published_or_final_version / Zoology / Master / Master of Philosophy

Type 2 Diabetes Mellitus (T2DM) is a metabolic disorder of global concern that is primarily characterized by insulin resistance, relative insulin deficiency, and hyperglycemia. G- Protein Coupled Receptors (GPCRs), important mediators of cellular signaling responses, have been prime targets of drug discovery efforts in various therapeutic areas. Human G-Protein Receptor 40 (HGPR40) is highly expressed in the pancreas and has been implicated in the regulation of glucose metabolism and pathophysiology of T2DM. For effective control of diabetes, combination therapy is being considered because no single drug can completely control diabetes and its associated complications. This necessitates the identification of novel drug targets including HGPR40 which might permit development of drugs which function differently from existing drugs. This project focused on the construction of two chimeric receptor proteins named HGPR40.1-715_43.709-1013 (Chimera I) and HGPR40.1-431_43.389-1013 (Chimera II) which were made by exchanging domains within trans-membranes regions 6 and 4, of HGPR40 and HGPR43 DNA, homologous receptors which vary in amino acid sequence but which have the same three-dimensional structure. After PCR amplification of sufficient quantities of the desired gene fragments they were ligated together to form the desired recombinant chimeric proteins which were cloned into the expression vector pcDNA3.1 in two successive cloning steps. The vector contains CMV promoter, multiple cloning sites, neomycin resistance gene etc for high-level expression in a wide range of mammalian cells. The two full-length chimeras were designed to be 6452 bp and 6483 bp by exchanging either the first or the first two external domains of HGPR40 with those of HGPR43. The creation of the correct chimeras was verified by both agarose gel electrophoresis and PCR analysis. Then chimeric DNA was transformed into Escherichia coli to obtain sufficient amounts of DNA for sequencing verification of the desired construct. Upon verification, the cloned DNA was to be transfected into cultured mammalian kidney (HEK293) cells for expression of the chimeric proteins. A (FLIPR) Fluorometric Imaging Plate Reader analysis by our collaborators at Eli Lilly would have been used to measure the extent of Ca2+ efflux from the endoplasmic reticulum testing a variety of stimulatory molecules to obtain an indication of which would activate the receptor. Activation would initiate the internal (second messenger cascade) G-protein signaling pathway and result in the secretion of insulin. However, transformation of the chimeric membrane receptors into E. coli resulted in altered sequences which could not be used for the FLIPR analysis. Expression of membrane proteins in E. coli can lead to such gene rearrangements. These experiments will be repeated in the future using another type of competent cell designed to prevent genetic re-arrangements. Construction of stable clones will permit us to test numerous potential ligands to aid in development of novel therapeutic drugs targeting HGPR40 to aid in combating diabetes. / Department of Biology

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The ability to sense and respond to environmental stimuli is essential for the survival of all living things. As a result, nature has evolved an uncountable number of ways to detect environmental signals. At the cellular level, G protein-coupled receptors (GPCRs) are used by eukaryotes, including fungi and humans, to convert extracellular molecular binding events into intracellular responses. Recently, synthetic biologists have shown that biological sensing systems can be repurposed to suit human needs, developing tools such as diagnostic devices and drug screening platforms. In this thesis, I present work exploring the potential of fungal GPCRs to be used as sensing elements in yeast-based biosensors. Chapter 1 gives background information related to synthetic biology, biosensors, and yeast signaling pathways. Chapter 2 describes the development of the baker's yeast Saccharomyces cerevisiae into a diagnostic device for detection of fungal pathogens, using fungal GPCRs. In Chapter 3 I demonstrate that the substrate specificity of fungal GPCRs can be altered using directed evolution. Chapter 4 describes experiments further probing the native binding abilities of fungal GPCRs, specifically examining protein ligands. Finally, in Chapter 5 we move beyond fungal GPCRs and engineer yeast to detect other stimuli, in the context of an engineered living material.

G蛋白偶聯雌激素受體（GPER，又名GPR30）乃最近於各種動物包括小鼠、大鼠、人類及斑馬魚中發現之新型跨膜雌激素受體。 GPER表達於脂肪組織及多種器官之中，其已被證明能與雌激素結合並介導各式快速反應及基因轉錄。針對GPER於成脂作用中角色之研究將達致對雌激素作用之更全面了解，且GPER亦有望成為治療肥胖症之一種新型標靶。 / 脂肪發育調控乃一複雜且精妙之排程，而雌激素已被證明能抑制脂肪形成，是故雌激素替代療法可舒減絶經後婦女之脂肪代謝問題。此項研究發現GPER於小鼠腹部脂肪組織及小鼠前脂肪細胞系3T3-L1中均有表達，且其信使RNA量於受誘導之3T3-L1成脂作用中錄得上調。 / 3T3-L1細胞分化作用會被名為G1之特異性GPER激動劑阻撓於克隆擴增階段（MCE），此即表明GPER有參與成脂調控之可能。通過油紅O染色發現，受G1處理之3T3-L1細胞於分化後所產生之油滴量實比其對照組為低，但此一效果能被特異性GPER小干擾RNA預處理抹除。另外，本研究以流式細胞儀及西方墨點法對細胞週期及細胞週期因子進行分析後，認為激活GPER能觸發對G1期細胞週期停滯之抑制。另一方面，受G1處理並分化中之3T3-L1細胞出現蛋白激酶B磷酸化效應，意味雌激素與GPER結合對成脂作用有雙向調節之可能性。 / 總而言之，本研究結果斷定GPER能介導雌激素對脂肪組織發育之影響，並為成脂作用之負調節因子，故此，一系列成果將有助肥胖症藥物之研發。 / A novel transmembrane estrogen receptor, G-protein coupled estrogen receptor (GPER, also known as GPR30), is recently identified in various animals including mouse, rat, human and zebrafish. GPER is expressed in many organs including fatty tissues, and has been demonstrated to mediate various rapid responses and transcriptional events upon estrogen binding. The study on the role of GPER in adipogenesis would lead to a more comprehensive understanding of estrogenic actions, with the view of identifying novel therapeutic targets for the treatment of obesity. / Regulation of adipose development is a complex and subtly orchestrated process. Estrogen has been shown to inhibit adipogenesis. Estrogen replacement therapy therefore affects fat metabolism in post-menopausal women. In this study, GPER is identified in mouse abdominal fatty tissues; and there is an up-regulation of GPER in the mouse preadipocyte cell line 3T3-L1 during induced adipogenesis. / Differentiation of 3T3-L1 cells is perturbed by the selective GPER agonist G1 at mitotic clonal expansion (MCE), indicating a possible involvement of GPER in the regulation of adipogenesis. By means of Oil-Red-O staining, the production of oil droplets in the G1-treated, differentiated 3T3-L1 cells is shown to be lower than the untreated control; and such effect is reversed by a specific siRNA knockdown of GPER. FACS analysis and Western blot analysis of cell cycle factors during MCE suggest that GPER activation triggers an inhibition of cell cycle arrest at the G1 stage. On the other hand, phosphorylation of Akt in G1-treated differentiating cells implies a possibility of bi-directional estrogenic regulation of adipogenesis via GPER. / To conclude, it is postulated that GPER mediates estrogenic actions in adipose tissues as a negative regulator of adipogenesis. These results provide insights into the development of therapeutic agents for the treatment of human obesity. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yuen, Man Leuk. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 144-166). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract (English version) --- p.I / Abstract (Chinese version) --- p.III / Acknowledgement --- p.V / Table of Contents --- p.VII / List of Abbreviations --- p.XI / List of Tables --- p.XII / List of Figures --- p.XIII / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1. --- Obesity and adipose tissue --- p.1 / Chapter 1.1.1. --- Obesity --- p.1 / Chapter 1.1.2. --- Fat deposition --- p.3 / Chapter 1.1.3. --- Origin and development of white adipose tissue --- p.5 / Chapter 1.2. --- Adipogenesis --- p.7 / Chapter 1.2.1. --- Origins of white adipocytes --- p.7 / Chapter 1.2.2. --- Signals for adipogenesis --- p.10 / Chapter 1.2.3. --- Regulation of gene expression during adipogenesis --- p.12 / Chapter 1.2.4. --- Common adipose cell lines --- p.16 / Chapter 1.2.5. --- Mechanism of in vitro adipogenesis --- p.21 / Chapter 1.2.5.1. --- Growth arrest --- p.23 / Chapter 1.2.5.2. --- Mitotic clonal expansion --- p.23 / Chapter 1.2.5.3. --- Early and terminal differentiation --- p.24 / Chapter 1.3. --- Estrogen and adipogenesis --- p.28 / Chapter 1.4. --- G-protein coupled estrogen receptor-1 --- p.33 / Chapter 1.4.1. --- General introduction of GPER --- p.33 / Chapter 1.4.2. --- Ligands of GPER --- p.36 / Chapter 1.4.3. --- Cellular signaling of GPER --- p.38 / Chapter 1.4.4. --- Metabolic actions of GPER: A brief introduction --- p.43 / Chapter 1.4.5. --- Metabolic actions of GPER on obesity and glucose metabolism --- p.48 / Chapter 1.4.6. --- Study objectives --- p.53 / Chapter Chapter 2: --- Expression profiles and cellular localization of Gper/GPER in mouse adipose, 3T3-L1 preadipocytes and 3T3-L1 mature adipocytes --- p.54 / Chapter 2.1. --- Introduction --- p.54 / Chapter 2.1.1. --- Expression and functional roles of GPER in adipose. --- p.55 / Chapter 2.1.2. --- Swiss mouse preadipocytes 3T3-L1 --- p.57 / Chapter 2.1.3. --- Study objectives --- p.57 / Chapter 2.2. --- Materials and Methods --- p.59 / Chapter 2.2.1. --- Reagents --- p.59 / Chapter 2.2.2. --- Animal tissues --- p.59 / Chapter 2.2.3. --- Cell culture --- p.60 / Chapter 2.2.4. --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.62 / Chapter 2.2.5. --- Quantitative real-time RT-PCR (qRT-PCR) --- p.66 / Chapter 2.2.6. --- SDS-PAGE and Western blot analysis --- p.68 / Chapter 2.2.7. --- Immunofluorescence assay --- p.69 / Chapter 2.2.8. --- Statistical analysis --- p.70 / Chapter 2.3. --- Results --- p.71 / Chapter 2.3.1. --- Expression of Gper/GPER in mouse visceral adipose tissues --- p.72 / Chapter 2.3.2. --- Expression profiles of Gper/GPER in undifferentiated 3T3-L1 preadipocytes and differentiated 3T3-L1 adipocytes --- p.73 / Chapter 2.3.3. --- Cellular localization of GPER in undifferentiated 3T3-L1 preadipocytes and differentiated 3T3-L1 adipocytes --- p.75 / Chapter 2.4. --- Discussion --- p.76 / Chapter Chapter 3: --- Rapid cellular responses induced by GPER activation in 3T3-L1 preadipocytes --- p.78 / Chapter 3.1. --- Introduction --- p.78 / Chapter 3.1.1. --- Rapid cellular response of estrogen via GPER --- p.79 / Chapter 3.1.2. --- Study objectives --- p.81 / Chapter 3.2. --- Materials and Methods --- p.82 / Chapter 3.2.1. --- Reagents --- p.82 / Chapter 3.2.2. --- Cell culture --- p.82 / Chapter 3.2.3. --- SDS-PAGE and Western blot analysis --- p.83 / Chapter 3.2.4. --- Statistical analysis --- p.84 / Chapter 3.3. --- Results --- p.86 / Chapter 3.3.1. --- Phosphorylation of p44/42 MAPK after time-dependent activation of GPER by ICI182,780 and G1 --- p.87 / Chapter 3.3.2. --- Phosphorylation of p44/42 MAPK after dose-dependent activation of GPER by a combination of chemical agents --- p.88 / Chapter 3.4. --- Discussion --- p.89 / Chapter Chapter 4: --- GPER activation on cell viability of 3T3-L1 preadipocytes --- p.90 / Chapter 4.1. --- Introduction --- p.90 / Chapter 4.1.1. --- Cell proliferation mediated by GPER --- p.90 / Chapter 4.1.2. --- Study objectives --- p.92 / Chapter 4.2. --- Materials and Methods --- p.93 / Chapter 4.2.1. --- Reagents --- p.93 / Chapter 4.2.2. --- Cell culture --- p.93 / Chapter 4.2.3. --- MTT assay for cell viability --- p.94 / Chapter 4.2.4. --- Statistical analysis --- p.95 / Chapter 4.3. --- Results --- p.96 / Chapter 4.3.1. --- Cell viability of 3T3-L1 after dose-dependent activation of GPER by 17β-estradiol, ICI182,780 and G1 --- p.97 / Chapter 4.4. --- Discussion --- p.99 / Chapter Chapter 5: --- GPER-mediated estrogenic action on lipid accumulation in the mature 3T3-L1 adipocytes --- p.101 / Chapter 5.1. --- Introduction --- p.101 / Chapter 5.1.1. --- Induction of differentiation in Swiss mouse preadipocyte 3T3-L1 --- p.101 / Chapter 5.1.2. --- Study objectives --- p.102 / Chapter 5.2. --- Materials and Methods --- p.103 / Chapter 5.2.1. --- Reagents --- p.103 / Chapter 5.2.2. --- Cell culture --- p.103 / Chapter 5.2.3. --- Oil-Red-O staining and measurement of absorbance --- p.105 / Chapter 5.2.4. --- Knockdown of Gper/GPER by siRNA --- p.107 / Chapter 5.2.5. --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.110 / Chapter 5.2.6. --- SDS-PAGE and Western blot analysis --- p.110 / Chapter 5.2.7. --- Statistical analysis --- p.110 / Chapter 5.3. --- Results --- p.112 / Chapter 5.3.1. --- GPER activation on 3T3-L1 differentiation --- p.114 / Chapter 5.3.2. --- Knockdown of Gper/GPER in Swiss mouse preadipocyte 3T3-L1 --- p.114 / Chapter 5.3.3. --- Phosphorylation of p44/42 MAPK in Gper/GPER-knockdown 3T3-L1 after time-dependent activation of GPER by G1 --- p.117 / Chapter 5.3.4. --- Action of drugs on differentiation of Gper/GPER-knockdown 3T3-L1 --- p.117 / Chapter 5.4. --- Discussion --- p.118 / Chapter Chapter 6: --- Role of GPER in regulating cell cycle progression during mitotic clonal expansion (MCE) stage in adipogenesis of 3T3-L1 --- p.120 / Chapter 6.1. --- Introduction --- p.120 / Chapter 6.1.1. --- Differentiation stages of Swiss mouse preadipocyte 3T3-L1 --- p.121 / Chapter 6.1.2. --- Apoptosis and cell cycle progression --- p.122 / Chapter 6.1.3. --- Study objectives --- p.126 / Chapter 6.2. --- Materials and Methods --- p.127 / Chapter 6.2.1. --- Reagents --- p.127 / Chapter 6.2.2. --- Cell culture --- p.127 / Chapter 6.2.3. --- Oil-Red-O staining and measurement of absorbance --- p.129 / Chapter 6.2.4. --- Trypan blue exclusion assay for cell viability determination --- p.129 / Chapter 6.2.5. --- SDS-PAGE and Western blot analysis --- p.131 / Chapter 6.2.6. --- Flow cytometry for analysis of cell cycle progression --- p.132 / Chapter 6.2.7. --- Statistical analysis --- p.133 / Chapter 6.3. --- Results --- p.134 / Chapter 6.3.1. --- Temporal effect of GPER activation on differentiation progress of Swiss mouse preadipocyte 3T3-L1 --- p.137 / Chapter 6.3.2. --- Effect of GPER activation on cell viability during adipogenesis --- p.139 / Chapter 6.3.3. --- Effect of GPER activation on apoptosis during adipogenesis --- p.139 / Chapter 6.3.4. --- Effect of GPER activation on cell cycle distribution during induced adipogenesis --- p.140 / Chapter 6.3.5. --- Effect of GPER activation on expression of cell cycle markers during induced adipogenesis --- p.142 / Chapter 6.3.6. --- Activation of PI3K/Akt pathway by GPER stimulation during induced adipogenesis --- p.143 / Chapter 6.4. --- Discussion --- p.144 / Chapter Chapter 7: --- Conclusions and Future Perspectives --- p.148 / References --- p.155

Ectopic and over-expression of G protein-coupled receptor (GPCR) have been reported to induce tumor formation. Mas protein is a member of GPCR family and was originally isolated from human epidermoid carcinoma. It was demonstrated that mas mRNA was abundantly expressed in human and rat brains by in situ hybridization and RNase protection assays. However, cellular mechanism that leads to such tumorigenic transformation is still an open question. / In order to identify the cellular mechanism of mas-induced tumor formation, a full-length mas cDNA was cloned into a mammalian expression vector pFRSV with dihydrofolate reductase gene as a selection marker. Detailed analyses of mas-transfected cell lines by Southern blot, Northern blot and tumorigenicity assay indicated that tumorigenicity of mas-transfected cells depended on the sites of chromosomal integration and the levels of mas expression. These results suggest that overexpression of mas is not sufficient to induce tumor formation. In line with the ability of mas-transfected cells Mc0M80 to form solid tumor in nude mice, MTT cell proliferation assay indicated that the mas-transfected cells Mc0M80 proliferated faster than vector-transfected cells. Moreover, mas-transfected cells Mc0M80 exhibited significantly increased anchorage-independent growth. Furthermore, mas-transfected cells Mc0M80 showed higher percentage cells in G2/M phase but lower in S-phase in comparison with vector-transfected cells. / Interestingly, Southern blot analysis of individual xenografted tumor tissue indicated that tumor was composed of cells not only derived from injected mas-transfected CHO cells but also cells from mouse tissues. The presence of mouse stromal cells in the tumor was confirmed by immunohistochemistry and in situ hybridization. Previously our laboratory had identified some up- and down-regulated genes in mas-transfected cells by fluorescent differential display (FluoroDD). Northern blot showed that these differential expressed genes were up- or down-regulated in mas-transfected cells and tumor samples, which might play an important role in cancerous growth. / Taken together, these results suggest that over-expression of GPCR mas up-regulated tumor-related genes, resulting in promoting excessive cell growth and tumorigenic transformation. In addition, when the tumor mass formed they secreted some growth factor(s) which altered the migration of mouse stromal cells into tumor mass. The interactions of transformed cells and stromal cells further aggravate the tumorigenicity process. / To complement our fluorescent differential display study and to compare changes of gene expression when transformed cells were exposed to the microenvironment in nude mice, protein expression profiles of mas over-expressing cells as well as tumor tissues were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. The 2D-PAGE analysis showed that a similar but distinct protein expression profiles in mas-transfected cells and in mas-induced tumor. Mass spectrometry analysis identified several cancerous growth-related proteins and they are involved in processes such as cell signaling, energy metabolism, transcription and translation and cytoskeleton organization. / Lin Wenzhen. / "December 2005." / Adviser: Cheung Wing Tai. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6381. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 222-240). / 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.

Bikkavilli Rama Kamesh. / "August 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 212-223) / 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.

Cysteinyl leukotriene receptor 2, a G-protein coupled receptor known to be expressed and functional on human platelets. However, it seems that upon ligand activation the cysteinyl leukotriene receptor 2 activates a variety of signaling pathways in multiple cell types among different species. Previously, a former laboratory member Vrinda Kulkarni found cysteinyl leukotriene receptor 2 to be expressed on the surface of adult zebrafish thrombocytes. In this work I studied the characteristics of aggregation in adult zebrafish thrombocytes with the knockdown of cysteinyl leukotriene receptor 2. I used a newly developed knockdown method to study the function of cysteinyl leukotriene receptor 2. Knockdown of the cysteinyl leukotriene was confirmed using RT-PCR results showed p=.001, reduced sell surface level of expression of the cysteinyl leukotriene receptor 2 results showed that p=.002. I found that the knockdown of cysteinyl leukotriene receptor 2 results in prothrombotic thrombocytes by using flow cytometry p=.0001.

Adhesion G-protein coupled receptors (GPCRs) are the second largest class of GPCRs, yet their functions and ligands remain predominantly unidentified. Polymorphisms in the gene encoding the adhesion GPCR latrophilin 3 (ADGRL3) have been associated with an increased risk for attention deficit hyperactivity disorder (ADHD) and substance use disorder (SUD) in various linkage and association studies. Disrupting the function of ADGRL3 homologs across mammalian and invertebrate model systems leads to changes in various dopaminergic phenotypes such as hyperactivity, sleep impairment, and changes in sensitivity to psychostimulants, suggesting that ADGRL3 contributes to behavior by modulating dopamine signaling. Here, I use behavioral and imaging studies to delineate an important role for Cirl, the Drosophila homolog of ADGRL3, in a recently characterized dopaminergic sleep circuit. Sleep impairment is a common symptom in both SUD and ADHD, and sleep studies are well established in Drosophila. Our work shows that fruit flies that carry a null mutation for Cirl are hyperactive and display a deficit in sleep that is enhanced by adult thermogenetic activation of dopamine neurons. Though Cirl displays high expression within dopamine neurons, conditional knockout of Cirl in dopamine neurons does not recapitulate sleep deficits seen in Cirl null flies, and specific rescue of Cirl in a knockout background does not ameliorate them. Intriguingly, activating dopamine neurons in Cirl null flies throughout development rescued the sleep deficits, indicating that this dopaminergic intervention induces lasting changes that can ameliorate lack of Cirl function. Imaging studies reveal that Cirl shows high expression in the central complex, which is involved in sleep and receives dense dopaminergic input. I demonstrate that Cirl functions within different populations of the central complex downstream of dopaminergic innervation to differentially affect night and daytime sleep through both dopaminergic and non-dopaminergic mechanisms. This work delineates a novel role for an adhesion GPCR in modulating sleep behavior, and further characterizes ADGRL3 as a potential therapeutic target for disorders characterized by dysregulation of dopaminergic neurotransmission.

Yeung, Barry Ho Sing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 137-154). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iv / Acknowledgements --- p.vii / Publications based on work in this thesis. --- p.ix / List of abbreviations --- p.x / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Dorsal root ganglion cells --- p.1 / Chapter 1.1.1 --- Primary sensory neurons --- p.1 / Chapter 1.1.2 --- Non-neuronal cells --- p.3 / Chapter 1.1.2.1 --- Satellite glial cells --- p.3 / Chapter 1.1.2.2 --- Schwann cells --- p.6 / Chapter 1.2 --- Peripheral sensitization --- p.8 / Chapter 1.3 --- Neuron-glia interactions --- p.9 / Chapter 1.4 --- Aim of Thesis --- p.11 / Chapter Chapter 2 --- "Materials, media, buffers and solutions" --- p.13 / Chapter 2.1 --- Materials --- p.13 / Chapter 2.2 --- "Culture media, buffer and solutions" --- p.19 / Chapter 2.2.1 --- Culture media --- p.19 / Chapter 2.2.2 --- General culture buffers and culture plate coating reagents --- p.19 / Chapter 2.3 --- Antibodies used for identifying DRG cells --- p.23 / Chapter 2.3.1 --- Primary antibodies --- p.23 / Chapter 2.3.2 --- Secondary antibodies --- p.23 / Chapter Chapter 3 --- Methods --- p.24 / Chapter 3.1 --- Preparation of DRG cell cultures --- p.24 / Chapter 3.2 --- Preparation of neuron-enriched and glial cell cultures --- p.25 / Chapter 3.3 --- Immunocytochemistry --- p.26 / Chapter 3.4 --- Immunohistochemistry --- p.27 / Chapter 3.4 --- Determination of [3H]cAMP production in DRG cells --- p.28 / Chapter 3.4.1 --- Principle of assay --- p.28 / Chapter 3.4.2 --- Loading DRG cells with [3H]adenine --- p.28 / Chapter 3.4.3 --- Column preparation --- p.28 / Chapter 3.4.4 --- Measurement of [3H]cAMP production in DRG cells --- p.29 / Chapter 3.4.5 --- Data analysis --- p.30 / Chapter Chapter 4 --- Identification of DRG cells in dissociated cultures --- p.31 / Chapter 4.1 --- Introduction --- p.31 / Chapter 4.2 --- Aim of study --- p.34 / Chapter 4.3 --- Results --- p.35 / Chapter 4.3.1 --- Identification of DRG cells in isolated cultures --- p.35 / Chapter 4.3.2 --- Activation and proliferation of glial cells in isolated cell cultures --- p.36 / Chapter 4.3.3 --- Identification of glial cells in cultures --- p.38 / Chapter 4.3.4 --- Modification of staining methods --- p.40 / Chapter 4.3.5 --- Immunohistochemistry to identify DRG cells in DRG slices --- p.42 / Chapter 4.3.6 --- Comparison of antibody staining in whole DRG and isolated DRG cells --- p.44 / Chapter 4.4 --- Discussion --- p.44 / Chapter 4.5 --- Summary --- p.53 / Chapter Chapter 5 --- Characterization of GPCRs in isolated DRG cultures --- p.69 / Chapter 5.1 --- Introduction --- p.69 / Chapter 5.1.1 --- G-protein coupled receptors --- p.69 / Chapter 5.1.2 --- Pharmacological characterization of prostanoid receptors on DRG cells --- p.73 / Chapter 5.1.3 --- Gs- and Gi/o-coupled GPCRs in DRG cells --- p.75 / Chapter 5.1.3.1 --- Gs-coupled GPCR: β-adrenoceptors --- p.76 / Chapter 5.1.3.2 --- Gs-coupled GPCR: CGRP receptors --- p.79 / Chapter 5.1.3.3 --- Gi/o-coupled GPCR: α2-adrenoceptors --- p.82 / Chapter 5.1.3.4 --- Gi/o-coupled GPCR: Cannabinoid receptors --- p.85 / Chapter 5.1.3.5 --- Gi/o-coupled GPCR: 5-HT1Areceptors --- p.88 / Chapter 5.1.3.6 --- Gi/o-coupled GPCR: opioid and opioid-receptor-like 1 receptors --- p.90 / Chapter 5.2 --- Aims of study --- p.93 / Chapter 5.3 --- Results --- p.94 / Chapter 5.3.1 --- Characterization of prostanoid receptors in isolated DRG cells --- p.94 / Chapter 5.3.2 --- Characterization of CGRP receptors in isolated DRG cells --- p.96 / Chapter 5.3.3 --- Investigation of the effect of CGRP8.37 on CGRP responses --- p.97 / Chapter 5.3.4 --- Characterization of β1-adrenoceptors in isolated DRG cells --- p.97 / Chapter 5.3.5 --- Characterization of β2-adrenoceptors in isolated DRG cells --- p.98 / Chapter 5.3.6 --- Identification of β-adrenoceptor subtype mediating isoprenaline-stimulated responses.. --- p.99 / Chapter 5.3.7 --- Characterization of α2-adrenceptors in isolated DRG cells --- p.100 / Chapter 5.3.8 --- Characterization of cannabinoid 1 receptors in isolated DRG cells ... --- p.100 / Chapter 5.3.9 --- Characterization of cannabinoid 2 receptors in isolated DRG cells --- p.101 / Chapter 5.3.10 --- Characterization of 5-HT1A receptors in isolated DRG cells --- p.101 / Chapter 5.3.11 --- Characterization of μ-opioid receptors in isolated DRG cells --- p.102 / Chapter 5.3.12 --- Characterization of opioid-receptor-like 1 receptors in isolated DRG cells --- p.102 / Chapter 5.3.13 --- Effect of nerve growth factor on DRG cells --- p.103 / Chapter 5.4 --- Discussion --- p.106 / Chapter 5.5 --- Summary --- p.114 / Chapter Chapter 6 --- Conclusion and further studies --- p.134 / References --- p.137