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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 孫錦虹 | zh_TW |
| dc.contributor.advisor | Chin-Hung Sun | en |
| dc.contributor.author | 吳祐佳 | zh_TW |
| dc.contributor.author | You-Jia Wu | en |
| dc.date.accessioned | 2024-08-19T16:20:49Z | - |
| dc.date.available | 2024-08-20 | - |
| dc.date.copyright | 2024-08-19 | - |
| dc.date.issued | 2024 | - |
| dc.date.submitted | 2024-08-12 | - |
| dc.identifier.citation | 1. Adam, R.D., Protozoa: Giardia lamblia, in Encyclopedia of Food Safety, Y. Motarjemi, Editor. 2014, Academic Press: Waltham. p. 37-44.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/94777 | - |
| dc.description.abstract | 早期認為梨形鞭毛蟲 (Giardia lamblia) 不具粒線體，不過近代研究於小胞器膜上發現鐵硫蛋白 (Iron-sulfur cluster, Isc)，說明梨形鞭毛蟲的祖先曾經擁有粒線體，只是在演化過程中逐漸捨棄許多原本應有的功能，發展出具有部分粒線體功能的囊泡，稱為mitosome，我們實驗室先前發現未知功能的骨髓性白血病因子 (Myeloid leukemia factor, MLF) 出現在未知的囊泡，利用IFA (Immunofluorescence assay) 來共染ISCU (mitosome marker) 和MLF的表現，發現兩種囊泡並沒有共定位，確認MLF定位於mitosome以外的囊泡中，而設計突變蛋白與MLF共染IFA則有共定位，可能MLF參與自噬作用和蛋白酶體 (proteasome) 降解，此外觀察到梨形鞭毛蟲在囊體形成過程中，MLF表現增加，推斷其與囊體形成有關；本次實驗透過選殖八種mitosome相關蛋白探究：(1) 八種mitosome相關蛋白在梨形鞭毛蟲滋養體與囊體形成過程時期中的表現，判斷這些蛋白功能是否與囊體形成有關；(2) 八種mitosome相關蛋白在蟲體的定位，以及其與MLF蛋白的位置關係；根據Western blot結果顯示，梨形鞭毛蟲在過度表現7188、8148、9296、10971、12229、16386、16891或22587蛋白的情況下，MLF蛋白表現上升，而過度表現所有八種蛋白的情況下，Bip蛋白表現增加，過度表達蛋白7188、9296、10971或16386讓CWP1 (Cyst wall protein 1) 表現下降，但其餘蛋白的高濃度表現讓CWP1上升，而IFA顯示的各自表達8148、9296、10971、12229、16386、16891或22587的囊泡和MLF所在囊泡有出現部分的共定位。 | zh_TW |
| dc.description.abstract | People used to believe that Giardia lamblia did not have mitochondria. However, recent studies have found iron-sulfur clusters (Isc) on its organelle membranes, suggesting that ancestors of Giardia once possessed mitochondria but gradually lost many of their original functions over the course of evolution, and then evolved vesicles called mitosomes, which retain some mitochondrial functions. Our lab identified an unknown molecule, named Myeloid Leukemia Factor (MLF) appearing in unidentified vesicles. They used Immunofluorescence Assay (IFA) to co-stain ISCU (mitosome marker) and MLF, finding no co-localization between two proteins. However, a mutant protein designed for co-staining with MLF in IFA showed co-localization, confirming that MLF is located in vesicles except mitosome and potentially involved in autophagy and proteasome degradation. In this study, we cloned eight mitosome-related proteins to investigate：(1) their expression in Giardia lamblia during trophozoite and encystation stage, assessing whether these proteins are involved in encystation, (2) their location within the organism and spatial relationship with MLF. According to the Western blot results, Giardia lamblia shows increased MLF expression levels upon overexpressing protein 7188, 8148, 9296, 10971, 12229, 16386, 16891 or 22587. In the case of overexpression of all eight proteins, Bip protein increases. An overexpressed protein 7188, 9296, 10971 or 16386 decreases CWP1, whereas overexpression of the other proteins increased CWP1 expression. The immunofluorescence analysis (IFA) showed partial colocalization between vesicles expressing 7188, 8148, 9296, 10971, 12229, 16386, 16891 or 22587 and the vesicles containing MLF. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2024-08-19T16:20:49Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2024-08-19T16:20:49Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員會審定書 I
致謝 II 摘要 III Abstract IV 目次 (contents) V 圖次 (list of figures) X 表次 (list of tables) XII 第一章 前言 (introduction) 1 1.1 簡介 1 1.1.1 歷史與分類 1 1.1.2 流行病學 1 1.1.3 生活史 1 1.1.4 結構 2 1.2粒線體介紹 2 1.2.1 演化 2 1.2.2 結構與功能 3 1.2.3 TOM/TIM complex (Translocase of the outer membrane / translocase of the inner membrane complex) 3 1.2.4 Iron-sulfur cluster (Isc) 4 1.2.5 梨形鞭毛蟲mitosome 5 1.2.6 mitosome相關未知蛋白分離 5 1.3 細胞壓力相關蛋白 6 1.3.1 囊壁蛋白 (Cyst wall protein；CWP1) 6 1.3.2 Binding immunoglobulin protein (Bip) 7 1.3.3 骨髓性白血病因子 (Myeloid leukemia factor, MLF) 7 1.3.4梨形鞭毛蟲的自噬作用 8 1.4 研究動機 11 第二章 材料與方法 (meterials and methods) 13 2.1 質體建構 (plasmid construction) 13 2.1.1 資料庫查詢 (database research) 13 2.1.2 引子設計 (primer design) 13 2.1.3 聚合酶連鎖反應 (Polymerase chain reaction；PCR) 13 2.1.4 DNA clean up (QIAquick® Gel Extraction Kit) 14 2.1.5 限制酶切割 (restriction enzyme digestion) 14 2.1.6 接合反應 (ligation) 16 2.2 質體轉化 (transformation) 16 2.3 質體純化 (plasmid purification) 17 2.4 定序 (sequencing) 17 2.5 轉染 (transfection) 18 2.6 細胞培養 (cell culture) 18 2.7 蛋白質沉澱 (protein precipitation) 18 2.8 蛋白質定量與西方墨點法 (coomassie blue stain and Western blot) 19 2.9 免疫螢光染色法 (immunofluorescence assay；IFA) 19 2.10 統計 (statistics) 20 第三章 結果 (result) 21 3.1 基因編號7188 21 3.1.1 UniProt BLAST與文獻分析 21 3.1.2 Pfam預測結構組成 21 3.1.3 鑑定HA標記的7188蛋白 21 3.1.4 不同蟲株之間的MLF、CWP1、Bip蛋白 22 3.1.5 在囊體形成過程中的蛋白變化 22 3.1.6 IFA 22 3.2 基因編號8148 22 3.2.1 UniProt BLAST與文獻分析 22 3.2.2 Pfam預測結構組成 23 3.2.3 鑑定HA標記的8148蛋白 23 3.2.4 不同蟲株之間的MLF、CWP1、Bip蛋白 23 3.2.5 在囊體形成過程中的蛋白變化 23 3.2.6 IFA 24 3.3 基因編號9296 24 3.3.1 UniProt BLAST、Pfam與文獻分析 24 3.3.2 鑑定HA標記的9296蛋白 24 3.3.3 不同蟲株之間的MLF、CWP1、Bip蛋白 25 3.3.4 在囊體形成過程中的蛋白變化 25 3.3.5 IFA 25 3.4 基因編號10971 25 3.4.1 UniProt BLAST、Pfam資料庫與文獻分析 25 3.4.2 鑑定HA標記的10971蛋白 26 3.4.3 不同蟲株之間的MLF、CWP1、Bip蛋白 26 3.4.4 在囊體形成過程中的蛋白變化 26 3.4.5 IFA 27 3.5 基因編號12229 27 3.5.1 UniProt BLAST、Pfam資料庫與文獻分析 27 3.5.2 鑑定HA標記的12229蛋白 28 3.5.3 不同蟲株之間的MLF、CWP1、Bip蛋白 28 3.5.4 在囊體形成過程中的蛋白變化 28 3.5.5 IFA 28 3.6 基因編號16386 29 3.6.1 UniProt BLAST與文獻分析 29 3.6.2 Pfam預測結構組成 29 3.6.3 鑑定HA標記的16386蛋白 29 3.6.4 不同蟲株之間的MLF、CWP1、Bip蛋白 29 3.6.5 在囊體形成過程中的蛋白變化 30 3.6.6 IFA 30 3.7 基因編號16891 30 3.7.1 UniProt BLAST與文獻分析 30 3.7.2 Pfam預測結構組成 31 3.7.3 鑑定HA標記的16891蛋白 31 3.7.4 不同蟲株之間的MLF、CWP1、Bip蛋白 31 3.7.5 在囊體形成過程中的蛋白變化 32 3.7.6 IFA 32 3.8 基因編號22587 32 3.8.1 UniProt BLAST與文獻分析 32 3.8.2 Pfam預測結構組成 32 3.8.3 鑑定HA標記的22587蛋白 33 3.8.4 不同蟲株之間的MLF、CWP1、Bip蛋白 33 3.8.5 在囊體形成過程中的蛋白變化 33 3.8.6 IFA 33 第四章 討論 (Discussion) 35 附圖 (figures) 37 附表 (Tables) 68 參考文獻 (reference) 72 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 梨形鞭毛蟲 | zh_TW |
| dc.subject | 粒線體 | zh_TW |
| dc.subject | MLF | zh_TW |
| dc.subject | 自噬作用 | zh_TW |
| dc.subject | mitosome | en |
| dc.subject | MLF | en |
| dc.subject | Giardia lamblia | en |
| dc.subject | autophagy | en |
| dc.title | 鑑定梨形鞭毛蟲mitosome相關因子與囊體形成之間的關係 | zh_TW |
| dc.title | Identification of the relationship between mitosome-related factors and the encystation of Giardia lamblia | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 112-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 詹迺立;蕭信宏 | zh_TW |
| dc.contributor.oralexamcommittee | Nei-Li Chan;Shin-Hong Shia | en |
| dc.subject.keyword | 梨形鞭毛蟲,粒線體,MLF,自噬作用, | zh_TW |
| dc.subject.keyword | Giardia lamblia,mitosome,MLF,autophagy, | en |
| dc.relation.page | 82 | - |
| dc.identifier.doi | 10.6342/NTU202404144 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2024-08-12 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 微生物學研究所 | - |
| 顯示於系所單位： | 微生物學科所 | |
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| ntu-112-2.pdf 未授權公開取用 | 3.51 MB | Adobe PDF |
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