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Includes bibliographical references (leaves 226-273.) Investigates the regulation of the expression of CYP27B1, CYP24 and vitamin D receptor (VDR) mRNA, both in the bone and in the kidney, with the aim to determine whether the regulation of the vitamin D metabolism in the bone is independent from that in the kidney. The effects of age, dietary calcium and vitamin D status on the expression of these genes in both the kidney and the bone, as well as on a number of biochemical factors known to regulate the renal metabolism of 1,25D, such as PTH, calcium and 1,25D itself, were examined. CYP27B1 mRNA expression was also studied in histological sections of rat femoral bone.

Includes bibliographical references. Electronic publication; Full text available in PDF format; abstract in HTML format. Investigates the regulation of the expression of CYP27B1, CYP24 and vitamin D receptor (VDR) mRNA, both in the bone and in the kidney, with the aim to determine whether the regulation of the vitamin D metabolism in the bone is independent from that in the kidney. The effects of age, dietary calcium and vitamin D status on the expression of these genes in both the kidney and the bone, as well as on a number of biochemical factors known to regulate the renal metabolism of 1,25D, such as PTH, calcium and 1,25D itself, were examined. CYP27B1 mRNA expression was also studied in histological sections of rat femoral bone. Electronic reproduction.[Australia] :Australian Digital Theses Program,2001.

Thesis (M.F.C.S.)--Bowling Green State University, 2010. / Document formatted into pages; contains vi, 59 p. Includes bibliographical references.

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Nuclear receptors (NRs) are ligand-activated transcription factors that regulate the expression of genes involved in all physiological activities. Disruption in NR function (e.g. mutations) can lead to a variety of diseases; making these receptors important targets for drug discovery. The ability to bind a broad range of 'drug-like' molecules also make these receptors attractive candidates for protein engineering, such that they can be engineered to bind novel small molecule ligands, for several applications. One application is the creation of potential molecular switches, tools that can be used for controlling gene expression. Gaining knowledge of specific molecular interactions that occur between a receptor and its ligand is of interest, as they contribute towards the activation or repression of target genes. The focus of this work has been to investigate the structural and functional relationships between the human vitamin D receptor (hVDR) and its ligands. To date, mutational assessments of the hVDR have focused on alanine scanning and residues typically lining the ligand binding pocket (LBP)that are involved in direct interactions with the ligand. A comprehensive analysis of the tolerance of these residues in the binding and activation of the receptor by its ligands has not been performed. Furthermore, residues not in contact with the ligand or that do not line the LBP may also play an important role in determining the activation profiles observed for NRs, and therefore need to be explored further. In order to engineer and use the hVDR in chemical complementation, a genetic selection system in which the survival of yeast is linked to the activation of a NR by an agonist, the hVDR gene was isolated from cDNA. To gain insight into how chemical and physical changes within the ligand binding domain (LBD) affect receptor-ligand interactions, libraries of hVDR variants exploring the role and tolerance of hVDR residues were created. To develop a comprehensive mutational analysis while also engineering the hVDR to bind a novel small molecule ligand, a rational and a random mutagenic approach were used to create the libraries. A variant, hVDRC410Y, that displayed enhanced activity with lithocholic acid (LCA), a known hVDR ligand, and novel activation with cholecalciferol (chole), a precursor of the hVDR's natural ligand known not to activate the wild-type hVDR, was discovered. The presence of a tyrosine at the C410 position resulting in novel activation profiles with both LCA and chole, and the fact that this residue does not line the hVDR's LBP led to interest in determining whether a physical or chemical property of the residue was responsible for the observed activity. When residue C410 was further assessed for its tolerance to varying amino acids, the results indicated that bulkiness at this end of the pocket is important for activation with these ligands. Both LCA and chole have reduced molecular volumes compared to the natural ligand, 1alpha, 25(OH)2D3. As a result, increased bulkiness at the C410 position may contribute additional molecular interactions between the receptor and ligands. Results obtained throughout this work suggest that the end of the hVDR's LBP consisting of two ligand anchoring residues, H305 and H397, and residue C410 tolerates structural variations, as numerous variants with mutations at these positions displayed enhanced activity. The receptor contains two tyrosines, Y143 and Y147, which were targeted for mutagenesis in one of the rationally designed libraries, located at the exact opposite end of the pocket. In an effort to gain further insight into the role of these residues at the other end of the LBP, mutagenesis assessing the tolerance of tyrosines 143 and 147 was performed. Overall, most changes at these positions proved to be detrimental to the function of the receptor supporting the hypothesis that this end of the LBP is less tolerant of structural changes, compared to the opposite end consisting of residues H305, H397 and C410. Overall, a better understanding of the structural and functional relationships between the human vitamin D receptor (hVDR) and its ligands was achieved. The effects of residue C410 on specificity and activation with the different ligands studied were unforeseen, as this residue does not line the receptor's ligand binding pocket (LBP). However, they serve as an example of the significant impact distant residues can have on receptor activation and also emphasize the important role physical properties of residues, such as volume, can play for specific ends of the LBP compared to chemical properties.

The human vitamin D receptor (hVDR) is a member of the nuclear receptor superfamily, involved in calcium and phosphate homeostasis; hence implicated in a number of diseases, such as Rickets and Osteoporosis. This receptor binds 1α,25-dihydroxyvitamin D3 (also referred to as 1,25(OH)2D3) and other known ligands, such as lithocholic acid. Specific interactions between the receptor and ligand are crucial for the function and activation of this receptor, as implied by the single point mutation, H305Q, causing symptoms of Type II Rickets. In this work, further understanding of the significant and essential interactions between the ligand and the receptor were deciphered, through a combination of rational and random mutagenesis. A hVDR mutant, H305F, was engineered with increased sensitivity towards lithocholic acid, with an EC50 value of 10 µM and 40 + 14 fold activation in mammalian cell assays, while maintaining wild-type activity with 1,25(OH)2D3. Furthermore, via random mutagenesis, a hVDR mutant, H305F/H397Y, was discovered to bind a novel small molecule, cholecalciferol, a precursor in the 1α,25-dihydroxyvitamin D3 biosynthetic pathway, which does not activate wild-type hVDR. This variant, H305F/H397Y, binds and activates in response to cholecalciferol concentrations as low as 100 nM, with an EC50 value of 300 nM and 70 + 11 fold activation in mammalian cell assays.