| 摘要 | 第1-13页 |
| ABSTRACT | 第13-17页 |
| Chapter Ⅰ:Introduction | 第17-33页 |
| ·The TTK protein kinase family | 第17-21页 |
| ·The roles for Mps1 | 第21-26页 |
| ·Mps1p in spindle pole duplication | 第21-22页 |
| ·Mps1 in the spindle assembly checkpoint | 第22-24页 |
| ·Mps1 corrects chromosome attachment errors | 第24-25页 |
| ·Other functions of Mps1 kinase | 第25-26页 |
| ·The SAC kinases Mps1 in cancer | 第26-28页 |
| ·Regulation of Mps1 | 第28-33页 |
| Chapter Ⅱ Cloning, expression and purification of recombinant proteins | 第33-50页 |
| ·Material and methods | 第34-40页 |
| ·Cell cultures | 第34页 |
| ·Chemicals and buffers | 第34-35页 |
| ·Mammalian expression constructs | 第35页 |
| ·Amplification of recombinant baculovirus encoding TTK-wt and TTK-d | 第35-36页 |
| ·Baculovirus expression of TTK-wt and TTK-d | 第36页 |
| ·Protein purification of TTK-wt and TTK-d | 第36-37页 |
| ·Western blot | 第37-38页 |
| ·3C protease expression and purification | 第38页 |
| ·Protein TTKA expression and purification | 第38-39页 |
| ·In vitro kinase assay | 第39-40页 |
| ·Results | 第40-48页 |
| ·Expression and purification of 3C protease | 第40-41页 |
| ·Expression and purification of TTKA | 第41-42页 |
| ·Purification of TTK-wt and TTK-d | 第42-43页 |
| ·Time course experiment of TTK kinase activity | 第43-44页 |
| ·Assay for different substrates phosphorylation by TTK | 第44-45页 |
| ·Comparison of kinase activity between TTK-wt and TTK-d | 第45-48页 |
| ·Discussion | 第48-50页 |
| Chapter Ⅲ Measuring the absolute abundance of the crucial protein kinases duringcell cycle in Hela cells | 第50-77页 |
| ·Materials and methods | 第51-60页 |
| ·Materials and reagents | 第51-52页 |
| ·Cell lines | 第52-53页 |
| ·Cell density and viability estimations | 第53页 |
| ·Cell synchronization | 第53页 |
| ·Cell cycle analysis | 第53-54页 |
| ·Cell culture and nocodazole treatment | 第54-55页 |
| ·Cell lysis and sample preparation | 第55-56页 |
| ·Quantification of cellular concentrations of TTK and Cdks | 第56-57页 |
| ·Densitometry using ImageJ | 第57-58页 |
| ·Data analysis and calculations for Coomassie-stained gels using Microsoft Excel | 第58-59页 |
| ·Data analysis and calculations for immunoblot data using Microsoft Excel | 第59-60页 |
| ·Results | 第60-71页 |
| ·The TTK level in Hela cells and sw480 cells | 第60-61页 |
| ·Analysis for cell cycle status by flow cytometry(FACScan) | 第61-63页 |
| ·Measuring GST-TTK protein standard concentration | 第63-64页 |
| ·Measuring GST-Cdk and CyclinB protein standards concentration | 第64-66页 |
| ·Determine cellular abundance of Mps1,Cdk1 and Cyclin B | 第66-68页 |
| ·Data analysis | 第68-71页 |
| ·Discussion | 第71-77页 |
| ·A method for accurately quantifying the absolute number of biological molecules | 第71-73页 |
| ·Cell cycle-dependent regulation of the steady state level of TTK in vivo | 第73-74页 |
| ·Accumulation of Mps1 at centrosomes is essential for centrosome duplication | 第74-75页 |
| ·Mitosis might require a higher threshold of TTK than centrosome duplication | 第75页 |
| ·Cdk attenuates degradation of the TTK at centrosome duplication | 第75-77页 |
| Chapter Ⅳ Kinetic study of TTK and the modulation of its kinase activity by thecarboxyl terminal tail | 第77-114页 |
| ·Materials and Method | 第78-87页 |
| ·Autokinase assay | 第78-79页 |
| ·In vitro kinase assays | 第79-80页 |
| ·Quantitating gels using Molecular dynamics densitometer and Image quant 5.0 | 第80-81页 |
| ·Which one is suitable substrate for kinetics studies? | 第81页 |
| ·Is product formation linear with time? | 第81-82页 |
| ·Does autophosphorylation increase transphosphorylation? | 第82页 |
| ·Is autophosphorylation of TTK intermolecular or intramolecular action? | 第82-83页 |
| ·How the reaction rate varied with enzyme concentration | 第83-84页 |
| ·Is there a product inhibition? | 第84页 |
| ·Is D domain of TTK responsible for its kinase activity? | 第84页 |
| ·Two-substrate kinetic analysis for TTK catalytic mechanism | 第84-86页 |
| ·What is role of D domain in TTK transphophorylation? | 第86页 |
| ·Substrate Analog Inhibitors Inhibition Analysis | 第86-87页 |
| ·Results | 第87-109页 |
| ·Suitable substrate for kinetics study | 第87-88页 |
| ·A Lag-phase in TTK Product Formation | 第88-89页 |
| ·Autophosphorylation enhances transphophorylation | 第89-91页 |
| ·Autophosphorylation is an intermolecular reaction | 第91-92页 |
| ·The reaction rate is varied with enzyme concentration | 第92-95页 |
| ·Interpretation of product inhibition experiments | 第95-97页 |
| ·D domain is critical for substrate phosphorylation of TTK | 第97-101页 |
| ·Two-substrate Kinetic Analysis for TTK-wt and TTK-d | 第101-108页 |
| ·Investigation of the TTK kinase activity using substrate analog inhibitor | 第108-109页 |
| ·Discussion | 第109-114页 |
| ·A lag-phase in TTK product formation and auto-phosphorylation enhances activity | 第109-111页 |
| ·Self association or clustering of Mpsl molecules may enhance autophosphorylation and activate its kinase activity | 第111-112页 |
| ·TTK catalysis transpires through a sequential kinetic mechanism | 第112-114页 |
| Conclusions | 第114-119页 |
| References | 第119-128页 |
| Acknowledgements | 第128-130页 |
| 攻读学位期间发表的学术论文 | 第130-131页 |
| 附录 | 第131-132页 |
| 学位论文评阅及答辩情况表 | 第132页 |
