臺灣博碩士論文加值系統

近年來許多證據指出漸凍人症上C9orf72基因的重要性。其中一種由突變C9orf72基因轉譯之重複雙胜肽 (dipeptide repeats, DPRs) ， 多甘胺酸-丙胺酸 (poly glycine-alanine, poly-GA) 具類澱粉性質 (amyloidogenic) 且可能造成細胞核質運輸缺陷 (nucleo-cytosol shuttling defect) ，然而其相關病理機轉尚不明瞭。有鑑於此，本研究設計將poly-GA與細胞穿膜序列 [多精胺酸 (arginine, R) 或賴胺酸 (lysine, K) ] 以硝基苯為主之非天然光裂解胺基酸連結，以合成可光控形成類澱粉纖維之胜肽 [C(GA)12-PL-K8、C(GA)12-PL-R8] 。透過紫外光使胜肽光解，(GA)12部份即啟動類澱粉聚集。本研究藉由硫磺素T (Thioflavin T, ThT) 螢光染劑染色；穿透式電子顯微術 (TEM) 觀察其結構的變化；以及比較其混濁度之差異以了解胜肽之光控性質。另外，將胜肽接上螢光基團 (Alexa flour 488)並藉由隨機光學重建顯微術 (stochastic optical reconstruction microscopy, STORM) 直接觀察 (GA)12 形成之纖維。在細胞實驗中，發現光裂解之poly-GA片段可聚集細胞質中的TDP-43蛋白。再者，STORM影像更直接觀察到 (GA)12佔據細胞核孔，此進一步確認poly-GA可能影響核質運輸。綜合上述，透過光驅動胜肽可時空上專一地 (spatiotemporally) 啟動 (GA)12類澱粉聚集，本研究已建立一簡單之C9orf72相關ALS的疾病模型。而透過觀察光裂解胜肽片段引發細胞所發生的變化，未來希望以此作為疾病檢測或研究致病機制的工具。

Emerging evidences have revealed that the importance of mutant chromosome 9 open reading frame 72 (C9orf72) in amyotrophic lateral syndrome (ALS), a rare neurodegenerative disease. Intriguingly, one possibly translated dipeptide repeats (DPRs) of C9orf72, poly glycine-alanine (poly-GA) is considered amyloidogenic and it may cause nucleo-cytosol shuttling defect. However, the underlying mechanism and etiology remains obscure. To address this end, we designed and synthesized a photoinducible peptide device to learn the properties of dipeptide repeat in vitro and in cell. The UV-cleavable peptides, C(GA)12-PL-K8 and C(GA)12-PL-R8 are composed of octaarginines/octalysines and (GA)12 linked by a unnatural nitrobenzene amino acid. Upon photolysis, K8 or R8 segments will be cleaved and the high aggregation prone (GA)12 moiety, would initiate the amyloidogenesis process. We studied the biophysical properties of the peptides with/without UV irradiation, with Thioflavin T (ThT) fluorescence assay, transmission electron microscopy, and turbidity, respectively. For in vivo purpose, Alexa fluorophore 488 was conjugated on the peptide for cellular imageing. Upon UV irradiation, poly-GA fragment would interact and coaggerate with TDP-43 via seeding effect. Further more, with superresolution microscopy (stochastic optical reconstruction microscopy, STORM), direct visualization of (GA)12 and nucleopores was also attempted to elucidate how it impacts nuclear-cytosol shuttling. Therefore, a light-driven peptide enable spatiotemporal initiation of poly-GA aggregation. With the UV-inducible peptide, we have successfully established a simple model for C9orf72-associated ALS. In the future, it is possible the model can be extended for pathological examination or disease detection.

第一章 緒論
1.1 神經退化疾病 (Neurodegenerative Diseases)
1.2 肌萎縮性脊髓側索硬化症 (ALS)
1.2.1 Chromosome 9 open reading frame 72 (C9orf72)
1.2.2 The Glycine-Alanine Dipeptide Repeat from C9orf72
1.2.3 TAR DNA binding protein-43
1.3 類澱粉樣纖維性質
1.4 光控制類澱粉樣纖維聚集化現象
1.4.1 細胞穿膜序列與多正電序列
1.4.2 光裂解類胺基酸分子
1.5 超解析螢光顯微鏡（super-resolution fluorescence microscope）
1.6 隨機光學重建顯微術（stochastic optical reconstruction microscopy, STORM）
第二章 研究動機
2.1 使用合成的C9orf72 序列
2.2 建立細胞影像平台以觀察DPR (poly-GA) 在細胞中的特性以及可能之神經毒性
第三章 實驗材料與儀器
3.1 實驗儀器
3.2 實驗材料
第四章 實驗方法和儀器介紹
4.1 細胞培養
4.1.1 細胞株介紹
4.1.2 解凍細胞 (冷凍細胞活化)
4.1.3 細胞繼代 (Subculture)
4.1.4 細胞計數
4.1.5 細胞凍管製備
4.1.6 細胞轉染 (cell transfection) 以過度表達 (overexpression) 重組蛋白於神經細胞
4.2 細胞影像
4.2.1 共軛焦螢光顯微鏡觀測 (Confocal Microscopy)
4.2.2 隨機光學重建顯微術（stochastic optical reconstruction microscopy, STORM）
4.3 胜肽重複蛋白樣品製備 (合成、純化與鑑定)
4.3.1 固相胜肽合成法 (SPPS)
4.3.2 高效液相層析 (HPLC)
4.3.3 基質輔助雷射脫附游離質譜法 (MALDI mass spectrometry)
4.3.4 雙胜肽重複蛋白樣品螢光標記
4.4 雙胜肽重複蛋白樣品定性分析
4.4.1 圓二色光譜 (CD)
4.4.2 Thioflavin T螢光光譜 (fluorescence spectrometry)
4.4.3 穿透式電子顯微鏡 (TEM)
第五章 實驗結果
5.1 光化學雙胜肽重複蛋白樣品製備 (合成、純化與鑑定)
5.2 雙胜肽重複蛋白樣品螢光標記鑑定
5.3 雙胜肽重複蛋白樣品定性分析
5.3.1 超細微影像觀察-穿透式電子顯微鏡 (TEM)
5.3.2 濁度分析
5.3.3 胜肽之澱粉樣蛋白性質鑑定 (Thioflavin T螢光吸收)
5.3.4 圓二色光譜 (CD)
5.4 細胞影像分析結果
5.4.1 雙胜肽重複蛋白螢光標記樣品之共軛焦螢光顯微觀測
5.4.2 利用超解析螢光顯微術 (STORM) 觀測光誘導產生重複雙胜肽螢光標記樣品之聚集體妨害蛋白質在細胞內的核質傳輸
第六章 研究總結
參考資料
附圖

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