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研究生： 林映秀
Ying-Siou Lin
論文名稱： ALS相關TDP-43胜肽之結構與功能研究
Structural and Functional Analysis of ALS-related TDP-43 Peptides
指導教授： 黃人則
Jen-Tse Huang
何明樺
Ming-Hua Ho
口試委員: 黃人則
Jen-Tse Huang
何明樺
mhho@mail.ntust.edu.tw
吳昆峯
Kuen-Phon Wu
學位類別： 碩士
Master
系所名稱： 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年： 2020
畢業學年度： 108
語文別： 中文
論文頁數： 79
中文關鍵詞： 漸凍症 、TDP-43蛋白質 、胜肽 、類澱粉纖維
外文關鍵詞： ALS, TDP-43, peptide, Amyloid fibril
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    • 摘要 II ABSTRACT III 圖表索引 VIII 第1章 緒論 1 1.1 肌萎縮性脊髓側索硬化症 1 1.2 TDP-43蛋白質 1 1.3 TDP-43蛋白質與漸凍症的關係 2 1.3.1 TDP-43蛋白質Ｃ端聚集化現象 2 1.3.2 TDP-43點突變與病症的關係 3 1.4 類澱粉纖維聚合途徑 4 1.5 研究目的 7 第2章 實驗儀器與材料 8 2.1 實驗儀器列表 8 2.2 實驗材料 9 2.2.1 胜肽合成與純化試劑配置 9 2.2.2 電子顯微鏡樣品製備材料 10 2.2.3 細胞材料 10 2.2.4 藥品表 10 第3章 實驗方法與使用儀器介紹 12 3.1 胜肽樣品製備純化與鑑定 12 3.1.1 固相胜肽合成法 12 3.1.2 高效液相層析 13 3.1.3 基質輔助雷射脫附游離法 14 3.1.4 紫外/可見光分光光譜儀 14 3.2 胜肽性質分析 15 3.2.1 圓二色光譜儀 15 3.2.2 衰減式全反射分析紅外線光譜儀 17 3.2.3 Thioflavin T (ThT)螢光光譜 18 3.3 無細胞系統（Cell-Free system）與西方墨點（Western Blot）之晶種效應 18 3.4 細胞毒性測試 20 3.5 胜肽之類澱粉蛋白之結構分析 21 3.5.1 穿透式電子顯微鏡 21 3.5.1.1 胜肽類澱粉纖維之前處理 21 3.5.1.2 胜肽類澱粉纖維上機觀察 21 3.5.2 冷凍電子顯微鏡 22 3.5.2.1 冷凍電顯樣品製備 24 3.5.3 電子顯微鏡之金屬網格前處理 25 3.5.3.1 輝光放電 26 3.5.3.2 氧化石墨烯支撐層覆蓋 26 3.5.4 Cryo-EM影像收集 28 3.5.4.1 Motion Correction分析 29 3.5.4.2 Contrast Transfer Function (CTF) Estimation分析 33 3.5.5 Cryo-EM之分析 37 3.5.5.1 結構解析軟體RELION 37 第4章 實驗結果與討論 39 4.1 胜肽合成、純化與鑑定 39 4.2 胜肽性質鑑定 (HPLC, MALDI-TOF MS) 39 4.2.1 胜肽之電顯細微影像觀測 43 4.2.2 以圓二色光譜 （CD spectrum）分析胜肽之類澱粉二級結構 45 4.2.3 以衰減式全反射分析紅外線光譜（ATR-IR）分析胜肽之類澱粉二級結構 46 4.2.4 以ThT dye binding assay分析胜肽之類澱粉性質 47 4.3 胜肽類澱粉纖維與其突變體的細胞毒性測試 47 4.4 胜肽類澱粉纖維對TDP-43蛋白質的引晶效應 48 4.5 胜肽類澱粉纖維與其突變體的結構分析 50 4.5.1 低階冷凍電子顯微鏡影像掃描 50 4.5.2 高階冷凍電子顯微鏡影像收集 51 4.5.3 RELION軟體結構分析 53 4.5.3.1 Manual-Picking 54 4.5.3.2 Particle Extraction 56 4.5.3.3 2D Classification 58 4.5.3.4 Auto-Picking 60 4.5.3.5 Initial 3D model generation: Cylinder 68 4.5.3.6 3D Classification 68 4.5.4 TDP-43307-322與TDP-43307-322-M311V結構比較 68 4.6 Cryo-EM解析類澱粉纖維結構之文獻回顧 70 總結 72 參考文獻 73 附錄 77

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