Cytotoxic T Lymphocyte Associate Antigen 4 (CTLA-4) is an important negative regulator of T-cell activation. The protein is expressed in activated T-cells and can also be found in regulatory T-cells. The mechanism of action of this protein remains controversial; it has typically been associated with a cell intrinsic negative signal however, there is increasing evidence that CTLA-4 may act as an effector molecule. Surprisingly, we find that blocking CTLA-4 in a model of T-cell activation driven by ligand-expressing transfectants has no effect on either proliferation or cytokine production, suggesting that CTLA-4 doesn’t inhibit in this setting. In contrast, blocking CTLA-4 in a dendritic cell based assay enhances proliferation and cytokine production, only when the amount of co-stimulation is limiting. In these experiments CTLA-4 function correlates with decreased expression of B7 ligands on dendritic cells consistent with the removal of ligands by CTLA-4. Furthermore, the addition of CTLA-4 transfected Jurkat cells acts to suppress T cell responses consistent with a role for CTLA-4 as an effector of suppression. Overall our data do not support a role for CTLA-4 in delivering a ligand-dependent cell-intrinsic negative signal and instead suggest a role for CTLA-4 as an effector molecule which inhibits co-stimulation by APC.

Epstein-Barr virus (EBV), a human gammaherpesvirus, possesses a unique set of latent genes whose constitutive expression in B cells leads to cell growth transformation. The initiation of this B-cell growth transformation programme depends on the activation of a viral promoter, Wp, present in each tandemly arrayed BamHI W repeat of the EBV genome. In order to examine the role of the BamHI W region in B cell infection and growth transformation, we constructed a series of recombinant EBVs carrying different numbers of BamHI W repeats and carried out B cell infection experiments. We concluded that EBV requires at least 2 copies of BamHI W repeats to be able to activate transcription and transformation in resting B cells in vitro. At least 5 copies of BamHI W were required for optimal transcription and transformation; while increasing the number beyond 5 copies had no further effect. Experiments to try to rescue the impaired virus indicated that the expression of sufficient levels of EBNA-LP and EBNA2 from Wp are the key determinants of virus-driven B cell transformation. We believe that EBV has evolved to contain multiple copies of BamHI W repeats to ensure high levels of Wp-initiated transcripts immediately post infection.

In cancer, loss of intercellular contact contributes to tumour progression and invasion. Desmosomal cadherins are essential constituents of desmosomes – intercellular junctions that confer significant adhesive strength to epithelial tissues and cardiac muscle. Although changes in desmosomal components have been noted in a variety of cancers previously, this investigation has shown for the first time altered desmocollin expression in colorectal cancer. Real-time PCR and western blotting were used to assess desmocollin expression in a series of colorectal cancer and matched normal tissue samples. Loss of desmocollin 2 expression was observed in the cancer samples. In addition, de novo expression of desmocollins 1 and 3, which are not normally expressed in the colon, was observed. Desmoglein gene expression was also altered in the cancer samples. Although classical cadherin switching is a hallmark of the epithelial-mesenchymal transition, desmocollin switching has not previously been reported. Further experiments, to investigate the effect of loss of desmocollin 2 and desmoglein 2 on the behaviour of cultured cells were performed. In addition, experiments were carried out to identify those transcription factors that regulate desmosomal cadherin gene expression in the colon. Transcription factors of the CCAAT/enhancer-binding proteins family act as transcriptional activators of desmosomal cadherin promoters in colonic cells.

This thesis summarises the author’s lifelong work to improve outcomes for patients with musculoskeletal tumours. It starts with analysing diagnostic features and steps that could be taken to improve the time to diagnosis of these rare tumours. It continues with a number of basic science projects which the author has either carried out or collaborated with. Benign bone tumours are common in children and are discussed. The author has probably one of the largest personal experiences of treating primary malignant bone and soft tissue tumours and this is evidenced by the papers on osteosarcoma, chondrosarcoma, Ewing’s sarcoma and soft tissue sarcoma. The indications for and outcomes of major ablative amputations are outlined followed by an extensive review of the outcomes of limb salvage surgery with endoprostheses and other techniques. The role of this type of surgery in metastatic disease is put in context. Finally the importance of follow up and guidelines is considered. The thesis represents an analysis of the huge improvements that have taken place in the past 25 years in musculoskeletal oncology but also reveals the many still unanswered questions in this evolving field.

Autotaxin is an extracellular enzyme that generates lysophosphatidic acid (LPA). LPA binds up to six different cell surface G protein-coupled receptors to initiate signaling resulting in cell survival, invasion and angiogenesis. For this reason autotaxin has emerged as a therapeutic target in several different malignancies. I have used immunohistochemistry to explore the expression of autotaxin and its correlation with clinico-pathological variables in bladder and renal cancer. I show that in bladder cancer, tumours from patients with muscle invasive disease were significantly more likely to show strong autotaxin expression than were those tumours from patients without evidence of muscle involvement (p=0.009). This observation is not only consistent with the known functions of autotaxin/LPA in promoting tumour invasion, but suggests that the potential use autotaxin inhibitors in preventing bladder cancer progression warrants further investigation. Although I failed to detect autotaxin expression in the tumour cells of patients with renal cancer, I did observe high-level expression of autotaxin on the tumour-associated vasculature, which in many cases was not apparent in blood vessels of matched normal renal tissues. This points to an important role for autotaxin in renal cancer-associated angiogenesis and suggests a potential role for autotaxin inhibition as an anti-angiogenesis therapy.

It is well established that adenoviruses inactivate the host cell DNA damage response to enhance viral DNA replication. This is achieved, in part, by the ability of viral E1B55K and E4ORF6 proteins to hijack host cell cullin-containing E3 ubiquitin ligase complexes and target key cellular proteins such as Mre11, p53 and DNA ligase IV for degradation via the ubiquitin-proteasome pathway. To assess the generality of this viral response, studies were undertaken using a panel of representative serotypes from adenovirus species A to E to determine how they interact with the host cell DNA damage response. Notably, serotypes from species B and D were unable to promote the degradation and/or relocalization of Mre11 and p53, although DNA ligase IV degradation was fundamentally conserved. Furthermore, species B and D serotypes induced the sustained overexpression of transcriptionally inactive p53, and induced both ATM and ATR kinase activity. As these events would typically be viewed as detrimental to virus survival, these data suggest that different adenovirus serotypes have evolved novel strategies in order to subvert the cellular DNA damage response during infection. Adenoviruses have long been utilised as useful tools for identifying, and characterizing, the function of fundamental cellular proteins such as tumour suppressors and those involved in the DNA damage response. Therefore, studies were also carried out to identify novel Ad12E1B54K-interacting proteins through mass spectrometric analysis, and subsequently to examine the functional significance of these interactions. Several putative novel Ad12E1B54K-interacting proteins were identified using this approach, one being the transcriptional intermediary factor 1γ (TIF1γ), a transcriptional regulator that has recently been identified as a tumour suppressor. Further studies determined that TIF1γ was relocalized to nuclear tracks in an E4ORF3-dependent manner early during adenovirus infection, and was subsequently degraded in a ubiquitin-mediated proteasome-dependent manner. Uniquely, TIF1γ degradation was shown to be E1B55K/E4ORF6-independent and E4ORF3-dependent. Data presented in this thesis also suggest that E4ORF3 does not utilise host cell cullin-based E3 ubiquitin ligases in order to promote TIF1γ degradation. The ability of E4ORF3 to target cellular substrates for degradation represents a novel way in which adenoviruses are able to target cellular substrates. Significantly, TIF1γ degradation was conserved during infection with Ad serotypes from species A to C which may highlight its importance for productive viral infection. It also appears that TIF1γ may have an as yet unidentified role in the DNA damage response since it was also found to interact with components of the ATR kinase pathway.

The outer membrane (OM) of Escherichia coli is an essential organelle. The OM allows E. coli to interact with its environment and has a critical function as a barrier to prevent the entry of toxic molecules into the cell. The OM is composed of phospholipids, lipoproteins, outer membrane β-barrel proteins (OMPs) and lipopolysaccharide (LPS). The correct ratio of these components is needed to ensure proper OM barrier function is maintained. Assembly of OMPs is performed by the Bam (β-barrel assembly machinery) complex, lipoproteins by the Lol (Localisation of lipoproteins) pathway and LPS by the Lpt (LPS transport) pathway. The factors responsible for the assembly of phospholipids at the OM are unknown. This study presents two key areas in understanding OM biogenesis. Firstly, a comprehensive mutagenesis screen was performed on the Bam complex member BamE. This analysis along with the structure of BamE has indicated crucial regions for BamE function. Secondly, we have performed a structure and function analysis on the previously uncharacterised protein, YraP. The structure of YraP has been solved and represents a novel fold. Additionally, we have obtained some functional evidence that suggest that YraP is involved in phospholipid biogenesis in the OM.

Thyroid cancers are the most common endocrine malignancies and their incidence continues to rise. Over-expression of the human pituitary tumor transforming gene (hPTTG) in thyroid carcinomas is a prognostic indicator of tumour recurrence. hPTTG is multifunctional with roles in mitotic control, DNA repair, genetic instability, cell transformation and apoptosis. Importantly, hPTTG transactivates expression of growth factors implicated in proliferation and angiogenesis, and represses the sodium iodide symporter (NIS), which is essential to radioiodine treatments in thyroid cancer. hPTTG interacts with a binding factor (PBF) which is an independent transforming gene and also represses iodine uptake. Work described in this thesis provides evidence for the existence of thyroidal autocrine regulatory pathways involving hPTTG and growth factors in vitro. We directly investigated the role of hPTTG in thyroid tumourigenesis through the generation and characterisation of a murine transgenic model with thyroid-targeted hPTTG over-expression (hPTTG-Tg mice). Unexpectedly, hPTTG over-expression was not sufficient for thyroid tumourigenesis. Investigations performed in hPTTG-Tg and Pttg-/- knockout mice indicated a particularly important relationship between hPTTG and the epidermal growth factor (EGF) in vivo. hPTTG and PBF were confirmed as repressors of NIS in vivo following studies in hPTTG-Tg and PBF-Tg mice respectively. The studies described in this thesis highlight the therapeutic potential of targeting hPTTG and PBF in thyroid cancer. To this effect, specific tyrosine kinase inhibitors prevented autocrine induction of hPTTG by growth factors, and siRNA depletion of PBF restored NIS function to normal levels in hPBF-Tg thyrocytes. Based on these data, hPTTG appears to play a dual role in endocrine tumourigenesis, being involved in both tumour initiation and subsequent progression towards more aggressive phenotypes.

Patient-level evidence obtained from clinical trials is essential in assessing the cost-effectiveness of health care technologies. Given the increasing demand for primary evidence and limited public resources for health care research, research funding organisations are routinely called to make decisions on which clinical trials to fund. Such decisions need to be informed by evidence on the likely costs and benefits of competing research programmes. Two main analytic approaches have been proposed to provide such evidence, ‘payback of research’ and ‘value of information’. This work applied the ‘payback’ and ‘value of information’ methodologies to case studies representing proposals for clinical trials in cancer. This application gave estimates of the value of undertaking the trials and offered an insight into the strengths, limitations and usefulness of the methods. ‘Payback of research’ and ‘value of information’ can help with different funding decisions in the context of different funding streams, they are practical to undertake and can be readily incorporated into the existing research funding processes. It is suggested that the methods should be used as part of existing deliberative processes, to provide additional assurance that limited public resources are allocated to clinical trials which are likely to result in benefits to the population.

We examined both the chromosomal copy number changes and differential RNA expression profiles in Nasopharyngeal carcinoma (NPC). Gene expression profiles identified a large number of differentially regulated genes involved in diverse functional processes, while genetic analysis detected extensive genomic abnormalities including large and small, discrete regions of copy number change and loci that exhibit uniparental disomy (UPD). The relationship between chromosomal copy number and level of gene expression were analysed. This revealed that the direct copy number/expression link applies in about 60% of the instances of copy number loss/down-expression and less than 35% of instances of copy number gain/up-expression that were examined. Signalling pathway analysis revealed that numerous components involved in the TGF-β, Wnt/β-catenin and Hedgehog pathways were universally upregulated in NPC tumours, and gene expression pattern of the C666-1 cell line approximated to other NPC tumours, indicating that it is a good tumour model. A preliminary in vitro investigation of signalling pathways revealed that the C666-1 cell line is intact in the activin and Hh signalling pathways but not in the TGF-β pathway. However, the C666-1 cells appear to resist activin-mediated cell growth inhibition