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Distribution and functional studies of the dyslexia-associated protein KIAA0319-Like.

Tsang, Wan Hong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 156-165). / Abstracts in English and Chinese. / Abstract --- p.iii / Abstract in Chinese (摘要) --- p.V / Acknowledgement --- p.vi / Table of Content --- p.vii / List of Figures and Tables --- p.xiv / List of Abbreviations --- p.xvii / Chapter Chapter 1 --- Introduction --- p.Page / Chapter 1.1 --- Developmental Dyslexia --- p.1 / Chapter 1.1.1 --- Study of Developmental Dyslexia --- p.1 / Chapter 1.1.2 --- Genetic Basis of Developmental Dyslexia --- p.2 / Chapter 1.1.3 --- Susceptibility Gene for Developmental Dyslexia --- p.3 / Chapter 1.2 --- Dyslexia-Susceptibility Gene KIAA0319-Like --- p.5 / Chapter 1.2.1 --- Association Studies of KIAA0319-Like --- p.5 / Chapter 1.2.2 --- KIAA0319-Like Gene and Protein --- p.6 / Chapter 1.2.3 --- Functional prediction of KIAA0319-Like Protein --- p.10 / Chapter 1.3 --- Potential Interacting Partners of KIAA0319-Like Protein --- p.12 / Chapter 1.3.1 --- Nogo Receptor 1 (NgR) --- p.12 / Chapter 1.3.2 --- SH2B Adaptor Protein 1 (SH2) --- p.13 / Chapter 1.3.3 --- FEM-1-like death receptor binding protein (FEM) --- p.14 / Chapter 1.3.4 --- Adaptor-related protein complex 2,mu 1 subunit (AP2) --- p.14 / Chapter 1.4 --- Notch Signaling Pathway and KIAA0319-Like --- p.16 / Chapter 1.5 --- Previous Research on KIAA0319-Like --- p.18 / Chapter 1.6 --- Research Objectives --- p.20 / Chapter Chapter 2 --- Materials and Methods --- p.Page / Chapter 2.1 --- Gene Cloning --- p.21 / Chapter 2.1.1 --- Cloning of Human KIAA0319-Like into expression vectors --- p.21 / Chapter 2.1.2 --- Gel Extraction of PCR Product --- p.22 / Chapter 2.1.3 --- Restriction enzyme digestion --- p.22 / Chapter 2.1.4 --- Ligation of gene products with vector --- p.23 / Chapter 2.1.5 --- "Cloning of Human SH2, FEM, AP2 and NgR gene" --- p.23 / Chapter 2.1.7 --- Preparation of chemically competent E.coli strain DH5a --- p.26 / Chapter 2.1.8 --- Transformation of the ligation product into competent cells --- p.26 / Chapter 2.1.9 --- Diagnostic PCR for confirmation of successful ligation --- p.27 / Chapter 2.1.10 --- Small scale preparation of bacterial plasmid DNA --- p.27 / Chapter 2.1.11 --- DNA sequencing of the cloned plasmid DNA --- p.28 / Chapter 2.1.12 --- Large scale preparation of target recombinant plasmid DNA --- p.28 / Chapter 2.2 --- Cell Culture --- p.30 / Chapter 2.2.1 --- Culture medium --- p.30 / Chapter 2.2.2 --- Cell lines --- p.30 / Chapter 2.2.3 --- Freezing and thawing cells --- p.31 / Chapter 2.3 --- DNATransfection --- p.32 / Chapter 2.3.1 --- Transfection of HEK293 Cells with LipofectaminéёØ Reagent --- p.32 / Chapter 2.3.2 --- Transfection of Neuronal Cells with Tranfas´tёØ Reagent --- p.32 / Chapter 2.4 --- Immunocytochemical Staining --- p.33 / Chapter 2.5 --- Immunohistochemical Staining --- p.34 / Chapter 2.6 --- Western Blotting --- p.36 / Chapter 2.6.1 --- Collection of Cell Lysate --- p.36 / Chapter 2.6.2 --- Collection of Mouse Tissue Lysate --- p.36 / Chapter 2.6.3 --- Protein concentration determination by Bradford Protein Assay --- p.36 / Chapter 2.6.4 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.37 / Chapter 2.6.5 --- Semidry Transfer to PVDF Membrane --- p.37 / Chapter 2.6.6 --- Blocking and Antibody Probing --- p.38 / Chapter 2.6.7 --- Enhanced chemiluminescence (ECL) --- p.38 / Chapter 2.6.8 --- Synthetic peptide based neutralization --- p.39 / Chapter 2.7 --- Quantitative Real-Time PCR --- p.40 / Chapter 2.7.1 --- RNA extraction from Cell culture --- p.40 / Chapter 2.7.2 --- Genomic DNA Elimination and Reverse Transcription --- p.40 / Chapter 2.7.3 --- Quantitative Real-Time PCR --- p.41 / Chapter 2.8 --- Coimmunoprecipitation --- p.42 / Chapter 2.9 --- Luciferase Reporter Assay --- p.43 / Chapter 2.10 --- Wound-Healing Assay --- p.44 / Chapter Chapter 3 --- Distribution of KIAA0319-Like Protein --- p.Page / Chapter 3.1 --- Study of the Distribution of KIAA0319-Like Protein --- p.45 / Chapter 3.1.1 --- Anti-KIAA0319-Like Antisera H635 --- p.45 / Chapter 3.1.2 --- Affinity Purification of Anti-KIAA0319-Like Antisera H635 --- p.45 / Chapter 3.2 --- C57/BL6 Mouse Tissue Expression Pattern of KIAA0319-Like --- p.48 / Chapter 3.3 --- C57/BL6 Mouse Brain Regional Expression Pattern of KIAA0319-Like --- p.50 / Chapter 3.4 --- Immunostaining of Sagittal Section of C57/BL6 Mouse Brain --- p.52 / Chapter 3.5 --- Mouse Brain Co-staining with KIAA0319-Like Protein and Markers --- p.54 / Chapter 3.6 --- Subcelluar Localization of KIAA0319-Like Protein in Human Neurons --- p.57 / Chapter 3.7 --- Discussion --- p.59 / Chapter 3.7.1 --- Affinity Purification of Anti-KIAA0319-Like Antisera --- p.59 / Chapter 3.7.2 --- Mouse Tissue Expression Pattern of KIAA0319-Like --- p.59 / Chapter 3.7.3 --- Mouse Brain Regional Expression Pattern of KIAA0319-Like --- p.59 / Chapter 3.7.4 --- Brain Cell Types expressing KIAA0319-Like Protein --- p.61 / Chapter 3.7.5 --- Subcellular Localization of KIAA0319-Like Protein --- p.62 / Chapter 3.7.6 --- Future Perspectives --- p.63 / Chapter Chapter 4 --- KIAA0319-Like Modulates Neuronal Migration --- p.Page / Chapter 4.1 --- Study of the effect of KIAA0319-Like on Neuronal Migration --- p.64 / Chapter 4.2 --- Establish Stable Cell Lines with Altered KIAA0319-Like Expression --- p.65 / Chapter 4.2.1 --- Cloning of siRNA into Lentiviral Vector pLVTHM --- p.65 / Chapter 4.2.2 --- Lentivirus packaging in HEK293 Cells --- p.67 / Chapter 4.2.3 --- Checking of Lentivirus by HEK293 Cells Transduction --- p.70 j / Chapter 4.2.4 --- Lentiviral Transductions to Neuronal Cells --- p.70 / Chapter 4.2.5 --- Fluorescent-Activated Cell Sorting of Transduced Cells --- p.70 / Chapter 4.2.6 --- KIAA0319-Like Expression Changes in Stable Cell Lines --- p.71 / Chapter 4.3 --- Effects of KIAA0319-Like Overexpression on Neuronal Migration --- p.75 / Chapter 4.4 --- Effects of KIAA0319-Like Knockdown on Neuronal Migration --- p.77 / Chapter 4.5 --- Effect of Anti-KIAA0319-Like Antibody on Neuronal Migration --- p.79 / Chapter 4.6 --- Discussion --- p.81 / Chapter 4.6.1 --- Stable Cell line with altered KIAA0319-Like Level --- p.81 / Chapter 4.6.2 --- Disruption of KIAA0319-Like expression affects Cell Migration --- p.81 / Chapter 4.6.3 --- Antibody Blockade of KIAA0319-Like Inhibits Cell Migration --- p.81 / Chapter 4.6.4 --- Possible existence of Multiple Regulatory Pathways --- p.82 / Chapter 4.6.5 --- Possible Limitations of Expression alternations by Transduction --- p.83 / Chapter 4.6.6 --- Possible Susceptibility Polymorphism of KIAA0319-Like --- p.83 / Chapter 4.6.7 --- Future Perspectives X --- p.84 / Chapter Chapter 5 --- Interacting Partners of KIAA0319-Like Protein --- p.Page / Chapter 5.1 --- Study of Interacting Partners of KIAA0319-Like Protein --- p.85 / Chapter 5.1.1 --- Identification of Potential Interacting Partners of KIAA0319-Like --- p.85 / Chapter 5.1.2 --- Identification of Domains involved in Interactions --- p.86 / Chapter 5.1.3 --- Subcloning of KIAA0319-Like Protein Domain Deletion Mutant --- p.86 / Chapter 5.2 --- Interaction between NgR and KIAA0319-Like Protein --- p.92 / Chapter 5.2.1 --- Reciprocal Coimmunoprecipitation of NgR for KIAA0319-Like --- p.92 / Chapter 5.2.2 --- Colocalization of NgR and KIAA0319-Like Protein --- p.94 / Chapter 5.2.3 --- Interaction between NgR and KIAA0319-Like Deletion Mutants --- p.97 / Chapter 5.3 --- Interaction between SH2 and KIAA0319-Like Protein --- p.99 / Chapter 5.3.1 --- Coimmunoprecipitation between SH2 and KIAA0319-Like --- p.» 99 / Chapter 5.3.2 --- Colocalization of SH2 and KIAA0319-Like Protein --- p.101 / Chapter 5.3.3 --- Interaction between SH2 and KIAA0319-Like Deletion Mutants --- p.103 / Chapter 5.4 --- Interaction between FEM and KIAA0319-Like Protein --- p.105 / Chapter 5.4.1 --- Coimmunoprecipitation between FEM and KIAA0319-Like --- p.105 / Chapter 5.4.2 --- Colocalization of FEM and KIAA0319-Like Protein --- p.107 / Chapter 5.4.3 --- Interaction between FEM and KIAA0319-Like Deletion Mutants --- p.109 / Chapter 5.5 --- Interaction between AP2 and KIAA0319-Like Protein --- p.111 / Chapter 5.5.1 --- Coimmunoprecipitation between AP2 and KIAA0319-Like --- p.111 / Chapter 5.5.2 --- Colocalization of AP2 and KIAA0319-Like Protein --- p.113 / Chapter 5.5.3 --- Interaction between AP2 and KIAA0319-Like Deletion Mutants --- p.115 / Chapter 5.6 --- Discussion --- p.117 / Chapter 5.6.1 --- The Interaction of KIAA0319-Like with NgR and SH2 Protein --- p.118 / Chapter 5.6.2 --- Interaction between FEM and KIAA0319-Like Protein --- p.121 / Chapter 5.6.3 --- Interaction between AP2 and KIAA0319-Like Protein --- p.121 / Chapter 5.6.4 --- Future Perspectives --- p.122 / Chapter Chapter 6 --- KIAA0319-Like Association with Notch Pathway --- p.Page / Chapter 6.1 --- Study of KIAA0319-Like association with Notch Pathway --- p.123 / Chapter 6.1.1 --- Luciferase Reporter System for quantifying y-Secretase Activity --- p.123 / Chapter 6.2 --- Effects of KIAA0319-Like Expression Alternations on y-Secretase Activity in HEK293 Cells --- p.126 / Chapter 6.3 --- Effects of Increasing KIAA0319-Like Protein Level in Culture Medium on y-Secretase Activity in HEK293 Cells --- p.128 / Chapter 6.4 --- Effects of KIAA0319-Like Expression Alternations on y-Secretase Activity in SH-SY5Y Cells --- p.130 i / Chapter 6.5 --- Effects of KIAA0319-Like Expression Alternations on y-Secretase Activity in Neuro-2a Cells --- p.132 / Chapter 6.6 --- Effect of Notch Blockade on SH-SY5Y Cells Migration Rate --- p.134 / Chapter 6.7 --- Effect of KIAA0319-Like Expression changes on Notch Inhibited SH-SY5Y Cells Migration Rate --- p.136 / Chapter 6.8 --- Discussion --- p.139 / Chapter 6.8.1 --- Luciferase Reporter System for quantifying y-Secretase Activity --- p.139 / Chapter 6.8.2 --- Cell-Type Specific association of KIAA0319-Like expression level and y-Secretase activity --- p.139 / Chapter 6.8.3 --- Entry Point of KIAA0319-Like to the Notch Signaling Pathway --- p.140 / Chapter 6.8.4 --- Functional relationship between KIAA0319-Like and Notch --- p.141 / Chapter 6.8.5 --- Future Perspectives --- p.142 / Chapter Chapter 7 --- Discussions and Conclusions --- p.Page / Chapter 7.1 --- Discussions --- p.143 / Chapter 7.1.1 --- Further Evidence for KIAA0319-Like Association with Dyslexia --- p.143 / Chapter 7.1.2 --- Possible Pathways connecting KIAA0319-Like with Cell Motility --- p.145 / Chapter 7.1.3 --- Nogo Receptor Pathway association with Cell Motility --- p.146 / Chapter 7.1.4 --- Notch Signaling Pathway association with Cell Motility --- p.146 / Chapter 7.1.5 --- Combined Effects of NgR and Notch Pathway on Cell Migration --- p.149 / Chapter 7.1.6 --- Cellular Compartment for Interactions of KIAA0319-Like --- p.151 / Chapter 7.2 --- Conclusions --- p.153 / Chapter Chapter 8 --- References --- p.156 / Chapter Chapter 9 --- Appendix --- p.166

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_327398
Date January 2011
ContributorsTsang, Wan Hong., Chinese University of Hong Kong Graduate School. Division of Biomedical Sciences.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xviii, 179 leaves : ill. (some col.) ; 30 cm.
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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