Previous studies have identified stem cell populations in articular cartilage using colony forming assays and mesenchymal stem cell (MSC) marker expression. The specificity of classical MSC markers for isolation of stem cells within articular cartilage is insufficient, with large and highly variable quantities being reported in the literature. This study has demonstrated, for the first time, a panel of stem cell markers specific for articular cartilage-derived stem cells (ACSC). ACSCs were isolated, quantified and cultured from healthy and OA joints. Stem cells were clonally-derived cell lines that proliferated beyond 50 population doublings whilst maintaining a phenotype, and demonstrated tri-lineage potential. We discovered that OA cartilage had a two-fold increase in stem cell number, consisting of two divergent stem cell sub-populations. These divergent populations varied in proliferative capacity with only 50% of stem cells from the OA joint capable of extended proliferation in vitro. Using transcriptomic next generation sequencing of culture-expanded chondrocytes and ACSCs we successfully identified differentially expressed genes and a panel of novel markers of cartilage-specific stem cells. Novel markers were validated using qPCR and protein labelling and, were specifically expressed in ACSCs, with no expression in the culture-expanded full-depth chondrocytes. Using immunofluorescence for novel stem cell markers we found articular cartilage-derived stem cells are localised within the transitional zone in normal cartilage and the superficial zone in OA cartilage. OA cartilage was found to contain a 2-fold increase in stem cells using immunofluorescence. Subsequently, we used the panel of novel markers and fluorescent active cell sorting to isolate a sub-population from full-depth cartilage with stem cell characteristics. These cells were plastic adherent, clonogenic, with proliferative capacity greater than 50PD and displayed tri-lineage potential, therefore meeting all criteria for classification as a MSC population. The use of specific markers to isolate ACSCs will allow for further characterisation of stem cells, including a more in-depth understanding of the mechanisms of proliferation, differentiation and degeneration within articular cartilage.

Aims: To fully characterise the RGC-5 cell line and determine whether it would be a suitable substitute for primary RGC cell culture. To optimise a CNTF Nogo-P4 inhibitory assay and establish whether (a) CNTF alone is capable of stimulating RGC neurite outgrowth and survival, or (b) an increase in intracellular cAMP is required for CNTF to be effective. To determine whether recombinant AAV2 viral constructs were capable of producing detectable levels of CNTF in HEK-293 transfected conditioned media. To optimise AAV2-eGFP delivery and establish whether AAV2-CNTF-hrGFP and AAV2-CNTF-shRhoA-hrGFP could promote RGC survival and regeneration after optic nerve crush surgery. Methods: The RGC-5 cell line was characterised using semi-quantitative PCR, sequencing and immunocytochemistry. RGC-5 cells were screened for a selection of neuronal, glial, progenitor, oligodendroglial lineages and cone photoreceptor cell markers to identify the cells origin. Retinal cultures were treated with recombinant CNTF and/or Forskolin to promote RGC neurite outgrowth and survival - this was quantified after 3 d. Retinal wholemounts were prepared to assess GFP transduction and survival after intravitreal delivery of AAV2-eGFP. Axonal regeneration and RGC survival were assessed through histological examination of optic nerves and retinal sections. Results: The RGC-5 cell line predominantly expressed oligodendroglial lineage markers and only weakly expressed \(\beta\)III-Tubulin mRNA. RGC-5 cells did not express mRNA for many of the phenotypic markers of RGC. CNTF was effective at stimulating RGC neurite outgrowth without the need for cAMP elevation - furthermore recombinant CNTF could disinhibit Nogo-P4 treated RGC in vitro. GFP transduction was low when injected alone, however, when administered with Pronase-E there was a significant increase in GFP expression. AAV2-CNTF-hrGFP and AAV2-CNTF-shRhoA-hrGFP did not promote RGC survival or regeneration 23 d post optic nerve crush. Conclusions: RGC-5 cells are not an appropriate substitute for primary retinal cell culture in vitro as they express many of the same markers as oligodendrocyte progenitors. CNTF is capable of stimulating RGC neurite outgrowth without an additional elevation of cAMP. AAV2-mediated GFP expression could be enhanced through the partial digestion of the inner limiting membrane - this seems to be the major obstacle in achieving optimal AAV2 transduction.

The natural angiogenic response of the vasculature to cardiovascular disease has been shown, at least in part, to involve circulating endothelial progenitor cells (EPCs). However, the native response is often insufficient to restore vascularity without additional intervention. In this study the angiogenic activity of EPCs, demonstrated by in vitro tubule formation, confirmed the suggested potential of EPCs to be used therapeutically. However, as EPCs are found in limited circulating numbers, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) were also investigated as sources of donor EPCs for transplantation. Here ESCs, but not iPSCs, were shown to generate cells with a genetic and proteomic profile, as well as an angiogenic potential, identical to natural EPCs. Using an in vivo mouse model of hindlimb ischemia, this investigation illustrated the preferential binding of transplanted EPCs at sites of angiogenic stimulation, and revealed the importance of platelets in the recruitment of circulating EPCs. In particular, using in vitro aggregation and flow-based adhesion assays, the adhesion molecule P-selectin was shown to play a significant role in this recruitment mechanism. In conclusion, this study has demonstrated that EPC transplantation has abundant potential for development into a viable and efficiacious therapeutic angiogenic treatment.

Factors contributing towards falls in older age during overground walking have been widely studied. Stepping behaviour, balance and head posture control during stair negotiation in young adults (YA) and older adults with either lower (LROA) or higher (HROA) risk of falling during midstair negotiation have not been investigated. The aims of the thesis were threefold. Firstly, age-related changes in gaze behaviour were investigated. The main finding was that older adults fixate stair edges for longer than YA. Secondly, the effect of manipulating visual information on stepping parameters and balance control was compared between YA, LROA and HROA. For stair ascent, stepping and balance control was preserved in LROA and HROA and highlighted stair edges led to increased foot clearance in all groups. For stair descent, HROA demonstrated smaller foot clearance than LROA and highlighted stair edges improved balance in LROA and HROA. Thirdly, head posture was studied in YA, LROA and HROA. Compared to walking, LROA and HROA demonstrated more variable head posture than YA. Overall the findings suggest that adults use visual and probably proprioceptive information about stair edge locations to negotiate stairs and HROA benefited from highlighted stair edges. HROA should be included in future stair negotiation studies.

Perhexiline is an anti-anginal drug that is thought to shift myocardial metabolism from \(\beta\)-oxidation of fatty acids to glucose utilisation. An associated improvement in energy efficiency may be beneficial in ischaemia-reperfusion as an adjunct to established techniques for myocardial protection during cardiac surgery. In this thesis, I conduct a prospective double-blind randomised placebo-controlled trial of oral perhexiline in patients undergoing coronary artery surgery, obtaining samples of serum, right atrium and left ventricle. I measure the concentration of perhexiline using high performance liquid chromatography and find that although highly concentrated in the heart, it may not have reached steady-state in the ventricular myocardium. I perform enzymatic colourimetry and ultra-high resolution mass spectrometry to detect changes in carbohydrate and lipid metabolism; however, the myocardial metabolic profiles of patients on perhexiline are indistinguishable from controls. On analysing the results of the clinical trial, I find no improvement in the primary endpoint, the incidence of a low cardiac output episode, or any secondary outcomes. I conclude that preoperative oral perhexiline does not improve clinical markers of myocardial protection and despite significant accumulation in the myocardium, it has no significant effect on the measurable metabolic profile of the heart at the time of surgery.

The process through which new blood vessels are formed within the body is known as angiogenesis. An essential part of our survival, it has also been implicated more recently in many diseases both in terms of induced growth, and abnormal vascular structure. Angiogenesis is characterized as two processes, the development of a vascular network during embryonic growth and the production of new blood vessels. This work focuses on the latter, and seeks to develop a robust, three-dimensional model for simulating blood vessel growth and the attendant processes of blood flow and mass transfer within the simulated system. A system was developed which utilises medical imaging scan data (specifically, MicroCT) as the initial conditions from which a network of vessels is grown. This is combined with GPU accelerated simulations of fluid dynamics, with the intention of providing a technique for future use in predictive medicine and therapeutic simulation.

Background: Quitting smokers gain weight, this offsets some advantages of quitting and may increase risk of type 2 diabetes above that of continuing smokers. The extent of weight gain, the associated characteristics, and management that will not hinder quit success are unclear. Method: Examination of weight gain in an 8year prospective cohort. Feasibility trial of smoking cessation combined with a very low calorie diet(VLCD) or individualised diet and physical activity planning(IDAP) with usual care. Results: Abstainers gained 9kg, 7kg more than smokers over 8years. Underweight and obese smokers gained most. Less weight gain (1.7kg) was associated with higher baseline alcohol consumption (14units/week vs. none). Recruitment from general practices was difficult and limited by VLCD contraindications. Following training, primary care nurses competently delivered specialist dietary interventions. The control condition was generally unacceptable. Half those on the VLCD were non-adherent. Mean weight change was +0.7kg(control), -1.3kg(IDAP), -7.1kg(VLCD) and +0.4kg for abstinence. We found lower cigarette cravings in the VLCD than control arm, but no difference in IDAP and unrelated to hunger. Relapse was greatest in the VLCD and least in the control. Conclusion: Weight gain after cessation is important and IDAP but not VLCD is a feasible approach for tackling this.

Total knee arthroplasty is a common elective orthopaedic procedure. The surgery itself causes soft tissue and bony trauma leading to a systemic response which includes endocrinological, immunological and haematological events. This thesis aims to investigate the potential association between total knee arthroplasty and such markers of inflammation, endothelium and coagulation. The study consisted of 4 groups; group 1 underwent an uncemented total knee arthroplasty; group 2 underwent a cemented total knee arthroplasty; group 3 underwent an uncemented total knee arthroplasty but received an intra-operative infiltration of local anaesthetic; group 4 underwent an uncemented total knee arthroplasty but had a post-operative drain for 24hours. Blood sampling was undertaken pre-operatively and at day 1 and day 7 post-operatively for the white cell count, platelets, neutrophils, C-reactive protein, interleukin 6, e-selectin, soluble CD40L, tissue plasminogen activator, von Willebrand factor, CD40 and CD1442a. Statistical analysis was undertaken in the form of pair sampled t-tests between group 1 and each of the other three groups. Although there some significant changes in one or two of the variables between the groups the only variable which demonstrated a significant difference in all comparisons was the CD1442a count. The exact role of CD1442a is unclear but there evidence to suggest that it may reflect the inflammatory and thrombotic process or contribute directly to the ongoing atherothrombogenesis. During the statistical analysis it was noted that the majority of the variables showed no clear statistical difference between the groups. In chapter 7 an ANOVA / Freidman analysis demonstrated that all but one of the variables, the CD1442a count, showed no statistical difference between all four groups. This allowed all the variables to be collated and presented as the single largest cohort study to date demonstrating the effects of total knee arthroplasty on the markers of inflammation, endothelium and coagulation. All the variables assessed showed a statistically significant change from pre-operative levels to day 7 post operation. 3 In summary our studies demonstrate that total knee arthroplasty results in activation of common markers of inflammation, endothelium and coagulation. These changes may explain the increased incidence of venous thrombosis and thrombo-embolism post-operatively as well as a potential risk of venous thrombo-embolism.

White adipose tissue is not just a storage organ. It is now recognised as an endocrine organ. It secretes many substances known as adipokines, which are thought to link obesity with type 2 diabetes (T2D). One of the most important adipokines is adiponectin. It is a peptide hormone consisting of 244 amino acids with molecular weight of 30 KD. It circulates in plasma in high concentrations (3-30 4g/ml). Adiponectin polymerises to form many bigger forms. Those are low molecular weight (LMW); middle molecular weight (MMW) and high molecular weight (HMW). The HMW adiponectin is the active form of the hormone. The concentrations of most adipokines are increased in obese people. Adiponectin is unusual in that its concentration is lower in obese people. Consequently its concentration is decreased in some related metabolic disorders. Its concentrations decrease in cardiovascular diseases, diabetes, dyslipidemia and insulin resistances. It is well known that exercise increases insulin sensitivity, also adiponectin was reported to regulate insulin. The effect of exercise on the adiponectin concentrations in plasma is controversial, but the extent to which the exercise regulates the interstitial adiponectin concentrations is not fully examined. The main site of adipokines secretion is adipose tissue. Therefore the study of these substances at the site of their production has a special interest. Recently, microdialysis techniques have been extended to become important in the measurement of substances in the extracellular fluid of many tissues such as subcutaneous adipose tissue. In particular, it has been used for measurement of adipokines. This thesis includes three studies. The first study was aimed at examining the effect of one hour of moderate exercise at 50% of maximum oxygen consumption v on adiponectin concentration in dialysate samples taken from subcutaneous abdominal adipose tissues (SCAAT). 15 healthy young female volunteers, age 22.8 ± 3.0 years (mean ± SD) participated in this experiment divided into two groups depending on their body mass index (BMI), a lean group BMI 22.2 ± 1.6 kg/m2 (mean ± SD) and an overweight group BMI 27.7 ± 1.9 kg/m2 (mean ± SD). The samples were collected using CMA 66 M. Alzwayi iii microdialysis catheters with membrane cut off 100 KD. Fitness assessment was done for all volunteers about one week before the main trials. The main trials were done on two consecutive days, a rest day and an exercise day. Each day lasted for 4 to 6 hours. On the first day the microdialysis catheter were inserted in abdominal subcutaneous tissue 4 cm lateral to the umbilicus on the left side. Dialysate samples were collected every 30 - 45 minutes. On the exercise day volunteers exercised for one hour at 50% 2 . V O max. All samples were analysed for adiponectin concentrations using Mercodia ELISA technique. The principle findings of this study were that CMA 66 microdialysis catheters worked effectively for two consecutive days for fluid recovery. Adiponectin concentrations were very low and varied, in same volunteer from time to time, and between volunteers. However, the statistical analysis showed no significant difference in adiponectin concentrations between lean and overweight groups. Adiponectin concentrations in the first two samples on the first day of the insertion were significantly higher than the first two samples on the second day of the insertion. Finally, adiponectin concentrations in dialysate samples recovered by 100 KD microdialysis catheters were very low. Therefore, the effect of the exercise was not clear. The second study aimed to compare the adiponectin concentrations in plasma and dialysate samples. Six healthy male volunteers age 32.8 ± 13.1 years and BMI 25.9 ± 3.3 kg/m2 (mean ± SD), were recruited for this study. The experiment was run for two consecutive days using the same microdialysis catheters CMA 66. Dialysate samples were collected as before. 2 ml of blood samples were collected using a cannula inserted into the anticubital vein. Samples were taken every hour for a period of five hours each day. The plasma and dialysate samples were analysed for adiponectin using the Mercodia kits. Adiponectin concentrations in plasma samples were 256 and 1791 times higher than the adiponectin concentrations in dialysate samples. The conclusion of the two studies was that the CMA 66 microdialysis catheter with cut off 100 KD membranes only recovers a small part of the total adiponectin present. M. Alzwayi iv Therefore a third study was designed to use plasma samples. The aim of the study was to investigate the effect of acute exercise at 50% 2 . V O max on HMW adiponectin, total adiponectin, interleukin (IL)-6, tumour necrosis factor alpha (TNF-α), insulin and glucose concentrations directly after the exercise, one hour after and 48 hours. 13 young healthy female volunteers age 24.3 ± 2.7 years and BMI 21.9 ± 2.2 kg/m2 (mean ± SD) contributed in this study. The volunteers were invited for five visits. Their fitness was measured on the first visit. Then they came for two main trials rest day and exercise day, which they were randomly assigned. The main trails lasted for two hours. Three blood samples were collected each day using same cannulated system in the second study. The volunteers followed 48 hours after each trial, one blood sample were collected each day. The 8 plasma samples were analysed for: total adiponectin and insulin concentrations via Mercodia ELISA kits, HMW adiponectin, IL-6 and TNF-α concentration via R&D systems and glucose concentration using the glucose oxidase colorimetric method. The results showed no statistical difference in total or HMW adiponectin, TNF-α and glucose concentrations under the effect of moderate exercise at 50% 2 . V O max either directly or 48 (p value > 0.05). IL-6 concentrations increased about two fold one hour after the exercise above the resting level (P value < 0.05). IL-6 concentrations return to the basal level 48 hour latter. Insulin concentrations show a decrease one hour after the exercise finished. The number of volunteers was small and the change was close to significance. A one way ANOVA returned a P value of < 0.05, but a two way ANOVA with repeated measures returned a P value of > 0.05. In conclusion, the acute exercise at 50% 2 . V O max changes IL-6 concentrations but it has no effect on adiponectin concentrations in dialysate or plasma samples. Low adiponectin concentration is related to obesity, insulin resistance and T2D. Therefore, increase in adiponectin concentration probably lies in weight loss and the exercise may play role, even if it has little direct action on adiponectin concentration.

The role of hydration in causing or alleviating suffering in patients with advanced cancer is poorly understood and remains controversial. Most dying cancer patients have reduced oral intake in the last days of life. This may be related to symptoms arising from the cancer or its treatment, such as dysphagia, anorexia, nausea, vomiting, or mechanical problems such as bowel obstruction. Accordingly, the subject of clinically assisted hydration (CAH) is emotive amongst patients and their carers, with the omission or withdrawal of CAH potentially viewed as hastening death in some instances. Current hydration assessment methods are limited in cancer patients approaching death. Bioelectrical impedance vector analysis (BIVA) is an accurate validated method of assessing body composition; however its clinical use in advanced cancer is uncertain. This study aims to measure hydration in advanced cancer patients using BIVA, in order, to determine the relationship between symptoms, physical signs and biochemistry. BIVA was used to evaluate hydration in advanced cancer patients within a hospice in the UK. Total body water (TBW) was estimated using the impedance index (Height2/Resistance [H2/R]). Regression analysis determined the predictive properties of clinical variables on H2/R. Assessed items included: performance status (ECOG), symptoms (Burge-4 score), physical signs (Morita Dehydration Score) and biochemistry. Ninety patients participated (recruitment rate = 76.3%). Hydration status was normal in 43 (47.8%), ‘more hydrated’ in 37 (41.1%) and ‘less hydrated’ in 10 (11.1%) patients. A multiple regression analysis was conducted. H2/R was significantly predicted by female gender (Beta = -13.85, p<.001), the Burge-4 score (Beta = -0.29, p=.04), the Morita dehydration score (Beta = -2.55, p=.02) and oedema (Beta = 2.55, p<.001). Median survival was significantly shorter in ‘less hydrated’ patients (44 vs. 68 days; p=.04) and in pre-renal failure (44 vs. 100 days; p=.003). Higher values of H2/R were associated with improved survival (HR=0.98 [95%CI= .96, .99], p=0.01). The results demonstrate that in advanced cancer, hydration status (as measured by H2/R and BIVA) relates to clinically measurable signs and symptoms. Lower TBW volume was associated with female gender and also linked with higher scores for symptoms and physical signs. Higher TBW was associated with oedema. H2/R, BIVA and pre-renal failure were independent predictors of survival. Further work is needed to determine how BIVA can be used to guide the management of fluid states in advanced cancer.