2012-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658497摘要：梨形鞭毛蟲是一種寄生於腸道之原蟲，可引起流行性的腹瀉疾病。其生活史中可以滋養體的形式寄生在小腸，滋養體也可以分化成囊體，以存活於體外並可感染新的宿主，形成囊體時可合成具有保護作用的囊壁，使囊體可存活於宿主外，並具有感染其他宿主的能力，然而目前對於滋養體分化成囊體時的機制並不清楚，尤其是形成囊壁蛋白(cyst wall proteins，CWPs)的調控機制有待了解。梨形鞭毛蟲的生活史已被建立於試管中，因此它成為一個很重要的模式，可以幫助了解其他在試管中尚未建立生活史的寄生蟲。除了醫學上的重要性外，梨形鞭毛蟲的研究可以幫助我們了解在真核生物演化上的重要過程，梨形鞭毛蟲的許多基因的轉錄起始因子組成相較於其他真核生物少了很多，許多基因的未轉譯區域明顯較短，而且啟動子缺乏TATA box及其他演化分支後期的高等真核生物所具有的cis-acting elements，但是梨形鞭毛蟲的許多基因的轉錄起始位置出現在具有多AT的序列，多AT的序列被認為功能是類似於高等真核生物的initiator element。E2F家族轉錄因子是真核生物中調控細胞增生及細胞週期重要的因子，細胞分化與細胞週期常是互相結合的，E2F也參與在細胞分化過程中。我們想了解E2F在梨形鞭毛蟲分化成囊體的過程中的角色，我們針對梨形鞭毛蟲的基因資料庫做Blast搜尋，找到2個可轉譯成具有可能是E2F 的DNA結合構造的基因(e2f1 和e2f2) 。初步的研究發現Epitope-tagged的E2F1蛋白質在滋養體生長時期及囊體化時期皆出現在細胞核，純化的E2F1重組蛋白可結合於thymidine kinase基因的啟動子，in vivo實驗中發現E2F可轉錄活化thymidine kinase基因的啟動子，這些結果顯示E2F可能是梨形鞭毛蟲的重要的轉錄因子。此計劃我們將以生化、細胞學及分子方法來研究E2F1及E2F2蛋白質之功能，研究結果對梨形鞭毛蟲囊體化及致病性提供相當寶貴的資訊，將來或許可以利用我們的研究結果來發展阻斷囊體的形成的藥物或是發展治療用藥。<br> Abstract: Giardia lamblia is a protozoan parasite causing water-borne intestinal diseaseworldwide. It parasitizes the host intestine and survives outside host for transmission byalternating between two distinct forms, trophozoite and cyst. Synthesis of a protective cystwall is required for survival outside the host and infection of G. lamblia. However, little isknown of the molecular mechanisms governing regulation of the cyst wall proteins (CWPs)during differentiation into dormant cysts. The completion of G. lamblia life cycle in vitromakes it a valuable model for other parasites whose differentiation has not been achieved invitro. In addition to its medical importance, G. lamblia is of biological interest inunderstanding the mechanisms of eukaryotic evolution. It has fewer cellular components fortranscription initiation. Unusually short 5’-flanking regions with no consensus TATA boxesor other cis-acting elements identified in late-branching eukaryotic promoters are sufficientfor gene expression. Instead, AT-rich sequences have been found around the transcriptionstart sites of many genes, functionally similar to the initiator element in late-branchingeukaryotes. E2F family transcription factors are important regulators of cell proliferation andG1/S cell cycle progression in many eukaryotes. Cell differentiation is often tightly linkedwith cell cycle control. E2Fs are also involved in cell differentiation. To understand the role ofE2Fs during Giardia differentiation into cyst, we did Blast searches of the Giardia genomedata base and identified two genes encoding putative E2F DNA-binding domains (e2f1 ande2f2). Epitope-tagged E2F1 localized to nuclei during both vegetative growth and encystation.Recombinant E2F1 specifically bound to the thymidine kinase gene promoter. We also foundthat E2F1 transactivated the thymidine kinase promoter in vivo. Our results suggest that E2F1may be an important transcription factor in G. lamblia. In this project, we will characterizeE2F1 and E2F2 to know their role using biochemical, cell biological, and molecularapproaches. The results will provide insight into encystation and pathogenicity of G. lambliaand help us to develop new drugs to inhibit the cyst formation or to treat giardiasis.囊體E2F2轉錄梨形鞭毛蟲cystE2F2transcriptionGiardia