published_or_final_version / abstract / Chinese Medicine / Master / Master of Philosophy

Orientador: Man Chin Chung / Coorientador: Cleverton Roberto de Andrade / Banca: Luiz Fernando Takase / Banca: Cíntia Duarte de Freitas Milagre / Banca: Renato Farina Menegon / Banca: Jean Leandro dos Santos / Resumo: O câncer colorretal (CCR) é um dos 10 tipos de câncer mais incidentes no Brasil. A terapia consiste na remoção cirúrgica e em alguns casos, a quimioterapia adjuvante, composta de 5-fluorouracil e outros fármacos associados. O 5-FU, apesar de amplamente utilizado, provoca uma série de efeitos adversos, relacionados à toxicidade renal, cardíaca, hepática e mucosite. Nesse contexto, a ferramenta do planejamento de pró fármacos é uma alternativa para a redução das propriedades indesejadas dos fármacos. Assim, o objetivo deste trabalho foi comprovar a hipótese da diminuição da toxicidade do 5-FU pela taurina, aminoácido com propriedades anti-inflamatórias e antioxidante, para a obtenção de pró-fármacos derivados. Para isso, ratos Wistar foram submetidos ao modelo de carcinogênese de cólon induzida por1,2-dimetilhidrazina (DMH), e tratados com a associação (5-FU+TAU) por 8 dias, na 20° semana pós indução. Os resultados demonstraram que a taurina inibe a formação de criptas aberrantes e atua sinergicamente com 5-FU reduzindo a quantidade das mesmas. Além disso, o grupo tratado com 5-FU isolado apresentou aumento de 28% no número de tumores, enquanto a TAU isolada promoveu 64% menos neoplasias que o controle. Na associação a redução na incidência alcança 86%, sendo nenhuma das neoplasias classificados como adenocarcinomas (tumor invasivo), enquanto TAU isolada apresenta 50%, e 5-FU 70% de adenocarcinomas entre as neoplasias. Os resultados demonstram que a TAU e o 5-FU apresentam efei... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Colorectal cancer is one of the ten most incident types of cancer in Brazil. Its treatment consists of surgical intervention and, in some cases, adjuvant chemotherapy, using 5-fluorouracil (5-FU) associated with other drugs. Although extensively used, 5-FU presents several adverse effects, related to renal, cardiac and hepatic toxicity and mucositis. In this context, the search of new drugs is an alternative to reduce some undesired properties of the drug. Thus, the objective of this work was to prove the hypothesis of the reduction of toxicity of 5-FU by taurine, amino acid with anti-inflammatory and antioxidant properties, to obtain derivative prodrugs. For this, Wistar rats were submitted to the colon carcinogenesis model induced by 1,2-dimethylhydrazine (DMH) and treated with the combination (5-FU + TAU) for 8 days at the 19th week post induction. The results have demonstrated that taurine inhibits the formation of aberrant crypts foci and acts synergistically with 5-FU. In addition, the group treated with 5-FU alone showed a 28% increase in the number of tumors, whereas the isolated TAU promoted 64% fewer neoplasms than the control. In the association, the reduction in incidence reaches 86%, none of the neoplasms classified as adenocarcinomas (invasive tumor), while isolated TAU presents 50%, and 5-FU 70% of adenocarcinomas between neoplasms. Thus, the association results have demonstrated that the two substances presented a chemotherapic effect and can be used to obtent... (Complete abstract click electronic access below) / Doutor

Experimentally naive rats show variance in their locomotor reactivity to novelty, some displaying higher (HR) while others displaying lower (LR) reactivity, associated with vulnerability to stress. LRHR phenotype is proposed as an antecedent to the development of stress hyper responsiveness. Results presented here show emergence of antidepressive-like behavior following peripubertal-juvenile exposure to chronic variable physical (CVP) and chronic variable social stress (CVS) in HR rats, and depressive-like behavior following CVP in the LRs. The antidepressive-like behavior in HR rats was accompanied by increased levels of acetylated Histone3 (acH3) and acetylated Histone4 (acH4) at the hippocampal brain-derived neurotrophic factor (BDNF) P2 and P4 promoters respectively. This effect may mediate increased mossy fibre (MF) terminal field size, particularly the suprapyramidal mossy fibre projection volume (SP-MF), in the HR animals following both stress regimens. These findings show that chronic variable stress during adolescence induces individual differences in molecular, neuromorphological and behavioral parameters between LRs and HRs, which provides further evidence that individual differences in stress responsiveness is an important factor in resistance or vulnerability to stress-induced depression and/or anxiety. / by Ozge Oztan. / Thesis (Ph.D.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Dorsal root ganglia (DRG) cell preparations are commonly used to study the properties of sensory neurons in relation to nociception. A typical DRG cell preparation contains both neurons and glial cells, and in addition to a conventional supportive role of glial cells, an increasing volume of literature has reported interactions between neurons and accompanying glial cells. A typical mixed DRG cell preparation can be separated into a neuron-enriched cell fraction and a preparation of purified glial cells. Using these purified cell fractions, we can study the relative contributions and interactions between neurons and glial cells in regulating neurite outgrowth and adenylyl cyclase-dependent cell signalling activity in vitro. / From our previous studies, pretreating DRG cell cultures with pertussis toxin (PTx) caused neurite retraction over a period of 2 h following the initial stimulus of removal from incubator. The purpose of the current study was to investigate whether this PIx-dependent response was specific to anyone of the three subpopulations of DRG neurons. Interestingly, no neurite retraction response was observed in enriched DRG cultures, including cultures enriched with isolectin B4 (IB4)-positive neurons or IB4-negative neurons. Addition of glial cells or conditioned medium from glial cells to IB4-negative cultures was necessary to restore the PTx-dependent neurite retraction response, which was then only observed in large diameter proprioceptive neurons. To conclude, glial cells constitutively release factor/s that stimulate neurite retraction in larger diameter neurons, and is counterbalanced by neuroprotective Gilo protein signalling pathway. / From our studies, we have provided evidence of bidirectional interactions between neurons and glial cells, with glial cells regulating neurite outgrowth and neurons regulating adenylyl cyclase activity in glial cells. These findings reveal the properties of glial cells in regulating neurite outgrowth and in producing prostanoid-stimulated responses. Moreover, our fmdings provide foundation to understand complex neuron-glia interactions in vivo which will eventually help to overcome obstacles in promoting neurite regeneration and in controlling pain. / In a parallel study, we proved that hyperalgesic agents such as prostaglandin E2 (PGE2) and the prostacyclin (PGI2) mimetic (cicaprost) stimulate cAMP production in DRG cell culture via EP4 and IP receptors, respectively. These prostanoids were presumed to act only on the neurons in typical mixed cell cultures, but since we had acquired purified glial cell preparation, we tested for involvement of glial cells in measurement of agonist-stimulated cAMP production. Interestingly, a purified glial cell cultures also produced EP4 and IP-dependent responses. The expression of EP4 and IP receptors by DRG glia was further confirmed by the detection of EP4 and IP-like immunoreactivity and mRNA. Moreover, these agonist-stimulated responses were greatest in the glial cell preparation, and surprisingly weakest in the neuron-enriched cell cultures. Furthermore, the presence of neurons significantly inhibited both EP4 and IP receptor-dependent signalling in glial cells, but was without effect on forskolin (agonist-independent) stimulation of adenylyl cyclase. In order to characterize this neuron-glia interaction, we tested the adenylyl cyclase activities in glial cell cultures which were treated with conditioned medium derived from neurons or were separated from physical contact with neurons plated on transwell membrane. These studies further suggest that the neuron-glia interactions were dependent on both soluble factors and cell-cell contact. / Ng, Kai Yu. / Adviser: Helen Wise. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 152-172). / 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.

Wei, Fangyuan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 182-211). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Lee Kai Woo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 90-104). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / Thesis Committee --- p.iii / Abstracts --- p.v / 論文槪要 --- p.vii / List of figures --- p.viii / List of abbreviations --- p.ix / Chapter Chapter one --- Introduction --- p.1 / Chapter 1.1 --- Drug metabolism and UGTs --- p.1 / Chapter 1.2 --- Natural substrates of UGTs --- p.4 / Chapter 1.3 --- Functions of UGT isoforms: roles of UGT polymorphisms --- p.6 / Chapter 1.4 --- Evolution of the UGT1 gene locus in vertebrates --- p.8 / Chapter 1.5 --- Multiple Variable First Exons: A Mechanism for Cell- and Tissue-Specific Gene regulation --- p.13 / Chapter 1.6 --- Evolutionary Origin of the Variable and Constant Genomic Organization --- p.14 / Chapter 1.7 --- Variable and Constant Genomic Organizations Exist in Mammalian UGTs --- p.20 / Chapter 1.8 --- The history of recombinant UGT expression --- p.20 / Chapter 1.9 --- UGT1A8 --- p.21 / Chapter 1.10 --- Licorice and its active component --- p.24 / Chapter 1.11 --- Enzyme induction in the liver --- p.25 / Chapter 1 12 --- Objectives --- p.28 / Chapter Chapter two --- Methods and Materials --- p.29 / Chapter 2.1 --- UGT1A8 induction studies --- p.30 / Chapter 2.1.1 --- Drug preparation --- p.30 / Chapter 2.1.2 --- Cell viability study with Neutral Red Assay Rat treatment --- p.30 / Chapter 2.1.3 --- Cell treatment --- p.31 / Chapter 2.1.4 --- Rat treatment --- p.31 / Chapter 2.1.5 --- RNA extraction from rat liver and cell culture --- p.31 / Chapter 2.1.6 --- Quantization of RNA --- p.32 / Chapter 2.1.7 --- Denaturing gel electrophoresis for RNA --- p.33 / Chapter 2.1.8 --- Northern hybridization --- p.33 / Chapter 2.1.9 --- Probe for Northern Blotting --- p.34 / Chapter 2.1.10 --- Agarose Gel analysis and Northern Blot analysis --- p.34 / Chapter 2.2 --- Recombinant expression of UGT1A8 in E.coli JM109 --- p.35 / Chapter 2.2.1 --- cDNA synthesis --- p.35 / Chapter 2.2.2 --- Polymerase chain reaction --- p.35 / Chapter 2.2.3 --- Agarose gel electrophoresis for DNA --- p.35 / Chapter 2.2.4 --- "Amplification of target gene, UGT1A8" --- p.36 / Chapter 2.2.5 --- Restriction enzyme digestion of plasmid and insert --- p.36 / Chapter 2.2.6 --- Ligation of plasmid and insert DNA --- p.37 / Chapter 2.2.7 --- Amplification of target plasmid --- p.37 / Chapter 2.2.8 --- Screening of target plasmid --- p.37 / Chapter 2.2.9 --- DNA sequencing --- p.38 / Chapter 2.2.10 --- Transformation of protein expression host --- p.38 / Chapter 2.2.11 --- Confirmation of transformation of protein expression host --- p.38 / Chapter 2.2.12 --- Protein expression --- p.39 / Chapter 2.2.13 --- Protein purification --- p.39 / Chapter 2.2.14 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis --- p.40 / Chapter 2.2.15 --- Confirmation of the protein --- p.40 / Chapter 2.3 --- Characterization of recombinant UGT1A8 --- p.41 / Chapter 2.3.1 --- UGT assay --- p.41 / Chapter 2.4 --- Routine experiment methods --- p.41 / Chapter 2.4.1 --- Determination of protein --- p.41 / Chapter 2.4.2 --- Nucleic acid purification --- p.42 / Chapter 2.4.3 --- Preparation of chemically competent bacterial cells --- p.42 / Chapter 2.4.4 --- Colony PCR --- p.43 / Chapter 2.4.5 --- Plasmid rescue by alkaline lysis --- p.44 / Chapter 2.4.6 --- Charging of His-tagged column --- p.44 / Chapter 2.4.7 --- Washing of His-tagged column --- p.45 / Chapter Chapter three --- Results --- p.46 / Chapter 3.1 --- UGT1A8 Expression in clone9 and H4IIE after treatment with licorice and 18 β glycyrrhentinic acid --- p.46 / Chapter 3.2 --- UGT1A8 induction in wistar and j/j rats after treatment --- p.63 / Chapter 3.3 --- Construction of pRset-UGT 1A8 Vector --- p.70 / Chapter 3.4 --- Purification of recombinant UGT1A8 --- p.75 / Chapter 3.5 --- Screening of substrate of the purified enzyme --- p.77 / Chapter Chapter four --- Discussion --- p.78 / Chapter 4.1 --- Effects of licorice and 18βglycyrrhetinic acid in the induction of UGT1A8 in different cell lines --- p.78 / Chapter 4.2 --- Comparison of wistar and j/j rats in the induction of UGT1A8 --- p.79 / Chapter 4.3 --- Comparison of licorice and 18(3 glycyrrhetinic acid in the induction of UGT1A8 in rats --- p.81 / Chapter 4.4 --- Comparison of in vivo and in vitro of drug treatment --- p.81 / Chapter 4.5 --- Expression of UGT1A7 after drug treatment in vitro --- p.82 / Chapter 4.6 --- Protein expression and purification --- p.83 / Chapter 4.7 --- Substrates of UGT1A8 --- p.83 / Chapter Chapter Five --- Conclusions --- p.86 / References --- p.90 / Appendix --- p.105

Further in vivo functional studies were also performed. The SLC26A3 antibody was injected into the BALB/C mice seminiferous tubules using micropipette. The animals were sacrificed after three days, and CASA, daily sperm production (DSP) were used to evaluate sperm motility and spermatogenesis. The results showed that sperm motility was increased while there was no significant difference between DSP. Our results indicate that SLC26A3 on sperm does not play a dominant role in spermatogenesis, epididymal maturation and sperm motility. / In the first part of study, guinea pig sperm which were incubated in medium with various concentrations of Cl- resulted in varied percentages of capacitated sperm, in a concentration dependent manner. Depleting Cl-, even in the presence of HCO3 -, abolished sperm capacitation and vice versa, indicating the involvement of both anions in the process. Capacitation-associated HCO 3- dependent events, including cAMP production, protein tyrosine phosphorylation and pHi increase also depend on Cl - concentrations. Similar Cl- dependence was observed for sperm hyperactivated motility and sperm-egg fusion. The capacitation-associated events could also be significantly reduced by inhibitors or antibodies of CFTR and SLC26A3, with a more potent effect observed for niflumate, an inhibitor more selective for SLC26A3, over that of DIDS, an inhibitor more selective for SLC4 exchangers. The expression and localization of CFTR and SLC26A3 in guinea pig sperm were also demonstrated using immunostaining and Western blot analysis. Our results indicate that Cl- is required for the entry of HCO3- necessary for sperm capacitation, implicating the involvement of SLC26A3 in transporting HCO3 - with CFTR providing the recycling pathway for Cl- . / In the second part of study, GC-1 spg cell line that expresses SLC26A6 but not SLC26A3 was used as a negative control. The cells and sperm were pretreated with anion exchanger inhibitors and SLC26A3 antibody, and then membrane potential and intracellular calcium were measured. Our results showed that DIDS could inhibit the HCO3- deficiency induced depolarization of GC-1 spg cells as well as the depolarization induced by Cl- or HCO3- deficiency in sperm. Niflumate could inhibit the HCO3- induced [Ca 2+] i increase of the sperm but not GC-1 spg cells. SLC26A3 antibody had no effect on the GC-1 spg cells but it could block the depolarization caused by C--deficiency in sperm. / Our previous study has demonstrated the involvement of Cystic fibrosis transmembrane conductance regulator (CFTR) in transporting bicarbonate necessary for sperm capacitation. However, whether its involvement is direct or indirect remains unclear. The present study is design to investigate: (1) the possibility of a Cl-/HCO3- exchanger, solute carrier family 26, number 3 (SLC26A3), operating with CFTR during sperm capacitation, (2) the role and the underlying mechanisms of SLC26A3 in other sperm post-testicular processes and spermatogenesis. / Taken together, our results demonstrate the involvement of SLC26A3 in sperm function, particularly in transporting HCO3- necessary for sperm capacitation, which appears to be working with CFTR providing the recycling pathway for Cl- in parallel. The present results also provide an explanation to the observed subfertility in patients with SLC26A3 mutations. Further in vitro and in vivo studies also have shown that SLC26A3 does not play a predominant role in spermatogenesis but may affect other post-testicular maturation processes. / Chen, Wenying. / "November 2009." / Adviser: H.C. Chan. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 101-109). / 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. / Abstract also in Chinese.

Hui, Pok Shun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 274-293). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgements --- p.vii / Publications Based on Work in this Thesis --- p.viii / Abbreviations --- p.ix / Table of Contents --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- The Mast Cell --- p.2 / Chapter 1.1.1 --- Origin and Development of Mast Cells --- p.3 / Chapter 1.1.2 --- Heterogeneity of Mast Cells --- p.5 / Chapter 1.1.2.1 --- Heterogeneity of Rodent Mast Cells --- p.5 / Chapter 1.1.2.2 --- Heterogeneity of Human Mast Cells --- p.6 / Chapter 1.1.3 --- Activation of Mast Cells --- p.8 / Chapter 1.1.3.1 --- IgE-dependent Activation of Mast Cells --- p.8 / Chapter 1.1.3.1.1 --- FceRI Aggregation and Tyrosine Residue Phosphorylation --- p.9 / Chapter 1.1.3.1.2 --- PLC Activation and Calcium Mobilization --- p.10 / Chapter 1.1.3.1.3 --- PKC and MAPK Activation --- p.11 / Chapter 1.1.3.2 --- IgE-independent Activation of Mast Cells --- p.14 / Chapter 1.1.3.2.1 --- Activation by IgG --- p.14 / Chapter 1.1.3.2.2 --- Activation by Basic Secretagogues --- p.14 / Chapter 1.1.3.2.3 --- Activation by Calcium Ionophores --- p.15 / Chapter 1.1.4 --- Mast Cell Mediators --- p.16 / Chapter 1.1.4.1 --- Preformed Mediators --- p.16 / Chapter 1.1.4.2 --- Newly Synthesized Lipid Mediators --- p.18 / Chapter 1.1.4.3 --- Cytokines and Chemokines --- p.19 / Chapter 1.1.5 --- Pathophysiological Roles of Mast Cells --- p.21 / Chapter 1.2 --- Arthritis --- p.23 / Chapter 1.2.1 --- Epidemiology of Arthritis --- p.23 / Chapter 1.2.2 --- Clinical Features of Arthritis --- p.25 / Chapter 1.2.2.1 --- Angiogenesis and Vasodilation --- p.25 / Chapter 1.2.2.2 --- Synovial Changes --- p.25 / Chapter 1.2.2.3 --- Cartilage Degradation and Bone Erosion --- p.26 / Chapter 1.2.3 --- Pathogenesis of Arthritis --- p.27 / Chapter 1.2.3.1 --- Roles of T Cells --- p.27 / Chapter 1.2.3.2 --- Roles of B Cells --- p.28 / Chapter 1.2.3.3 --- Roles of Mast Cells --- p.28 / Chapter 1.2.3.4 --- Roles of Cytokines --- p.31 / Chapter 1.2.4 --- Treatments of Arthritis --- p.32 / Chapter 1.2.4.1 --- NSAIDs --- p.33 / Chapter 1.2.4.2 --- Glucocorticoids --- p.34 / Chapter 1.2.4.3 --- DMARDs --- p.35 / Chapter 1.2.4.4 --- New Drugs --- p.36 / Chapter 1.3 --- Proteinase-Activated Receptor (PAR) --- p.38 / Chapter 1.3.1 --- Introduction to PARs --- p.38 / Chapter 1.3.2 --- Discovery of PARs --- p.39 / Chapter 1.3.2.1 --- PAR1 --- p.39 / Chapter 1.3.2.2 --- PAR2 --- p.39 / Chapter 1.3.2.3 --- PAR3 --- p.40 / Chapter 1.3.2.4 --- PAR4 --- p.41 / Chapter 1.3.3 --- Structure of PARs --- p.43 / Chapter 1.3.4 --- Activation of PARs --- p.43 / Chapter 1.3.4.1 --- Serine Proteinases --- p.44 / Chapter 1.3.4.1.1 --- Thrombin --- p.44 / Chapter 1.3.4.1.2 --- Trypsin --- p.46 / Chapter 1.3.4.1.3 --- Mast Cell Tryptase --- p.46 / Chapter 1.3.4.2 --- PAR Activating Peptides (PAR-APs) --- p.47 / Chapter 1.3.4.3 --- Proteinase Binding and the Tethered Ligand Mechanism --- p.49 / Chapter 1.3.5 --- Signaling of PARs --- p.50 / Chapter 1.3.5.1 --- Signaling of PAR1 --- p.51 / Chapter 1.3.5.2 --- Signaling of PAR2 --- p.52 / Chapter 1.3.5.3 --- Signaling of PAR 3 and PAR4 --- p.53 / Chapter 1.3.6 --- Termination of Signals and Antagonism of PARs --- p.53 / Chapter 1.3.6.1 --- Termination of Signals by Proteolysis --- p.53 / Chapter 1.3.6.2 --- Termination of Signals by Receptor Desensitization --- p.54 / Chapter 1.3.6.3 --- Antagonism of PARs --- p.55 / Chapter 1.3.7 --- Roles of PARs in Immune Responses --- p.56 / Chapter 1.3.7.1 --- PARs and Mast Cells --- p.57 / Chapter 1.3.7.2 --- PARs and A rthritis --- p.58 / Chapter 1.4 --- Aims of Study --- p.60 / Chapter Chapter 2 --- Materials and Methods --- p.62 / Chapter 2.1 --- Materials --- p.63 / Chapter 2.1.1 --- Materials for Study of PAR Gene Expression in Mast Cells by RT-PCR --- p.63 / Chapter 2.1.1.1 --- Materials for RNA Extraction --- p.63 / Chapter 2.1.1.2 --- Materials for cDNA Synthesis by Reverse Transcription --- p.63 / Chapter 2.1.1.3 --- Materials for Gene Amplification by PCR --- p.64 / Chapter 2.1.1.4 --- Materials for Agarose Gel Electrophoresis --- p.64 / Chapter 2.1.1.5 --- Miscellaneous --- p.64 / Chapter 2.1.2 --- Materials for Study of Histamine Release from RPMCs and LAD2 Cells --- p.65 / Chapter 2.1.2.1 --- Drugs --- p.65 / Chapter 2.1.2.1.1 --- Peptides --- p.65 / Chapter 2.1.2.1.2 --- Serine Proteinases --- p.65 / Chapter 2.1.2.1.3 --- Mast Cell Secretagogues --- p.66 / Chapter 2.1.2.1.4 --- Other Drugs --- p.66 / Chapter 2.1.2.2 --- Materials for Rat Sensitization --- p.66 / Chapter 2.1.2.3 --- Materials for LAD2 Cell Culture --- p.66 / Chapter 2.1.2.4 --- Materials for Buffers --- p.67 / Chapter 2.1.2.5 --- Materials for Spectrofluorometric Analysis of Histamine Contents --- p.67 / Chapter 2.1.2.6 --- Miscellaneous --- p.68 / Chapter 2.1.3 --- Materials for Histological Study of Synovial Mast Cells --- p.69 / Chapter 2.1.3.1 --- Drugs --- p.69 / Chapter 2.1.3.2 --- Chemicals --- p.69 / Chapter 2.1.3.3 --- Miscellaneous --- p.69 / Chapter 2.1.4 --- Materials for Study of Rat Knee Joint Inflammation --- p.70 / Chapter 2.1.4.1 --- Drugs --- p.70 / Chapter 2.1.4.1.1 --- Peptides --- p.70 / Chapter 2.1.4.1.2 --- Other Drugs --- p.70 / Chapter 2.1.4.2 --- Materials for Assessment of Vascular Permeability --- p.71 / Chapter 2.1.4.3 --- Miscellaneous --- p.71 / Chapter 2.2 --- Methods --- p.72 / Chapter 2.2.1 --- Study of PAR Gene Expression in Mast Cells by RT-PCR --- p.72 / Chapter 2.2.1.1 --- Animals --- p.72 / Chapter 2.2.1.2 --- LAD2 Cell Culture --- p.72 / Chapter 2.2.1.3 --- Preparation of Buffers --- p.73 / Chapter 2.2.1.4 --- RNA Extraction --- p.73 / Chapter 2.2.1.5 --- Heparinase and DNase Treatments --- p.74 / Chapter 2.2.1.6 --- cDNA Synthesis by Reverse Transcription --- p.75 / Chapter 2.2.1.7 --- Gene Amplification by PCR --- p.75 / Chapter 2.2.1.8 --- Agarose Gel Electrophoresis --- p.77 / Chapter 2.2.2 --- Study of Histamine Release from RPMCs and LAD2 Cells --- p.77 / Chapter 2.2.2.1 --- Rat Sensitization --- p.77 / Chapter 2.2.2.2 --- Preparation of Buffers --- p.75 / Chapter 2.2.2.3 --- Preparation of Stock Solutions --- p.78 / Chapter 2.2.2.3.1 --- Stock Solutions of Peptides --- p.75 / Chapter 2.2.2.3.2 --- Stock Solutions of Serine Proteinases --- p.79 / Chapter 2.2.2.3.3 --- Stock Solutions of Mast Cell Secretagogues and Other Drugs --- p.79 / Chapter 2.2.2.4 --- Preparation of Mast Cells --- p.80 / Chapter 2.2.2.4.1 --- Isolation and Purification of RPMCs --- p.80 / Chapter 2.2.2.4.2 --- Preparation of LAD2 Cells --- p.81 / Chapter 2.2.2.4.3 --- Determination of Cell Number and Viability --- p.81 / Chapter 2.2.2.5 --- General Protocol for Histamine Release Assay --- p.82 / Chapter 2.2.2.5.1 --- RPMC Experiments --- p.52 / Chapter 2.2.2.5.2 --- LAD2 Cell Experiments --- p.53 / Chapter 2.2.2.6 --- Spectrofluorometric Analysis of Histamine Contents --- p.83 / Chapter 2.2.2.6.1 --- Manual Analysis --- p.85 / Chapter 2.2.2.6.2 --- Automated Analysis --- p.85 / Chapter 2.2.2.7 --- Data Analysis --- p.86 / Chapter 2.2.2.7.1 --- Calculation of Histamine Release --- p.86 / Chapter 2.2.2.7.2 --- Data Presentation and Statistical Analysis --- p.87 / Chapter 2.2.3 --- Histological Study of Synovial Mast Cells --- p.88 / Chapter 2.2.3.1 --- Preparation of Buffers and Chemicals --- p.88 / Chapter 2.2.3.2 --- Preparation of Drugs --- p.88 / Chapter 2.2.3.3 --- Intra-peritoneal Injections of Compound 48/80 --- p.88 / Chapter 2.2.3.4 --- Fixation --- p.89 / Chapter 2.2.3.5 --- Processing --- p.89 / Chapter 2.2.3.6 --- Embedding --- p.90 / Chapter 2.2.3 --- Sectioning --- p.90 / Chapter 2.2.3.8 --- Staining --- p.90 / Chapter 2.2.4 --- Study of Rat Knee Joint Inflammation --- p.91 / Chapter 2.2.4.1 --- Animals --- p.91 / Chapter 2.2.4.2 --- Preparation of Drugs --- p.92 / Chapter 2.2.4.3 --- Induction of Anaesthesia --- p.92 / Chapter 2.2.4.4 --- Intra-articular Injection of Drugs --- p.93 / Chapter 2.2.4.5 --- Topical Administration of Drugs --- p.93 / Chapter 2.2.4.6 --- Assessment of Mechanical Allodynia --- p.93 / Chapter 2.2.4.7 --- Assessment of Joint Oedema --- p.94 / Chapter 2.2.4.8 --- Assessment of Hyperaemia --- p.95 / Chapter 2.2.4.9 --- Assessment of Vascular Permeability --- p.95 / Chapter 2.2.4.10 --- Data Analysis --- p.96 / Chapter Chapter 3 --- Studies of Roles of PAR in Mast Cells --- p.97 / Chapter 3.1 --- Introduction --- p.98 / Chapter 3.2 --- Materials and Methods --- p.103 / Chapter 3.2.1 --- Study of PAR Gene Expression in Mast Cells by RT-PCR --- p.103 / Chapter 3.2.2 --- Study of Effects of PAR Agonists on Histamine Release from Mast Cells --- p.103 / Chapter 3.2.3 --- Study of Signaling Pathways Induced by PAR Agonists in Mast Cells --- p.104 / Chapter 3.3 --- Results --- p.105 / Chapter 3.3.1 --- Study of PAR Gene Expression in Mast Cells by RT-PCR --- p.105 / Chapter 3.3.1.1 --- PAR Gene Expression in RPMCs --- p.105 / Chapter 3.3.1.2 --- PAR Gene Expression in LAD2 Cells --- p.105 / Chapter 3.3.2 --- Study of Effects of PAR Agonists on Histamine Release from Mast Cells --- p.106 / Chapter 3.3.2.1 --- Effects of Serine Proteinases on Histamine Release from RPMCs --- p.106 / Chapter 3.3.2.1.1 --- Thrombin --- p.106 / Chapter 3.3.2.1.2 --- Trypsin --- p.106 / Chapter 3.3.2.1.3 --- Tryptase --- p.107 / Chapter 3.3.2.2 --- Effects of PAR-APs on Histamine Release from RPMCs --- p.107 / Chapter 3.3.2.2.1 --- TFLLR-NH2 (PAR1-AP) --- p.107 / Chapter 3.3.2.2.2 --- SLIGRL-NH2 (PAR2-AP) --- p.108 / Chapter 3.3.2.2.3 --- 2-Furoyl-LIGRLO-NH2 (PAR2-AP) --- p.108 / Chapter 3.3.2.2.4 --- SFNGGP-NH2 (PAR3-AP) --- p.109 / Chapter 3.3.2.2.5 --- AYPGKF-NH2 (PARrAP) --- p.110 / Chapter 3.3.2.3 --- Effects of PAR Control Peptides on Histamine Release from RPMCs --- p.111 / Chapter 3.3.2.4 --- Effects of PAR-APs on Histamine Release from LAD2 Cells --- p.111 / Chapter 3.3.3 --- Study of Signaling Pathways Induced by PAR Agonists in Mast Cells --- p.112 / Chapter 3.3.3.1 --- Effect of PTX on PAR-AP-induced Histamine Release from RPMCs --- p.112 / Chapter 3.3.3.2 --- Effect of BAC on PAR-AP-induced Histamine Release from RPMCs --- p.113 / Chapter 3.4 --- Discussion --- p.115 / Chapter 3.5 --- Figures and Tables --- p.132 / Chapter Chapter 4 --- Studies of Roles of PAR in Rat Knee Joint Inflammation --- p.175 / Chapter 4.1 --- Introduction --- p.176 / Chapter 4.2 --- Materials and Methods --- p.181 / Chapter 4.2.1 --- Histological Study of Synovial Mast Cells --- p.181 / Chapter 4.2.2 --- Study of Rat Knee Joint Inflammation Induced by Intra-articular Injections of PAR-APs --- p.181 / Chapter 4.2.3 --- Study of Rat Knee Joint Blood Flow Changes Induced by Topical Administration of PAR-APs --- p.182 / Chapter 4.2.4 --- Study of the Involvement of Bradykinin B2 Receptors in Rat Knee Joint Inflammation Induced by PAR-APs --- p.183 / Chapter 4.3 --- Results --- p.184 / Chapter 4.3.1 --- Histological Study of Synovial Mast Cells --- p.184 / Chapter 4.3.2 --- Study of Rat Knee Joint Inflammation Induced by Intra-articular Injections of PAR-APs --- p.185 / Chapter 4.3.2.1 --- Intra-articular Injections of Carrageenan and Ovalbumin --- p.185 / Chapter 4.3.2.2 --- Intra-articular Injections of PAR-APs --- p.187 / Chapter 4.3.2.2.1 --- TFLLR-NH2 (PARrAP) --- p.187 / Chapter 4.3.2.2.2 --- 2-Furoyl-LIGRLO-NH2 (PAR2AP) --- p.187 / Chapter 4.3.2.2.3 --- SFNGGP-NH2 (PARrAP) --- p.189 / Chapter 4.3.2.2.4 --- AYPGKF-NH2 (PAR4-AP) --- p.190 / Chapter 4.3.2.3 --- Intra-articular Injections of PAR Control Peptides --- p.191 / Chapter 4.3.3 --- Study of Rat Knee Joint Blood Flow Changes Induced by Topical Administration of PAR-APs --- p.191 / Chapter 4.3.3.1 --- Topical Administration of 2-Furoyl-LIGRLO-NH2 (PAR2-AP) --- p.191 / Chapter 4.3.3.2 --- Topical Administration of A YPGKF-NH2 (PAR4-AP) --- p.192 / Chapter 4.3.4 --- Study of the Involvement of Bradykinin B2 Receptors in Rat Knee Joint Inflammation Induced by PAR-APs --- p.193 / Chapter 4.3.4.1 --- Effect of HOE 140 on Rat Knee Joint Inflammation Induced by Bradykinin --- p.193 / Chapter 4.3.4.2 --- Effect of HOE 140 on Rat Knee Joint Inflammation Induced by 2-Furoyl-LIGRLO-NH2 (PAR2-AP) --- p.194 / Chapter 4.3.4.3 --- Effect of HOE 140 on Rat Knee Joint Inflammation Induced by AYPGKF-NH2 (PARrAP) --- p.195 / Chapter 4.4 --- Discussion --- p.196 / Chapter 4.5 --- Figures and Tables --- p.209 / Chapter Chapter 5 --- General Discussions and Concluding Remarks --- p.261 / Chapter 5.1 --- General Discussions --- p.262 / Chapter 5.2 --- Further Studies --- p.267 / Chapter 5.3 --- Conclusion --- p.271 / References --- p.274

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

published_or_final_version / Anaesthesiology / Doctoral / Doctor of Philosophy