| Table of Contents | 第9-15页 |
| List of Tables in Part I | 第15-16页 |
| List of Tables in Part II | 第16-17页 |
| List of Figures in Part I | 第17-19页 |
| List of Figures in Part II | 第19-22页 |
| Part I A systems biology approach toidentifying the signaling networkregulated by Rho-GDI-γ duringneural stem cell differentiation | 第22-127页 |
| ABSTRACT | 第23-25页 |
| 摘要 | 第25-27页 |
| 研究背景 | 第27-32页 |
| 研究结果一 | 第32-38页 |
| 讨论一 | 第38-40页 |
| 研究结果二 | 第40-44页 |
| 讨论二 | 第44-45页 |
| CHAPER I Introduction | 第45-61页 |
| 1.1 Neural stem cells | 第45-49页 |
| 1.2 Small GTPases | 第49-58页 |
| 1.2.1 Rho GTPase (guanosine triphosphates)subfamily | 第50页 |
| 1.2.3 Rho-GDIs (Rho-GDP-dissociation inhibitors) | 第50-52页 |
| 1.2.4 Structure-function relationships | 第52-57页 |
| 1.2.5 Involvement of the Rho proteins in neuronal differentiation | 第57-58页 |
| 1.2.6 Role of Rho GTPase proteins in neuronal migration | 第58页 |
| 1.3 Transcription factor decoy | 第58-61页 |
| 1.3.1 Advantages and disadvantages of the ODN decoy strategy | 第59页 |
| 1.3.2 Application of transcription factor decoy in disease | 第59-61页 |
| CHAPTERII Materials and Methods | 第61-70页 |
| 2.1 C17.2 cell culture | 第61页 |
| 2.2 Mouse neural stem cell culture | 第61页 |
| 2.3 EMSA | 第61-62页 |
| 2.4 HNF4-1 and MAZ-1 dODNs and nonsense control dODNs | 第62页 |
| 2.5 Gene transfection | 第62页 |
| 2.6 Decoy incorporation studies and cytotoxicity and proliferation assays | 第62-64页 |
| 2.7 Immunofluorescence analysis | 第64页 |
| 2.8 Total RNA extraction, cDNA synthesis, and real-time PCR analysis | 第64-67页 |
| 2.9 In vitro scratch assay | 第67页 |
| 2.10 Brain tissue immunohistochemistry | 第67页 |
| 2.11 Animal behaviour | 第67-68页 |
| 2.11.1 Y-maze | 第67-68页 |
| 2.11.2 Morris water maze(MWM) | 第68页 |
| 2.11.3 Probe trial | 第68页 |
| 2.12 Data for signaling pathways regulated by Rho-GDI-γ | 第68页 |
| 2.13 Identification of signaling pathways regulated by Rho-GDI-γduring the differentiation of NSCs | 第68-69页 |
| 2.14 Western blot | 第69页 |
| 2.15 Statistical analysis | 第69-70页 |
| CHAPTER III Regulation of neural stem cell differentiation and migration by transcription factors HNF4-1 and MAZ-126 | 第70-94页 |
| 3.1 Introduction | 第70-72页 |
| 3.2 Results | 第72-91页 |
| 3.2.1 The decoy regulating strategy of Rho-GDI-γ | 第72-74页 |
| 3.2.2 HNF4-1 and MAZ-1 specifically bind to the decoy ODNs | 第74-76页 |
| 3.2.3 HNF4-1 and MAZ-1 dODNs significantly knock down Rho-GDI-γ gene transcription | 第76-78页 |
| 3.2.4 HNF4-1 and MAZ-1 dODNs induce changes in cell morphology but do not affect cell proliferation | 第78页 |
| 3.2.5 HNF4-1 and MAZ-1 dODNs lead to NSCs differentiation towards a neuronal phenotype | 第78-82页 |
| 3.2.6 Modulation of Rho-GDI-γ-mediated gene expression | 第82-85页 |
| 3.2.7 Downregulation of Rho-GDI-γenhances C17.2 cell migration | 第85-87页 |
| 3.2.8 The expression of Rho-GDI-γ mRNA was regulated by decoy in vivo | 第87-89页 |
| 3.2.9 Injection in vivo does not affect the spatial learning and memory ability | 第89-91页 |
| 3.3 Discussion | 第91-94页 |
| Chapter IV A systems biology approach to identifying the signaling network regulated by Rho-GDI-γ during neural stem cell differentiation | 第94-112页 |
| 4.1 Introduction | 第94-95页 |
| 4.2 Results and discussions | 第95-111页 |
| 4.2.1 Prediction of the signaling network regulated by Rho-GDI-γ during differentiation of NSCs | 第95-103页 |
| 4.2.2 Experimental validation of the signaling network regulated by Rho-GDI-γ | 第103-108页 |
| 4.2.3 Ywhah signalling pathway is regulated by Rho-GDI-γ during the differentiation of NSCs | 第108-111页 |
| 4.3 Conclusions | 第111-112页 |
| Reference | 第112-127页 |
| Part II Identification of CDC50A-P4-ATPase Interacting Network & Characterization of the ATP11 Subfamily of P4-ATPases | 第127-259页 |
| ABSTRACT | 第128-130页 |
| 摘要 | 第130-132页 |
| 研究背景 | 第132-141页 |
| 研究结果1 | 第141-147页 |
| 讨论1 | 第147-151页 |
| 研究结果2 | 第151-155页 |
| 讨论2 | 第155-157页 |
| CHAPTER Ⅰ Introduction | 第157-176页 |
| 1.1 Biological Membranes | 第157-162页 |
| 1.1.1 Membrane Lipid Asymmetry | 第158-162页 |
| 1.1.2 Lipid Asymmetry Function | 第162页 |
| 1.2 Phospholipid Flippases | 第162-166页 |
| 1.2.1 Classification of Phospholipid Transporters | 第163-164页 |
| 1.2.2 Structure of Phospholipid Flippases | 第164-166页 |
| 1.3 β-Subunit of Flippase, the CDC50 Family | 第166-169页 |
| 1.4 Mechanism of Lipid Transport | 第169-171页 |
| 1.4.1 P-type ATPase Transport Cycle | 第169页 |
| 1.4.2 Possible Mechanism for Lipid Transport | 第169-171页 |
| 1.5 Progress in P4-ATPase Research | 第171-176页 |
| 1.5.1 ATP8A Subfamily | 第171-172页 |
| 1.5.2 ATP8B Family | 第172-173页 |
| 1.5.3 ATP10A | 第173-174页 |
| 1.5.4 ATP11 Family | 第174-176页 |
| CHAPTER Ⅱ Methods | 第176-193页 |
| 2.1 Materials | 第176页 |
| 2.2 Cell Culture and Transfection | 第176页 |
| 2.3 Cell Lysis | 第176-177页 |
| 2.4 Membrane Fragment Preparation | 第177页 |
| 2.5 ATPase- pcDNA3 Constructs | 第177页 |
| 2.6 Gene Expression by RT-PCR | 第177页 |
| 2.7 Immunofluorescence Microscopy | 第177-178页 |
| 2.8 Isolation of Erythrocytes and Erythrocyte Membrane Preparation | 第178页 |
| 2.9 Isolation of Tissue Membranes | 第178页 |
| 2.10 Purification of Atpases | 第178-179页 |
| 2.11 Reconstitution of ATPases into Lipid Vesicles | 第179页 |
| 2.12 ATPase Activity Assay | 第179页 |
| 2.13 Flippase Assay | 第179-180页 |
| 2.14 PCR and GST Fusion Protein Vector Cloning | 第180页 |
| 2.15 Expression and Purification of GST-Fusion Proteins | 第180-181页 |
| 2.16 Injection of Mice | 第181页 |
| 2.17 Myeloma and Mouse Spleen Fusions | 第181页 |
| 2.18 Secondary Screen, Western Blot Strips | 第181-182页 |
| 2.19 Determining Antibody Isotype | 第182页 |
| 2.20 Preparing Immunoaffinity Matrix | 第182页 |
| 2.21 Polyclonal Antibody | 第182页 |
| 2.22 Buffers | 第182-193页 |
| CHAPTER Ⅲ Proteomic Studies of P4-ATPases Binding to CDC50A | 第193-225页 |
| 3.1 Introduction | 第193-195页 |
| 3.2 Results | 第195-221页 |
| 3.2.1 Purification of CDC50A Complex by Immunoaffinity Chromatography | 第195-203页 |
| 3.2.2 Proteomic Analysis of the Protein Complex | 第203-205页 |
| 3.2.3 Confirmation of ATP8A2 Protein in Different Tissues | 第205-210页 |
| 3.2.3.1 Immunolocalization of the ATP8A2 Protein in Retina, Brain and Testis | 第205-210页 |
| 3.2.4 Confirmation of ATP8A1 Protein in Different Tissues | 第210-215页 |
| 3.2.4.1 Specificity of a Commercial α-ATP8A1 Antibody | 第210-211页 |
| 3.2.4.2 Immunolocalization of ATP8A1 Protein in Retina, Brain and Testis | 第211-215页 |
| 3.2.5 Confirmation of ATP11C Protein in Different Tissues | 第215-221页 |
| 3.2.5.1 Domain analysis, Topology of ATP11C, and GST-fusion fragments | 第215-216页 |
| 3.2.5.2 GST-Fusion Protein Antigen Purification | 第216-217页 |
| 3.2.5.3 Secondary Screen- Western Blotting on PVDF Membrane Strips: | 第217-218页 |
| 3.2.5.4 3D8 mAb Specificity: | 第218-219页 |
| 3.2.5.5 Immunolocalization of ATP11C in Retina, Testis | 第219-221页 |
| 3.3 Discussion | 第221-225页 |
| CHAPTER Ⅳ Expression and Characterization of ATP11A and ATP11C | 第225-246页 |
| 4.1 Introduction | 第225-227页 |
| 4.2 Results | 第227-242页 |
| 4.2.1 ATP11A, ATP11B, ATP11C Gene Expression by RT-PCR | 第227-228页 |
| 4.2.2 Human ATP11A, Mouse ATP11B and Mouse ATP11C Cloning and Sequencing | 第228-229页 |
| 4.2.3 Co-immunoprecipitation of ATP11A, ATP11B, or ATP11C with or without CDC50 (A or B) on a 1D4 Immunoaffinity Column | 第229-233页 |
| 4.2.4 Localization of ATP11A, ATP11C in Transfected COS-7 Cells | 第233-236页 |
| 4.2.5 ATPase Activity Assay | 第236-238页 |
| 4.2.6 The Effect of Varying PS Concentration on the Velocity of ATP Hydrolysis in the Presence of PC and 5mM ATP | 第238-239页 |
| 4.2.7 The Effect of Varying PE Concentration on the Velocity of ATP Hydrolysis in the Presence of PC and 5mM ATP | 第239-240页 |
| 4.2.8 The Effect of Varying ATP Concentration on the Velocity of ATP Hydrolysis of ATP11A and ATP11C in the Presence of 10% PS | 第240-241页 |
| 4.2.9 Phospholipid Flippase Activities of ATP11A and ATP11C | 第241-242页 |
| 4.3 Discussion | 第242-246页 |
| Future Direction | 第246-248页 |
| References | 第248-259页 |
| 作者在攻读博士学位期间公开发表的论文 | 第259-260页 |
| ACKNOWLEDGEMENTS | 第260-261页 |
