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Murine macrophages were induced to over-express iNOS by treatment with a combination of cytokines, mixed with HT1080 and HCT116 human tumour cells and the toxicity of AQ4N was determined under normoxic or hypoxic conditions. The normoxic toxicity of AQ4N towards tumour cells was not affected through co-culturing with macrophages. However, under hypoxic conditions, the induction of iNOS activity in the macrophages was associated with an increase in AQ4N metabolism and a substantial increase in tumour cell toxicity, which was dependent upon the proportion of macrophages in the culture. This study is the first demonstration of tumour associated macrophage mediated pro-drug activation to result in bystander killing of human tumour cells. The oxygenase domain of the iNOS enzyme is responsible for the production of nitric oxide (NO), a potent biological mediator, whose complex role in tumour biology is still not fully understood as it seems to have both anti- and protumour effects. We were able to use the co-culture model of NO-producing macrophages and cancer cells to demonstrate that NO is also a potent tumour radiosensitiser under hypoxic conditions with a sparing effect on well-oxygenated tumour cells, also a characteristic of normal tissues. Additionally, NO produced by the macrophages resulted in a significant induction of hypoxia inducible factor-1 (HIF-1) driven transcriptional activity in the co-cultured tumour cells under both normoxic and hypoxic conditions. We therefore, suggest the use of HIF-1 inhibitors in combination with NO-based therapies and radiation to favour tumour regression.

In this thesis we use mathematical techniques to model two biological systems. First, we examine the growth dynamics of the antibiotic producing bacteria Streptomyces coelicolor and present a system of PDEs. We study the system both numerically and analytically. Due to oscillations in the numerical solution when solved using NAG, which uses a finite difference discretization, we change to a finite element discretization which corrects the oscillations. S. coelicolor also produces anticancer drugs, these can be encapsulated during the self-assembly of nanometre-sized vesicles, BPVs (biomimetic polymer vesicles) which are used as a novel targeted cancer therapy. We present a system of ODEs that focuses on the binding kinetics between cell-surface receptors and targeting molecules (ligands) on the BPV. We solve the system numerically, showing there is an optimal number of ligands per BPV for optimal uptake by tumour cells. We extend the model to allow for the infiltration of BPVs into tumour spheroids. Numerical solutions show that the growth of the spheroid is linear if the therapeutic BPVs are absent, and slows in the other case (for some parameter values). Using large time asymptotics we explore regions of parameter space where either steady states or travelling waves will occur.

Purpose: To investigate the biochemical pathways underlying therapy-induced changes in FDG uptake in tissue culture cells. Methods: Breast cancer cell lines MCF-7 (wild-type (WT), clones of 5-FU-resistant cells and a clone with stably transfected dominant negative p53 (DD)) and T47D were studied. Colon cancer cell lines, SW620 and HCT-8 were also studied. Experiments were performed with tumour cells treated with IC50 chemotherapy determined using the 3-(4,5-dimehtylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MCF-7 and T47D cells were treated with tamoxifen, doxorubicin and doectaxel; DD with doxorubicin, cisplatin and 5-FU; SW620 with 5-FU, oxaliplatin and irinotecan and HCT-8 cells with irinotecan and cetuximab. 18F-FDG incorporation was determined following treatment for 24, 48 or 72 hours and related to cell cycle changes, glucose transport, hexokinase activity and ATP content. Results: MCF-7 cells showed decreased 18F-FDG incorporation and ATP levels with each 72 hour treatment. Hexokinase activity was reduced in tamoxifen-treated cells whilst glucose transport decreased with doxorubicin and tamoxifen. T47D cells showed decreased 18F-FDG incorporation and ATP levels with each 72 hour treatment. 18F-FDG incorporation decreased and glucose transport increased in MCF-7FU1 and MCF-7FU5 cells, compared with sensitive cells. DD cells showed increased ATP levels, 18F-FDG incorporation and glucose transport, compared with WT cells. 18F-FDG incorporation decreased in cisplatin and doxorubicin-treated DD cells and cisplatin-treated WT cells. SW620 cells showed decreased 18F-FDG incorporation and hexokinase activity with each 72 hour treatment. 18F-FDG incorporation by HCT-8 cells decreased following treatment with irinotecan and cetuximab alone and combined. Conclusion: 18F-FDG uptake is mainly influenced by the type of cells, the length of exposure to a drug and the type of drug administered.

It is now understood that CD4+ T cells play a central role in antitumour immunity; they are important in the priming of CTL and can augment cytotoxicity; they are also responsible for enhancing antitumour responses via indirect mechanisms such as recruitment of accessory cells to the tumour site and they are crucial in the formation of memory T cells. CD4+ T cells recognise antigen in the context of peptide presented on MHC class II molecules, therefore recent research has focused on the identification of these peptides in order to formulate more effective immunotherapeutic strategies. As such, the aim of this study was to identify novel MHC class II HLA-DR1 and HLA-DR4 restricted immunogenic peptides derived from the MART-1 and Tyrosinase tumour antigens. A computer algorithm (SYFPEITHI; available on the World Wide Web) was used to predict immunogenic peptides from the two tumour antigens; these peptides were then used to immunise HLA-DR1 and HLA-DR4 transgenic mice in order to assess their immunogenicity. At the same time efforts were made to optimise the screening process by fully characterising the BM-DC used in proliferation assays and employing antioxidants in conjunction with T cell culture. A second aspect of the project utilised p53 peptides in order to investigate the effects of protein specific T cell help on the generation of effector and memory CTL. Efforts were made to maximise the efficiency of the MHC class II peptide screening method by optimising expression of co-stimulatory molecules and cytokine production by BM-DC. Testing of BM-DC derived from FVB/N-DR1, C57bl/6-DR4 and C57bl/6 HHD II HLA-A2 transgenic mice revealed that differing maturation protocols were required to generate BM-DC with the optimal T cell stimulatory capacity, depending upon the strain of transgenic mice employed. The screening process was further optimised by the use of Vitamin E in conjunction with T cell culture; this antioxidant was found to increase peptide specific proliferative responses against the immunised peptide. Using the optimised screening protocols, immunisation of transgenic mice with predicted epitopes led to the discovery of the novel HLA-DR1 restricted MART-129-43 and the HLA-DR1/DR4 restricted Tyrosinase147-161 peptides. Further experiments also indicated that the Tyrosinase protein was processed by murine dendritic cells to produce the Tyrosinase147-161 peptide. This study demonstrated that HLA-DR restricted responses to novel peptide can be obtained in HLA-DR1 and HLA-DR4 transgenic mice.

It was hypothesised that genetic modification of viruses to encode for an extracellular matrix degrading enzyme would improve virus spread through solid tumours. Initially, the effect of extracellular matrix degrading enzymes on adenovirus infection was investigated in 3-dimensional spheroid cell cultures. Hyaluronidase demonstrated good enhancement of adenovirus infection with limited toxicity and was therefore investigated further. The oncolytic adenovirus, vKH6, was modified to encode the sperm-associated hyaluronidase SP AMi, however no hyaluronidase activity was detectable from infected cells. A replication-deficient adenovirus encoding SP AMi was also engineered and found to produce hyaluronidase activity. A vaccinia virus with attenuating deletions of thymidine kinase and vaccinia growth factor was engineered and was demonstrated to produce detectable levels of SPAMi activity. The efficacy of this virus was tested in an immunocompetent murine model however no enhancement of tumour inhibition was detected.

The insulin receptor (IR) exists as two isoforms IR-A and IR-B, due to alternative splicing of exon 11. It is reported that IR-A is over-expressed in a number of cancers. This PhD aimed to assess the ratio of IR isoforms in prostate cancer cell lines and to manipulate the IR isoform ratio in an attempt to investigate the function of each isoform. I successfully manipulated IR isoforms by using siRNA to silence the IR-B isoform, exposing cells to different levels of glucose, IGFs, insulin or an insulin-sensitizing drug called metformin. I discovered the IR-B ratio was up-regulated under hyperglycaemic conditions, while in hypoglycaemic conditions, the IR-A ratio was up-regulated. In prostate cancer patients IR-A isoform was up-regulated in cancer regions compared to benign regions. This study also supports the theory that the IR-A isoform has a higher affinity for IGF-II and is involved in prostate cancer progression. Androgen receptor (AR) alterative splicing is involved in the development of prostate cancer resistance to androgen deprivation therapy. This study also assessed changes in AR alternative splicing in prostate cancer cell lines and cancer patient samples. And data suggest that the AR was over expressed in hyperglycaemic conditions in cell lines, and the study in prostate cancer patient samples suggest that AR variant 7 (AR-V7) is associated with cancer progression. This finding could have imp0l1ant implication to therapeutic strategies III prostate cancer patients presenting with diabetes.

The transcription factor Activator Protein 1 (AP-1) is a dimeric DNA binding protein, which plays an important role in the development and progression of numerous cancers, and has consequently attracted interest as a target in cancer therapy. Of particular importance to the function of AP-1 is the leucine zipper (LZ) domain, a 32 residue sequence motif contained within the protein that allows for dimerisation to take place. As this is a requirement for AP-1 to bind to DNA, this is a promising strategy for its inhibition. In recent years, peptides have been of growing interest as pharmaceutical agents, owing to their advantageous properties and these have shown promise in previous research concerning AP-1 inhibition. Using a combination of experimental and computational approaches, this thesis explores strategies used to design inhibitors of AP-1 . It was demonstrated, using a library screen protein fragment complementation assay (peA), coupled with rational library design, that truncation of an inhibitor template could yield a shorter sequence which retains the inhibitory function of the template I sequence. This demonstrates a semi-rational approach to the design of LZ based peptide inhibitors. Using sequence data mining, residue selection preferences were studied for less well understood solvent exposed residues in LZ domains in order to derive selection rules for these residues. Finally, design rules for LZ domains were combined into an algorithm which aims to predict inhibitors towards sequences. The results of these computational studies are not generally successful and so this thesis speculates that there are shortcomings in the rules presently used to design and predict LZ domains de novo. In particular design rules used for rational design need to make sequence context considerations, as opposed to single residue pairing decisions. Once derived however, such rules can be simply incorporated into the design process, giving scope for future enhancement.

High intensity focused ultrasound (HIFU) can be used to induce thermal ablation and cavitation for therapy. This paper considers thermal ablation. Despite intensive exploration of this topic, the relative contribution of HIFU's thermal effects for tumour destruction is not yet fully defined, because of a lack of standardised parameters. Additionally, observation of the extent of tissue damage on ex vivo specimens tends to be limited to histology. The present work began with the design and fabrication of a focused ultrasonic (925 kHz) transducer, and then sought to examine the feasibility of generating lesions in ex vivo poultry tissue, submerged in degassed water. Basic characterisation of the transducer and the 'ultrasonic field was carried out, with peak intensity determined to be approximately 300 W.cm⁻², followed by development of an appropriate experimental system to house both the transducer and the tissue.