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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林晉玄(Ching-Hsuan Lin) | |
dc.contributor.author | Yu-Chia Chen | en |
dc.contributor.author | 陳昱嘉 | zh_TW |
dc.date.accessioned | 2021-06-17T01:35:44Z | - |
dc.date.available | 2020-08-14 | |
dc.date.copyright | 2017-08-14 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-08-01 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67518 | - |
dc.description.abstract | 念珠菌屬 (Candida species) 包括白色念珠菌 (Candida albicans) 及熱帶念珠菌 (Candida tropicalis) 皆屬於伺機性致病真菌,可以在不同的型態間轉換以適應變化的環境。其中 yeast-hyphae 的型態轉換對念珠菌的毒性、寄主免疫系統反應等形態特徵高度相關,也是念珠菌生物膜形成的重要步驟。生物膜是微生物在自然界中主要增生的方式,也是造成人體永久感染的主因之一。熱帶念珠菌是台灣僅次於白色念珠菌常見的念珠菌;儘管如此,對抗真菌藥物 fluconazole 耐受性菌株產生的速度卻比白色念珠菌快。因此本研究欲瞭解與熱帶念珠菌致病力高度相關的菌絲生成及生物膜形成的相關機制。過去研究顯示,白色念珠菌主要由包含 CaBcr1、 CaBrg1、 CaEfg1、 CaNdt80、 CaRob1與 CaTec1的六個轉錄因子組成生物膜生成控制網;除了 CaBcr1 之外的五個轉錄因子也參與菌絲生長的調控。因此,我們猜測熱帶念珠菌的六個同源轉錄因子在調控生物膜及菌絲生成同樣扮演重要角色。首先實驗構築熱帶念珠菌六個同源轉錄因子基因的突變株及互補株,觀察其生物膜及菌絲生成變化。六個基因突變株生物膜生成皆有缺失,顯示熱帶念珠菌六個同源基因皆能正向調控生物膜的形成;而除了 CtNdt80 抑制菌絲生成外,其餘五個轉錄因子皆促進菌絲生成。利用即時聚合酶鏈式反應分析六個基因在菌絲生成時的表現量,發現在最適菌絲生成條件下,只有 CtROB1 的表現量明顯上升;然而,與菌絲生成高度相關的基因 CtUME6 的表現量在熱帶念珠菌六個同源基因突變株中,皆顯著下降。為了探尋六個基因調控生物膜及菌絲生成的可能機制,我們將熱帶念珠菌六個同源基因分別送入白色念珠菌相對應的基因突變株內。在 Cabcr1、 Caefg1 或 Catec1 突變株分別表現 CtBCR1、 CtEFG1 或 CtTEC1,可以恢復生物膜及菌絲生成能力;而表現 CtBRG1 或 CtROB1 對 Cabrg1 或 Carob1 突變株的生物膜或菌絲生長能力沒有影響。CtNDT80 雖然無法回復 Candt80 突變株的生物膜生成缺失,但在 Spider 液態培養基刺激下,可觀察到菌絲的生長 [Candt80Δ(0%)、Candt80Δ::CtNDT801 (8.46%)];除此之外,在 Ctndt80 突變株中表現 CaNDT80,菌絲的生成比例反而會顯著下降 [ Ctndt80Δ(63.10%)、Ctndt80Δ::CaNDT801 (36.48%)]。根據上述實驗結果,本研究發現 C. tropicalis 及 C. albicans 的六個同源基因調控生物膜及菌絲生成具有保守性功能;然而,NDT80對菌絲的調控在兩物種間有所不同,暗示著菌種間演化出複雜的菌絲生成調控迴路,而使單一基因保守性功能無法彰顯。 | zh_TW |
dc.description.abstract | Candida albicans and Candida tropicalis are opportunistic fungi, which can undergo morphological transition to adapt different environments. The yeast-hyphae transition is considered to be highly related to Candida properties in virulence, and host-fungus interactions. In addition, the transition from yeast to hyphae is an important step for Candida biofilms, which are a major growth form in natural environments and a leading cause of persistent infections. C. tropicalis is not only the 2nd common commensal pathogen among Candida species in Taiwan, but also develops resistant strains against fluconazole faster than C. albicans. It has been known that regulatory network composed of CaBcr1, CaBrg1, CaEfg1, CaNdt80, CaRob1 and CaTec1 is involved in biofilm development in C. albicans. Except for CaBcr1, other five genes hold the ability to regulate filamentous gowth as well. In this study, we speculated the functions of six homologous genes in C. tropicalis are the same as those genes in C. albicans. Six gene mutants and complementary strains were constructed in C. tropicalis to observe the differences of hyphae and biofilm formation. Six mutant strains were deficient in biofilm development, which means they positively regulate biofilm formation in C. tropicalis. Besides, while CtBcr1, CtBrg1, CtEfg1, CtRob1 and CtTec1 promoted hyphae formation in C. tropicalis, CtNdt80 inhibited filamentation. RT-qPCR showed that only CtROB1 was highly expressed during filamentation, but CtUME6 expression level, which is one of determining factors for hyphae formation, was downregulated in all six mutant genes. To understand the possible mechanisms of six genes in regulating biofilm and filamentation in C. tropicalis, we transformed C. tropicalis homologous genes into C. albicans mutans strains, separately. Biofilm and filament ability could be recovered by expression of CtBCR1, CtEFG1 and CtTEC1 in Cabcr1, Caefg1 and Catec1 mutants; however, there were no differences in biofilm and hyphae development when expressing CtBRG1 or CtROB1 in Cabrg1 or Carob1 mutants. CtNdt80 could not recover biofilm deficiency of Candt80 mutant; nevertheless, Candt80 mutant would form hyphae when expressing CtNDT80 in Spider medium [Candt80Δ(0%)、Candt80Δ::CtNDT80 (8.46%)]. Besides, the percetange of filaments became lower when CaNDT80 expressed in Ctndt80 mutant [ Ctndt80Δ(63.10%)、Ctndt80Δ::CaNDT80 (36.48%)]. Taken together, our study demonstrates that each single transcriptional gene hold the conserved function in biofilm and in filamental growth; however, the filament regulatory circuit of Ndt80 is different in two species, implying that species may have evolved a sophisticated regulatory network for the control of hyphae formation, leading to contrain or varnish over a single gene’s function. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T01:35:44Z (GMT). No. of bitstreams: 1 ntu-106-R04b22064-1.pdf: 3196558 bytes, checksum: 018b545e4767c4ceec85fe934447b446 (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 誌謝 i
中文摘要 ii 英文摘要 iv 目錄 vi 圖目錄 ix 表目錄 x 前言 1 念珠菌 (Candida species) 與念珠菌菌血症 (Candidemia) 1 白色念珠菌 (C. albicans) 1 白色念珠菌菌絲型態轉換調控 2 白色念珠菌生物膜特性 4 白色念珠菌活體外 (in vitro) 的生物膜生成調控 5 熱帶念珠菌生物膜形成與菌絲生成調控 6 實驗目的 8 材料與方法 9 實驗藥品與培養基 9 DNA 聚合酶鏈式反應 (Polymerase chain reaction, PCR) 9 洋菜膠體電泳分析 (Agarose gel electrophoresis) 9 DNA 片段回收 10 DNA 接合(Ligation)反應 10 大腸桿菌菌株轉形作用(Transformation) 10 大腸桿菌質體 DNA 抽取 11 質體建構 11 構築表現 C. tropicalis 同源基因的 C. albicans轉形菌株 14 構築大量表現 C. tropicalis 基因 (Ptet – Ctgene) 之 C. albicans 菌株 16 構築 C. tropicalis 六個同源基因互補菌株 16 構築表現 C. albicans 同源基因 C. tropicalis 轉形菌株 17 刺激菌絲 (hyphae) 生長 17 活體外 (in vitro) 生物膜(Biofilm)生成 18 RNA 抽取 19 反轉錄聚合酶鏈式反應 (Reverse Transcription-PCR) 19 在菌絲生成條件下大量表現 C. tropicalis 基因 20 定量即時聚合酶鏈鎖反應 (Quantitative real-time polymerase chain reaction) 20 結果 21 壹、C. tropicalis 六個同源基因與生物膜及菌絲生成關聯性之探討 21 1. PCR 鑑定 Ctbcr1、 Ctbrg1、 Ctefg1、 Ctndt80、 Ctrob1、 Cttec1突變株及互補株 21 2. CtBCR1、 CtBRG1、 CtEFG1、 CtNDT80、 CtROB1、 CtTEC1六個轉錄因子正向調控生物膜生成。 21 3. CtBCR1、 CtBRG1、 CtEFG1、 CtROB1、 CtTEC1正向調控菌絲生長。然而,剔除CtNDT80 會導致 C. tropicalis 菌絲大量生成。 22 4. C. tropicalis 六個轉錄因子基因在serum 刺激的條件下, CtROB1表現量顯著上升。 23 5. C. tropicalis 六個同源基因突變株 CtUME6 表現量與野生株相比,明顯下降。 24 貳、測試 C. tropicalis 同源基因對 C. albicans 突變株生物膜與菌絲生成影響 25 1. PCR 鑑定C. tropicalis 六個同源基因送入 C. albicans 相對應突變株。 25 2. C. tropicalis BCR1、EFG1、TEC1 能恢復 C. albicans bcr1、efg1、tec1 突變株缺失的生物膜生成能力。 26 3. 在 serum 刺激下,表現 CtEFG1、 CtTEC1能增加 Caefg1、 Catec1 突變株的菌絲生成比例。 27 4. 以 Spider 液態培養基測試發現 CtNDT80 能部分回復Candt80 突變株的菌絲生成能力。 28 參、測試 C. albicans 同源基因對 C. tropicalis 突變株生物膜與菌絲生成影響 29 1. PCR 鑑定CaNDT80基因送入 Ctndt80突變株。 29 2. CtNdt80 正向調控 C. albicans 生物膜及菌絲生成 29 討論 31 未來研究方向 37 圖表 38 參考文獻 59 附錄一 70 附錄二 75 | |
dc.language.iso | zh-TW | |
dc.title | 熱帶念珠菌六個同源基因對菌絲及生物膜生成的影響探討 | zh_TW |
dc.title | Investigation of six conserved transcriptional factors on the regulation of hyphal formation and biofilm development in Candida tropicalis | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 呂俊毅(Jun-Yi Leu),羅秀容(Hsiu-Jung Lo),薛雁冰(Yen-Ping Hsueh),陳穎練(Ying-Lien Chen) | |
dc.subject.keyword | 熱帶念珠菌,生物膜形成,yeast-hyphae 型態轉換,轉錄因子,白色念珠菌, | zh_TW |
dc.subject.keyword | Candida tropicalis,biofilm,yeast-hyphae transition,transcription factor,Candida albicans, | en |
dc.relation.page | 76 | |
dc.identifier.doi | 10.6342/NTU201701794 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-08-02 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
顯示於系所單位: | 生化科技學系 |
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