研究生: |
游主依 Chu-Yi Yu |
---|---|
論文名稱: |
以光引導富含甘胺酸及丙胺酸之探針形成類澱粉纖維,並以超解析影像觀測 Glycine-Alanine dipeptide repeat containing probe enables superresolution imaging of light-induced amyloid fiber formation |
指導教授: |
黃人則
Jen-Tse Huang |
口試委員: |
廖仲麒
Jung-Chi Liao 何明樺 Ming-Hua Ho |
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: | 2017 |
畢業學年度: | 105 |
語文別: | 中文 |
論文頁數: | 110 |
中文關鍵詞: | 重複雙胜肽 、光控探針 、超解析影像 、類澱粉纖維 |
外文關鍵詞: | dipeptide, superresolution imaging, light-induced, amyloid fiber |
相關次數: | 點閱:225 下載:0 |
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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.
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