The unique situation where there is persistent clinical suspicion of prostate cancer (PCa) despite repeated benign histology from conventional prostate biopsy is a diagnostic dilemma for clinicians and patients. Transperineal template guided saturation biopsy (TTSB) has a high cancer yield in this group, but is associated with unacceptably high morbidity particularly high rates of acute urinary retention (AUR). As localised PCa rarely involves periurethral area at the base, we hypothesised that urethral trauma from extensive sampling of this area is a major trigger factor for AUR. This thesis presents data from modified periurethral sparing TTSB technique to determine whether the risk of AUR is reduced compared with rates reported in the literature, without compromising cancer yield and investigates biochemical predictors of pathological outcome and role of pre-biopsy MRI in this group. Three hundred and three men with persistent clinical suspicion of PCa despite a median of 2 (range 1-6) sets of negative biopsies were investigated. Patients prospectively completed a questionnaire evaluating bleeding, pain (visual analogue scale, range 0-10) and analgesic requirements which were assessed at 1 hour post biopsy and on days 1, 3 and 7. Serum PSA, PSAD and %fPSA were documented and evaluated for their ability to predict PCa diagnosis, Gleason score and cancer volume. Furthermore, a pre-biopsy magnetic resonance imaging (MRI) was performed and abnormalities in 4 anatomical quadrants were compared with TTSB to assess whether pre-biopsy MRI could defer need for TTSB or allow a more targeted biopsy regime. Median age was 64 years (range 43-85). PCa was diagnosed in 167 of 303 men (55.1%) from median of 29 cores [range 16-43, Gleason 6 (29.9%), 7 (45.5%) and 8-10 (24.6%)] and 140 (83.8%) were clinically significant with 77.2% of cancers involving the anterior region. AUR occurred in 23 men (7.6%). On multivariate analysis, only prostate volume predicted AUR (P=0.004). Pain was minimal (peak on day 1, mean 0.8 out of 10), requiring simple analgesia in 27% on day 1, mostly for mild perineal discomfort. Haematuria (75%) and rectal bleeding (20%) significantly decreased over one week, with 33% still experiencing haematospermia at this stage. PSAD (AUC 0.76) was more predictive of cancer diagnosis compared to 0.71 for %fPSA and pre-TTSB PSA respectively. The overall sensitivity and specificity of MRI for cancer diagnosis were 65% and 77% respectively with multiparametric MRI showing more accuracy with sensitivity and specificity of 83% and 95% respectively compared to other MRI sequences. Of the 24 false negative MRI cases, 16 (67%) were clinically significant. So complete prostate mapping should continue at present. In conclusion, this thesis demonstrates that clinical suspicion of PCa despite negative histology from conventional TRUS Biopsy should not be disregarded, as a significant proportion harbour aggressive disease. Modified TTSB is well tolerated with low risk of AUR. Presentations of aspects of this thesis at key regional, national and international urology meetings have generated interest and helped set up dedicated units in the region for PCa diagnosis in this group. Questions raised in this study provide backbone for future research in this field.

Following five decades of kidney transplantation, increasingly high risk immunological kidney transplantation (which previously was considered as sub-optimal) are carried out. The risk stratification with the current available assays have allowed safe transplantation in low risk non-sensitised patients and direct transplantation in high risk highly sensitised patients by removal of circulating donor specific antibodies (DSA) with reasonable outcomes. However, a large number of patients with chronic kidney disease and with low or intermediate antibody levels measured by current assay, the best way forward is uncertain resulting in denial of transplantation in some cases. Whilst in other cases, the solid phase Luminex assay may under or overestimate the risks of rejection and graft failure following direct kidney transplantation. Currently only IgG-class of DSA is considered immunologically important and routinely measured in clinical laboratories. Other bio-physiological characteristics such as class, sub-class and binding kinetics of DSA may be more specific for risk stratification of immunological risks. In this thesis, we studied effect of de novo IgM class of HLA-specific antibodies on outcome of kidney transplantation and characterised binding kinetics and strength of HLA-specific antibodies. De novo IgM or IgG HLA-specific responses alone were not associated with adverse outcomes following kidney transplantation. Presence of both IgM and IgG responses, however, was associated with poor graft function at 36 months. There was no temporal relationship of antibody response and episodes of rejections. De novo Donor specific responses were less frequent compared to non-specific responses. A shorter follow-up and use of modern triple immunosuppressant therapy (Tacrolimus, Mycophenolate and Steroid) may explain this. Binding kinetics measured by biosensor assay- surface plasmon resonance (SPR) on purified monoclonal HLA-specific antibodies showed binding kinetics and strength differed between HLA alleles despite same epitope and paratope interactions. There was a tendency towards higher affinity and faster association rate for HLA protein that was the initial immunizing antigen for the corresponding monoclonal HLA-specific antibodies. The dissociation constant (KD) of human monoclonal HLA-specific antibodies range between 10-8 to 10-10 M. Thermodynamic analysis showed higher Gibbs free energy released for interactions with higher binding strength. The binding strength of mixed monoclonal HLA-specific antibodies is generally average of the strength of individual monoclonal HLA-specific antibodies. Enriched polyclonal HLA-specific antibodies from clinical sample gave distinct binding response on bio-sensor based on SPR assay. Quantification of polyclonal HLA-specific antibodies using sandwich ELISA and SPR allowed quantitative measurement of binding kinetics and strengths. A range of binding strength was observed between patients and within same patient antibodies of different affinities was observed. Thus the antibodies could be grouped in four groups based on the strength of binding and this can serve as additional biomarker for risk stratifications.

Cutaneous fatty acid-binding protein (C-FABP) or FABP5, is a FABP family member that can bind to long chain fatty acids with high affinity. C-FABP was identified by our research group as a gene involved in malignant progression of prostate cancer and able to promote the growth of primary tumours and induce metastasis when transfected into rat benign Rama 37 model cells. It was demonstrated that C-FABP was a prognostic marker for patient outcome and a target of tumour-suppression for prostate cancer. As an initial step to understanding the complex molecular mechanisms involved in the cancer promoting activity of C-FABP, this study investigated the possible relationship between tumorigenicity-promoting activity of C-FABP and its fatty acid-binding capability. After single and double mutations were generated in the fatty acid binding motif of the C-FABP cDNA, wild type and mutant C-FABP cDNAs were excised from the mammalian vector pIRES2-EGFP and inserted into pBluescript cloning vector to generate a complimentary restriction sites at both ends of the cDNAs. The C-FABP cDNAs were excised from the pBluescript vector with KpnI and PstI and inserted into pQEs expression vector to form three constructs that express the wild type and two mutant C-FABPs (C-FABP-WT, C-FABP-R109A and C-FABP-R109/129A), respectively. SDS-PAGE and sequence analysis confirmed the correct insertion of C-FABPs into expression vector pQE. The pure recombinant proteins were subsequently produced and purified. The importance of fatty acid-binding activity to the cancer promoting function was assessed by comparing the cancer promoting abilities exerted by C-FABPs with different fatty acid-binding capabilities. To test whether the recombinant proteins produced were biological active, the fatty acid binding ability of wild type and mutant C-FABPs were tested. When fatty acid binding ability of the wild type C-FABP is set at 1, the average fatty acid binding ability the C-FABP-R109A and C-FABP-R109/129A were significantly reduced to 0.32% and 0.09%, respectively (Student t-test, P<0.005). These results suggested that fatty acid binding ability of C-FABP depends on the structured integrity of the binding motif. Thus, changing one amino acid in the motif significantly reduced the fatty acid binding ability by 68%, and changing two amino acids almost completely abolished the fatty acid binding ability of C-FABP. To access the importance of the structural integrity of the fatty acid binding motif to the tumorigenicity-promoting activity of C-FABP in prostate cancer, the effect of wild type and mutant C-FABPs on cell proliferation, invasion and colony formation as indication of tumourigencity were analysed. The average growth rate of cells stimulated with C-FABP-WT was significantly increased by 17% (Student t-test, P<0.05) when compared to control cells. Whereas, the average growth rate of cells stimulated with C-FABP-R109A and C-FABP-R109/129A were significantly reduced by 33% and 47%, respectively (Student t-test, P<0.005) when compared to control cells. The invasiveness of cells stimulated with C-FABP-WT, C-FABP-R109, C-FABP-R109/129A and the control cells were 256±40, 163±32, 80±26 and 96.6±15.2, respectively. The number of invaded cells stimulated with C-FABP-WT was the highest in all cell lines and more than 2.6-fold higher than the number of invaded cells from control (Student t-test, P<0.05). The average number of colonies produced in the soft agar by selected cells stimulated with C-FABP-WT, C-FABP-R109A, C-FABP-R109/129A and control were 1050±132.29, 283±76.38, 157±38.1 and 155±68.74, respectively. In comparison with the control, the average number of colonies produced by C-FABP-R109A stimulated cells was increased by 45% (Student t-test, P<0.01) whereas the average number of colonies produced by C-FABP-R109/129A stimulated cells was at same level as the control cells (Student t-test, P>0.5). The most significant change occurred in C-FABP-WT stimulated cells that produced more than a 6.7-fold (85%) increase in the number of colonies formed in soft agar when compared to controls (Student t-test, P<0.001). These results showed that the increased wild type C-FABP stimulation in prostate cell line significantly increased cell proliferation, invasiveness, and tumorigenicity. Whereas, the increased expression of both mutant forms of C-FABP did not significantly affect these characteristics. Overall, this study has confirmed that the biological potential of C-FABP to promote tumourigencity of prostate cancer cells depends on its ability of binding to fatty acids. Thus, C-FABP may facilitate cancer development through a mechanism involving transportation of intracellular fatty acids into cells. These results were supported by our recent data obtained from in-vivo studies performed in a nude mouse model.

Sustained, toxic elevations of pancreatic acinar cell cytosolic free calcium ion concentration ([Ca2+]C), such as those observed with supramaximal secretagogue stimulation (CCK) are implicated in acute pancreatitis. However, Cholecystokinin (CCK) has been thought to act only indirectly on human pancreatic acinar cells via vagal nerve stimulation, rather than by direct CCK receptor activation as observed in rodent pancreatic acinar cells. However, in the series of experiments presented here using human pancreatic acinar cells, CCK at physiological concentrations (1-20 pM) elicited rapid, robust, oscillatory rises of the cytosolic Ca2+ ion concentration ([Ca2+]C), showing apical to basal progression in acinar cells, in the presence of atropine and tetrodotoxin. The [Ca2+]C rises were followed by increases in mitochondrial ATP production and secretion, concluding that CCK acts directly on acinar cells in the human pancreas. The earliest pathological mechanisms, such as sustained, toxic elevations of the acinar cytosolic free calcium ion concentration ([Ca2+]C), incriminated in experimental pancreatitis have been previously demonstrated by non-oxidative metabolites of ethanol (FAEE’s), as well as bile salts, at supramaximal concentrations. However, in the clinical situation such hyperstimulation is unlikely to occur. To simulate a more clinically relevant stimulus, pancreatic acinar cells were stimulated with lower doses of FAEE’s and/or bile salts in combination with physiological doses of secretagogues - a process which may precipitate pancreatitis clinically. Illustrated here, the toxic transformation of secretagogue induced physiological Ca2+ signalling occurs with the perfusion of low doses of TLCS and POAEE resulting in cell injury. The intracellular second messengers implicated are IP3, cADPR and NAADP with the IP3 receptor channel pivotal with both toxins. However, as previously demonstrated with supramaximal concentrations of POAEE, if supplementary ATP is added to the intracellular milieu, cellular injury is avoided with continued extrusion of large quantities of Ca2+ from the cytosol indicating functional Ca2+ ATPase pumps. This is not observed in cells which do not receive supplementary ATP. The toxic sustained Ca2+ elevation is also be prevented by the removal of external Ca2+ or blockade of IP3 receptor using caffeine and cell injury is again avoided. Therefore, it may be concluded, that it is the large, sustained toxic [Ca2+]c load which impairs mitochondrial function and ATP production leading to Ca2+ATPase pump failure and ultimately cell death. Lowering sustained intracellular [Ca2+]c by blockade of IP3 receptor channels may reduce cell injury in clinical acute pancreatitis.

Chronic lymphocytic leukaemia (CLL) cells are derived from mature B lymphocytes and are distinctive with respect to overexpression of the classical protein kinase C isoform protein kinase CβII (PKCβII), which is encoded by PRKCB. Expression of PKCβII in CLL plays a vital role in the pathogenesis of the malignant cells in this disease, and within the microenvironment cells where it provides signals for the production of factors which support the survival of CLL cells. In CLL cells PRKCB transcription is stimulated by vascular endothelial growth factor (VEGF) through a mechanism involving activated PKCβII. However, at the beginning of this thesis the molecular regulatory mechanism(s) governing expression of the PKCβ gene were poorly described. Thus, to characterise the factors regulating PRKCB transcription in CLL cells I used different approaches including mithramycin treatment, a drug which intercalates into GC-rich areas of DNA to inhibit binding of specificity protein 1 (Sp1), specific Sp1 siRNA, promoter function assays and site directed mutagenesis and chromatin immunoprecipitation (ChIP). Experiments using these techniques showed that Sp1 has a direct role in driving expression of the gene coding for PKCβII in CLL cells. My results also show that Sp1 is highly associated with the PRKCB promoter in CLL cells compared to that in normal B cells, and suggest that this is likely because of the presence of histone marks permissive of gene activation. Examination of other transcription factors such as Sp3, MITF, RUNX1 and E2F1 that potentially bind the PRKCB promoter showed that they have static or indirect effects in regulating transcription of this gene. The exception to this is STAT3 which my data suggests plays a role in suppressing PKCβ gene expression in CLL cells. Exploration of the mechanism through which VEGF induces PRKCB transcription revealed that this growth factor stimulates increased association of Sp1 and decreased association of STAT3 with the PRKCB promoter. Thus, VEGF-stimulated activation of PKCβII may play a role in this process. Taken together, Sp1 is the major driver for overexpression of PKCβII in CLL cells, and because this transcription factor is also overexpressed in these cells, the mechanisms I describe controlling PRKCB transcription potentially provide a foundation for further study of other genes contributing to the phenotype of CLL cells that are regulated by this pleiotropic transcription factor.

The accuracy of dose calculation algorithms used for radiotherapy treatment planning play a significant role in the clinical outcome of various treatment regimens. Heterogeneities in human anatomy such as lung, air cavities, bone, soft tissue and fat present challenges to the dose calculation algorithms as they are prone to disrupt the charged-particle equilibrium. Monte Carlo (MC) based dose calculation algorithms are proven to be superior to all the current analytical algorithms owing to their ability to account for all the physical interactions that are involved in radiation transport. Numerous publications have examined the differences in physical doses calculated by analytical algorithms when compared to MC in dealing with heterogeneities. However, before this work the clinical significance of these differences in physical dose has never been investigated in detail. An EGSnrc, BEAMnrc and DOSXYZnrc based MC dose calculation engine was set up in a parallel computing environment to simulate three-dimensional conformal radiotherapy (3DCRT) and intensity modulated radiation therapy (IMRT). A Varian 2100 C/D accelerator head was modeled and validated to match measurements of open and dynamic wedged fields in a homogeneous water phantom which was found to be in good agreement with measurements within 2%/2mm and 3%/3mm respectively. In addition, MC calculated doses in a heterogeneous lung phantom were compared to radiochromic film measurements. Overall, there was good agreement between the two, although large differences of upto 16% were found in some cases. This dose calculation system was used to perform MC simulations on computed tomography (CT) images. The clinical impact of the differences in absolute doses calculated by various photon dose calculation algorithms for two clinical tumour sites was investigated. The tumour control probability (TCP) and normal tissue complication probability (NTCP) were estimated using well established bio-mathematical radiobiological models. This work includes the analysis of 7 convolution (i.e. pencil-beam) and convolution-superposition (CS) based photon dose algorithms available in commercial treatment planning systems (TPSs) as well as MC, in treatment plans of non-small cell lung carcinoma (NSCLC) and nasopharyngeal carcinoma (NPC). In both NSCLC and NPC, the convolution algorithms overestimate the dose to the tumour and hence overestimate the TCP to up to 45%. Some of the CS algorithms were comparable to MC though others exhibit significant differences. In NSCLC, the absolute differences in the NTCP values with radiation pneumonitis and rib fracture as end points were not as large as the differences found in the TCPs. On the other hand, in NPC, the overestimation of probability of occurrence of xerostomia by some TPS algorithms may be preventing dose escalation. Parameters for the TCP model were derived by fitting the TCP predictions to published outcome for four widely varying dose-fractionation regimens for a patient cohort undergoing radical radiotherapy treatment for NSCLC. The derived parameter sets strongly depend on the accuracy of the dose calculation algorithm involved. Parameters derived based on dose-distribution data sets obtained using one particular dose calculation algorithm may not hold good when evaluating treatment plans calculated with a different algorithm. In this sub-study, the influence of dose calculation algorithms on TCP model parameters was evaluated. Significant differences were found in TCPs when calculated with inconsistent parameters. Hence, the choice of dose calculation algorithm is crucial and although some algorithms generally perform close to MC in handling inhomogeneities, it is necessary to understand how the underlying differences affect the predicted clinical outcome.

The ability to therapeutically manipulate regulatory T (Treg) cell survival/function would have far reaching implications; with the potential to limit immune pathology in autoimmune disease, allergy and transplantation; and to reduce regulation of anti-tumour responses in cancer. This study has established a method to study the survival and function of antigen specific Treg cells in vivo, adapting an existing approach in which an endogenous naïve T cell population is expanded and tracked. Multiple immunisation of antigen, and an agonistic anti-DR3 antibody were used to ensure a sufficient number and proportion of Treg cells could be expanded. Further to this, an assay for investigating Treg cell function in vivo was applied to this system. This approach revealed that the tumour necrosis factor receptors OX40 and CD30 may play a role in Treg function, as well as expansion. Unexpectedly, these data also revealed that in the absence of OX40 there is a gross defect in the function of CD4 T cells. A regulatory role of group 3 Innate Lymphoid cells is emerging in the literature, and in accordance with this, this study demonstrates that Treg cell expansion is grossly impaired in mice which lack RORγt, their lineage defining transcription factor.

Cytomegalovirus (CMV) is a β-herpes virus that infects the majority of the world’s population. Tyrosine kinase inhibitors (in particular imatinib, dasatinib and nilotinib) have been successfully used in the treatment of chronic myeloid leukaemia, as they target the \(Abl\) kinase, which is constitutively activated in the disease. Although thought of as targeted therapies, they have significant “off target” effects including inhibition of \(Src\) family kinases important in T cell receptor mediated activation. I demonstrated that CMV infection is associated with significant alterations in the immune repertoire in imatinib-treated patients; in particular with expansions of differentiated CD8 T cells and Vδ1 γδ T cells. Furthermore, dasatinib treatment is associated with evidence of subclinical CMV reactivation and marked expansions of terminally differentiated CD8 T cells and Vδ1 γδ T cells. These atypical Vδ1 γδ T cells have activity against CMV infected fibroblasts, and sequencing of their TCRs demonstrated remarkable oligoclonality suggestive of antigen driven proliferation. In a second group of patients that underwent reduced intensity allogeneic stem cell transplant for myeloid malignancies, CMV seropositivity of patient or donor is associated with increased lymphocyte counts at 3 months post transpant, particularly of CD8 and Vδ1 γδ T cell subsets. Survival analysis of these patients revealed that CMV seropositivity is associated with improved overall survival, due to a decreased relapse risk.

This thesis investigates the role of CD151 in modulating the form and function of its integrin partners, α3β1 and α6β1/β4. Stable depletion of CD151 in the MDA-MB-231 (MDA-231) cell line, changed the glycosylation profile of α3β1, but not α6β1/β4 integrins. Glycosylation of CD151, tight association between CD151 and α3β1 integrin and recruitment into tetraspanin enriched microdomains (TERM) are all required for this effect and the intervention occurs during the first mannose trimming step within the ER. Further analysis showed that CD151 preferentially associates with α3β1 over α6β1/β4. CD151 mediated changes to glycosylation of α3β1 integrin decreased their ability to migrate towards Lm332 by ~90%. Sucrose density gradient assays showed α3β1 and α6β1/β4 are recruited by CD151 into the light fractions as part of a TERM as well as separate entities. Glyco-analysis of the tetraspanins themselves showed CD9 and possibly CD63 and CD82 to be phospho-mannosylated. Castanospermine treatment dramatically reduced the level of CD151, α3β1 and α6β1/β4 in these cells, indicating a novel therapeutic use. Depletion of various tetraspanins in MDA-231 altered their cell surface glycotope presentation and the cleavage profile of α6β1/β4 integrin; highlighting the role played by tetraspanins in post-translational modification of their partners.

The incidence of oesophageal adenocarcinoma (OAC) is increasing at an alarming rate in the Western World. Despite advances in surgical technique and patient selection, overall survival remains abysmal. Understanding the molecular and genetic events in the evolution of OAC is crucial to improving outcome. The role of iron in the carcinogenesis of OAC is supported by epidemiological and experimental evidence. Oesophageal cancers are iron loaded and aggressively capture systemic iron to potentiate a malignant phenotype. This project aimed to establish that OAC cells are also capable of acquiring haem and to explore the potential of iron chelation therapy in the treatment of oesophageal cancer. Haem import proteins are sequentially over expressed in the evolution of OAC. Culturing OAC cells with supplementary haem significantly enhances viability, proliferation, migration and anchorage independent growth. Abrogation of haem import protein expression reverses the stimulatory effect of supplementary haem and significantly reduces tumour burden in-vivo. Different classes of iron chelators exhibit potent in-vitro and in-vivo anti-neoplastic action in oesophageal malignancy. Iron chelators demonstrate chemo-sensitising properties and are able to overcome chemo-resistance. Haem import proteins are potential therapeutic targets in the treatment of oesophageal malignancy. Iron chelation therapy represents an effective, predictable and well tolerated adjunct to standard chemotherapy and should be considered for clinical trial.