Introduction Bladder Cancer (BC) is the 10th most common cancer in the UK, with about 10,000 new cases annually. It affects more men than women (ratio 3:1). Major risk factors include tobacco, chemical carcinogens, schistosomiasis infection and age. About 75-85% of BC are non-muscle invasive (NMIBC), which is associated with high recurrence and progression rates (50-60% within 7-10 years). Currently, diagnosis, treatment and management of BC is via clinical procedures such as transurethral resection of the bladder tumour (TURBT) and endoscopy. Concerning laboratory investigations, there are no routine biomarkers currently available for identifying BC patients at increased risk of developing recurrence and progression. By monitoring changes in selective biomarkers post-TURBT, any sustained changes may be a predictor of cancer recurrence or progression. The main-focus of this research study was to evaluate changes in selective novel biomarkers and their association with recurrence and progression in BC. Materials & Methods In this research, 40 patients (n=40) scheduled for TURBT at the Wrexham Maelor Hospital, North Wales were recruited after written informed consent. Ethical approval for the project was granted via IRAS (REC4: 14/WA/0033). Venous blood samples were taken at baseline (pre-operative) and following TURBT surgery at 1, 3 and 6 months post-operatively. Bladder tumour samples were also taken during TURBT according to standard procedure. Selective biomarkers to assess inflammation, angiogenesis and tumour growth, were measured using commercially available ELISA and BioPlex multiplex assay kits. Tissue immunoreactivity of novel biomarkers were also assessed in BC tissues using immunohistochemistry, with clinical outcome measures being recorded for all patients. Results Significant increases in serum Cluster of differentiation 31 (CD31) (p=0.003) and Stem Cell Factor (SCF) (p=0.032) concentration, as well as trends of increasing concentration of serum basic Fibroblast Growth Factor (bFGF) (p=0.14), Vascular Endothelial Growth Factor Receptor-1 and 2 (VEGFR-1) (p=0.15), VEGFR-2 (p=0.15) and Follistatin (p=0.40) were observed in BC patients up to 6 months post-operative. There were also significant decreases in serum Macrophage Inflammatory Protein -2 (MIP-2) (p=0.001), Platelet Derived Growth Factor (PDGF) (p=0.012), Matrix Metalloproteinase-9 (MMP-9) (p=0.002) and Vascular Endothelial Growth Factor C (VEGF-C) (p=0.04) serum concentration. Trends of decreasing concentration in MMP-2 (p=0.79), MMP-3 (p=0.15), interleukin-6 (IL-6) (p=0.26), interleukin-8 (IL-8) (p=0.15) and tumour necrosis factor-α (TNF-α) (p=0.69) were observed in BC patients up to 6 months post-operative. There was significant immunoreactivity of CD31 (p< 0.001), CD34 (p< 0.001), Human epidermal growth factor receptor-2 (HER-2) (p=0.032), S100P (p< 0.001), Cyclooxygenase-2 (COX-2) (p< 0.001), VEGFR-3 (p< 0.001), SOX-2 (p< 0.001) and thrombomodulin (p=0.010) in bladder tumours. Although recurrence was significantly associated with cancer grade, there was no association with antibody immunoreactivity. Conclusion Findings from the present study may indicate an alternative approach in the monitoring and management of patients with BC. It is proposed that by allowing urological surgeons access to laboratory markers such as MIP-2, MMP-9, PDGF, SCF, HER-2, Thrombomodulin and CD31 (biomarker profile), potentially, in the future, these biomarkers may be used in addition to, or in combination with, currently used scoring systems to predict cancer recurrence and progression. However, verification and validation of these biomarkers are needed using larger cohorts.

This thesis is composed of a series of papers, all of which have been published in peer reviewed publications. The papers use the recognised process of qualitative risk assessment in a range of scenarios in the field of wildlife health and welfare in both in situ and ex situ environments. Chapter 1 discusses the challenges faced regarding availability of empirical data in field of wildlife and zoological health and welfare and justifies the exploration of techniques to assist with decision making. The development of risk analysis and its integration with risk management and risk communication to become risk assessment is described before being put into the specific context of wildlife and zoological disease. Chapters 2 and 3 consider two scenarios where disease risk assessment is well established as a tool, importation across national borders and in conservation interventions. Chapter 2 develops the standard import risk assessment approach to include multiple species and multiple diseases. Chapter 3 reviews developments made over the last 25 years and proposes best practice approaches to implement. Chapter 4 describes how the risk assessments formulated as described in Chapter 3 are used for licensing purposes emphasising the importance of risk management and communication. This theme is continued in Chapter 6 where the integration of risk assessment and evidence based decision making is considered in the broad context of a strategic approach to wildlife health bringing together the outcomes and processes described in Chapters 2, 3, 4 and 5. The papers in Chapters 2,5 and 8 are focused on how risk analysis aids in development of disease control approaches and policy. The evidence base is composed primarily of peer-reviewed literature supported by expert review of the finalised assessment. Chapter 7 uses risk assessment in an applied scenario, taking the recognised process and modifying it to structure an active disease investigation demonstrating the versatility of the technique. Chapter 9 takes this a step further by again adapting the methodology which, has historically been used primarily for infectious diseases, to consider reproduction and assess risks to welfare rather than purely health. The paper in Chapter 9 builds on the methodology by combining existing peer-reviewed literature with data collected specifically for the purpose of feeding into the assessment and utilising a stakeholder and expert opinion elicitation workshop to obtain data too. These process are proposed and described in Chapter 3. The final chapter critically reviews risk assessment, highlighting three key areas of potential weakness and proposing approaches to address these criticisms. The value of the approach in wildlife and zoological health and welfare as demonstrated by this series of papers is described

Aptamers are biomolecular ligands composed of nucleic acids. They can be selected to bind specifically to a range of target molecules and subsequently exploited in a fashion analogous to more traditional biomolecules such as antibodies. In this study a method for selecting new aptamers which specifically bind whole live bacterial cells is described. A non-pathogenic strain of Escherichia coli K12 was used to develop the method. A DNA library containing 100 bases long random nucleotide sequences was produced and the aptamer selection process was repeated nine times. An enzyme-linked technique was first used to detect bound aptamers thereafter fluorimetry and fluorescence microscopy methods were used for the detection. The aptamers were cloned and sequenced and the cloned aptamers produced with fluorescent labels. The E. coli K12-binding aptamers were used to demonstrate the detection of the bacterial cells in a complex food matrix, namely probiotic yogurt, by using fluorescence based detection method. The aptamer selection method with some modifications was also used to select aptamers with specificity for the food pathogens E. coli O157, Listeria monocytogenes, L. innocua, S. typhimurium and S. enteritidis. The aptamers against E. coli O157 and S. typhimurium were cloned and the sequences and the binding properties of these aptamers were analysed. The use of E. coli K12 as a target organism and the aptamer sequences presented in this study, have not previously been published in scientific literature. This is also the first report where the aptamers have been used in detection of live bacterial cells in yogurt.

The transport of ornamental marine fish may cause stress, which to date has been the subject of limited research. The present study aimed to characterise the behavioural and physiological responses to simulated transport stress in the common clownfish Amphiprion ocellaris (Cuvier, 1830) with the additional goal of validating non-invasive measures of water cortisol in a marine teleost for the first time. Behaviour and physiology of the animals was measured at different stages of transport (from initial capture and handling up to 72 hours transport time) and water quality measurements were taken from the transport water at key sampling points. In a second experiment biological filtration materials (“Bioballs” with denitrifying bacteria) were added to the transport bag to determine if stress was reduced when water quality was improved. The results of the study suggest that capture, handling and transport are stressful for clownfish, and the stress response appears to peak between 24 and 48 hours after the onset of the stressor. Water-borne cortisol was found to be a valid alternative to invasive methods of sampling, although only an average of 53% cortisol was recovered from sea water. Although handling and confinement appeared to be highly significant factors in eliciting the stress response water quality measurements revealed that fish are temporarily subjected to relatively high concentrations of ammonia as transport time increased, which may contribute to long-term effects on the health of the animals. This was reflected in an increased latency to feed and reduced social behaviours in fish transported for 24 hours or longer. Improving water quality did reduce the concentration of ammonia present; however, fish still exhibited elevated cortisol excretion suggesting that water quality is not the primary stressor associated with transport. Thus, the duration of transport should be restricted to a maximum of 24 hours to reduce the stress associated with this practice. A separate study investigated the potential for beauty treatment ‘fish spas’ to elicit stress in the freshwater cyprinid fish Garra rufa (Heckel, 1843). Water cortisol was measured non-invasively to determine if stress was reduced through the provision of environmental enrichment / furnishings, and whether stocking density influenced stress. Water quality was monitored to determine the effects of stocking density on environmental parameters (pH, ammonia, nitrite and nitrate). Finally, the effect of ii human hands being placed into the aquarium was investigated, to determine whether this influenced stress. Three hundred G. rufa were used in total, with groups being allocated randomly to one of four treatment groups: OP/B (optimum stocking density / barren tank – i.e. no enrichment); OP/E (optimum stocking density / enriched tank); OS/B (overstocked / barren tank); and OS/E (overstocked / enriched tank). Human hands were placed in each tank, and water samples were collected before and after for measurement of cortisol by ELISA, and to determine water quality parameters. The results revealed that overstocking tanks with G. rufa produced relatively higher baseline cortisol levels, suggesting that stocking density may have a significant effect on stress levels. The addition of furnishings into the aquarium did not reduce baseline cortisol levels in the fish prior to the introduction of human hands. However, fish maintained under enriched conditions exhibited a greater cortisol response when compared to individuals in barren tanks. It is hypothesised that the provision of enrichment reduces the available space for fish following the introduction of human hands, thereby increasing stress. Further studies are required to attempt to determine the effect of enrichment based upon the results of the present study. Feeding on human hands resulted in an elevated cortisol response from three out of the four treatment groups (with the exception of OP/B), with the results suggesting that either 1) the lower (i.e. optimal) stocking density and lack of enrichment in holding tanks is preferable for G. rufa fish welfare, or 2) the elevated cortisol reflected a response to a rewarding stimulus and is linked to increased foraging. Overall, the results of these studies have shown that water cortisol measurements are a valid means of assessing physiological stress in two species of fish in different contexts. This negates the need for invasive sampling and is an important refinement to existing protocols where fish are killed for plasma or whole body samples. The results also highlight the welfare impacts of transport and overstocking of ornamental fish providing valuable evidence that may be relevant to improving the husbandry and guidelines with respect to the ornamental fish industry.