Lui, Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 163-171). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.v / Acknowledgements --- p.vii / Abbreviations --- p.ix / Table of Content --- p.x / Chapter Chapter 1. --- Introduction / Chapter 1.1 --- Overview and Endocrinology of hormones and hormone receptors --- p.1 / Chapter 1.2 --- Hormone receptors: membrane bounded receptors --- p.3 / Chapter 1.3 --- Hormone receptors: steroid nuclear receptors --- p.4 / Chapter 1.4 --- "Estrogen, estrogen receptor alpha and beta (ERa, ERβ) and prostate gland" --- p.6 / Chapter 1.5 --- Orphan nuclear receptors --- p.10 / Chapter 1.6 --- The first orphan receptors identified-estrogen receptor related receptors --- p.12 / Chapter 1.6.1 --- Estrogen receptor related receptor alpha (ERRα) --- p.13 / Chapter 1.6.2 --- Estrogen receptor related receptor alpha (ERRβ) --- p.17 / Chapter 1.6.3 --- Estrogen receptor related receptor alpha (ERRγ) --- p.19 / Chapter 1.7 --- Aim of study --- p.21 / Figure 1.1 Mechanism of activation of classical nuclear receptor by ligand --- p.23 / Figure 1.2 Distribution of ERa and ERβ in human body --- p.24 / Chapter Chapter 2. --- Methods and Materials / Chapter 2.1 --- Origin and supply of Noble rats --- p.25 / Chapter 2.2 --- Cell culture / Chapter 2.2.1 --- Cell lines and culture media --- p.26 / Chapter 2.2.2 --- Cell culture onto cover slips for immunohistochemistry --- p.27 / Chapter 2.3 --- RNA preparation / Chapter 2.3.1 --- Total RNA extraction --- p.27 / Chapter 2.3.2 --- mRNA extraction by Oligote´xёØ procedure --- p.29 / Chapter 2.3.3 --- mRNA extraction by Fast Track 2.0 procedure --- p.30 / Chapter 2.4 --- Molecular cloning by Rapid Amplification of cDNA Ends (RACE) / Chapter 2.4.1 --- Molecular cloning of rERRα --- p.31 / Chapter 2.4.2 --- Molecular cloning of rERRβ --- p.36 / Chapter 2.4.3 --- Molecular cloning of rERRγ --- p.42 / Chapter 2.5 --- Molecular cloning into pCRII TOPO cloning vector --- p.47 / Chapter 2.6 --- Sequencing analysis of DNA sequence by dRodamine® or BigDye® --- p.47 / Chapter 2.7 --- DNA sequence analysis --- p.49 / Chapter 2.8 --- Reverse transcription and RT-PCR --- p.49 / Chapter 2.9 --- Southern blotting analysis / Chapter 2.9.1 --- Preparation of DNA blot membrane --- p.51 / Chapter 2.9.2 --- Purification of DNA fragment from agarose gel for DIG-DNA labeling --- p.52 / Chapter 2.9.3 --- Preparation of the DIG-labeled DNA probe --- p.53 / Chapter 2.9.4 --- Membrane hybridization and colorimetric detection --- p.53 / Chapter 2.10 --- In-situ hybridization histochemistry / Chapter 2.10.1 --- Linearization of DNA plasmid --- p.55 / Chapter 2.10.2 --- Synthesis of riboprobe --- p.56 / Chapter 2.10.3 --- Hybridization and detection --- p.56 / Chapter 2.11 --- Western blotting analysis / Chapter 2.11.1 --- Protein extraction --- p.59 / Chapter 2.11.2 --- Casting of SDS-PAGE electrophoresis --- p.59 / Chapter 2.11.3 --- Polyacrylamide gel electrophoresis --- p.61 / Chapter 2.11.4 --- Protein blotting analysis --- p.61 / Chapter 2.12.1 --- Immunohistochemistry / Chapter 2.12.1 --- Histological preparation --- p.63 / Chapter 2.12.2 --- Immunohistochemistry --- p.64 / Table 1. List of culture media --- p.66 / Table 2. Primer sequences for RACE-PCR --- p.67 / Table 3. PCR conditions for RT-PCR --- p.68 / Table 4. Primer sequences for RT-PCR --- p.68 / Table 5. Reagent mixtures for linearization of the plasmid DNA --- p.69 / Table 6. Riboprobe synthesis by in-vitro transcription --- p.70 / Chapter Chapter 3. --- Results / Chapter 3.1 --- Cloning of full-length cDNA of rERRs by RACE-PCR --- p.71 / Chapter 3.2 --- Cloning of full-length cDNA of rERRα from rat ovary cDNA library --- p.72 / Chapter 3.3 --- Cloning of full-length cDNA of rERRβ from rat ventral prostate --- p.76 / Chapter 3.4 --- Cloning of full-length cDNA of rERRγ from rat prostate --- p.80 / Chapter 3.5 --- Expression distribution of ERRs detected by RT-PCR --- p.83 / Chapter 3.6 --- mRNA expression of ERRs detected by in-situ hybridization --- p.86 / Chapter 3.7 --- Protein expression of ERRa and ERRγ detected by western blotting --- p.87 / Chapter 3.8 --- Expression of ERRa and ERRγ detected by immunohistochemistry --- p.88 / Figure 3.1 Full-length DNA sequence of rERRα --- p.92 / Figure 3.2 Predicted amino acid sequence of rERRα --- p.93 / "Figure 3.3 DNA sequence alignment of rat, mouse and human ERRα" --- p.94 / "Figure 3.4 Amino acid sequence alignment analysis of rat, mouse and human ERRα" --- p.95 / Figure 3.5 Full-length DNA sequence of rERRβ --- p.96 / Figure 3.6 Predicted amino acid sequence of rERRβ --- p.97 / "Figure 3.7 DNA sequence alignment of rat, mouse and human ERRβ" --- p.98 / "Figure 3.8 Amino acid sequence alignment analysis of rat, mouse and human ERRβ" --- p.99 / Figure 3.9 Full-length DNA sequence of rERRγ --- p.100 / Figure 3.10 Predicted amino acid sequence of rERRγ --- p.101 / "Figure 3.11 DNA sequence alignment of rat, mouse and human ERRγ" --- p.102 / "Figure 3.12 Amino acid sequence alignment analysis of rat, mouse and human ERRγ" --- p.103 / Figure 3.13 Restriction enzyme cutting of full-length plasmids --- p.104 / Figure 3.14 Expression pattern of rERRα in male sex accessory sex glands by RT-PCR --- p.105 / Figure 3.15 Expression pattern of rERRα in urinary system and female sex organs by RT-PCR --- p.106 / Figure 3.16 Tissue expression of rERRα by RT-PCR --- p.107 / Figure 3.17 In-situ hybridization of ERRα in ovary --- p.108 / Figure 3.18 Western blotting of ERRα --- p.109 / Figure 3.19 Immunohistochemistry of ERRα in ovary --- p.110 / Figure 3.20 Expression pattern of rERRβ in male sex accessory sex glands by RT-PCR --- p.111 / Figure 3.21 Expression pattern of rERRβ in urinary system and female sex organs by RT-PCR --- p.112 / Figure 3.22 Tissue expression of rERRβ by RT-PCR --- p.113 / Figure 3.23 In-situ hybridization of ERRβ in rat prostate --- p.114 / Figure 3.24 Negative control of in-situ hybridization of ERRβ in rat prostate --- p.115 / Figure 3.25 Expression pattern of rERRγ in male sex accessory sex glands by RT-PCR --- p.116 / Figure 3.26 Expression pattern of rERRy in urinary system and female sex organs by RT-PCR --- p.117 / Figure 3.27 Tissue expression of rERRγ by RT-PCR --- p.118 / Figure 3.28 Expression pattern of rERRγ in different prostatic cancer cell lines and xenografts by RT-PCR --- p.119 / Figure 3.29 In-situ hybridization of ERRγ in rat prostate --- p.120 / Figure 3.30 Negative control of in-situ hybridization of ERRβ in rat prostate --- p.121 / Figure 3.31 Western blotting of ERRγ --- p.122 / Figure 3.32 Immunohistochemistry of ERRγ in ERRy-transfected MCF-7 cells --- p.123 / Figure 3.33 Immunohistochemistry of ERRγ in ventral prostate of rat --- p.124 / Figure 3.34 Immunohistochemistry of ERRγ in lateral prostate of rat --- p.125 / Figure 3.35 Immunohistochemistry of ERRγ in dorsal prostate of rat --- p.126 / Figure 3.36 Immunohistochemistry of ERRγ in testis of rat --- p.127 / Figure 3.37 Immunohistochemistry of ERRγ in epididymis of rat --- p.128 / Figure 3.38 Immunohistochemistry of ERRγ in brown adipose tissues of rat --- p.129 / Figure 3.39 Immunohistochemistry of ERRγ in brain of rat --- p.130 / Figure 3.40 Immunohistochemistry of ERRγ in brain of rat --- p.131 / Chapter Chapter 4. --- Discussion / Chapter 4.1 --- Sequence analysis of the full-length cDNA sequences of the rat estrogen receptor-related receptors (ERRs) --- p.132 / Chapter 4.2 --- Ligand independence and constitutive self-activation of estrogen receptor-related receptors --- p.133 / Chapter 4.3 --- Board expression pattern of estrogen receptor-related receptors --- p.138 / Chapter 4.3.1 --- Board expression pattern of estrogen receptor-related receptor alpha --- p.138 / Chapter 4.3.2 --- Board expression pattern of estrogen receptor-related receptor beta --- p.140 / Chapter 4.3.3 --- Board expression pattern of estrogen receptor-related receptor gamma --- p.141 / Chapter 4.4 --- Expression of ERRs in the prostate gland --- p.143 / Chapter 4.5 --- Expression of ERRs in the prostatic cell lines and cancer xenografts --- p.147 / Chapter 4.6 --- Expression of ERRs in the ERRγ-transfected MCF-7 cells --- p.149 / Chapter 4.7 --- Expression of ERRs in the testis and epididymis --- p.149 / Chapter 4.8 --- Expression of ERRs in the adipose tissue --- p.150 / Chapter 4.9 --- Expression of ERRs in the ovary --- p.151 / Chapter 4.10 --- Expression of ERRs in the brain --- p.153 / Figure 5.1 Map of full-length clone of rERRα --- p.155 / Figure 5.2 Map of full-length clone of rERRβ --- p.156 / Figure 5.3 Map of full-length clone of rERRα --- p.157 / Figure 5.4 Comparison of the homology of amino acid sequences amongst ERs and ERRs --- p.158 / Figure 5.5 Phylogeny tree of nuclear receptors --- p.159 / Figure 5.6 Relationship of different prostatic cell lines and xenografts --- p.160 / Chapter Chapter 5. --- Summary --- p.161 / References --- p.163-171

Antibodies specific for DNA, with varying degrees of sequence specificity, are common in many autoimmune diseases including systemic lupus erythematosus. The presence of anti-DNA antibodies is a useful determinant in arriving at a prognosis in these conditions. Given the prevalence of these diseases in both the developing and developed world and the difficulty that often accompanies diagnosis of autoimmune diseases, it is desirable to have sensitive, rapid, and inexpensive diagnostic tools for these diseases. Because of the great sensitivity of electrochemical techniques and their potential utility in characterizing interactions between macromolecules, electrochemistry has great potential as a diagnostic tool for any disease involving antibodies. Anti-DNA antibodies are present in many autoimmune diseases, notably systemic lupus erythematosus. Since DNA is a stable and well-characterized antigen, an electrochemical-based assay is particularly useful for diagnosis of these diseases. <p>The impedance of a gold surface was measured in the presence and absence of single- and double-stranded DNA monolayers. The DNA monolayer was diluted with butanethiol in order to provide a surface with more accessible binding sites than an undiluted monolayer. The change in impedance of the DNA monolayer following exposure to various small molecules and macromolecules was assessed. The molecules used included polyamines that induce conformational changes in DNA, proteins which bind DNA specifically, proteins which bind DNA non-specifically, and proteins which do not bind DNA. The presence of a DNA monolayer, whether single- or double-stranded, increased the impedance of the gold surface and dilution of the monolayers by butanethiol decreased the impedance, as expected. When exposed to polyamines, the impedance of the DNA monolayer decreased further. This could be due to lowered charge repulsion, to DNA condensation, or to a combination of both. When methylated bovine serum albumin was exposed to the monolayer, there was an increase in impedance. Conversely, when bovine serum albumin was exposed to the monolayer, the impedance was only increased at very high concentrations of protein. The increase following exposure to high concentrations of bovine serum albumin was likely due to deposition of protein on to the monolayer. The specificity of these interactions was illustrated by experiments with the antibody Hed 10, which binds single-stranded but not double-stranded DNA. Exposure to Hed 10 only caused a significant change in impedance when exposed to monolayers of single-stranded DNA.<p>The decreased impedance of the DNA monolayer caused by the presence of polyamines is consistent with the known structural perturbations induced by these molecules as measured with other methods. Similarly, the increase in impedance caused by the presence of proteins which bind DNA is consistent with increased steric interference by the protein-DNA complex. The failure of proteins which do not bind DNA to affect the impedance of the monolayer indicated that the effects in the experiments with DNA-binding proteins were due to protein binding and not other factors. The specificity of the assay as demonstrated by the results of the experiments with Hed 10 suggest that impedance-based measurements may provide the basis for a reliable, sensitive, and inexpensive assay for detecting the presence of anti-DNA antibodies in the serum of autoimmune disease patients.

Antibodies specific for DNA, with varying degrees of sequence specificity, are common in many autoimmune diseases including systemic lupus erythematosus. The presence of anti-DNA antibodies is a useful determinant in arriving at a prognosis in these conditions. Given the prevalence of these diseases in both the developing and developed world and the difficulty that often accompanies diagnosis of autoimmune diseases, it is desirable to have sensitive, rapid, and inexpensive diagnostic tools for these diseases. Because of the great sensitivity of electrochemical techniques and their potential utility in characterizing interactions between macromolecules, electrochemistry has great potential as a diagnostic tool for any disease involving antibodies. Anti-DNA antibodies are present in many autoimmune diseases, notably systemic lupus erythematosus. Since DNA is a stable and well-characterized antigen, an electrochemical-based assay is particularly useful for diagnosis of these diseases. <p>The impedance of a gold surface was measured in the presence and absence of single- and double-stranded DNA monolayers. The DNA monolayer was diluted with butanethiol in order to provide a surface with more accessible binding sites than an undiluted monolayer. The change in impedance of the DNA monolayer following exposure to various small molecules and macromolecules was assessed. The molecules used included polyamines that induce conformational changes in DNA, proteins which bind DNA specifically, proteins which bind DNA non-specifically, and proteins which do not bind DNA. The presence of a DNA monolayer, whether single- or double-stranded, increased the impedance of the gold surface and dilution of the monolayers by butanethiol decreased the impedance, as expected. When exposed to polyamines, the impedance of the DNA monolayer decreased further. This could be due to lowered charge repulsion, to DNA condensation, or to a combination of both. When methylated bovine serum albumin was exposed to the monolayer, there was an increase in impedance. Conversely, when bovine serum albumin was exposed to the monolayer, the impedance was only increased at very high concentrations of protein. The increase following exposure to high concentrations of bovine serum albumin was likely due to deposition of protein on to the monolayer. The specificity of these interactions was illustrated by experiments with the antibody Hed 10, which binds single-stranded but not double-stranded DNA. Exposure to Hed 10 only caused a significant change in impedance when exposed to monolayers of single-stranded DNA.<p>The decreased impedance of the DNA monolayer caused by the presence of polyamines is consistent with the known structural perturbations induced by these molecules as measured with other methods. Similarly, the increase in impedance caused by the presence of proteins which bind DNA is consistent with increased steric interference by the protein-DNA complex. The failure of proteins which do not bind DNA to affect the impedance of the monolayer indicated that the effects in the experiments with DNA-binding proteins were due to protein binding and not other factors. The specificity of the assay as demonstrated by the results of the experiments with Hed 10 suggest that impedance-based measurements may provide the basis for a reliable, sensitive, and inexpensive assay for detecting the presence of anti-DNA antibodies in the serum of autoimmune disease patients.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 186-201). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (p. 103-113).

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 190-204). Available also in a digital version from Dissertation Abstracts.

The mouse node is the structural equivalent of Spemann's Organizer. The transcription factor Foxa2 (HNF3beta) is required for node and notochord specification and its enhancer has been characterized to a 500bp element that drives the expression of a reporter gene to the node and notochord in transgenic embryos. / The aim of the study was to identify sequence elements, within this enhancer, required to drive node/notochord specific-expression. Since, in the Xenopus organizer, Siamois activates organizer-specific gene expression, it was tested whether this factor could activate expression from the Foxa2 node-specific enhancer. Using deletion analysis, the response element was mapped to a 33bp region and it was shown that this element was both necessary and sufficient for reporter gene activation by X-Siamois . Furthermore, it was shown that X-Siamois can bind this DNA element and two sequence motifs required for binding and transactivation by X-Siamois were identified. Preliminary results suggest that the 33bp element, within the 500bp enhancer, is required for the maintenance of expression in the notochord of transgenic mice.

This thesis gives insight into the way that transcription of the insulin gene is regulated by nutrients. This is achieved primarily by characterising a MAP kinase pathway which links glucose metabolism to the activation of a beta cell transcription factor IUF1. An understanding of the precise mechanisms by which nutrients control beta cell function may be invaluable for the development of artificial cell lines that can be used for gene replacement therapy. A study of the E2 element of the rat II promoter illustrated that at least three factors bound to the region. These were identified as IUF1 (complex D5), USF (complex D4) and an uncharacterised factor D3. IUF1 is a beta cell specific transcription factor that has been implicated previously in glucose responsive insulin gene transcription. IUF1 binds to the insulin promoter in response to high levels of extracellular glucose. USF has been shown to be involved in the carbohydrate responsive transcription of various hepatic genes. The recently characterised stress activated (Reactivating Kinase) MAP kinase pathway was clearly shown to be involved in mediating the link between glucose metabolism within the beta cell and the binding activity of IUF1. Phosphorylation of the factor serves to induce an alteration in protein structure, which converts the factor to an active form that shows a high affinity for its DNA binding site, thus activating transcription. The RK pathway may prove to be a crucial link between nutrient metabolism and the activity of other physiological processes.

This thesis is concerned with structural aspects of the recognition and effector functions of antibody molecules. The recognition process is investigated in the dinitrophenyl (DNP) binding mouse IgA produced by the myeloma MOPC 315. The studies on effector functions utilize a DNP binding mouse hybridoma IgG2a to examine the role of N-glycosylation in IgG. The combining site of protein 315. The involvement of tyrosyl residues in the combining site of protein 315 was examined by preparing specifically nitrated NO₂-Tyr-33_H and NO₂-Tyr-34_L derivatives of the Fv fragment of this protein. The ionizations of tnese derivatives were studied in the presence and absence of various DNP-ligands. Perturbations to the nitrotyrosine ionizations were found to be caused by the side chains of certain of these ligands, allowing an indication of the distance of these tyrosines from the bound hapten. On examination of the compatibility of these data with the model of the combining site of protein 315 proposed by Dower <en>et al. (1977) (Biochem. J. 165, 207-225) it was found that while the location of Tyr-33_H is consistent with this model, the position of Tyr-34_L is not. A remodelled combining site using the modified ring-current treatment of Perkins and Dwek (1980) (Biochemistry 19, 245-258) is presented. This allows a better rationalization of the nitration data and of previous experimental observations on protein 315. The role of the conserved C 2 domain oligosaccharide of IgG. This was examined by a functional comparison of native IgG with an aglycosylated IgG preparation. Aglycosylation was acheived by cell culture of the hybridoma cells in the presence of the glycosylation inhibitor tunicamycin. The conditions for preparation and purification of this aglycosyl IgG are described. Aglycosylated IgG is found to be correctly assembled as an H₂L₂ unit. It retains the antigen binding and Staphylococcal protein A binding abilities of the native glycosylated molecule. Using an assay system designed specifically to overcome certain problems in comparing Clq binding to different preparations of IgG it was found that the aglycosylated preparation showed only slightly reduced affinity for Clq. In addition the aglycosylated IgG is able to activate bound Cl. The above results are consistent with the structure of the Fc region being only minimally altered in the absence of oligosaccharide. The structural integrity of the aglycosylated molecule may be compromised however, as its ability to bind to monocyte Fc receptor is significantly reduced. In addition the aglycosylated molecule becomes much more susceptible to proteolytic digestion. A computational model-building analysis of the quaternary structure of Fc allows an explanation of at least some of the effects of aglycosylation in terms of reduced conformational stability of the C_H2 domains.

No description available.