白色念珠菌是一伺機性致病菌,共生在人體中。它對健康人體是無害的，但對免疫低下的病患經常造成嚴重生命威脅。微量元素-銅(Cu)可維持生命體的基本生理機能，例如酵素活性。雖然銅對於細胞生長很重要，但是當過量銅存在便會對細胞造成毒性。此外，銅的恆定與人類疾病也有關。舉例來說，當人體銅的調節失衡，會造成緬克斯症候群和威爾森氏症。最近，許多研究指出銅也與真菌致病力有關。宿主可藉由累積銅在吞噬體中的機制來殺死微生物以抵抗致病菌入侵。由此可見，對細胞來說，銅的調控是非常重要的。為了解白色念珠菌對銅的調控以及銅與其致病性的關係，本研究以一個(尚未被證實)轉錄因子Cup2作為研究對象。研究發現，Cup2基因剔除的菌株在高銅環境(YPD培養基加銅)生長較遲緩，然而，在正常(YPD培養基)與低銅(YPD加銅螯合劑)中生長並無太大的影響。另外我們發現Cup2在高銅中是轉錄活化因子，並且調控銅運送以及去毒性相關的基因表現。當細胞沒有此轉錄因子時，細胞內的銅以及活性氧分子會大量增加。在我們的實驗中發現，Cup2並沒有直接影響到白色念珠菌的致病力。總而言之，本篇研究發現白色念珠菌Cup2 對於真菌體內銅的調節是有關聯的。

Abstract I
中文摘要 II
Table of Contents III
1. Introduction 1
1.1 Candida albicans and its significance in healthcare 1
1.2 Metals in biological processes 2
1.2.1 Metal homeostasis 2
1.2.2 Interaction of metals and microorganisms 3
1.2.3 The significance of copper in biology 3
1.3 Copper homeostasis: uptake and distribution 4
1.4 Copper tolerance and virulence 6
1.5 The aim of this study 7
2. Materials and Methods 8
2.1 C. albicans strains and growth conditions 8
2.2 Strain construction 8
2.2.1 CaCUP2 gene deletion and reconstitution 8
2.2.2 CaCUP2 one hybrid 10
2.2.3 CaCUP2 promoter analysis 10
2.2.4 Construction of green fluorescent protein (GFP)-tagged Cup2 11
2.3 Genomic DNA isolation 11
2.4 Southern blot analysis 12
2.5 RNA isolation, RT-PCR 13
2.6 Spot assay 14
2.7 Fluorescence microscopy 14
2.8 β-galactosidase assays 14
2.9 Measurement of intracellular copper 15
2.10 Measurement of intracellular ROS 16
2.11 Measurement of superoxide dismutase (SOD) activity 17
2.12 Virulence assay 17
2.13 Statistical analysis 18
3. Results 19
3.1 C. albicans Cup2 is similar to S. cerevisiae Cup2 (a.k.a. Ace1) 19
3.2 C. albicans CUP2-deleted mutants are defective in growth at a high copper condition 19
3.3 C. albicans Cup2 functions as a transcription activator in response to copper availability 20
3.4 C. albicans Cup2 activates copper homeostasis–related genes expression at a high copper condition 21
3.5 Detection of Cup2 binding on copper homeostasis-related gene promoter 22
3.6 C. albicans Cup2 location in response to copper. 22
3.7 Intracellular copper accumulation in CUP2-deleted mutants in a high copper condition. 23
3.8 Intracellular ROS levels CUP2-deleted mutants in a high copper condition 23
3.9 The role of Cup2 in C. albicans Virulence 24
4. Discussion 25
5. References 28
6. Appendix 59

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