Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 6 uppsatser. Med sammanfattning på svenska.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

Regulators of G protein signaling (RGSs) are negative regulators of G protein coupled receptors (GPCRs). Our lab has demonstrated that yeast Saccharomyces cerevisiae is a useful system to study RGS and G protein signaling. Mammalian RGSs can be expressed in yeast and favored to interact with mammalian GPCRs as well. / Based on the observation that human RGS1 causes yeast cell growth arrest, I therefore used RGS1 expressing yeast cells to screen a mouse T cell cDNA library in order to find potential interacting proteins. From the screen, I identified a mouse sphingomyelin synthase 1 (SMS1) cDNA. By using a series of different apoptotic stimuli, such as hydrogen peroxide, osmotic stress, exogenous ceramide and its precursors, high temperature etc., SMS1 expression was found to suppress cell growth arrest and prevent viability decline, indicating that SMS1 represents an anti-apoptotic protein that functions by decreasing the intracellular level of pro-apoptotic ceramide. / Gene analysis further indicated that the SMS1 gene consists of 16 exons spread over a 256kb portion of mouse chromosome 19. It is alternatively spliced to produce 4 different transcripts (SMS1alpha1, SMS1alpha2, SMS1beta and SMS1gamma) and encode 3 different proteins (SMS1alpha, SMS1beta and SMS1gamma). Notably, I found that SMS1beta protein does not interfere with SMS1alpha anti-apoptotic function, although both of these two proteins contain the protein-protein interaction domain, sterile alpha motif (SAM), at their N-terminus. / I also carried out a study to examine GPCR-RGS interactions using the yeast expression system. Our lab had noticed that there was an extra RGS5 related protein that was detected by western blot analysis in the protein extracts prepared from yeast and HEK293 cells expressing RGS5. The size of the band was approximately 2 times the molecular weight of RGS5, indicating the possibility that RGS5 forms a dimer. To further examine this hypothesis, I, therefore, performed a series of experiments, included yeast 2 hybrid assays, to demonstrate that RGS5 does interact with itself. This is the first report that RGS can form a dimer. The implications for this finding are discussed in detail.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

No description available.

Lau Pui Ngan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 171-181). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgement --- p.vii / Abbreviations --- p.viii / Publications Based on work in this thesis --- p.xii / Chapter Chapter 1 --- Introduction and project overview --- p.1 / Chapter 1.1 --- Ghrelin structure and its synthesis --- p.3 / Chapter 1.2 --- Types of growth hormone secretagogues (GHSs) --- p.6 / Chapter 1.3 --- Characterization of GHS-R --- p.7 / Chapter 1.3.1 --- Cloning of GHS-Rla and GHS-Rlb --- p.7 / Chapter 1.3.1.1 --- GHS-R subtypes --- p.7 / Chapter 1.3.1.2 --- Properties of GHS-R subtypes --- p.7 / Chapter 1.3.1.3 --- Evidence of non-GHS-Rla stimulated by ghrelin and GHSs --- p.9 / Chapter 1.3.1.4 --- Distribution of GHS-R --- p.10 / Chapter 1.3.2 --- Signal transduction pathways of GHS-R --- p.11 / Chapter 1.3.3 --- Comparison between human and seabream GHS-R --- p.12 / Chapter 1.4 --- Is adenosine a partial agonist at GHS-Rla? --- p.15 / Chapter 1.5 --- Physiological effects of ghrelin --- p.17 / Chapter 1.6 --- Apoptosis --- p.19 / Chapter 1.6.1 --- Introduction --- p.19 / Chapter 1.6.2 --- Apoptosis versus necrosis --- p.19 / Chapter 1.6.3 --- Mechanisms of apoptosis --- p.20 / Chapter 1.6.4 --- Methods to study apoptosis --- p.23 / Chapter 1.6.5 --- Different types of apoptotic inducers --- p.24 / Chapter 1.7 --- Apoptotic and anti-apoptotic pathways regulated by GPCRs --- p.27 / Chapter 1.7.1 --- Bcl-2 family pathway --- p.27 / Chapter 1.7.2 --- Caspase pathway --- p.27 / Chapter 1.7.3 --- ERK pathway --- p.28 / Chapter 1.7.4 --- PI3K/Akt pathway --- p.29 / Chapter Chapter 2 --- Materials and solutions --- p.31 / Chapter 2.1 --- Materials --- p.31 / Chapter 2.2 --- "Culture medium, buffer and solutions" --- p.37 / Chapter 2.2.1 --- Culture medium --- p.37 / Chapter 2.2.2 --- Buffers --- p.37 / Chapter 2.2.3 --- Solutions --- p.38 / Chapter Chapter 3 --- Methods --- p.41 / Chapter 3.1 --- Maintenance of cell lines --- p.41 / Chapter 3.1.1 --- Human Embryonic kidney (HEK293) cells --- p.41 / Chapter 3.1.2 --- HEK293 cells stably expressing black seabream growth hormone secretagogues receptors (HEK-sbGHS-Rla and HEK-sbGHS-Rlb) --- p.41 / Chapter 3.2 --- Preparation of plasmid DNA --- p.42 / Chapter 3.2.1 --- Preparation of competent E. coli --- p.42 / Chapter 3.2.2 --- Transformation of DNA into competent cells --- p.42 / Chapter 3.2.3 --- Small-scale and large-scale plasmid DNA preparation --- p.43 / Chapter 3.2.4 --- Confirmation of the purity and the identity of the plasmid DNA --- p.43 / Chapter 3.3 --- Transient transfection of mammalian cells --- p.45 / Chapter 3.4 --- Development of stable cell lines --- p.46 / Chapter 3.4.1 --- Determination of the optimum concentration of each antibiotic used in selection of clones --- p.46 / Chapter 3.4.2 --- Development of monoclonal stable cell line --- p.46 / Chapter 3.4.3 --- Confirmation the expression of 2myc-hGHS-Rla and myc-hGHS-Rlb --- p.48 / Chapter 3.5 --- Measurement of phospbolipase C activity --- p.49 / Chapter 3.5.1 --- Introduction --- p.49 / Chapter 3.5.2 --- Preparation of columns --- p.49 / Chapter 3.5.3 --- [3 H]-inositol phosphate assay --- p.49 / Chapter 3.5.4 --- Measurement of [3H]-inositol phosphates production --- p.50 / Chapter 3.5.5 --- Data analysis --- p.50 / Chapter 3.6 --- Determination of transient transfection efficiency --- p.51 / Chapter 3.7 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.52 / Chapter 3.7.1 --- RNA extraction and first strand cDNA production --- p.52 / Chapter 3.7.2 --- PCR and visualization of amplicons --- p.52 / Chapter 3.7.3 --- Real-time PCR --- p.59 / Chapter 3.7.3.1 --- Construction of standard curve --- p.60 / Chapter 3.7.3.2 --- Data analysis --- p.60 / Chapter 3.8 --- Measurement of caspase-3 activity --- p.65 / Chapter 3.8.1 --- Determination of caspase-3 activity using colorimetric assay --- p.65 / Chapter 3.8.1.1 --- Introduction --- p.65 / Chapter 3.8.1.2 --- Induction of apoptosis --- p.65 / Chapter 3.8.1.3 --- Preparation of cell lysates --- p.65 / Chapter 3.8.1.4 --- Quantification of caspase-3 activity by measuring pNA absorbance --- p.66 / Chapter 3.8.1.5 --- Data analysis --- p.67 / Chapter 3.8.2 --- Determination of caspase-3 activity using bioluminescence resonance energy transfer (BRET2) assay --- p.67 / Chapter 3.8.2.1 --- Introduction --- p.67 / Chapter 3.8.2.2 --- Quantification of caspase-3 activity using BRET2 assay --- p.68 / Chapter 3.8.2.3 --- Data analysis --- p.69 / Chapter 3.8.3 --- Determination of caspase-3 activity using fluorescence resonance energy transfer (FERT) assay --- p.70 / Chapter 3.8.3.1 --- Introduction --- p.70 / Chapter 3.8.3.2 --- Quantification of caspase-3 activity using FRET assay --- p.70 / Chapter 3.8.3.3 --- Data analysis --- p.71 / Chapter Chapter 4 --- Results --- p.72 / Chapter 4.1 --- Characterization of GHS-R --- p.72 / Chapter 4.1.1 --- Properties of GHS-Rla --- p.72 / Chapter 4.1.1.1 --- Constitutively active receptor --- p.72 / Chapter 4.1.1.2 --- Characterization of epitope-tagged hGHS-Rla --- p.73 / Chapter 4.1.2 --- Properties of GHS-Rlb --- p.75 / Chapter 4.1.3 --- Conclusions --- p.75 / Chapter 4.2 --- Effect of co-transfection of HEK293 cells --- p.85 / Chapter 4.2.1 --- Effect of balancing DNA concentrations transfected into HEK293 cells --- p.85 / Chapter 4.2.2 --- Effect of balancing DNA concentration using another Gq-coupled receptor --- p.87 / Chapter 4.2.3 --- Effect of Gi- and Gs-coupled receptor on GHS-Rla signaling --- p.88 / Chapter 4.2.4 --- Potentiating effect of co-transfection appeared using different transfection reagents --- p.88 / Chapter 4.2.5 --- Co-transfection improves transfection efficiency --- p.89 / Chapter 4.2.6 --- Discussions --- p.91 / Chapter 4.3 --- Development of cell lines stably expressing hGHS-Rla or hGHS-Rlb --- p.102 / Chapter 4.3.1 --- Advantages of using a monoclonal cell line --- p.102 / Chapter 4.3.2 --- Sensitivity of HEK293 cells to antibiotics --- p.102 / Chapter 4.3.3 --- Production of polyclonal stable cell line --- p.103 / Chapter 4.3.4 --- Monoclonal stable cell line selection --- p.104 / Chapter 4.3.5 --- Discussions --- p.105 / Chapter 4.4 --- Effect of adenosine on GHS-Rla signaling --- p.111 / Chapter 4.4.1 --- Adenosine acts as partial agonist --- p.111 / Chapter 4.4.2 --- Effect of substance P analog on adenosine-mediated GHS-Rla signaling --- p.112 / Chapter 4.4.3 --- Effect of adenosine deaminase (ADA) on adenosine- and ghrelin-stimulated GHS-Rla signaling --- p.113 / Chapter 4.4.4 --- Specificity of ADA --- p.115 / Chapter 4.4.5 --- Conclusions --- p.116 / Chapter 4.5 --- Role of GHS-R in apoptosis --- p.124 / Chapter 4.5.1 --- Different methods to measure caspase-3 activity --- p.124 / Chapter 4.5.1.1 --- Colorimetric assay --- p.124 / Chapter 4.5.1.1.1 --- Time course for staurosporine and etoposide in HEK293 cells --- p.125 / Chapter 4.5.1.1.2 --- Effect of 2myc-hGHS-Rla on staurosporine- and etoposide-induced caspase-3 activity --- p.127 / Chapter 4.5.1.1.3 --- Time course for staurosporine and etoposide in sbGHS-R monoclonal stable cell line --- p.128 / Chapter 4.5.1.1.4 --- Effect of sbGHS-Rs on staurosporine- and etoposide- induced caspase-3 activityin HEK 293 cells --- p.129 / Chapter 4.5.1.1.5 --- Effect of sbGHS-Rs on staurosporine- induced caspase-3 activity in sbGHS-R monoclonal stable cell line --- p.130 / Chapter 4.5.1.1.6 --- Differences between epitope-tagged and non-tagged sbGHS-Rs in staurosporine- induced caspase-3 activity --- p.131 / Chapter 4.5.1.1.7 --- The role of epitope-tagged sbGHS-Rlbin staurosporine-induced caspase-3 activity --- p.132 / Chapter 4.5.1.1.8 --- Effect of staurosporine and etoposide on GHS-Rla signaling --- p.133 / Chapter 4.5.1.2 --- BRET2 assay --- p.135 / Chapter 4.5.1.3 --- FRET assay --- p.136 / Chapter 4.5.1.4 --- Conclusions --- p.136 / Chapter 4.6 --- Determination of GHS-R amount in terms of mRNA --- p.155 / Chapter 4.6.1 --- Determination of GHS-R amount in stable cell lines --- p.155 / Chapter 4.6.2 --- Transfected DNA amount match with stable cell lines --- p.155 / Chapter Chapter 5 --- "Discussion, Conclusions and Future Plan" --- p.159 / Chapter 5.1 --- General Discussion and Conclusions --- p.159 / Chapter 5.2 --- Future Plan and Experimental Design --- p.168 / References --- p.171

In a phage binding assay, phage clone (3p5A190) expressing a surrogate mas ligand displayed punctate binding and were internalized in cell expressing native mas and GFP-tagged variants. However, the number of bound and internalized phages in cells expressing mas-GFP was substantially less than the cells expressing mas-(Gly10Ser5)GFP and native mas. In parallel, biotinylation experiment quantitatively showed that the extent of mas-(Gly10Ser 5)-GFP translocation was higher than that of mas-GFP. Consistently, cells expressing mas-(Gly10Ser5)-GFP and native mas showed a rapid and sustained increase of intracellular calcium levels upon MBP7 stimulation. By contrast, cells expressing mas-GFP only response to higher concentration of MBP7 challenge and showed a delayed increase of intracellular calcium level. Moreover, cells expressing native mas had a higher proportion (80%) of cells responsive to MBP7 stimulation; in contrast to only 10∼20% of cells expressing mas fusion proteins. / MBP7-like motif was identified in human facilitative GLUT1 and GLUT7 indicating that mas might interact with glucose transporter (GLUT) and regulate cellular glucose uptake. GLUT4 was found to be expressed endogenously in the CHO cell by RT-PCR, but expression of insulin receptor was not detectable. Although no statistical difference was detected in basal glucose uptake among control cells Vc0M80 and cells with different levels of mas expression, cells expressing mas-(Gly10Ser5)-GFP showed a high glucose uptake in response to insulin. Furthermore, basal 2-DOG uptake in Mc0M80 cells was not affected by pretreatment with various kinase inhibitors or transient expression of Rho variants. By contrast, MBP7 was found to induce a significant elevation of glucose uptake specifically in Mc0M80 cells transiently transfected with GLUT1. / Orphan G protein-coupled receptor (GPCR) mas was initially isolated from a human epidermal carcinoma. Previous study from our lab identified a surrogate ligand---MBP7 (mas binding peptide 7) for mas, and suggested that GFP tagging might affect the receptor activity of mas. In this project, three stable CHO cell lines expressing native mas, mas-GFP and mas-(Gly10Ser 5)-GFP were used to characterize receptor activity of mas. / To summarize, direct GFP tagging at the C-terminus of mas decreased its interactions with ligand and downstream signaling molecules. Partial recovery of mas receptor activity by adding a peptide linker was confirmed by phage binding, membrane fusion protein translocation and calcium response. In addition, mas was possibily coupled with GLUT1 to affect cellular glucose uptake via signaling pathways yet to be fully characterized. / Sun, Jingxin. / Adviser: Cheung Wing Tai. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0104. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 150-170). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Animals rely on the integration of a variety of external cues to understand and respond appropriately to their environment. The relative amounts of food and constitutively secreted pheromone detected by the nematode C. elegans determines how it will develop and grow. Starvation conditions cause the animal to enter a protective stage, termed dauer. Dauer animals are non-eating, long-lived and stress resistant. Yet, when these animals are introduced to food replete conditions they will recover from dauer and proceed into normal development. Furthermore, food restriction has been demonstrated to extend the lifespan of a wide-range of species including C. elegans. However, the exact mechanism by which food signals are detected and transduced by C. elegans to influence development and longevity remains unknown. Here, we identify a G protein-coupled receptor (GPCR) DCAR-1 that acts in two chemosensory neurons to mediate food signaling in an autophagy-related manner. The DCAR-1 ligand Dihydrocaffeic acid (DHCA) competes with dauer-inducing pheromone to promote growth. DHCA is a key intermediate in the shikimate pathway, which is required to synthesize folate and aromatic amino acids. We report that dcar-1 mutations influence dauer formation and extend wildtype lifespan via a mechanism of dietary restriction. Moreover, we show that the lifespan extension of dcar-1 mutants is completely dependent on autophagy gene atg- 18. Furthermore, our data suggests metabolites derived from shikimate are food signals that control aging and dauer development through GPCR signaling in C. elegans. These studies will contribute to the delineation of mechanisms behind the beneficial effects of dietary restriction in eukaryotic organisms. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

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.