Doctorat en sciences médicales / info:eu-repo/semantics/nonPublished

Abstract The hormonally active form of vitamin D, 1,25(OH)2D3, is involved in many biological functions throughout the body, such as regulation of calcium and phosphate homeostasis, bone remodeling and controlling cell proliferation and differentiation. Vitamin D receptor (VDR), a member of the nuclear hormone receptor (NHR) super family, mediates those genomic actions of 1,25 (OH)2D3 by actively repressing or activating its target genes. In the present study recombinant human nuclear VDR and its ligand binding domain (LBD) were expressed in Spodoptera frugiperda (Sf9) insect cells and in E.coli. Recombinant proteins were purified and their biochemical and biophysical properties were characterized. Recombinant VDR was shown to bind to the vitamin D response element (VDRE) of osteopontin and osteocalcin genes as a homodimer or as a heterodimer with the retinoid X receptor (RXR)-αΔAB. Full-length VDR and its LBD were demonstrated to bind natural ligand 1,25 (OH)2D3 with high affinity. The binding affinities of several vitamin D analogs were also determined. Ligand binding induced conformational change within the receptor was studied using several methods such as partial proteolytic digestion, small angle neutron scattering (SANS), native gel electrophoresis and circular dichroism (CD) spectroscopy. Results indicate that ligand binding induces conformational change within VDR and different 1,25(OH)2D3 analogs might induce a somewhat different conformation within the receptor. This is seen as an unequal capacity of analogs to stabilize receptor against proteases or heat and as differences in the promotion of receptor homodimerization. Compared to other nuclear hormone receptors, VDR presents a large insertion region at the N-terminal part of the LBD between helices H1 and H3, encoded by an additional exon. In the present study this additional exon was deleted and the properties of mutated LBD were compared to the wild type LBD. Biochemical analyses indicated that the mutant protein exhibits the same ligand binding, dimerization with RXR and transactivation properties as the wild-type VDR, suggesting that the insertion region does not affect these main functions. Furthermore, solution studies by small angle X-ray scattering indicated that the insertion region in the VDR locates on the surface of molecule and it is not structurally well ordered.

ANALYSIS OF PREVALENCE OF VITAMIN D DEFICIENCY IN ADULTS Author: Jana Nováková Supervisor: PharmDr. Eva Zimčíková, PhD. INTRODUCTION Estimated vitamin D deficiency occurs in up to 1/3 of the population. Vitamin D deficiency occurs in a number of diseases and in different countries of the world. AIMS The aim of the theoretical part of the diploma thesis was to get acquainted with vitamin D and its deficit in general. The objective of the practical part was to map vitamin D levels in adults, to assess prevalence of vitamin D deficiency, to determine if vitamin D levels fluctuate over the course of the year depending on age or gender and how they are related to individual illnesses. METHODS PubMed and Embase database search was performed to find evidence on vitamin D deficiency in adults. Only studies in humans, published in English in the period from 2000 to 2016 were included. Non-systematic review was provided, the quality of the studies was not evaluated. RESULTS Overall, 84 studies were included. Most of the studies enrolled healthy adults (n = 34). Among the studies concerning various diseases, musculoskeletal disorders were the most prevalent (n = 13). The highest prevalence of vitamin D deficiency was seen in the study from USA (in healthy adults) using cut-off value for deficiency ≤ 50...

Class of 2015 Abstract / Objectives: The primary objective of the study was to evaluate for change in vitamin D levels and regimens in cystic fibrosis (CF) patients following implementation of the 2012 Cystic Fibrosis Foundation (CFF) vitamin D guidelines. Secondary endpoints included clinician adherence to guideline recommendations for treatment and management of vitamin D deficiency. Methods: This retrospective chart review included CF patients with 25-hydroxy vitamin D (25(OH)D) levels from University of Arizona Medical Center (UAMC) between April 1, 2011-March 31, 2012 and July 1, 2012-June 30, 2013. Total 25(OH)D levels and vitamin D regimens were collected along with data on respiratory cultures, pulmonary function, and hospitalizations. Data were analyzed by Student’s T-tests and chi square analyses. Results: A total of 62 patients were included in the study. Mean 25(OH)D levels did not significantly differ between the study periods (28.9±10.5 ng/mL pre-guideline and 27.0±9.1 ng/mL post-guideline, p=0.158). Cholecalciferol use increased post-guideline (57.1%) versus pre-guideline (75.8%, p=0.027). Post-guideline cholecalciferol doses increased to 2836.5±2669.4 international units [IU] daily compared to 1518.0±912.0 IU daily pre-guideline (p<0.001). Clinician adherence to dose titration recommendations resulted in significant 25(OH)D level elevations (28.3±8.9 ng/mL versus 24.7±9.0, p=0.047). Conclusions: The prescribing pattern of clinicians significantly changed to reflect vitamin D regimens suggested by CFF guidelines. This finding suggests that had sufficient time been allowed following guideline implementation, a significant difference in 25(OH)D levels would have resulted. Additional research is needed concerning the effect of the guidelines on vitamin D status, clinical outcomes, and comorbidities.

M.Sc. (Biochemistry) / The vitamin D receptor (VDR) has been implicated in communicable diseases such as tuberculosis (TB), HIV infection and leprosy, as well as development or prognosis of noncommunicable diseases such as multiple sclerosis, lupus, type I diabetes mellitus, and osteoporosis. The burden of these diseases is often higher in specific population groups, for instance the higher TB burden of African as compared to White South Africans. Besides genetics, epigenetic factors play a role in differential disease susceptibility and prognosis between population groups. To uncover the cause of differential susceptibility to VDR-related diseases, it is crucial that population group-specific variations in genetic and epigenetic mechanisms that regulate VDR are identified. Increases in predictive power of in silico tools, and the recent explosion in availability of genome-wide genetic and epigenetic data made bioinformatics an attractive option to preselect regions to study in the VDR...

M.Sc. / Various researchers have found that supplementing extremely high levels of dietary vitamin D₃ for a limited time prior to slaughter improved meat tenderness due to the increase in blood calcium levels caused by vitamin D, which play an important role in activating the calpain protease system. Vitamin D₃ is metabolised in the liver into 25-hydroxyvitamin D₃ , which controls calcium and phosphate homeostasis, a process regulated by parathyroid hormone. It is hypothesised that ultra-high vitamin D₃ supplementation should alleviate negative effects of beta-agonist supplementation (which reduces meat tenderness), but these levels might be toxic for human consumption. The aim of this study was to determine the levels of vitamin D₃ and its metabolites after feeding of high levels of vitamin D₃ to beef animals to alleviate the effects of beta-agonist supplementation in feedlot cattle and to determine the safety of tissues after ultra-high levels of supplementation of vitamin D₃ . In this study, 20 young steers received neither beta agonist nor vitamin D₃ (control, C), 20 animals each received zilpaterol hydrochloride (Z) and vitamin D₃ . Various levels of vitamin D₃ was administered (xM=x million units), for a given number (y) of days (yD) in some cases withdrawn (N) for a period of z days prior to slaughter (zN). Thus the following treatment groups were examined in this study: C, Z, Z3D7M, Z6D7M, Z6D7M7N and Z9D1M. Samples of liver, meat and fat were analysed for vitamin D₃ and 25-hydroxyvitamin D₃ . An HPLC method for quantification of vitamin D₃ and its metabolites was developed and used to quantify and compare vitamin D₃ and its metabolites from liver, meat and fat. Blood serum was analysed for calcium, phosphorus and parathyroid hormone. Serum calcium concentrations were analyzed using a colorimetric assay kit whereas plasma parathyroid hormone levels (PTH) were determined by an electrochemiluminescence immunoassay employing a sandwich test principle. Levels of vitamin D₃ and 25- hydroxyvitamin D₃ differed with the amount fed - levels in the meat were generally lower than the RDA. Vitamin D₃ was most abundant in liver, especially in the group supplemented with 7 million IU of vitamin D₃ for six days (Z6D7M), accumulating at an average level of 74 μg/100 g, followed by fat (8.39 μg/100 g accumulated vitamin D₃ ), which is higher than the average RDA. Vitamin D₃ supplementation resulted in significant lowering of the M. longissimus lumborum Warner-Bratzler shear force (WBS) and myofibril fragmentation (MFL). There were several positive and negative ix correlations between levels of 25-hydroxyvitamin D₃ and calcium, phosphate, parathyroid hormone and vitamin D₃ , WBS and MFL of M. longissimus lumborum and the muscle proteolytic degradation system (calpastatin, μ-calpain and mcalpain). Controversy remains regarding the upper limit (toxic level) of vitamin D₃ for human consumption. New studies indicate that the current RDA of vitamin D is too low for sufficient health benefits. Vitamin D₃ levels measured in this study were higher than the current RDA, but within the limit proposed by recent clinical studies. Vitamin D₃ as a tool for a meat tenderizer seems not providing reliable results because in this study the control group exhibited lower shear force than the treated groups who received vitamin D₃ zilpaterol supplementation. This is an indication that the tenderazation mechanism by vitamin D₃ still needs further elucidation before vitamin D₃ supplementation can confidently be used as a tool for meat tenderization.

M.Sc. (Biochemistry) / Tuberculosis (TB) is an infectious disease that worldwide, is more prevalent in Africans compared to Caucasians. TB also affects males to a greater extent than females. Genetic associations between VDR sequence variants and TB is often inconsistent across different populations. Epigenetic and environmental factors, as well as ethnicity may confound associations found between VDR and TB...

M.Sc. / Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis). TB is a multifactorial disease, influenced by both environmental and genetic factors. Susceptibility varies among individuals and is likely explained by differential environmental exposures and genetic factors. For example, Africans are more susceptible to TB than non-Africans, likely attributed to their generally lower socioeconomic status and possible higher frequencies of ‘susceptible’ genetic variants. Similarly, males are more susceptible to TB than females, presumably as a consequence of gender-based sociocultural differences as well as biological and/or genetic differences. From a host genetic perspective, TB is a complex disease associated with variants from several genes. The Vitamin D Receptor (VDR) gene, coding for the VDR protein, has received much attention as a candidate gene; the VDR mediates vitamin D functions, of which one is to restrict M. tuberculosis survival in macrophages. Several studies attempting to associate VDR single nucleotide polymorphisms (SNPs) with TB susceptibility have given inconsistent results. Factors suggested to contribute to these inconsistencies include confounding environmental factors as well as higher VDR genetic/haplotypic diversity and less linkage disequilibrium (LD) in African populations compared to non-African populations. However, epigenetic variation has not yet been considered as an additional confounding factor leading to inconsistencies in genetic association studies for TB and VDR. vi Epigenetic factors are heritable and pivotal to regulate gene transcription. Moreover, epigenetic factors are highly susceptible to environmental influences and have been shown to be the underlying factor in certain disease aetiologies. Not only are epigenetic factors susceptible to environmental influences, but also to genetic factors acting in cis or in trans. An example is the formation or elimination of a methylatable CpG by a SNP. On the other hand, epigenetic factors may influence the genotype through formation of methylation-induced SNPs. The synergistic effect of genetic variants, epigenetic variants and the environment on disease is known as the common disease genetic epigenetic (CDGE) hypothesis. The CDGE hypothesis supports the study of both genetic and epigenetic variants to provide a better understanding of disease aetiologies and to increase the power of association studies.

Objectives: To assess the association between serum 25(OH)D and body mass index (BMI) in pre- and early-adolescents and to determine whether this association varies by demographic/clinical characteristics. Methods: Vitamin D status was determined using serum 25(OH)D in healthy pre- and early adolescents in Pittsburgh, PA (deficiency=/mL, insufficiency=12-/mL, sufficiency=≥20 ng/mL). Adiposity was quantified using BMI percentile (normal=<85th, overweight=>85th-95th, obese=>95th). The relationship between serum 25(OH)D and adiposity was assessed in the total population and after stratification by gender, race, Fitzpatrick skin type, age, and Tanner stage. Results: 294 children (mean age 10.2 + 2.1 years; 60% African American; median serum 25(OH)D=27.0 ng/mL) were studied. Serum 25(OH)D was significantly lower in obese (n=72) vs. overweight (n=48) and normal weight (n=171) participants at 23.6, 29.5, and 28.2 ng/mL, respectively; p=0.015. This trend remained significant for early adolescents but did not differ after stratification by other demographic/clinical characteristics. A significant negative correlation was found between BMI and serum 25(OH)D (r = -0.315; p=0.000). Regression analysis predicted that 25% of the variance in serum 25(OH)D levels was attributed to BMI, gender, race, skin type, age, pubertal status, daily vitamin D and calcium intake, sun exposure, and sunscreen use, with Tanner stage being the only significant independent predictor. Conclusions: A significant inverse association between serum 25(OH)D and adiposity was observed in a population of pre- and early adolescents. This relationship was stronger in early adolescents. A meta-analysis to further explore this association in pediatric populations is warranted.

Receptor interacting protein kinase 1 (RIPK1) is an enzyme acting downstream of tumor necrosis factor alpha to control cell survival and death. RIPK1 expression has been reported to cause drug resistance in cancer cells; but so far, no published studies have investigated the role of RIPK1 in vitamin D action. In the present study, we investigated whether RIPK1 played any role in 1,25-dihydroxyvitamin D3 (1,25D3)-induced growth suppression. In our studies, RIPK1 decreased the transcriptional activity of vitamin D receptor (VDR) in luciferase reporter assays independently of its kinase activity, suggesting a negative role of RIPK1 in 1,25D3 action. RIPK1 also formed a complex with VDR and deletion analyses mapped the RIPK1 binding region to the C-terminal ligand-binding domain of VDR. Subcellular fractionation analyses indicated that RIPK1 increased VDR retention in the cytoplasm, which may account for the inhibition of VDR transcriptional activity. Consistent with the reporter analyses, 1,25D3-induced growth suppression was more pronounced in RIPK1-null mouse embryonic fibroblasts (MEF) and RIPK1 knockdown ovarian cancer cells than control cells. We have also shown that VDR was involved in RIPK1-mediated cell death pathway in a cell line specific manner. In vivo study showed that VDR deletion delayed the necroptotic response to tumor necrosis factor alpha in mice. Western blot analyses of platinum sensitive and resistant cell lines showed a correlation between RIPK1 expression and drug resistance, suggesting a possible role of RIPK1 in drug resistance. In conclusion, this study is the first to define RIPK1 as a VDR repressor, projecting RIPK1 depletion as a potential strategy to increase the potency of 1,25D3 and its analogs for cancer intervention.