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Vitamin D deficiency is a serious public health concern. Natural dietary sources and ultraviolet-B (UVB) cutaneous synthesis remains inadequate to maintain year-round 25-hydroxyvitamin D (25(OH)D) concentration (> 25 nmol/L). A systematic review of the literature demonstrated the validity of vitamin D biofortification via feed and/or UVB exposure, but highlighted clear knowledge gaps regarding the impact of biofortified foods, particularly meat, on consumer vitamin D status. Therefore, this thesis adopted a farm-to-fork approach, which aimed to investigate the efficacy of pork meat as a potential vehicle for vitamin D biofortification. Daily UVB exposure, coupled with 2000 IU/kg vitamin D in feed, resulted in higher vitamin D3, 25(OH)D3 and total vitamin D activity (vitamin D3 + [25(OH)D3 x 5]) in pork loin compared to that from control pigs (no UVB). Cooking increased the vitamin D concentration in pork loin, mince and sausage. Total vitamin D activity retention varied between pork products and cooking methods (90.5-130.3%) but did not significantly differ from 100%. A novel acute crossover postprandial study (n=14) showed a significant time*treatment effect of vitamin D3 biofortified pork (5 μg/portion), dose-matched vitamin D3 supplement and control pork; however, there was no significant difference between the three treatments regarding bioavailability to increase 25(OH)D3 concentrations. Evidently, UVB exposure should be further optimised in on-farm interventions to ensure provision of a greater single-dose of vitamin D3 to fully understand food matrix effects of biofortified meat. Using UK population data, dietary modelling demonstrated that increasing the vitamin D content by 50-200% in biofortified pork and pork products would theoretically increase consumer intakes by 5-20%, respectively. In conclusion, the multidisciplinary research within this thesis provides clear rationale for vitamin D biofortification of pork meat as an important and novel food-based strategy to increase vitamin D supply within the food system and, ultimately, helping to reduce prevalence of hypovitaminosis D.

Amphibians are declining worldwide in response to pressures that are too numerous, difficult and rapid to ameliorate in the wild before some taxa become extinct. Ex situ conservation, whereby animals are maintained in captivity until threats in the wild have been resolved, is the only means of saving up to five hundred amphibian species. Amongst political, financial and practical hurdles, the most fundamental problem for these programmes is lack of knowledge about how to maintain species successfully in captivity. Captive populations have failed to survive or reproduce and, furthermore, captive husbandry may produce animals unsuitable for reintroduction through intra or inter-generational changes. These problems entirely undermine initiatives and if ex situ programs are to succeed, evidence based captive husbandry is needed to support captive breeding programs. In this thesis, I quantify our ignorance of amphibian requirements in captivity. Furthermore, I present investigations into fundamental areas ofamphibian husbandry, about which we currently know very little. I investigate the relationship between amphibians and Ultraviolet B (UVB) radiation and examine the relationship between UVB provision and calcium provision and evaluate routes of dietary calcium supplementation. I also present data on the effects of enrichment and rearing environment on the growth, fitness, behaviour and dermal bacterial communities of captive amphibians. These results, from a range of areas of amphibian husbandry, together demonstrate the power of the captive environment to influence the phenotype and therefore the fitness of amphibians, even within single generations. Additionally, I provide some of the first data addressing key areas of amphibian husbandry that until now have been led mostly or entirely by anecdote and hearsay.

The skin is exposed to a variety of external stressors, from irritants to toxic agents to ultraviolet radiation (UVR). While mice and human volunteers can be used for research studies, there are limitations in the range of environmental stressors that can be studied using these models. Therefore, our research team has been investigating the use of human skin explants as a model to study the immediate effects of environmental stressors ex vivo. Although this method has been successful, obtaining fresh human skin for these studies is difficult due to limited availability. To overcome this issue, we are evaluating the possibility of using porcine skin explants in our model systems. The primary advantage of using porcine skin is that it is readily available in large quantities, and its structure bears many similarities to that of human skin. In our research, we have found that various stressors, including UVB radiation, can trigger the production of Platelet-Activating Factor (PAF) in the skin. This, in turn, activates the enzyme acid sphingomyelinase (aSMase), leading to the shedding of subcellular microvesicle particles (MVP). In our research studies, we have used various models, such as cell lines, mice, human skin explants, and limited studies in human volunteers. Currently, we are examining the possibility of using porcine skin explants as a model to study MVP release in response to different types of stressors that are relevant to the skin. The application of the carbamyl PAF agonist PAF (CPAF) or a phorbol ester topically to the skin leads to an increase in MVP release. Similarly, exposing the skin to different UVB fluences also results in a higher release of MVP. Our experiments on porcine skin explants show similar results to human skin explants in terms of MVP release, although porcine skin appears to be less reactive to these agents. Although porcine skin shares many structural similarities with human skin, unlike human skin and cell lines, the combined impact of these stressors on porcine skin did not produce any synergistic results. Similar physiology does not necessarily imply that the skin will react to external stimuli in the same way. As the use of porcine skin in this type of skin model is relatively new, further research is needed to evaluate all parameters before considering porcine skin as a viable substitute for human skin models.

Skin cancer is the most prevalent human malignancy and is primarily caused byultraviolet (UV) wavelengths of sunlight. However, the fact that most skin cancers occurin people over the age of 60 indicates that advantaged age is a second skin cancer riskfactor. Why geriatric skin is prone to developing skin cancers is not clear, but severalstudies have shown that dermal fibroblasts in geriatric skin express lower levels of thehormone insulin-like growth factor-1 (IGF-1) than young adult skin and that deficientIGF-1 signaling negatively impacts how epidermal keratinocytes respond to UVBradiation. A major regulator of the cellular response to UVB-induced DNA damageresponse is the tumor suppressor protein p53, and a previous study indicated that p53 isnot properly activated in UVB-irradiated human keratinocytes deficient in IGF-1signaling. Using cultured human keratinocytes in vitro, we show here that severaldownstream transcriptional targets of p53, including the cell cycle-dependent kinaseinhibitor p21, the translesion synthesis polymerase pol eta, and the DNA repair factorsXPC and DDB, are not properly induced following UVB exposure in cells deprived ofIGF-1. Using discarded human skin from routine panniculectomies, we show that the ivtopical application of an IGF-1 receptor antagonist similarly abrogates the ability of UVBexposure to properly induce pol eta and p21. Because these various p53 target geneproducts are involved in delaying entry into S phase, accurately replicating UVBphotoproducts, and removing UVB photoproducts from genomic DNA, our findingsindicate that the inability of IGF-1-deficient geriatric skin properly activate p53-responsegenes may predispose geriatric skin to mutagenesis and carcinogenesis.

Skin cancer is the most common type of cancer in the United States and it is primarily caused by the exposure to ultraviolet-B radiation. Non-melanoma skin cancers, such as basal cell carcinoma and squamous cell carcinoma comprise the majority of skin cancer cases. Ultraviolet-B radiation induces a host of changes in the skin, the primary target of UVB, including inflammation, DNA damage, and eventually skin carcinogenesis. UVB radiation can also induce immunosuppression. This immunosuppression makes it less likely that the immune system will be able to respond efficiently to immunogenic tumors such as squamous cell carcinomas. Therefore, we would predict that factors that can affect cell-mediated immunity would also have a high likelihood of affecting SCC tumor development as well. Psycho-physiological stress is powerful modulator of immune system function as well. While short-term (acute) stress enhances cell-mediated immunity long-term (chronic) stress suppresses cell-mediated immunity. We have used these stressors in combination with UVB radiation to understand the effects of psychological stress on UVB induced inflammation, DNA damage, and ultimately SCC skin carcinogenesis. Using techniques such as immunohistochemistry, real-time PCR, flow cytometry, and ELISA, we found that acute stress reduced DNA damage, enhanced gene expression of Th1 cytokines and chemokines in the skin, and enhanced leukocyte traffic, primarily T cell, into the skin and sites of tumor formation. As a result of this, acute stress reduced SCC tumor development. In contrast to these results, we found that chronic stress increased DNA damage, decreased the gene expression of Th1 cytokines and chemokines in the skin, and decreased T cell infiltration into the skin and sites of tumor formation. As a result of this, chronic stress increased SCC tumor development. Finally, we examined anxiety levels in our mice and because we considered high anxiety as a type of innate chronic stress, we predicted that anxiety would produce the same types of effects in response to UVB as chronic stress. This was, in fact, the case as the high anxiety mice had reduced levels of Th1 cytokines and chemokines and also reduced T cell infiltration which caused increased SCC tumor development.