<p>Mast cells are key effector cells in allergic and inflammatory diseases. However, their primary role is most likely in host defence against parasitic and bacterial infections. Mast cells are a particularly rich source of serine proteases. These proteases belong to the chymase or the tryptase family, which are encoded from the mast cell chymase and the multigene tryptase loci, respectively. To better understand the biological functions and the molecular evolution of these enzymes we have studied the organisation of these two loci in species ranging from fish to human. We show that the mast cell chymase locus has evolved from a single founder gene to a complex locus during the past 200 Myr of mammalian evolution. Forty-five fish candidate genes for hematopoietic serine proteases were also identified. However, in phylogenetic analyses none of them grouped with individual branches holding mammalian mast cell chymase locus genes, indicating an independent parallel evolution in fish. </p><p>Studies of the evolution of the multigene tryptase locus showed that this locus has been highly conserved between marsupials and eutherians. However, no genes belonging to the individual subfamilies identified in eutherians could be identified in fish, amphibians or in birds, which also here indicates parallel evolution.</p><p>To study the evolution of specific cleavage specificities associated with these proteases, the extended cleavage specificity of opossum α-chymase was determined and found to be nearly identical to human mast cell chymase and the major mouse mast cell chymase mMCP-4. This indicates a strong pressure to maintain this specificity during mammalian evolution.</p><p>Basophils are rare blood cells with functions similar to mast cells that when mature almost completely lack mRNA. To study the proteome and to primarily characterize the granule protein content of basophils, an <i>in vitro</i> purification protocol was developed to obtain transcriptionally active umbilical cord blood-derived basophil precursors.</p>

Mast cells stem from the bone marrow and migrate via the blood as mast cell progenitors. Upon arrival in peripheral tissues, they develop into mast cells. These rare immune cells have numerous granules that contain large amounts of pro-inflammatory mediators. Mast cells accumulate at certain sites in the asthmatic lung, and once activated they release mediators that are thought to induce symptoms. In mouse models of allergic airway inflammation, the increase in lung mast cells in asthma can be mimicked and is mainly caused by the recruitment of mast cell progenitors to the lung. However, whether other types of lung inflammation stimulate the recruitment of mast cell progenitors to the lung was unknown until now. Here, using a murine model of influenza A virus infection, this type of virus was demonstrated to trigger an extensive recruitment of mast cell progenitors to the lung, most likely through the induction of VCAM-1 expression in the lung endothelium. Thereafter, some influenza-induced mast cell progenitors developed into an intermediate mast cell stage before they matured into mast cells. However, upon the resolution of inflammation, the mast cells that accumulated in the lung upon influenza infection were gradually lost. Because the recruitment of mast cell progenitors started early after influenza infection, the role of innate immune signals in inducing the recruitment of mast cell progenitors was addressed. The intranasal administration of either Poly I:C or IL-33 was sufficient to induce an increase in lung mast cell progenitors in a TLR3- or ST2-dependent fashion. However, the influenza-induced recruitment of mast cell progenitors to the lung occurred independently of TLR3 and ST2. VAAT/SLC10A4 is a member of the solute carrier family of proteins that is expressed in nerve cells and mast cells. In this study, murine VAAT was localized to mast cell granules and regulated the IgE/antigen-mediated release of granule-associated mediators and ATP. However, the absence of VAAT did not affect IgE/antigen-mediated de novo synthesis of cytokines and lipid mediators. Additionally, mice lacking VAAT had attenuated passive cutaneous anaphylaxis reactions and scratched less frequently in response to compound 48/80 injections, suggesting that VAAT regulates reactions for which mast cells are implicated in vivo.

Kam Man Fai Afia. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves [195]-224). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.v / Publications --- p.vi / Abbreviations --- p.vii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- The Mast Cell --- p.2 / Chapter 1.1.1 --- Historical Perspective --- p.2 / Chapter 1.1.2 --- Mast Cell Origin and Development --- p.3 / Chapter 1.1.3 --- Mast Cell Heterogeneity --- p.5 / Chapter 1.1.3.1 --- Rodent Mast Cell Heterogeneity --- p.5 / Chapter 1.1.3.2 --- Human Mast Cell Heterogeneity --- p.7 / Chapter 1.1.4 --- Mast Cell Mediators --- p.10 / Chapter 1.1.4.1 --- Preformed Mediators --- p.11 / Chapter 1.1.4.2 --- Newly Synthesized Lipid Mediators --- p.14 / Chapter 1.1.4.3 --- Cytokines --- p.16 / Chapter 1.1.5 --- Mast Cell Activation --- p.17 / Chapter 1.1.5.1 --- Immunological Activation --- p.19 / Chapter 1.1.5.1.1 --- FcεIR Activation and Protein Tyrosine Phosphorylation --- p.19 / Chapter 1.1.5.1.2 --- Activation of Phospholipases --- p.20 / Chapter 1.1.5.1.3 --- The Role of Calcium --- p.22 / Chapter 1.1.5.1.3.1 --- Intracellular Calcium Mobilization --- p.23 / Chapter 1.1.5.1.3.2 --- Calcium Influx --- p.24 / Chapter 1.1.5.1.3.3 --- Mechanisms of Action of Calcium in Mast Cells --- p.28 / Chapter 1.1.5.1.4 --- The Role of G-proteins --- p.30 / Chapter 1.1.5.1.5. --- The Role of Cylic AMP --- p.33 / Chapter 1.1.5.1.2.1 --- Mechanisms of Action of Cyclic AMP in Mast Cells --- p.36 / Chapter 1.1.5.1.2.2 --- Implications for the Inhibitory Role of Cyclic AMP in Mast Cell Activation --- p.37 / Chapter 1.2 --- The Cyclic Nucleotide Phosphodiesterases --- p.39 / Chapter 1.2.1 --- Introduction --- p.39 / Chapter 1.2.2 --- Classification and Structure --- p.41 / Chapter 1.2.3 --- Distribution and Physiological Functions of the Different PDE Families --- p.45 / Chapter 1.2.4 --- Phosphodiesterase Inhibitors --- p.49 / Chapter 1.2.4.1 --- Non-selective PDE Inhibitors --- p.50 / Chapter 1.2.4.2 --- Selective PDE Inhibitors --- p.52 / Chapter 1.2.4.2.1 --- PDE1 and PDE2 Inhibitors --- p.52 / Chapter 1.2.4.2.2 --- PDE3 Inhibitors --- p.53 / Chapter 1.2.4.2.3 --- PDE4 Inhibitors --- p.54 / Chapter 1.2.4.2.4.1 --- PDE5 Inhibitors --- p.56 / Chapter 2. --- Materials and Methods --- p.59 / Chapter 2.1 --- Materials --- p.60 / Chapter 2.1.1 --- Drugs --- p.60 / Chapter 2.1.1.1 --- Phosphodiesterase Inhibitors --- p.60 / Chapter 2.1.1.2 --- Mast Cell Secretagogues --- p.61 / Chapter 2.1.2 --- Materials for Rat Peritoneal Mast Cell Experiments --- p.61 / Chapter 2.1.2.1 --- Materials for Rat Sensitization --- p.61 / Chapter 2.1.2.2 --- Materials for Buffers --- p.62 / Chapter 2.1.2.3 --- Materials for Histamine Assay --- p.62 / Chapter 2.1.2.4 --- Miscellaneous --- p.63 / Chapter 2.1.3 --- Materials for RBL-2H3 Cell Line Experiments --- p.63 / Chapter 2.1.3.1 --- Materials for Cell Culture --- p.63 / Chapter 2.1.3.2 --- Materials for Cell Sensitization and Enzyme Release --- p.64 / Chapter 2.1.3.3 --- Materials for β-Hexosaminidase Assay --- p.64 / Chapter 2.1.3.4 --- Miscellaneous --- p.64 / Chapter 2.2 --- Rat Peritoneal Mast Cell Experiments --- p.65 / Chapter 2.2.1 --- Preparation of Buffers --- p.65 / Chapter 2.2.2 --- Preparation of Stock Solutions --- p.66 / Chapter 2.2.2.1 --- Mast Cell Secretagogue Stock Solutions --- p.66 / Chapter 2.2.2.2 --- Phosphodiesterase Inhibitor Stock Solutions --- p.66 / Chapter 2.2.3 --- Animals and Cell Isolation --- p.71 / Chapter 2.2.3.1 --- Animals --- p.71 / Chapter 2.2.3.2 --- Sensitization of Animals --- p.71 / Chapter 2.2.3.3 --- Cell Isolation --- p.71 / Chapter 2.2.3.4 --- Cell Purification --- p.72 / Chapter 2.2.3.5 --- Determination of Cell Number and Viability --- p.73 / Chapter 2.2.4 --- General Protocol for Histamine Release and Histamine Measurement --- p.75 / Chapter 2.2.4.1 --- Histamine Release --- p.75 / Chapter 2.2.4.2 --- Spectrofluorometric Determination of Histamine Content --- p.76 / Chapter 2.2.4.2.1 --- Manual Histamine Assay --- p.76 / Chapter 2.2.4.2.2 --- Automated Histamine Assay --- p.78 / Chapter 2.2.4.3 --- Calculation of Histamine Levels --- p.78 / Chapter 2.2.4.4 --- Presentation and Statistics --- p.79 / Chapter 2.3 --- RBL-2H3 Cell Line Experiments --- p.80 / Chapter 2.3.1 --- Preparation of Stock Solutions --- p.80 / Chapter 2.3.2 --- Preparation of Materials for Enzyme Release and Assay --- p.81 / Chapter 2.3.2.1 --- Cell Culture --- p.81 / Chapter 2.3.2.2 --- Preparation of Cells for β-Hexosaminidase Release Experiments --- p.82 / Chapter 2.3.2.3 --- β-Hexosaminidase Release --- p.82 / Chapter 2.3.2.4 --- β-Hexosaminidase Assay --- p.83 / Chapter 3. --- Effects of Phosphodiesterase Inhibitors on Mediator Release from Rat Mast Cells --- p.84 / Chapter 3.1 --- Introduction --- p.85 / Chapter 3.2 --- Materials and Methods --- p.87 / Chapter 3.2.1 --- Rat Peritoneal Mast Cells --- p.87 / Chapter 3.2.1.1 --- Experiments Employing Immunological Stimulus in RPMCs --- p.87 / Chapter 3.2.1.2 --- Experiments Employing Non-Immunological Stimuli in RPMCs --- p.88 / Chapter 3.2.2 --- Rat Basophilic Leukemia Cells --- p.88 / Chapter 3.3 --- Results --- p.89 / Chapter 3.3.1 --- Rat Peritoneal Mast Cells --- p.89 / Chapter 3.3.1.1 --- Immunologically Activated Rat Peritoneal Mast Cells --- p.89 / Chapter 3.3.1.1.1 --- Effects of Non-Selective PDE Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.89 / Chapter 3.3.1.1.2 --- Effects of Selective PDE1 and PDE2 Inhibitors on Anti-IgE- Mediated Histamine Release from RPMCs --- p.90 / Chapter 3.3.1.1.3 --- Effects of Selective PDE3 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.90 / Chapter 3.3.1.1.4 --- Effects of Selective PDE4 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.91 / Chapter 3.3.1.1.5 --- Effects of Selective PDE5 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.91 / Chapter 3.3.1.2 --- Non-Immunologically Activated Rat Peritoneal Mast Cells --- p.92 / Chapter 3.3.1.2.1 --- Effects of Selective PDE Inhibitors on Compound 48/80- Mediated Histamine Release from RPMCs --- p.92 / Chapter 3.3.1.2.2 --- Effects of Selective PDE Inhibitors on Histamine Release from RPMCs Stimulated by Calcium Ionophores --- p.93 / Chapter 3.3.2 --- Rat Basophilic Leukemia Cells --- p.93 / Chapter 3.3.2.1 --- Effects of Non-Selective PDE Inhibitors on Antigen-Mediated β-Hexosaminidase Release from RBL-2H3 Cells --- p.93 / Chapter 3.3.2.2 --- Effects of Selective PDE Inhibitors on Antigen-Mediated β-Hexosaminidase Release from RBL-2H3 Cells --- p.94 / Chapter 3.4 --- Discussion --- p.95 / Chapter 3.4.1 --- Rat Peritoneal Mast Cells --- p.95 / Chapter 3.4.1.1 --- Immunologically Activated RPMCs --- p.95 / Chapter 3.4.1.2 --- Non-Immunologically Activated RPMCs --- p.99 / Chapter 3.4.2 --- Rat Basophilic Leukemia Cells --- p.103 / Chapter 4. --- Combined Effects of Selective Phosphodiesterase Inhibitors on Immunologically Induced Histamine from Rat Mast Cells --- p.143 / Chapter 4.1 --- Introduction --- p.144 / Chapter 4.2 --- Materials and Methods --- p.144 / Chapter 4.2.1 --- Simultaneous Addition of PDE3 and PDE4 Inhibitors --- p.145 / Chapter 4.2.2 --- Sequential Addition of PDE3 and PDE4 Inhibitors --- p.145 / Chapter 4.3 --- Results --- p.146 / Chapter 4.3.1 --- Effects of the Selective Inhibitors for PDE3 and PDE4 Alone: Calculation of the Expected Inhibition Curve --- p.146 / Chapter 4.3.2 --- Effects of the Simultaneous Addition of PDE3 and PDE4 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.148 / Chapter 4.3.2.1 --- Rolipram and Siguazodan --- p.148 / Chapter 4.3.2.2 --- Ro 20-1724 and Siguazodan --- p.149 / Chapter 4.3.2.3 --- Rolipram and Quazinone --- p.149 / Chapter 4.3.2.4 --- Ro 20-1724 and Quazinone --- p.150 / Chapter 4.3.3 --- Effects of the Sequential Addition of PDE3 and PDE4 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.150 / Chapter 4.3.3.1 --- Rolipram and Siguazodan --- p.150 / Chapter 4.3.3.2 --- Ro 20-1724 and Siguazodan --- p.151 / Chapter 4.3.3.3 --- Rolipram and Quazinone --- p.151 / Chapter 4.3.3.4 --- Ro 20-1724 and Quazinone --- p.152 / Chapter 4.4 --- Discussion --- p.153 / Chapter 5. --- Future Directions --- p.191 / Chapter 5.1 --- Future Directions --- p.192 / References --- p.195

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

Mast cells are key effector cells in allergic and inflammatory diseases. However, their primary role is most likely in host defence against parasitic and bacterial infections. Mast cells are a particularly rich source of serine proteases. These proteases belong to the chymase or the tryptase family, which are encoded from the mast cell chymase and the multigene tryptase loci, respectively. To better understand the biological functions and the molecular evolution of these enzymes we have studied the organisation of these two loci in species ranging from fish to human. We show that the mast cell chymase locus has evolved from a single founder gene to a complex locus during the past 200 Myr of mammalian evolution. Forty-five fish candidate genes for hematopoietic serine proteases were also identified. However, in phylogenetic analyses none of them grouped with individual branches holding mammalian mast cell chymase locus genes, indicating an independent parallel evolution in fish. Studies of the evolution of the multigene tryptase locus showed that this locus has been highly conserved between marsupials and eutherians. However, no genes belonging to the individual subfamilies identified in eutherians could be identified in fish, amphibians or in birds, which also here indicates parallel evolution. To study the evolution of specific cleavage specificities associated with these proteases, the extended cleavage specificity of opossum α-chymase was determined and found to be nearly identical to human mast cell chymase and the major mouse mast cell chymase mMCP-4. This indicates a strong pressure to maintain this specificity during mammalian evolution. Basophils are rare blood cells with functions similar to mast cells that when mature almost completely lack mRNA. To study the proteome and to primarily characterize the granule protein content of basophils, an in vitro purification protocol was developed to obtain transcriptionally active umbilical cord blood-derived basophil precursors.

Made available in DSpace on 2014-12-17T14:03:31Z (GMT). No. of bitstreams: 1 JaniceSC.pdf: 2257338 bytes, checksum: 87ae25d4af8c910f8c675e38f8bfe103 (MD5) Previous issue date: 2005-04-08 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Berberine is an alkaloid used as a fluorochrome in the identification of heparin and DNA. Enerback, 1974, described the technique used until today to study granules rich in heparin of vertebrate mast cells. Santos et al., 2003, studied mast cells of the mollusk Anomalocardia brasiliana using biochemical and histological analysis. This work used the fluorescent dye berberine technique to improve characterization of these cells. Mollusk organs (ctenidium and mantle) were processed with routine histological techniques. Tissue sections were treated with berberine 0,02% in redistilled water acidified to pH 4, by the addition of citric acid for 20 minutes. The visualization was made through fluorescence microscopy with ultraviolet region emission. The mast cell fluorescence had a strong yellow color, where cell nuclei appeared more greenish. This result was very similar to the ones reported before. Mast cells are location at the epithelium surface is the same in both organs, mantle and ctenidium. The fluorescence was easily observed in the granules. Therefore, this technique showed to be good and sensitive to study mast cell of invertebrates / A berberina ? um alcal?ide usado como fluorcromo na identifica??o de heparina e DNA. Enerb?ck, em 1974, descreveu a t?cnica at? hoje empregada deste fluorcromo no estudo dos gr?nulos ricos em heparina dos mast?citos de vertebrados. Santos et al., em trabalhos anteriores, utilizando abordagens bioqu?mica e histol?gica, descreveram a presen?a de mast?citos no marisco Anomalocardia brasiliana. Baseado nestes estudos, testamos a t?cnica de fluoresc?ncia com berberina para a caracteriza??o adicional dessas c?lulas. Destarte, mantos e cten?deos dos esp?cimes foram submetidos a t?cnicas rotineiras de fixa??o e inclus?o em parafina. Os cortes histol?gicos foram desparafinados, hidratados e incubados com cloridrato de berberina (0,02%) em ?cido c?trico (1%) por 20 minutos, sendo o excesso retirado com ?cido c?trico (pH 4) por 5 minutos. A visualiza??o das se??es foi feita por microscopia de fluoresc?ncia com emiss?o no espectro ultravioleta. Pudemos evidenciar c?lulas com conte?do citoplasm?tico fluorescente amarelo-brilhante, correspondentes ?quelas descritas anteriormente como mast?citos, localizadas nos epit?lios dos ?rg?os. Algumas esp?cimes, logo ap?s a disseca??o foram submetidas a tratamento com desgranulantes e depois processadas como citado acima. O uso da fluoresc?ncia tornou mais f?cil a delimita??o do conte?do granular destas c?lulas em rela??o ao n?cleo (que emite fluoresc?ncia esverdeada). Sendo assim, a t?cnica apresentada mostrou ser bastante sens?vel e promissora para o estudo dos mast?citos de invertebrados

This diploma thesis is focused on the study olephylic formulations – ointments and cream type of V/O. This thesis gives out information about production, use and properties 3 cosmetic ointments and about properties baby´s skin cream and the study of stability of these products in the presence of antioxidants - tocopherol acetate and rosemary extract. Ointments are made from hemp oil, olive oil and shea butter and chamomile extract. Each ointment is produced in two versions. Once the use of vitamin E as an antioxidant, a second is used as an antioxidant extract from rosemary. Among other important substances with positive effects on human skin include, for example, saturated fatty acids such as palmitic acid and stearic acid, unsaturated fatty acids such as oleic, linoleic, and more. Ointments are suitable for greasing dry to very dry skin hands, face and other body parts. The thesis also discusses the current legislation, which producer of cosmetics must comply, the details which must be placed on the label of a cosmetic product and it contains proposals of cosmetic labels Further thesis includes studying methods for determination of lipid characterizations were used for study of behaviour of ointments during time and during storage at different temperatures. They include iodine, acid, peroxide and saponification values. The ester value and the content of glycerol were counted.

Mast cells contribute to the activities of innate and adaptive branches of the immune system. They participate in pro-inflammatory responses to a wide range of pathogens, such as parasites, bacteria, and other foreign agents. These beneficial properties are in contrast to the contribution of mast cells to certain pathologies, such as asthma, allergy, autoimmune disorders, anaphylaxis, and systemic mastocytosis. Thorough knowledge of mast cell biology in health and disease is critical for the development of new therapeutic approaches. However, molecular mechanisms that control mast cell development and function are still incompletely defined. Our preliminary data indicate that the transcription factor C/EBP is a key player in mast cell biology. Here, using in vitro and in vivo models, we determine how C/EBP regulates the commitment of hematopoietic progenitors towards mast cells, and modulates mast cells function. These efforts provide novel insights to the role of C/EBP in hematopoiesis, and contribute to a better understanding of the mechanisms governing mast cell biology. Key words Mast cells, C/EBP, transcription factors, bone marrow-derived mast cell cultures, mast cell development, Cebpg conditional knockout mice

The γ-ray characterizing possibility of the neutron collimated flux monitor (in short, Neutron Camera) at the Mega Ampere Spherical Tokamak (MAST) is explored. Typically used to monitor neutron emission, the Neutron Camera has excellent neutron/γ-ray discrimination properties and thus presents the opportunity to measure spatially and temporally resolved γ-ray emission - a possibility of an additional fusion diagnostics method with already existing equipment. An Online Data Analysis (ODA) code was used to analyze the data on γ-rays from several plasma discharges with similar plasma parameters. A high statistics temporal distribution of the γ-ray emission and a lower statistics spatial distribution were analyzed. However, the low energy resolution and range for the Neutron Camera γ-ray measurements revealed few conclusive results on the origin of the higher energy γ-rays. Detection systems with higher energy resolution and range are suggested for an extensive analysis of γ-ray emission at MAST Upgrade.

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