O efeito antiproliferativo do calcitriol foi detectado principalmente em linhagens de carcinoma mamário expostas in vitro a alta concentração hormonal (10 - 100nM), que é associado com hipercalcemia em seres humanos. Nossa hipótese era que a administração intratumoral de calcitriol permitiria maior concentração do hormônio e ativação da via genômica. Para testar esta hipótese, um modelo de enxerto tumoral que reproduz o mais próximo das características moleculares do tumor primário, foi estabelecida. As amostras de câncer de mama recolhidos foram enxertados em camundongos nude e depois da sexta semana, semana, os enxertos tumorais foram tratados semanalmente com injeções intra-tumorais de veículo (controle) ou o calcitriol 0,06 mcg (dose que pode permitir que picos séricos de calcitriol na faixa terapêutica prevista) durante seis semanas. A proliferação e apoptose do enxerto tumoral Veículo (controlo) ou o calcitriol 0,06 mcg (dose que pode permitir que o soro de pico, assim como, a expressão dos genes alvos foram avaliadas através de reações imunohistoquímica ou RT-PCR. A expressão de VDR foi detectada em todas as amostras, assim como uma tendência para maior expressão de mRNA CYP24A1 (indução 10-18 vezes) em amostras tratadas com calcitriol, indicando que a via genómica foi induzida pelo hormonio. O elevado índice proliferativo, avaliado pela expressão de Ki67, foi detectado. No entanto, não havia diferenças na expressão de marcadores de proliferação (incorporação de BrdU, Ki67 e CDKN1B expressão) nem marcadores de apoptose (caspase-3 clivada e BCL2 expressão) entre os enxertos tumorais tratados por veículo e calcitriol tratado. Além disso, não houve diferença entre os grupos detectada na expressão de mRNA do CDKN1A. Em resumo, os efeitos antitumorais não foram observados neste modelo de enxerto tumoral. A indução do gene alvo CYP24A1 pode ter em parte impedido os efeito antitumorais da vitamina D / Antiproliferative effects of calcitriol were mainly detected in breast carcinoma lineages exposed in vitro to high hormone concentrations (10-100 nM), which is associated with hypercalcemia in human beings. Our hypothesis was that intra-tumoral administration of calcitriol would allow higher issue concentration of the hormone and activation of the genomic pathway. To test this hypothesis, a tumorgraft model, that more closely reproduces the molecular characteristics of the primary tumor, was established. Freshly collected breast cancer samples were grafted in nude mice and after the 6th week, tumorgrafts were treated weekly with intra-tumoral injections of vehicle (control) or calcitriol 0.06 mcg (dose that may allow peak serum calcitriol levels in the predicted therapeutic range) for six weeks. Tumorgraft proliferation and apoptosis, as well as expression of target genes, were evaluated through immnunohistochemistry reactions or RT-PCR. VDR expression was detected in all samples as well as a trend towards higher expression of CYP24A1 mRNA (10-18 fold induction) in calcitriol treated samples, indicating that the genomic pathway was induced by the hormone. A high proliferative index, evaluated by Ki67 expression, was detected. However, there were neither differences in the expression of proliferation markers (BrdU incorporation, Ki67 and CDKN1B expression) nor in apoptosis markers (cleaved caspase 3 and BCL2 expression) between vehicle and calcitriol treated tumorgrafts. In addition, no difference between groups was detected for the expression of CDKN1A mRNA. In summary, calcitriol antitumoral effects were not observed in this tumorgraft model. Calcitriol induction of the target gene CYP24A1, might have in part, precluded vitamin D antitumoral effects

Chan Chi-keung. / Thesis (M.Sc.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 93-107). / Abstracts in English and Chinese. / List of Tables --- p.ix / List of Figures --- p.x / Chapter 1. --- Literature Review --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Oncogenic anatomy of the prostate gland --- p.1 / Chapter 1.3 --- Characteristics of prostate cancer --- p.7 / Chapter 1.4 --- Incidences of prostate cancer --- p.8 / Chapter 1.5 --- Risk factors for prostate cancer --- p.14 / Chapter 1.5.1 --- Endogenous risk factors --- p.14 / Chapter (A) --- Age --- p.14 / Chapter (B) --- Race --- p.16 / Chapter (C) --- Family history --- p.21 / Chapter (D) --- Hormonal factors --- p.24 / Chapter (I) --- Androgen --- p.24 / Chapter (II) --- Vitamin D --- p.32 / Chapter 1.5.2 --- Exogenous risk factors --- p.41 / Chapter (A) --- Dietary factors --- p.41 / Chapter (B) --- Body Mass Index & physical condition --- p.44 / Chapter (C) --- Occupation --- p.46 / Chapter (D) --- Vasectomy --- p.47 / Chapter (E) --- Others --- p.48 / Chapter 2. --- Introduction to the project --- p.49 / Chapter 3. --- Objectives --- p.50 / Chapter 4. --- Materials and Methods --- p.51 / Chapter 4.1 --- Prostate cancer cases --- p.51 / Chapter 4.2 --- Controls --- p.52 / Chapter (A) --- Benign prostatic hyperplasia --- p.52 / Chapter (B) --- Population control --- p.52 / Chapter 4.3 --- DNA extraction --- p.53 / Chapter 4.4 --- Amplification of target DNA --- p.54 / Chapter 4.5 --- Allele typing --- p.55 / Chapter 4.6 --- Statistical analysis --- p.55 / Chapter 5. --- Results --- p.60 / Chapter 5.1 --- Optimization of DNA extraction --- p.60 / Chapter 5.2 --- Optimization of PCR condition --- p.61 / Chapter 5.3 --- Allele typing --- p.65 / Chapter 5.4 --- Characteristics of subjects samples --- p.68 / Chapter 5.4.1 --- Age of subjects and tumor grading --- p.68 / Chapter 5.4.2 --- Genotype typing --- p.69 / Chapter (A) --- Bsm genotype --- p.69 / Chapter (B) --- Fok genotype --- p.69 / Chapter 6. --- Discussions --- p.73 / Chapter 6.1 --- Technical issues --- p.73 / Chapter (A) --- DNA extraction --- p.73 / Chapter (B) --- Primer design --- p.76 / Chapter (C) --- Determination of the optimal PCR condition --- p.77 / Chapter (D) --- Restriction enzyme digestion --- p.82 / Chapter 6.2 --- Age distribution of prostate cancer patients --- p.83 / Chapter 6.3 --- Genotype frequency --- p.84 / Chapter 6.4 --- Histopathological samples of case and control --- p.87 / Chapter 6.5 --- Vitamin D receptor genotypes and prostate cancer --- p.89 / Chapter 7. --- Conclusions --- p.92 / Chapter 8. --- References --- p.93

As hiperhomocisteinemias (HHcy), leve e moderada são consideradas um fator de risco para doenças cardiovasculares e cerebrais, entretanto os mecanismos e as complicações decorrentes dessa condição ainda não estão bem estabelecidos. Ela ocorre em 5-10% da população geral e em 40% dos pacientes com doenças vascular periférica e doenças cerebrovasculares. Estudos recentes têm demonstrado que a vitamina D (calcitriol) possui efeitos protetores em diversos modelos experimentais que enfatizam suas possíveis ações antioxidantes. O objetivo principal dessa tese de doutorado foi estabelecer um protocolo experimental com diferentes concentrações de homocisteína em fatias de cortex cerebral e coração e em cultura de astrócitos de ratos adultos. Seguindo esses modelos experimentais, investigamos alguns parâmetros bioquímicos em córtex cerebral e coração de ratos. Posteriormente foi analisado o efeito protetor do calcitriol. No primeiro capítulo da presente tese, observamos que a incubação de 30 μM de Hcy por 30 min e 60 min em fatias de coração, alterou a função e a permeabilidade mitocondrial, o estado redox e a atividade das enzimas da cadeia respiratória; o calcitriol foi capaz de prevenir a maioria dos efeitos da Hcy. No segundo capítulo, vimos que em fatias de córtex cerebral a Hcy prejudica o metabolismo energético, aumentando a morte neuronal e induzindo estresse oxidativo. Todavia, o calcitriol atenuou esses efeitos deletérios induzidos pela Hcy através da ativação do receptor de vitamina D. No último capítulo desta tese, realizamos um estudo em cultura primária de astrócitos corticais de ratos Wistar adultos. Nossos resultados demonstram que a Hcy ativa a via do fator nuclear kappa B (NFκB), inibindo a expressão de heme oxigenase 1 (HO-1), promovendo alterações morfológicas, aumentando a resposta inflamatória e diminuindo as defesas antioxidantes e a atividade da Na+, K+ - ATPase. Em resumo, em todos modelos experimentais estudados nesta tese, a Hcy, mesmo em concentrações leves e moderadas causou alterações na homeostasia celular. A vitamina D preveniu parte destes efeitos, tornando-se um possível ferramenta terapêutica no intuito de atenuar os efeitos da Hcy. / Hyperhomocysteinemia (HHcy), mild and moderate are a risk factor for cardiovascular and cerebral diseases, but the mechanisms and complications of this condition are not yet well established. It occurs in 5-10% of the general population and 40% of patients with peripheral vascular and cerebrovascular disease. Recent studies have shown that vitamin D (calcitriol) has protective effects in various experimental models which emphasize their potential antioxidant actions. The main objective of this PhD thesis was to establish an experimental with different concentrations of homocysteine in slices of cerebral cortex and heart in adult rat astrocyte cultures. Following this experimental model, we investigated some biochemical parameters in the cerebral cortex and heart of rats. It was subsequently examined the protective effect of calcitriol. In the first chapter of this thesis, we found that incubation of 30 μM of Hcy for 30 min and 60 min in heart slices change the function and mitochondrial permeability, redox state and activity of the enzymes of the respiratory chain; calcitriol was able to prevent most of the effects of Hcy. In the second chapter, we demonstrated that Hcy in the cerebral cortex slices impairs energy metabolism, increasing neuronal death and inducing oxidative stress. However, calcitriol attenuated these Hcy-induced deleterious by activation of vitamin receptor D. In the last chapter of this thesis, we conducted a study in primary culture of cortical astrocytes. Our results demonstrate that the Hcy active the pathway of nuclear factor kappa B (NFκB) inhibiting heme oxygenase expression 1 (HO-1), promoting morphological changes, increasing the inflammatory response and decreased antioxidant defenses and activity of the Na +, K + - ATPase. In summary, in all experimental models studied in this PhD thesis, Hcy, even in mild and moderate concentrations caused deleterious actions in cellular homeostasis. Vitamin D warned of these effects, becoming a potential therapeutic target in order to attenuate the effects of Hcy.

Background: The role of nutrients in mental health has recently been recognized and investigated. Vitamin D has been known to play a role in a wide range of diseases, such as bone, cardiovascular, and autoimmune diseases, and cancers. Recently, its role in cognitive function and mental health has been reported. Vitamin D receptor and hydroxylases have been mapped throughout the brain, suggesting a role for vitamin D in brain tissue. An inverse association between vitamin D and depression was observed in European epidemiologic studies. There is a paucity of data on the association between vitamin D concentrations and depression in the U.S. population. Objective: The objective of this study was to investigate the association between serum vitamin D concentrations and depression in a large, nationally representative sample survey, the third National Health and Nutrition Examination Survey 1988-1994 (NHANES III). Methods: The study sample included 7970 adults, ages 15-39 years, who completed the Diagnostic Interview Schedule for depression and had vitamin D concentrations measured. SAS and SUDAAN statistical software packages were used in data analysis. Multivariate logistic regression was used to estimate the likelihood of having depression in vitamin D deficient persons in relation to vitamin D sufficient persons, after taking several confounding variables into consideration. Significance was set at α < 0.05. Results: The prevalence of vitamin D deficiency was higher in women than in men (24 % vs. 15%), higher in African-Americans than in whites (60% vs. 10%), higher in people living in metropolitan rather than in rural areas (25% vs. 14%), and higher in subjects below the poverty threshold than in higher income subjects (29% vs. 14%). The prevalence of vitamin D deficiency increased as BMI increased. The diagnostic variables for depression did not show an association with vitamin D deficiency after adjusting for several confounding factors. However, subjects having a depressive episode at the time of the interview, were significantly more likely to exhibit vitamin D deficiencies (OR = 1.85; P = 0.0210). Conclusions: This is the first large epidemiologic study on the association between vitamin D and depression in a US representative sample survey. A significant positive association was found between subjects having an episode of depression and vitamin D deficiency. However, a causal relationship could not be established due to the cross-sectional nature of the study. Further studies need to investigate the mechanistic and causal relation between vitamin D and depression.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 65-82).

The CYP27B1 enzyme that synthesizes 1alpha,25-(OH) 2D, is expressed in chondrocytes, suggesting that local production of 1alpha,25-(OH)2D could play an autocrine or paracrine role in the differentiation of these cells. To test this hypothesis, we have engineered mutant mice that do not express the Cyp27b1 gene in chondrocytes. This led to increased width of the hypertrophic zone of the growth plate at E15.5, increased bone mass in neonatal long bones, and increased expression of the chondrocytic differentiation markers Indian Hedgehog and PTH/PTHrP receptor. VEGF mRNA levels were decreased, accompanied by decreased PECAM-1 immunostaining, suggesting a delay in vascularization. We have also engineered mice overexpressing a Cyp27b1 transgene in chondrocytes. The transgenic mice showed a partial mirror image phenotype compared to the tissue-specific inactivation model. These results support an autocrine/paracrine role of 1alpha,25-(OH) 2D in endochondral ossification and chondrocyte development in vivo. / The CYP24A1 enzyme is involved in the catabolic breakdown of 1alpha,25-(OH)2D but also synthesizes the 24R,25-(OH) 2D metabolite. Studies in chicken suggest a role for 24R,25-(OH) 2D in fracture repair. We induced stabilized transverse mid-diaphysial fractures of the tibia in four-month-old wild-type and Cyp24a1-deficient mice. Examination of the callus sections showed delayed hard callus formation in the homozygous mutant animals compared to wild-type littermates. RT-qPCR showed perturbed levels of type X collagen transcripts in mutant mice at 14 and 21 days post-fracture, reflecting the delayed healing. Rescue of the impaired healing by subcutaneous injection of 24R,25-(OH)2D3 normalized the histological appearance of the callus, static histomorphometric index and type X collagen mRNA expression, while 1alpha,25-(OH)2D 3 did not. These results show that Cyp24a1 deficiency delays fracture repair and strongly suggest that vitamin D metabolites hydroxylated at position 24, such as 24R,25-(OH)2D, play an important role in the mechanisms leading to normal fracture healing.

Vitamin D is a precursor to a steroid hormone, 1,25 dihydroxyvitamin D (1,25(OH)2D). After its discovery and the characterization of its receptor, the vitamin D receptor (VDR), it was initially thought only to be involved in calcium homeostasis, but further research revealed an important role for vitamin D in the regulation of cell growth and differentiation of such cells as osteoblasts and bone marrow adipocytes. 1,25(OH)2D has also been shown to be a strong inhibitor and pro-differentiator of keratinocytes. The anti-proliferative and pro-differentiative properties of this hormone have led to studies where 1,25(OH)2D anticancer properties were assessed and initial findings that showed a requirement of other factors beyond VDR to induce 1,25(OH)2D signaling led to the identification of the retinoid X receptor, a common heterodimeric partner for several hormone receptors. The focus of thesis was to further elucidate the structure-function relationship of both the vitamin D receptor and the retinoid X receptor. Additionally, contributions to work directed towards further identifying the effects of vitamin D on osteoblast differentiation and survival. Interactions of 1,25(OH) 2D3 with its cognate receptor, identifying a key amino acid (Tryptophan 286) required for ligand contact and transcriptional activation, are described in Chapter 2. Mechanisms of vitamin D action on mesenchymal stem cell differentiation, promotion of osteoblast induction and maturation, and inhibition of adipocyte differentiation, are eluicidated in Chapter 3. Chapter 4 illustrates the effects of RAS/RAF/Mitogen-activated protein kinase mediated RXRalpha phosphorylation on the three-dimensional structure of the RXR/nuclear receptor partner heterodimers. Furthermore, this chapter reveals the inhibitory effect of the phosphorylation of a critical amino acid (serine 260) on the interaction of the AF-2 domain of the RXR with several coactivators, resulting in a decrease in the signaling potential of multiple steroid hormone receptors. The findings of this thesis further the knowledge of several areas of vitamin D biology, including both the canonical areas of bone formation, and the non-canonical area of vitamin D and cancer.

Background: The role of nutrients in mental health has recently been recognized and investigated. Vitamin D has been known to play a role in a wide range of diseases, such as bone, cardiovascular, and autoimmune diseases, and cancers. Recently, its role in cognitive function and mental health has been reported. Vitamin D receptor and hydroxylases have been mapped throughout the brain, suggesting a role for vitamin D in brain tissue. An inverse association between vitamin D and depression was observed in European epidemiologic studies. There is a paucity of data on the association between vitamin D concentrations and depression in the U.S. population. Objective: The objective of this study was to investigate the association between serum vitamin D concentrations and depression in a large, nationally representative sample survey, the third National Health and Nutrition Examination Survey 1988-1994 (NHANES III). Methods: The study sample included 7970 adults, ages 15-39 years, who completed the Diagnostic Interview Schedule for depression and had vitamin D concentrations measured. SAS and SUDAAN statistical software packages were used in data analysis. Multivariate logistic regression was used to estimate the likelihood of having depression in vitamin D deficient persons in relation to vitamin D sufficient persons, after taking several confounding variables into consideration. Significance was set at α < 0.05. Results: The prevalence of vitamin D deficiency was higher in women than in men (24 % vs. 15%), higher in African-Americans than in whites (60% vs. 10%), higher in people living in metropolitan rather than in rural areas (25% vs. 14%), and higher in subjects below the poverty threshold than in higher income subjects (29% vs. 14%). The prevalence of vitamin D deficiency increased as BMI increased. The diagnostic variables for depression did not show an association with vitamin D deficiency after adjusting for several confounding factors. However, subjects having a depressive episode at the time of the interview, were significantly more likely to exhibit vitamin D deficiencies (OR = 1.85; P = 0.0210). Conclusions: This is the first large epidemiologic study on the association between vitamin D and depression in a US representative sample survey. A significant positive association was found between subjects having an episode of depression and vitamin D deficiency. However, a causal relationship could not be established due to the cross-sectional nature of the study. Further studies need to investigate the mechanistic and causal relation between vitamin D and depression.

Ulm, Univ., Diss., 2008.

Dissertation (D.P.H.)--University of Michigan.