Platelets are involved in many processes ranging from fighting microbial infections and triggering inflammation to promoting tumor angiogenesis and metastasis. Nevertheless, the primary physiological function of platelets is to act as essential mediators in maintaining homeostasis of the circulatory system by forming hemostatic thrombi that prevent blood loss and maintain vascular integrity. CLEC-2 is a C-type lectin-like receptor that is highly expressed in platelets and lesser extent, in other cell types such as activated dendritic cells and B cells. Rhodocytin was the first ligand used to identify CLEC-2 receptor and it’s signaling on platelets. In the first chapter we identified a new agonist for CLEC-2 receptor. Fucoidan, a sulfated polysaccharide from fucus vesiculosus, decreases bleeding time and clotting time in hemophilia, possibly through inhibition of tissue factor pathway inhibitor. However, its effect on platelets and the receptor by which fucoidan induces cellular processes has not been elucidated. In this study, we demonstrate that fucoidan induces platelet activation in a concentration-dependent manner. Fucoidan-induced platelet activation was completely abolished by the pan-Src family kinase (SFK) inhibitor, PP2, or when Syk is inhibited. PP2 abolished phosphorylation of Syk and Phospholipase Cγ−2. Fucoidan-induced platelet activation had a lag phase, which is reminiscent of platelet activation by collagen and CLEC-2 receptor agonists. Platelet activation by fucoidan was only slightly inhibited in FcRγ chain null mice, indicating that fucoidan was not acting primarily through GPVI receptor. On the other hand, fucoidan-induced platelet activation was inhibited in platelet-specific CLEC-2 knock-out murine platelets revealing CLEC-2 as a physiological target of fucoidan. Thus, our data show fucoidan as a novel CLEC-2 receptor agonist that activates platelets through a SFK-dependent signaling pathway. Furthermore, the efficacy of fucoidan in hemophilia raises the possibility that decreased bleeding times could be achieved through activation of platelets. Lipid rafts are distinct areas of the plasma membrane implicated in the regulation of signaling in a variety of cells including platelets. A previous study C-type lectin like receptor 2 (CLEC-2) has been reported to activate platelets through a lipid raft-dependent manner. Secreted ADP potentiates CLEC-2-mediated platelet aggregation. We have investigated whether the decrease in CLEC-2-mediated platelet aggregation, previously reported in platelets with disrupted rafts, is a result of the loss of agonist potentiation by ADP. We disrupted platelet lipid rafts with methyl-β-cyclodextrin (MβCD) and measured signaling events downstream of CLEC-2 activation. Lipid raft disruption decreases platelet aggregation induced by CLEC-2 agonists. The inhibition of platelet aggregation by the disruption of lipid rafts was rescued by the exogenous addition of epinephrine but not 2-methylthioadenosine diphosphate (2MeSADP), which suggests that lipid raft disruption effects P2Y12-mediated Gi activation but not Gz. Phosphorylation of Syk (Y525/526) and PLCγ2 (Y759), were not affected by raft disruption in CLEC-2 agonist-stimulated platelets. Furthermore, tyrosine phosphorylation of the CLEC-2 hemi-ITAM was not effected when MβCD disrupts lipid rafts. Lipid rafts do not directly contribute to CLEC-2 receptor activation in platelets. The effects of disruption of lipid rafts in in vitro assays can be attributed to inhibition of ADP feedback that potentiates CLEC-2 signaling. Tyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an Immune Tyrosine Activation Motif (ITAM) and hemi-ITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In chapter 3, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation. CLEC-2-mediated, but not GPVI-mediated, platelet activation and Syk phosphorylation were abolished by inhibition of PI3-Kinase, which demonstrates that PI3-Kinase regulates Syk downstream of CLEC-2. Ibrutinib, a Tec family kinase inhibitor, also completely abolished CLEC-2-mediated aggregation and Syk phosphorylation in human and murine platelets. Furthermore, embryos lacking both Btk and Tec exhibited cutaneous edema associated with blood-filled vessels in a typical lymphatic pattern similar to CLEC-2 or Syk-deficient embryos. Thus our data show, for the first time, that PI3-Kinase and Tec family kinases play a crucial role in the regulation of platelet activation and Syk phosphorylation downstream of CLEC-2 receptor. / Physiology

Receptors and signal transduction complexes are assembled in a precise manner at specific subdomains of the plasma membrane. Recent research has implicated scaffolding proteins in organizing these receptor and signaling complexes. One well characterized example is the C. elegans LIN-2/LIN-7 /LIN-1 0 complex. This complex is essential in the proper localization of LET -23, the EGFR ortholog, to the basolateral membrane surface of vulval epithelial cells. The mammalian orthologs of the LIN-2/LIN-7 /LIN-10 complex have been identified. CASKIVELI!Mintl/Xllalpha function as a tripartite complex in neurons, presynaptically and postsynaptically. Presynaptically, the multi protein complex aids in linking cell adhesion to ion influx, synaptic vesicle fusion with the presynaptic membrane. and subsequent neurotransmitter release. At the post-synaptic membrane, the CASKIVELI!Mintl/Xllalpha complex is hypothesized to function in the sorting and proper localization of the NMDA type glutamate receptor, reflecting the function of the C. elegans orthologs in receptor localization. We have identified the Drosophila orthologs ofLIN-2/CASK, LIN-7NELI, and LIN-10/Mintl/Xllalpha, termed CMG, dVELI and dMINT. respectively. These proteins were found to be highly conserved among species. The Drosophila YELl protein was initially identified by the McGlade laboratory, University of Toronto, where it was found to bind phosphorylated Drosophila EGFR (DER). We have mapped the chromosomal location of dveli, determined RNA transcript distribution and protein localization, and initiated a P-element mutagenesis screen to generate a dveli mutant. Furthermore, candidate genes for other proteins known to associate with LIN-7 (PALS) have been identified by sequence analysis. dVELI expression begins early in the larval stage. It is concentrated mostly in neuropil areas, sites of synaptic connections. This expression pattern continues into adult development. Within the larval CNS, dVELI protein is localized to the neuropil areas of the ventral nerve cord and brain. NMJ staining further localizes dVELI almost exclusively to the post-synaptic density. This post-synaptic localization resembles that of mammalian YELls, wherein the complex is thought to aid in glutamate receptor sorting and localization. The similarity in structure and expression patterns of dVELI to that of its mammalian orthologs suggests a model in which the Drosophila complex aids in the localization of receptors to post -synaptic specializations in neurons. / Thesis / Master of Science (MSc)

Plants have developed specific responses to external stimuli such as drought, cold, high salinity in soil, and precipitation in addition to internal developmental stimuli. These stimuli trigger signal transduction pathways in plants, leading to cellular adaptation. A signal transduction pathway is a network of entities that interact with one another in response to given stimulus. Such participating entities control and affect gene expression in response to stimulus . For computational purposes, a signal transduction pathway is represented as a network where nodes are biological molecules. The interaction of two nodes is a directed edge. A plethora of research has been conducted to understand signal transduction pathways. However, there are a limited number of approaches to explore and integrate signal transduction pathways. Therefore, we need a platform to integrate together and to expand the information of each signal transduction pathway. One of the major computational challenges in inferring signal transduction pathways is that the addition of new nodes and edges can affect the information flow between existing ones in an unknown manner. Here, I develop the Beacon inference engine to address these computational challenges. This software engine employs a network inference approach to predict new edges. First, it uses mutual information and context likelihood relatedness to predict edges from gene expression time-series data. Subsequently, it incorporates prior knowledge to limit false-positive predictions. Finally, a naive Bayes classifier is used to predict new edges. The Beacon inference engine predicts new edges with a recall rate 77.6% and precision 81.4%. 24% of the total predicted edges are new i.e., they are not present in the prior knowledge. / Master of Science

Mitogen-activated protein kinases are part of an evolutionarily conserved protein phosphorylation cascade which serves essential regulatory functions in eukaryotic organisms. Although the role of MAPKs in the regulation of a plant’s response to environmental stress and plant defense has been well established, very little is known about their role in the regulation of plant developmental processes. In order to examine the role of MAPKs in plant growth and development, a strong mammalian MAPK phosphatase (MKP-1), which is known to inactivate MAPKs in plants, was introduced into tobacco plants. In tobacco plants, MKP-1 overexpression altered plant responses to the phytohormones, ethylene and cytokinin. Tobacco plants expressing MKP-1 flowered earlier and senesced later than wild-type. Additionally, these plants exhibited similar floral morphology as flowers from ethylene-insensitive tobacco plants. These observed phenotypes seem to depend on the protein phosphatase activity, as transgenic lines expressing an inactive form of MKP-1 (MKPCS) did not show the same phenotypes. Furthermore, both tobacco and Arabidopsis MKP-1 transgenic plants exhibited increased shoot regeneration when compared to wild-type plants, suggesting increased cytokinin sensitivity. In an attempt to elucidate the mechanism by which MKP-1 affects plant growth and development, expression of selected genes were analyzed using RT-PCR. MKP-1 transformed tobacco plants exhibited downregulated expression of an ethylene biosynthesis gene (NtACO) and upregulated expression of a pathogenesis-related gene (PR-1b), similar to gene expression studies previously conducted in plants with increased production of cytokinin. The same MKP-1 transgenic plants also exhibited upregulated expression of the flowering time gene, FT. Results from this study indicate that constitutive expression of MKP-1 may interfere with ethylene-related MAPK pathways, which normally serves to restrict plant growth during times of environmental stress. The reduced responses to ethylene resulted in elevated sensitivity to cytokinin, promoting an enhanced shoot regeneration phenotype.

All of the above findings demonstrated that eosinophil activation in allergic inflammation could be sensitively regulated by diverse stimuli. Besides, highly redundant functional effects and underlying signaling mechanisms were observed among different mediators. Elucidation of the mechanisms of eosinophil activation could improve our understanding of its complex and active role in the pathogenesis of allergic diseases, thereby providing biochemical basis for the development of more effective therapeutic strategies for treating the diseases. (Abstract shortened by UMI.) / Apart from the cytokines produced by T lymphoctyes, leptin, a cytokine produced by adipocytes, was also demonstrated to activate eosinophils. It was reported that the plasma levels of leptin were elevated in both obese and allergic patients. We found that leptin could activate eosinophils for survival enhancement, adhesion and migration, and secretion of cytokines and chemokines. Besides, we showed that the MAPKs and NF-kappaB pathways were involved in eosinophil adhesion, migration and mediator release induced by leptin, while Janus kinases (JAK)-signal transducers and activators of transcription (STAT) pathway was responsible for leptin-induced eosinophil survival. Our study indicated a potential correlation between obesity and exacerbation of allergic inflammation. / Eosinophilia is a hallmark pathological feature of allergic diseases and it has been targeted as a novel therapeutic strategy for allergic diseases. / In atopic dermatitis (AD), dermal infiltration of eosinophils is one of the pathological features of this disease. IL-31 is a novel Th2 cytokine reported to induce pruritus and skin dermatitis resembling human AD. Our study on the co-culture system of eosinophils and keratinocyte cell line HaCaT illustrated the production of pro-inflammatory cytokines and chemokines from the co-culture system of eosinophils and HaCaT cells, under the stimulation of IL-31. In co-culture system, surface expression of CD18 and intercellular adhesion molecule (ICAM)-1 on eosinophils and HaCaT cells was also up-regulated respectively, implying a direct interaction between the two cell types through their cell surface adhesion molecules. The interaction of eosinophils and HaCaT cells under IL-31 stimulation was shown to be mediated through MAPKs, NF-kappaB and PI3K pathways. These findings therefore elucidate the immunological roles of IL-31, eosinophils and keratinocytes in AD. / In the present study, we investigated the mechanisms of eosinophil activation induced by various stimuli including novel T helper type 2 (Th2) cytokines, adipokine, microbial products and direct interaction with tissue cells. The activation of eosinophils was studied in terms of survival enhancement, modulation of adhesion and migration, and the release of inflammatory mediators including cytokines, chemokines, granular proteins and superoxide. Using pharmacological and molecular approaches, we further investigated the intracellular signaling mechanisms regulating the eosinophil activation mediated by various stimuli. / Increasing evidence has indicated that bacterial and viral infections could intensify allergic responses. Our findings demonstrated that eosinophil activation could be elicited by microbes through toll-like receptors (TLRs), the recently discovered receptors for the recognition of conserved motifs in pathogens. We found that eosinophils could be activated by the ligands of TLR2, 5 and 7 in enhancing survival, adhesion and migration, release of pro-inflammatory cytokines, chemokines, granular proteins and superoxides. These stimulatory effects, mediated by TLR2, 5 and 7, were differentially regulated by MAPKs, NF-kappaB and phosphatidylinositol 3-kinase (PI3K) pathways. Moreover, an important finding of our study is the common involvement of focal adhesion kinase (FAK)-dependent extracellular-regulated protein kinase (ERK) phosphorylation in the signaling of TLR 2, 5 and 7, implying a special role of FAK in linking TLR signaling with MAPKs cascade in human eosinophils. Our study on microbe-induced eosinophil activation provided a potential explanation for linking infection with exacerbation of allergic diseases. / The interleukin (IL)-17 family is a newly discovered group of cytokines which was reported to be important in allergic inflammation. We studied the roles of two IL-17 family members, IL-175E/IL-25 and IL-17F on eosinophil activation. Both cytokines were found to induce the secretion of inflammatory cytokines and chemokines from eosinophils, in which IL-25 could also enhance eosinophil survival and adhesion. Besides, we found that the stimulatory effects induced by both IL-25 and IL-17F were mediated through mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-kappaB) pathways. The main source of IL-17F has recently been reported to be a novel T lymphocyte population, Th17, which is specifically driven by IL-23. An important finding of our present study was the synergistic effects of IL-17F and its potent inducer, IL-23, on cytokine release from eosinophils. Since IL-23 was produced by macrophages and dendritic cells upon microbial stimulation, the synergistic effect of IL-17F and IL-23 on eosinophil activation might imply a potential role in linking microbial infection and allergic inflammation. Our findings also provide further support to the crucial role of the IL-17 family and Th17 lymphocytes in the amplification of allergic diseases. / Cheung, Fung Yi. / Source: Dissertation Abstracts International, Volume: 69-03, Section: B, page: 1552. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 205-221). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Liang, Fengting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 87-101). / Abstracts in English and Chinese. / DECLARATION --- p.I / ACKNOWLEDGEMENT --- p.II / ABBREVIATIONS --- p.III / ABSTRACT IN ENGLISH --- p.IV / ABSTRACT IN CHINESE --- p.VI / TABLE OF CONTENTS --- p.VIII / LIST OF FIGURES --- p.XI / LIST OF TABLES --- p.XIII / Chapter CHAPTER I- --- INTRODUCTION / Chapter 1.1 --- Roles of human bronchial epithelial cells --- p.1 / Chapter 1.2 --- Role of epithelium in airway inflammation --- p.3 / Chapter 1.3 --- Pathology of asthma --- p.5 / Chapter 1.4 --- The role of eosinophils in asthma --- p.7 / Chapter 1.5 --- "Effects of poly-L-arginine, a MBP analogue, on airway epithelium" --- p.10 / Chapter 1.6 --- Inflammatory pathways involved in epithelial cytokine production --- p.12 / Chapter 1.7 --- Roles and function of IL-6 and IL-8 in epithelial cells --- p.16 / Chapter 1.8 --- P2 receptors and inflammation --- p.18 / Chapter 1.9 --- Objectives --- p.20 / Chapter CHAPTER II- --- MATERIALS AND METHODS / Chapter 2.1 --- Materials and regents --- p.21 / Chapter 2.2 --- Cell culture --- p.22 / Chapter 2.3 --- RNA extraction and Real-time PCR --- p.23 / Chapter 2.4 --- Measurement of cytokine secretion by antibody array --- p.24 / Chapter 2.5 --- Quantification of IL-6 and IL-8 secretion --- p.27 / Chapter 2.6 --- Western Blotting --- p.28 / Chapter 2.7 --- NF-kB translocation assay --- p.29 / Chapter 2.8 --- Data analysis --- p.30 / Chapter CHAPTER III- --- RESULTS / Chapter 3.1 --- Poly-L-arginine-induced IL-6 and IL-8 release from 16HBE 14o- --- p.31 / Chapter 3.2 --- Signaling pathways involved in poly-L-arginine-induced IL-6 and IL-8 release --- p.34 / Chapter 3.2.1 --- "Effects of p38 MAPK, ERK1/2 and NF-kB inhibitors on poly-L-arginine-induced IL-6 and IL-8 release" --- p.35 / Chapter 3.2.2 --- Poly-L-arginine induces p38 MAPK and ERK1/2 phosphorylation --- p.43 / Chapter 3.2.3 --- Poly-L-arginine activates NF-kB translocation from cytoplasm to nucleus --- p.49 / Chapter 3.3 --- Effects of MAPK and NF-kB inhibitors on IL-6 and IL-8 mRNA expression on poly-L-arginine-challenged 16HBE14o- cells --- p.52 / Chapter 3.4 --- P2 receptors modulate poly-L-arginine-induced IL-6 and IL-8 ^ production --- p.55 / Chapter 3.4.1 --- Extracellular nucleotides modulate IL-6 and IL-8 production --- p.56 / Chapter 3.4.2 --- Effects of P2Y6 antagonist on poly-L-arginine-induced IL-6 and IL-8 production --- p.61 / Chapter 3.4.3 --- Effects of MAPKs inhibitors on UDP-induced IL-6 and IL-8 secretion --- p.64 / Chapter 3.4.4 --- UDP induces NF-kB translocation in 16HBE14o- cells --- p.67 / Chapter CHAPTER IV- --- DISCUSSION / Chapter 4.1 --- Involvement of p3 8 MAPK and NF-kB in poly-L-arginine-induced IL-6 and IL-8 secretion --- p.70 / Chapter 4.2 --- Involvement of p38 MAPK and NF-kB in poly-L-arginine-induced IL-6 and IL-8 mRNA elevation --- p.72 / Chapter 4.2.1 --- Regulation of NF-kB on IL-6 and IL-8 mRNA --- p.73 / Chapter 4.2.2 --- Regulation of p38 MAPK on IL-6 and IL-8 mRNA --- p.75 / Chapter 4.2.3 --- Crosstalk between NF-kB and p38 MAPK --- p.77 / Chapter 4.3 --- Extracellular nucleotides mediate IL-6 and IL-8 production in 16HBE14o- --- p.79 / Chapter 4.3.1 --- P2Y6 receptor is linked to poly-L-arginine-induced IL-6 and IL-8 release --- p.80 / Chapter 4.3.2 --- P2Y6 receptor regulates IL-6 and IL-8 secretion via p38 MAPK and NF-kB --- p.83 / Chapter 4.4 --- Summary --- p.86 / Chapter CHAPTER V- --- References --- p.87 / Publications --- p.102

Zhang, Siwei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 87-98). / Abstracts in English and Chinese. / Chapter 1. --- General introduction --- p.1 / Chapter 1.1. --- "Types, structure and function of human hemoglobin" --- p.1 / Chapter 1.1.1. --- Structure and functions of human hemoglobin --- p.1 / Chapter 1.1.2. --- Types of human hemoglobin --- p.2 / Chapter 1.2. --- Regulatory mechanism of human hemoglobin expression --- p.3 / Chapter 1.2.1. --- The human a and β locus --- p.3 / Chapter 1.2.2. --- Development of globin genes switching concept --- p.4 / Chapter 1.2.3. --- Factors that regulate globin gene expression --- p.5 / Chapter 1.2.3.1. --- The locus control region (LCR) --- p.5 / Chapter 1.2.3.2. --- The cis-regulatory elements --- p.5 / Chapter 1.2.3.3. --- The trans-acting factors --- p.6 / Chapter 1.3. --- The human hemoglobinopathies --- p.8 / Chapter 1.3.1. --- α-thalassemia --- p.8 / Chapter 1.3.2. --- β-thalassemia --- p.9 / Chapter 1.3.3. --- Sickle cell anemia --- p.10 / Chapter 1.4. --- Current approaches towards β-thalassemia treatment --- p.11 / Chapter 1.4.1. --- Blood transfusion --- p.11 / Chapter 1.4.2. --- Bone marrow transplantation --- p.12 / Chapter 1.4.3. --- Drug-induced activation of fetal hemoglobin production --- p.12 / Chapter 1.4.3.1. --- Hydroxyurea --- p.12 / Chapter 1.4.3.2. --- Butyrate and short-chain fatty acids --- p.13 / Chapter 1.4.3.3. --- "Mutagens, DNA methyltransferase inhibitors and other HbF inducible agents" --- p.13 / Chapter 1.4.3.4. --- Cucurbitacin D --- p.14 / Chapter 1.4.4. --- Gene therapy --- p.14 / Chapter 1.5. --- Research Objectives --- p.15 / Chapter 2. --- "Analysis of CuD, Hydroxyurea and other inducers on the induction of α, β, γ, δ， ε，ζ BP-1 genes and fetal hemoglobin induction" --- p.16 / Chapter 2.1. --- Introduction --- p.16 / Chapter 2.1.1. --- Properties of human K562 cell line --- p.16 / Chapter 2.1.2. --- Induction and measurement of fetal hemoglobin --- p.16 / Chapter 2.1.3. --- "Induction of α, β, γ, δ, ε , ζ and BP-1 gene and Real-time RT-PCR analysis" --- p.17 / Chapter 2.2. --- Materials --- p.18 / Chapter 2.2.1. --- Chemicals and reagents --- p.18 / Chapter 2.2.2. --- Kits --- p.19 / Chapter 2.2.3. --- Buffers and solutions --- p.19 / Chapter 2.2.4. --- Cell lines --- p.20 / Chapter 2.3. --- Experimental procedures --- p.20 / Chapter 2.3.1. --- Hemoglobin quantity measurement by HbF ELISA --- p.20 / Chapter 2.3.1.1. --- MTT assay --- p.21 / Chapter 2.3.1.2. --- Preparation of capture-antibody coated ELISA plates --- p.21 / Chapter 2.3.1.3. --- Plate blocking --- p.22 / Chapter 2.3.1.4. --- Sample and standard preparation --- p.22 / Chapter 2.3.1.5. --- HRP antibody and colorimetric detection --- p.23 / Chapter 2.3.1.6. --- Statistical analysis --- p.23 / Chapter 2.3.2. --- Preparation of mRNA extract from K562 cells --- p.23 / Chapter 2.3.3. --- Reverse transcription and Real-time PCR analysis --- p.24 / Chapter 2.4. --- Results --- p.25 / Chapter 2.4.1. --- CuD significantly upregulates HbF expression in K562 cells --- p.25 / Chapter 2.4.2. --- "CuD augments α, β, γ, δ, ε , ζ and BP-1 genes at different level in K562 cells" --- p.28 / Chapter 2.4.3. --- Cucurbitacin D-induced γ-globin gene activation requires12-24 hours in K562 cells --- p.31 / Chapter 2.5. --- Discussion --- p.33 / Chapter 2.5.1. --- Enhancement of fetal hemoglobin production using different chemical compounds --- p.33 / Chapter 2.5.2. --- CuD increased HbF synthesis by increasing γ-globin mRNA amount --- p.35 / Chapter 2.5.3. --- CuD and HU down-regulated the BP-1 gene expression --- p.36 / Chapter 3. --- Determination of potential signal transduction pathways during CuD and HU-mediated fetal hemoglobin production --- p.36 / Chapter 3.1. --- Introductions --- p.36 / Chapter 3.1.1. --- The p38 MAPK family --- p.37 / Chapter 3.1.2. --- The JAK2-STAT3 pathway --- p.38 / Chapter 3.1.3. --- Fundamentals on inhibition assay of p38 MAPK and JAK2-STAT3 pathway --- p.39 / Chapter 3.1.4. --- Fundamentals on nuclear translocation of STAT3 --- p.41 / Chapter 3.2. --- Materials --- p.41 / Chapter 3.2.1. --- Chemicals and reagents --- p.41 / Chapter 3.2.2. --- Kits --- p.44 / Chapter 3.2.3. --- Buffers and solutions --- p.44 / Chapter 3.3. --- Experimental procedures --- p.45 / Chapter 3.3.1. --- Detection of p3 8 MAPK phosphorylation status --- p.46 / Chapter 3.3.1.1. --- Preparation of cytosolic protein extracts --- p.46 / Chapter 3.3.1.2. --- Quantitative measurement of phospho-p38 and pan-p38 by ELIS A method --- p.46 / Chapter 3.3.1.2.1. --- Antigen adsorption and establishment of standard curves --- p.46 / Chapter 3.3.1.2.2. --- Plate washing and application of detection antibody --- p.47 / Chapter 3.3.1.2.3. --- Plate washing and application of secondary antibody --- p.47 / Chapter 3.3.1.2.4. --- Plate washing and chromogen detection --- p.48 / Chapter 3.3.2. --- Detection of signal cascade on JAK2-STAT3 pathway --- p.48 / Chapter 3.3.2.1. --- Preparation of cytosolic protein extracts for Western Blot detection --- p.48 / Chapter 3.3.2.2. --- Gel running and Western Blot detection --- p.48 / Chapter 3.3.3. --- Quantitative measurement of phospho-STAT3-Tyr705 using ELISA method --- p.50 / Chapter 3.3.3.1. --- Preparation of cytosolic protein extracts --- p.50 / Chapter 3.3.3.2. --- Reconstitution and Dilution of STAT3 [pY705] Standard --- p.50 / Chapter 3.3.3.3. --- Measurement of STAT3 [pY705] concentration in cell lysates --- p.51 / Chapter 3.3.4. --- Inhibitor assay of JAK2-STAT3 and p38 MAPK pathway --- p.52 / Chapter 3.3.4.1. --- Establishment of inhibitor assay --- p.52 / Chapter 3.3.4.2. --- HbF ELISA detection --- p.53 / Chapter 3.3.5. --- Detection of STAT3 nuclear translocation and DNA binding affinity --- p.53 / Chapter 3.3.5.1. --- Preparation of nuclear extract from K562 cells --- p.53 / Chapter 3.3.5.2. --- EMS A detection of transcriptional factors binding to γ-promoter region --- p.54 / Chapter 3.3.5.2.1. --- 3´ة end-labeling of EMS A probes --- p.54 / Chapter 3.3.5.2.2. --- Dot blotting for labeling efficiency estimation --- p.56 / Chapter 3.3.5.2.3. --- EMSA binding reaction and non-denaturing gel electrophoresis --- p.57 / Chapter 3.3.5.2.4. --- Membrane development and chemiluminescence detection --- p.58 / Chapter 3.3.5.3. --- Preparation of K562 samples for immunofluorescence detection --- p.60 / Chapter 3.3.5.3.1. --- Slide coating for cell capture --- p.60 / Chapter 3.3.5.3.2. --- Preparation of cell slide --- p.60 / Chapter 3.3.5.3.3. --- Sample fixation and antibody probing treatment --- p.60 / Chapter 3.3.5.3.4. --- Sample imaging and immunofluorescence detection --- p.61 / Chapter 3.4 --- Results --- p.62 / Chapter 3.4.1. --- Activation of p38 MAPK pathway and STAT3 phosphorylation by hydroxyurea --- p.62 / Chapter 3.4.1.1. --- "The p38 MAPK pathway is activated by hydroxyurea, but not activated by Cucurbitacin D" --- p.62 / Chapter 3.4.1.2. --- Increased p38 phosphorylation level elicits STAT3 phosphorylation at Ser727 site --- p.64 / Chapter 3.4.2. --- Activation of JAK2 and STAT3 phosphorylation by Cucurbitacin D --- p.66 / Chapter 3.4.2.1. --- Cucurbitacin D promotes JAK2 activation --- p.66 / Chapter 3.4.2.2. --- Cucurbitacin D and hydroxyurea promote STAT3 phosphorylation at Tyr705 site --- p.66 / Chapter 3.4.3. --- Basal activity of signal transduction pathways is essential for HbF induction --- p.69 / Chapter 3.4.3.1. --- Activation of γ-globin gene requires presence of basal phosphorylation level of p38 MAPK --- p.69 / Chapter 3.4.3.2. --- Inhibition on JAK2-STAT3 pathway results in reduced fetal hemoglobin production --- p.71 / Chapter 3.4.4. --- Translocation and DNA binding of STAT under Cucurbitacin D induction --- p.72 / Chapter 3.4.4.1. --- Cucurbitacin D and hydroxyurea both enhance binding affinity of transcriptional factors to the Gγ/Aγ promoter --- p.72 / Chapter 3.4.4.2. --- Cucurbitacin D and hydroxyurea induces nuclear translocation of STAT3 --- p.75 / Chapter 3.5. --- Discussion --- p.77 / Chapter 3.5.1. --- The role of p38 MAPK activation during γ-globin gene activation --- p.77 / Chapter 3.5.2. --- STAT3 phosphorylation at Ser727 site promotes transcription factor activity and γ-globin gene expression --- p.77 / Chapter 3.5.3. --- The role of JAK2-STAT3 activation during γ-globin gene activation --- p.78 / Chapter 3.5.4. --- Inhibitor assay --- p.79 / Chapter 3.5.5. --- Relations between STAT3 nuclear translocation and enhanced fetal hemoglobin production --- p.82 / Chapter 4. --- Summery and Prospect --- p.83 / Chapter 5. --- References --- p.87

Zhong, Lan. / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 122-154). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016). / Zhong, Lan.

Lam, Kwok Ho. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 149-159). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thesis (Ph.D.)--University of Minnesota, 1999. / Includes bibliographical references (leaves 74-98). Also available on the World Wide Web as a PDF file.