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Graduate School of Ajou University
Department of Neuroscience and Technology Course
3. Theses(Master)
Tissue inhibitor of Metalloproteinases-3(TIMP-3) 단백질에 의한 신경세포 고사과정 조절기전에 관한 연구
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 곽병주 | - |
| dc.contributor.author | 이재근 | - |
| dc.date.accessioned | 2019-10-21T07:11:56Z | - |
| dc.date.available | 2019-10-21T07:11:56Z | - |
| dc.date.issued | 2007-08 | - |
| dc.identifier.other | 2456 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/17156 | - |
| dc.description | 학위논문(박사)----아주대학교 일반대학원 :신경과학기술과정,2007. 8 | - |
| dc.description.tableofcontents | ACKNOWLEDGEMENTS i ABSTRACT ii TABLE OF CONTENT iv LIST OF FIGURES viii LIST OF TABLES x ABBREVIATION xi I. INTRODUCTION 1 A. Proteomic approach to identify target proteins of apoptosis 1 B. Tissue inhibitors of matrix metalloproteinases (TIMPs) 4 1. Overview 4 2. The role of TIMPs in nervous system 4 3. Distinctive features of tissue inhibitor of matrixmetalloproteinases (TIMP-3) 6 C. Potential function and mechanism of TIMP-3 in neuronal apoptosis 7 D. Aims of study 10 II. MATERIALS AND METHODS 11 A. Materials 11 1. Reagents 11 2. Human postmortem tissues 11 3. Animals 12 B.Methods 12 1. Cell biological method 12 (A) Mouse cortical cell culture 12 (B) Neuro2A cell line culture 13 (C) Assay of neuronal death 13 (D) Assay of neuro2A cell death 14 2. Biochemical methods 15 (A) 2-D PAGE 15 1) Preparation of protein samples 15 2) 2D gel electrophoresis 15 3) MALDI-TOF MS protein identification 16 4) Database search 16 5) Qualitative and quantitative analysis of differential expression profiles of proteoms related to apoptosis 17 (B) Western blot analysis 17 (C) Immunoprecipitation 19 (D) Immunocytochemistry 19 (E) Immunohistochemistry 20 3. Molecular biological methods 21 (A) RNA interference 21 1) Plasmid Construction 21 2) Generation of siRNA mixtures for RNAi 22 3) Transfection and apoptosis induction 23 4.In vivo methods 23 (A) Tissue preparation 23 III. RESULTS 24 A. Quantitative 2-D analysis of proteins sensitive to serum deprivation in neuron-rich cortical cell cultures 24 B.TIMP-3 mediates neuronal apoptosis and neuronal loss of neurodegenerative diseases 30 1.TIMP-3 levels increase during serum deprivation-induced neuronal apoptosis (SDIA) 30 2. Neuronal expression of TIMP-3 is increased in Alzheimer’s disease and an animal model of ALS 35 3. TIMP-3 contributes to Fas activation and SDIA through interaction with MMP-3 42 4. TIMP-3 mediates SDIA 54 IV. DISCUSSION 66 A. Differential proteome analysis of SDIA 66 B. The proapoptotic function of TIMP-3 in neuronal death 68 V. SUMMARY AND CONCLUSION 72 BIBLIOGRAPHY 75 국문요약 97 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | Tissue inhibitor of Metalloproteinases-3(TIMP-3) 단백질에 의한 신경세포 고사과정 조절기전에 관한 연구 | - |
| dc.title.alternative | Lee Jae-Keun | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.alternativeName | Lee Jae-Keun | - |
| dc.contributor.department | 일반대학원 신경과학기술과정 | - |
| dc.date.awarded | 2007. 8 | - |
| dc.description.degree | Master | - |
| dc.identifier.localId | 566934 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000002456 | - |
| dc.subject.keyword | Neuronal Apoptosis | - |
| dc.subject.keyword | Serum deprivation | - |
| dc.subject.keyword | 2-D PAGE | - |
| dc.subject.keyword | AD | - |
| dc.subject.keyword | ALS | - |
| dc.subject.keyword | TIMP-3 | - |
| dc.subject.keyword | MMP-3 | - |
| dc.subject.keyword | Fas | - |
| dc.subject.keyword | FADD | - |
| dc.subject.keyword | Caspase | - |
| dc.description.alternativeAbstract | Apoptosis as well necrosis plays a role in neuronal loss in various neurological diseases including hypoxic-ischemia, Alzheimer’s disease, and Parkinson’s disease. Up to now, caspase-mediated signaling pathways have been proposed as a common route of apoptosis. We applied proteomic approach to draw expression profiles of proteins specifically altered in the process of neuronal apoptosis. Cortical neurons deprived of serum underwent widespread apoptosis over the next 24 hr, which was sensitive to cycloheximide, a protein synthesis inhibitor, that was administered within 8 - 10 hr after serum deprivation. Protein samples were obtained from cortical neurons deprived of serum for 8 hr and subjected to two-dimensional gel electrophoresis (2-DE). The first dimension separation was performed on an immobilized pH gradient strip, which was then separated on SDS-PAGE for the second dimension. Thousands of protein spots were visualized by silver staining, analysed by image processing, and identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). We have identified 49 proteins that are mainly associated with metabolism, transcription, development, and synthetic pathways Of them, tissue inhibitor of metalloproteinases-3 (TIMP-3 Tissue inhibitor of metalloproteinases-3 (TIMP-3), a pro-apoptotic protein in various cancer cells, was increased during serum deprivation-induced apoptosis (SDIA), but not during necrosis induced by excitotoxicity or oxidative stress. Levels of TIMP-3 were markedly increased in degenerating neurons in a transgenic model of familial amyotrophic lateral sclerosis and in brains of patients with Alzheimer’s disease. Upregulation of TIMP-3 following serum deprivation was accompanied by activation of the Fas death pathway. SDIA and activation of the Fas pathway were prevented by addition of an active MMP-3. Timp-3 deletion by RNA interference attenuated SDIA in N2a cells. These findings provide evidence that TIMP-3 is an upstream mediator of neuronal apoptosis and likely contributes to neuronal loss in neurodegenerative diseases such as amyotrophic lateral sclerosis or Alzheimer’s disease. | - |
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