Severe isolated patellofemoral arthritis is a highly debilitating disease. Total knee arthroplasty is considered the gold standard treatment, however, patellofemoral arthroplasty has certain advantages. This ‘less invasive’ procedure preserves the tibiofemoral joint and cruciate ligaments, facilitating a more rapid recovery and allows for a relatively straightforward revision if required in the future. As the use of patellofemoral arthroplasty steadily gains popularity in the orthopaedic community, it is important to establish a consensus on which treatment should be the primary intervention of choice. Through background reading and expert opinion, three areas of research were chosen for further investigation: 1. Extensor mechanism efficiency 2. Survival and complication proportions following patellofemoral arthroplasty and total knee arthroplasty 3. Assessment of differences in function and quality of life outcomes following patellofemoral arthroplasty and total knee arthroplasty The purpose of this thesis was to further inform the debate between the choice of total knee arthroplasty and patellofemoral arthroplasty for the treatment of severe isolated patellofemoral arthritis. Study I: The cadaveric biomechanics study compared the extensor mechanism efficiency of the native knee, patellofemoral arthroplasty, cruciate-retaining total knee arthroplasty and posterior-stabilising total knee arthroplasty. Patellofemoral resultant force, peak pressure and contact area were also analysed. The data produced a bimodal distribution during the flexion-extension cycle for all four conditions. The results showed patellofemoral arthroplasty produced the greatest extensor mechanism efficiency in the range of mid flexion to extension (50° to 0°). Further research is required to determine whether this efficiency translates to the clinical setting. Study II: The systematic review compared the survival and complication proportions of patellofemoral arthroplasty and total knee arthroplasty. The patellofemoral arthroplasty studies were divided into seven groups depending on femoral component design. Thirty-six out of the forty studies identified were uncontrolled retrospective case series’ and therefore subject to reporting and selection biases and overall provided low quality evidence. A meta-analysis could not be performed due to high clinical heterogeneity. Other limitations included variations in study design and length of follow-up. Despite, these weaknesses the review established inlay designs produced the poorest survival and complication outcomes. Malpositioning/misalignment and disease progression were the most common complications. Study III: The double-blind randomised clinical trial assessed for differences in function and quality of life outcomes between patellofemoral arthroplasty and total knee arthroplasty. The trial failed to show evidence of a difference between the two interventions. Complication rates were overall low but greater in the total knee arthroplasty group. Tests for significance were not performed due to the small numbers involved. Although, the study was underpowered, the data did not support superiority of patellofemoral arthroplasty over total knee arthroplasty. Therefore, future studies should test for non-inferiority and involve multiple centres to increase generalizability to the wider orthopaedic community.

The Adaptive Foam Reticulation (AFR) technique, a combination of foam reticulation and freeze casting, has been investigated for producing bone repair bioscaffolds from hydroxyapatite (HA), titanium (Ti) and titanium-aluminium-vanadium (Ti-6Al-4V). Scaffolds have a network of macropores of diameter between 94 and 546 mm, with struts 20 to 118 mm thick. The structure was dependent on the template from which structures were produced, the number of coats of slurry applied to the template and the sintering temperature. The struts contained numerous micropores, the size of which was increased from 2-5 to 20-30 mm by decreasing the freezing temperature. Whilst the size of individual micropores was independent of the amount of porogen in the slurry, there was some coalescence at higher percentages. Scaffolds exhibited porosities of between 76 and 96%, with porosity consistently decreased by increasing the number of coats from one to five. The mechanical strength of all samples was determined theoretically by the novel incorporation of a shape factor conventially used for microporous structures into an existing equation used to calculate the yield stress of porous structures. In most cases this agreed with the experimentally obtained yield stress. With compressive yield stresses of 0.002 to 0.18MPa and 0.002 to 1.8 MPa respectively, HA and Ti structures are only suitable in non-load bearing situations. However Ti-6Al-4V scaffolds had yield stresses of 0.21-13.7 MPa, within the range of cancellous bone. AFR-fabricated HA scaffold offered greater in-vitro cell viability than a commercially available porous HA disc. Including a porogen offered no improvement in viability compared to structures fabricated without porogen, except at the highest inclusion where a statistically significant increase was observed. The weak compressive strength of scaffolds needs improving, and fabrications require in-vivo analyses. However, AFR could offer a viable alternative to other manufacturing techniques.

Barrett’s oesophagus is a common condition in which the normal stratified squamous oesophageal epithelium is replaced by metaplastic reflux-induced glandular (“columnar”) mucosa (Jankowski, Barr, Wang et al. 2010;Playford 2005). Over the last three decades, the incidences of oesophageal adenocarcinoma (OA) and Barrett’s oesophagus have risen to the point that OA is now common in the United Kingdom, with Scotland having one of the highest rates in the world (Jankowski, Provenzale, & Moayyedi 2002). Unfortunately most cancers present at an advanced stage with five year survival less than 30% (Holmes and Vaughan 2007). Barrett’s oesophagus is associated with malignant progression via a recognised metaplasia-dysplasia-carcinoma sequence (Jankowski, Wright, Meltzer et al. 1999). The premalignant nature of Barrett’s oesophagus has powered intense clinical interest in the hope of eventually having an impact on the earlier diagnosis and treatment of dysplasia, and ultimately the prognosis of oesophageal adenocarcinoma. Despite years of research interest, Barrett’s oesophagus remains an enigmatic condition. The exact incidence is unknown, and it is recognised that not all patients with Barrett’s oesophagus will progress to adenocarcinoma. Current strategies aim to ascertain the presence of dysplasia, the current gold standard marker of malignant progression. However although Barrett’s mucosa is visible at endoscopy, the presence of dysplasia is difficult to diagnose as these areas tend to be focal and inconspicuous to the naked eye. Current systematic biopsy regimes are recommended, but can be fraught with sampling errors. Furthermore, the molecular mechanisms underlying Barrett’s metaplasia and progression to dysplasia remain unclear. Molecular risk biomarkers have been sought with modest success, and at present dysplasia remains the most reliable clinical marker. However dysplasia itself is not without limitations: focal dysplasia can be difficult to ascertain, with many biopsies sometimes necessary to detect it reliably (Abela, Going, Mackenzie et al. 2008). Inter-observer variability may cause over or under diagnosis, especially regarding LGD (Flejou 2005). Moreover, although patients with HGD are at elevated risk of progression to OA, few studies provide reliable data on rates of progression from HGD to OA, with estimates varying between 16-59% at five years (Reid, Blount, Feng et al. 2000;Schnell, Sontag, Chejfec et al. 2001;Shaheen and Richter 2009;Spechler SJ 2011). There is a real need, therefore, to be able to identify and treat those patients at greatest risk of malignant transformation, and reassure those at low risk. Without an improved molecular understanding of Barrett's metaplasia and progression to neoplasia, clinically useful prognostic biomarkers (allowing appropriate targeting of surveillance and therapy) will be delayed. The current challenges associated with Barrett’s oesophagus are 1) to accurately determine the rate of malignant progression of Barrett’s oesophagus and identify clinical risk factors, 2) to improve the endoscopic detection of dysplasia and early neoplasia allowing earlier diagnosis and treatment and, 3) to understand the molecular mechanisms involved in the initiation of Barrett’s metaplasia, and the pathways involved in disease progression. In an attempt to improve the care of patients with Barrett’s oesophagus within the West of Scotland, my thesis will address each of the main challenges associated with this puzzling condition at clinical, endoscopic and molecular levels. The hypotheses of my thesis are threefold - a) Patients with Barrett's oesophagus in the West of Scotland have high rates of progression to high grade dysplasia and oesophageal adenocarcinoma. b) The WavSTAT optical biopsy system will be able to correctly identify non-dysplastic and dysplastic Barrett's oesophagus. c) The Wnt signalling pathway is upregulated in Barrett's oesophagus and dysplasia. The aims of my thesis are as follows: 1)To present a general overview of the Barrett’s literature highlighting current clinical challenges 2)To examine the incidence of dysplasia and oesophageal adenocarcinoma in the West of Scotland by analysing a cohort of patients undergoing surveillance endoscopy 3)To review the current endoscopic imaging adjuncts for the diagnosis of Barrett’s oesophagus and dysplasia, and assess the role of optical biopsy forceps in determining the presence of dysplasia 4)To evaluate the role of Wnt signalling in Barrett’s oesophagus, from metaplasia to carcinoma in a mouse model, with complementary human studies Chapter 1 introduces the reader to Barrett’s oesophagus and highlights current areas of clinical challenge and debate. A universal definition of Barrett’s oesophagus does not exist and Chapter 2 explores the need for the presence of intestinal metaplasia in the diagnosis of Barrett’s oesophagus. Chapters 3 and 4 present original data from a West of Scotland Barrett’s oesophagus database, specifically analysing rates of dysplasia and adenocarcinoma and cause of death. This study suggests patients with Barrett’s oesophagus in the West of Scotland are at high risk of disease progression with almost 10% of patients dying from oesophageal adenocarcinoma. The results highlight the importance of a comprehensive surveillance in our “high risk” population - an ideal niche for future chemopreventative and molecular studies. In an attempt to improve the diagnosis of dysplasia in our West of Scotland population, Chapter 5 reviews current endoscopic imaging adjuncts used in research and clinical practice while Chapter 6 presents original data from a pilot study assessing the use of innovative optical biopsy forceps in the endoscopic diagnosis of dysplasia. While this technology is in its infancy and further changes in the algorithm are required, the optical forceps could be a promising tool for ongoing surveillance in high risk Barrett’s patients. Chapter 7 summarises the role of biomarkers in Barrett’s oesophagus, reviewing the literature and highlighting the lack of clinically useful markers of disease progression to date. The Wnt signalling pathway plays an important role in normal oesophageal (and intestinal) development, yet when aberrantly activated leads to carcinogenesis. To date, very little is known about the role of Wnt signalling in Barrett’s oesophagus. Chapter 8 presents the results of a mouse model of upregulated Wnt signalling and the interesting finding of dysplasia within the oesophageal mucosa. Chapter 9 therefore translates these results to the human population by assessing the role of Wnt signalling in Barrett’s metaplasia and dysplasia by immunohistochemical analysis of a panel of markers. The results suggest Wnt signalling is upregulated in Barrett’s dysplasia, particularly in high grade, and this may have a future role as a biomarker. Chapter 10 summarises the main findings of the thesis, and presents future directions.

Renal cancer is a malignancy which is not only increasing in incidence but there has also been an increase in mortality rates. There are various prognostic factors in renal cell cancer. We have demonstrated that some of these such as nuclear grading, tumour necrosis and systemic inflammatory response can be further refined to aid in prognosis but cannot be utilised at present to assess which would benefit from therapeutic agents when recurrence occurs. We investigated if SFK members are expressed in renal cancer. Eight SFK members were found to be expressed in renal cancer and were present to varying degrees. Furthermore, expression differed in organ confined disease and metastatic disease. Immunohistochemistry was employed to assess protein expression and activation of c-Src and SFK activity as well as the downstream marker FAK Y861. Analysis demonstrated that c-Src expression was associated with improved survival and expression of the downstream marker FAK Y861 was associated with poor survival and demonstrated a positive relationship with known prognostic factors. This would suggest that another SFK member was associated with poor survival. Dasatinib, a SFK inhibitor was utilised on renal cell lines, demonstrating a dose dependant reduction on cellular metabolic activity as well an increase in apoptotic rates. This would support that Dasatinib may be a useful therapeutic drug for RCC. Treatment with Dasatinib also demonstrated that expression of c-Src, SFK activity and FAK Y861 reduced in a dose dependant manner. It was necessary to further assess that another SFK member was responsible for poor prognosis and this was undertaken by silencing c-Src. Cellular metabolic activity rates increased following silencing c-Src and assessment of SFK activity (Src Y416) and FAK Y861 on cell pellets demonstrated no change suggesting that another SFK member is responsible for the phosphorylation of FAK Y861 and therefore responsible for poor survival. This would suggest that another SFK inhibitor and not c-Src inhibitors may play a role in the treatment of renal cell cancer and further work is required to ascertain which SFK member is responsible so that this can be targeted for treatment.

Colorectal cancer is the second commonest cause of cancer death in the western world. In addition to tumour factors such as depth of invasion, lymph node involvement and venous invasion it is increasingly recognised that host factors, are important determinants of survival. In particular the host local and systemic inflammatory responses are stage independent predictors of survival in operable disease. The present thesis further examines the prognostic importance of host and tumour factors in colorectal cancer, specifically: 1. An examination of the prognostic importance of venous invasion (detected using elastica stains) in colorectal cancer. 2. Detailed analysis of the determinants (including age, comorbidity and deprivation) of the systemic inflammatory response and their relationship with survival. 3. The application and validation of a prognostic score providing a measure of the local inflammatory response in colorectal cancer. 4. Detailed analysis of the determinants (including all white cells, lymphocytes and macrophages) of the local inflammatory response and their relationship with survival. 5. The inter-relationships between the local and systemic inflammatory responses in colorectal cancer specifically: early stage disease (node negative) and in patients receiving adjuvant chemotherapy. 6. Mediators (including immunological parameters and vitamin antioxidants) of the local and systemic inflammatory responses and their relationship with survival.

A successful kidney transplant is the best treatment for established renal failure, yet around 300 patients per annum are denied transplants because they have antibodies, most notably directed against donor HLA or ABO in their blood, which have the potential to cause acute and chronic rejection of the transplant. Such antibodies are present in 25% (roughly 1750 of the 7000 on the kidney transplant waiting list) of the patients listed for a deceased donor transplant. Programmes to remove antibody and transplant patients across HLA antibody barriers have been developed, but are limited by a high rate of acute rejection. This thesis explores the factors which may impact upon the pathogenicity of HLA-specific antibodies and also aims to enhance the understanding of the techniques used in the laboratory to define these antibodies. A range of studies were carried out examining factors such as the IgG subclass composition of the anti-HLA response. Assay variations were designed to enable a higher definition of antibody specificity to be achieved, and for the first time in the literature the direct quantification of HLA-specific antibodies in patient sera was performed. In addition, proof of principle design and testing was carried out on a novel prototype therapeutic device for the selective depletion of HLA-specific antibodies directly from patient plasma and sera. The antibodies isolated from this approach were also used in studies to examine the factors which determine serum cytotoxicity.

This project highlighted that Src and Src kinase family (SFK) members play a definitive role in breast cancer. Due to the paucity of translational studies, we investigated if SFK members are expressed in human breast tissue. Eight SFK members were present with distinct mRNA expression patterns in normal, non-malignant and malignant breast tissue. Immunohistochemistry was employed to investigate protein expression and activation of Src and SFK members. Survival analysis revealed that c-Src and activated Y419Src were associated with worse patient outcome, confirming current in vitro literature, whereas a different phosphorylation site of Src (Y215) and expression of Lck was associated with improved clinical outcome. Dasatinib was employed in different breast cancer cell lines to establish its effect on those phosphorylation sites. Decreased expression of c-Src and Y419Src was observed, whilst Y215Src expression stayed unchanged, providing a rationale for using this Src kinase inhibitor in clinical trials.

The reconstruction of critical-size bone defects following tumour resection or bone loss due to trauma is topical today and relates to the complexity of the treatment involved and poor healing outcomes. In bone bioengineering, the current trends are to explore novel methods of repairing these defects by using various bone substitutes. Various graft materials have been used for the restoration of these defects. A graft ideally needs to promote osteogenesis, osteoinduction and osteoconduction. The aim of this investigation was to assess the histological, radiographic and mechanical properties of the tissue regenerate following the application of tricalcium phosphate (TCP) scaffolding and recombinant human bone morphogenetic protein 7 (rhBMP-7) for the reconstruction of a critical-size osteoperiosteal mandibular continuity defect in the rabbit model. Highly purified and freeze dried recombinant human BMP-7 was used. It was produced by Chinese hamster ovary cells in culture and purified from the culture media. All the TCP samples had a porosity of 80% and average pore size of 100 – 500µm. For the rhBMP-7 loaded scaffolds; rhBMP-7 was reconstituted according to a recommended specification and 400ng were loaded by adsorption into the TCP scaffolds. Nine adult New Zealand white rabbits (3.0-4.0kg) were used for the planned study. In each case a unilateral osteoperiosteal mandibular body critical-size defect was created. In six cases the critical-size defect was filled with the rhBMP-7 on the TCP scaffolding, and in three cases the TCP was used alone. Assessments were made with plain radiographs at 0, 4, 8, and 12 weeks follow-up. Three months post-operatively the animals were sacrificed, the mandibles removed and the surgical sites were assessed with cone beam CT radiography, tested mechanically and analysed histologically. More bone regeneration was seen radiographically and histologically within the mandibles that received rhBMP-7 in the TCP, with evidence of both woven and lamellar bone formation. Union was obtained at the surgical site with no cartilage formation. The regenerated bone was confined to the area that had received the scaffold, with no calcification of the surrounding soft tissues. The TCP was also resorbed more completely in this experimental group. Very little bone was formed in the cases where the defect was filled with TCP alone. The mechanical properties of the regenerate in the group that received the rhBMP-7 and TCP were also significantly superior to those of the cases that received TCP alone. Histologically the overall mean of the percentage regenerated bone volume in the rhBMP-7 and TCP cases was 29.41% ± 6.25, while that for the TCP alone cases was 6.35% ± 3.08. The difference between the groups was statistically significant (p = 0.014). Mechanically the failure moments for the TCP alone cases were found to be very low (0-48mNm) while those for the rhBMP-7 and TCP cases were higher but there was considerable variation between the cases (55-2115mNm). Some of the cases in this group achieved failure moments comparable to normal untreated bone. In conclusion TCP scaffolding and rhBMP-7 can be used successfully for the reconstruction of critical-size mandibular defects in the rabbit model and TCP loaded with rhBMP-7 was significantly superior in its capacity for bone regeneration histologically when compared to TCP alone. The resultant bony regenerate could also at times have mechanical properties similar to those of natural bone. But due to the variability of the mechanical properties further investigations are required before clinical application.

The aim of this research was to investigate the blood supply to the lower abdomen. This is a commonly used donor site for autologous reconstruction following breast cancer and the flap of tissue used is based on the deep inferior epigastric circulation (DIEP flap) or the superficial inferior epigastric circulation (SIEA flap). A pilot study investigated the feasibility of assessing the vascular territory of multiple blood vessels in the lower abdomen, and also observed the timing of changes in skin blood supply after free flap transfer. Further studies included sampling using microdialysis catheters, from different areas of the flap, around the theoretical time of opening of choke vessels between angiosomes. Manipulation of skin blood flow was initially investigated using capillary malformations as a model, observing current clinical use of EMLA and AMETOP topical anaesthetic pre-laser treatment.

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