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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳韻如(Yun-Ru Chen) | |
| dc.contributor.author | Xin-Peng Lin | en |
| dc.contributor.author | 林昕芃 | zh_TW |
| dc.date.accessioned | 2021-07-09T15:52:09Z | - |
| dc.date.available | 2025-08-19 | |
| dc.date.copyright | 2020-08-28 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-17 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76424 | - |
| dc.description.abstract | 阿茲海默症是一種慢性神經退化性疾病，並佔失智症病例的60-70%，乙型類澱粉蛋白 (Amyloid β) 聚集而成的老年斑塊以及tau蛋白不正常堆積形成的神經纖維糾結 (Neurofibrillary tangles, NFTs) 是阿茲海默症的兩大病理特徵。Tau蛋白具有穩定微管的功能，但不正常折疊的tau會脫離微管並堆積沉澱形成神經纖維糾結，先前的研究證明突變型ΔK280相較於野生型 (wild-type) 的tau更易形成纖維狀沉積，近期的研究也指出tau蛋白和prion蛋白相似具有在細胞間轉移的性質，不正常tau蛋白的轉移會造成疾病的惡化。本研究透過對tau單體與纖維狀沉積物進行分析，了解野生型與突變型ΔK280在結構上的差異，此外為了進一步了解野生型與突變型ΔK280在細胞轉移上的差異，本研究也建立一個平台可以分析tau蛋白轉移的現象，該平台利用螢光共振能量轉移 (Fluorescence resonance energy transfer, FRET) 偵測tau的交互作用，或是以流式細胞儀分析tau在細胞間的轉移率，而為了進一步研究O-GlcNAcylation對tau的影響，我們也完成初步O-GlcNAc tau純化系統的建立。我們的實驗結果指出在tau單體的結構上突變型ΔK280較野生型鬆散，而在細胞實驗中也發現突變型ΔK280較野生型更易在細胞間轉移，此外加入tau的纖維種子 (fibril seeds) 也會增加tau蛋白的轉移作用。了解tau在細胞間的轉移特性以及結構上的差異可以幫助我們對tau的病理機制有進一步的認識，並且對於減緩阿茲海默症的惡化提供可能的治療方向。 | zh_TW |
| dc.description.abstract | Alzheimer’s disease (AD) is a chronic neurodegenerative disease. It is known that β-amyloid plaques and tau neurofibrillary tangles (NFTs) are two main hallmarks of AD. Tau protein is a soluble microtubule-associated protein that stabilizes microtubules under normal physiological conditions. In brain of AD patients, tau becomes abnormally hyperphosphorylated and loses its ability to bind microtubules. The detached tau accumulates and aggregates into insoluble NFTs. Previous studies indicated ΔK280 mutant tau has higher tendency to form aggregates. Another research found tau protein transmits from cell to cell as prion-like behavior. Abnormal tau spreads throughout the brain may lead to disease progression. In this study, we investigated the structural differences between wild-type (WT) and ΔK280 tau in monomer or aggregated tau. To further study cellular transmission properties of WT and ΔK280 tau, we established a cell-to-cell tau transmission platform. Forster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) was used to confirm interaction of tau and flow cytometry for tau transmission rate. In order to investigate the effect of O-GlcNAcylation on tau in the future, we also established an O-GlcNAc tau purification protocol. In this study, we found ΔK280 tau monomer is structurally looser than WT tau. Furthermore, ΔK280 mutant tau has higher transmission propensity, and tau fibril seeds may increase tau transmission. Characterization of tau transmission and structure can help us to understand more about tau pathology and could provide a possible therapeutic strategy to slow down AD progression. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-09T15:52:09Z (GMT). No. of bitstreams: 1 U0001-1708202013163300.pdf: 4256952 bytes, checksum: 886c8471ca3b305188a1aaf5580acf7c (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 誌謝 i 中文摘要 ii ABSTRACT iii CONTENTS iv LIST OF FIGURES vii ABBREVIATIONS ix Chapter 1 Introduction 1 1.1 Alzheimer’s disease 1 1.2 Tau protein 2 1.2.1 Overview 2 1.2.2 Function and dysfunction of tau 3 1.2.3 Tau isoforms 4 1.3 Mutations of tau 5 1.3.1 Tau mutants 5 1.3.2 ΔK280 mutant 6 1.4 Structure of tau 7 1.5 Transmission of tau 8 1.6 Modification of tau 9 1.6.1 Phosphorylation 9 1.6.2 O-GlcNAcylation 10 1.7 Aims of the research 11 Chapter 2 Materials and methods 12 2.1 Materials 12 2.1.1 Primer for cloning 12 2.1.2 Buffer for tau purification 13 2.1.3 Gel electrophoresis 13 2.1.4 Western blot and dot blot 15 2.1.5 Cross-linking mass 16 2.1.6 Cell lines 16 2.2 Methods 17 2.2.1 Expression and purification of tau 17 2.2.2 Cross-linking mass spectrometry 18 2.2.3 Thioflavin T assay 20 2.2.4 Preparation of tau fibril seeds 20 2.2.5 Circular dichroism spectroscopy 21 2.2.6 Congo red absorbance 21 2.2.7 Western blot 21 2.2.8 Transmission electron microscopy (TEM) 22 2.2.9 Tau cloning 22 2.2.10 Filter retardation assay 23 2.2.11 Detection of tau interaction by FLIM-FRET 24 2.2.12 Tau transmission assay with FRET 24 2.2.13 Tau transmission assay by flow cytometry 25 2.2.14 Purification and enrichment of O-GlcNAc modified tau 27 2.2.15 Dot blot 28 Chapter 3 Results 29 3.1 Tau purification 29 3.1.1 Purification of WT tau 29 3.1.2 Purification of ΔK280 tau 29 3.2 Cross-linking mass spectrometry 30 3.3 Characterization of tau fibril 31 3.3.1 Aggregation of tau 31 3.3.2 Thermal stability of tau fibril 32 3.3.3 Congo red absorbance 32 3.3.4 Western blot 33 3.3.5 TEM imaging 34 3.3.6 Induction of intracellular tau aggregates 34 3.4 Establishment of cellular transmission platform 35 3.4.1 FLIM-FRET 35 3.4.2 Tau transmission assay with FRET 36 3.4.3 Tau transmission assay by flow cytometry 36 3.2.4 Transmission assay treated with heparin 37 3.4.5 Transmission assay treated with tau fibril seeds 38 3.4.6 Transmission assay treated with Aβ40 fibril seeds 38 3.5 O-GlcNAcylation of tau 39 3.5.1 Transmission assay with OGT inhibitor 39 3.5.2 Transmission assay with different glucose concentration 39 3.5.3 O-GlcNAc tau purification 40 Chapter 4 Discussion 41 4.1 Structure of tau monomer and fibril 41 4.2 Cellular transmission properties of tau 43 4.3 Future work in O-GlcNAc tau 45 FIGURE 47 REFERENCE 81 Appendix 88 | |
| dc.language.iso | en | |
| dc.subject | cross-linking質譜 | zh_TW |
| dc.subject | 阿茲海默症 | zh_TW |
| dc.subject | Tau蛋白 | zh_TW |
| dc.subject | Tau結構 | zh_TW |
| dc.subject | Tau蛋白轉移 | zh_TW |
| dc.subject | Tau structure | en |
| dc.subject | Alzheimer’s disease | en |
| dc.subject | Tau transmission | en |
| dc.subject | Cross-linking mass spectrometry | en |
| dc.title | 研究Tau的結構與在神經退化性疾病中的細胞轉移現象 | zh_TW |
| dc.title | Structure and Cellular Transmission of Tau protein in Neurodegenerative Diseases | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 楊健志(Chien-Chih Yang),廖憶純(Yi-Chun Liao) | |
| dc.subject.keyword | 阿茲海默症,Tau蛋白,Tau結構,Tau蛋白轉移,cross-linking質譜, | zh_TW |
| dc.subject.keyword | Alzheimer’s disease,Tau structure,Tau transmission,Cross-linking mass spectrometry, | en |
| dc.relation.page | 90 | |
| dc.identifier.doi | 10.6342/NTU202003728 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2020-08-18 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| dc.date.embargo-lift | 2025-08-19 | - |
| 顯示於系所單位： | 生化科技學系 | |
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