Siu-wai Michael Sung. / Thesis submitted in: December 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 97-111). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abbreviations --- p.v / Table of Contents --- p.vii / Chapter Chapter 1 --- General Introduction / Chapter l. 1 --- Drosophila as a model for studying development --- p.1 / Chapter l .2 --- The formation of the body axis in Drosophila --- p.2 / Chapter l .3 --- Maternal genes are essential for development --- p.9 / Chapter 1.4 --- Maternal gene bicoid is essential for formation of the anterior structures in the embryo --- p.13 / Chapter 1.5 --- Establishment of an anterior to posterior bicoid protein gradient --- p.13 / Chapter 1.6 --- The bicoid protein gradient controls the downstream zygotic target genes in a concentration-dependent manner --- p.17 / Chapter 1.7 --- Bicoid protein acts as transcriptional regulators \9 --- p.19 / Chapter 1.8 --- Bicoid protein acts as transcriptional regulators --- p.21 / Chapter 1.9 --- The anterior localization of bcd mRNA --- p.21 / Chapter 1.10 --- Components required for bcd mRNA localization at anterior pole of oocyte / Chapter 1.10.1 --- Cis-acting elements --- p.22 / Chapter 1.10.1.1 --- BLE1 at 3' UTR directs localization of bcd mRNA --- p.23 / Chapter 1.10.2 --- Trans-acting elements / Chapter 1.10.2.1 --- "Exuperantia, swallow, and staufen are necessary for localization for bcd mKNA" --- p.27 / Chapter 1.10.2.2 --- exu protein is an absolute requirement for localization for bcd mRNA --- p.30 / Chapter 1.10.2.3 --- Microtubules dependence of localization --- p.31 / Chapter 1.11 --- Functions of exu in localization of bcd mRNA --- p.32 / Chapter 1.12 --- Characteristics of Bicoid protein and Bic-D gene --- p.33 / Chapter 1.13 --- Aim of Project --- p.36 / Chapter CHAPTER 2 --- Materials and Methods / Chapter 2.1 --- Fly Food --- p.37 / Chapter 2.2 --- Conditions in maintaining the fly stocks and working stocks --- p.37 / Chapter 2.3 --- Localization of exu protein and other intracellular elements by indirect immunofluorescence detection / Chapter 2.3.1 --- Immunohistrochemical distribution of exu and Bic-D protein --- p.38 / Chapter 2.3.2 --- Immunohistrochemical distribution of β-tubulin --- p.39 / Chapter 2.4 --- Preparation of total protein from the female and male flies --- p.41 / Chapter 2.5 --- Analysis of interactions between exu and trans-acting elements / Chapter 2.5.1 --- 35S-methionine metabolic labelling and immunoprecipitation by RIPA buffer --- p.41 / Chapter 2.5.2 --- 35S-methionine metabolic labelling and immunoprecipitation by Mach and Lehmann buffer system --- p.43 / Chapter 2.6 --- Co-immunoprecipitation of exu and Bic-D protein / Chapter 2.6.1 --- Co-immunoprecipitation of exu and Bic-D protein synthesized by in vitro coupled transcription and translation system with modified Mach and Lechmann buffer system --- p.44 / Chapter 2.7 --- in vivo ovary extract co-immunoprecipitation / Chapter 2.7.1 --- in vivo ovary extraction co-immunoprecipitation of exu and Bic-D protein with modified Mach and Lehmann buffer system supplemented with recombinant exu protein --- p.45 / Chapter CHAPTER 3 --- Results / Chapter 3.1 --- Analysis of co-localization of exu and Bic-D protein by double immuno-fluorescence staining on w1118 flies --- p.47 / Chapter 3.2 --- Analysis of co-localization of exu protein and β-tubulin protein by double immuno-fluorescence staining on w1118 flies --- p.51 / Chapter 3.3 --- Analysis of co-localization of exu and Bic-D protein by double immuno-fluorescence staining on Bic-D mutants --- p.55 / Chapter 3.4 --- Co-immunoprecipitation of exu and Bic-D protein synthesized by in vitro coupled transcription and translation system --- p.61 / Chapter 3.5 --- 35S-Methionine metabolic labelling and co-immunoprecipitation of exu and Bic-D protein with RIP A buffer system --- p.65 / Chapter 3.6 --- 35S-Methionine metabolic labelling and co-immunoprecipitation of exu and Bic-D protein with Mach and Lehmann buffer system --- p.68 / Chapter 3.7 --- in vivo ovary extract co-immunoprecipitation of exu and Bic-D protein with modified Mach and Lehmann buffer system supplemented with recombinant exu protein --- p.71 / Chapter CHAPTER 4 --- Discussion / Chapter 4.1 --- Analysis of co-localization of exu protein and other intracellular elements by indirect double immunofluorescence staining detection --- p.74 / Chapter 4.2 --- Analysis of co-localization of exu and BicD protein by double immuno- fluorescence staining on Bic-D mutants --- p.78 / Chapter 4.3 --- Co-immunoprecipitation of exu and BicD protein synthesized by in vitro coupled transcription and translation system --- p.79 / Chapter 4.4 --- Analysis of interactions between exu and trans-acting elements by 35S- Methionine metabolic labelling and immunoprecipitation --- p.82 / Chapter 4.5 --- "in vivo ovary extract coimmunoprecipitation of exu and Bic-D protein with modified Mech and Lehmann buffer system, supplemented with recombinant exu protein" --- p.84 / Chapter 4.6 --- Recent developments on the concept of ribonucleoprotein --- p.86 / Appendix A Supplementary protocols --- p.91 / Appendix B Reagents --- p.95 / Reference --- p.97

by Lau Hoi Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 83-96). / Abstract also in Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / Table of contents --- p.vi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Endocrine control of normal and abnormal growth of prostate --- p.1 / Chapter 1.1.1 --- Androgen regulation of prostate gland --- p.1 / Chapter 1.1.2 --- Estrogen regulation of prostate gland --- p.4 / Chapter 1.2 --- Gonadotropin-releasing hormone plays a central role in reproduction --- p.6 / Chapter 1.2.1 --- GnRH gene --- p.7 / Chapter 1.2.2 --- GnRH receptor --- p.9 / Chapter 1.3 --- Therapeutic strategies using GnRH analogs to treat prostate cancer --- p.12 / Chapter 1.4 --- Expression of GnRH or its receptor in reproductive tissues --- p.12 / Chapter 1.4.1 --- Expression of GnRH in reproductive --- p.13 / Chapter 1.4.2 --- Expression of GnRH and its receptor in pituitary and reproductive tissues --- p.13 / Chapter 1.5 --- Animal models for the study of prostate cancer --- p.15 / Chapter 1.5.1 --- Nobel rat inducible model --- p.15 / Chapter 1.5.2 --- Androgen dependent rat Dunning prostatic tumor --- p.16 / Chapter 1.5.3 --- Androgen-independent prostatic carcinoma line of Noble rat --- p.18 / Chapter 1.6 --- Aim of study --- p.18 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Origin and supply of Nobel rat --- p.20 / Chapter 2.2 --- Induction of dysplasia in Nobel rat prostate gland by long-term treatment with steroids --- p.20 / Chapter 2.2.1 --- Chemicals --- p.20 / Chapter 2.2.2 --- Preparation of steroid hormone-filled Silastic tubings --- p.20 / Chapter 2.2.3 --- Surgical implantation of Silastic® tubings --- p.21 / Chapter 2.2.4 --- Protocols of hormonal treatments --- p.21 / Chapter 2.3 --- Androgen- dependent Dunning rat prostatic adenocarcinoma --- p.22 / Chapter 2.4 --- Androgen- independent prostatic carcinoma line (ALT) of Noble rat --- p.22 / Chapter 2.5 --- Detection of mRNA expression of gonadotropin- releasing hormone (GnRH) in normal and neoplastic rat prostates --- p.23 / Chapter 2.5.1 --- Preparation of tissue for total RNA extraction --- p.23 / Chapter 2.5.2 --- Total RNA extraction --- p.24 / Chapter 2.5.3 --- Reverse-transcription Polymerase Chain Reaction (RT-PCR) --- p.25 / Chapter 2.5.4 --- Purification of DNA fragments from agarose gels --- p.27 / Chapter 2.5.5 --- Subcloning of DNA into vector --- p.27 / Chapter 2.5.6 --- Nucleotide sequencing --- p.30 / Chapter 2.5.7 --- Southern blot analysis --- p.32 / Chapter 2.5.7.1 --- Southern blotting --- p.32 / Chapter 2.5.7.2 --- Preparation of α-32P-dCTP labelled GnRH probe --- p.32 / Chapter 2.5.7.3 --- Hybridization --- p.33 / Chapter 2.6 --- Detection of mRNA expression of gonadotropin-releasing hormone receptor (GnRH-R) in normal and neoplastic rat prostates --- p.34 / Chapter 2.6.1 --- Cloning of GnRH-R cDNA and synthesis of its probe --- p.34 / Chapter 2.6.2 --- Detection of GnRH receptor mRNA expression in normal and dysplastic Nobel rat prostates by Southern blot --- p.36 / Chapter 2.6.3 --- Detection of GnRH receptor mRNA expression in Dunning tumor --- p.37 / Chapter 2.6.4 --- Detection of the GnRH receptor mRNA expression in AIT tumor by RT-PCR --- p.37 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Detection of mRNA expression of gonadotropin-releasing hormone (GnRH) in normal and neoplastic rat prostates --- p.38 / Chapter 3.1.1 --- Reverse -transcription Polymerase Chain Reaction (RT-PCR) --- p.38 / Chapter 3.1.2 --- Purification of DNA fragments amplified by PCR from the agarose gel --- p.38 / Chapter 3.1.3 --- Subcloning of DNA into vector --- p.39 / Chapter 3.1.4 --- Nucleotide sequencing --- p.39 / Chapter 3.1.5 --- Southern-blot analysis --- p.39 / Chapter 3.2 --- Detection of gonadotropin-releasing hormone receptor mRNA expression in normal and neoplastic rat prostates --- p.40 / Chapter 3.2.1 --- Cloning of gonadotropin-releasing hormone receptor (GnRH) cDNA and synthesis of probe from the normal Noble rat pituitary gland --- p.40 / Chapter 3.2.2 --- Detection of GnRH receptor mRNA expression in normal and dysplastic Nobel rat prostates --- p.42 / Chapter 3.2.3 --- Detection of GnRH receptor mRNA expression in rat Dunning tumor by PCR --- p.43 / Chapter 3.2.4 --- Detection of GnRH receptor mRNA expression in AIT tumor --- p.43 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Detection of mRNA expression of gonadotropin-releasing releasing hormone(GnRH) in normal and neoplastic rat prostates --- p.69 / Chapter 4.1.1 --- Expression of GnRH mRNA in normal Nobel rat prostate gland --- p.69 / Chapter 4.1.2 --- Expression of GnRH mRNA in dysplastic Nobel rat prostate --- p.71 / Chapter 4.1.3 --- Expression of GnRH mRNA in androgen-dependent rat Dunning prostatic tumor --- p.72 / Chapter 4.1.4 --- Expression of GnRH mRNA in AIT tumor --- p.74 / Chapter 4.2 --- Detection of GnRH receptor in normal and dysplastic rat prostates --- p.75 / Chapter 4.2.1 --- Negative expression of GnRH receptor in normal and dysplastic Nobel in rat prostates --- p.75 / Chapter 4.2.2 --- Positive expression of GnRH receptor mRNA in rat Dunning tumor --- p.77 / Chapter 4.2.3 --- Negative expression of GnRH receptor mRNA in ALT tumor --- p.78 / Chapter Chapter 5 --- Summary and Conclusions --- p.80 / References --- p.83

Diabetic nephropathy (DN) is one of the leading causes of end stage renal disease (ESRD) in western world and has a trend to spread in developing countries. Pathogenesis of DN is not fully elucidated. Studies of recent years showed that podocyte loss and activation of the rennin-angiotensin system (RAS), especially intra-renal RAS, played important roles in this process. Although renal biopsy is currently the most common way used to determine the expression pattern of podocyte and RAS associated molecules in DN, this invasive procedure has its own risk and is not practical for serial monitoring. We hypothesized that measurement of messenger ribonucleic acid (mRNA) expression of related genes in the urinary sediment might be a useful way to assess the severity of DN. / Firstly, we found that urinary mRNA expressions of podocyte-associated molecules nephrin, podocin, synaptopodin, Wilm's tumor-1 (WT-1) and alpha-actinin-4 were higher in patients with DN than in healthy controls, and urinary nephrin, podocin and synaptopodin expression was related to proteinuria and baseline renal function. In addition, there was a close relationship between urinary mRNA expression of type 2 angiotensin converting enzyme (ACE2), a key element of RAS, and the degrees of proteinuria, renal function and rate of decline of glomerular filtration rate (GFR). Urinary mRNA expression of ACE also inversely correlated with the rate of renal function decline. / In the next step, we studied the change in urinary mRNA expression of nephrin, podocin, synaptopodin, ACE and ACE2 in patients with DN treated with angiotensin converting enzyme inhibitor (ACEI) and addition of angiotensin receptor blocker (ARB). We found that urinary mRNA expression of podocin, synaptopodin and propably nephrin increased with disease progression, and percentage change in urinary podocin expression negatively correlated with rate of decline of GFR. Furthermore, serial measurement of urinary expression of nephrin and possibly synaptopodin may reflect therapeutic response to ARB in these patients. Urinary mRNA expression of ACE and ACE2, however, remained unchanged during the study duration and did not correlate with therapeutic response. / In this series of work, we investigated (i) the relation between the gene expression profile of podocyte-associated molecules and RAS related molecules in the urinary sediment and the severity of DN, including clinically defined parameter of disease severity, histological scarring, and the degree of intra-renal podocyte loss, (ii) the relation between urinary and intra-renal gene expression of patients with DN, (iii) the application of urinary gene expression on the monitoring of disease progression and therapy response of DN. The urinary mRNA expression of related genes was quantified by real-time quantitative polymerase chain reaction (RT Q-PCR). The intra-renal mRNA expression of related genes was studied from the histologic specimens of kidney biopsy by laser catapult microdissection (LCM) and RT Q-PCR. The degree of renal scarring was determined by morphometric analysis. Glomerular podocyte number was determined by stereological study on serial sections of renal biopsy specimen. / Taken together, our results suggest that although urinary mRNA expression of podocyte and RAS associated molecules is not related to intra-renal expression, urinary expression has the potential to be used as a non-invasive tool to assess the severity and progression of DN, and serial measurements of urinary gene expression of podocyte associated molecules may be used to reflect therapy response for patients with DN. Our findings also indicate that the information from urinary gene expression is supplementary to, but not a surrogate of, the data obtained from renal biopsy. / We then examined the relation between urinary gene expression and histological changes in the kidney. We found that urinary WT-1 expression correlated with the degree of kidney fibrosis. Unlike intra-renal expression, urinary mRNA expression of podocyte associated molecules did not correlate with glomerular podocyte number. There was also no association between urinary and intra-renal mRNA expression. / Wang, Gang. / Adviser: Cheuk Chen Szeto. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3423. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 156-180). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Balding causes widespread psychological distress but is poorly controlled. The commonest treatment, minoxidil, was originally an antihypertensive drug that promoted unwanted hair. We hypothesized that another serendipitous discovery, increased eyelash growth side-effects of prostamide F(2alpha)-related eyedrops for glaucoma, may be relevant for scalp alopecias. Eyelash hairs and follicles are highly specialized and remain unaffected by androgens that inhibit scalp follicles and stimulate many others. Therefore, we investigated whether non-eyelash follicles could respond to bimatoprost, a prostamide F(2alpha) analog recently licensed for eyelash hypotrichosis. Bimatoprost, at pharmacologically selective concentrations, increased hair synthesis in scalp follicle organ culture and advanced mouse pelage hair regrowth in vivo compared to vehicle alone. A prostamide receptor antagonist blocked isolated follicle growth, confirming a direct, receptor-mediated mechanism within follicles; RT-PCR analysis identified 3 relevant receptor genes in scalp follicles in vivo. Receptors were located in the key follicle regulator, the dermal papilla, by analyzing individual follicular structures and immunohistochemistry. Thus, bimatoprost stimulates human scalp follicles in culture and rodent pelage follicles in vivo, mirroring eyelash behavior, and scalp follicles contain bimatoprost-sensitive prostamide receptors in vivo. This highlights a new follicular signaling system and confirms that bimatoprost offers a novel, low-risk therapeutic approach for scalp alopecias.