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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/10101完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 孫錦虹 | |
| dc.contributor.author | Bo-Chi Lin | en |
| dc.contributor.author | 林柏齊 | zh_TW |
| dc.date.accessioned | 2021-05-20T21:02:00Z | - |
| dc.date.available | 2013-10-07 | |
| dc.date.available | 2021-05-20T21:02:00Z | - |
| dc.date.copyright | 2011-10-07 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-07-19 | |
| dc.identifier.citation | Adam RD. Clin Microbiol Rev 2001; 14:447–75. Biology of Giardia lambia.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/10101 | - |
| dc.description.abstract | 梨形鞭毛蟲是具有鞭毛的單細胞生物，它是全世界造成腹瀉的常見原因之一。根據16s rRNA序列分析，梨形鞭毛蟲是非常原始的真核生物。已經有研究發現在梨形鞭毛蟲的基因體存在有拓樸異構酶-II的基因。拓樸異構酶-II的功能是當基因進行轉錄或是DNA複製時負責對DNA進行解螺旋和恢復超螺旋結構。先前研究發現梨形鞭毛蟲Myb2蛋白是一個重要的轉錄因子之一，負責調節囊體化時期大量表現之基因。而我們也在梨形鞭毛蟲拓樸異構酶-II基因的啟動子上找到了Myb2蛋白的結合位置，所以本研究目標是想要了解在梨形鞭毛蟲囊體化過程中，拓樸異構酶所扮演的角色。我們發現拓樸異構酶-II基因在囊體化時期mRNA的表現上升，而且拓樸異構酶-II基因的啟動子活性也在囊體化時期上升，表示拓樸異構酶-II在囊體化時期表現量提高，可能在這個時期有某些功能或作用。而梨形鞭毛蟲拓樸異構酶-II的序列也和真核生物的拓樸異構酶-II相似。從電泳位移分析實驗中得知拓樸異構酶-II和其C端部分也具有結合雙股DNA的能力，並且也具有切割雙股DNA的能力。從分解ATP活性分析中得知拓樸異構酶-II和其N端部分也具有分解ATP能力。Etoposide是一種抗腫瘤藥物，在藥物治療上此藥物屬於拓樸異構酶-II的抑制物。以添加濃度為400μM的抑制劑-eoposide，觀察梨形鞭毛蟲的生長情形，發現加藥後3~24小時滋養體數目明顯少於對照組。表示eoposide可以抑制滋養體的生長。在進行囊體化24小時期間添加濃度400μM的eoposide，也發現囊體數目明顯少於對照組，表示eoposide也會抑制梨形鞭毛蟲從滋養體分化成囊體。而在過度表現拓樸異構酶-II細胞株中我們發現細胞形成囊體的能力有上升的情形，且一些與囊體化相關的基因表現都有上升，例如cwp1、cwp2、myb，表示拓樸異構酶確實在梨形鞭毛蟲囊體化過程中扮演一個重要的角色。 | zh_TW |
| dc.description.abstract | Giardia lamblia is a flagellated unicellular eukaryotic microorganism that commonly causes diarrheal disease throughout the world. According to the 16s rRNA sequence analysis, G. lamblia has been proposed as an early branching eukaryote. A gene encoding type II DNA topoisomerase was identified in G. lamblia genome. Topoisomerases are enzymes that unwind and wind DNA, in order for genes to be transcribed and to facilitate DNA replication. Myb2 protein is a transcription factor that regulates some genes that were highly expressed during encystation stage. We found that a Myb2 binding site is present in the topoisomerase II gene promoter. The aim of this study is to understand the role of topoisomerase II in Giardia encystation. We found that the endogenous topoisomerase II mRNA levels increases significantly during encystation. The topoisomerase II activity tested by a luciferase reporter system also increased during encystation. The results suggest that topoisomerase II gene may be upregulated during encystation stage and it may have some function in this stage. The sequence of the G. lamblia topoisomerase II is similar to that of the eukaryotic topoisomerase II. EMSA data indicate that topoisomerase II and C-terminal part of topoisomerase II can bind to double stranded DNA, and topoisomerase II and C-terminal part of topoisomerase II have double stranded DNA cleavage ability. ATPase data indicate that topoisomerase II and N-terminal part of topoisomerase II can hydorlyze ATP. Etoposide is an anti-cancer chemotherapy drug. This medication is classified as a topoisomerase II inhibitor. We added etoposide in trophozoite cultures at 400μM concentration and observed cell growth, and found that the cell number decreased significantly 3~24 h after treatment. The results suggest that topoisomerase II may lead to inhibition of G. lamblia cell growth. Addition of 400μM etoposide also resulted a significant decrease of cyst number during 24h encystation, suggesting that topoisomerase II may inhibit G. lamblia differentiation into cysts. Overexpression of topoisomerase II resulted in an increase in the levels of cyst formation and expression of genes for encystation, including cwp1, cwp2 and myb genes, suggesting that topoisomerase II plays an important role in the encystation of G. lamblia. | en |
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| dc.description.tableofcontents | 口試委員會審定書……………………………...……………………………………..i
誌謝……………………………………………………………….……..…………….ii 中文摘要………………………………………………….…………..………………iii 英文摘要……………………………………….……………………….…………….iv 第一章 前言…………………………………………………………………………1 1.1 簡介……………………………….…………………………………………1 1.2 拓樸異構酶…………………………...................………………………..…2 1.3 研究動機………………………………………………….…………………5 第二章 材料與方法……………………………………………………………….….7 2.1梨形鞭毛蟲細胞株的培養………………………………...…………………7 2.2轉殖質體的建構………………………………………………………….….7 2.2.1 5’△5N-Pac………………………………………………………..…7 2.2.2 pPgTopoIIWT…………………………………………………….…7 2.2.3 pPgTopoIIm1………………………………………………..………8 2.2.4 pPgTopoIIm2…………………………………………………..……8 2.2.5 pPgTopoIIm3…………………………………………………..……9 2.3重組gTopoII以及突變gTopoII蛋白質的表現載體建構……………..……9 2.3.1 gTopoII…………………………………………………………...…9 2.3.2 gTopoII-N…………………………………………………….……10 2.3.3 gTopoII-C……………………………………………………….…10 2.3.4 gTopoII-Cm1………………………………………………………10 2.3.5 gTopoII-Cm2………………………………………………………11 2.3.6 gTopoII-Cm3………………………………………………………11 2.4 轉殖質體的轉型與萃取………………………………………………...…11 2.4.1 質體的轉型 (transformation) …………………………………...…11 2.4.2 質體的萃取…………………………………………………………12 2.5重組蛋白的表現與純化……………………………………………………12 2.6 梨形鞭毛蟲的轉染與選殖……………………………………………...…13 2.7 反轉錄聚合酶鏈式反應 (RT-PCR) ………………………………………13 2.8 西方墨點法 (Western blot) 與Coomassie blue染色…………….………14 2.8.1 西方墨點法…………………………………………………...….…14 2.8.2 Coomassie blue染色………………………………………………15 2.9 免疫螢光染色 (Immunofluorescence assay) ………………………..……15 2.10 Electroretic Mobility Shift Assays (EMSA) ………………………………16 2.11切割DNA活性分析………………………………………………………16 2.12水解ATP活性分析……………………………………………………..…16 第三章 實驗結果……………………………………………………………………18 3.1 梨形鞭毛蟲gtopoII基因序列及胺基酸序列分析…………………..……18 3.2 以RT-PCR及luciferase偵測滋養體時期和囊體時期gtopoII基因的mRNA 表現量與啟動子活性………………………………………………………19 3.3 梨形鞭毛蟲gtopoII基因的表現………………………………………..…20 3.4 梨形鞭毛蟲拓樸異構酶、N端、C端切割雙股DNA活性分析…………20 3.5 梨形鞭毛蟲拓樸異構酶、N端、C端ATPase活性分析………………..21 3.6 梨形鞭毛蟲拓樸異構酶、N端、C端結合雙股DNA活性分……………22 3.7 利用不同基因的啟動子序列鑑定gTopoII-C端蛋白與DNA結合能…22 3.8 抗腫瘤藥物etoposide抑制梨形鞭毛蟲生長……………………………23 3.9 抗腫瘤藥物etoposide抑制滋養體時期梨形鞭毛蟲Cwp1表現………25 3.10 抗腫瘤藥物etoposide抑制梨形鞭毛蟲形成囊體…………………...…25 3.11 鑑定突變gTopoII的表現以及切割和結合DNA的能力………………26 3.12 分析gTopoII以及gTopoII突變蛋白對於囊體化相關基因的調控和囊體 形成的影響………………………………………………………..………27 第四章 討論…………………………………………………………………………29 4.1 梨形鞭毛蟲TopoII蛋白功能區域分析……………………………...……29 4.2 gTopoII具有與鎂離子結合區域…………………………………………...30 4.3 gTopoII偏好與AT-rich序列做結合………………………………….……31 4.4 Etoposide對梨形鞭毛蟲的生長和分化的影響……………………………31 4.5 gTopoII促進梨形鞭毛蟲行囊體化…………………………………...……32 4.6 myb基因與gtopoII基因的表現之互相調控關係……………………..……32 附圖………………………………………………………………………………..…34 參考文獻…………………………………...…………………………………...……68 | |
| dc.language.iso | zh-TW | |
| dc.title | 梨形鞭毛蟲拓樸異構酶-II的鑑定與功能分析 | zh_TW |
| dc.title | Characterization and Functional Study of Topoisomerase II in Giardia lamblia | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李財坤,詹迺立 | |
| dc.subject.keyword | 梨形鞭毛蟲,拓墣異構酶,囊壁蛋白,囊體化,etoposide, | zh_TW |
| dc.subject.keyword | Giardia lamblia,topoisomerase II,cyst wall protein,etoposide,encystation, | en |
| dc.relation.page | 74 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2011-07-19 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-100-1.pdf | 7.52 MB | Adobe PDF | 檢視/開啟 |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。