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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16061完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 孫錦虹 | |
| dc.contributor.author | Chia-Wei Chen | en |
| dc.contributor.author | 諶加偉 | zh_TW |
| dc.date.accessioned | 2021-06-07T17:59:39Z | - |
| dc.date.copyright | 2012-09-19 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-08 | |
| dc.identifier.citation | Adam RD. Biology of Giardia lambia. 2001. Clin Microbiol Rev. 14(3):447-75.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16061 | - |
| dc.description.abstract | 梨形鞭毛蟲是常見的腸道病原體，其傳播需要囊體形成。囊體在結構上具有一層囊體壁 (cyst wall)以幫助梨形鞭毛蟲抵抗外界逆境，囊體壁主要由蛋白質及多醣類所組成，目前已知的囊體壁蛋白 (Cwp, cyst wall protein)有3種，分別為Cwp1、Cwp2及Cwp3。本研究探討2個影響囊體形成的分子，分別是鋅手指蛋白及細胞凋亡因子。第1部分是關於探討鋅手指蛋白質，鋅手指 (zinc finger)蛋白質為含有可與鋅離子結合以穩定其蛋白質構型的鋅手指結構。依據與鋅離子結合的胺基酸數量與排列順序可分成很多種類型，其中又以C2H2鋅手指蛋白質最常見。在真核生物中，C2H2鋅手指蛋白質屬於數量最多的調控蛋白質，約有3%的人類基因會表現C2H2鋅手指蛋白質。C2H2鋅手指蛋白質可與DNA、RNA、蛋白質結合，並參與許多細胞功能的轉錄調節，例如：細胞分化、發育。從梨形鞭毛蟲的基因組資料庫中，我們發現1個鋅手指蛋白質，經過序列及SMART軟體分析，發現該蛋白質具有6個C2H2鋅手指結構，並將此蛋白質命名為C2H2-ZFP1。在梨形鞭毛蟲的滋養體時期及囊體化時期中，我們發現內生性C2H2-ZFP1基因在mRNA層級上囊體化時期表現量較高。我們將HA tag接到C2H2-ZFP1轉染到梨形鞭毛蟲，透過免疫螢光染色分析發現，在滋養體時期及囊體化時期，C2H2-ZFP1均表現於梨形鞭毛蟲的細胞核中，蛋白質層級的表現也是囊體化時期較高。從電泳位移分析實驗中得知C2H2-ZFP1具有與囊體壁蛋白質 (cwp1、cwp2和cwp3)基因啟動子結合的能力，且大量表現C2H2-ZFP1基因會促進Cwp1蛋白質、cwp1和cwp2基因表現量及囊體形成數目，若將C2H2-ZFP1上的鋅手指結構破壞則會導致此現象消失，推測C2H2-ZFP1可能為參與囊體化相關之cwp基因轉錄調控的轉錄因子，且其功能與鋅手指結構的完整性有重要關係。
第2部分所探討的為細胞凋亡因子，細胞凋亡是一種細胞程序性死亡，在細胞分化中扮演重要角色，過程中需要許多基因參與及調控。PDCD5是1個與細胞凋亡有關的蛋白質，又被稱為TFAR19。PDCD5在演化過程中具有高度保留性，在許多物種間均可找到具有高度同源性的基因。利用人類PDCD5在梨形鞭毛蟲基因組資料庫進行BLAST搜尋，找到1個蛋白質並將其命名為PDCD5。在梨形鞭毛蟲的滋養體時期及囊體化時期中，我們發現內生性PDCD5基因在mRNA層級上囊體化時期表現量較高，透過免疫螢光染色分析發現，在滋養體時期及囊體化時期，PDCD5均表現於梨形鞭毛蟲的細胞質及細胞核中，且大量表現PDCD5會促進梨形鞭毛蟲Cwp1蛋白質和cwp1基因表現量及囊體形成數目，當加入會造成梨形鞭毛蟲死亡的藥物，包括etoposide、metronidazole及curcumin時，我們發現PDCD5的表現量會上升，推測PDCD5可能為參與囊體化相關基因及凋亡調控的因子。 | zh_TW |
| dc.description.abstract | Giardia lamblia is a common protozoan parasite that parasitizes the small intestine of humans. It survives outside of the host by differentiation of trophozoites into cysts.
The cyst wall (CW), which is required for survival of G. lambla. The cyst wall is composed of specific proteins and polysaccharide. Key differentiation genes encode three cyst wall structural proteins (Cwp1, Cwp2, Cwp3). This thesis describes identification and characterization two Giardia factors, a zinc finger protein and an apoptosis-related factor. The first part describes functional characterization of a zinc finger protein. C2H2 zinc finger proteins constitute a large family of DNA-binding transcription factors and typically contain several fingers that make tandem contacts along the DNA. They participate in a variety of cellular activities, such as cell growth, differentiation, apoptosis and development. We found one C2H2 zinc finger-like open reading frame from G. lamblia genome data base and we named it C2H2-ZFP1. C2H2-ZFP1 has six C2H2 zinc finger domains as predicted by SMART program. We found that the mRNA expression levels of endogenous C2H2-ZFP1 gene during 24 hour encystation were higher than that during vegetative growth in G. lamblia. To understand the function of C2H2-ZFP1, we transfected a construct which expressed HA-tagged C2H2-ZFP1 gene into G. lamblia. The results from immunofluorescence assays and Western blot analysis revealed that the HA-tagged C2H2-ZFP1 was localized to nuclei and expressed at higher protein levels during encystation stage. Using electrophoretic mobility shift assay, we found that C2H2-ZFP1 can bind to the promoter of cwp1, cwp2, and cwp3 genes which are important during encystation in G. lamblia. Overexpression of C2H2-ZFP1 resulted in a significant increase of levels of Cwp1 protein, cwp1 and cwp2 mRNA and cyst formation. Using mutated plasmids and transfection assays, we found that the levels of Cwp1 protein and cyst formation in the C2H2-ZFP1-mutant overexpressing cell line decreased significantly relative to the levels in the wild type C2H2-ZFP1 overexpressing cell line. We suggest that C2H2-ZFP1 may be an important transcriptional activator in regulation of the cwp genes, and the C2H2 zinc finger domains may contribute to the function of C2H2-ZFP1. The second part describes functional characterization of an apoptosis-related factor. Apoptosis is a genetically controlled program of cellular self destruction, which is of central importance to the cellular development and cellular differentiation.The Human PDCD5 (programmed cell death 5) is an apoptosis-related factor, which is also designated as TF-1 cell apoptosis-related gene-19 (TFAR19). PDCD5 protein is conservative in the process of evolution and has significant homology to the corresponding proteins of species from yeast to mouse. A putative PDCD5 gene has been identified in G. lamblia genome. We found that the mRNA expression levels of endogenous PDCD5 gene during 24 hour encystation were higher than that during vegetative growth in G. lamblia. To understand the function of PDCD5, we transfected a construct which expressed HA-tagged PDCD5 gene into G. lamblia. Immunofluorescence assay showed that the HA-tagged PDCD5 was both localized to nuclei and cytosol during encystation stage. Overexpression of PDCD5 resulted in a significant increase of levels of Cwp1 protein, cwp1 mRNA and cyst formation. Using mutated plasmids and transfection assays, we found that the levels of Cwp1 protein and cyst formation in the PDCD5-mutant overexpressing cell line decreased significantly relative to the levels in the wild type PDCD5 overexpressing cell line. While we treated G. lamblia with lethal compounds, including etoposide, metronidazole and curcumin, PDCD5 expression increased. We suggest that PDCD5 may be an important factor in regulation of the cwp1 gene and apoptosis in G. lamblia. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-07T17:59:39Z (GMT). No. of bitstreams: 1 ntu-101-R99445203-1.pdf: 3857601 bytes, checksum: 3c5264f82cb5fcb52d8451bb9c4eb3e8 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 口試委員審定書 I
致謝 II 摘要 III Abstract V 目錄 VIII 第一部分 1 第一章 前言 2 1.1 梨形鞭毛蟲生活史與簡介 2 1.2 鋅手指蛋白質簡介 3 1.3 研究動機 5 第二章 材料與方法 6 2.1 梨形鞭毛蟲細胞株的培養 6 2.2 轉殖質體的建構 6 2.2.1 pP5pac5n 6 2.2.2 pPC2H2-ZFP1 6 2.2.3 pPC2H2-ZFP1-m1 7 2.2.4 pPC2H2-ZFP1-m2 7 2.2.5 pPC2H2-ZFP1-m3 8 2.3重組C2H2-ZFP1以及突變C2H2-ZFP1蛋白質的表現質體建構 11 2.3.1 C2H2-ZFP1 11 2.3.2 C2H2-ZFP1-m 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 即時定量反轉錄聚合鏈式反應 (Q-PCR) 14 2.9 西方墨點法 (Western blot) 與Coomassie blue染色 15 2.9.1 西方墨點法 15 2.9.2 Coomassie blue染色 16 2.10 免疫螢光染色 (Immunofluorescence assay) 16 2.11 Electroretic Mobility Shift Assays (EMSA) 16 第三章 實驗結果 18 3.1 梨形鞭毛蟲C2H2-ZFP1基因序列及胺基酸序列分析 18 3.2 以RT-PCR及Q-PCR偵測滋養體時期和囊體化時期C2H2-ZFP1基因mRNA表現量 18 3.3 梨形鞭毛蟲C2H2-ZFP1蛋白質在細胞中的表現 19 3.4 鑑定C2H2-ZFP1和DNA結合能力 19 3.5 鑑定C2H2-ZFP1與cwp1基因啟動子cwp1-45/-1序列之結合位置 20 3.6 利用distamycin A鑑定C2H2-ZFP1蛋白質與DNA的次要溝槽結合的能力 21 3.7 利用methyl green鑑定C2H2-ZFP1蛋白質與DNA的主溝槽結合的能力 21 3.8 分析C2H2-ZFP1蛋白質與cwp1基因啟動子的結合位置 22 3.9 梨形鞭毛蟲C2H2-ZFP1突變蛋白質在細胞中的表現 22 3.10 分析轉染C2H2-ZFP1蛋白質對囊體化相關基因的影響 23 第四章 討論 25 4.1 梨形鞭毛蟲C2H2-ZFP1蛋白質功能區域分析 25 4.2 梨形鞭毛蟲C2H2-ZFP1蛋白質的表現位置及促進囊體化進行 25 4.3 梨形鞭毛蟲C2H2-ZFP1蛋白質偏好結合AT-rich序列 26 4.4 梨形鞭毛蟲C2H2-ZFP1蛋白質偏好結合DNA主溝槽 26 附圖 28 附表 49 第二部分 50 第一章 前言 51 1.1 PDCD5蛋白質簡介 51 1.2 研究動機 52 第二章 材料與方法 53 2.1 梨形鞭毛蟲細胞株的培養 53 2.2 轉殖質體的建構 53 2.2.1 pP5pac5n 53 2.2.2 pPPDCD5 53 2.2.3 pPPDCD5-m1 53 2.2.4 pPPDCD5-m2 54 2.2.5 pPPDCD5-m3 55 2.3 轉殖質體的轉型與萃取 55 2.3.1 質體的轉型 (transformation) 55 2.3.2 質體的萃取 55 2.4 梨形鞭毛蟲的轉染與選殖 56 2.5 反轉錄聚合酶鏈式反應 (RT-PCR) 56 2.6 即時定量反轉錄聚合鏈式反應 (Q-PCR) 57 2.7.1 西方墨點法 58 2.7.2 Coomassie blue染色 59 2.8 免疫螢光染色 (Immunofluorescence assay) 59 第三章 實驗結果 60 3.1 梨形鞭毛蟲PDCD5基因序列及胺基酸序列分析 60 3.2 分析梨形鞭毛蟲滋養體時期及囊體化時期PDCD5蛋白質的表現量 60 3.3 梨形鞭毛蟲PDCD5蛋白質在細胞中的表現 61 3.4 分析轉染PDCD5蛋白質對囊體化相關基因的影響 61 3.5 梨形鞭毛蟲PDCD5突變蛋白質在細胞中的表現 61 3.6 分析轉染PDCD5及突變蛋白質對囊體化相關基因的影響 62 3.7 分析不同藥物在滋養體時期對囊體化相關蛋白質及PDCD5的影響 63 第四章 討論 65 4.1 梨形鞭毛蟲PDCD5蛋白質功能區域分析 65 4.2梨形鞭毛蟲PDCD5蛋白質的表現位置及促進囊體化進行 65 4.3 etoposide、metronidazole及curcumin對梨形鞭毛蟲PDCD5蛋白質表現的影響 66 附圖 68 參考文獻 83 | |
| dc.language.iso | zh-TW | |
| dc.subject | Cwp1 | zh_TW |
| dc.subject | Cyst wall protein 1 | zh_TW |
| dc.subject | C2H2-ZFP1 | zh_TW |
| dc.subject | 梨形鞭毛蟲 | zh_TW |
| dc.subject | TFAR19 | zh_TW |
| dc.subject | PDCD5 | zh_TW |
| dc.subject | C2H2 | zh_TW |
| dc.subject | 細胞凋亡 | zh_TW |
| dc.subject | 鋅手指 | zh_TW |
| dc.subject | apoptosis | en |
| dc.subject | Cwp1 | en |
| dc.subject | PDCD5 | en |
| dc.subject | TFAR19 | en |
| dc.subject | Giardia lamblia | en |
| dc.subject | zinc finger | en |
| dc.subject | C2H2 | en |
| dc.subject | C2H2-ZFP1 | en |
| dc.subject | Cyst wall protein 1 | en |
| dc.title | 鑑定梨形鞭毛蟲的C2H2鋅手指蛋白質及PDCD5蛋白質 | zh_TW |
| dc.title | Characterization of a Novel C2H2 Zinc Finger Protein and a PDCD5 Protein in Giardia lamblia | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 余明俊,陳佑宗 | |
| dc.subject.keyword | 梨形鞭毛蟲,鋅手指,細胞凋亡,C2H2,C2H2-ZFP1,Cyst wall protein 1,Cwp1,PDCD5,TFAR19, | zh_TW |
| dc.subject.keyword | Giardia lamblia,zinc finger,C2H2,C2H2-ZFP1,Cyst wall protein 1,Cwp1,PDCD5,TFAR19,apoptosis, | en |
| dc.relation.page | 92 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2012-08-08 | |
| 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 | |
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