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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蕭信宏 | |
| dc.contributor.author | De-Chang Lin | en |
| dc.contributor.author | 林德昌 | zh_TW |
| dc.date.accessioned | 2021-06-15T04:45:01Z | - |
| dc.date.available | 2013-09-09 | |
| dc.date.copyright | 2010-09-09 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-06 | |
| dc.identifier.citation | Anderson, K. V., and C. Nusslein-Volhard. 1984. Information for the dorsal--ventral pattern of the Drosophila embryo is stored as maternal mRNA. Nature 311 (5983):223-227.
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Jeffrey, P. Wang, C. Chu, D. M. Koepp, S. J. Elledge, M. Pagano, R. C. Conaway, J. W. Conaway, J. W. Harper, and N. P. Pavletich. 2002. Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex. Nature 416 (6882):703-709. Zhu, J., K. Miura, L. Chen, and A. S. Raikhel. 2003. Cyclicity of mosquito vitellogenic ecdysteroid-mediated signaling is modulated by alternative dimerization of the RXR homologue Ultraspiracle. Proc Natl Acad Sci U S A 100 (2):544-549. Zollman, S., D. Godt, G. G. Prive, J. L. Couderc, and F. A. Laski. 1994. The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila. Proc Natl Acad Sci U S A 91 (22):10717-10721. Zou, Z., S. W. Shin, K. S. Alvarez, V. Kokoza, and A. S. Raikhel. Distinct melanization pathways in the mosquito Aedes aegypti. Immunity 32 (1):41-53. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45715 | - |
| dc.description.abstract | 在蚊子的免疫系統中，只有innate immunity而沒有adaptive immunity，現在已知蚊子對抗病原的免疫機制包括了phagocytosis、encapsulation、melanization以及分泌性的antimicrobial peptide (AMP)等。埃及斑蚊(Aedes aegypti)體內的AMP有五種，分別為Cecropin、Defencin、Attacin、Diptericin以及Gambicin，而這些AMP的產生是由蚊子的Toll和Imd pathway來調控。此外，產卵和發育的過程都是蚊子重要的生理現象，對於控制蚊子傳播疾病的研究都是重要的參考依據。在岡比亞瘧蚊(Anopheles gambiae)的cDNA microarray分析結果發現，A. gambiae感染bacteria後，有一部份的基因會受到調控，其中我們對CAS1、ELMO和F-box/LRR較有興趣。CAS1在人類扮演acetyltransferase的角色與acetylation有關，在一種真菌的莢膜同是如此。ELMO被認為在果蠅(Drosophila melanogaster)的發育以及穩定細胞骨架占有重要角色，在mammalian則被認為跟small GTPase活化有關，也可能影響phagocytosis。F-box/LRR則被認為參與protein-protein的interaction，目前認為其在ubiquitylation中扮演重要角色。因此我們的研究目標在釐清埃及斑蚊ELMO及F-Box/LRR是否參與Toll及Imd pathway的調控，以及ELMO是否涉及調控埃及斑蚊的phagocytosis，同時也探討這些基因對vitellogenesis和metamorphosis的影響。首先，我們利用RNA interference (RNAi)的方式來抑制埃及斑蚊ELMO及F-Box/LRR的表現，再分別去感染Staphylococcus aureus和Escherichia coli，最後計算埃及斑蚊之存活率。實驗結果顯示埃及斑蚊的ELMO及F-Box/LRR被抑制之後，感染Staphylococcus aureus的存活率和控制組比較起來有明顯的上升，因此我們推測ELMO及F-Box/LRR參與埃及斑蚊體內的Toll pathway的調控且可能是負調控子。接著我們再分析Cecropin A的表現情形，實驗結果顯示當ELMO表現被抑制時，Cecropin A的mRNA會有過量的表現，說明ELMO可能是Cecropin A的負調控子。我們也利用螢光標定的S. aureus和E. coli來觀察ELMO對於埃及斑蚊phagocytosis能力的影響，實驗結果顯示ELMO的抑制會加強埃及斑蚊對於S. aureus的phagocytosis，說明了ELMO扮演負向調控phagocytosis能力的角色。另外利用RNAi的方式分別抑制CAS1和F-box再讓蚊子吸血去計算產卵的情形，結果顯示抑制CAS1的蚊子產卵率明顯下降，且與卵成熟有關的卵黃素生成前驅物Vitellogenin表現量也受到影響。最後我們knockdown pupae的ELMO和F-box，去計算其羽化成成蚊的比例，結果顯示抑制F-box的pupae羽化率會降低，且在成蚊時期部分metamorphosis gene包括broad、E75b和JHA15明顯也受到影響。 | zh_TW |
| dc.description.abstract | It has been widely accepted that invertebrates contain only innate immunity but no adaptive immunity. The innate immunity includes phagocytosis, encapsulation, melanization and secretion of antimicrobial peptides (AMPs). There are five AMPs called Attacin, Cecropin, Defencin, Diptericin and Gambicin in the mosquito Aedes aegypti and these AMPs are regulated by Toll or Imd pathway. In mosquitoes, vitellogenesis and metamorphosis are important life cycle that can be researches decreasing vectore-borne diseases. Previous cDNA microarray analysis revealed that CAS1, ELMO and F-Box/LRR are simultaneously up-regulated with Inhibitor of Apoptosos 2 (IAP2), an important regulator of IMD pathway, upon bacteria challenge in Anopheles gambiae. CAS1 was associated with acetylation in hunan and fungus－Cryptococcus neoformans. It is also called acetyltransferase. ELMO was shown to be involved in D. melanogaster development and cytoskeleton stability. It was demonstrated to affect phagocytosis in mammals. Previous research showed that F-box may play important role in protein-protein interaction and it is an important factor in ubiquitylation. Therefore, we ought to explore the functions of CAS1, ELMO and F-box in the mosquito A. aegypti. First, we made use of RNA interference (RNAi) technique to silence the mRNA expression of ELMO and F-box in A. aegypti, followed by the challenge of Staphylococcus aureus or Escherichia coli. The survival assay was performed to analyze the mosquito resistance to bacteria. Our results revealed that Aedes aegypti showed resistance to S. aureus in the absence of ELMO and F-Box. Therefore, we speculated that ELMO and F-box may serve as negative regulators in Toll pathway. Next, the expression of Cecropin A, a downstream target of Toll pathway, was examined. The results showed that silencing of ELMO resulted in the over-expression of Cecropin A upon S. aureus challenge, suggesting that ELMO negatively regulateof the expression of Cecropin A. Then we made use of FITC-labeled bacteria to observe the effect of phagocytosis in A. aegypti. The result showed that silencing of ELMO can increase the phagocytic ability to S. aureus in the mosquito. It’s suggested that ELMO played a negative role in phagocytosis to Gram positive bacteria. In addition, we made use of RNAi technique to silence the mRNA expression of CAS1 and F-box in A. aegypti, followed by feeding blood meal. The result showed that silencing of CAS1 decreased vitellogenin expression and egg production. Finally we use RNAi technique to silence the ELMO and F-box in larvalstage of Aedes aegypti and calculate the ratio of mosquito emergence. The result revealed that silencing of F-box resulted in the decrease of metamorphosis rate in larval stage and metamorphosis genes (broad,E75b, JHA15) expression in adult mosquitoes. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T04:45:01Z (GMT). No. of bitstreams: 1 ntu-99-R97445205-1.pdf: 3079051 bytes, checksum: aef3fd9068e5c9773368ad825d193f56 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 誌謝………………………………………………………...…..i
中文摘要………………………………………………..…….iii 英文摘要……………………………………………..………..V 第一章 緒論 1.1 文獻探討－蚊子傳染疾病（Mosquito-borne diseases）……………..….…..1 1.2 防治方法及衍生問題……………………………………………..…..…...…..2 1.3 成蚊發育………………………..………………………………………....…...3 1.3.1 蚊子變態過程 (metamorphosis)及相關調控…………………………...3 1.3.2 成蚊卵黃生成機制 (vitellogenesis)……………………………..……...6 1.4 成蚊免疫系統………………………………………………………….….…...8 1.4.1 細胞性免疫反應……………………………………………………...….9 1.4.1.1 Hemocytes………………………………...…………………………..9 1.4.1.2 吞噬作用 (phagocytosis)…………………………….……….……..11 1.4.1.3 黑化反應 (melanization)和包封反應 (encapsulation)…………….12 1.4.2 體液免疫反應—抗微生物胜肽(antimicrobial peptide)調控與產生….12 1.5 候選基因的發現－CAS1、ELMO和F-box/LRR…………...……………….14 1.5.1 CAS1 domain containing protein 1………………………..………..…..14 1.5.2 ELMO (engulfment and cell motility) domain containing protein 1…...15 1.5.3 F-box leucine-rich repeat protein………………………………..……...19 1.6 實驗動機和假說……………………………………………………………...20 第二章 材料與方法 2.1 埃及斑蚊培養……………………………………………………..………….22 2.2 cDNA preparation…………………………………………………….……....22 2.3 clone的製備………………………………………………………..…………23 2.4 質體的轉型與萃取………………………………………………….….…….27 2.5 雙股RNA (double strand RNA)製備與純化…………………….………..…27 2.6 雙股RNA純化……………………………………………………….………28 2.7 雙股RNA傳送…………………………………………………………….…28 2.8 感染埃及斑蚊………………………………………………………….……..29 2.9 即時核酸序列定量（Real-Time PCR）………………………….……………29 2.10 西方點墨法（Western blotting）……………………………………………....31 2.11 活體內吞噬作用實驗…………………………………………………...……32 2.12 產卵實驗…………………………………………………………….….…….33 2.13 蛹變態實驗法……………….………………………………………….…….33 2.14 pfam和boxshade比對分析之方法…………………………………………33 2.15 蛋白質3D結構預測方法……………………………………….……………34 2.16 演化樹分析之方法………………………………………………………..….34 2.17 統計分析方法…………………………………………………………...……34 第三章 結果 3.1 CAS1的保留區域 (Conserved domain of CAS1)…………………………...35 3.2 CAS1的演化樹分析 (phylogenetic tree of CAS1)………………………….35 3.3 ELMO的保留區域 (Conserved domain of ELMO)………………………....35 3.4 ELMO的演化樹分析 (phylogenetic tree of ELMO)……………………..…36 3.5 F-box/LRR的保留區域 (Conserved domain of F-box/LRR)……………….36 3.6 F-box/LRR的演化樹分析(phylogenetic tree of F-box/LRR)……………..…37 3.7 感染共生和非共生菌後埃及斑蚊的存活率……………………………..….37 3.8 ELMO和F-box對感染E. coli或S. aureus後埃及斑蚊存活率影響……….38 3.9 ELMO及F-box調控抗微生物胜肽(antimicrobial peptide)的能力………....39 3.10 Cecropin和Defencin對感染E. coli或S. aureus埃及斑蚊存活率影響…...40 3.11 ELMO影響埃及斑蚊體內吞噬作用 (phagocytosis)的能力……………....41 3.12 CAS1、ELMO和F-box在埃及斑蚊不同時期的表現量…………………...42 3.13 CAS1和F-box對埃及斑蚊產卵的影響…………………………………....44 3.14 CAS1和F-box對埃及斑蚊vitellogenin的影響……………………………45 3.15 探討ELMO和F-box對埃及斑蚊變態 (metamorphosis)的影響………...…45 第四章 討論 4.1 CAS1、ELMO和F-box在不同物種間的演化相關性……………………….47 4.2 ELMO和F-box對埃及斑蚊存活率之探討………………………………….48 4.3 ELMO與吞噬作用…………………………………………………….…..…50 4.4 CAS1和F-box對埃及斑蚊產卵和變態的影響之探討……………………..51 4.5 結論…………………………………………………………………………...52 圖表與說明………………………………………………………………………...53 參考文獻……………………………………………………..…………………….81 附錄…………………………………………………………..……………………...98 | |
| dc.language.iso | zh-TW | |
| dc.subject | 變態過程 | zh_TW |
| dc.subject | 核醣核酸干擾 | zh_TW |
| dc.subject | Toll pathway | zh_TW |
| dc.subject | 卵黃生成 | zh_TW |
| dc.subject | 埃及斑蚊 | zh_TW |
| dc.subject | metamorphosis | en |
| dc.subject | Aedes aegypti | en |
| dc.subject | RNA interference | en |
| dc.subject | Toll pathway | en |
| dc.subject | CAS1 | en |
| dc.subject | ELMO | en |
| dc.subject | F-Box | en |
| dc.subject | vitellogenesis | en |
| dc.title | 埃及斑蚊CAS1、ELMO和F-box/LRR之功能性分析 | zh_TW |
| dc.title | Functional analysis of CAS1、ELMO and F-box/LRR in the yellow fever mosquito, Aedes aegypti | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 徐立中,顧家綺,蔡坤憲 | |
| dc.subject.keyword | 埃及斑蚊,核醣核酸干擾,Toll pathway,卵黃生成,變態過程, | zh_TW |
| dc.subject.keyword | Aedes aegypti,RNA interference,Toll pathway,CAS1,ELMO,F-Box,vitellogenesis,metamorphosis, | en |
| dc.relation.page | 100 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-08-07 | |
| 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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