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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 蕭信宏(SHIN-HONG SHIAO) | |
dc.contributor.author | Chiao-Fan Hsu | en |
dc.contributor.author | 許巧帆 | zh_TW |
dc.date.accessioned | 2021-06-08T02:18:30Z | - |
dc.date.copyright | 2020-09-03 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-08-15 | |
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Inhibition of protein kinase C promotes dengue virus replication. Virol J. 2016; 13:35. Newton AC. Protein kinase C: structure, function, and regulation. J Biol Chem. 1995; 270(48):28495-28498. Ojani R, Liu P, Fu X, Zhu J. Protein kinase C modulates transcriptional activation by the juvenile hormone receptor methoprene-tolerant. Insect Biochem Mol Biol. 2016; 70:44–52. Omodior O, Luetke MC, Nelson EJ. Mosquito-borne infectious disease, risk-perceptions, and personal protective behavior among U.S. international travelers. Prev Med Rep. 2018; 12:336–342. Park R, Baines JD. Herpes simplex virus type 1 infection induces activation and recruitment of protein kinase C to the nuclear membrane and increased phosphorylation of lamin B. J Virol. 2006; 80(1):494–504. Petersen LR, Jamieson DJ, Powers AM, and Honein MA. Zika Virus. N Engl J Med. 2016; 374:1552-63. Salazar M, Richardson J, Sa´ nchez-Vargas I, Olson K, and Beaty B. Dengue virus type 2: replication and tropisms in orally infected Aedes aegypti mosquitoes. BMC Microbiol. 2007; 7(9) Screaton G, Mongkolsapaya J, Yacoub S, and Roberts C. New insights into the immunopathology and control of dengue virus infection. Nat Rev Immunol. 2015; 15:745-59. Söderhäll K. Invertebrate immunity. Dev Comp Immunol. 1999; 23:263-6. Song BH, Yun SI, Woolley M, and Lee YM. Zika virus: History, epidemiology, transmission, and clinical presentation. J Neuroimmunol. 2017; 308:50-64. Tjaden NB, Thomas SM, Fischer D, Beierkuhnlein C (2013) Extrinsic Incubation Period of Dengue: Knowledge, Backlog, and Applications of Temperature Dependence. PLoS Negl Trop Dis. 7(6): e2207. Tolle MA. Mosquito-borne diseases. Curr Probl Pediatr Adolesc Health Care. 2009; 39:97-140. Wang L, Liang R, Gao Y, et al. Development of Small-Molecule Inhibitors Against Zika Virus Infection. Front Microbiol. 2019; 10:2725. Wang LL, Liang RY, Gao YN, et al. 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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19776 | - |
dc.description.abstract | 茲卡(Zika)與登革熱同樣都是藉由埃及斑蚊傳播的重要疾病。孕婦感染茲卡病毒甚至可能導致胎兒出現小頭症,再加上現今並無疫苗及藥物可使用,因此積極開發新穎的防疫策略是各個國家重視的議題之一。過去已知蛋白質的磷酸化會影響其構造和活性,進而調控細胞內訊息傳遞的過程,而蛋白激酶(Protein kinase)為主要負責蛋白質磷酸化的酵素。現在已知的蛋白激酶種類非常多,其中Protein kinase C (PKC)被認為是一類可以透過磷酸化serine以及threonine的烴基(hydroxyl group)而控制其功能的蛋白激酶。過去在哺乳類動物的研究發現,當宿主體內的PKC受到抑制時,會使登革病毒的非結構性蛋白(non-structural protein 5, NS5)無法被磷酸化,進而影響登革病毒的複製,但病媒蚊Protein kinase對於病毒調控的機制至今仍所知甚少。因此在本研究中,我們想探討埃及斑蚊的PKC是否對於病毒的複製扮演重要角色。我們首先使用PKC的抑制劑—Calphostin C對埃及斑蚊細胞株(ATC10)進行抑制,我們的結果顯示病毒感染前加入抑制劑可有效抑制病毒E蛋白(E protein)的產生,且使病毒的感染力下降。接著我們比對人類的PKC與埃及斑蚊的PKC,發現埃及斑蚊PKC3與人類影響登革病毒複製的PKC相似程度較高,因此我們利用過表現的方式使埃及斑蚊細胞株表現帶有HA標記的PKC3,根據實驗結果顯示,當PKC3過表現時,茲卡病毒的表現量也隨之上升,而病毒的感染力下降,因此我們推測PKC3可能影響病毒進入與釋出細胞的階段。我們也使用RNA干擾的方式抑制蚊子體內PKC3和PKC5的表現,結果顯示當埃及斑蚊PKC5表現被抑制後,E protein含量相對於控制組有減少的趨勢。根據目前的研究結果,我們推測埃及斑蚊蛋白激酶可能參與調控茲卡病毒的複製,未來我們將深入探討茲卡病毒與埃及斑蚊PKC之間的交互作用,同時將進一步探討宿主蛋白激酶影響茲卡病毒的詳細分子機制。 | zh_TW |
dc.description.abstract | Zika virus (ZIKV) infection is currently one of the world’s most important mosquito-borne diseases. ZIKV infection during pregnancy may result in microcephaly and other congenital abnormalities in the newborn. There is yet to be any effective drug or vaccine available for ZIKV. Therefore, intensive study for potential host factors in the mosquito is essential for establishment of novel alternative strategies for the disease control. Previous studies have indicated that Protein kinase (PK) is responsible for protein phosphorylation in the change of protein structure or activity. Protein kinase C (PKC) is a family of PK that is involved in functional control of other proteins through phosphorylating the serine and threonine amino acid residues of the target protein. Previous studies revealed that the phosphorylation of dengue virus non-structral protein 5 (NS5) is impaired when PKC is inhibited in mammalian cell, thereby affecting the replication of dengue virus. However, the mechanism of PKC in the regulation of the virus remains unclear. Hence, the aim of this study is to investigate the role of Protein kinase C in ZIKV replication in the mosquito Aedes aegypti. Our results showed that the ZIKV E protein production and virus infectivity was inhibited while the PKC was inhibited via PKC inhibitor—Calphostin C in the Aedes aegypti cell line. Moreover, the human PKCα and the Aedes aegypti PKC3 showed high level of sequence similarity. We also demonstrated that the viral genome and the E protein expression of ZIKV was up-regulated in the PKC3 overexpressed mosquito cell line post ZIKV infection whereas the virus infectivity was suppressed post ZIKV infection. We speculated that PKC3 is crucial for the entry and release of ZIKV. We further demonstrated that silencing of PKC5 resulted in the inhibition of ZIKV E protein production in vivo. Taken together, our results suggested that the protein kinase C of Aedes aegypti is involved in the regulation of Zika virus replication. In the future, we will further elucidate the detail interaction between ZIKV and PKC. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T02:18:30Z (GMT). No. of bitstreams: 1 U0001-1408202017402100.pdf: 8767990 bytes, checksum: dfe46a74b24a8945bf50a04abd6b34ca (MD5) Previous issue date: 2020 | en |
dc.description.tableofcontents | 中文摘要 i Abstract ii 目錄 iii 圖目錄 vi 表目錄 vii 第一章 緒論 1 1.1. 病媒蚊傳播疾病 (Mosquito-borne disease) 1 1.1.1. 瘧疾 (Malariae) 1 1.1.2. 登革熱 (Dengue fever) 2 1.1.3. 其他疾病 3 1.2. 茲卡病毒感染症 (Zika virus infection) 4 1.2.1. 全球之流行病學 4 1.2.2 茲卡病毒 (Zika virus;ZIKV) 4 1.2.3. 臨床症狀與治療 5 1.3. 埃及斑蚊生活史 6 1.4. 茲卡病毒生活史 7 1.4.1. 宿主細胞內之複製 7 1.4.2. 病媒蚊體內之複製 7 1.5. 蛋白激酶C (Protein kinase C) 8 1.6. Protein kinase C inhibitor -- Calphostin C 9 1.7. 實驗動機與假說 9 第二章 實驗材料與方法 11 2.1. 埃及斑蚊飼養與繼代 11 2.2. 細胞培養 (Cell culture) 11 2.3. 病毒製備 12 2.4. RNA萃取 (RNA extraction) 12 2.5. 反轉錄作用 (Reverse transcription, RT) 13 2.6. 聚合酶連鎖反應 (Polymerase chain reaction, PCR) 13 2.7. 即時定量聚合酶連鎖反應 (Real-time PCR, Quantitative PCR) 13 2.8. Protein kinase C inhibitor -- Calphostin C實驗 14 2.9. 雙股RNA (double-stranded RNA, dsRNA) 製備 14 2.9.1. 質體建構 (plasmid construction) 14 2.9.2. 雙股RNA合成 (dsRNA synthesis) 15 2.10. 埃及斑蚊顯微注射 (Microinjection) 16 2.10.1. 病毒注射 (virus injection) 16 2.10.2. dsRNA 注射 (dsRNA injection) 16 2.11. 西方點墨法 (Western blotting) 16 2.12. Focus forming assay (FFA) 17 2.13. 免疫螢光染色實驗(Immunofluorescent assay;IFA) 18 2.14. 轉染(Transfection) 19 2.14.1. 質體建構 (plasmid construction) 19 2.14.2. Liposome合成 20 2.15. 實驗試劑製備 20 第三章 結果 24 3.1. 埃及斑蚊體內蛋白激酶C序列結構分析 24 3.2. 蛋白激酶C在埃及斑蚊ATC10細胞之表現狀況 24 3.3. 蛋白激酶C抑制劑—Calphostin C對茲卡病毒基因表現之影響 24 3.4. 蛋白激酶C抑制劑—Calphostin C對茲卡病毒E protein表現之影響 26 3.5. 蛋白激酶C抑制劑—Calphostin C對茲卡病毒感染力之影響 26 3.6. 埃及斑蚊ATC10細胞之PKC3過表現對茲卡病毒複製之影響 27 3.7. 埃及斑蚊體內體內Protein kinase C表現量 28 3.8. 埃及斑蚊體內PKC3及PKC5對茲卡病毒複製之重要性 29 第四章 討論 30 4.1. 埃及斑蚊Protein kinase C在不同組織之表現與發育之影響 30 4.2. 埃及斑蚊Protein kinase C對茲卡病毒複製之影響 30 4.3. 埃及斑蚊Protein kinase C對茲卡病毒進入細胞之影響 31 4.4. 埃及斑蚊Protein kinase對茲卡病毒離開細胞之影響 32 附圖 33 圖 一 以Pfam軟體預測之埃及斑蚊PKC結構 33 圖 二 埃及斑蚊PKC在ATC10細胞內之表現量 34 圖 三 以Calphosin C抑制ATC10細胞之PKC影響茲卡病毒基因複製 37 圖 四 以Calphostin C抑制ATC10細胞之PKC影響茲卡病毒E protein表現 38 圖 五 以Calphostin C抑制ATC10細胞之PKC影響茲卡病毒效價 41 圖 六 PKC3-HA過表現影響茲卡病毒之基因複製及E protein表現 45 圖 七 埃及斑蚊成蚊母體內各組織之PKC表現量 48 圖 八 埃及斑蚊成蚊母體內PKC對茲卡病毒複製之重要性 50 附表 51 附錄 54 參考文獻 65 | |
dc.language.iso | zh-TW | |
dc.title | 探討埃及斑蚊蛋白激酶C對於茲卡病毒複製之影響 | zh_TW |
dc.title | Effects of mosquito protein kinase C on Zika virus replication in Aedes aegypti | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 劉旻禕(MIN-YI LIU),徐立中(Li-Chung Hsu) | |
dc.subject.keyword | 埃及斑蚊,茲卡病毒,蛋白激酶,病毒複製, | zh_TW |
dc.subject.keyword | Aedes aegypti,Zika virus,Protein kinase,virus replication, | en |
dc.relation.page | 70 | |
dc.identifier.doi | 10.6342/NTU202003470 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2020-08-17 | |
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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