Cancers of the upper gastrointestinal tract present at an advanced stage and carry a poor prognosis. Oesophageal and gastric tumours have a rich stroma composed of vascular cells, immune cells, and myofibroblasts, which promotes tumour growth, invasion and metastasis. In addition, mesenchymal stromal cells (MSCs) are recruited from the bone marrow to the tumour stroma; the mechanisms underpinning this have not yet been defined. Extracellular proteases play a role in cell migration, invasion, and cell signalling and are known to influence cancer growth in conflicting ways. Myofibroblasts present in normal tissue differ from those found in cancer. Cancer-associated myofibroblasts (CAMs) are known to modulate extracellular protease activity by secreting plasminogen activator inhibitor-1 (PAI-1), an inhibitor of the serine protease urokinase plasminogen activator, and matrix metalloproteinases (MMPs). This investigation studies the role of PAI-1 in gastric cancer and assesses the contribution of myofibroblast-derived MMPs to tumour growth. Finally, the role of chemerin in recruiting MSCs has been investigated. The expression of PAI-1 in myofibroblasts was found to be higher than in gastric cancer cells. Overexpression of PAI-1 in gastric cancer cells resulted in decreased cell adhesion and decreased tumour growth in an in vivo subcutaneous xenograft model of gastric tumour growth. The addition of gastric CAMs potentiated the growth of gastric cancer subcutaneous xenografts. This was not accounted for by differences in cell proliferation rate, apoptosis or final stromal content. Xenografts containing CAMs suppress the growth of a contralateral xenograft without CAMs, demonstrating that a long-range signal can be generated as a result of stromal-epithelial interactions. MMP and cathepsin activity was compared between xenografts containing myofibroblasts to those without. MMP activity is increased in xenografts injected with CAMs, compared to those injected with myofibroblasts taken from normal stomach or those with gastric cancer cells alone. In an organotypic co-culture system, MMP inhibition resulted in a decrease in gastric cancer cell invasion. The injection of fluorescently labelled MSCs injected resulted in homing of these cells to subcutaneous oesophageal tumours containing CAMs. Antagonism at the ChemR23 receptor inhibited this MSC homing to oesophageal xenografts containing CAMs. This work emphasises the importance of assessing the contribution of specific proteases and their inhibitors in gastric cancer. The stroma is an important contributor to extracellular protease activity and myofibroblasts contribute both proteases and their inhibitors to the tumour microenvironment, resulting in the modulation of tumour growth and cell adhesion. MSCs are recruited to oesophageal tumours via a novel signalling pathway.

This volume presents the research carried out in partial fulfilment of the Doctorate in Clinical Psychology at the University of Liverpool. It contains three papers, addressing the area of cancer patients‘ trust in care. In times of distress, patients engaged in a course of psychological therapy wish to feel safe, contained and to trust that the clinician is working in their best interests. The same holds true in a medical setting, where being diagnosed with a life-threatening illness such as cancer evokes feelings of vulnerability, helplessness and intense fear. Clinicians can be viewed as attachment figures in this time of stress, being the cancer patient‘s main hope for creating safety in the face of threat. Whilst a relationship with the clinician characterised by trust is beneficial at this time, unfortunately not all patients experience the relationship in this way. Before we can suggest means to improve cancer patients‘ sense of trust in the clinician, we need to understand the factors which prevent patients from developing trust in their clinician. Unlike the field of psychotherapy, where patient factors influencing the relationship with the therapist have been extensively researched, the medical field has focused its efforts on attempting to highlight the contribution of the clinician. This thesis aims to address this current dearth of literature, and focus on patient factors which could impede them from having trust in cancer care. Paper 1: Literature review The review paper provides the backdrop for the research. A specific sub-section of the background literature that informed the development of the research is considered here. A general review of current knowledge relating to all aspects of cancer patients‘ experiences highlighted an area of relative dearth in research. From this, a specific research question was developed: what is the role of patients‘ experiences in trust in cancer care? A focussed literature review was conducted in response to this pre-determined question, investigating whether patient factors of trauma, abuse and attachment style shape patients‘ trust in cancer care. The review adopted a structured approach to interrogating the evidence base, and the search terms and eligibility criteria for inclusion of papers are outlined. This section presents the identified papers, comparing and contrasting aims, design, methodology and findings, and explores the key themes that arose. The collective limitations, inconsistencies and gaps in this literature base are explored, and future directions to address these points are suggested. The review paper presents a picture of the current knowledge in this area, enabling the reader to locate the research study in its broader context. Paper 2: Empirical paper Building upon paper one, the second paper describes the main features of the research study, presented according to author guidelines set out for the journal Psycho-Oncology (Appendix A). Whilst a trusting patient/clinician relationship is repeatedly highlighted as important in healthcare, there is a tendency in the research to neglect the patient‘s contribution to this interaction. Attempts to identify individual patient factors that contribute to the sense of clinical relationship are dwarfed by extensive research focussing upon the clinician‘s competence, skill, communication and interpersonal style. The little research that has been carried out in this area has predominantly sampled women with breast cancer, leaving unexplored questions about the generalisability to other populations. In an attempt to redress balance, this research investigated patient factors which affect trust in the clinician, in a sample of gynaecological cancer survivors. The process of developing and conducting the study took two and a half years, whilst recruitment commenced after ethical approval was sought, and spanned 52 weeks. The key features of the study are outlined in this section. Paper 3: Concluding discussion The third section draws out the main findings of the research, and discusses the wider relevance. Further attention is given to the relative strengths and weaknesses of the study, and the study‘s applicability to clinical practice is considered. A short lay summary is presented, which was written for dissemination of the research to the participants who had requested feedback. This discusses the key contributions of the research to the literature base, and states what will be done with the findings. Finally, a research proposal describing a possible follow up study is outlined, taking into account the limitations of this research and considering how it can be extended. This concludes the thesis.

Introduction: Colorectal cancer is the second most common UK cancer. Biomarkers which predict survival may be valuable for targeting adjuvant therapy and can provide insights into tumour biology. Small and early cancers are being diagnosed more commonly in the UK population due to the introduction of population-based colorectal cancer screening in 2005. Analysis of resected small (≤20mm across) tumours in Liverpool has established that flat and depressed morphology can predict advanced stage at presentation. Proteomic analysis of small cancers was conducted with the aim of generating biomarkers which correspond to morphology, stage and patient survival. Patients and Methods: Laser capture microdissection was used to procure enriched matched benign and malignant colorectal epithelial cell populations. Laser captured proteins were extracted into lysis buffer, normalised against a reference standard, separated using 2D SDS-PAGE and visualized with silver staining. Comparison was made between the tumour gels, (n=10) and matched normal colonic gels, (n=9) by two different observers and gel analysis software, Progenesis SameSpots. Differentially expressed proteins were identified using tandem mass spectrometry and included redox proteins peroxiredoxin 2, peroxiredoxin 6 and SH3 binding glutamic acid-rich protein-like 3; and cytoskeletal protein cofilin1. Also identified were the anti-apoptotic protein heat shock protein 27 and inflammatory protein S100A8, which had been previously identified in 2D gel analysis of undissected colorectal cancer in our Institution (n=12 gels) and previously validated in a small cohort of paraffin-embedded colorectal cancers (n=98). In this study, HSP27 was further evaluated in a large cohort of paraffin-embedded colorectal cancer tissue (n=404). S100A8 and related proteins S100A9 and Smad4 were similarly evaluated in a large cohort (n=313). Results: High HSP27 levels were strongly associated with poor cancer-specific survival in rectal cancer (n=205, P=0.0063) but not colon cancer; (n=199, P=0.7385). Multivariate Cox regression confirmed nodal metastases (P=0.0001) and HSP27 expression (P=0.0233) as independent markers of survival in rectal cancer. HSP27 levels remained unchanged in the majority of cases 65/80 (81%) between diagnostic biopsies and matched surgical samples, regardless of whether patients had undergone preoperative radiotherapy. S100A8 expression co-localised with a subset of S100A9-positive monocytes. S100A9 was co-expressed with CD14 in tumour-associated monocytes, but not with CD68 in tissue macrophages. Smad4 was expressed in the tumour cytoplasm of 262/304 (14%) tumours. Loss of Smad4 expression correlated with a reduction in the stromal S100A8-positive, but not S100A9-positive cell count, (P=0.034, Mann-Whitney U test) and was associated with a poorer overall survival in patients with stage I-II disease, but not stage III disease. Antibodies to cofilin1 and cofilin-phospho(ser3) were assessed in colorectal cancer cell and tissue lysate and found to be specific on 1D and 2D western blot. Conclusion: Elevated HSP27 is an independent marker of poor prognosis in rectal cancer whose expression is not altered by neo-adjuvant radiotherapy. Smad4-negative tumours are associated with fewer infiltrating S100A8 positive stromal monocytes. In node-negative tumours, loss of Smad4 expression in associated with a poorer prognosis. These findings provide a sound platform for further investigation of both S100A8 and HSP27 proteins in colorectal cancer.

The Wnt/β-catenin signalling pathway is involved in regulating cellular transcription of numerous target genes in chronic myeloid leukaemia (CML). A previous report using granulocyte-macrophage progenitors (GMPs) showed that elevated levels of unphosphorylated (active) β-catenin, alongside mis-splicing of glycogen synthase kinase 3β (GSK3β), correlated with disease progression. It was of interest to determine if using the more readily available peripheral blood mononuclear cells (MNCs) also showed any correlation with patient outcome when β-catenin and GSK3β were measured in this source material. Further investigation in these cells addressed GSK3β activity regulation, possible regulation of c-Myc degradation by GSK3β, and investigation of Wnt transcription factors in CML. Results indicated that levels of β-catenin (and its phosphorylated variants) could not be used to distinguish patient treatment outcomes using CML MNCs. However the levels of BCR-ABL1 induced β-catenin-Tyr654 phosphorylation showed a significant decrease in patients in blast crisis compared to chronic phase (P=0.012), as did the levels of GSK3β – which demonstrated significantly decreased activity in blast crisis patients via a significant increase in Ser9 phosphorylation and a significant decrease in Tyr216 phosphorylation in blast crisis compared to chronic phase (P=0.026 and <0.001 respectively). This decrease in GSK3β activity was not reflected by changes in the levels of β-catenin and, as such, this led to the question of whether alternative mechanisms were influencing regulation of GSK3β activity other than Wnt/β-catenin signalling. Results showed that protein phosphatase 2A (PP2A) was regulating the inhibition of GSK3β in CML. This is a dual mechanism with GSK3β also affecting PP2A inhibition. GSK3β is known to have numerous substrates, which include c-Myc. Due to the role of c-Myc as an oncogene in CML and the role of GSK3β in c-Myc degradation, it was of interest to determine if the changes in GSK3β activity were reflected in the degradation of c-Myc. Results showed that neither GSK3β nor PP2A (also involved in the mechanism) were the rate limiting components of c-Myc degradation. Analysis of the Wnt signalling pathway revealed up regulation of WNT1, WNT8A, WNT9A and FZD7 mRNAs in chronic phase MNCs (P=0.011, 0.045, 0.037 and 0.037 respectively) - which was inconsistent with β-catenin findings. These unexpected results can be explained by internal regulation of the pathway by the negative regulator NKD1, which is also significantly higher in samples from chronic phase patients (P=0.013). Finally, investigation into Wnt transcription factors showed that factors TCF1, TCF4 and LEF1 mRNA levels were significantly higher in blast crisis samples compared to chronic phase (P=0.001, 0.016, and 0.003 respectively). This suggests a possible mechanism for Wnt-independent activation of these transcription factors in the blast crisis phase of the disease. In conclusion, it was determined that β-catenin levels cannot be used to either; distinguish between patient response cohorts, or be a factor of disease progression in CML MNCs. The activity of GSK3β is significantly down-regulated in blast crisis. PP2A is involved in this regulation. However when investigating c-Myc degradation, neither GSK3β nor PP2A act as the regulating factors in CML. There may be an internal regulation of the pathway by which NKD1 prevents an upregulation of pathway activity via WNT1, WNT8A, WNT9A and FZD7 reaching β-catenin (CTNNB1). Finally, the transcription factors mRNA expression is significantly upregulated in blast crisis, but they could be acting independently of the Wnt pathway.

GRP78 is a known cyto-protective gene which is induced in microenvironments typical of tumours with low glucose and hypoxia. Furthermore up-regulation of GRP78 has been linked to chemo- and radioresistance as well as poor survival outcome. This study is the first to confirm that GRP78 is up-regulated in tumours of the oropharynx, larynx and hypopharynx compared with matched histologically normal tissue (p(Χ2)<0.001). Up-regulation of GRP78 may be important for tumour development and therefore we have investigated the consequences of inhibition of GRP78 in cells derived from laryngeal squamous cell carcinomas (LSCC). EGF-SubA is a novel drug consisting of EGF covalently attached to the A subunit of an E.coli derived AB5 toxin which can cleave GRP78. EGF-SubA was able to induce EGFR-dependent cytotoxicity in a panel of seven laryngeal SCC cells with an IC50 range of between 4-100pM. EGF-SubA treatment induced G1 cell cycle arrest as well as apoptosis. Therefore apoptosis may be the mechanism by which EGF-SubA causes cell death. In vitro studies demonstrated that EGF-SubA enhances the effects of clinically relevant genotoxic agents. Two Gy survival fractions (SF2) were significantly reduced with EGF-SubA pre-treatment (p<0.03). In addition EGF-SubA in combination with the primary head and neck cancer chemo-therapeutic agent cisplatin, resulted in IC50 drug combination indexes (CI) as low as 0.542, which is suggestive of a synergistic effect. The potency of EGF-SubA appears to be substantially dependent on EGFR membrane expression since cells expressing higher EGFR levels were associated with increased sensitivity to EGF-SubA where Spearman’s rank correlation coefficient = 0.919 (p=0.003). Furthermore pre-incubation of LSCC cells with sub-toxic doses of cetuximab, a therapeutic monoclonal antibody to EGFR, completely rescued cells from the cytotoxic effects of EGF-SubA (p(t test)≤0.005). This is an important proof of principal for a proposed combined toxin-protectant therapeutic strategy that would permit topical use of EGF-SubA peri- or post-operatively after tumour resection in order to kill any remaining tumour cells, or as an oral rinse in patients who present with pre-malignant lesions of the oral cavity, with any potential systemic toxicity being abrogated by cetuximab. In summary this study has found that GRP78 is up-regulated in head and neck cancers suggesting that this protein may be important for tumour development and survival. Thus inhibition of GRP78 through EGF-SubA may offer a novel approach to cancer therapy. In addition to suppressing the growth of LSCC cell, EGF-SubA was found to enhance the effects of relevant genotoxic agents in vitro of cisplatin and radiation. Further work is now warranted in order to assess the efficacy and toxicity of EGF-SubA, in vivo, before phase I clinical trials can commence.

Neuroblastoma is a paediatric cancer that arises from the sympathetic ganglia and adrenal medulla. Tumours with MYCN amplification have the worst prognosis, and make up around 30% of neuroblastoma diagnoses. Neuroblastoma exhibits an unusually high propensity for spontaneous regression, which occurs most frequently in the youngest patients (under 18 months of age), perhaps because developmental cues still present prompt belated differentiation of the tumour cells. This led to the hypothesis that factors from an appropriate embryonic environment may be capable of activating the correct molecular switches to encourage the differentiation and/or apoptosis of neuroblastoma cells, reprogramming the tumour to a benign phenotype. To test this hypothesis, EGFP-labelled MYCN-amplified Kelly cells were injected into the extra-embryonic vitelline veins of embryonic day 3 (E3) and E6 chick embryos, and the responses of injected cells were analysed at E10 and E14. Kelly cells injected at E3 respond to neural crest migratory cues and integrate into neural crest-derived tissues: some neural, notably the sympathetic ganglia and enteric nervous system, although never the adrenal gland; and others non-neural, such as the meninges and tail. Cells injected at E6 do not show such targeting, integrating into various tissues such as the liver, kidney and meninges. The cells respond to their respective microenvironments, and in sympathetic ganglia some cells differentiate, show reduced cell division, and crucially such cells have undetectable MYCN expression by E10. In non-neural locations, cells form more rapidly dividing clumps and continue to express MYCN. The downregulation of MYCN is dependent on continuous and direct interaction with the sympathetic ganglion environment. Kelly cells’ morphology, behaviour and gene expression are altered by the sympathetic ganglia microenvironment. Taking these key observations, we speculate that the Kelly cells’ MYCN amplicon may likely contain the required DNA regulatory sequences to enable MYCN expression to be altered in response to the embryonic environment. If the factors responsible for MYCN repression can be elucidated, they may represent effective new therapeutic targets.

Introduction: Treating breast cancer is based on a combination of therapies: surgery, radiotherapy, chemotherapy, as well as hormonal and biological agents. The full impact of the disease and its treatment at a human level is often underestimated, and the benefits of holistic cancer care are increasingly recognised. Furthermore, patients often face a frightening and uncertain journey that presents a variety of needs. Moreover, recovery is not necessarily the end-point of the cancer experience. The many complexities and challenges in the identification of patient issues along this journey can lead to unmet needs. This can be particularly difficult in the confines of a busy clinic, where time constraints, together with an over-reliance on verbal communication, can pose significant barriers to effective consultations. A novel tool, known as the patient concerns inventory (PCI), has been successfully developed and introduced for use in patients with head and neck cancer. In this setting, it has helped to formulate an individualized record of patient concerns, needs, and priorities, thereby structuring outpatient consultations, and promoting and facilitating a multidisciplinary approach. This study aimed to develop and assess a PCI specific to breast cancer and to evaluate its impact on patient care; that is, to provide a “proof of concept” for a breast cancer PCI. Methods: This was a four-phase study, as follows. (1) Item generation through a literature review, input from clinicians (n = 10), four patient focus groups (n = 24), and national breast cancer charities (n = 3). (2) A survey of breast cancer patients (n = 200) for cross-sectional validation, to compare the PCI with an established quality of life tool and to look at the relative frequency of items and any associations. (3) A pilot, before and after study, assessing the PCI in a clinical setting with breast cancer patients (n = 53). (4) Semi-structured interviews with a breast surgeon (n = 1) and specialist nurses (n = 2) who used the PCI during clinics, to identify the perceived benefits of using the PCI. Results: In total 277 patients responded and participated in this work. The literature review identified 164 items; following input from clinicians, focus groups, and national charities, 56 items remained. The cross sectional study (phase 2; n=200, 80 % response rate) revealed that patients wanted to discuss the following: breast sensitivity or pain (46 %), fatigue (46 %), hot flushes (44 %), sleep (34 %); breast appearance (30 %), unable to control weight (28 %), mastectomy appearance (19 %), overall physical appearance (17 %); fear of recurrence (62 %), fear of cancer spreading (39 %), fear about the future (32 %), or one or more of these (72 %); ‘mood’ (15 %), ‘anxiety’ (21 %), ‘depression’ (17 %), or one or more of these (35 %); Phase 3 found that the PCI resulted in a focused consultation and no increase in consultation time. All the patients from phase 3 wanted to see a breast surgeon. Phase 4 revealed that clinicians involved with the PCI supported its use, and stated several advantages. In its final format, the breast cancer specific PCI had 57 items over several domains, with 16 referral options. Conclusions: The PCI could identify issues that patients would like to discuss in the breast oncology clinic. The routine use of the PCI in follow-up clinics could ultimately improve care for women with breast cancer; however, the clinical environment continues to make it difficult to screen for issues related to intimacy, relationship, and sex. Further research is essential to evaluate the breast cancer specific PCI. A larger patient cohort, a longitudinal approach, qualitative input, and a link to possible interventions, would each improve our understanding of the issues faced by breast cancer patients.

The malignant cells of chronic lymphocytic leukemia (CLL) over-express PKCβII, a feature pathogenically important because the TCL1 mouse model of CLL fails to develop disease when PKCβ expression is disrupted. The purpose of this project was to generate transgenic mice in which PKCβII is over-expressed only in B cells. The central hypothesis of this thesis was that over-expression of PKCII in developing B cells will shift development to favour the generation of the B-1 and MZ B cell populations and eventually lead to the development of a CLL-like disease in the mouse. To construct the expression plasmid, an Eµ promoter was used to direct B cell-specific expression of PKCβII in transgenic mice (Eµ-PKCβII tg mice). PKCβII was tagged with haemagglutination antigen (HA) for identification in Western blots and immunohistochemistry (IHC). Also, mCherry was integrated within the expression plasmid construct in order to visualize transgenic B cells. Over-expressed PKCβII and mCherry fluorescence were detected when this plasmid was tested in A20 cells, a mouse B lymphoma cell line. The construct was then injected into pro-nuclei of fertilized ova isolated from pregnant mice. The injected ova were then transferred to recipient mice to generate potential founder mice. Sequential crossings of transgenic litters born from a single founder mouse led to the generation of homozygous Eµ-PKCβII tg mice. In the spleen of Eµ-PKCβII tg mice, the expression of the transgenic PKCIIHA was detected and quantification of PKCβII, using Western blotting, showed that PKCβII was over-expressed in Eµ-PKCβII tg mice compared with wild type mice. However, the mCherry could not be detected; this might be due to the fact that expression of the secondary gene(in this case mCherry) was not always efficient because of variable transcription from the IRES sequence. IHC analysis of the spleen of Eµ-PKCβII tg mice confirmed the expression of PKCIIHA in B cell-rich tissues where the staining for either HA or for PKCII showed the high expression of these proteins in the white pulp of the spleen specifically to the follicular region. There was no notable development of disease, CLL-like or otherwise in aged Eµ-PKCβII tg mice, but Flow cytometry analysis pointed out that the over-expression of PKCII resulted in a reduction in the proportion of follicular B cells and an increase in the proportion of MZ B cells in the spleen, and in B-1 cells in peritoneum and periphery of Eµ-PKCβII tg mice. This expansion of MZ B cells in the spleen was confirmed by H&E stains showing an enlarged marginal zone within the structure of the spleen, and IgM stains showed that this expanded MZ consists mainly of IgM+ cells. Thus, the generation of a mouse that over-expresses PKCII specifically within the B cell compartment led to an expansion in the populations of IgM+ B cells (MZ and B-1 B cells) and a reduction of follicular (mature) B cells. This mouse may be useful for the study of human CLL because of the potential to accelerate disease progression, and would therefore serve as a useful model for drug testing.

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.