<p>Upper respiratory tract infection (URTI), which presents clinically as the common cold, is the most common form of respiratory illness experienced by humans and is a major cause of morbidity and hospitalizations. Currently, URTI prevention focuses on hand hygiene with some consideration given to other lifestyle factors such as sleep, nutrition, and exercise. Identifying additional interventions for the prevention of URTI could reduce the burden of this disease.</p> <p>In this thesis, I examine the role of vitamin D3 supplementation and tap water gargling for the prevention of URTI. I employ experimental and observational study designs to assess the effect of these interventions on the risk of URTI in the context of a randomized controlled trial of 600 participants, and a longitudinal cohort of 416 participants. Further, I investigate the association between modifiable lifestyle factors and risk of URTI using data from the longitudinal cohort. Data from this study is also used to explore statistical methods for the analysis of repeated events.</p> <p>When evaluating self-reported, clinical URTI, all analyses supported the use of vitamin D3 supplementation to reduce the risk of URTI. However, this finding was only statistically significant in the analysis of the longitudinal cohort study; results from the RCT indicated that vitamin D3 supplementation statistically significantly reduced the risk of laboratory confirmed infections but had a non-significant benefit for clinical infections. Gargling did not reduce the risk of clinical or laboratory confirmed infections.</p> / Doctor of Philosophy (PhD)

<p><strong>Background:</strong> Despite strong evidence, strategies for improving bone health are underutilized. Knowledge translation (KT) interventions aim to improve uptake of evidence-based practices, however the feasibility and effectiveness of such strategies require further evaluation within Long-term Care (LTC). In this thesis, we examined the impacts of a province-wide osteoporosis strategy and a more intensive multifaceted KT strategy including expert-led educational meetings, audit/feed-back, and action planning for quality improvement. Both studies targeted interdisciplinary LTC teams (physicians, nurses, pharmacists, dietician, and other staff).</p> <p><strong>Methods: </strong>In the first thesis study, we examined the impact of the <em>Ontario Osteoporosis Strategy for LTC</em> by investigating changes in facility-level prescribing rates (vitamin D, calcium, osteoporosis medications) before and after its implementation (2007 versus 2012). The second study was a pilot cluster randomized trial evaluating the feasibility and effectiveness of a 12-month, multifaceted, interdisciplinary KT intervention [Vitamin D and Osteoporosis Study (ViDOS)]. Prescribing outcomes included: vitamin D ≥800 IU (primary), calcium ≥500 mg/day, and osteoporosis medications (high-risk residents only). Feasibility outcomes included recruitment, retention, data collection, intervention fidelity, and process changes. We analyzed resident level data using the generalized estimating equations (GEE) technique, adjusting for clustering.</p> <p><strong>Results:</strong></p> <p>In both studies, significant improvements were observed for vitamin D and calcium prescribing. In the first study, prescribing increased by 38% and 4%, respectively, between 2007 and 2012. In the ViDOS trial, the 12-month intervention resulted in an absolute improvement of 15% and 7%, respectively (intention to treat cohort). There was no significant effect for prescribing of osteoporosis medications in either study. In the ViDOS study, recruitment and retention rates were 22% and 63%, respectively; good intervention fidelity was achieved and intervention homes reported several process changes.</p> <p><strong>Conclusion:</strong></p> <p>This thesis study demonstrated that KT interventions targeting evidence-based osteoporosis and fracture prevention strategies were feasibly and effectively applied with interdisciplinary LTC teams.</p> / Doctor of Philosophy (PhD)

<p>Leptin, an adipose derived hormone, has emerged as a regulator of bone metabolism. Recent findings support a role of leptin in the process of fetal bone remodeling during pregnancy; however, the link between maternal leptin during pregnancy and offspring bone status is undocumented. Evidence exists that the intrauterine environment plays a role in programming peak bone mass that is achieved in late adolescence and thus osteoporosis risk later in life. We investigated the association between maternal leptin during the third trimester and offspring bone mass at 3 years of age.</p> <p><strong>Method:</strong> Based on a sub-sample of a prospective birth cohort study, we conducted analysis on 425 mothers from whom maternal blood samples in pregnancy were analyzed for leptin and 25- hydroxyvitamin D, and whole body bone mass by dual energy x-ray absorptiometry were available for both mother and child at 3 years. Data were collected for maternal prepregnancy body mass index (BMI), lifestyle, and nutrition during pregnancy, as well as the child’s nutrition and physical activity at 3 years.</p> <p><strong>Results:</strong> Women obese on entering pregnancy have a two-fold greater circulating leptin during pregnancy than women with normal weight BMIs. Maternal age and skinfold thickness were positively associated with maternal leptin status. However, maternal leptin status was not a significant predictor of offspring BMC z-score at 3 years of age, when adjusted for relevant maternal and child variables. Maternal vitamin D status was also not a predictor of offspring bone status. Rather the key predictors of child BMC z-score were maternal bone mineral density z-score and child’s weight and vitamin D intake at 3 years.</p> <p><strong>Conclusion:</strong> While maternal leptin status during pregnancy is highly variable among women of different BMI categories, in utero exposure to leptin is not a significant factor that influences child bone status at 3 years of age when adjusted for other relevant variables.</p> / Master of Science (MSc)

Resistance exercise training (RET) and protein supplementation are potent nonpharmacological countermeasures against sarcopenic muscle and strength loss, however other exercise modalities and isolated nutritional supplements are effective in combating additional deleterious age-related changes, such as reduced cardiometabolic health. Accordingly, in Study 1 we assessed the 48-hour integrated muscle protein synthesis (MPS) response to a single session of RE, aerobic exercise, or high-intensity interval exercise (HIIE) in a group of healthy older men using the novel heavy water method. The results of Study 1 indicated that both RE and HIIE were capable of significantly elevating myofibrillar MPS above resting rates, with the most substantial effect observed following RE. In Studies 2 and 3 we evaluated whether daily consumption of a nutritional supplement which comprised whey protein, creatine, vitamin D/calcium, and omega-3 polyunsaturated fatty acids could: augment strength, physical function, and lean tissue mass (Study 2), and also improve glycemic control, lipidemia, and systemic inflammation (Study 3) in healthy older men following 6 weeks of supplementation in the absence of exercise; and enhance exercise training-induced improvements in the same outcomes following a 12-week RET + HIIT program. Six weeks of multi-ingredient nutritional supplementation stimulated gains in strength (~6%) and lean mass (~1%), roughly equivalent to one year's worth of age-related decline, as well as reduced circulating concentrations of lipids and inflammatory markers. Twelve weeks of combined RET + HIIT simultaneously improved strength, aerobic fitness, and glucose handling in the same group of older men. Further improvements in systemic inflammation and glucose handling were observed when multi-ingredient nutritional supplementation was combined with exercise training. Collectively, these studies demonstrate that multiple exercise modalities and nutritional supplements can be employed concurrently to alleviate various aspects age-related physiological decline. / Thesis / Doctor of Philosophy (PhD) / Aging is associated with a variety of deleterious physiological changes including loss of skeletal muscle mass and strength, reduced aerobic fitness, dyslipidemia, impaired glycemic control, and increased systemic inflammation. Broadly, this thesis explores how multiple exercise modalities and nutritional supplements can be used in combination to simultaneously alleviate several of these negative aspects of aging. This series of studies demonstrates that, in older men, consumption of a multi-ingredient nutritional supplement containing whey protein, creatine, vitamin D, calcium, and fish oil: A) stimulates significant improvements in lean mass, strength, plasma lipids, and systemic inflammation over a relatively short period of time (6 weeks) in the absence of exercise training; and B) enhances exercise training-induced gains in strength and glycemic control, as well as reductions in systemic inflammation. The findings of this thesis challenge the relatively common practice of targeting individual facets of aging with singular exercise or nutrition interventions.

Exploration of the internal structures of teeth is complex and has the potential to add greatly to existing information about the lifecourse of archaeological individuals, but has yet to realize its full interpretative value as an avenue of bioarchaeological inquiry. This thesis consists of three papers that focus on the potential for internal dental structures to provide important information on chronological age, and physiological alterations linked to vitamin D deficiency. The first paper used SEM, microscopic imaging, and histological investigation of tooth dentin to determine the presence of mineralisation defects, observed as interglobular dentin (IGD) (spaces following incremental lines) in living (with known medical history) and archaeological individuals with clear healed rickets. This paper demonstrated that incremental bands of IGD are indicative of vitamin D deficiency. The second paper expands identification of those with deficiency by quantifying morphological changes in pulp chambers of living and archaeological individuals. Pulp chambers were radiographed, evaluated histologically, and measured. Those with evidence of past vitamin D deficiency displayed constricted or chair shaped pulp horns. This radiographic technique provides a non-destructive tool to identify individuals that experienced childhood vitamin D deficiency. The role vitamin D plays in the development of IGD over the lifecourse requires that accurate age estimates be conducted on older as well as younger adults. The third paper used a new version of pulp/tooth area ratios to provide an accurate estimation of age-at-death in older adults (50+). ImageJ software was used to calculate areas on sectioned teeth and results provided a mean absolute error (MAE) of ±3.9 years in older adults. The results described in this thesis contribute to broader topics of discussion in anthropology, such as investigating health and metabolic disease in human populations, and adds to the ongoing discussion and evaluation of age-at-death techniques used to extend our ability to study the lifecourse of archaeological individuals. / Dissertation / Doctor of Philosophy (PhD) / Teeth record life events and the three papers in this thesis use dental structures to provide methodological foundations to evaluate the occurrence and severity of vitamin D deficiency in early life. The potential long-term consequences of such events are investigated through accurate recognition of older adults. Vitamin D regulates skeletal health by mediating calcium absorption and phosphorous homeostasis and deficiency is recognised as an important health concern. Accurate identification of older adults is also a widely recognised problem in skeletal studies. Age-at-death estimation in older individuals was calculated and the exploration of abnormal pulp chamber shape and mineralisation defects in tooth dentin was done to determine vitamin D status in both younger and older individuals. This research established that internal dental structures enables past episodes of vitamin D deficiency to be recognized in cases where skeletal indicators are not clear and permits increased precision in age-at-death estimations in the older individual.

This research contributes to understandings of the occurrence of and associations between skeletal evidence of vitamin D deficiency and chronic respiratory infections across the life course based on human skeletal material from the Roman period sites of Isola Sacra in Italy (1st - 3rd centuries AD) and Ancaster in the United Kingdom (3rd - 4th centuries AD). Modern clinical data demonstrate a positive association between these two conditions that affects the ways in which they are experienced today, and may extend into the past. Macroscopic, radiographic, and histological evidence for skeletal manifestations of vitamin D deficiency and chronic respiratory infections were considered in the context of archaeological and historical evidence available for the Roman period in order to elucidate patterns in disease occurrence that reflect the unique local biologies of these two assemblages. Differing prevalence values for active and healed lesions caused by both conditions, as well as variation in age at death distributions and the relationship of lesions associated with vitamin D deficiency and chronic respiratory infections with one another and with age at death, provide information on the experience of both conditions and the potential interactions between them. Skeletal lesions caused by both conditions are present in individuals throughout the life course at Ancaster and Isola Sacra, with particular implications for disease experiences during infancy, adolescence, and pregnancy in the Roman period. These results point to a picture of morbidity and mortality at Ancaster that involves longer term survival of and more efficient immune responses to chronic disease processes, with higher levels of skeletal lesions indicating the presence of more "survivors" at this site. The combination of lower frequencies of skeletal lesions and higher mortality at Isola Sacra, on the other hand, suggests that fewer individuals may have survived to the point where they were able to mount a skeletal response to disease. / Dissertation / Doctor of Philosophy (PhD)

High sugar-sweetened beverage (SSB) intake and poor vitamin D status have both been associated with increased risk of elevated systolic blood pressure (SBP) in previous research. However, these associations have never been investigated in the same study population, leaving the question of a possible interaction uninvestigated. One potential mechanism for an interaction is that SSB intake may increase serum uric acid (UA) and UA may interfere with utilization of vitamin D. This study examined these relationships in a sample of men and women (n=2,875) aged 20-74 using data collected in the 2003-2006 NHANES survey. No statistically significant association was found between SSB intake and risk of elevated SBP (defined as SBP>120mmHg) in whole group analysis. In subgroup analysis by gender, women (n=1,550) showed a 68% (OR: 1.68, 95% CI: 1.12-2.50, p-value 0.011) increased risk of elevated SBP in the highest SSB intake quartile (mean intake of 3.27 servings/day) compared to the lowest (mean intake of 0.03 servings/day) after adjustment for age, race, BMI, alcohol use, physical activity, and smoking, but no association was found in men (n=1,325). A statistically significant association was found between 25(OH)D and SBP, with a 30% decrease in risk of elevated SBP (OR: 0.70, 95% CI: 0.55-0.90, p-value 0.005) for those in the highest serum 25(OH)D group (>75nmol/L) compared to the lowest (<50nmol/L) in the fully adjusted model. However, no association was found between SSB intake and serum UA. Assessing potential effect modification between SSB and vitamin D in their impact on blood pressure using a multiplicative term and stratified analysis did not provided evidence of an interaction effect.

This systematic review's goal is to evaluate the efficacy of vitamin D supplementation in helping to manage the nutritional needs of patients diagnosed with prostate cancer. A systematic literature search following the PRISMA guidelines using Scopus, PubMed, and Cochrane databases was conducted to review randomized controlled trials and interventional studies up to 2023. The search strategy targeted randomized controlled trials and intervention studies. The selection process involved screening for study characteristics (study design), participant demographics (prostate cancer patients receiving treatment), intervention details (vitamin D assessment methods, dosages), outcome measures (progression, prognosis, quality of life), and risk estimates (hazard ratios, odds ratios, relative risks) along with covariates adjusted for in the analysis. Data analysis and synthesis included studies assessing vitamin D supplementation's impact on prostate-specific antigen (PSA) levels, tumor progression, osteomalacia, overall survival rates, and quality of life assessments. The literature search yielded a total of 3575 documents. After a preliminary screening of titles and abstracts, 34 full-text studies were examined. In total, nine studies were determined to meet the inclusion criteria. The findings of nine studies suggest a modest but significant association between vitamin D supplementation, reduced PSA levels, slower progression of localized prostate cancer, and improved bone loss. Due to the various treatment options, the overall effects of supplementation on advanced prostate cancer and overall survival were inconclusive. However, this research highlights the potential role of vitamin D in prostate cancer management.

A vitamina D (VD) pode estar envolvida no controle da proliferação, diferenciação e apoptose em linhagens mamárias. Existem evidências de que mulheres com câncer de mama apresentam menor concentração sérica de 25(OH)D3 ou de 1,25(OH)2D3 em relação às mulheres sem câncer. Por outro lado, pouco se sabe se a concentração sérica de VD pode influenciar o desenvolvimento de câncer de mama em cadelas e se o hormônio pode ter efeito quimiopreventivo, inibindo o aparecimento de tumores ou mesmo efeito terapêutico, reduzindo a proliferação de células malignas. Logo, nossos objetivos foram comparar a concentração sérica de 25(OH)D3 em animais com e sem tumor mamário e analisar as ações de 1,25(OH)2D3 em glândulas mamárias normais e tumorais de cadelas, utilizando como modelo a cultura de tecidos. Inicialmente foram incluídas 39 cadelas portadoras de tumor de mama e 64 cães sem tumor (controle), sendo que 50 eram fêmeas e 14 eram machos. Observamos que os animais do grupo tumoral possuíam idade mais avançada (mediana de 108 meses) em relação ao grupo controle (mediana de 36 meses para os machos e 24 meses para as fêmeas). No grupo controle, a concentração sérica foi maior nos machos (32,5 ± 19,3 ng/mL) do que nas fêmeas (22,8 ± 9,6 ng/mL), mas não houve diferença em relação ao grupo tumor (26,62 ± 14,25 ng/mL). Em relação à dieta, a concentração sérica de 25(OH)D3 foi maior nas fêmeas do grupo controle que se alimentavam de ração em comparação às que se alimentavam de comida caseira e ração. Entretanto, não houve diferença em relação à exposição ao sol e características da pelagem em todos os animais. No grupo tumoral, houve correlação inversa da concentração sérica de 25(OH)D3 em relação à idade, mas não houve diferença quanto ao tipo histológico ou estadiamento da doença. Foram coletadas 70 amostras de tumor de mama e de tecido mamários normal de cadelas, as quais foram cultivadas em fatias. Dos tecidos tumorais, 50% eram positivos para receptor de estrógeno (acima de 10% de células marcadas) e 44% eram positivos para HER-2 (método HercepTest). Detectou-se expressão gênica e proteica do receptor da vitamina D (VDR) em tecido mamário normal e tumoral, sendo identificado três padrões na imunoistoquímica: I - células epiteliais e mioepiteliais (mais frequentemente encontrada em tecido normal), II - marcação predominante em células mioepiteliais (mais comum em tecido tumoral e III - marcação predominante em células epiteliais. As amostras foram tratadas com 1,25(OH)2D3 nas concentrações 0,228 nM, 2 nM e 100 nM (concentração fisiológica, farmacológica que não induz hipercalcemia e farmacológica que induz hipercalcemia, respectivamente). O conteúdo de VD tecidual avaliado por cromatografia líquida foi crescente de acordo com as concentrações de VD utilizadas, indicando penetração do hormônio nas fatias. Observou-se indução da expressão de CYP24A1, que variou de 27 a 158 vezes dependendo da concentração utilizada, indicando ativação genômica da via da VD e que o tecido permanece metabolicamente ativo em cultura. Entretanto, não houve diferença da expressão gênica de outros genes envolvidos com o metabolismo da VD (CYP27B1), genes envolvidos no controle da proliferação (CDKN1A e CDKN1B) e genes envolvidos com a imunidade (CD14). O tratamento com calcitriol nas diferentes concentrações não induziu a apoptose (expressão proteica de caspase-3 clivada) e não alterou a proliferação nos tecidos normais (expressão proteica de Ki-67), mas diminuiu a proliferação nos tecidos tumorais. Não foi observada correlação entre a indução da apoptose e redução da proliferação com os padrões de expressão proteica de VDR. Concluindo, não observamos diferença na concentração sérica de 25(OH)D3 entre cadelas com tumor de mama e animais controle. Detectamos que o calcitriol em concentração fisiológica ativa a via genômica de VD em mama normal e tumoral e reduz o índice de proliferação (expressão de Ki-67) nos tumores de mama / Vitamin D (VD) may be involved in the control of proliferation, differentiation and apoptosis of mammary cell lines exposed to high concentrations of the hormone. There is some evidence that women with breast cancer present lower serum level of 25(OH)D3 or 1,25(OH)2D3 compared to women without cancer. Moreover, little is known if serum concentration of VD can influence the development of mammary tumors in dogs and if the hormone may have chemopreventive effect by inhibiting the appearance of tumors or therapeutic effect, reducing the proliferation of malignant cells. Therefore, our goals were to compare the serum 25(OH)D3 level in animals with and without mammary tumor and to analyze 1,25(OH)2D3 effects in normal and tumoral canine mammary glands, using as a model the tissue culture. At first, 39 bitches with mammary tumor and 64 dogs without tumor (control), of which 50 were females and 14 were males were included. Animals in tumor group were older (median 108 months) compared to control group (median 36 months for males and 24 months for females). In control group, serum concentration was higher in males (32.5 ± 19.3 ng/mL) than in females (22.8 ± 9.6 ng/mL), but there was no difference when compared to tumor group (26.62 ± 14.25 ng/mL). Regarding diet, serum 25(OH)D3 level was higher in control bitches fed commercial pet food compared to those fed homemade and commercial pet food combined. However, there was no difference of serum 25(OH)D3 concentration, sun exposure and coat features. In tumor group, there was an inverse correlation between serum 25(OH)D3 and age, but there was no difference in 25(OH)D3 concentration among bitches with different histological type or clinical stage of the disease. Seventhy bitches diagnosed with mammary tumors had tumor and mammary samples collected, sliced and cultured. In tumor tissues, 50% were positive for estrogen receptor (over 10% of cells stained), and 44% were positive for HER-2 (HercepTest method). Vitamin D receptor (VDR) protein and genic expression was detected in normal and tumoral samples. Three patterns of VDR were detected by immunohistochemistry: I - localizated in epithelial and myoepithelial cells (more often in normal tissues), II - predominantly in myoepithelial cells (most common in tumor tissues) and III - predominantly in epithelial cells. Normal anmammary slices were treated with 1,25(OH)2D3 0.228 and 100nM concentrations (concentration physiological and pharmacological, respectively) and mammay tumor slices were treated with 1,25(OH)2D3 2 nM concentrations (drug concentration which does not induce hypercalcemia) and 100 nM, for 24 hours. VD tissue content measured by liquid chromatography was higher in samples exposed to high VD concentration, indicating penetration of the hormone in slices. VD treatment induced CYP24A1 expression, 27 to 158 fold depending on the concentration used, and indicating activation of the VD genomic pathway. This result also suggest that the tissue remains metabolically active in culture. However, no difference in gene expression of other target genes such as CYP27B1, genes involved in proliferation as CDKN1A and CDKN1B and genes involved in immunity, such as CD14. Calcitriol treatment at different concentrations did not induce apoptosis (protein expression of cleaved caspase-3) and did not alter proliferation in normal tissues (expression of protein Ki -67), but decreased proliferation in tumor tissues. No correlation was observed between the induction of apoptosis and reduction of proliferation with the protein expression patterns of VDR. In conclusion, no difference in serum 25(OH)D3 between bitches with mammary tumor and control animals was observed. In normal and tumoral mammary samples calcitriol physiologic concentration activated VD genomic pathway and in tumor samples calcitriol reduced the proliferation index (Ki-67)

胰腺的胰島具有重要的生理功能，表現在系列的荷爾蒙，特別是能夠控制血糖穩態的胰島素的合成和分泌。胰島素的功能受到各種分子信號及環境的調節。在過去的十年裡，腎素血管緊張素系統(RAS)被發現除了調節血壓和體液穩態之外還具有局部性的生理功能。根據我們最近的發現，胰島存在自有的腎素血管緊張素系統並且可能在胰島生理作用和糖尿病方面發揮新穎的作用。同時，越來越多的研究發現一些與臨床相闊的調節因子在胰島的功能和糖尿病中起著關鍵的作用。這些調節因子促進胰島素分泌並且可以調節胰島細胞的生長和凋亡。其中一些調節因子顯示出極大的研究價值。胰高血糖素樣肽-1(GLP-1)能通過它在胰島上的受體改善胰島的功能和血糖的控制;另一方面， 維生素D 也可以通過它在胰島B細胞上的受體來起到調節胰島素分泌及控制糖尿病的作用。像胰島局部RAS一樣， GLP-1 和維生素D 都可以通過它們在同一個靶器官--胰島細胞上的受體來發揮它們的功能。因此，不難想象這三種調節因子之前具有潛在的聯系並且直接或間接地影響胰島功能。此研究可以分為三部分以闡述這三種調節因子在胰島上的新穎作用(1) GLP-l 和RAS 在胰島功能上的潛在協同作用; (2)維生素D 對於胰島RAS 表達的調節作用及對膜島功能的影響；(3) 維生素D 缺乏下的胰島RAS 表達以及胰島功能的改變。 / 在第一部分的研究裡，我們檢測了阻斷血管緊張素一型受體(纈沙坦)和增強GLP-l 作用(DPPIV 抑制劑LAF237) 的復合效應對二型糖尿病小鼠(db/db) 血糖控制和胰島功能方面的影響。我們比較了接受單一給藥和聯合給藥的db/db 小鼠的胰島功能。所有的藥物處理都改善了db/db 小鼠的血糖穩態，而聯合給藥組在增加胰島B細胞面積，減少細胞凋亡，促進增殖以及降低膜島氧化應激和膜島纖維化方面體現出復合效應。另外，短期的聯合給藥顯著促進分離出來的胰島細胞的胰島素分泌。這些結果顯示了血管緊張素型受體阻斷劑和DPPIV 抑制劑在改善胰島的結構與功能以及治療二型糖尿病方面具有復合效應。 / 據研究，維生素D 是種具有抗糖尿病和高血壓作用的荷爾蒙，而不適合的RAS活性能夠減少胰島功能和糖耐量。維生素D 對腎臟腎素的直接抑制作用表明維生素D 可能可以調節胰島得局部RAS 活性進而調節胰島的生理作用。因此第二部分的實驗旨在研究維生素D 是否能夠抑制分離培養的胰島中非正常表達的胰島局部RAS組分並且改善胰島且細胞功能。維生素D 受體存在於胰島且細胞的核與質中，計量依賴性地調節受體對活性維他命D-骨化三醇的反應。骨化三醇的刺激可以通過增加維生素D24羥化黣激發胰島局部維他命D 系統的反饋機制。在分離的胰島中，長期處於高糖的環境，胰島局部RAS 的異常表達可以一定濃度的骨化三醇治療和預防。然而，骨化三醇的送科治療效果，並沒有在生理正常糖濃度的情況下被發現。另外，在高糖環境下，骨化三醇增加胰島素前體合成以及葡萄糖刺激的服島素分泌。這些結果顯示骨化三醇能夠調節以及保護高糖環境引起的異常胰島RAS 組分表達並通過增加胰島素的合成與分泌來改善胰島的功能，為在高血糖和糖尿病情況下的維生素D 與胰島功能關系提供了新的機制。 / 循環中的維生素D 水平與血糖濃度以及糖尿病的患病風險成反比。第二部分的實驗結果現實了維生素D 具有潛在的調節胰島RAS 進而調節胰島功能的作用。因此，在第三部分的實驗裡，我們假設不充足的維生素D 水平可能引起異常的胰島RAS 表達進而引起胰島功能障薇。為了這個目的，我們使用了維生素D 受體缺失的基因敲除小鼠和維生素D 缺乏小鼠來檢測糖代謝，膜島形態以及局部RAS 組分的表達。結果顯示，在缺乏維生素D 以及正常的維生素D 作用的情況下，胰島局部RAS 組分異常表達。而這個維生素D 導致的RAS 異常表達的作用可能發生在高血糖現象之前，從而導致了胰島功能障礙，異常的糖代謝以及減弱的胰島且細胞本身的胰島素作用。這些結果為在生理情況下，維生素D 可以通過調節胰島局部RAS 的表達進而調節胰島功能提供了有力的支持。 / 總括來說，胰島局部RAS 在持續高糖環境下的胰島功能中有著關鍵的作用。GLP-l 和維生素D 都與胰島RAS 具有潛在的生物相關性並可以影響RAS 的表達，進而調節胰島功能和自細胞體積。我們的實驗數據顯示了這三種調節系統共同作用並調節目突島細胞功能以及血糖穩態，進一步提議了它們在二型糖尿病治療中的價值。 / Pancreatic islets perfonn critical biological activities by means of synthesizing and releasing islet peptide honnones, notably insulin that controls our glucose homeostasis. The insulin secretory function is, in turn, governed by various conditions and signaling molecules. In the past decade, it is recognized that the renin-angiotensin system (RAS) has local function rather than the maintenance of blood pressure and fluid homeostasis. With our recent recognition of an islet RAS, it is believed that it has novel roles in islet physiology and diabetes. Meanwhile, more and more clinically relevant regulators that have pivotal roles in islet function and diabetes have been well investigated; such regulators have positive action on insulin secretion, B-cell replication and cell apoptosis/proliferation balance. Of great interest in this context is the glucagon-like peptide I (GLP-I) that improves islet function and glycemic control via its islet specific receptors located on the islets. On the other hand,vitamin D also regulates islet insulin secretion and diabetes via its mediation of receptors on islet B-cells. Like islet RAS, GLPI and vitamin D exert their biological effects via mediation of respective receptors located on the common target, i.e. the islet beta-cells. As such, it is plausible to propose that all these three regulators have potential interactions so as to affect islet functions in a direct or an indirect manner. Accordingly, the primary objective of this study is to examine the potential roles oflocal RAS, GLP-I and vitamin D system in pancreatic islet function. The present study is thus divided into three main parts addressing the issues of these three novel regulators in islet function: (1) the potential synergism of GLP-I and RAS in islet function; (2) the modulatory effects of vitamin D on islet RAS expression and function; (3) The altered islet RAS and islet function under a hypovitaminosis D condition. / In the first part of our study, we examined the combined effect of blocking islet A Tl receptor (ATl receptor blocker: valsartan) and enhancing GLP-l actions (DPP IV inhibitor: LAF237) on islet function and glycemic control in a mouse model with type 2 diabetes, db/db mice. We compared the islet function in db/db mice with either valsartan or LAF237 mono treatment or combined treatment. Consistently, all these treatments improved glucose homeostasis in db/db mice while combined treatment resulted in a significant increase in islet B-cell area by decreasing cell apoptosis and increasing proliferation, together with marked decreases of islet oxidative stress and fibrosis. In addition, a short-term effect on stimulating insulin secretion was also observed in isolated islets with combined treatment. These results indicate that the combination treatments with ATl receptor blocker and DPP IV inhibitor has beneficial additive effects on islet structure and function in type 2 diabetes, compared with their monotherapeutic treatments. / It is reported that vitamin D is a hormone with anti-diabetic and anti-hypertension effects in human while inappropriate RAS activity has been known to reduce islet function and glucose tolerance. The direct suppressive effect of vitamin D on renal renin activity indicates vitamin D may acts as a regulator in RAS activity thus modulate islet physiology. In the second part of our study, it was aimed to study whether vitamin D vitamin D downregulation of abnormal islet RAS activity improves B-cell function using an isolated pancreatic islet model. VDR was localized in islet B-cell nuclei and cytoplasm, mediated responses to active form of vitamin D calcitriol in a dose-dependent manner. This islet local vitamin D system may have its own feedback system as a marked increase ofCYP24 transcription was triggered by calcitriol stimulation. In isolated islets exposed to prolonged high glucose environment, abnormal expressed islet RAS components could be reversed or protected by calcitriol at a specific concentration. However, the inhibition effect of calcitriol on islet RAS were not observed at physiological glucose concentrations. In additon, calcitriol increased islet proinsulin synthesis and insulin secretion with hyperglycemia. These results indicated that calcitriol modulate or protect the abnormal isolated islet RAS component expression against hyperglycemia and improve islet function via increasing insulin synthesis and secretion, which might provide an alternative mechanism by which vitamin D availability enhances islet function in hyperglycemia or diabetes. / The circulating vitamin D level is inversely related to blood glucose level and risks of diabetes. Results in the second part of experiments suggested the potential RAS modulatory effect of vitamin D in isolated islets Therefore, in the third part of our study, we hypothesize that the insufficient vitamin D levels may lead to the inappropriate regulation of islet RAS expression and thus result in islet dysfunction. To achieve this, we examined the potential islet RAS-mediated effect of vitamin D on islet function by accessmg glucose homeostasis, islet histomorphology, and local RAS expression and function by means of using a vitamin D receptor knockout and diet-induced vitamin D deficiency mouse models. Results showed that the islet RAS components were abnormally expressed when lacking a sufficient vitamin D level and normal vitamin D action. These observed effects of insufficient vitamin D might occur prior to onset of hyperglycemia thus modulating islet RAS expression, which in turn lead to islet failure and dysfunctional glucose homeostasis, together with decreased insulin actions in islet B-cells. These results provide supports for the view that vitamin D physiologically exerts modulatory effects on islet function by downregulating islet RAS expression and function. / In summary, islet local RAS may have a central role in islet function under prolonged hyperglycemic stress. GLP-l and vitamin D have biological interactions with the islet RAS by downregulation of its expression and function, thereby affecting islet cell function and cell mass. Our data indicate that all three regulators work together in the regulation of pancreatic islet B-cell functions and glucose homeostasis, further suggestive of their potential values in the treatment of type 2 diabetes. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheng, Qianni. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves [205]-243). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.v / Acknowledgements --- p.viii / List of Publications --- p.x / Table of Contents --- p.xii / List of Abbreviations --- p.xvi / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Endocrine Pancreas --- p.2 / Chapter 1.1.1 --- The structure and composition of endocrine pancreas --- p.3 / Chapter 1.1.2 --- Functions of endocrine pancreas --- p.4 / Chapter 1.1.3 --- Insulin structure and insulin receptors --- p.8 / Chapter 1.1.4 --- Mechanisms of insulin secretion --- p.11 / Chapter 1.1.5 --- Mechanisms of insulin actions --- p.18 / Chapter 1.1.6 --- Disorders of the endocrine pancreas --- p.22 / Chapter 1.2 --- Diabetes mellitus --- p.23 / Chapter 1.2.1 --- Type 1 diabetes mellitus (TlDM) --- p.24 / Chapter 1.2.2 --- Type 2 diabetes mellitus (T2DM) --- p.26 / Chapter 1.2.3 --- Other types of diabetes mellitus --- p.29 / Chapter 1.2.4 --- Islet dysfunction and T2DM --- p.30 / Chapter 1.3 --- Renin-angiotensin system (RAS) --- p.33 / Chapter 1.3.1 --- Components ofRAS --- p.33 / Chapter 1.3.2 --- Tissue local RAS --- p.42 / Chapter 1.3.3 --- Pancreatic local RAS --- p.43 / Chapter 1.4 --- Glucagon like peptide-l (GLP-l) and pancreatic islet function --- p.54 / Chapter 1.4.1 --- Gastrointestinal incretin honnones --- p.54 / Chapter 1.4.2 --- GLP-l and pancreatic islet function --- p.56 / Chapter 1.4.3 --- Incretin based therapies for T2DM --- p.59 / Chapter 1.4.4 --- GLP-lIRAS axis and pancreatic islet function --- p.62 / Chapter 1.5 --- Vitamin D and pancreatic islet function --- p.64 / Chapter 1.5.1 --- Vitamin D synthesis and metabolism --- p.65 / Chapter 1.5.2 --- Vitamin D physiological functions and pancreatic islets --- p.67 / Chapter 1.5.3 --- Vitamin D and diabetes mellitus --- p.68 / Chapter 1.5.4 --- Vitamin D and RAS --- p.70 / Chapter 1.6 --- Objectives --- p.71 / Chapter Chapter 2 --- Materials and Methods --- p.73 / Chapter 2.1 --- Experimental animal models --- p.74 / Chapter 2.1.1 --- Animal model ofT2DM --- p.74 / Chapter 2.1.2 --- Animal model for pancreatic islet isolation --- p.75 / Chapter 2.1.3 --- Vitamin D receptor knockout mice (VDRKO mice) --- p.75 / Chapter 2.1.4 --- Animal model for vitamin D deficiency --- p.76 / Chapter 2.2 --- Pancreatic islet isolation and culture --- p.76 / Chapter 2.2.1 --- Mice pancreatic islet and single B-cell isolation --- p.77 / Chapter 2.2.2 --- Primary culture of isolated pancreatic islets: --- p.78 / Chapter 2.3 --- Physiological assay for pancreatic islet function --- p.78 / Chapter 2.3.1 --- Measurement of blood glucose and glucose tolerance test --- p.78 / Chapter 2.3.2 --- Measurement of glucose-induced insulin secretion --- p.79 / Chapter 2.3.3 --- Measurement of (pro )insulin biosynthesis --- p.80 / Chapter 2.4 --- Detection ofmRNA expression --- p.80 / Chapter 2.4.1 --- Design of primers --- p.81 / Chapter 2.4.2 --- mRNA extraction and cDNA synthesis --- p.82 / Chapter 2.4.3 --- Detection of mRN A expression by conventional peR --- p.83 / Chapter 2.4.4 --- SYBR Green real-time peR --- p.83 / Chapter 2.4.5 --- Real-time peR analysis using the comparative eT method --- p.84 / Chapter 2.5 --- Detection of protein expression --- p.84 / Chapter 2.5.1 --- Western blot analysis --- p.84 / Chapter 2.5.2 --- Immunostaining assessment --- p.85 / Chapter 2.6 --- In situ detection of oxidative stress, proliferation and apoptosis --- p.88 / Chapter 2.6.1 --- Detection of islet reactive oxygen species --- p.88 / Chapter 2.6.2 --- Detection of cell proliferation --- p.89 / Chapter 2.6.3 --- Measurement of cell apoptosis --- p.90 / Chapter 2.7 --- Statistical data analysis --- p.90 / Chapter Chapter 3 --- Combination of DPP-IV Inhibitor LAF237 with ATl Receptor Antagonist Valsartan Enhances Pancreatic Islet Morphology and Function in a Mouse Model of Type 2 Diabetes (This work has been published in J Pharmacal Exp Ther, 327: PI-9) --- p.91 / Chapter 3.1 --- Abstract --- p.92 / Chapter 3.2 --- Introduction --- p.94 / Chapter 3.3 --- Materials and Methods --- p.96 / Chapter 3.4 --- Results --- p.103 / Chapter 3.4.1 --- Effects of acute treatment with GLP-I and valsartan on insulin secretion in isolated islets --- p.103 / Chapter 3.4.2 --- Effects of LAF237 and valsartan on pancreatic --- p.105 / Chapter 3.4.3 --- Effects of LAF237 and valsartan on --- p.107 / Chapter 3.4.4 --- Effects ofLAF237 and valsartan on islet apoptosis --- p.109 / Chapter 3.4.5 --- Effects of LAF237 and valsartan on islet fibrosis --- p.110 / Chapter 3.4.6 --- Effects of LAF237 and valsartan on pancreatic islet superoxide and nitrotyrosine expression --- p.113 / Chapter 3.4.7 --- Effects of LAF237 and valsartan on bood glucose concentration and glucose tolerance in db/db diabetic mice --- p.116 / Chapter 3.5 --- Discussion --- p.119 / Chapter Chapter 4 --- The Role of Calcitriol in Modulating the Expression and Function of Islet Renin-Angiotensin System in Isolated Mouse Pancreatic Islets --- p.124 / Chapter 4.1 --- Abstract --- p.125 / Chapter 4.2 --- Introduction --- p.127 / Chapter 4.3 --- Materials and Methods --- p.130 / Chapter 4.4 --- Results --- p.135 / Chapter 4.4.1 --- The expression of islet VDR under different glucose conditions and the effects of calcitriol --- p.135 / Chapter 4.4.2 --- The effect of calcitriol on high glucose-modulated islet RAS component expression --- p.140 / Chapter 4.4.3 --- The protective effect of calcitriol against high glucose on islet RAS component expression --- p.144 / Chapter 4.4.4 --- The effect of calcitriol on (pro )insulin biosynthesis and insulin release in isolated islets --- p.148 / Chapter 4.5 --- Discussion --- p.151 / Chapter Chapter 5 --- Altered Islet Local Renin-Angiotensin System and Islet Function in Mice with Hypovitaminosis D --- p.158 / Chapter 5.1 --- Abstract --- p.159 / Chapter 5.2 --- Introduction --- p.160 / Chapter 5.3 --- Materials and methods --- p.163 / Chapter 5.4 --- Results --- p.168 / Chapter 5.4.1 --- Glucose homeostasis and islet morphology in VDR KO mice --- p.168 / Chapter 5.4.2 --- Expression of vitamin D receptor and major RAS components in the pancreatic islets of WT and VDR KO mice --- p.170 / Chapter 5.4.3 --- Vitamin D deficiency in mice on a vitamin D deficient diet --- p.172 / Chapter 5.4.4 --- Altered glucose homeostasis in vitamin D deficient mice --- p.174 / Chapter 5.4.5 --- Islet histomorphology in vitamin D deficient mice --- p.176 / Chapter 5.4.6 --- Regulation of islet RAS components expression in vitamin D deficient mice --- p.179 / Chapter 5.4.7 --- Transcriptional regulation of islet insulin receptor and its substrates in vitamin D deficient mice --- p.181 / Chapter 5.4.8 --- Effect of calcitriol treatment on glucose tolerance in vitamin D deficient mice --- p.183 / Chapter 5.5 --- Discussion --- p.185 / Chapter Chapter 6 --- General Discussion --- p.191 / Chapter 6.1 --- Combination effects of blocking islet RAS components and enhancing incretin activity on improving islet function in type 2 diabetes --- p.193 / Chapter 6.2 --- Potential modulatory effect of vitamin D on islet RAS expression and action --- p.196 / Chapter 6.3 --- The role of vitamin D in modulating islet RAS in glucose homeostasis and islet function --- p.199 / Chapter 6.4 --- The significance ofRAS, GLP-l and vitamin D in the management of T2DM --- p.201 / Chapter 6.5 --- Conclusion --- p.202 / Chapter 6.6 --- Future studies --- p.202 / Chapter Chapter 7 --- Bibliography --- p.205