Clear cell renal cell carcinoma (ccRCC) is an aggressive malignancy characterized by inactivation of the von Hippel-Lindau tumour suppressor gene, the protein product of which mediates degradation of the transcription factor hypoxia-inducible factor (HIF). GAL3ST1 is a sulfotransferase which catalyzes the production of sulfatide, a plasma membrane sulfolipid previously implicated in metastasis. We observed GAL3ST1 overexpression in primary ccRCC tumours relative to matched-normal tissue and subsequently asked if GAL3ST1 was a HIF-responsive gene that facilitates ccRCC metastasis. GAL3ST1 expression was suppressed in ccRCC cells by stable reconstitution of wild-type VHL and also siRNA-mediated knockdown of HIF1alpha and HIF2alpha. Dual luciferase assays and chromatin immunoprecipitation revealed a hypoxia-response element in the GAL3ST1 5’-UTR that appeared to be crucial for HIF-mediated upregulation. Finally, stable knockdown of GAL3ST1 significantly impeded ccRCC cell invasion through an in vitro basement membrane mimic. These results suggest GAL3ST1 is a HIF-responsive gene that promotes tumour cell invasion.

A brief introduction to cancer biology and treatment is presented with a focus on current clinical advances in the delivery of chemotherapy and antiangiogenic therapies. Mathematical oncology is then surveyed with summaries of various models of tumor growth, tumor angiogenesis and other relevant biological entities such as angiogenic growth factors. Both strictly time-dependent ordinary differential equation (ODE)-based and spatial partial differential equation (PDE)-based models are considered. These biological models are first developed into an ODE model where various treatment options can be compared including different combinations of drugs and dosage schedules. This model gives way to a PDE model that includes the spatially heterogeneous blood vessel distribution found in tumors, as well as angiogenic growth factor imbalances. This model is similarly analyzed and implications are summarized. Finally, including the effects of interstitial fluid pressure into an angiogenic activity model is performed. This model displays the importance of factor convection on the angiogenic behaviour of tumours.

A brief introduction to cancer biology and treatment is presented with a focus on current clinical advances in the delivery of chemotherapy and antiangiogenic therapies. Mathematical oncology is then surveyed with summaries of various models of tumor growth, tumor angiogenesis and other relevant biological entities such as angiogenic growth factors. Both strictly time-dependent ordinary differential equation (ODE)-based and spatial partial differential equation (PDE)-based models are considered. These biological models are first developed into an ODE model where various treatment options can be compared including different combinations of drugs and dosage schedules. This model gives way to a PDE model that includes the spatially heterogeneous blood vessel distribution found in tumors, as well as angiogenic growth factor imbalances. This model is similarly analyzed and implications are summarized. Finally, including the effects of interstitial fluid pressure into an angiogenic activity model is performed. This model displays the importance of factor convection on the angiogenic behaviour of tumours.

Light delivery and sample handling systems are essential for any high performance imaging application. The custom design for two such devices with medical imaging applications are presented. The first device, a galvanometer-stage combination, is for general use optical coherence tomography and can be configured to scan over a large range of sample sizes and types. The second device, constructed in parallel, a rotation-linear stage combination, has been carefully designed for a specific imaging task: assessing tumour margins. The design of the two devices is driven by operational requirements and although requirements vary greatly from application to application, there are several common parameters that must be considered for every system. In this thesis, parameters like total scan time, scan resolution, sampling rate, and sample type flexibility are analysed and are some of the primary factors that influence the viability of a system for further development. This work's contribution to medical imaging research is the design of two light delivery systems and an analysis process that can be applied to future iterations of scan systems. The devices are shown to be flexible enough for use in test-bed systems, while providing the necessary functionality to meet the needs of medical histology and pathology. Controlling the light delivery and sample positioning of an imaging device adds important functionality to a scan system and is not a trivial task when high spatial-resolution scan spacing is required. The careful design of an imaging system to meet the unique requirements of the application enables better information and better resulting decision making. Advanced imagery provides new insights and perspectives to everyday scenes. It is these new perspectives that allow for re-evaluation and examination of problems with a fresh eye.

Glioblastoma multiforme (GBM) is one of the most common and aggressive primary brain tumours, with a median patient survival time of 6-12 months in adults. It has been recently suggested that a typically small sub-population of brain tumour cells, in possession of certain defining properties of stem cells, is responsible for initiating and maintaining the tumour. More recent experiments have studied the interactions between this subpopulation of brain cancer cells and tumour microenvironmental factors such as hypoxia and high acidity. In this thesis a computational approach (based on Gillespie’s algorithm and cellular automata) is proposed to investigate the tumour heterogeneities that develop when exposed to various microenvironmental conditions of the cancerous tissue. The results suggest that microenvironmental conditions highly affect the characterization of cancer cells, including the self-renewal, differentiation and dedifferentiation properties of cancer cells.

<p>The electrochemical treatment (EChT) of tumours entails thattumour tissue is treated with a continuous direct currentthrough two or more electrodes placed in or near the tumour.Promising results have been reported from clinical trials inChina, where more than ten thousand patients have been treatedwith EChT during the past ten years. Before clinical trials canbe conducted outside of China, the underlying destructionmechanism behind EChT must be clarified and a reliabledose-planning strategy has to be developed. One approach inachieving this is through mathematical modelling.</p><p>Mathematical models, describing the physicochemical reactionand transport processes of species dissolved in tissuesurrounding platinum anodes and cathodes, during EChT, aredeveloped and visualised in this thesis. The consideredelectrochemical reactions are oxygen and chlorine evolution, atthe anode, and hydrogen evolution at the cathode. Concentrationprofiles of substances dissolved in tissue, and the potentialprofile within the tissue itself, are simulated as functions oftime. In addition to the modelling work, the thesis includes anexperimental EChT study on healthy mammary tissue in rats. Theresults from the experimental study enable an investigation ofthe validity of the mathematical models, as well as of theirapplicability for dose planning.</p><p>The studies presented in this thesis have given a strongindication of the destruction mechanism involved in EChT. It isshown by the modelling work, in combination with theexperiments, that the most probable cause of tissue destructionis acidification at the anode and alkalisation at the cathode.The pH profiles obtained from the theoretical models have showngood correlation with the experimentally measured destructionzones, assuming that a pH above and below certain values causetissue destruction. This implies that the models presented inthis thesis could be of use in predicting the tumourdestruction produced through EChT, and thereby provide a basisfor a systematic dose planning of clinical treatments.Moreover, the models can serve as valuable tools in optimisingthe operating conditions of EChT.</p><p>Modelling work of theanode processes has explained the roleof chlorine in the underlying destruction mechanism behindEChT. It is found that the reactions of chlorine with tissueplay important roles as generators of hydrogen ions. Thecontribution of these reactions to the acidification of tissue,surrounding the anode, is strongly dependent on the appliedcurrent density and increases with decreasing currentdensity.</p><p><b>Keywords:</b>cancer, direct current, dose planning,electrochemical treatment (EChT), electrotherapy, mathematicalmodelling, tumour.</p>

Within solid tumours, a microenvironment exists that causes resistance to chemotherapy. New drugs that target cells within this microenvironment are required, the first step in this process being the identification of new targets. The aim of this thesis was to characterise changes in the transcriptome and proteome within specific regions of multicell-tumour spheroids (MCTS), an experimental model that mimics many of the features of the tumour microenvironment. HT29 MCTS were separated by sequential trypsinisation into 3 main regions; the outer surface layer (SL) the peri-necroric region (PN) and the necrotic core (NC). Using an iTRAQ quantitative proteomics approach, the proteome of the different MCTS regions was investigated. A 2 dimensional separation approach using Agilent's OffGel system and RP-nano HPLC was incorporated prior to MS analysis. MS analysis was done using both MALDI-TOF-TOF (Bruker Ultraflex II) and ESI-Q-TOF (Agilent 6530 QTOF LC/MS) instruments. Gene expression profiles of the different MCTS were investigated and compared using Agilent's one-color oligonucleotide based microarrays. Transcriptomic and proteomic analysis identified several key differences in the proteins involved in cell metabolism between the SL and PN/NC regions. Similar metabolic changes were also noted between autophagic and normal monolayer cells. Many highlighted proteins represented established cancer associated proteins. Interestingly, a number of proteins were highlighted which have no previous association with cancer and may upon further validation, provide attractive leads for therapeutic intervention.

Tumour cell dissemination is a major issue with the treatment of cancer, thus new therapeutic strategies which can control this process are needed. Antagonism of integrins highly expressed in tumours is one potential strategy. The integrins are transmembrane glycoprotein adhesive receptors. Two of the integrins, αVβ3 and αIIbβ3, are highly expressed in a number of tumours and induce bi-directional signalling through their interaction with extracellular matrix proteins, and growth factor receptors. Through this signalling they play an important role in a number of cellular processes that are involved in tumour dissemination such as tumour growth, migration, invasion, metastasis and angiogenesis. Dual αIIbβ3 and αVβ3 integrin antagonism will have a direct effect on β3-expressing tumour cells that leads to the inhibition of cell migration and dissemination. Furthermore, through targeting tumour cell interaction with endothelial cells and platelets, this will also lead to inhibition of angiogenesis and metastasis. The aim of this project was to characterise the expression of αVβ3 and αIIbβ3 integrin in a panel of tumour cell lines and in human tumour xenograft samples, and to develop and utilise cell-based models to investigate potential novel β3 antagonists. The expression of αV and β3 subunits was detected in xenograft tissue using immunoblotting techniques. A panel of cell lines of different tumour types including melanoma, prostate, breast, colon and non small cell lung carcinoma was then characterised for αVβ3 and αIIbβ3 integrin expression using immunoblotting and immunocytochemistry. Melanoma cell lines demonstrated the strongest αVβ3 expression. No αIIbβ3 integrin expression was seen in any of the cell lines evaluated. A selection of cell lines with varying αVβ3 expression were then used to develop a functional test for cell migration, the scratch wound healing assay. Migration of tumour cells that expressed αVβ3 integrin was inhibited by the known β3 antagonists, cRGDfV peptide and LM609 antibody. A panel of 12 potential novel β3 integrin antagonists was screened for cytotoxicity and activity in the validated scratch assay. ICT9055 was the most effective antagonist in inhibition of M14 cell migration as determined by the scratch assay, with an IC₅₀ of < 0.1 μM. Therefore the work presented in this thesis has established models and tools for evaluating potential novel β3 integrin antagonists, and identified a promising molecule to progress for further preclinical evaluation.

The methods presented in this study are aimed at the identification of subpopulations (clones) of genetically similar cells within tissue samples through measurement of loci-specific Fluorescence in-situ hybridization (FISH) spot signals for each nucleus and analyzing cell spatial distributions by way of Voronoi tessellation and Delaunay triangulation to robustly define cell neighbourhoods. The motivation for the system is to examine lung cancer patient for subpopulations of Non-Small Cell Lung Cancer (NSCLC) cells with biologically meaningful gene copy-number profiles: patterns of genetic alterations statistically associated with resistance to cis-platinum/vinorelbine doublet chemotherapy treatment. Current technologies for gene-copy number profiling rely on large amount of cellular material, which is not always available and suffers from limited sensitivity to only the most dominant clone in often heterogeneous samples. Thus, through the use of FISH, the detection of gene copy-numbers is possible in unprocessed tissues, allowing identification of specific tumour clones with biologically relevant patterns of genetic aberrations. The tissue-wide characterization of multiplexed loci-specific FISH signals, described herein, is achieved through a fully automated, multicolour fluorescence imaging microscope and object segmentation algorithms to identify cell nuclei and FISH spots within. Related tumour clones are identified through analysis of robustly defined cell neighbourhoods and cell-to-cell connections for regions of cells with homogenous and highly interconnected FISH spot signal characteristics. This study presents experiments which demonstrate the system’s ability to accurately quantify FISH spot signals in various tumour tissues and in up to 5 colours simultaneously or more through multiple rounds of FISH staining. Furthermore, the system’s FISH-based cell classification performance is evaluated at a sensitivity of 84% and specificity 81% and clonal identification algorithm results are determined to be comparable to clone delineation by a human-observer. Additionally, guidelines and procedures to perform anticipated, routine analysis experiments are established.

The presence of inflammation in the progression of fatty liver disease induced by fasting was determined in mink. Tumour necrosis factor alpha (TNF-?), and monocyte chemoattractant protein 1 (MCP-1) liver mRNA levels were quantified by real-time PCR. Mink fasted for 5 and 7 days had significantly higher levels of TNF-? and MCP-1 liver mRNA, compared to mink fasted for 0, 1, and 3 days. Mink fasted for 7 days, but re-fed for 28 days had the lowest mRNA levels of both TNF-?, and MCP-1 demonstrating the liver’s ability to restore homeostasis post-fasting. TNF-? mRNA levels were correlated with MCP-1 liver mRNA and liver fat percent. To confirm the physical presence of inflammation, slides stained with haematoxylin and eosin were analyzed for bile ducts resulting in no significant differences. Results indicate that elevated MCP-1 and TNF-? expression are associated with fasting induced fatty liver in mink.