage. Furthermore, this work identifies a new role of the Fanconi anaemia protein FANCD2 in limiting replication fork progression and genomic instability in human cancer cells lacking BRCA2. This function of FANCD2 is vital for BRCA2-deficient cell survival and affects treatment responses. Finally, the data presented here reveal a synthetic lethal interaction between MRE11 nuclease and BRCA2. Characterisation of a novel chemical MRE11 nuclease inhibitor with activity on 2D and 3D cell culture models for BRCA2 deficiency highlights the clinical relevance for the use of MRE11 inhibitors for targeting BRCA2-deficient tumours. In summary, this report describes novel roles of BRCA1 and BRCA2 relevant for selective targeting of BRCA-deficient tumour cells.