Colorectal cancer (CRC) is the third most common cancer in the UK with 41,000 new cases diagnosed in 2011. Despite undergoing potentially curative resection, a significant amount of patients develop recurrence. Biomarkers that aid prognostication or identify patients who are suitable for adjuvant treatments are needed. The TNM staging system does a reasonably good job at offering prognostic information to the treating clinician, but it could be better and identifying methods of improving its accuracy are needed. Tumour progression is based on a complex relationship between tumour behaviour and the hosts’ inflammatory responses. Sustained tumour cell proliferation, evading growth suppressors, resisting apoptosis, replicative immortality, sustained angiogenesis, invasion & metastasis, avoiding immune destruction, deregulated cellular energetics, tumour promoting inflammation and genomic instability & mutation have been identified as hallmarks. These hallmarks are malignant behaviors are what makes the cell cancerous and the more extreme the behaviour the more aggressive the cancer the more likely the risk of a poor outcome. There are two primary genomic instability pathways: Microsatellite Instability (MSI) and Chromosomal Instability (CI) also referred to as Microsatellite Stability (MSS). Tumours arising by these pathways have a predilection for specific anatomical, histological and molecular biological features. It is possible that aberrant molecular expression of genes/proteins that promote malignant behaviors may also act as prognostic and predictive biomarkers, which may offer superior prognostic information to classical prognostic features. Cancer related inflammation has been described as a 7th hallmark of cancer. Despite the systemic inflammatory response (SIR) being associated with more aggressive malignant disease, infiltration by immune cells, particularly CD8+ lymphocytes, at the advancing edge of the tumour have been associated with improved outcome and tumour MSI. It remains unknown if the SIR is associated with tumour MSI and this requires further study. The mechanisms by which colorectal cancer cells locally invade through the bowel remain uncertain, but connective tissue degradation by matrix metalloproteinases (MMPs) such as MMP-9 have been implicated. MMP-9 has been found in the cancer cells, stromal cells and patient circulation. Although tumoural MMP-9 has been associated with poor survival, reports are conflicting and contain relatively small sample sizes. Furthermore, the influence of high serum MMP-9 on survival remains unknown. Src family kinases (SFKs) have been implicated in many adverse cancer cell behaviors. SFKs comprise 9 family members BLK, C-SRC, FGR, FYN, HCK, LCK, LYN, YES, YRK. C-SRC has been the most investigated of all SFKs, but the role of other SFKs in cellular behaviors and their prognostic value remains largely unknown. The development of Src inhibitors, such as Dasatinib, has identified SFKs as a potential therapeutic target for patients at higher risk of poor survival. Unfortunately, clinical trials so far have not been promising but this may reflect inadequate patient selection and SFKs may act as useful prognostic and predictive biomarkers. In chapter 3, the association between cancer related inflammation, tumour MSI, clinicopathological factors and survival was tested in two independent cohorts. A training cohort consisting of n=182 patients and a validation cohort of n=677 patients. MSI tumours were associated with a raised CRP (p=0.003). Hypoalbuminaemia was independently associated with poor overall survival in TNM stage II cancer (HR 3.04 (95% CI 1.44 – 6.43);p=0.004), poor recurrence free survival in TNM stage III cancer (HR 1.86 (95% 1.03 – 3.36);p=0.040) and poor overall survival in CI colorectal cancer (HR 1.49 (95% CI 1.06 – 2.10);p=0.022). Interestingly, MSI tumours were associated with poor overall survival in TNM stage III cancer (HR 2.20 (95% CI 1.10 – 4.37);p=0.025). In chapter 4, the role of MMP-9 in colorectal cancer progression and survival was examined. MMP-9 in the tissue was assessed using IHC and serum expression quantified using ELISA. Serum MMP-9 was associated with cancer cell expression (Spearman’s Correlation Coefficient (SCC) 0.393, p<0.001)) and stromal expression (SCC 0.319, p=0.002). Serum MMP-9 was associated with poor recurrence-free (HR 3.37 (95% CI 1.20 – 9.48);p=0.021) and overall survival (HR 3.16 (95% CI 1.22 – 8.15);p=0.018), but tumour MMP-9 was not survival or MSI status. In chapter 5, the role of SFK expression and activation in colorectal cancer progression and survival was studied. On PCR analysis, although LYN, C-SRC and YES were the most highly expressed, FGR and HCK had higher expression profiles as tumours progressed. Using IHC, raised cytoplasmic FAK (tyr 861) was independently associated with poor recurrence free survival in all cancers (HR 1.48 (95% CI 1.02 – 2.16);p=0.040) and CI cancers (HR 1.50 (95% CI 1.02 – 2.21);p=0.040). However, raised cytoplasmic HCK (HR 2.04 (95% CI 1.11 – 3.76);p=0.022) was independently associated with poor recurrence-free survival in TNM stage II cancers. T84 and HT29 cell lines were used to examine the cellular effects of Dasatinib. Cell viability was assessed using WST-1 assay and apoptosis assessed using an ELISA cell death detection assay. Dasatinib increased T84 tumour cell apoptosis in a dose dependent manner and resulted in reduced expression of nuclear (p=0.008) and cytoplasmic (p=0.016) FAK (tyr 861) expression and increased nuclear FGR expression (p=0.004). The results of this thesis confirm that colorectal cancer is a complex disease that represents several subtypes of cancer based on molecular biological behaviors. This thesis concentrated on features of the disease related to inflammation in terms of genetic and molecular characterisation. MSI cancers are closely associated with systemic inflammation but despite this observation, they retain their relatively improved survival. MMP-9 is a feature of tissue remodeling during inflammation and is also associated with degradation of connective tissue, advanced T-stage and poor outcome when measured in the serum. The lack of stromal quantification due to TMA use rather than full sections makes the value of tumoural MMP-9 immunoreactivity in the prognostication and its association with MSI unknown and requires further study. Finally, SFK activation was also associated with SIR, however, only cytoplasmic HCK was independently associated with poor survival in patients with TNM stage II disease, the group of patients where identifying a novel biomarker is most needed. There is still some way to go before these biomarkers are translated into clinical practice and future work needs to focus on obtaining a reliable and robust scientific technique with validation in an adequately powered independent cohort.

AMP-activated protein kinase (AMPK) is a key regulator of cell energy homeostasis. More recently, it has become apparent that AMPK regulates cell proliferation, migration and inflammation. Previous evidence has suggested that AMPK may influence proliferation and invasion by regulating the pro-proliferative mitogen-activated protein kinases (MAPKs). However, the mechanisms underlying this crosstalk between AMPK and MAPK signalling are not fully understood. As AMPK activation has been reported to have anti-proliferative effects, there has been increasing interest in AMPK activation as a therapeutic target for tumourigenesis. The aim of this study was to investigate whether AMPK activation influenced prostate cancer (PC) cell line proliferation, migration and signalling. Therefore, different PC cell lines were incubated with two structurally-unrelated molecules that activate AMPK by different mechanisms, AICAR and A769662. Both chemicals activated AMPK in a concentration- and time-dependent manner in PC3, DU145 and LNCaP cell lines. AMPK activity as assessed by AMPK activating phosphorylation as well as phosphorylation of the AMPK substrate ACC increased along with tumour severity in PC biopsies. Furthermore, both activators of AMPK decreased cell proliferation and migration in the androgen-independent PC cell lines PC3 and DU145. Inhibition of proliferation by A769662 was attenuated in AMPK α1-/- AMPK α2-/- knockout (KO) mouse embryonic fibroblasts (MEFs) compared to wild type (WT) MEFs, and the inhibitory effect on migration of AICAR lost significance in PC3 cells infected with adenoviruses expressing a dominant negative AMPK α mutant, indicating these effects are partially mediated by AMPK. Furthermore, long-term activation of AMPK was associated with inhibition of both the phosphatidylinositol 3’-kinase/protein kinase B (PI3K/Akt) signalling pathway in addition to the extracellular signal-regulated kinase 1/2 (ERK1/2) signalling pathway. Indeed, the actions of AMPK activators on PC cell line viability were mimicked by selective inhibitors of Akt and ERK1/2 pathways. In contrast to the effects of prolonged incubation with AMPK activators, short-term incubation with AMPK activators had no effect on epidermal growth factor (EGF)-stimulated ERK1/2 phosphorylation in PC cell lines. In addition, AMPK activation did not influence phosphorylation of the other MAPK family members p38 and JNK. Interestingly, both AICAR and A769662 decreased EGF-stimulated ERK5 phosphorylation in PC3, DU145 and LNCaP cells as assessed with an anti-phospho-ERK5 antibody. Further characterisation of this effect indicated that prior stimulation with the AMPK activators had no effect on ERK5 phosphorylation stimulated by transient transfection with a constitutively active ERK5 kinase (MEK5DD), which represents the only known canonical kinase for ERK5. Intriguingly, the pattern of EGF-stimulated ERK5 phosphorylation was distinct from that mediated by MEK5DD activation of ERK5. This finding indicates that AMPK activation inhibits EGF-stimulated ERK5 phosphorylation at a point at or above the level of MEK5, although why EGF and constitutively active MEK5 stimulate markedly different immunoreactive species recognised by the anti-phospho-ERK5 antibody requires further study. A769662 had a tendency to reduce EGF-stimulated ERK5 phosphorylation in WT MEFs, yet was without effect in MEFs lacking AMPK. These data indicate that AMPK may underlie the effect of A769662 to reduce EGF-stimulated ERK5 phosphorylation. Prolonged stimulation of PC cell lines with AICAR or A769662 inhibited EGF-stimulated Akt Ser473 phosphorylation, whereas only incubation with A769662 rapidly inhibited Akt phosphorylation. This difference in the actions of the different AMPK activators may suggest an AMPK-independent effect of A769662. Furthermore, AICAR increased phosphorylation of Akt in WT MEFs, an effect that was absent in MEFs lacking AMPK, indicating that this effect of AICAR may be AMPK-dependent. Taken together, the data presented in this study suggest that AMPK activators markedly inhibit proliferation and migration of PC cell lines, reduce EGF-stimulated ERK1/2 and Akt phosphorylation after prolonged incubation and rapidly inhibit ERK5 phosphorylation. Both AMPK activators exhibit a number of effects that are likely to be independent of AMPK in PC cell lines, although inhibition of ERK1/2, ERK5 and Akt may underlie the effects of AMPK activators on proliferation, viability and migration. Further studies are required to understand the crosstalk between those signalling pathways and their underlying significance in PC progression.

Metal-on-metal (MoM) hip joint replacements have been increasingly used for younger and more active patients in recent years due to their improved wear performance compared to conventional metal-on-polymer bearings. MoM bearings operate at body temperature within a corrosive joint environment and therefore are inevitably being subjected to wear and corrosion as well as the combined action of tribo-corrosion. Issues such as metal sensitivity/metallosis associated with high levels of metal ion release triggered by the wear and corrosion products remain critical concerns. During the past few decades, significant research has been conducted into understanding the wear/lubrication mechanisms within the MoM hip joints in order to improve their performance and thereby prolonging their life. However, not much attention has been given to the combined effect of wear and corrosion of such devices in the hip joint environment, in addition, the role of third body particles and the effects of proteins have not been well understood. In this work, a systemic approach is presented for the first time for the mapping of abrasion and tribo-corrosion performance of a cast CoCrMo (F75) in simulated hip joint environments. The effects of third body particles have been studied in the MoM context using 4 μm SiC, 1 μm and 300 nm Al2O3, as well as sub-micron BaSO4. Modified tribo-testers (micro-abrasion, nanoindenter/scratching) incorporating a novel electrochemical cell have been used to monitor the abrasion-corrosion behaviour of the alloy in situ. The effects of solution chemistry, abrasives size / concentration and presence of proteins on the wear / corrosion level, wear-corrosion mechanisms, and the depassivation/repassivation kinetics of the CoCrMo have been explored. A variety of surface and sub-surface characterization techniques have been employed to identify the microstructual wear mechanism interactions. Results show that the change of protein concentration (0, 25% and 50% bovine serum) and pH (pH 7.4 and pH 4.0) of the test solutions can significantly influence the protein adsorption behaviour, which subsequently influence the wear rates (synergy), wear mechanisms as well as the wear-induced corrosion currents of the CoCrMo. For abrasion-corrosion tests, reducing abrasive size from 4 μm to 300 nm and/or abrasive volume concentration from 0.238 vol% to 0.006 vol% results in different abrasion-corrosion wear mechanisms (rolling or grooving abrasion) and the average wear-induced corrosion currents show a linear correlation with wear rates for 4 μm and 1 μm abrasives. For low volume concentration (< 0.03 vol%) slurries containing bovine serum, organo-metallic conglomerates have been found within the wear scars. These conglomerates help separate the surfaces, impose less damage to the surface passive film and polish the wear scars through a chemical mechanical polishing mechanism. In addition, tribo-corrosion tests at micro-/nano- scales reveal the effects of single abrasive particle on the surface/sub-surface microstructual change. This investigation has revealed the nanoscale wear mechanisms that generate nanoscale wear debris, the mechanical mixing of the surface nanostructure with adsorbed denatured protein and also the slip/dislocation systems that are present near and on abraded surfaces that are likely to disrupt the surface passive films. The findings give a better understanding of the evolution of the sub-surface nanocrystalline structures and tribo-layers formation seen for the retrieved implants. This near surface nanostructure layer and phase transformation might offer better wear resistance through these inherent self-protecting mechanisms (i.e. increased hardness); conversely, it may become the precursors to debris ejection and enhanced ion-release into the CoCrMo joints. This work established an experimental technique that gives greater understanding of the tribocorrosion behaviour of cast CoCrMo in simulated hip joint environments. In particular, the roles of third body abrasive particles and proteins have been addressed, which are relevant to clinical applications. The material multi-scale wear mechanisms as well as the evolution of the surface / subsurface microstructures and tribo-layers have been elucidated, which provide new insights into the in vivo wear mechanisms of CoCrMo. The findings of this study may provide some important indications for improved MoM joint materials, design, manufacture and evaluation.

There is an increasing demand for total knee replacements (TKR). Young patients are placing increasing functional demands on modern TKR. Clinical experience has also shown the need for high flexion in patients after TKR. In this study, assessment of TKR performance subjected to deep knee bend was investigated. Patellar resurfacing in TKR is assumed to release pain and restore knee function. Despite the recent advance and success in TKR operation, patellar resurfacing has been associated with an increase in complications at the patello-femoral joint, and hence revisions following TKR. Complications include poor tracking, instability, wear, loosening and fractures. These complications have been attributed in part to the component design features (e.g. sagittal radius, depth, and orientation of the trochlear groove of the femur and the geometry of the patellar component surface) and surgical technique (e.g. component alignment and ligament balance). However, the influence of these factors on the overall performance of TKR has not been investigated extensively. The objective of the study was to determine the variation of patellar kinematics (tracking motion) and contact mechanics (contact force, area, pressure and stress) induced by component design and surgical technique. A three-dimensional finite element (FE) model of a PFC-Sigma TKR, including the tibio-femoral and patello-femoral joints was developed. Explicit FE analysis was used to simulate TKR under a deep knee flexion. The models predicted substantial increase in patellar pressure and stress with nonconforming patello-femoral articulating surfaces. Femoral groove orientation affected patellar tracking and contact mechanics. Extending femoral groove distally reduced patello-femoral contact stress at high flexion angles. Also, externally rotating the femoral component and adjusting the line of action of quadriceps pull would be beneficial by reducing patellar lateral force. The FE model used in the current study provided insight into the effect of component design parameters and surgical technique on patellofemoral kinematics and contact mechanics.

Human organs are the most valuable gifts of life. Until today, through organ transplantation, thousands of lives have been saved and many more blessed with hope and happiness through a better quality of living. However, rapid developments in transplant technology will be meaningless if supply of the needed organs remains scarce and organ transplantation procedures cannot take place accordingly. This global problem of organ shortage is also faced by Malaysia. Despite campaigns and initiatives introduced by the Malaysian authorities, the problem remains unresolved and the situation is worsening. Malaysia is reported to have less than one donor for every one thousand of the population (Lela Yasmin Mansor, 2007). However, statistics from the National Transplant Registry Malaysia confirm a steady increase in the number of registered potential donors each year. This suggests that certain factors must be preventing potential donors from becoming actual donors. Therefore, this study will not only discuss the current scenario of the organ shortage problem in Malaysia, highlighting its underlying factors, but will also scrutinise legal and social factors causing actual donations to remain relatively small, despite the promising number of potential donors registering each year. The study will suggest practical solutions to help solve organ shortages in Malaysia, particularly by utilising brain-dead patients from serious road traffic accidents as a potential source of cadaveric organs. Clarification on the Islamic perspective concerning organ donation is also included, as Islam is the main religion professed in Malaysia.

Acrylic bone cement has been used for fixation of load-bearing orthopaedic implants for over five decades, and continues to be the 'gold standard' for elderly patients and those with systemic disease. Aseptic loosening remains a major indication for revision of cemented hip implants, and has been associated with mechanical degradation of the cement mantle via the initiation and coalescence of fatigue micro-cracks. Microstructural defects such as voids and agglomerates of radiopacifier particles have been implicated in this damage accumulation process. Improved understanding of the relative effects of these features on the mechanisms of fatigue crack initiation and failure within the cement is required in order to inform the development of more robust cement formulations and thus increase the longevity of the cement mantle in vivo. The present study utilised micro-computed tomography (μ-CT) and scanning electron microscopy (SEM), in conjunction with mechanical testing, to provide a systematic, quantitative assessment of the effect of cement formulation and microstructure (including voids and radiopacifiers) on the in vitro fatigue failure of four commercial, vacuum-mixed cement formulations. Results were compared with μ-CT data and fractographic analysis of an ex vivo cement specimen. This novel 'data rich' methodology enabled non-destructive, three-dimensional analysis of defect populations in terms of the size, morphology and spatial density of individual microstructural features, and the identification and characterisation of crack-initiating defects. The inclusion of barium sulphate as a radiopacifier was found to have a negative effect on the fatigue life of cement; radiopacifier particles showed a tendency to form numerous large agglomerates, which readily initiated fatigue cracks; furthermore, fatigue life scaled consistently with initiating defect size. In contrast, cement containing zirconium dioxide as a radiopacifier demonstrated superior fatigue performance, and failure in these cement formulations was dominated by crack initiation from voids. In all four cement formulations, void populations were found to be bi-modal, and the largest voids (> 0.5 mm equivalent spherical diameter) were surrounded by secondary satellite voids in both in vitro and ex vivo cement specimens. Extensive void formation was also noted in both moulded specimens and cement mixing gun stubs, in addition to ex vivo cement. Optimisation of cement formulations and vacuum-mixing techniques may therefore be advantageous in order to reduce the formation of barium sulphate agglomerates and large voids, and thus minimise their potential crack initiation effects in vivo.

This thesis documents the work of Andrew I. M. Greer undertaken for the fulfilment of the requirements for the Degree of Doctor of Philosophy. The project, funded by the EPSRC and MRC, is to develop a nanofabrication processing strategy compatible with titanium based orthopaedic implants. Such a development will facilitate the translation from current and historical in vitro analysis of cell-stimulating nanotopographical cues to in vivo studies upon an implant relevant material. The work presented opens by summarising the social motives and consequences before contextualising the project aims with reference to existing approaches in the field. The thesis progresses through a series of different nanofabrication approaches until an effective strategy satisfying the goals of the project is devised. Thereafter the strategy is explored with its results characterised from a material level through to a biological level. Ultimately the primary goal of the project is realised through the development of novel sol-gel chemistry capable of retaining a nanopattern and transforming into titania, the natural composition at the surface of a titanium based implant. Furthermore, nanofeatures previously too stringent to fabricate for a comprehensive biological study are readily achievable using the documented strategy and fundamental studies have been carried out which indicate that the features concerned are highly effective at up-regulating early indicators of bone formation.

RésuméLe présent travail a été réalisé dans et autour de la forêt de montagne du Parc National de Kahuzi-Biega (PNKB), à l’Est de la RD Congo. Ce site naturel de patrimoine mondial fut créé principalement pour protéger la sous-espèce de gorille, le Gorilla beringei graueri, une espèce en danger critique d’extinction. L’objectif de cette thèse est d’analyser le double usage des espèces végétales qui sont utilisées dans la médecine traditionnelle et dans le régime alimentaire des gorilles pour une gestion durable de la biodiversité, et en particulier, des ces espèces végétales les plus sollicitées par les tradipraticiens et qui sont utiles dans l’alimentation et potentiellement l’automédication des gorilles. Pour réaliser cette étude, nous avons abordé les tradipraticiens spécialistes ;nous avons fait des observations directes de gorilles de 4 groupes et nous avons évalué la végétation de leur habitat en forêt d’altitude du parc. L’analyse des données a été réalisée grâce aux approches de l’ethnobotanique quantitative et de l’écologie numérique.Les résultats montrent que, sur la base de l’usage de 77 espèces médicinales inventoriées, la typologie des tradipraticiens n’est pas basée sur leurs origines ethniques et géographiques mais sur leurs spécialités. Ils utilisent les mêmes plantes pour soigner les mêmes maladies. La forte convergence de leurs pratiques et le degré de consensus très élevé sur les plantes qu’ils utilisent pour traiter les maladies traduisent une tradition médicinale solidement ancrée au sein des groupes ethniques vivant dans cette région du Parc. Cependant, 83% des espèces utilisées par les tradipraticiens sont forestières. Les usages des plantes médicinales comparés aux choix alimentaires des gorilles montrent que 78 % des espèces végétales consommées par ces primates sont utilisées par les tradipraticiens, avec une similarité de 80% d’usage des parties de plantes. Ce chevauchement très élevé d’usages traduit une compétition potentielle pour les plantes à double usage. Il met en danger à la fois les plantes, les gorilles et la médecine locale.Cette thèse montre ainsi la nécessité de mieux comprendre les différentes spécialités médicinales au sein des ethnies et leur recours à certaines plantes spécifiques d’une part, et d’autre part ,le besoin de mieux étudier les pratiques d’automédication du gorille de Grauer au PNKB.Ces enseignements contribueront à la conservation de ces espèces végétales à double usage à la fois utiles pour l’homme et pour les grands singes.AbstractThis work was carried out in the mountain region of Kahuzi-Biega National Park (KBNP) in eastern DR Congo. That natural site was primarily created to protect the lowland gorilla, Gorilla beringei graueri, a species in critical danger of extinction. The aim of this thesis is to analyze the dual-use of plants that are used both in traditional medecine and by gorillas in order to sustain the conservation management of plants most used by traditional healers and at the same time as food or for medication purposes by gorillas. For this study, we addressed traditional healers recognized as specialists in Batwa, Havu, Bashi and Tembo ethnic groups; we made direct observations of 4 groups of gorillas and assessed the vegetation within gorilla habitat in the mountain forest. Data analysis was conducted with two approaches: quantitative ethnobotany and numerical ecology. For 77 medicinal species identified, the results show that the typology of traditional healers relies on their specialisation than on ethnic and geographical membership. They use the same plants to treat the same diseases.The strong convergence of their practices and the high degree of consensus on plants they use to treat diseases reflect a consolidated medicinal tradition among ethnic groups living in this area of the Park. However, 83% of species used by traditional healers grow in the forest.The uses of medicinal plants compared to gorilla’s food choices show that 78% of plant species consumed by these primates are also used by traditional healers, with a similarity of 80% of use of plant parts. This high overlap of uses reflects a potential competition for dual-use plants, that endangers both plants, gorillas and local medicine.The topic thus shows the need of further studies for better understanding the different specialisation within local medicine and the use of specific plants on the one hand. The other hand, the self-medication practices of Grauer's gorilla in KBNP need to be investigated.These insights will contribute to the conservation of these dual-use plant species which are useful for both humans and great apes.Keywords: Traditional medicine – Traditional healers- Gorilla Grauer - Kahuzi-Biega- National Park- DR Congo. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

Prostatic diseases are common; benign prostate hyperplasia (BPH) is almost ubiquitous in elderly men and 899,000 men were diagnosed with prostate cancer worldwide in 2008. The incidence of both is increasing and expected to continue to rise. Therefore, prostatic diseases represent a considerable economic burden, but there are currently no reliable markers available to accurately differentiate indolent from aggressive disease nor to predict who will benefit from treatment for either BPH or prostate cancer. This results in over and under-treatment of both diseases with consequent patient related morbidity and mortality. The molecular mechanisms underlying the natural history of prostatic diseases remain elusive. It is accepted that prostate cell growth and survival are exquisitely dependent upon activation of the androgen receptor (AR) by androgens. Following ligand binding, AR undergoes further phosphorylation at serine residues, which inhibit proteolytic degradation, stabilise AR and influence AR transactivation. It is therefore plausible that alterations in AR phosphorylation may drive prostatic disease progression. However, few studies have explored the significance of AR phosphorylation, or the kinases driving AR serine phosphorylation in the clinical setting. The over-riding objective of this study was to establish the clinical relevance of AR serine phosphorylation status in prostate tissue in both BPH and prostate cancer. The specific aims of the current study were: • To firstly establish and validate a panel of AR phosphospecific antibodies. • To evaluate site specific AR serine phosphorylation expression levels in prostate cancer and BPH patient cohorts, with full clinical data and follow-up. • To investigate the expression of candidate kinases mediating such phosphorylation. This involved establishing tissue banks with linked comprehensive clinical databases, and utilising this tissue to establish AR phosphorylation expression profiles for each patient. Six AR phosphospecific antibodies (pARS81, pARS94, pARS213, pARS515, pARS578, pARS650) were verified using peptide competition assays and western blotting. Cdk1, ERK1/2, Akt and PKC were identified as putative kinases mediating AR phosphorylation using the online kinase search tool Scansite 2.0. Immunohistochemistry was performed on hormone naïve diagnostic prostate cancer tissue relating to 90 patients. High expression levels of AR phosphorylation at serine sites 81, 515 and 578 were each associated with a poorer clinical outcome. Following cox regression analysis, cytoplasmic pARS515 expression (p=0.038, HR 4.5 (95% CI 1.1–20.6)) and pARS81 nuclear expression (p=0.030, HR 0.033 95% CI 0.002-0.721) were independently associated with shorter time to biochemical relapse and shorter disease specific survival respectively. Cdk1 and/or pCdk1161 were significantly associated with pARS81 and pARS515 as predicted by Scansite 2.0. Similarly, nuclear PKC expression was significantly associated with pARS578 expression both in the cytoplasm and the nucleus. In patients with PSA at diagnosis ≤20ng/ml, high cytoplasmic pARS515 expression was associated with significantly shorter time to biochemical relapse (p=0.019). This translated into significantly shorter disease-specific survival (p<0.001, 10y survival 38.1% vs 100%). Prostate cancer patients with a low serum PSA level at diagnosis may be suitable for delayed radical treatment via active surveillance. An investigation was therefore undertaken in 51 prostate cancer patients treated by active surveillance. Active surveillance is a deferred radical treatment approach which provides a potential solution to the problem of over treatment as a result of over-diagnosis. However some patients harbour occult aggressive disease and delay in treatment may result in disease progression and failure of radical therapy. Although none of the individual AR serine phosphorylation sites were associated with clinical outcome measures on univariate analysis, high expression of total AR in the cytoplasm (p=0.021, HR 4.6 (95% CI 1.3-16.8)) and presence of perineural invasion in the tumour specimen (p=0.003, HR 8.6 (95% CI 2.1-35.7)) were deemed independent with regards to shorter time to treatment intervention in a cox regression analysis. Validation of the results seen in the first active surveillance prostate cancer cohort was undertaken in a second prospectively collected cohort consisting of 84 active surveillance patients. The results in the first cohort were not replicated in the second. Although cytoplasmic pARS81 was associated with time to intervention (p=0.032) and pARS515 expression trended towards an association (p=0.072), an increase in patient numbers in both cohorts may have provided more reliable results. However even with the numbers available in contrast to the first active surveillance cohort, but in line with the pilot prostate cancer cohort, Cdk1 was associated with pARS515 expression, and pCdk1161 trended towards an association. BPH is also an androgen driven disease dependent upon the AR. Previous research into predictive and prognostic markers in BPH is scant. Therefore a comprehensive analysis of clinical and novel pathological factors, including markers of inflammation, was performed in 336 BPH patients. Following this a complete panel of AR serine phosphorylation sites, and associated kinases, was analysed with reference to clinical outcome measures in the BPH cohort. Low expression levels of total AR and AR phosphorylated at Ser-81, 515 and 650 were associated with poorer clinical outcomes. Low expression of smooth muscle pARS515 (p=0.029, HR 0.31 (95% CI 0.10-0.94)) and older age (p=0.004, HR 5.13 (95% CI 1.43-18.41)) were deemed independent on cox regression analysis with regards to shorter time to postoperative acute urinary retention (AUR). Furthermore, low expression of pARS515 in the smooth muscle was associated with increased incidence of postoperative AUR in patients over 70 years old (25.1% vs 2.8% at 10 years following transurethral resection of prostate (TUR)), (p=0.002, HR 0.20 (95% CI 0.06-0.62)). This may have important clinical implications in postoperative counselling of these patients. In addition it may influence the decision to commence early postoperative medical treatment (with 5-alpha-reductase inhibitors and/or alpha blockers) on a prophylactic basis in these patients. Cytoplasmic pARS650 expression (p=0.010, HR 0.50 (95% CI 0.29-0.86)) and PSA at diagnosis (p=0.018, HR 1.89 (95% CI 1.11-3.16)) were independently associated with time to failure of surgical intervention. Furthermore, low expression of pARS650 in the cytoplasm was associated with increased failure of surgical intervention in patients with PSA ≥4ng/ml at diagnosis (45.5% vs 13% at 5 years post TUR), (p=0.026, HR 0.52 (95% CI 0.29-0.93)). This comprehensive study on immunohistochemical expression of site specific AR serine phosphorylation and associated kinases fills a gap in the current literature. It has demonstrated the clinical significance of AR serine phosphorylation in prostate cancer and BPH and uncovered potentially exciting new avenues for future investigation. Site specific serine phosphorylation of the AR may serve as a prognostic and predictive biomarker in prostatic disease and has potential as a future target for therapeutic intervention.

Orthopaedic infections can be polymicrobial existing as a microbiome. Infections often incorporate staphylococcal species, including Staphylococcus aureus. Such infections can lead to life threatening illness and implant failure. Furthermore, biofilm formation on the implant surface can occur, increasing pathogenicity, exacerbating antibiotic resistance and altering antimicrobial mechanism of action. Bacteria change dramatically during the transition to a biofilm growth state: phenotypically; transcriptionally; and metabolically, highlighting the need for research into molecular mechanisms involved in biofilm formation. Metabolomics can provide a tool to analyse metabolic changes which are directly related to the expressed phenotype. Here, we aimed to provide greater understanding of orthopaedic infection caused by S. aureus and biofilm formation on the implant surface. Through metagenome analysis by employing: implant material extraction; DNA extraction; microbial enrichment; and whole genome sequencing, we present a microbiome study of the infected prosthesis to resolve the causative species of orthopaedic hip infection. Results highlight the presence of S. aureus as a primary cause of orthopaedic infection along with Enterococcus faecium and the presence of secondary pathogen Clostridium difficile. Although results were hindered by the presence of host contaminating DNA even after microbial enrichment, conclusions could be made over the potential increased pathogenicity caused by the presence of a secondary pathogen and highlight method and sample preparation considerations when undertaking such a study. Following this finding, studies were focused on an orthopaedic clinical isolate of S. aureus and a metabolome extraction method for staphylococcal biofilms was developed using cell lysis through bead beating and solvent metabolome extraction. The method was found to be reproducible when coupled with liquid chromatography-mass spectrometry (LC-MS) and bioinformatics, allowing for the detection of significant changes in metabolism between planktonic and biofilm cultures to be identified and drug mechanism of actions (MOA) to be studied. Metabolomics results highlight significant changes in a number of metabolic pathways including arginine biosynthesis and purine metabolism between the two cell populations, evidence of S. aureus responding to their changing environment, including oxygen availability and a decrease in pH. Focused investigations on purine metabolism looking for biofilm modulation effects were carried out. Modulation of the S. aureus biofilm phenotype was observed through the addition of exogenous metabolites. Inosine increased biofilm biomass while formycin B, an inosine analogue, showed a dispersal effect and a potential synergistic effect in biofilm dispersal when coupled with gentamycin. Changes in metabolism between planktonic cells and biofilms highlight the requirement for antimicrobial testing to be carried out against planktonic cells and biofilms. Untargeted metabolomics was used to study the MOA of triclosan in S. aureus. The triclosan target and MOA in bacteria has already been characterised, however, questions remain over its effects in bacteria. Although the use of triclosan has come under increasing speculation, its full effects are still largely unknown. Results show that triclosan can induce a cascade of detrimental events in the cell metabolism including significant changes in amino acid metabolism, affecting planktonic cells and biofilms. Results and conclusions provide greater understanding of orthopaedic infections and specifically focus on the S. aureus biofilm, confirming S. aureus as a primary cause of orthopaedic infection and using metabolomic analysis to look at the changing state of metabolism between the different growth states. Metabolomics is a valuable tool for biofilm and drug MOA studies, helping understand orthopaedic infection and implant failure, providing crucial insight into the biochemistry of bacteria for the potential for inferences to be gained, such as the MOA of antimicrobials and the identification of novel metabolic drug targets.