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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 陳進庭(Chin-Tin Chen) | |
| dc.contributor.author | Man Shen | en |
| dc.contributor.author | 沈縵 | zh_TW |
| dc.date.accessioned | 2021-06-17T07:10:40Z | - |
| dc.date.available | 2024-07-24 | |
| dc.date.copyright | 2019-07-24 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-07-21 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72928 | - |
| dc.description.abstract | 念珠菌是造成院內感染的重要致病菌，在臨床上念珠菌抗藥性問題也益發嚴重。目前所使用的抗真菌藥物種類不多，加上這些抗真菌劑之副作用強而造成使用上的限制，因此開發新作用機制的抗真菌藥物有其必要與急迫性。目前研究指出粒線體可能是一個良好的抗真菌藥物標的，其原因是由於粒線體參與在真核細胞內廣泛的生理調控角色，如各式各樣的生理代謝，包括了致病因子的調控。在確認小分子化合物BMVC-12C-P是能夠專一的進入粒線體並以粒線體做為主要攻擊標的而造成白色念珠菌粒線體的失能後，我們利用BMVC-12C-P作為工具，並配合白色念珠菌突變菌株庫的使用來探討白色念珠菌粒線體的失能對菌體造成的生物效應。本研究已經建立有效篩選突變菌株庫的篩選模式並在轉錄因子和激酶之突變菌株庫篩選出會受到BMVC-12C-P所影響的突變菌株。由於對於粒線體與致病機制的關係較感興趣，故將篩選出的突變菌株依其所參與的致病機制做歸類，結果以參與在菌絲的生成相關機制為大宗，故本研究進一步挑選菌絲作為探討主軸。我們發現BMVC-12C-P能夠抑制菌絲與生物膜生成之能力，並進一步以Q-PCR確認BMVC-12C-P能透過抑制菌絲轉錄因子EFG1、BRG1、CPH1、CPH2和TEC1的mRNA表現，然而其不透過Hog1MAPK訊息傳遞路徑去調控這些受到BMVC-12C-P所影響的菌絲轉錄因子。未來希望能夠更進一步了解粒線體是透過怎樣的訊息傳遞路徑來調控這些可能參與在菌絲生成的轉錄因子，並且去了解這些轉錄因子在白色念珠菌菌絲生成機制中所扮演的角色，以利未來我們能更深入了解白色念珠菌粒線體參與在菌絲調控的致病機制，更可以做為未來作為新抗真菌藥物發展之重要基石。於此，本研究亦探討BMVC-12C-P對不同標準念珠菌屬及來自臨床檢體中所篩選出來的具抗藥性之念珠菌屬的藥物感受性，發現BMVC-12C-P具有相當優異的殺菌能力，具有發展成良好抗真菌劑之潛力。 | zh_TW |
| dc.description.abstract | Candida species are important pathogens causing nosocomial infections and the prob-lem of antifungal drugs resistance in clinical is also a serious problem. At present, we do not have many choices for antifungal agents to use so it is necessary and urgent to develop new antifungal drugs. In this study, we explored BMVC-12C-P can specifically enter the mito-chondria to cause the mitochondrial dysfunction and we also confirmed the mitochondria is the main target of BMVC-12C-P. Therefore, BMVC-12C-P was used as a tool and com-bined with C. albicans mutant library to investigate the biological effects of mitochondrial dysfunction induced by BMVC-12C-P. We had established the optimal screening model to screen and selected the candidates from mutant library of transcription factors and kinases. Because we are interested in the relationship between mitochondria and pathogenic mecha-nisms, candidates are classified according to the pathogenic mechanisms involved. As a re-sult, the pathogenic mechanism of candidates involved in the formation of hyphae was most, so we selected the regulation of hyphae to explore. We confirmed BMVC-12C-P can inhibit the formation of hyphae and biofilm. We also found the ability of hyphae switch was sup-pressed which correlated with the reduced mRNA expressions of genes involved in hyphal formation (EFG1、BRG1、CPH1、CPH2 and TEC1) and not through Hog1MAPK pathway to regulate them. In this way, we hope to further understand the mechanism of BMVC-12C-P on the regulation of hyphae and can be used as an important cornerstone for the development of new antifungal drugs in the future. Moreover, we also found that BMVC-12C-P displayed the strongest antifungal activities to against Candida species and even Fluconazole-resistant clinical isolates of Candida species that indicated BMVC-12C-P can be used as a highly potential antifungal agent. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T07:10:40Z (GMT). No. of bitstreams: 1 ntu-108-D03B22007-1.pdf: 5848122 bytes, checksum: a62980d74c8e719cfd43b13c00ad6ee0 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 致謝 I
摘要 II Abstract III 目錄 IV 圖目錄 IX 表目錄 XI 第一章 文獻探討 1 1.1白色念珠菌 1 1.2白色念珠菌致病能力 1 1.2.1菌絲與侵入性過程 2 1.2.2菌絲型態調控機制 2 1.2.2.1 Protein kinase A signaling (PKA) 2 1.2.2.2 Mitogen-activated protein kinase (MAPK) 5 1.2.2.3 Chromatin modification 5 1.2.2.3.1 組蛋白乙醯化酶 (histone acetyltransferase，HAT) 5 1.2.2.3.2 组蛋白去乙醯化酶 (histone deacetylase，HDAC) 6 1.2.3菌絲與生物膜之關係 6 1.3臨床抗真菌劑使用及缺點 8 1.3.1抗真菌藥物種類 8 1.3.2抗真菌藥物的缺點 8 1.4 白色念珠菌粒線體 9 1.4.1粒線體調控菌絲生成 10 1.4.2粒線體調控抗藥性能力 10 1.4.2.1 藥物輸出幫浦的活性 10 1.4.2.2 細胞膜的穩定性 11 1.4.2.3 細胞壁的完整性 11 1.5 BMVC-12C-P 11 第二章 研究動機 13 第三章 材料與方法 15 3.1 菌種來源與保存 15 3.1.1菌種來源 15 3.2 藥品與儀器 15 3.2.1白色念珠菌培養試劑 15 3.2.2藥品 16 3.2.3儀器 17 3.3 實驗方法 18 3.3.1 BMVC-12C-P原液配製 18 3.3.2以雷射共軛焦顯微鏡觀察BMVC-12C-P於白色念珠菌位置 18 3.3.3以雷射共軛焦顯微鏡觀察白色念珠菌粒線體之結構 18 3.3.4建立rho-之突變細胞 19 3.3.5藥物毒殺效果測試 19 3.3.6 Q-PCR定量分析 19 3.3.7白色念珠菌突變菌株庫培養 19 3.3.8建立篩選白色念珠菌突變菌株庫方式 20 3.3.9誘發菌絲型態觀察 20 3.3.10 mRNA表達分析 21 3.3.10.1 RNA萃取 (RNA extraction) 21 3.3.10.2反轉錄 (Reverse Transcription, RT) 21 3.3.10.3 Q-PCR定量分析 22 3.3.11西方墨點法 (Western bloting) 22 3.3.12粒線體活性分析 (MTT assay) 24 3.3.13生物膜形成能力 24 3.3.14 BMVC-12C-P對於標準念珠菌菌株的藥物感受性測試 25 3.3.15 統計分析 25 第四章 結果 26 4.1 BMVC-12C-P對白色念珠菌粒線體之影響 26 4.1.1 BMVC-12C-P作用於白色念珠菌之位置 26 4.1.2 BMVC-12C-P破壞白色念珠菌粒線體結構 26 4.1.3粒線體為BMVC-12C-P之主要殺菌作用標的 26 4.1.4 BMVC-12C-P影響粒線體複製能力 27 4.2 BMVC-12C-P的作用機轉探討 (Mechanism of Action，MOA) 28 4.2.1以突變菌株庫為工具探討BMVC-12C-P因應粒線體失能而受到影響 (活化或抑制) 的分子機轉 (包括訊息傳遞路徑及轉錄因子)。 28 4.2.1.1 BMVC-12C-P對於突變菌株庫之標準株的藥物感受性測試 28 4.2.1.2 建立以BMVC-12C-P篩選白色念珠菌突變菌株庫之適當方式 29 4.2.1.3 以BMVC-12C-P篩選白色念珠菌轉錄因子突變菌株庫 30 4.2.1.4 文獻探討以及分析候選基因的致病能力歸類 30 4.2.2 BMVC-12C-P造成粒線體失能與菌絲生成調控相關分子機轉 (包括訊息傳遞路徑及轉錄因子) 之探討。 31 4.2.2.1 BMVC-12C-P對菌絲的影響 31 4.2.2.2 BMVC-12C-P調控菌絲之Kinase cascade 32 4.2.2.3 BMVC-12C-P以乙醯化機制調控菌絲之角色 33 4.2.2.4 Anacardic acid對白色念珠菌之影響 34 4.2.2.4.1 Anacardic acid與BMVC-12C-P合併對白色念珠菌敏感性之影響 34 4.2.2.4.2 Anacardic acid與BMVC-12C-P合併對白色念珠菌粒線體功能之影響 35 4.2.2.4.3 Anacardic acid與BMVC-12C-P合併對白色念珠菌粒線體活性之影響 35 4.2.2.5 BMVC-12C-P對生物膜的影響 36 4.3 BMVC-12C-P對於標準念珠菌菌株的藥物感受性測試 36 第五章 討論 38 5.1 BMVC-12C-P造成粒線體失能的機制探討 38 5.2 BMVC-12C-P對菌絲的影響 39 5.3 BMVC-12C-P對Hog1MAPK路徑的影響 41 5.4 BMVC-12C-P是否利用甲基化進而調控菌絲 42 5.5 BMVC-12C-P具殺菌效果之抗真菌劑 42 第六章 結論 44 第七章 未來研究方向 45 7.1 BMVC-12C-P造成粒線體失能的機制探討 45 7.2探討BMVC-12C-P所造成的死亡路徑 45 7.3探討BMVC-12C-P抑制菌絲之路徑 46 7.4利用突變菌株庫發現所得探索菌絲的調控機制 46 7.5 BMVC-12C-P對白色念珠菌致病機制之功能性影響 47 7.6 BMVC-12C-P是否能夠透過乙醯化進而調控菌絲 48 7.7 BMVC-12C-P-β-環糊精的明膠海綿敷料 48 圖 50 表 79 附圖 82 參考文獻 93 | |
| dc.language.iso | zh-TW | |
| dc.title | BMVC-12C-P誘導白色念珠菌粒線體失能導致之生物效應及相關分子機制探討 | zh_TW |
| dc.title | Biological consequences and mechanistic mechanisms of mitochondrial dysfunction induced by BMVC-12C-P in Candida albicans | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.coadvisor | 林晉玄(Ching-Hsuan Lin) | |
| dc.contributor.oralexamcommittee | 張大釗(Ta-Chau Chang),陳宜君(Yee-Chun Chen),吳?承(Hsuan-Chen Wu) | |
| dc.subject.keyword | 白色念珠菌,粒線體,BMVC-12C-P,菌絲, | zh_TW |
| dc.subject.keyword | Candida albicans,mitochondria,BMVC-12C-P,hyphae, | en |
| dc.relation.page | 104 | |
| dc.identifier.doi | 10.6342/NTU201901707 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2019-07-22 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| dc.date.embargo-lift | 2300-01-01 | - |
| Appears in Collections: | 生化科技學系 | |
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