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Kawasaki disease (KD) is a multisystem vasculitis that results in localized coronary artery elastin breakdown and aneurysm formation. It is the leading cause of acquired heart disease of children in North America. Despite conventional treatment, a significant proportion of patients continue to develop coronary sequelae. The mechanisms of arterial aneurysm formation in KD are not known. Using a murine model of KD, Lactobacillus casei cell wall extract-induced coronary arteritis, the processes leading to coronary aneurysm formation were examined. Vessel damage occurred as a result of the increased enzymatic activity of the elastase, matrix metalloproteinase (MMP)-9. MMP-9 protein and activity levels were elevated in the heart post-disease induction. Expression and activity were specific for and localized to inflamed coronary arteries. The pro-inflammatory cytokine, tumour necrosis factor (TNF)-α, was required for increasing local MMP-9 expression. Importantly, MMP-9-deficient animals had a significantly reduced incidence of elastin breakdown. Furthermore, in a cohort of KD patients, serum MMP-9 did not correlate with coronary outcome, highlighting the importance of local expression of this elastase. Intravenous immunoglobulin (IVIG) and aspirin/salicylate are therapeutic agents in current use for the treatment of KD, though their exact mechanisms of action in KD are not known. The biologic effects of IVIG and salicylate on critical stages of disease development were examined. IVIG and salicylate had differential effects on TNF-α expression, with therapeutic concentrations of IVIG inhibiting, and salicylate inducing, TNF-α expression leading to an indirect modulation of MMP-9 expression. Interestingly, TNF-α expression and MMP-9 activity were both directly inhibited by the metal-chelating drug doxycycline. Treatment of affected mice with doxycycline significantly improved coronary outcome. Inhibiting both the inflammatory response as well as the downstream effects of inflammation were of therapeutic value in this model of KD. These results taken together demonstrate the importance of MMP-9 in the pathogenesis of coronary artery aneurysms in KD. Targeting MMP activity holds the promise of transforming KD from the leading cause of acquired heart disease to a self-limited febrile illness.

Hyaluronic acid (HA) is one of the major components of the extracellular matrix; and may exhibit different biological functions, dependent on polymer molecular weight (MW). The signalling events performed by HA are mediated through interactions with its main cell receptors: CD44 and RHAMM. However, the direct effect between the HA MW and the expression of CD44 and RHAMM remains unclear. This study aimed to investigate whether different HA polymer MW alters the proliferation of tumour-derived cell lines, and whether different HA-sized has an effect on the regulation of the expression of CD44 and RHAMM. In order to determine size-specific responses of tumour cells of defined fragment MW, this investigation was undertaken using HA-tethered culture surfaces. Four surfaces were constructed, coated with polymers of different MWs. HA (4, 234, 2590 kDa) and an oligomer mixture were tethered onto an aminosilane (AHAPTMS)-treated glass surfaces using a carbodiimide reaction. Surfaces were analysed using a toolbox of in situ characterisation techniques, including wettability measurements, QCM, AFM and confocal microscopy. Using the constructed surfaces was demonstrated that HA-polymer MW modulates cell proliferation of human bladder (RT112 and T24) and prostate (PC3 and PNT1A) cell lines, with low HA MW (HA4) increasing proliferation, whereas a decrease is seen in the presence of medium (HA234) and high MW fragments (HA2590). The proliferation stimulus performed by HA was found to be phenotype dependent, with HA4 surfaces stimulating an increased proliferation in those less invasive cell lines (T24 and PNT1A), while HA234 and HA2590 inducing a sharper decrease in the most malignant tumour cell lines (RT112 and PC3). It was also demonstrated that the regulation of CD44 and RHAMM transcripts expression appears to be phenotype dependent but not HA-MW dependent. HA down-regulates CD44 and RHAMM in the most malignant cell lines; with up-regulation of the expression of the cell receptors in the less invasive cell lines. In addition, the presence of exogenous HA was shown to be involved in the regulation of the expression of CD44 variants expression. The results obtained for the CD44 and RHAMM protein expression were also found to be correlated with the obtained transcripts expression. However, the significance of these findings in tumourigenesis remains unclear. Findings from this investigation may help in the design and development of biocompatible implants with controlled surface properties to be used in cancer therapeutics; with medium and large HA polysaccharides being potential biopolymer candidates, useful for the development of novel therapies for highly invasive cancer. In addition, implications from this work can serve as a base for future research, and can lead to ideas for drugs and methods to be used in cancer therapeutic approaches.

The transcription factor Interferon Regulatory Factor-1 (IRF-1) has been demonstrated to suppress tumour growth through the regulation of many anti-oncogenic genes. Pro- and anti-apoptotic factors, cell cycle control genes, DNA damage response genes and prometastatic factors are all under the control of IRF-1, which effects both transcriptional activation and repression. In addition to these cell autonomous tumour suppressor activities, IRF-1 is also a key regulator of the immune system and, as such, mediates immune surveillance of tumours. Numerous studies have confirmed that loss or mis-regulation of IRF-1 is a key factor in several different types of cancer. Despite strong evidence for the crucial role of IRF-1 in cancer, and frequent assertions that this protein warrants further investigation as a drug target, very little is known about its regulation. Furthermore, since recent studies have linked upregulation of IRF-1 to the development of autoimmune diseases, it is particularly important that drugs be able to decouple autoimmune and anti-cancer functions of IRF-1 to avoid harmful side effects. This thesis describes how phosphorylation of IRF-1 in its regulatory C-terminal Mf1 domain modulates transactivatory and tumour suppressor activity. Phosphospecific antibodies were developed as tools to study the C-terminal phosphorylation. Using these, it was shown that treatment of cells with Interferon-γ(IFN-γ) not only causes accumulation of IRF-1 protein, but also results in phosphorylation of IRF-1 at two sites in the C-terminal Mf1 domain. Phosphomimetic mutants demonstrated that these phosphorylations enhanced the transactivatory activity of IRF-1 at various promoters, but did not affect repressor activity. Gel shift assays revealed that dual phosphorylation of IRF-1 (IRF-1 D/D) promoted DNAbinding and suggested this was through increased interaction with the cofactor/histone acetylase p300 which induces a conformational change in IRF-1, favouring DNA-binding. Acetylation by p300 appears to be important although it is not yet clear whether this directly or indirectly affects IRF-1 activity. Since the tumour suppressor activity of IRF-1 is of particular interest, the effect of phosphorylation was examined in clonogenic and invasion assays. IRF-1 D/D more efficiently suppressed colony formation in both anchorage dependent and independent assays, and may improve inhibition of invasion in Transwell assays. Thus, cell treatment with the therapeutic agent IFN-γ nduces phosphorylation of IRF-1, resulting in enhanced DNA binding of IRF-1 through improved p300 binding. In cells the outcome is more effective tumour suppression and inhibition of metastasis.

The human epidermal growth-factor like receptor (HER) family of receptor tyrosine kinases are important targets for cancer therapy. The family consists of four members - EGFR, HER2, HER3 and HER4 - that normally transfer stimulatory signals from extracellular growth factors to the intracellular signalling network. Over-activation of these receptors leads to uncontrolled cell proliferation and is seen in several types of tumours. The aim of the studies reported in this thesis was to study the uptake and effects of affibody molecules against EGFR, HER2 and HER3 in cultured cells. Affibody molecules are affinity proteins originally derived from one of the domains of protein A, and their small size and robust structure make them suitable agents for tumour targeting and therapy. Papers I and II of this thesis concern EGFR-specific affibody molecules, which were shown to be more similar to the antibody cetuximab than the natural ligand EGF in terms of cellular uptake, binding site and internalisation rate. In addition, fluorescence-based methods for the quantification of internalisation were evaluated. In the studies reported in papers III and IV, HER2-specific affibody molecules were utilised as carriers of radionuclides. Paper III reports that different cell lines exhibit different radiosensitivities to 211At-labelled affibody molecules; radiosensitivity was found to correlate with cell geometry and the rate of internalisation. Paper IV discusses the use of 17-AAG, an agent that induces HER2 internalisation and degradation, to force the internalisation of 211At- and 111In-labelled affibody molecules. Papers V and VI describe the selection and maturation of HER3-specific affibody molecules, which were found to compete with the receptor’s natural ligand, heregulin, for receptor binding. These affibody molecules were demonstrated to inhibit heregulin-induced HER3 activation and cell proliferation. The studies summarised in this paper will hopefully contribute to a better understanding of these affibody molecules and bring them one step closer to being helpful tools in the diagnosis and treatment of cancer.

Biomedical imaging at microwave frequencies has shown potential for breast cancer detection and monitoring. The advantages of microwave imaging over current imaging techniques are that it is relatively inexpensive, and uses low-energy, non-ionizing radiation. It also provides a quantitative measurement of the dielectric properties of tissues, which offers the ability to characterize tissue types. Microwave imaging also comes with significant drawbacks. The resolution is poor compared to other imaging modalities, which presents challenges when trying to resolve fine structures. It is also not very sensitive to low contrast objects, and the accuracy of recovered tissue properties can be poor. This thesis shows that the use of prior information in microwave imaging inversion algorithms greatly improves the resulting images by minimizing mathematical difficulties in reconstruction that are due to the ill-posed nature of the inverse problem. The focus of this work is to explore novel methods to obtain and use prior information in the microwave breast imaging problem. We make use of finite element contrast source inversion (FEM-CSI) software formulated in two and three dimensions (2D, 3D). This software has the ability to incorporate prior information as an inhomogeneous numerical background medium. We motivate the usefulness of prior information by developing a simulated annealing technique that segments experimental human forearm images into tissue regions. Tissue types are identified and the resulting map of dielectric properties is used as prior information for the 2D FEM-CSI code. This results in improvements to the reconstructions, demonstrating the ability of prior information to improve breast images. We develop a combined microwave tomography/radar algorithm, and demonstrate that it is able to reconstruct images of superior quality, compared to either technique used alone. The algorithm is applied to data from phantoms containing tumours of decreasing size and can accurately monitor the changes. The combined algorithm is shown to be robust to the choice of immersion medium. This property allows us to design an immersion medium-independent algorithm, in which a numerical background can be used to reduce the contrast. We also develop a novel march-on-background technique that reconstructs high quality images using data collected in multiple immersion media. / October 2016

Multicellular tumour spheroids (TS) are an in vitro model of avascular tumours, and have been widely used to investigate tumour growth, metabolism and hypoxia. The geometry of the TS lends itself to mathematical representation, and theoretical models of TS growth and the development of hypoxia are abundant. With some notable exceptions however, these models have been developed independently of the biological data collection process and are overwhelmingly based upon data from multiple sources. Thus, whilst mathematical modeling has the potential to help explain and guide biological experiments, without reliable data it is unlikely to live up to this expectation. In this thesis, a combination of experimental and theoretical approaches was used to characterize the relationship between proliferation, hypoxia and metabolism during the growth of TS derived from the DLD-1 human colon adenocarcinoma cell line. Experimental data were collected over the entire period of TS growth, generating a high volume of predominantly imaging data. To facilitate the extraction of quantitative information from this, a suite of image analysis software, which is readily applicable to other data sets, was developed. During growth, the DLD-1 TS maintained a macroscopic spherical geometry but at the microscale level the TS boundary was increasingly irregular, with TS disintegrating rapidly after 20 days. Immunofluorescence (IF) studies showed that hypoxia developed soon after TS initiation, followed by the characteristic onset of necrosis. Reduced proliferation was found to be concomitant with the development of hypoxia, although some cells retained proliferative capacity even under severely hypoxic conditions. Towards the end of culture, TS were primarily comprised of severely hypoxic and necrotic cells, a probable cause of disintegration. Mathematical simulation of oxygen gradients in TS using literature-based values for the maximal rate of oxygen consumption was used to estimate the partial oxygen pressure (pO₂) at which the IF marker of hypoxia was bound. Assuming a spatially-invariant rate of oxygen consumption, the model predicted that the onset of hypoxic binding occurs at pO₂ levels similar to those reported in the literature, however the onset of necrosis was overestimated. Mathematical simulations predicted that oxygen consumption decreases as TSs increase in size, supporting previous observations. The Warburg Effect, where glucose metabolism is favoured even under aerobic conditions, is a hallmark of tumours. Although development of the glycolytic phenotype during TS growth was observed in the form of an elevated activity of the lactate dehydrogenase V (LDHV) enzyme, the activity and expression of other glycolytic enzymes, such as hexokinase II (HKII), was unaltered. Whilst the spatial distribution of HKII was unrestricted throughout the TS's viable fraction, LDHV expression was elevated in regions of hypoxia, suggesting constant adaptation of tumour cells to their microenvironment. In addition to the above findings, the data generated have been collected and analysed in the context of the requirements of theoretical modelling at each step; thus, they can be used to parameterise and inform more sophisticated models of tumour metabolism.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoma subtype with heterogeneous clinical outcome and a significant number of patients still die of their disease. Characterising lymphoma patients’ autoantibody repertoires represent one approach to improve the understanding of their disease biology. Our hypothesis was that characterisation of antigens eliciting humoural immune responses in lymphoma patients may provide insights into mechanisms of lymphomagenesis and identify novel diagnostic/therapeutic targets. HIP1R was validated as a novel B-NHL autoantigen by immunoblotting with patients’ sera. No response was identified to the related HIP1 protein. Consistent with this finding, more widespread expression of HIP1R, compared to HIP1, was observed in lymphoma cell lines. Expression studies, at both transcript (qRT-PCR and analysis of microarray datasets) and protein levels (blotting and immunolabelling), identified abundant HIP1R in normal B cells and low level expression in a poor prognosis activated B-cell (ABC)-like DLBCL subtype. Upregulation of HIP1R expression was observed in activated non-malignant B cells at both transcript and protein levels, suggesting that downregulation of HIP1R expression in ABC-DLBCL might be a disease-related process. Despite its potential for DLBCL subtyping, HIP1R protein expression was not statistically significantly associated with patients’ survival in a series of 256 DLBCL. Short FOXP1 isoforms were identified as one mechanism repressing HIP1R transcription in an ABC-DLBCL cell line. Phenotypic analysis of HIP1R-depleted B-cell lymphoma cells indicated that HIP1R silencing could increase the surface levels of B-cell receptor (BCR) components such as IgM and CD79b. HIP1R represents a novel lymphoma autoantigen and cell-of-origin marker for distinguishing germinal centre- versus ABC-DLBCL subtypes. As ABC-DLBCL survival is dependent on chronic BCR signalling, future studies will address whether HIP1R silencing plays a fundamental role in disease pathogenesis by promoting BCR signalling.

The effectiveness of radiotherapy treatment could be significantly improved if tumour cells could be rendered more sensitive to ionising radiation without altering the sensitivity of normal tissues. However, many of the key mechanisms that determine intrinsic tumour radiosensitivity are largely unknown. This thesis is concerned with the identification of novel determinants of tumour radiosensitivity. A siRNA screen of 200 genes involved in DNA damage repair was conducted using γH2AX foci post-irradiation as a marker of cell damage. This screen identified POLQ as a potential tumour-specific contributor to radioresistance. Subsequent investigations demonstrated that POLQ knockdown resulted in radiosensitisation of a panel of tumour cell lines, whilst having little or no effect on normal tissue cell lines. It was subsequently shown that POLQ depletion rendered tumour cells significantly more sensitive to several classes of cytotoxic agents. Following exposure to etoposide, it was found that tumour cells depleted of POLQ had reduced RAD51 foci formation, suggesting that POLQ is involved in homologous recombination. A homologous recombination assay was used to confirm that POLQ depletion does indeed result in reduced homologous recombination efficiency. These findings led to the investigation of the clinical significance of tumour overexpression of POLQ. The clinical outcomes of patients with early breast cancer were correlated with tumour expression levels of POLQ. It was found that POLQ overexpression was correlated with ER negative disease and high tumour grade, both of which are associated with poor clinical outcomes. POLQ overexpression was associated with extremely poor relapse free survival rates, independently of any other clinical or pathological feature. The mechanism that causes this adverse outcome may in part arise from resistance to adjuvant chemotherapy and radiotherapy treatment. These findings, combined with the limited normal tissue expression of POLQ, make it an appealing target for possible clinical exploitation.

Alterations in energy metabolism due to factors including cellular stress from the hostile tumour micro-environment are a emerging cancer hallmark. Distinct hypoxic and quiescent cell populations develop, which are resistant to chemotherapy due to lack of proliferation, drug inactivity in the altered redox status of the cell and enhanced drug biotransformation. The present study characterises the metabolic strategies employed by these distinct populations of cancer cells. The in vitro 3-dimensional tumour spheroid model, which reflects tumour architecture and behaviour, cultured under different micro-environmental conditions was utilized in this study. Metabolic enzyme activity and expression, overall metabolic flux rates for nutrients, metabolomics profiles of specific pathways and tissue status were assessed. Metabolic adaptations consistent with the Warburg effect were observed in fully oxygenated, proliferative tumour spheroids, with glucose being metabolised to produce lactate. Additionally, metabolomics investigations determined glucose was metabolised by the pentose phosphate pathway, demonstrated by high enrichment of glucose-derived carbon in 6-phophogluconate. The extraction of 39.7 ± 7.6 μ moles (mg protein) ^-1 glutamine from the medium over 24 hours was observed in these spheroids, consistent with glutaminolysis pathway activity. A 2-fold higher rate of glycolytic flux (measured by production of 3h2O from 5-³H-glucose) was measured in hypoxic tumour spheroids, despite reduced levels of glycolytic enzymes being determined. Surprisingly, although lower rates of glycolysis (2.6-fold) were measured in quiescent spheroids, increased glycolytic enzyme activities (HK 1.9 fold, PK 2 fold and LDH 1.8 fold), glucose (1.9 fold over 24 hours) and glutamine uptake (5.5 fold over 12 hours) as well as lactate production (1.8 fold) were measured, relative to their proliferating counterparts. This study demonstrates that metabolic strategies employed by tumour spheroids differ upon exposure to distinct micro-environmental stresses, additionally identifying hexokinase as a potential therapeutic target for the inhibition of glycolysis under all micro-environmental stress conditions analysed.