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Introduction: Upper respiratory illness (URI) is the most frequently reported illness in athletes, resulting in lost training time and reduced success at competition. Due to the high physical demands on elite swimmers, identification of risk factors associated with URI and individuals at increased risk of illness would be paramount. Individual athletic monitoring of mucosal immunity and identifying risk factors of URI, could enable modification of training and other illness preventative strategies (Gleeson & Pyne, 2016). Vitamin D levels are known to naturally decrease in winter, further increasing risk of seasonal illnesses. However, few studies have examined the effect of vitamin D supplementation on mucosal immunity and the prevention of URI in athletes. Methods: Study one (Chapter 4) investigated the effect of training load, sleep and EBV status on mucosal immunity and self-reported URI symptoms, for 8-months leading into the 2018 Commonwealth Games. The periodised training load plan for each week was classified by coaches as low, moderate, and high. When this was compared against swimmer's perceived weekly training intensity, it was significantly correlated. Weekly sIgA was examined to investigate changes in mucosal immunity over different training loads and leading into major competition. Blood samples were taken to determine EBV status and self-reported URI symptoms were quantified using an adapted illness questionnaire. In addition to monitoring self-perceived sleep, quantitative sleep parameters were measured use wrist-worn actigraphy at three different time points during the study period. Chapter 5 presents the development and optimisation of an in-house Enzyme Linked Immunosorbent Assay (ELISA). Assay development and validation protocols were carried out in order to produce an ELISA for determination of sIgA, from the elite swimmer samples in Chapter 4. The final study (Chapter 6) provides a met-analysis and systematic review on the effectiveness of vitamin D supplementation in prevention of URI and the effect on immune markers. Results: Over the 8-month observation (Chapter 4), 70 upper respiratory symptom (URS) episodes were recorded, leading to 34 days of missed training. Significantly more URS were reported, with increased duration and severity during high training loads. In total, 61% of swimmers had evidence of past infection with EBV. However, EBV seropositivity had no correlation with the number of episodes, severity, or duration of symptoms. When sIgA values were normalised to each individual's mean, relative sIgA concentration was significantly lower during URS (25%) than when no symptoms were present. No relationship was found for sIgA against training load, and incidence, duration, or severity of URS. Sleep duration was significantly lower during high training loads, plus sleep efficiency was poor throughout the study period. Validation and optimisation of the ELISA was conducted in Chapter 5, which included successful analysis of sensitivity, accuracy, precision, linearity of dilution, matrix effects, range, and intra-variation. Additionally, results from the in-house ELISA were significantly correlated with those from a commercially available assay. The meta-analysis (Chapter 6) found no effect on sIgA, or URS duration and severity following vitamin D supplementation. Practical Application: The current thesis highlights the importance of individual athlete monitoring for coaches and physiologists to identify athletes at increased illness risk. Identification of risk factors associated with URS, such as increased training load, lowered sIgA and inadequate sleep, may allow for modifications in training or other illness preventative strategies. Overall, elite swimmers showed inadequate sleep; therefore promoting the use of sleep hygiene strategies and napping. There was high prevalence of EBV seropositivity for the elite swimmers; however it was not a predictor of URS. Low participant numbers could be considered for the lack of significant findings with EBV serostatus; there is ongoing debate that individual data and trends may be more useful in elite athletic research, compared to group statistical analysis. Secondly, thesis work presenting ELISA optimisation and validation was successful in offering a good basis for future work. Lastly, findings from the meta-analysis regarding the effect of vitamin D supplementation on URI contrasted with many large cohort studies from the general population. The small number of studies included, low participant numbers, and differences in study design may have influenced these findings. Thus, a clear gap in research is evident and more well-designed control trial studies are needed to establish the relationship between supplementation of vitamin D and URI in athletes.

Background: The inhibition of hepatic cytochrome P450 activity is one of the most significant mechanisms of a drug interaction which can result in a change in the pharmacokinetic behaviour of the drug. Severe adverse events have been associated with drug interactions caused during coadministration. COVID-19 infection has rapidly grown into a worldwide pandemic in 2020, and this has had a significant impact on human health. Dexamethasone, aspirin, ibuprofen, remdesivir, and omeprazole are some of the drugs being used in the treatment of COVID-19 during the pandemic. Aim: This research aims to determine the inhibitory effects of aspirin, ibuprofen, remdesivir, and omeprazole on dexamethasone metabolism (CYP3A2 activity) and the effect of testosterone on Vitamin D metabolism (CYP2C11 activity) in rat liver microsomes using High-Performance Liquid chromatography. Methods: A Dexamethasone and 6β-hydroxydexamethasone assay was developed and validated, and inhibition parameters were calculated using Lineweaver and Michaelis-Menten plots in Chapter 2. Using the previously developed HPLC assay, inhibition parameters were calculated using Lineweaver and Michaelis-Menten plots in Chapter 3. In chapter 4, a Vitamin D3 and Vitamin D2 and metabolites (25(OH)D3 and 25(OH)D2) assay was developed and validated on HPLC using the isocratic mode of elution. Results: The linearity (r2 > 0.99), intra and interday precision (< 15%), accuracy and recovery (80-120%), and stability study values of the newly developed methods for CYP3A2 and CYP2C11 substrates and metabolites were following International Conference on Harmonization (ICH) guidelines. Our inhibition study data showed that aspirin is a competitive inhibitor (weak) with the Ki = 95.46 ± 4.25 µM and IC50 = 190.92 ± 8.50 µM for the CYP3A2 assay. The results also showed that ibuprofen acts as a non-competitive inhibitor for CYP3A2 activity with Ki = 224.981 ± 1.854 µM and IC50 = 230.552 ± 2.020 µM although remdesivir showed a mixed inhibition pattern with a Ki = 22.504 ± 0.008 µM and IC50 = 45.007 ± 0.016 µM. Additionally, omeprazole uncompetitively inhibits dexamethasone metabolism by the CYP3A2 enzyme activity with a Ki = 39.175 ± 0.230 µM and IC50 = 78.351 ± 0.460 µM. Moreover, a study of inhibition parameters was not conducted due to the presence of interferences and extremely low concentrations of Vitamin D and metabolites. Conclusion: The findings of this PhD research represent that there is a minimal risk of toxicity when dexamethasone is co-administrated with aspirin, ibuprofen, remdesivir, and omeprazole and a very low risk of toxicity and drug interaction with drugs that are a substrate for CYP3A2 in healthcare settings.

Vitamin D has been shown to play an essential role during pregnancy for maternal and child health. Whilst a vitamin D status of >75 nmol/L is needed for optimal musculoskeletal health, a status of >100 nmol/L has been suggested for non-skeletal functions including neurodevelopment. The main source of vitamin D is via sun exposure, and it can be obtained from limited dietary sources including fish. The overall aim of this thesis was to examine associations between early life vitamin D status and child neurodevelopmental outcomes. A systematic review and meta-analysis investigated associations between prenatal and neonatal vitamin D status and child diagnosis of autism spectrum disorder (ASD) and highlighted that vitamin D deficiency during early pregnancy may be a risk factor for ASD diagnosis. Associations between vitamin D status of maternal and cord samples in an equatorial (Seychelles, 4°S), high fish-eating population, and in school children in a high latitude country (Northern Ireland 55°N), with child neurodevelopment outcomes were examined. Overall, trends from these three studies suggest that a vitamin D status of >75 nmol/L is beneficial for child neurodevelopment. The effects of fish consumption on the vitamin D status of women of childbearing age in Northern Ireland during winter was assessed. Results showed that 1-2 portions of fish were not effective at increasing or maintaining vitamin D status during winter compared to no fish consumption in a vitamin D insufficient population. Overall, this thesis demonstrates the importance of vitamin D during early life for neurodevelopmental outcomes in children. It adds to the current evidence that a status of >75 nmol/L is optimal for beneficial neurodevelopment outcomes, and that fish consumption should be supplemented with safe sun exposure and supplement use to achieve an adequate status. Future studies are required to confirm the optimal vitamin D status at the optimal time during early life for extra-skeletal outcomes such as neurodevelopment.

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.

Atherosclerosis-related cardiovascular diseases (CVDs) are a major burden on human health. Many studies have reported the central role of dietary nutrients in modulating the inflammatory and oxidative processes involved in the development of atherosclerosis. Selenium (Se) through its incorporation into selenoproteins is an important antioxidant in the body. When dietary Se is suboptimal (i.e. in most European countries) it may be insufficient to maximize cellular selenoenzyme activities leading to lowered protection against oxidative damage and increased risk of oxidative-damage associated diseases including CVD. This study examined the potential for another dietary nutrient, vitamin D, to enhance Se status and to potentially provide protection against oxidative stress and stress-related cellular damage. Vitamin D is a known modulator of inflammation in addition to its classical effects on calcium and bone homeostasis. In the present study, results showed that vitamin D increased selenoenzyme activity within monocyte cells when Se levels were suboptimal. In addition, vitamin D also augmented the reduction in cytokine-stimulated adhesion of monocytes to endothelial cells (ECs), an in vitro model of atherosclerosis. Investigation of the mechanism involved showed there is a reciprocal interaction of vitamin D with Se that acts through regulation of the enzymes 25-hydroxyvitamin D 1-alpha-hydroxylase (CYP27B1) and cystathionine--synthase (CBS). CYP27B1 converts circulating levels of vitamin D form to biologically active vitamin D whereas CBS is required to incorporate organic forms of dietary Se into selenoproteins. Results from this study provide the first evidence to suggest that the impact of suboptimal Se status may be partly alleviated by maintaining adequate vitamin D levels and conversely, Se may act to enhance localized levels and action of active vitamin D. These interactions may have important consequences both for health and for diseases such as atherosclerotic CVDs.

Vitamin D is a secosteriod hormone with a key role in increasing the intestinal absorption of calcium and plays a key role in bone metabolism. Older adults, in particular those residing in care homes, are susceptible to vitamin D deficiency owing to lack of sunlight exposure, poor dietary intake of vitamin D, medication use, compromised renal function and lack of supplementation. The overall aims of this thesis were to determine, for the first time, the vitamin D status and prevalence of vitamin D deficiency in care home residents in Northern Ireland (NI), investigate factors which impact vitamin D status and to determine the association between vitamin D, musculoskeletal health outcomes and inflammatory cytokines. Dietary contribution of vitamin D and other nutrients important for bone health was investigated within care home menus in NI. Additionally, given recent evidence highlighting a role for n-3 long chain polyunsaturated fatty acids (LCPUFA) in benefiting bone heath, an investigation into the associations of long chain polyunsaturated fatty acids with bone mineral density and bone turnover markers in postmenopausal women was completed. An extensive critical review of the literature on the vitamin D status of older adults in care homes across the globe outlined a high prevalence of vitamin D deficiency and identified that those residents that were severely deficient, deficient or insufficient require remedial supplementation prior to maintenance supplementation to improve vitamin D status. In addition, promising findings in relation to intervening with vitamin D and calcium fortified foods for resultant benefits to health outcomes in care home residents were summarized identifying that more research is required in this area. An observational study in care home residents in NI identified vitamin D deficiency and insufficiency in 34.8% and 17.4% of residents. 25-hydroxyvitamin D (25(OH)D) concentration was not an independent predictor of T-score, muscle strength, timed up and go (TUG) or inflammatory cytokines albeit, higher 25(OH)D concentration was associated with lower bone turnover. Vitamin D ± calcium supplementation was a significant predictor of a higher 25(OH)D concentration albeit many residents were not taking any form of vitamin D supplementation. Building on this, nutrient contribution of care home menus in NI identified low dietary contribution of vitamin D (3.7μg vitamin D/day) and that the inclusion of vitamin D fortified milk would increase intakes by 40.5%. Secondary analysis of a well-defined cohort of postmenopausal women, a vulnerable group also at risk of osteoporosis similarly to care home residents, showed that a low n-6:n-3 ratio was associated with higher femur bone mineral density (BMD) and a higher n-3 LCPUFA (ALA) was associated with lower bone resorption. In this thesis, the benefit of both vitamin D and n-3 LCPUFA for the prevention of bone loss in care home residents and postmenopausal women has been demonstrated, thus the introduction of fortified milk or rich dietary sources of vitamin D and n-3 LCPUFA could be a protentional intervention to improve dietary intake of vitamin D in these groups. In addition, mandatory vitamin D ± calcium supplementation for care home residents within NI may be required to ensure vitamin D deficient residents reach sufficiency.

Poor oral health, in particular dental caries and periodontal disease, is a common problem in the UK population. Previous research has suggested an association between vitamin D deficiency and increased risk of poor oral health. The main source of vitamin D for people resident in the UK is via exposure to solar irradiance. This study aims to determine if an association exists between sunshine exposure and better oral health, also specifically asking if increased exposure to UVB light between the wavelengths 290-315 nm approximately, from natural or artificial sources, affects the incidence of caries and periodontal disease. The findings of the study aim to inform novel public health measures to improve oral health. Following a review of the existing literature and a scoping review of previous relevant studies, a protocol was developed and a systematic review undertaken. The findings supported the presence of a positive association, but highlighted the lack of good quality observational trials and cohort studies. The scoping review highlighted a number of ecological studies, which further supported the existence of an association, but were outdated and limited in their methodology. These studies inspired the empirical studies undertaken in this thesis which used secondary data from modern epidemiological studies to investigate the hypothesis further. The findings of these studies suggested that increased exposure to sunshine is associated with a small, but consistent, reduction in the presence of dental caries in children in the UK. The evidence did not support an association between sunshine hours and a reduction in the prevalence of periodontal disease. Further research could focus on repeating these analyses using other datasets to see if relationships are replicated. Furthermore, the relationship between sunshine and other pertinent oral health outcomes such as oral cancer should be considered. Longer term studies could be undertaken using more accurate personal exposure measures and personalised health data, a viable possibility with the increasing use of wearables for data collection. This research undertaken uses a novel approach to investigate the relationship between sunlight, UVB and oral health, and to inform future public guidance, policy and health interventions.

Much of the morbidity and mortality associated with obesity is related to its metabolic complications. Obese individuals are at higher risk of microvascular disease and premature cardiovascular disease (CVD). Bariatric surgery is currently accepted as the most effective treatment for obesity and related co-morbidities. It reduces the incidence of CVD in a weight-independent manner conferring beneficial mechanisms that have remained elusive. However, bariatric surgery has also been associated with nutritional deficiencies which can cause metabolic disturbance, such that observed deteriorations in bone health have been linked to post-operative vitamin D deficiency. This thesis examined the association between different metabolic factors that are clustered in obesity to elucidate how CVD risk is increased and subsequently reduced following bariatric surgery. There were, in the studies presented, reductions in insulin resistance, inflammatory cytokines, atherogenic lipoproteins and dysglycaemia following bariatric surgery. Consequently, this thesis demonstrated the use of bariatric surgery as a model to further understand the basis of the metabolic syndrome (Reaven's hypothesis), provided evidence of how the metabolic syndrome augments CVD risk and showed that the risk factors are reduced following bariatric surgery. Studies in this thesis found post-bariatric-surgical improvements in high-density lipoprotein functionality (in a multi-faceted manner) and levels of autoantibodies to apolipoprotein A-1, both of which, in recent years, have been associated with enhanced CVD risk. The increases in HDL function were related to reductions in inflammation following bariatric surgery. Furthermore, this thesis quantitatively demonstrated reductions in diabetic neuropathy (using corneal confocal microscopy) and diabetic kidney disease but not diabetic retinopathy after bariatric surgery. Additionally, sexual dysfunction in obese men was shown to be related to microvascular disease and not hormonal dysfunction; further evidence of the adverse metabolic environment in obesity. Moreover, this thesis highlighted the prevention of vitamin D deficiency following bariatric surgery by using a carefully implemented post-operative programme of nutritional supplementation.

The broad aims of this thesis were to investigate the influence of vitamin D supplementation on URTI burden in military recruits (Chapter 3), to examine the influence of psychosocial and behavioural factors on URTI risk in marathon runners (Chapter 4), and to investigate the influence of psychological stress and anxiety on the mucosal immune response to exercise, in both a controlled, lab-based setting and a field-based marathon (Chapter 5). Firstly, we found that vitamin D sufficiency lowered URTI burden in military recruits during Army training (Chapter 3). Specifically, in 1,644 military recruits, we found that vitamin D sufficient recruits were less likely to have a clinician-diagnosed URTI during training, compared to those with serum 25(OH)D < 50 nmol·L-1(Study 1). Then, in a randomised-control-trial (RCT), including 249 men, we found that vitamin D supplementation by oral D3 or simulated sunlight, which achieved vitamin D sufficiency in almost all, reduced URTI burden regardless of supplementation type: 21% lower URTI peak severity and 36% fewer days with URTI (Study 2). And that individuals beginning supplementation with 25(OH)D < 50 nmol·L-1 benefitted from greater reductions in URTI burden: 33% shorter URTI duration. However, we did not find any influence of vitamin D supplementation on mucosal immunity. In Chapter 4, a prospective, cohort study of 305 marathon runners, we examined the influence of psychosocial factors and sleep on URTI prevalence. We found that runners were more likely to report a URTI during the two weeks before or after a marathon if they reported higher psychological stress, anxiety or neuroticism, early life adverse experience, or lower perceived sleep quality. Runners with early life adverse experience were over two times more likely to report a URTI during the two weeks before the marathon (OR: 2.33) and runners with poorer perceived sleep quality were two times more likely to report a URTI during the two weeks after the marathon (OR: 0.48). In the final empirical chapter, we examined the influence of psychological stress and anxiety on the mucosal immune response to a moderate, lab-based bout of exercise (Study 1) and a field-based marathon (Study 2; Chapter 5). We found that perceived psychological stress, trait anxiety and state anxiety influenced the mucosal immune response to exercise. In men, perceived psychological stress, trait anxiety and state anxiety were negatively correlated with the mucosal immune response to exercise. In response to a controlled, moderate bout of exercise, in individuals with predominantly low-moderate psychological stress and anxiety, men with lower psychological stress and anxiety experienced an increase in saliva SIgA SR, whereas those with moderate stress and anxiety saw little change. Then, in a field-based marathon, when including individuals reporting high stress and anxiety, men with higher perceived psychological stress and trait anxiety had a greater reduction in mucosal immunity in response to a marathon. These findings support the recommendation that exercise physiologists should account for psychological stress and anxiety when examining the immune response to exercise. Taken together, Chapters 4 and 5 indicate a key role of psychological factors in immunity in exercising individuals. Specifically, individuals with higher psychological stress and anxiety appear to be at greater risk of URTI and experience greater reduction in mucosal immunity following exercise.

Castrate resistant prostate cancer (CRPC) relies on the supply of potentandrogens such as dihydrotestosterone (DHT) from intratumoral conversionfrom the precursor dehydroepiandrosterone (DHEA) for growth. Vitamin D hasbeen proposed as a chemotherapeutic agent by way of vitamin D receptor(VDR) activation. The mechanistic enzymes of the intracrine pathway areinfluenced by VDR activation and thus can have a beneficial effect of theoutcome of androgen synthesis, by reducing the active androgens produced. In this thesis, I aimed to explore the metabolic outcomes of vitamin D treatmentupon a CRPC translation model, by assessing the androgen intracrineconversions using mass spectrometry-based techniques and genetic changes. Employing an in vitro based assay, effects of vitamin D treatment wereassessed for metabolic changes within the CRPC steroid pathway, using a semiquantitative method. Further evaluation of key metabolic gene changes wasalso undertaken. Findings confirm that vitamin D treatment decreased the levelsof potent androgens, with increased hydroxylation, linked to the upregulation ofthe gene CYP3A4.An in vivo prostate cancer xenograft was also evaluated for vitamin D treatmentupon castration and DHEA introduction. Spatial distribution of endogenousandrogens in prostatic cancerous tissue were assessed using massspectrometry imaging. A method of chemical derivatisation for neutralendogenous androgens was developed using the ImagePrep device, enhancingionisation efficiency for on-tissue analysis. Biological assays were alsoemployed to determine any effects exhibited by the vitamin D treatment, such agene expression analysis and total androgen quantitation. Vitamin D was foundto induce changes in slowing growth and lowering levels of potent androgens. To further explore vitamin D treatment assay capabilities, a novel chemicalderivatisation method for mass spectrometry imaging was developed byscreening numerous reagents for off and on-tissue analysis. Ion efficiency wasenhanced for on-tissue endogenous vitamin D metabolites with successfulimaging of derivatised 25-(OH)-D2 and 1,25-(OH)2-D3 in mouse kidneys.

The accurate determination of biologically active compounds in the skin is of considerable importance when evaluating the penetration of skin health products through different layers of the skin. This thesisreportsonthecharacterisation andquantitation ofbiologically active compoundsinsimple model mixtures and complex mixtures which mimic that of a typical skin product, through the use of qNMR, pure shift NMR and DOSY techniques complemented by semi-automated software packages. Characterisation and quantitation conditions were acquired over several heterogeneous samples allowingfor analysisofhowdynamicrangeand complexity of different sample mixtures affect the Limits of Detection (LOD) and Limits of Quantitation (LOQ) of biologically active compounds. NMR is of particular value inthis task, as it is non-destructive, uses a primary ratio method for quantification, and tolerates a wide variety of hydrophilic and hydrophobic components within a given matrix. In this investigation we have attained a trueness level < 10%, repeatability values of < 1% and brought the limit of quantitation down to 100nM ( ̃limit of baseline range of several key biomarkers in the skin per litre seen in vivo), while commenting on the limitations observed, such as peakoverlap and sensitivity limits. Pure shift optimised sequences allow ustoreducepeakoverlapping, allowing further characterisation of individual compounds and the separation of complex mixtures using NMR. These validated methodologies are then all brought together to develop a new methodology for ex-vivo analysis of skin layers. This analysis allowed for characterisation and quantitation of natural moisturising factors (NMF), biomarkers of hydration and skin health alongside construction of permeation profiles for common topical components in different formulations. Data analysis demonstrates resultscorroboratewithresults seen in previous validated methodologies which havemore complex and time-consumingpreparation and data processingthan the proof of concept study presented here.

A low vitamin D status (determined by 25(OH)D concentration) has been identified as an association risk factor in the aetiology of numerous chronic diseases, with older adults identified as generally more deficient than younger populations. Accumulating data is suggestive that physical activity and exercise may influence 25(OH)D and vitamin D metabolites downstream in the complex metabolic pathway. Specifically, exercise has been shown to act as a direct and indirect stimulus on the intracellular vitamin D receptor (VDR), which mediates the effects of vitamin D and initiates genomic and non-genomic signalling responses. The role of physical activity and exercise on 25(OH)D concentration and VDR expression is not yet recognised in a healthy human population. This thesis aimed to establish whether there is a link between physical activity status and 25(OH)D concentration, and if there is a role of age and exercise on 25(OH)D concentration and VDR expression (as quantified in circulating systemic T lymphocytes).The main results demonstrate that 25(OH)D concentration is not influenced by age or cardiorespiratory fitness (CRF), however VDR expression declines with older age and a higher CRF predicts a greater expression of the VDR. It was found that a single bout of exercise acutely increases VDR expression in circulating T lymphocytes, with exercise modality appearing to influence the response. There was a more pronounced response observed following an endurance compared to a resistance exercise bout. The impact of the exercise-induced lymphocyte response on the observed changes in T cell VDR expression was explored, however they appear to be independent. In conclusion, the findings of this thesis support the notion that exercise could be used as a short-term strategy to increase cellular VDR expression in a human population.

The combined effects of maternal obesity and low vitamin D status have adverse implications for maternal and fetal health. The overall aim of this PhD thesis was to investigate the association between maternal body weight and vitamin D status throughout pregnancy. From the systematic review, this thesis reports that few observational studies have assessed vitamin D status across BMI categories. Obese or obese class 2 pregnant women had lower vitamin D status compared to non-obese pregnant women. Maternal vitamin D status was negatively associated with maternal BMI and positively associated with infants vitamin D status. Vitamin D status was lower in infants born to obese mothers than infants born to normal weight mothers. When we examined data from a previous cohort of pregnant women in Northern Ireland (from 2006) who were not taking vitamin D supplements in pregnancy, we were able to examine the true association between BMI and vitamin D status in this unique cohort. Our results demonstrate that pregnant women with obesity had significantly lower vitamin D status than those who were normal weight, this was evident particularly during the winter months. There was a high prevalence of vitamin D insufficiency (<50 nmol/L) among pregnant women living in Northern Ireland in 2006. When we assessed the association between BMI and vitamin D status in early pregnancy in a cohort that were recruited for the purposes of this PhD work, we found that pregnant women with overweight and obesity had significantly lower vitamin D status compared to pregnant women of normal weight in early pregnancy despite 62% of women reporting to take a vitamin D supplement. Maternal BMI was found to be a significant negative predictor of vitamin D status and a high prevalence of vitamin D insufficiency still exists among pregnant women living in Northern Ireland in 2017. This insufficiency was highest among non-supplement users during winter months. We assessed the effect of supplementation with 10μg vs 20μg vitamin D3/d throughout pregnancy on vitamin D status of normal weight, overweight and obese pregnant women and on the cord blood of their infants. We report that vitamin D status increased throughout the pregnancy in both treatment groups, with a significantly higher increase observed in the 20μg group. In the 20μg group, maternal and cord vitamin D status reached and maintained sufficiency (≥50 nmol/L) throughout pregnancy, even in those who started pregnancy with an insufficient status. In obese women who started pregnancy with an insufficient vitamin D status, the related cord blood vitamin D status was deficient (<25 nmol/L) in both treatment groups (4). Overall, maternal obesity was negatively associated with maternal and infant vitamin D status and the current recommendation of 10μg/d is inadequate to reach and maintain sufficiency in those who start pregnancy with an insufficient status.

Urbanisation and the associated nutrition transition have been linked with the recent rise in osteoporotic fragility fracture incidence in many countries. Predictions indicate that hip fracture incidence will increase 6-fold in Africa and Asia by 2050, partially attributed to demographic transition and population ageing. Differences in areal bone mineral density (aBMD) between rural and urban locations indicate that urban regions of high-income countries (HIC) have lower aBMD and a higher incidence of hip fracture. The few studies conducted in low and middle-income countries (LMIC) provide inconsistent results; in contrast to HIC, most have found higher aBMD in urban populations. To investigate the impact of migrating to an urban environment, detailed studies of bone phenotype and factors affecting bone health have been conducted in two groups of pre-menopausal Gambian women: urban migrant (n=58) and rural (n=81). Both groups spent their formative years in the same rural setting of Kiang West, urban women were known to have migrated to coastal districts, concentrated in Brikama and Kanifing, when aged ≥16 years. Bone phenotype (bone mineral content (BMC); bone area (BA); aBMD, and size-adjusted BMC (adjusted for height, weight and BA) of the whole-body, lumbar spine and hip) was measured by dual energy x-ray absorptiometry (DXA), with further characterisation by peripheral quantitative computed tomography (pQCT). Data were also collected on anthropometry, body composition, food and nutrient intakes, physical activity, socio-demographic characteristics, vitamin D status, and 24hr urinary mineral outputs (Na, K, P, and Ca). Mean age and height of rural and urban migrant groups were not significantly different (p > 0.05). Urban migrant women were significantly heavier (p < 0.01). Significant differences in BMC and aBMD were found between groups at all skeletal sites, with urban women having higher BMC and aBMD; BA was not significantly different. The greatest difference in BMC was found at the lumbar spine (8.5% ± SE 3.0, p < 0.01), a meaningful difference, equivalent to 0.76 of rural SD. T- Scores were also calculated using a young adult (white, female) reference population, mean T- scores were -1.03 and -0.22, for rural and urban groups respectively. After adjusting for size, differences in whole-body and hip BMC were mostly attenuated (p > 0.05), but difference in spine BMC remained significant (6.2% ± SE 2.1, p < 0.01). These results indicate that rural-to-urban migration is associated with higher BMC; BA and height were similar, and difference in body weight could not fully account for higher BMC at the lumbar spine. Calcium intakes were low in both groups, urban migrant 294mg/d (IQR: 235 to 385) and rural 305mg/d (IQR: 222 to 420). Urban women had significantly lower intakes of potassium, magnesium and dietary fibre (p < 0.01), related to lower consumption of fruit, green leafy vegetables and groundnuts. 25-hydroxy vitamin D status was good in both groups, urban migrant 64.0 ± 14.2nmol/L and rural 68.3 ± 15.7nmol/L (M ± SD, p > 0.05). Implications for bone health of the nutrition and demographic transition, principally future fracture risk and other non-communicable diseases require further research in LMICs. ORIGINAL CONTRIBUTION TO KNOWLEDGE To my knowledge, this is the first study investigating the impact of rural-to-urban migration on bone health to be conducted in sub Saharan Africa. It is the first study of bone health and determinants of bone health in an urban population in The Gambia.

Colorectal cancer (CRC) is common, with >1 million annual incidence worldwide and is associated with significant morbidity and mortality. Prevention is a particularly appealing strategy to combat CRC, but there is a paucity of well-founded mechanistic research in the area. CRC is a complex disorder, with both genetic and environmental factors influencing incidence. The heritable component of CRC variance is estimated to be ~35%, with ~5% due to highly penetrant mutations. Common genetic variance likely makes up the majority of the heritable component, yet a large proportion of heritability remains unexplained. One possible explanation is gene-environment interactions (GxE) where-by both genetic and environmental factors interact to influence risk. Observational data implicate vitamin D as an environmental risk factor in CRC aetiology and in-house data indicates that genotype at the GWAS identified CRC risk locus rs9929218 influences this association, i.e. a GxE. The rs9929218 locus is intronic within CDH1, a tumour suppressor gene, and present evidence suggests a gene-environment interaction model of vitamin D-induced CDH1 transcription dependent on genotype at the rs9929218 locus and mediated by VDR and FOXO transcription factors and SIRT1, a FOXO regulator. To test this model, two broad approaches were employed - an observational approach to assess the association between human plasma vitamin D status, rs9929218 genotype and normal colorectal mucosa CDH1 expression and an interventional approach treating cell lines, organoids and human subjects with vitamin D to assess genotype-specific effects. Observational analysis of vitamin D level (25OHD) in CRC cases identified a significant influence of age, gender, BMI and selected vitamin D pathway genetic variation, while analysis of 424 normal colorectal mucosa samples from CRC cases and cancer-free subjects demonstrated strong sampling, gender, age and site differences in gene expression. 25OHD was not significantly associated with mucosa gene expression at individual gene level. However, association with a number of pathways relevant to tumourigenesis, including 'cell adhesion', 'migration' and 'cell death' was seen, providing possible mechanism to the published observational data. Circulating 25OHD level was not associated with mucosa CDH1 expression, yet crucially, analysis demonstrated a strong additive gene-environment interaction effect between plasma 25OHD, the rs9929218 genotype and NM expression of VDR, FOXO and SIRT1 explaining ~70% of the variance of mucosal CDH1 expression. The interventional approach first investigated vitamin D effects on CRC cell lines and human colorectal mucosa organoids. Calcitriol, the active form of vitamin D, induced CDH1 expression in 4 CRC cell lines, with interaction effects explaining 66% of the variance of CDH1 expression. CDH1 induction was replicated in human colorectal epithelial organoids, a non-aberrant tissue model, while gene enrichment analysis from both cells and organoids implicate vitamin D in a number of processes relevant to CRC tumourigenesis including regulation of cell proliferation, differentiation, migration and apoptosis, consistent with the pleiotropic effects of vitamin D reported in the published literature. Finally, a novel human intervention study was undertaken to investigate the impact of vitamin D supplementation in human blood and rectal mucosa. Short-term high-dose supplementation of 50 participants significantly induced CDH1 expression in rectal mucosa, with significant gene interaction effects between 25OHD, rs992818 genotype and CDH1, VDR and FOXO expression, thus independently replicating the same gene interaction effects seen in the human observational study. Meanwhile, transcriptome profiling identified numerous pathways relevant to tumourigenesis significantly enriched after supplementation, validating several pathways also associated with vitamin D status in the observational study. In summary, the research presented in this thesis demonstrates that vitamin D treatment of cells, epithelial organoids and human subjects induces CDH1 expression, and that strong gene interaction effects involving the colorectal cancer risk locus rs9929218 modulate this effect. FOXO transcription factors influence the gene interaction effect, consistent with the proposed model of ligand dependent regulation of FOXO by VDR and transcription activation of the CDH1 gene by the FOXO complex dependent on rs9929218 genotype. These data provide support for rectal CDH1 expression as an intermediate biomarker for vitamin D chemopreventive studies and suggest that gene environment interactions underlie some of the missing heritability of CRC.

Introduction Vitamin D status is an important public health issue due to the high prevalence of vitamin D insufficiency and deficiency, especially in high latitude areas. Furthermore, it has been reported to be associated with a number of diseases. In a previous umbrella review of meta-analyses of randomized clinical trials (RCTs) and of observational studies, it was found that plasma/ serum 25-hydroxyvitamin D (25(OH)D) or supplemental vitamin D has been linked to more than 130 unique health outcomes. However, the majority of the studies yielded conflicting results and no association was convincing. Aim and Objectives The aim of my PhD was to comprehensively explore the association between vitamin D and multiple outcomes. The specific objectives were to: 1) update the umbrella review of meta-analysis of observational studies or randomized controlled trials on associations between vitamin D and health outcomes published between 2014 and 2018; 2) conduct a systematic literature review of previous Mendelian Randomization studies on causal associations between vitamin D and all outcomes; 3) conduct a systematic literature review of published phenome wide association studies, summarizing the methods, results and predictors; 4) create a polygenic risk score of vitamin D related genetic variants, weighted by their effect estimates from the most recent genome wide association study; 5) encode phenotype groups based on electronic medical records of participants; 6) study the associations between vitamin D related SNPs and the whole spectrum of health outcomes, defined by electronic medical records utilising the UK Biobank study; 7) explore the causal effect of 25- hydroxyvitamin D level on health outcomes by applying novel instrumental variable methods. Methods First I updated the vitamin D umbrella review published in 2015, by summarizing the evidence from meta-analyses of observational studies and meta-analyses of RCTs published between 2014 and 2018. I also performed a systematic literature review of all previous Mendelian Randomizations studies on the effect of vitamin D on all health outcomes, as well as a systematic review of all published PheWAS studies and the methodology they applied. Then I conducted original data analysis in a large prospective population-based cohort, the UK Biobank, which includes more than 500,000 participants. A 25(OH)D genetic risk score (weighted sum score of 6 serum 25(OH)D-related SNPs: rs3755967, rs12785878, rs10741657, rs17216707, rs10745742 and rs8018720, as identified by the largest genome wide association study of 25(OH)D levels) was constructed to be used as the instrumental variable. I used a phenotyping algorithm to code the electronic medical records (EMR) of UK Biobank participants into 1853 distinct disease categories and I then ran the PheWAS analysis to test the associations between the 25(OH)D genetic risk score and 950 disease outcome groups (i.e. outcomes with more than 200 cases). For phenotypes found to show a statistically significant association with 25(OH)D levels in the PheWAS or phenotypes which were found to be convincing or highly suggestive in previous studies, I developed an extended case definition by incorporating self-reported data collected by UK Biobank baseline questionnaire and interview. The possible causal effect of vitamin D on those outcomes was then explored by the MR two-stage method, inverse variance weighted MR and Egger's regression, followed by sensitivity analyses. Results In the updated systematic literature review of meta-analyses of observational studies or RCTs, only studies on new outcomes which had not been covered by the previous umbrella review were included. A total of 95 meta-analyses met the inclusion criteria. Among the included studies there were 66 meta-analyses of observational studies, and 29 meta-analyses of RCTs. Eighty-five new outcomes were explored by meta-analyses of observational studies, and 59 new outcomes were covered by meta-analyses of RCTs. In the systematic review of published Mendelian Randomization studies on vitamin D, a total of 29 studies were included. A causal role of 25(OH)D level was supported by MR analysis for the following outcomes: type 2 diabetes, total adiponectin, diastolic blood pressure, risk of hypertension, multiple sclerosis, Alzheimer's disease, all-cause mortality, cancer mortality, mortality excluding cancer and cardiovascular events, ovarian cancer, HDL-cholesterol, triglycerides and cognitive functions. For the systematic literature review of published PheWAS studies and their methodology, a total of 45 studies were included. The processes for implementing a PheWAS study include the following steps: sample selection, predictor selection, phenotyping, statistical analysis and result interpretation. One of the main challenges is the definitions of the phenotypes (i.e., the method of binning participants into different phenotype groups). In the phenotyping step, an ICD curated phenotyping was widely used by previous PheWAS, which I also used in my own analysis. By applying the ICD curated phenotyping, 1853 phenotype groups were defined in the participants I used. In PheWAS, only phenotype groups with more than 200 cases were analysed (920 phenotypes). In the PheWAS, only associations between rs17216707 (CYP24A1) and "calculus of ureter" (beta = -0.219, se = 0.045, P = 1.14*10-6), "urinary calculus" (beta = -0.129, se = 0.027, P = 1.31*10-6), "alveolar and parietoalveolar pneumonopathy" (beta = 0.418, se = 0.101, P = 3.53*10-5) survived Bonferroni correction. Nine outcomes, including systolic blood pressure, diastolic blood pressure, body mass index, risk of hypertension, type 2 diabetes, ischemic heart disease, depression, non-vertebral fracture and all-cause mortality were explored in MR analyses. The MR analysis had more than 80% power for detecting a true odds ratio of 1.2 or larger for binary outcomes. None of explored outcomes were statistically significant. Results from multiple MR methods and sensitivity analyses were consistent. Discussion Vitamin D and its association with multiple outcomes has been widely studied. More than 230 outcomes have been linked with vitamin D by meta-analyses of observational studies and RCTs. On the contrary, evidence from Mendelian Randomization studies is lacking. In particular I identified only 20 existing MR studies and only 13 outcomes were suggested to be causally related to vitamin D. In the systematic literature review of previous PheWAS studies, I summarized the applied methods, predictors and results. Although phenotyping based on ICD codes provided good performance and was widely applied by previous PheWAS studies, phenotyping can be improved if lab data, imaging data and medical notes can be incorporated. Alternative algorithms, which takes advantage of deep learning and thus enable high precision phenotyping, needs to be developed. From the PheWAS analysis, the score of vitamin D related genetic variants was not statistically significantly associated with any of the 920 phenotypes tested. In the single variant analysis, only rs17216707 (CYP24A1) was shown to be associated with calculus outcomes statistically significantly. Previous studies reported associations between vitamin D and hypercalcemia, hypercalciuria, nephrolithiasis and nephrocalcinosis, may be due to the role of vitamin D in calcium homeostasis. In the MR analysis, I found no evidence of large to moderate (OR > 1.2) causal associations of vitamin D on a very wide range of health outcomes. These included SBP, DBP, hypertension, T2D, IHD, BMI, depression, non-vertebral fracture and allcause mortality which have previously been proposed to be influenced by low vitamin D levels. Further, even larger studies, probably involving the joint analysis of data from several large biobanks with future IVs that explain a higher proportion of the trait variance, will be required to exclude smaller causal effects which could have public health importance because of the high population prevalence of low vitamin D levels in some populations.

In humans, the epidermis is the main site for the synthesis of Vitamin D3 (cholecalciferol) from 7-dehydrocholesterol. Cholecalciferol undergoes further hydroxylation in the liver and kidney to produce the active form of the circulating hormone 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). In target cells, 1,25(OH)2D3 interacts with the specific intracellular vitamin D receptor (VDR), a member of the nuclear receptor superfamily. However, epidermal keratinocytes, in addition to being target cells, have enzymes required for autocrine production of 1,25(OH)2D3. They can convert cholecalciferol to 1,25(OH)2D3 via 25-hydroxylase (CYP2R1) and 1α-hydroxylase (CYP27B1). Another enzyme, 24-hydroxylase (CYP24A1), regulates local levels by inactivating 1,25(OH)2D3. While recent studies have shown that absence of VDR or 1,25(OH)2D3 impairs formation of granulation tissue during wound healing in mice, little is known about the autocrine and paracrine regulation of biologically active vitamin D3 by human dermal fibroblasts during cutaneous wound healing. Primary cultures of human keratinocytes and fibroblasts expressed VDR and all the cytochrome enzymes necessary for autocrine production of vitamin D. The relative expression of VDR mRNA was higher in dermal fibroblasts than donor-matched keratinocytes. In contrast, epidermal keratinocytes had a higher mRNA expression of vitamin D3 metabolising enzymes. A scratch wound assay confirmed that 1,25(OH)2D3 stimulated keratinocyte migration, but paradoxically inhibited fibroblast migration as early as 4h, yet neither cholecalciferol nor 25-hydroxyvitamin D3 had any effect. VDR knockdown using small interfering RNA (siRNA) abolished the inhibitory effect of 1,25(OH)2D3 on fibroblast migration, demonstrating the requirement for the VDR in this response. Immunofluorescent staining revealed that 1,25(OH)2D3 increased nuclear VDR protein expression, without a corresponding increase in VDR mRNA transcription only in mechanically wounded dermal fibroblasts, indicating activation of the receptors. Incubation with either 1,25(OH)2D3, cholecalciferol or 25(OH)D3 up-regulated CYP24A1 transcription. This response was most pronounced with 1,25(OH)2D3, suggesting a tightly regulated feedback control on 1,25(OH)2D3 bioavailability within the dermis. In addition, cholecalciferol also increased CYP2R1 and CYP27B1 mRNA expression in scratched dermal fibroblasts, providing evidence for autocrine regulation of 1,25(OH)2D3 by dermal fibroblasts. Expression of α-SMA protein was up-regulated in cultured dermal fibroblasts following scratching, which was down-regulated in the presence of 1,25(OH)2D3. These observations suggest that 1,25(OH)2D3 may restrict differentiation of wounded dermal fibroblasts into pro-fibrotic myofibroblasts. 1,25(OH)2D3 also down-regulated MMP-2 secretion and collagen type I to III ratio in scratched dermal fibroblasts. Using a human ex vivo wound healing model, it was demonstrated that 1,25(OH)2D3, but not cholecalciferol, stimulated the rate of wound closure. In summary, this study has confirmed that human dermal fibroblasts express the transcriptional machinery for autocrine production of 1,25(OH)2D3, and a higher VDR expression suggests they are more responsive than keratinocytes. Changes in CYP and VDR expression in the presence of cholecalciferol, 25-hydroxyvitamin D3 or 1,25(OH)2D3 indicate fine-tuning of the bioavailability of vitamin D in the dermis after wounding. Down-regulation of α-SMA, MMP-2 secretion and the collagen type I to III ratio by 1,25(OH)2D3 highlight an important role for 1,25(OH)2D3 in modulating wound healing and the scarring process.

Given the strong evidence for a beneficial role of vitamin D in diabetes and CVD pathogenesis, and the prevalence of vitamin D deficiency, vitamin D supplementation has been advocated for the prevention of cardiometabolic disease. To provide information on the effects of 5,000IU (125µg) vitamin D3 on cardiometabolic risk, a double blind, RCT in a cohort of overweight and obese UK adult males with plasma 25(OH)D concentration < 75nmol/L for a duration of 8 weeks was conducted. To the best of my knowledge, this is the first RCT to investigate the effect of 5,000IU (125µg) vitamin D3 on cardiometabolic markers in vitamin D insufficient, non-hypertensive and non-diabetic overweight and obese adult males.

There is a paucity of longitudinal data investigating the role of vitamin D in the maintenance of health in ethnic minority groups. Specifically, little is known about the role of vitamin D on muscle strength, how this affected by emigrating from low to high latitudes. This PhD thesis provides insight into the relationship between vitamin D status and muscle strength in healthy adults migrating from sunny climates (45°N to 45°S to the equator) to higher latitude (Aberdeen, 57°N) (first study) and also sunlight behaviours in both low and high latitudes (second study). A total of 71 people of ethnic minority aged 19-42 years took part in the first study with 56% were seen within 3 months of arriving in Aberdeen (newcomers) and the remainder having been resident for any longer period than this (residents). Participants attended visits every 3 months for 15 months. At each visit, fasted blood samples were collected for analysis of serum 25-hydroxyvitamin D [25(OH)D] by dual tandem-mass spectrometry, parathyroid hormone (PTH), carboxy-terminal collagen crosslinks (CTX) and N-terminal propeptide of type I collagen (P1NP) by electrochemiluminescent immunoassay. Maximal voluntary contraction (MVC) was measured using a Takei digital grip dynamometer (both arms) and a Biodex dynamometer (right knee) for isometric knee extension and isokinetic knee extension and flexion. Mean baseline 25(OH)D concentrations were higher in the newcomers (35.2 nmol/L) than the residents (25.0 nmol/L, p<0.01). After adjusting for the season of measurement at baseline, there was a trend for higher vitamin D status only between newcomers arriving in spring (33.5 nmol/L) and residents (26.8 nmol/L, p=0.09), but not between the newcomers arriving in autumn (36.1 nmol/L) and residents in autumn (35.2 nmol/L, p=0.81). Notably, few newcomers (13%) and residents (7%) had 25OHD concentrations >50 nmol/L. There was a small seasonal variation in 25(OH)D concentration with the difference between zenith (summer/autumn) and nadir (winter/spring) was 5 nmol/L, 6 nmol/L, and 12 nmol/L among the newcomers arriving in spring, autumn and residents, respectively. There was no seasonal variation in bone turnover markers. There was a positive, albeit small, association between vitamin D and knee extensor isometric torque. Mixed modelling predicted that each 1 nmol/L increase in 25(OH)D would increase peak torque by 1 Nm (p=0.04). If extrapolated to higher 25(OH)D, the change in muscle strength would be clinically meaningful. In the second study, 117 Asians in Kuala Lumpur (3°N) and 110 non-Europeans (78% Asians) in Aberdeen were recruited and monitored for one year [every 3 months for participants in Aberdeen and during Southwest monsoon (SWM; July-Sept) and Northeast monsoon (NEM; Nov-Feb) in Kuala Lumpur]. Personal sunlight exposures were assessed by polysulphone film badges and a web-based questionnaire. Asians living in their usual environment (Kuala Lumpur) had similar sunlight behaviours to Asians living in Aberdeen. Sunlight was the major source of vitamin D in Kuala Lumpur during both seasons (median % cutaneous in SWM = 61%; NEM = 59%), but only during summer in Aberdeen (52%). Dietary intakes provided the most source of vitamin D in Aberdeen during autumn (94%), winter (97%) and spring (56%). This thesis has added evidence to the current limited observations on vitamin D and muscle strength, particularly in the cohort of young adults migrating from low to high latitudes. Intervention studies are yet to be conducted with a speculation that an increase in vitamin D status may show a positive impact on muscle strength. The extent of low sunlight behaviour among healthy adults in Kuala Lumpur is of concern.

Firstly, the aims of the study were to investigate the influences of various factors, sex differences, Cytomegalovirus/Epstein-Barr virus (CMV/EBV) serostatus and vitamin D concentrations on respiratory illness incidence and immune function during the winter months in a student cohort of endurance athletes. In Chapter 3, the findings of the study concur with recent reports of illness incidence at major competitive games which indicate that female athletes may be more susceptible than their male counterparts to upper respiratory tract illness (URTI) symptoms and that lower oral-respiratory mucosal immunity may, in part, account for this. It was also found that previous coinfection with CMV and EBV might promote protective immune surveillance to lower the risk of URTI. In addition, it can be concluded that athletes with low plasma vitamin D concentrations may have a higher risk of URTI and suffer more severe symptoms when URTI is present. This may be due to impaired mucosal and systemic immunity as secretory immunoglobulin A (SIgA) secretion, cathelicidin levels and antigen-stimulated pro-inflammatory cytokine production appear to be increased by vitamin D-dependent mechanisms. A series of follow-up studies were also conducted to examine the effect of vitamin D on mucosal and systemic immunity in athletes. In Chapter 4, it was reported that the influence of vitamin D on circulating cytokines might be different in athletes compared with non-athletes and that both pro-inflammatory and anti-inflammatory cytokine production by multi-antigen stimulated whole blood culture were not influenced by 1,25-dihydroxy vitamin D (1, 25(OH)2D) iconcentrations within the normal healthy range. In Chapter 5, it was found that 5000 IU of vitamin D3 supplementation daily appears to have a beneficial effect in up-regulating the expression of SIgA and cathelicidin in athletes during a winter training period. Nevertheless, the findings reported in Chapter 6 showed that there were no significant effects of vitamin D status and a 4-week period of daily high does vitamin D3 supplementation on salivary antimicrobial protein (AMP) responses to prolonged exercise. In conclusion, a series of studies in this thesis have demonstrated the influence of various factors (sex differences, CMV/EBV serostatus and vitamin D concentrations) on susceptibility to URTI among athletes. Moreover, it was suggested that vitamin D3 supplementation could have a positive effect on immune function and lead to decreased incidence of respiratory infections.

Skeletal muscle is a direct target for the group of seco-steroids collectively termed Vitamin D. Skeletal muscle expresses both CYP27A1 and CYP27B1 encoding for the hydroxylases required to convert Vitamin D to 25[OH]D and subsequently the bioactive 1α-25-dihydroxyvitamin D3 (1α-25[OH]2D3) (Girgis et al., 2014b). Crucially, the Vitamin D receptor (VDR) is also present in skeletal muscle (Srikuea, Zhang, Park-Sarge, & Esser, 2012) and upon exposure, binds to its ligand 1α-25[OH]2D3 and initiates genomic and non-genomic rapid signalling responses. At present there is a global prevalence of low serum Vitamin D (25[OH]D) concentrations due to a lack of sun exposure (the primary route for Vitamin D synthesis) as a function of latitude and/or an indoor lifestyle coupled with few dietary sources of Vitamin D (Chen et al., 2007). Accumulating data are now suggestive that low 25[OH]D may be associated with perturbations in contractile activity and the regeneration of human skeletal muscle (Owens, Fraser, & Close, 2014), although a definitive causal relationship is yet to be established. Therefore, this thesis aimed to establish a more precise role for Vitamin D in human skeletal muscle function and regeneration. There were four overarching aims: 1. Explore the role of Vitamin D in human skeletal muscle contractile properties in humans in vivo. 2. Identify the role of Vitamin D in human skeletal muscle contractile properties ex vivo. 3. Investigate the role of Vitamin D in skeletal muscle regeneration following eccentric exercise induced muscle damage in vivo. 4. Elucidate cellular mechanisms of the muscle regeneration process that are responsive to Vitamin D in vitro. The main findings from this work imply that serum 25[OH]D concentrations across a broad range from 18 → 100 nmol.L-1 do not affect skeletal muscle contractile properties. Conversely elevating serum 25[OH]D from < 50 nmol.L-1 to > 75 nmol.L-1 resulted in significant improvements in the recovery of maximal voluntary contraction force following a bout of eccentric exercise. Implementing an in vitro model of muscle regeneration also identified potential cellular mechanisms for these observations: Muscle derived cells treated with a total amount of 10 nmol 1α-25[OH]2D3 following a mechanical scrape improved migration dynamics and fusion capability of skeletal muscle derived cells. Taken together, low Vitamin D concentrations are highly prevalent but can be easily corrected with supplementation of Vitamin D3. This may have the advantage of optimising the regenerative capacity of skeletal muscle amongst other health benefits previously characterised by others.

Introduction: Patients with Systemic Lupus Erythematosus (SLE) have increased cardiovascular risk, endothelial dysfunction, and abnormal endothelial repair mechanisms. Vitamin D deficiency is common in SLE and has been associated with active disease and increased vascular stiffness. Myeloid angiogenic cells (MACs) repair damaged vessels by secretion of angiogenic factors and may be a target for vitamin D. Vitamin D may therefore be a novel therapy to improve cardiovascular risk in SLE patients. This study aimed to determine the effects of vitamin D on endothelial function and repair in patients with SLE. Methods: The effects of the active form of vitamin D (1,25(OH)2D3) were studied on MACs from vitamin D deficient SLE patients ex vivo. Functional models were developed to study MAC migration, adhesion and interaction with endothelial cells. Additional experiments used a model of healthy MACs treated with IFN-alpha. An observational study of clinically stable vitamin D deficient SLE patients being treated with high dose vitamin D over 3 months was used to investigate the effects of vitamin D on endothelial function as measured by flow-mediated dilatation (FMD). Results: MACs expressed markers consistent with an M2 macrophage phenotype and they enhanced endothelial network formation in vitro. SLE patients had an increased number of MACs; however, these were dysfunctional compared to healthy controls. Vitamin D increased the number, changed the phenotype and improved the functional capacity of SLE MACs ex vivo. In addition, the angiogenic capacity of SLE MACs was restored toward that of healthy controls via a reduction in the anti-angiogenic cytokine IP10. Vitamin D-treated MACs were also more able to protect endothelial cells against TNF-mediated down-regulation of endothelial nitric oxide synthase (eNOS). In SLE patients treated with vitamin D, there was a strong correlation between the change in serum 25(OH)D and the change in the ratio of endothelium-dependent:independent dilatation (r=-0.650, p=0.006). This was accompanied by an increase in the number of MACs at 3 months (p=0.015). These observations were independent of changes in serum PTH, calcium or lupus disease activity. Conclusions: Vitamin D can target MACs and therefore offers a novel approach to improve endothelial repair in patients with SLE. In addition, vitamin D treatment in lupus patients resulted in an improvement in endothelial function, related to the change in vitamin D status. These results suggest that vitamin D could improve surrogate markers of cardiovascular disease and thus reduce cardiovascular risk in this patient group.

Multiple sclerosis is a complex immune mediated condition of the central nervous system characterized by myelin loss and progressive neurodegeneration. The risk of developing MS is influenced by both genetic and environmental agents and, among them, several lines of evidence support a role for vitamin D deficiency, Epstein-Barr virus (EBV) infection and smoking in the aetiology of this disease. The aim of this work was to further elucidate how nature and nurture act in the causal cascade leading to MS. In chapter 1, I show that the main genetic factor in adult MS (the HLA-DRB1*1501 allele) plays an equally important role in paediatric cases of MS (PMS) and that EBV negative PMS patients represent a separate entity characterized by lower age at disease onset, lower female to male ratio and a trend towards a lower frequency of the HLA-DRB1*1501 allele. In chapter 2, I provide evidence in support of month of birth having a role on MS risk and T cell production and that vitamin D may underlie this effect. In chapter 3 I demonstrate the presence of a link between vitamin D deficiency and the immune response against EBV, whereby the proportion of EBV seropositive MS patients and controls increases with increasing latitude and high dose vitamin D supplementation appears to reduce the level of antibodies against this virus. In chapter 4, I show that MS associated genetic variants are located in genomic regions that exert a regulatory function and are active in immune cell types. In chapter 5, I illustrate how vitamin D receptor binding is also located within active regulatory regions in immune cells and that this is particularly evident near MS associated genes. Finally, in chapter 6, I use chromatin data on more than 100 different cell types and conclude that MS associated genetic variants are particularly active in T helper, T cytotoxic and B cells. Further work is needed to elucidate how genetic and environmental agents play a role in the cause of MS and to develop effective strategies for disease treatment and prevention.

Introduction: Vitamin D is a micronutrient that possesses immunomodulatory actions. Higher vitamin D status has been associated with decreased incidence of acute respiratory infections (ARI) in a number of observational studies. However, mechanistic in vitro work investigating effects of vitamin D on the immune response to ARIs is lacking, especially for rhinovirus, which is the most common respiratory pathogen. Results of clinical trials of vitamin D supplementation in the prevention of ARIs have also been conflicting, in that some demonstrate a protective effect of this intervention against ARI, while others do not. Methods: An immunological assay of ex vivo stimulation with TLR ligands and pathogens in blood samples from participants with asthma, COPD or neither condition in three randomised controlled trials of vitamin D supplementation for the prevention of ARI and exacerbations was developed. This assay was used in conjunction with cellular profiling of clinical trial blood and sputum samples, and a rhinovirus-infected human alveolar cell line (A549 cells) to determine the effects of vitamin D in the protection against acute respiratory infections. Results: The main finding of cell culture experiments was that A549 cells pre-treated with physiological concentrations of 25-hydroxyvitamin D (25[OH]D, the major circulating vitamin D metabolite) had increased resistance to rhinovirus infection, which was associated with attenuation of rhinovirus-induced intercellular adhesion molecule-1 (ICAM-1) and platelet-activating factor receptor (PafR) expression. Immunological analysis of clinical trial samples did not demonstrate any consistent effect of bolus-dose vitamin D supplementation on circulating or pathogen-stimulated inflammatory profiles, or on inflammatory indices in induced sputum. Conclusions: Co-incubation with 25(OH)D was associated with transient protection against rhinovirus infection in a respiratory epithelial cell line in vitro, but these findings did not translate to any changes in cellular profile or inflammatory mediator release in clinical trials samples following in vivo vitamin D supplementation.

The accurate assessment of human diabetic somatic polyneuropathy (DSPN) is important to define at risk patients, predict deterioration, and assess the efficacy of pathogenetic treatments. Corneal confocal microscopy (CCM) has been proposed as a surrogate endpoint for DSPN. Approximately 50% of patients with DSPN experience neuropathic pain or symptoms and the underlying reasons are not clearly elucidated. Vitamin D deficiency has been associated with diabetic complications including DSPN and diabetic retinopathy (DR). However there is a paucity of data regarding the interaction of vitamin D status with diabetic complications. This thesis shows that CCM can readily detect small fibre neuropathy prior to large fibre involvement and assess rapidly progressive nerve fibre loss prior to conventional thermal threshold testing. CCM has a superior diagnostic capabilities compared to intra-epidermal nerve fibres and correlates better with nerve conduction studies. Patients with LADA have a greater prevalence of small fibre neuropathy compared to matched patients with type 2 diabetes. Vitamin D deficiency is highly prevalent in patients with diabetes and despite relatively aggressive replacement regimens are inadequate in raising vitamin D levels in a significant proportion of patients. Vitamin D deficiency is not associated with DR but there is a strong association between painful DSPN and vitamin D insufficiency and more so with overt deficiency.

Genome-wide association studies in multifactorial diseases have contributed to our understanding of genetic risk and defined specific disease-associated loci in particular populations. However, risk cannot be fully explained by genetics and evidence points to both genetic and environmental factors being important in causation and pathophysiology. The role of vitamin D in calcium homeostasis is well established. Over the last 30 years it has become clear that vitamin D has a wider role in physiology and disease, notably in autoimmune, cancer and infectious conditions. However, the molecular mechanisms and possible causal role of these associations are poorly understood. Here I propose that the role of vitamin D in immune and inflammatory responses is significant, that genetic variation partly determines the response to vitamin D and that integrative analysis can yield important insights for disease mechanisms. For this I investigate the relationship between vitamin D and genetic risk involving the immune system by focusing on multiple sclerosis and sepsis, conditions classically defined as autoimmune and inflammatory respectively. I describe data resolving genetic variation associated with autoimmune diseases in vitamin D receptor binding sites; the association to multiple sclerosis of a genetic variant lying within a VDR binding site; the correlation of plasma vitamin D with genotype and cell specific transcriptomes in healthy volunteers; and the extent of vitamin D deficiency in severe sepsis and septic shock, its association with survival, correlation with gene expression and use in sub-classification to identify patients at higher risk of death. The limitations of each study and future work are discussed. Integrating epidemiological and clinical observations with genetic and functional genomics techniques has the potential to reveal interactions in population specific disease susceptibility that may lead to an improved understanding of disease mechanisms and clinical translation. The work I present here bridges molecular analysis, candidate and genome- wide, with phenotypic observations that are important in our understanding of disease.

Cardiovascular disease varies by ethnicity in the UK. South Asians (SA) have higher coronary heart disease (CHD) and diabetes prevalence, while African-Caribbeans (AfC) have greater stroke, but intriguingly lower CHD rates despite higher blood pressures and diabetes risk than Europeans. Conventional risk factors do not fully explain such differences. This cross-sectional study tested the hypothesis that the hormones, vitamin D measured as 25(OH)D and aldosterone, would be independently associated with intermediate cardiovascular outcome markers in these ethnic groups. Community-dwelling men 40-80 years old (AfC: n=67, 55±10yr; SA: n=68, 55±10yr; European: n=63, 57±8yr) were sampled from Greater Manchester’s multi-ethnic population. The intermediate markers examined were aortic pulse wave velocity (aPWV), left ventricular (LV) mass and function, and carotid intima media thickness (CIMT), measured non-invasively by ultrasound, and hemodynamic profiling methods (the Arteriograph) in the total sample and by magnetic resonance imaging (MRI) in a subsample of 50. Adjusted for age, systolic blood pressure and diabetes, mean(SE) aPWV by the Arteriograph, was 0.5(0.2) m/s higher in SA than AfC and Europeans (p=0.01), which paralleled known cross-ethnic CHD risk differences in the UK. By MRI, aPWV along the descending aorta in SA was 0.7(0.3) and 0.8(0.3) m/s higher than that in AfC and Europeans, but aPWV along the aortic arch was not significantly different. Unlike aldosterone, 25(OH)D was independently and inversely correlated with aPWV (unstandardised B(SE)=-0.013[0.004] m/s, p<0.001), and partly explained the ethnic variation in aPWV. Similar inverse correlations were found between 25(OH)D and LV concentricity measured by echocardiography and MRI. Compared to Europeans, SA and AfC, had 21(3) and 14(3) nmol/L lower mean(SE) 25(OH)D, respectively (p<0.01). Mean(SE) of relative wall thickness, an index of LV concentricity by echocardiography, was 0.05(0.01) higher in SA and AfC than Europeans. Lower 25(OH)D levels were also associated with higher myocardial deformation rates measured by MRI myocardial tagging (n=50), supporting previous animal experimental evidence. A one standard deviation (SD) decrease in 25(OH)D was associated with a 0.38 SD increase in absolute systolic strain rate (p=0.003) and 0.22 SD rise in diastolic strain rate (p=0.04). Right and left CIMT showed different relations with 25(OH)D and aldosterone. Left-right CIMT differences varied by ethnicity and were related to SA ethnicity and aldosterone levels. Two related technical studies investigated the relatively new method of hemodynamic profiling, the Arteriograph, used here. The results suggested a standardisation method of aortic length estimation for purely central aPWV, which significantly improved aPWV agreement between the Arteriograph and MRI (reference method here), and was used for calibrating the Arteriograph aPWV in the above-mentioned results for the total sample. Future well-designed trials are necessary to investigate any cause-effect relationship between vitamin D deficiency and the unfavourable cardiovascular intermediate outcomes found here in a cross-sectional design and multi-ethnic background.

Background: Previous research suggests that vitamin D deficiency during pregnancy may be associated with suboptimal fetal growth, but direct evidence is lacking. Our objectives were 1) to develop a method for measurement of the fetal sphenoidal fontanelle area (FA) and femur volume (FV) using 3D ultrasound; 2) to create normal charts for FA and FV; and 3) to correlate FA and FV with maternal vitamin D concentration. Methods: FA measurement in 3D was evaluated in vitro and in vivo. Different segmentation methods for FV measurement were explored. A novel FV method was described which consists of three linear measurements and a volume equation; this was validated in vitro and also by comparing FV measured sonographically to the true volume assessed by computed tomography (CT), in 6 cases following pregnancy termination. A cohort of 868 uncomplicated pregnancies was selected on the basis of strict inclusion criteria; participants underwent serial ultrasound scans for FV and multilevel modeling was used for the creation of a “prescriptive” FV chart. Finally, a different cohort of 357 healthy pregnant women had serum vitamin D levels and FV ultrasound at 34 weeks gestation and dual emission x-ray absorptiometry (DEXA) of their neonates in order to investigate the prenatal determinants of fetal bone mass. Results: FA measurement was accurate in vitro, but unreliable in vivo and was therefore abandoned. A novel FV method had excellent agreement with CT and superior repeatability compared with segmentation-based methods. A normal FV chart was created and the regression equations for the median and percentile values were presented. Vitamin D demonstrated a significant correlation with FV. Conclusions: FV is a reliable sonographic marker of skeletal growth. Maternal vitamin D deficiency is associated with reduced FV. This finding has public health implications as reduced bone mass may increase the lifetime risk of osteoporosis, through fetal programming.