Micro-RNAs (miRNAs) are a class of non-coding RNA which post-transcriptionally regulate gene expression. Epstein-Barr Virus (EBV) transforms resting B-cells in vitro to establish continuously proliferating lymphoblastoid cell lines (LCLs) and is aetiologically linked to lymphomas. Little is known about the contribution of miRNAs to the transformation of B cells. We initially examined the regulation of the oncogenic miR-155, which is highly expressed in Hodgkin’s lymphoma but was reportedly absent in Burkitt’s lymphoma. We found that miR-155 was up-regulated by EBV-LMP1 expression, and that a reported defect of miR-155 processing in Burkitt’s lymphoma was a misinterpretation of data. Next, to identify cellular miRNAs and genes modulated during EBV-induced transformation, we compared the expression profiles of resting B cells and B cells either infected with EBV or stimulated to proliferate with CD40L and IL4. This revealed that a large proportion of miRNAs and genes differentially regulated by EBV and not by CD40L/IL4 were modulated by EBV interaction with its CD21 receptor complex, but these changes were maintained or amplified in LCLs; and included a set of tumour suppressor genes down-regulated by EBV. In addition, bioinformatics analysis indicated that EBV modulates the expression of multiple miRNAs predicted to target the same cellular genes.

The flattening filter is a conical shaped piece of metal sitting within the treatment head of a linear accelerator, used to produce a flat, uniform beam of X-rays from the forward-peaked distribution exiting the target. Despite their routine use since the introduction of the linac in the 1950’s, however, there are still several unresolved issues surrounding their use. The photon scatter and electron contamination introduced by modifying the fluence are difficult to model, as is the variation in energy spectrum caused by differential absorption across the field. Leakage radiation also causes increased whole body doses to the patient, and the filter itself causes acts as an amplifier for beam bending and steering issues. With advances in tumour imaging, dose optimisation and in-room image-guidance it is now possible to locate a tumour accurately in space and to design radiation fields to conform to its shape, avoiding adjacent normal and critical tissues. This active production of non-flat fields means that the prerequisite for flat fields no longer exists, and the filter is potentially no longer a necessary component. This thesis reports on research to produce a filter-free linear accelerator, from basic operation and optimisation, dosimetric characterisation and beam modelling, through to treatment planning and dose delivery. FFF beams have been shown reduce many of the problems seen with the current generation of linear accelerators, producing beams that are inherently more stable, simple to model and with reduced patient leakage (leading to reduced secondary cancers). The increase in dose rate also translates into shorter treatment times for many treatments, aiding patient comfort and reducing problems associated with intra-fraction motion.

Allogeneic stem cell transplantation (SCT) is the only curative treatment option for many haematological malignancies. Adenovirus (Ad) infections are a significant cause of morbidity and mortality post SCT. Lack of effective anti-viral treatment for Ad disease has led to the development of adoptive immunotherapy of Ad-specific T-cells as a promising therapeutic option for patients in this setting. The aim of this project was to establish preclinical criteria for the development of a clinical trial comparing two T-cell enrichment methods- multimer selection and cytokine secretion selection to enrich Ad-specific T-cells for the purposes of adoptive transfer directly without the need for in vitro culture. Eight pHLA tetramers containing HLA class I restricted Ad epitopes were generated and their ability to identify and enrich Ad-specific T-cells investigated. HLA A*01 TDL tetramer consistently detected T-cells in all (13/13) healthy adult donors screened. Frequency and enrichement of Ad-specific T-cells by cytokine secretion and selection was also investigated. Despite the low frequency of Ad-specific T-cells, clinical grade enrichment was feasible by both methods. T-cells selected by both methods were then characterised for homing and proliferative potential. Ad-specific T-cells identified by either method had a high proliferative potential, possessed a novel minimally differentiated memory phenotype, were cytotoxic towards Ad species responsible for infections in SCT recipients and capable of limiting virus replication. In conclusion, Ad-specific T-cells enriched by multimer selection or cytokine secretion selection are suitable for adoptive transfer to patients with Ad infection following HSCT. Both methods also allow the monitoring of Ad-specific immune reconstitution after adoptive transfer.

Lymphocytes express antigen receptors which are formed by re-arrangement of gene segments. Mutations acquired during this process, predominantly in the complementarity determining regions (CDRs), result in generation of non-germline sequences. Through analysing the CDR3 sequence, this study attempts to determine whether editing of the lymphocyte repertoire is present in an HLA-dependant manner. Data presented demonstrates a decrease frequency of CDR3-derived HLA-A2 binding peptides in HLA-A2+ donors (0.03% (SYFPEITHI) and 0.35% (BIMAS)) compared with HLA-A2- donors (0.24% (SYFPEITHI, p=0.01) and 0.54% (BIMAS, p=0.19)). Trends similar to those seen in HLA-A2 were observed in other HLA alleles as well suggesting that there may be a process by which potentially dangerous B cell populations are edited from the B cell repertoire. Similar analysis of the TCR CDR3 did not reveal any such process in all of the HLA alleles tested suggesting that there is no immunoediting of the T cell repertoire. Simultaneously, this study attempts to determine the processing and presentation of CDR3-derived peptides at the cell surface using lymphocyte antigen receptor models containing CDR3-encoded viral epitopes. The apparent presence of these peptides on the cell surface leads to the hypothesis that antibodies enter the antigen processing pathway and potentially deliver an immunogenic peptide to a target cell. Using antibodies specific for B cells, this study has shown that cells labelled with an antibody-peptide complex are targeted and lysed by cytotoxic CD4+ T cells in a peptide-specific manner. The use of such technology in antibody immunotherapy may be of considerable therapeutic benefit.

Recent years have seen important advances in the area of T cell-based therapy for human malignancies. Epstein Barr virus-associated tumours like Hodgkin’s Lymphoma provide important models in this field. If a T cell-based therapy is to be effective however, T cells must be capable of trafficking to the tumour. To address this, the molecular mechanisms of T cell homing to Hodgkin’s Lymphoma were explored. Chemokine and adhesion receptors were examined on infiltrating T cells. CXCR3, CXCR4 and CCR7 were expressed on major T cell populations with CXCR5, CXCR6, CCR4 and CCR5 on minor populations. Tumour cells expressed CXCL10, CXCL12 and CCL21. Vessels expressed ICAM-1, CXCL12, CCL17 and CCL21. Tumour cell lines secreted factors that mediated chemotaxis of lymphoblasts in vitro and TIL demonstrated chemotaxis to CXCL12 but not CCL17. VAP-1 was expressed on vessels and a tissue-binding assay was evaluated to examine VAP-1 function. T cell clones generated as part of an existing clinical trial of adoptive T cell therapy were found to express a polarised Tc1 phenotype (CXCR3, CXCR6 and ccr5), which was typically independent of target antigen specificity, CD4/CD8 and donor status. However, lack of CCR7 expression and an inability to capture to VCAM-1 in a chemokine dependent manner suggested that clones expanded in vitro using existing protocols may be inefficient at trafficking to tumour tissue and thus may require modification of their homing phenotype.

Dynamic Nuclear Polarisation (DNP) is a method used to increase the signal available for nuclear magnetic resonance (NMR) experiments. DNP is one of many hyperpolarisation methods and one implementation, so called ex-situ DNP, sees the sample polarised with a stable radical doping agent at low temperature and with microwave irradiation in a magnetic field before transfer to a second higher field NMR magnet to acquire a liquid state NMR spectrum. The primary goal of this project has been to gain an understanding of the underlying reasons for unpredictable ex-situ DNP polarisation failures and to develop methods to overcome these limitations. In the course of the work an additional polarisation mechanism arising from hindered methyl rotors was discovered. This thesis describes the preparation of DNP samples and the way in which DNP-NMR experiments are performed have been optimised. The concept of a co-polarisation agent has been introduced and methods employing a Nuclear Overhauser Effect have been implemented. Additionally the identification and characterisation of a quantum tunnelling effect that is a variant of the commonly known Haupt effect. Finally these methods have been combined in a number of situations to give results that would have otherwise been unobtainable.

During lytic cycle replication EBV expresses at least three genes; BNLF2a, BILF1 and BGLF5, which individually act to inhibit efficient processing and presentation of CD8+ T cell epitopes. This thesis sets out to assess the relative contribution of these potential immune-modulating proteins to the evasion from CD8+ T cells at different stages of EBV lytic cycle. Lentiviral vectors for shRNAs were used to silence expression of these individual viral genes in EBV-transformed B-cells, which were then probed with CD8+ T cell effector clones of specificities for epitopes derived from the three phases of the EBV lytic cycle; allowing us to determine the contribution each immune evasion gene makes towards the inhibition of antigen presentation during lytic cycle. Cells replicating viruses lacking BNLF2a were more efficiently recognised by CD8+ T cells specific for immediate early and early expressed antigens relative to those lacking BGLF5 and BILF1. Conversely, cells lacking the expression of BILF1 were better recognised by CD8+ T cells specific for early and late lytic antigens. These data suggest that whilst the role BNLF2a plays in interfering with antigen presentation diminishes as lytic cycle progresses (IE>E>>L), BILF1 plays a more active role with the progression of lytic cycle (IE<E<<L).

Three areas of investigation regarding a novel class of anti-cancer therapeutics, triple-helicate dinuclear compounds known as ‘cylinders’, have been addressed. An improved protocol for the synthesis of a ruthenium cylinder has been developed, utilising microwave synthesis rather than reflux techniques. The use of Sephadex C-25 as a solid phase for column chromatography led to shorter purification protocols. Experiments to determine the cellular localisation of fluorescent ruthenium cylinder using confocal microscopy showed fluorescence within MDA-MB-231 cells, but not within SKOV-3 cells, suggesting preferential uptake between cell lines. Co-localisation experiments suggested localisation of the cylinder within cell nuclei. Synchrotron radiation has been used to image iron cylinder within SKOV-3 cells. High levels of iron are found near the cell membrane, corresponding to areas of high calcium concentration. XANES spectra show that the iron is in an environment closer to that of ferric iron as opposed to the ferrous iron found within the cylinder. The cellular effects of cylinder treatment have been imaged, showing that cell motility is compromised by cylinder treatment. Iron cylinder causes production of NO within MDA-MB-231 cells. Control images showed low levels of fluorescence within MDA-MB-231 cells, suggesting the presence of endogenous NO, which had been debated within literature.

Adrenocortical carcinoma (ACC) is an aggressive malignancy with high recurrence rates and poor response to chemotherapy. With this work, we have evaluated a potential new treatment target focusing on the mitochondrial NADPH generator Nicotinamide Nucleotide Transhydrogenase (NNT). NNT has a central role within the mitochondrial antioxidant pathways, which protect cells from oxidative stress. Our hypothesis was that NNT silencing will expose cells to cytotoxic levels of oxidative stress. We knocked down NNT transiently in NCJ-H295R ACC cells in vitro; this led to an increase in cellular oxidative stress and a strong cytotoxic and cytostatic effect. With stable NNT knockdown, we observed the emergence of a partially compensated phenotype over the course of time, with restored redox balance. Surprisingly, steroidogenesis was stimulated by transient NNT loss, challenging current perceptions about the impact of oxidative stress on steroidogenesis. In our clinical study, we evaluated a new diagnostic tool for biochemical detection of ACC recurrence. Serial post-operative urine samples were collected from a large cohort of patients who had undergone complete ACC resection. Standardised review of longitudinal steroid measurements resulted in detection of disease recurrence prior to or concurrently with imaging with high sensitivity in cases where a pre-operative steroid profile had been provided.

Intracellular signalling cascades are mediated by a plethora of receptors, enzymes, adaptors and small molecules. The Protein Tyrosine Phosphatase (PTP) Shp2 is a highly conserved enzyme involved in a myriad of cellular processes including growth, differentiation and apoptosis. Shp2 is multi-domain protein composed of two SH2 domains in tandem, a PTP catalytic domain and a C-terminal tail containing multiple phosphorylation sites and a proline-rich region. The majority of biophysical research has utilised X-ray crystallography to study interactions and effects of mutations at the structural level. To gain a further understanding of Shp2 ligand binding and perturbations caused by disease relevant mutations, a structural investigation was performed with Nuclear Magnetic Resonance (NMR) spectroscopy and Small-angle X-ray Scattering (SAXS) in solution. The NMR signals from the backbone of both SH2 domains were assigned and residue-level interactions and differential SH2 domain specificities with peptides from the novel receptor G6b-B were delineated. In addition, the E76K point mutation that causes Noonan Syndrome and leukaemia was found to have increased conformational dynamics, the first experimental evidence of this phenomenon at the structural level.