In a HEK 293 cell line stably expressing seabream GHS-R1a (sbGHS-R1a), we found that a synthetic growth hormone secretagogue (GHS) increased [ 3H]-inositol phosphate production, clearly indicating coupling of this receptor to Gq/11-proteins. Using Western blotting, we found that GHS could also stimulate extracellular signal-regulated kinases 1 and 2 (ERK1/2), and that this response was inhibited by the MEK inhibitor U0126. For both the [3H]-inositol phosphate and ERK1/2 assays, the presence of the GHS-R antagonist D-Lys(3)-GHRP-6 significantly inhibited the GHS-stimulated activities, and in addition inhibited basal activities by 50% and 40%, respectively. These results showed that sbGHS-R1a is a constitutively active receptor and the antagonist D-Lys(3)-GHRP-6 is an inverse agonist. We also proposed that the expression of sbGHS-Rs was involved in the regulation of cell apoptosis. / Oligomerization of the human GHS-Rs (hGHS-Rs) was explored by transient transfection of the hGHS-Rs in HEK 293 cells followed by co-immunoprecipitation of differentially epitope-tagged forms of the receptors and bioluminescence resonance energy transfer 2 (BRET2) studies. (Abstract shortened by UMI.) / The concept that G protein-coupled receptors (GPCRs) exist and potentially function as dimers and/or higher oligomers has progressed from hypothesis to being widely accepted recently. Oligomerization of GPCRs has been increasingly noted in the regulation of the biological activity of the receptors. The growth hormone secretagogue receptor 1a (GHS-R1a) is a GPCR which principally regulates the pulsatile release of growth hormone from the pituitary gland. The GHS-R exists in two forms: GHS-R1a being a constitutively-active GPCR with 7 transmembrane (TM) domains, and GHS-R1b being a truncated version of type 1a but having only 5 TM domains. The endogenous agonist for GHS-R1a is ghrelin which exerts a wide range of physiological actions, but the function of GHS-R1b is still unclear. Since the tissue distribution patterns of the two isoforms of GHS-R are different, the objective of the present study is to explore the mechanisms of cell signalling of GHS-R1a and to determine the extent and importance of interactions between these two receptor isoforms. / Leung Po Ki. / "July 2005." / Adviser: Helen Wise. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3728. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 189-210). / 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. / School code: 1307.

Kwan Hiu Yee. / "June 2006." / 2+ in the title is superscript. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 131-150). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Wong, Chun Kit. / "September 2010." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 104-118). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Acknowledgements --- p.ii / Contents --- p.iii / Declaration --- p.vi / Abstract --- p.vii / 摘要 --- p.x / Abbreviations --- p.xi / List of Figures --- p.xiii / Chapter CHAPTER ONE: --- INTRODUCTION / Chapter 1.1 --- Embryonic Stem Cells (ESCs) / Chapter 1.1.1 --- Characteristics of ESC --- p.1 / Chapter 1.1.2 --- Therapeuticotential of ESCs --- p.2 / Chapter 1.2 --- 17β-estradiol (E2) / Chapter 1.2.1 --- Genomic Actions of E2 --- p.3 / Chapter 1.2.2 --- Non-genomic Actions of E2 --- p.5 / Chapter 1.2.3 --- hysiological Roles of E2 on Early Mammalian Development --- p.9 / Chapter 1.2.4 --- E2 and Cell Proliferation --- p.10 / Chapter 1.3 --- Ca2+ homeostasis / Chapter 1.3.1 --- Overview --- p.11 / Chapter 1.3.2 --- Ca2+ Signaling in mESCs --- p.14 / Chapter 1.4 --- Store-operated Ca2+ Entry (SOCE) / Chapter 1.4.1 --- Overview --- p.15 / Chapter 1.4.2 --- Store Depletion --- p.15 / Chapter 1.4.3 --- Activation of SOCE --- p.16 / Chapter 1.5 --- Molecular Identities of Store-operated Ca2+ Channels (SOCCs) on plasma Membrane / Chapter 1.5.1 --- TRPC Channels --- p.17 / Chapter 1.5.2 --- ORAI Channels --- p.18 / Chapter 1.5.3 --- Regulation of SOCCs at Different Levels --- p.18 / Chapter 1.5.4 --- Regulation of SOCE --- p.19 / Chapter 1.6 --- Nuclear Factor of Activated T-cells (NFAT) / Chapter 1.6.1 --- Overview --- p.20 / Chapter 1.6.2 --- Mechanisms of Action --- p.21 / Chapter 1.6.3 --- Functions --- p.22 / Chapter 1.7 --- Aims of the Study --- p.23 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS / Chapter 2.1 --- Maintenance of mESCs --- p.24 / Chapter 2.2 --- Cell proliferation Assay and Viability Test --- p.24 / Chapter 2.3 --- "RNAreparation, Reverse Transcription (RT) and Quantitative Polymerase Chain Reaction (qPCR)" --- p.25 / Chapter 2.4 --- Totalrotein Extraction --- p.27 / Chapter 2.5 --- Measurement of protein Concentration --- p.27 / Chapter 2.6 --- De-phosphorylation Assay --- p.28 / Chapter 2.7 --- Western Blot --- p.28 / Chapter 2.8 --- Ca2+ Measurement by Confocal Microscopy --- p.30 / Chapter 2.9 --- Ca2+ Measurement by Flow Cytometry --- p.31 / Chapter 2.10 --- siRNA Transfection --- p.31 / Chapter 2.11 --- DNAlasmid Transfection --- p.32 / Chapter 2.12 --- Molecular and Fluorescence Imaging --- p.33 / Chapter 2.13 --- Statistical Analysis --- p.34 / Chapter 2.14 --- Primers used in the Study (Table 1:Primers List) --- p.34 / Chapter 2.15 --- Drugs used in the Study (Table 2: Drugs List) --- p.36 / Chapter 2.16 --- Antibodies used in the Study (Table 3: Antibodies List) --- p.37 / Chapter CHAPTER THREE: --- RESULTS / Chapter 3.1 --- Expression of SOCE in mESCs --- p.38 / Chapter 3.2 --- SOCC Blockers Attenuated mESCroliferation --- p.43 / Chapter 3.3 --- E2 Increased mESCroliferation --- p.48 / Chapter 3.4 --- E2 Increased Intracellular Ca2+ ([Ca2+]i) Level in mESCs --- p.48 / Chapter 3.5 --- E2 Increased the Amplitude of SOCE --- p.51 / Chapter 3.6 --- Increase in mESC proliferation and SOCE Caused by E2 Could be Reversed by SOCC Blocker --- p.51 / Chapter 3.7 --- Relative Expression of SOCC Candidates at mRNA Level Under the Treatment of E2 --- p.56 / Chapter 3.8 --- E2 Down-regulated the Expression of ORAI3 --- p.56 / Chapter 3.9 --- Knockdown of ORAI3 in mESCs --- p.61 / Chapter 3.10 --- Identification of NFATc3 Specific Bands --- p.63 / Chapter 3.11 --- E2 Increased the phosphorylation of NFATc3 --- p.67 / Chapter 3.12 --- Effects of 2-APB on NFATc3 phosphorylation Status --- p.67 / Chapter 3.13 --- Identification of NFATc4 Specific Bands ？ --- p.72 / Chapter 3.14 --- E2 Increased the Translocation of GFP-NFATc4 From the Cytoplasm to the Nucleus and This Effect Could be Reversed by 2-APB --- p.80 / Chapter 3.15 --- CsA Reversed E2-induced Increase in proliferation --- p.82 / Chapter CHAPTER FOUR: --- DISCUSSION / Chapter 4.1 --- Expression of SOCE in mESCs --- p.84 / Chapter 4.2 --- proliferation of mESCs Depends on SOCE --- p.85 / Chapter 4.3 --- E2 Acts an Extrinsic Factor for Stimulatingroliferation of mESCs Via SOCE --- p.87 / Chapter 4.4 --- roposed Mechanism to Show an Increment of SOCE Can be Due to a Down-regulation of ORAI3 --- p.89 / Chapter 4.5 --- Experiments Aiming to Knockdown ORAI3 --- p.92 / Chapter 4.6 --- roposed Mechanism to Show an Increment of SOCE by Other SOCC Candidates Rather than ORAI3 --- p.93 / Chapter 4.7 --- Activation of NFATc3 and NFATc4 by E2 in mESCs --- p.94 / Chapter 4.8 --- possible Downstream Targets of NFAT Responsible for E2-induced mESCs proliferation --- p.96 / Chapter CHAPTER FIVE: --- FUTUREERSPECTIVES --- p.98 / Chapter CHAPTER SIX: --- CONCLUSION --- p.100 / REFERENCES --- p.104

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / Typescript. Includes bibliographical references. Also available on the Internet.

Thesis (Ph.D.)--Memorial University of Newfoundland, Faculty of Medicine, 2001. / Typescript. Bibliography: leaves 188-241.

Thesis (M.S.)--West Virginia University, 2000. / Title from document title page. Document formatted into pages; contains ix, 70 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 63-68).

Neuronal connections in cat visual cortex are highly susceptible to visual experience at early postnatal age and thus serve as a useful model of neural plasticity. The biochemical mechanisms underlying this cortical plasticity remain unclear. In this thesis, the development of several elements in calcium-related signal transduction systems, including the type-1 muscarinic and alpha-1 adrenoceptor systems as examples of cell surface receptors and protein kinase C. calcium/calmodulin dependent kinase II and inositol 1,4,5 phosphotate receptors as second messenger targets, were investigated using the methods of immunocytochemistry and autoradiography. The results show that each receptor develops with its own time-table and laminar distribution; the various elements all culminate and display the maximal colocalization during the critical period; and, only at this age, the cortical levels of the receptors and kinases are dependent on subcortical afferents. The results suggest that cell surface receptors and their second messenger targets develop in specific temporal and spatial patterns, which may be both genetically and environmentally determined, and this specific sequence of development of the molecules for signal transduction results in a series of modifications in the morphology and physiology of the developing cortex leading to its maturation. / Medicine, Faculty of / Graduate

Tang, Yongjiang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 159-202). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Ng Sin Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 120-135). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.iii / Abstract --- p.vi / 撮要 --- p.ix / Publications --- p.xi / Table of contents --- p.xiii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Allergic Diseases and Allergic Inflammation --- p.1 / Chapter 1.1.1 --- Prevalence of Allergic Diseases --- p.1 / Chapter 1.1.2 --- Common Allergic Diseases: Allergic Asthma --- p.1 / Chapter 1.1.3 --- Common Allergic Diseases: Atopic Dermatitis --- p.2 / Chapter 1.1.4 --- Allergic Inflammation --- p.3 / Chapter 1.2 --- The Inflammatory Leukocytes: Mast Cells and Eosinophils --- p.6 / Chapter 1.2.1 --- Characteristics of Mast Cells --- p.6 / Chapter 1.2.2 --- Mast Cells Distribution --- p.8 / Chapter 1.2.3 --- Mast Cells Subtypes --- p.8 / Chapter 1.2.4 --- HMC-1 Cells --- p.9 / Chapter 1.2.5 --- Characteristics of Eosinophils --- p.12 / Chapter 1.3 --- Adhesion Molecules in Allergic Diseases --- p.15 / Chapter 1.3.1 --- Adhesion Molecules and Leukocyte Migration --- p.15 / Chapter 1.3.2 --- Selectin --- p.17 / Chapter 1.3.3 --- Intermolecular Adhesion Molecules --- p.17 / Chapter 1.3.4 --- Integrin --- p.18 / Chapter 1.4 --- Cytokines and Chemokines in Allergic Diseases --- p.18 / Chapter 1.4.1 --- IL-6 --- p.20 / Chapter 1.4.2 --- CXCL1 --- p.21 / Chapter 1.4.3 --- CXCL8 --- p.21 / Chapter 1.4.3 --- CCL2 --- p.22 / Chapter 1.5 --- Intercellular Signal Transduction Pathways in Inflammation --- p.24 / Chapter 1.5.1 --- RAS-RAF-mitogen-activated Protein Kinases --- p.24 / Chapter 1.5.2 --- Janus Kinase/ Signal Transducers and Activators of Transcriptions Pathway --- p.27 / Chapter 1.5.3 --- Nuclear Factor-KB Pathway --- p.29 / Chapter 1.5.4 --- Phosphoinositide 3-Kinase Pathway --- p.31 / Chapter 1.6 --- Aims and Scope of the Study --- p.33 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- HMC-1 Cell Line --- p.35 / Chapter 2.1.2 --- Human Buffer Coat --- p.35 / Chapter 2.1.3 --- Human Mast Cell Chymase and TLR ligands --- p.35 / Chapter 2.1.4 --- Media and Reagents for Cell Culture --- p.36 / Chapter 2.1.5 --- Reagents and Buffers for Purification of Human Eosinophils --- p.37 / Chapter 2.1.6 --- Reagents and Buffers for Flow Cytmetry --- p.38 / Chapter 2.1.7 --- Reagents and Buffers for Total RNA Extraction --- p.41 / Chapter 2.1.8 --- Reagents and Buffers for Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.42 / Chapter 2.1.9 --- Reagents and Buffers for Agarose Gel Electrophoresis --- p.45 / Chapter 2.1.10 --- Reagents and Buffers for Sodium Dodecyl Sulfate -polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.46 / Chapter 2.1.11 --- Reagents and Buffers for Western Blot Analysis --- p.48 / Chapter 2.1.12 --- Chemotactic Migration --- p.51 / Chapter 2.1.13 --- Signaling Transduction Inhibitors and Protein Synthesis Inhibitors --- p.51 / Chapter 2.2 --- Methods --- p.52 / Chapter 2.2.1 --- HMC-1 Cell Cultures --- p.52 / Chapter 2.2.2 --- Purification of Buffy Coat Eosinophils by MACS and Eosinophil Culture --- p.52 / Chapter 2.2.3 --- Total Cellular RNA Extraction --- p.53 / Chapter 2.2.4 --- RT-PCR --- p.54 / Chapter 2.2.5 --- Agarose Gel Electrophoresis --- p.55 / Chapter 2.2.6 --- Flow Cytometry Analysis --- p.55 / Chapter 2.2.7 --- Protein Array Analysis of Cytokine Release --- p.57 / Chapter 2.2.8 --- Quantitative Analysis ofCXCLl --- p.58 / Chapter 2.2.9 --- Total Protein Extraction --- p.58 / Chapter 2.2.10 --- SDS-PAGE --- p.58 / Chapter 2.2.11 --- Western Blot Analysis --- p.59 / Chapter 2.2.12 --- Chemotactic Migration Analysis --- p.60 / Chapter 2.2.13 --- Statistical Analysis --- p.60 / Chapter Chapter 3 --- Effects of Mast Cell Derived Chymase on Human Eosinophils and the Signaling Mechanisms: Implication in Allergic Inflammation / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Results --- p.65 / Chapter 3.2.1 --- Effects of Chymase on Eosinophil Survival --- p.65 / Chapter 3.2.2 --- Effects of Chymase on the Adhesion Molecule Expression of Eosinophils --- p.68 / Chapter 3.2.3 --- Effects of Chymase on the Chemokinetic Properties on Eosinophils --- p.71 / Chapter 3.2.4 --- Effects of Chymase on the Release of Chemokines and IL-6 from Eosinophils --- p.73 / Chapter 3.2.5 --- Signal Transduction Mechanism Involved in Regulating Chymase-induced Effects on Eosinophils --- p.78 / Chapter 3.3 --- Discussion --- p.71 / Chapter Chapter 4 --- TLR-mediated Effects and Signal Transduction Mechanism of HMC-1 Cells / Chapter 4.1 --- Introduction --- p.92 / Chapter 4.2 --- Results --- p.97 / Chapter 4.2.1 --- Expression of Adhesion Molecules on HMC-1 Cells --- p.95 / Chapter 4.2.2 --- TLR Expression Profile on HMC-1 Cells --- p.97 / Chapter 4.2.3 --- Effects of TLR ligands on HMC-1 Cell Adhesion Molecule Expressions --- p.99 / Chapter 4.2.4 --- TLR7-induced Phosphorylation of ERK and Effects of PD98059 on TLR7-induced ERK Phosphorylation --- p.104 / Chapter 4.2.5 --- Effect of TLR7 Ligand on HMC-1 Cells Cytokine Release --- p.108 / Chapter 4.3 --- Discussion --- p.110 / Chapter Chapter 5 --- Conclusions and Future Perspectives / Chapter 5.1 --- Conclusions --- p.115 / Chapter 5.2 --- Future Perspectives --- p.117 / References --- p.120 / Appendix --- p.136

Decapentaplegic (Dpp), a member of the TGF-[Beta] superfamily, acts as a morphogen to direct cell differentiation, determine cell fate and promote cell survival and proliferation in Drosophila wing development. To investigate the role of Dpp in Drosophila wing development, three aspects of the patterning role of Dpp have been analyzed. First, I investigated the cellular responses to Dpp signaling by a loss of function strategy. The consequences of lacking Dpp signal transduction on cell morphology and tissue integrity were analyzed. Second, I investigated whether Dpp signaling is down-stream of Hh signaling to maintain the normal cell segregation at the A/P boundary by clonal analysis. Third, I investigated whether cross talk among the Hh, Dpp and Wg signaling pathways exists and what its relevance for wing patterning is. To investigate the role of Dpp in Drosophila wing development, the general strategies are to look at the phenotypes of loss-of-function and gain-of-function. Mutant clones lacking Dpp signal transduction by knock down Dpp receptor Thick veins (Tkv) do not survive in wing blade due to JNK dependent apoptosis. To get larger mutant clones for analysis, JNK pathway was inhibited by knock down bsk (encodes JNK) in mutant clones lacking Dpp signaling using FLP-FRT system. Clones double mutant for tkv and bsk did not undergo apoptosis, but recovered at very low frequencies compared to sibling clones. Here, I showed that the low recovery of tkv bsk double mutant clones are due to the extrusion of mutant cells. The extrusion of tkv bsk double mutant cells correlated with changes in the actin cytoskeleton and a dramatic loss of the apical microtubule web normally present in these cells. These results suggest that Dpp signaling is required for cell morphogenesis in Drosophila wing development. We propose that Dpp acts as a survival factor in the wing disc epithelium by orchestrating proper cytoskeletal organization and maintaining normal cell-cell contact. Drosophila wing is subdivided into anterior (A) and posterior (P) compartments. This developing into adjacent compartments is crucial for the patterning of Drosophila wing. Previous study has shown that Hedgehog (Hh) signaling is required in A cells to maintain the A/P boundary and is sufficient to specify A type cell sorting. A previous study has in addition implicated the signaling molecule Decapentaplegic (Dpp) in maintaining the A/P boundary. However, this study did not address whether and in which cells, A and/or P, Dpp signal transduction was required to maintain this boundary. Here, I have analyzed the role of components of the Dpp signal transduction pathway and the relation of Dpp and Hh signaling in maintaining the A/P boundary by clonal analysis. I showed that Dpp signaling mediated by the Dpp target gene, T-box protein Optomotor-blind (Omb), is required in A cells, but not in P cells, to maintain the normal position of the A/P boundary. During patterning formation, it is essential for cells to receive precise positional information to pattern the tissue. It has been proposed for a long time that different signaling pathways such as Hedgehog (Hh), Dpp and Wingless (Wg) signaling pathways provide positional information for tissue patterning in an integrated manner. Recently, evidence of interactions between Hh and Dpp as well as Wg and Hh signaling pathways has been reported in Drosophila wing. Here, I have identified additional interactions among Hh, Dpp and Notch/Wg signaling. We propose that the selector gene engrailed, Hh and Dpp signaling interact with each other to regulate target genes expression and thus to pattern the wing along the A/P axis. Further more, I showed that Dpp signaling is also participating in the patterning along the D/V axis by interaction with the selector gene apterous and Notch/Wg signaling.