Retinoic acid (RA) exerts important effects in the processes of vertebrate development, cellular growth and differentiation, and homeostasis. However, the mechanisms of action of RA in the control of cellular and developmental processes are incompletely understood, as the retinoid target genes have not been fully characterized. The goal of these studies described herein was to contribute towards a greater understanding of the cellular effects of retinoids through the identification and characterization of an RA-induced gene from mouse P19 embryonal carcinoma cells. The predicted amino acid sequence of GRASP is characterized by several putative protein-protein interaction motifs, suggesting that GRASP may function in cell signaling pathways. Towards the goal of identifying which signaling pathways GRASP may participate in, a yeast two-hybrid screen was performed using GRASP as a bait to identify protein interaction partners. The general receptor for phosphinositides 1 (GRP1), a guanine nucleotide exchange factor for the ADP-ribosylation factor 6 (ARF6) GTPase, was identified as a GRASP interaction partner. GRASP was shown to colocalize with endogenous ARFs in cells and enhance GRP1 association with the plasma membrane, suggesting that GRASP may function as a scaffold protein in the recruitment of GRP1 and ARF6 to plasma membrane loci. Overexpression of GRASP was observed to induce accumulation of GRASP in the endosomal compartment where GTP-binding deficient mutants of ARF6 reside, suggesting that GRASP induced a block in an ARF6 plasma membrane recycling pathway. Coexpression of GRP1, but not a catalytically inactive mutant, dramatically reduced the accumulation of GRASP in this compartment. Furthermore, GRP1 mutants that lack the region of interaction with GRASP failed to prevent accumulation of GRASP in the endosomal compartment, suggesting that GRASP recruits GRP1 to the endosomal compartment where GRP1 stimulates nucleotide exchange on ARF6 and recycling. Results described herein demonstrate that GRASP functions in the ARF6 regulated plasma membrane recycling pathway, and that upon overexpression, induces a block in recycling. Our results suggest a role for GRASP as an adapter or scaffold protein that may link cell surface receptors to the ARF6 recycling pathway, resulting in modulation of signal transduction events at the cell surface. / Graduation date: 2002

Lee, Man Yuen. / "March 2008." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 103-128). / Abstracts in English and Chinese. / Title Page --- p.i / Acknowledgements --- p.ii / Table of Content --- p.iii / List of Figures --- p.vii / List of Graphs --- p.viii / List of Tables --- p.x / Abbreviations --- p.xii / Abstract --- p.xiii / Abstract (Chinese) --- p.xv / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- Diabetes mellitus --- p.2 / Chapter 1.1.1 --- Type 1 diabetes mellitus --- p.3 / Chapter 1.1.2 --- Type 2 diabetes mellitus --- p.4 / Chapter 1.1.3 --- Gestational diabetes --- p.5 / Chapter 1.2 --- Diabetic pregnancy --- p.6 / Chapter 1.3 --- Etiology of diabetes-induced malformations --- p.9 / Chapter 1.3.1 --- Hyperglycaemia --- p.10 / Chapter 1.3.2 --- Hyperketonaemia and somatomedin inhibitors --- p.10 / Chapter 1.3.3 --- Oxidative stress --- p.11 / Chapter 1.3.4 --- Deficiency of myo-inositol and arachidonic acid --- p.12 / Chapter 1.4 --- Vitamin A --- p.13 / Chapter 1.5 --- Retinoic acid --- p.14 / Chapter 1.5.1 --- RA signaling on embryo development --- p.15 / Chapter 1.5.2 --- RA teratogenicity --- p.15 / Chapter 1.5.3 --- RA regulation --- p.17 / Chapter 1.6 --- RA and maternal diabetes-induced caudal regression share similar pathogenic mechanisms --- p.19 / Chapter 1.7 --- Strategy of the thesis --- p.21 / Chapter Chapter 2: --- General Materials and Methods / Chapter 2.1 --- Animal --- p.25 / Chapter 2.2 --- Induction of diabetes --- p.25 / Chapter 2.3 --- Preparation of retinoic acid for mouse injection --- p.26 / Chapter 2.4 --- RA responsive cell line --- p.26 / Chapter 2.4.1 --- Cell culture --- p.27 / Chapter 2.4.2 --- Seeding and adding sample to 96-well plate --- p.28 / Chapter 2.4.3 --- Staining of cells --- p.28 / Chapter 2.5 --- Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) --- p.29 / Chapter 2.5.1 --- Collection and storage of tissues --- p.29 / Chapter 2.5.2 --- Total RNA extraction --- p.29 / Chapter 2.5.3 --- Reverse transcription --- p.30 / Chapter 2.5.4 --- Polymerase chain reaction --- p.30 / Chapter 2.5.5 --- Preparation of cDNA standard --- p.31 / Chapter 2.5.6 --- Mini-scale preparation of plasmid DNA --- p.32 / Chapter Chapter 3: --- Effects of Maternal Diabetes on RA Catabolism in the Tail Bud Region / Chapter 3.1 --- Introduction --- p.34 / Chapter 3.2 --- Experimental design --- p.37 / Chapter 3.3 --- Materials and methods --- p.38 / Chapter 3.3.1 --- Preparation of RA stock solution for cell culture --- p.38 / Chapter 3.3.2 --- Preparation of RA standard solutions --- p.38 / Chapter 3.3.3 --- Characterization of RA responsive cell line --- p.39 / Chapter 3.3.3.1 --- Determining optimal culture time for maximum response --- p.39 / Chapter 3.3.3.2 --- Testing toxicity of DMSO --- p.39 / Chapter 3.3.3.3 --- Detection of β-galactosidase activity by β_gal Assay Kit --- p.40 / Chapter 3.3.4 --- In vitro assay of enzymatic degradation of RA --- p.41 / Chapter 3.3.4.1 --- Testing toxicity of enzyme cofactor-reducing agent --- p.41 / Chapter 3.3.4.2 --- Collection of tail bud --- p.42 / Chapter 3.3.4.3 --- In vitro enzymatic reaction --- p.43 / Chapter 3.3.5 --- In vivo assay of enzymatic degradation of RA --- p.44 / Chapter 3.3.5.1 --- Determining the optimal time for maximum release of RA from the tail bud into the medium --- p.44 / Chapter 3.3.5.2 --- Monitoring of RA remained in the tail bud after injection of exogenous RA --- p.45 / Chapter 3.3.6 --- Statistical analysis --- p.45 / Chapter 3.4 --- Results --- p.46 / Chapter 3.4.1 --- Optimization of RA responsive cell line --- p.46 / Chapter 3.4.1.1 --- Effect of incubation time with RA on β-galactosidase expression level --- p.46 / Chapter 3.4.1.2 --- Toxicity of RA and DMSO --- p.47 / Chapter 3.4.1.3 --- Comparison of X-gal staining assay and β_gal Assay Kit for detection of β-galactosidase expression --- p.48 / Chapter 3.4.2 --- In vitro assay of RA catabolic activity in the tail bud --- p.49 / Chapter 3.4.2.1 --- Toxicity of enzyme cofactor-reducing agent --- p.49 / Chapter 3.4.2.2 --- RA catabolic activity in lysed and intact tail buds --- p.50 / Chapter 3.4.2.3 --- Effect of enzyme cofactor and inhibitor --- p.50 / Chapter 3.4.2.4 --- Comparsion of in vitro RA catabolic activity of diabetic and non-diabetic groups --- p.51 / Chapter 3.4.3 --- In vivo assay of RA catabolic activity in the tail bud --- p.52 / Chapter 3.4.3.1 --- Optimal time for maximum release of RA from the tail bud into the medium --- p.53 / Chapter 3.4.3.2 --- Comparison of RA remained in the tail bud of embryos of diabetic and non-diabetic mice --- p.53 / Chapter 3.5 --- Discussion --- p.55 / Chapter Chapter 4: --- Analysis of Cyp26 Expression in the Tail Bud Region / Chapter 4.1 --- Introduction --- p.60 / Chapter 4.2 --- Experimental design --- p.63 / Chapter 4.3 --- Materials and methods --- p.64 / Chapter 4.3.1 --- Sample collection --- p.64 / Chapter 4.3.2 --- Real-time quantitative RT-PCR --- p.64 / Chapter 4.3.3 --- Statistical analysis --- p.66 / Chapter 4.4 --- Results --- p.67 / Chapter 4.4.1 --- "Relative expression levels of Cyp26al, Cyp26bl and Cyp26cl" --- p.67 / Chapter 4.4.2 --- Molecular changes in Cyp26al in tail bud region after maternal RA treatment --- p.68 / Chapter 4.5 --- Discussion --- p.72 / Chapter Chapter 5: --- Comparison of Cyp26al Heterozygous and Wild-type Embryos in Diabetic and Non-diabetic Pregnancies / Chapter 5.1 --- Introduction --- p.76 / Chapter 5.2 --- Experimental design --- p.79 / Chapter 5.3 --- Materials and methods --- p.81 / Chapter 5.3.1 --- Animal --- p.81 / Chapter 5.3.2 --- DNA genotyping --- p.81 / Chapter 5.3.3 --- Measurement of RA remained in the tail bud after RA treatment --- p.82 / Chapter 5.3.4 --- Analysis of extent of caudal regression --- p.83 / Chapter 5.3.5 --- Real-time quantitative RT-PCR --- p.83 / Chapter 5.3.6 --- Statistical analysis --- p.84 / Chapter 5.4 --- Results --- p.85 / Chapter 5.4.1 --- Comparsion of pregnancy outcome of mating with ICR or Cyp26al+/- males --- p.85 / Chapter 5.4.2 --- Expression levels of Cyp26al determined by real-time quantitative RT-PCR --- p.85 / Chapter 5.4.3 --- Determination of RA catabolic activity --- p.86 / Chapter 5.4.4 --- Extent of caudal regression --- p.90 / Chapter 5.5 --- Discussion --- p.93 / Chapter Chapter 6: --- Conclusion and Future Perspectives --- p.96 / References --- p.102 / Figures / Graphs

Graduation date: 2004

Lee, Man Yuen. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 191-215). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

by Tse Kam Wah. / "September 1998." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 123-145). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

All-trans retinoic acid (atRA) is an important morphogen in many developmental processes, including apoptosis, growth, organogenesis and differentiation. During the early embryonic development, atRA is synthesized in an irreversible reaction from all-trans retinal (atRAL), catalyzed mainly by retinal dehydrogenase 2 (RALDH2). The upstream metabolic pathway, including the redox reaction between all-trans retinol (atROL) and atRAL, mediated by short-chain dehydrogenase/reductase, however, is less understood during embryonic development. / Previously a Xenopus laevis short-chain dehydrogenase/reductase 3 (dhrs3) was identified as a gene differentially expressed in the Spemann-Mangold Organizer. In this study, dhrs3 was found to be expressed in the circumblastoporal ring, neuroectoderm and pronephros region, and was up-regulated by atRA signalling. By using loss-of-function and gain-of-function approaches, it was found that the phenotype induced by knockdown of dhrs3 mimicked those with an elevated level of atRA signalling, and overexpression of dhrs3 enhanced the phenotype of cyp26a1, which functions in degradation of atRA. In dhrs3 knock-down embryos (morphants), expression domain of the mesoderm markers brachyury was disrupted, and that of organizer marker lim1 were significantly expanded, suggesting altered mesoderm induction. Overexpression of dhrs3, on the other hand, exerted an opposite effect on lim1 by reducing its expression. dhrs3 also rescued the phenotype following raldh2 overexpression induced by exogenous atRAL, suggesting that dhrs3 competed with raldh2 for the same substrate, atRAL. In line with these findings, expression of the mid-brain, hindbrain and neural crest markers was posteriorized in dhrs3-overexpressing embryos, similar to the phenotype of atRA-deficient embryos induced by cyp26a1. These findings indicate that dhrs3 participates in the retinoid metabolism by reducing atRAL to atROL. / Xenopus dhrs3 morphants displayed a shortened anteroposterior axis, similar to that of atRA toxicity. Examination of convergent extension (CE) markers papc indicated a defect in the CE movement, which was also evidenced by the disrupted bra and not expression. Overall, the results of the present study suggest that dhrs3 regulates proper mesoderm patterning through regulating the CE movement. / Kam, Kin Ting. / Advisers: Yu Pang Eric Cho; Wood Yee Chan; Hui Zhao. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves [158]-184). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.