Ischaemic stroke has become one of the leading causes of death and disability in the world. Protease activated receptors (PARs, PAR-1 to PAR-4) belong to G protein coupled receptors that can be self-activated by tethered ligands (TL) revealed through proteolytic cleavage. Based on these TL, many activating peptides (APs) and antagonists have been synthesized to investigate PARs actions. / In the present study, the roles of PARs were examined in two models of ischaemic stroke. For the in vivo model, transient middle cerebral artery occlusion (tMCAO) was performed to establish cerebral ischaemia in rats. For the in vitro model, oxygen and glucose deprivation (OGD) was used to mimic an ischaemia insult in primary cultured rat embryonic cortical neurones. / Western blot studies showed that expressions of PAR-1 and PAR-2 were increased in the rat ischaemic brain cortex, whereas PAR-1 was reduced in the rat cortical neurones subjected to OGD. Pretreatments of PAR-1 AP (SFLLRN-NH₂) and PAR-2 AP (SLIGRL-NH₂) produced significant protection against ischaemia-induced damage. Pretreatment of PAR-3 AP (SFNGGP-NH₂) only improved ischaemic symptoms in in vivo but not in in vitro model. When treated after ischaemia, only PAR-1 AP produced significant reductions on ischaemia-induced damage. Protective actions of PAR-1 and PAR-2 APs were inhibited by PAR-1 antagonist (BMS-200261) and PAR-2 antagonist (ENMD-1068) respectively, but PAR-1 antagonist did not affect posttreatment effects of PAR-1 AP in in vitro model. Pre- and posttreatments of thrombin, and pretreatment of trypsin also protected ischaemia-induced damage in the two models. / PAR-1 AP produced marked increase in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and ratio of bcl-2/bax, but reduced contents of reactive oxygen species (ROS), nitric oxide (NO) and malondialdehyde (MDA) in both ipsilateral ischaemic brain cortices and in rat cortical neurones subjected to OGD. In the in vitro model, PAR-1 AP greatly decreased caspase-3 activity and TUNEL positive cells, while markedly increased mitochondrial membrane potential (MMP). All these protective actions were inhibited by its antagonist, which suggests it was mediated via activation of PAR-1. / In MCA isolated from normal and ischameic rats, PAR-2 AP and trypsin produced vasodilatation while PAR-3 AP elicited vasoconstriction. However, another PAR-3 AP had no effect in the two types of MCA. A high concentration of PAR-1 AP relaxed MCA isolated form ischaemic rats, and it was not inhibited by a PAR-1 antagonist. The vasodilator action of PAR-2 AP was inhibited by one of two PAR-2 antagonists tested. The vasodilator actions induced by PAR-1 and PAR-2 APs involved NO production since L-NAME was effective in inhibiting their actions. / In conclusion, PAR-1 AP was found to be the most efficacious in protecting the brain from ischaemia-induced damage when administered either before or after ischaemia insults. The protective actions were likely to be attributed to its anti-oxidant properties in the ischaemic brain that reduced apoptosis of brain cells. Therefore, PAR-1 was identified as a promising target for development of novel prophylactic and therapeutic treatments of ischaemic brain disease. / 缺血性腦中風已經成為全世界導致死亡和殘疾的最主要的疾病之一。蛋白酶激活受體（PARs, PAR-1 to PAR-4）屬於G蛋白偶聯受體並且可以通過蛋白水解生成系鎖配體（TL）從而作用於受體本身而激活信號通路。根據TL的序列已經合成了很多激活肽和拮抗劑，它們可以作為有價值的工具藥進行PAR的作用研究。 / 當前，PAR的作用在兩個缺血性腦中風模型中進行研究。體內模型是通過大鼠大腦中動脈阻塞手術而建立；體外模型是通過對大鼠胚胎大腦皮層神經元進行氧糖剝奪模擬缺血性損傷。 / 蛋白質印跡法的實驗表明PAR-1和PAR-2的表達在缺血側大腦皮層中有所增多，而PAR-1在氧糖剝奪的大鼠皮層神經元中表達卻有所降低。預處理PAR-1（SFLLRN-NH₂）和PAR-2（SLIGRL-NH₂）的激活肽顯著改善了缺血導致的損傷。預處理PAR-3激活肽（SFNGGP-NH₂）僅僅改善了體內缺血症狀，卻對體外缺血模型沒有效果。然而，當這些激活肽在缺血后給予的時候，只有PAR-1的激活肽顯著改善了缺血損傷。PAR-1的拮抗劑（BMS-200261）和PAR-2的拮抗劑（ENMD-1068）抑制了PAR-1和PAR-2激活肽的保護作用，但是體外實驗後處理PAR-1激活肽的保護作用卻未收影響。預處理及後處理凝血酶，預處理胰酶都在這兩個模型中顯示出保護缺血性損傷的作用。 / PAR-1激活肽在缺血同側大腦皮層以及經受氧糖剝奪的大鼠皮層神經元中，顯著提高了超氧化物歧化酶（SOD）、過氧化氫酶（CAT）、谷胱甘肽過氧化物酶（GSH-Px）的活力以及bcl-2/bax的比例，同時顯著降低了活性氧自由基（ROS）、一氧化氮（NO）以及丙二醛（MDA）的含量。在體外模型中，PAR-1激活肽還顯著降低了caspase-3的活力以及TUNEL陽性細胞的比例，同時顯著提高了線粒體膜電位（MMP）。所有這些作用都可以被拮抗劑抑制，說明PAR-1激活肽的保護作用是通過激活PAR-1介導的。 / 不管是從正常還是缺血的大鼠中分離出來的大腦中動脈，PAR-2激活肽和胰酶都可以使之舒張，PAR-3激活肽卻對其有收縮作用。然而，另外一種PAR-3激活肽卻未顯現出對血管活性的影響。高劑量的PAR-1激活肽只可以在分離于缺血大鼠的大腦中動脈中引起舒張，但此作用不能被其拮抗劑所抑制。PAR-2激活肽導致的血管舒張只可以被檢測的兩個拮抗劑中的其中一個所抑制。PAR-1和PAR-2激活肽引起的血管舒張與NO的產生有關，因為L-NAME可以有效抑制它們的作用。 / 總之，不管是預處理還是後處理的給藥方式，PAR-1的激活肽在保護大腦的缺血性損傷中都是最有效果的。保護作用可能可以歸因于其抗氧化以及抗凋亡的特性。所以，PAR-1是研究防治缺血性腦疾病的發展中富有希望的一個靶點。 / Zhen, Xia. / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 194-206). / Abstracts also in Chinese. / Title from PDF title page (viewed on 11, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

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