白色念珠菌是人體的共生性真菌，然而它同時也是一種伺機性感染病原菌。在免疫缺失的患者身上，它會造成各種不同感染包括致命的系統性感染。再者，白色念珠菌在近年的院內感染報告中感染率也是名列前茅。在疾病進程中，白色念珠菌時常需要面對來自人體先天性免疫系統及抗真菌藥物治療的多種壓力。舉例來說，先天免疫系統中的巨噬細胞及嗜中性白血球等會對白色念珠菌進行吞噬作用，並產生活性氧化物質(ROS)來殺死真菌細胞。另外，許多的抗真菌藥物的作用機制已證實包含誘導產生活性氧化物質進而達成殺菌。因此，抗氧化能力對於白色念珠菌而言，可說是其生存及致病的關鍵。在這篇研究中，我們主要探討在白色念珠菌中參與胺基酸生合成的一個酵素Aro1的功能。當ARO1的表現受到抑制時，我們發現白色念珠菌會提高在面對氧化壓力時的生存比率。本研究結果連結細胞的生理代謝與其抗氧化能力之間的關係，並進一步提供對於白色念珠菌的受壓反應的新觀點。

Candida albicans is a commensal fungus of humans. However, it is also an opportunistic pathogen and can cause severe life-threatening systemic infection in immunosuppressed patients. Moreover, C. albicans is one of the most prevalent causes of nosocomial infection. In the disease progress, C. albicans commonly faces diverse challenges such as human innate immunity and antifungal therapy. For example, both macrophages and polymorphonuclear leukocytes (PMNs) can engulf the pathogen and generate reactive oxygen species (ROS) to destroy the fungal cells. Moreover, induction of ROS is a mechanism of action for many antifungal drugs. Therefore, antioxidative activity is critical for C. albicans survival and pathogenesis. In this work, we studied the functions of C. albicans Aro1, an enzyme involves in amino acid biosynthesis. A knockdown of the ARO1 gene affected cell susceptibility to oxidative stress. Our findings correlate cell metabolism with antioxidative activity, and provide a new insight for cell stress response in C. albicans.

Abstract I
中文摘要 II
Contents III
List of Tables V
List of Figures VI
1. Introduction 1
1.1 Candida albicans and its threat to human health 1
1.2 Virulence and pathogenicity of Candida albicans 2
1.3 Antioxidants and their role in C. albicans virulence 3
1.3.1 Superoxide dismutases (Sods) 3
1.3.2 Catalases 4
1.3.3 The glutathione system 5
1.4 Nutrient availability and stress response 7
1.4.1 Nutrient availability affects virulence factors and fitness attributes in C. albicans 7
1.4.2 Possible connections between amino acid starvation, reactive oxygen species (ROS) and C. albicans virulence 8
(1) C. albicans encounters phagocytes. 8
(2) C. albicans encounters ROS-generating antifungal drugs. 9
1.5 The shikimate pathway, aromatic amino acids and the pentafunctional AROM enzyme Aro1 10
1.6 Aim of this study 10
2. Materials and Methods 12
2.1 Yeast strains and growth media 12
2.2 Growth conditions 12
2.3 C. albicans DNA microarray analysis 12
2.4 RNA preparation and reverse transcription (RT) real-time quantitative PCR (qPCR) 13
2.5 Flow cytometry 14
2.5.2 Dichlorodihydrofluorescein diacetate (H2DCFDA) staining 14
2.6 Cell susceptibility assays 15
2.7 Total protein extraction and quantification 15
2.8 Total antioxidative capacity toward H2O2 15
2.9 Catalase enzyme activity assay 16
2.10 Sod activity assay by a colorimetric method 16
2.11 Spot assay 17
2.12 Glutathione assay by a colorimetric method 17
2.12.1 Total glutathione (GSH) content 17
2.12.2 The oxidized form of glutathione (GSSG) 18
2.13 Glutathione peroxidase activity assay by a colorimetric method 18
2.14 Glutathione S-transferase activity assay by a colorimetric method 19
2.15 Lipid peroxidation assay 19
2.16 Scanning Electron Microscopy (SEM) 19
2.17 Assay for C. albicans killing by macrophage 20
2.18 Statistical analysis 21
3. Results 22
3.1 DNA microarray analysis revealed the potential role of Aro1 in regulation of oxidative stress response. 22
3.2 Knockdown of ARO1 led to cell resistance to oxidative stress 23
3.3 The ARO1-knockdown strain had a lower ROS level and exhibited a higher total antioxidant capacity 23
3.4 ARO1 influenced multiple antioxidative mechanisms in C. albicans 24
3.4.1 Antioxidants to detoxify hydrogen peroxide 24
3.4.2 Antioxidants to detoxify superoxide 27
3.5 Measurement of the oxidative damage 28
3.6 The ARO1-knockdown strain showed a higher ability to survive in macrophages 28
4. Discussion 29
5.Future Perspectives 32
6.References 33
List of Tables
Table 1. Strains used in this study. 42
Table 2. Oligonucleotides used in this study. 43
Table 3. Fold changes of antioxidant related genes from DNA microarray analysis. 44
Abstract I
中文摘要 II
Contents III
List of Tables V
List of Figures VI
1. Introduction 1
1.1 Candida albicans and its threat to human health 1
1.2 Virulence and pathogenicity of Candida albicans 2
1.3 Antioxidants and their role in C. albicans virulence 3
1.3.1 Superoxide dismutases (Sods) 3
1.3.2 Catalases 4
1.3.3 The glutathione system 5
1.4 Nutrient availability and stress response 7
1.4.1 Nutrient availability affects virulence factors and fitness attributes in C. albicans 7
1.4.2 Possible connections between amino acid starvation, reactive oxygen species (ROS) and C. albicans virulence 8
(1) C. albicans encounters phagocytes. 8
(2) C. albicans encounters ROS-generating antifungal drugs. 9
1.5 The shikimate pathway, aromatic amino acids and the pentafunctional AROM enzyme Aro1 10
1.6 Aim of this study 10
2. Materials and Methods 12
2.1 Yeast strains and growth media 12
2.2 Growth conditions 12
2.3 C. albicans DNA microarray analysis 12
2.4 RNA preparation and reverse transcription (RT) real-time quantitative PCR (qPCR) 13
2.5 Flow cytometry 14
2.5.2 Dichlorodihydrofluorescein diacetate (H2DCFDA) staining 14
2.6 Cell susceptibility assays 15
2.7 Total protein extraction and quantification 15
2.8 Total antioxidative capacity toward H2O2 15
2.9 Catalase enzyme activity assay 16
2.10 Sod activity assay by a colorimetric method 16
2.11 Spot assay 17
2.12 Glutathione assay by a colorimetric method 17
2.12.1 Total glutathione (GSH) content 17
2.12.2 The oxidized form of glutathione (GSSG) 18
2.13 Glutathione peroxidase activity assay by a colorimetric method 18
2.14 Glutathione S-transferase activity assay by a colorimetric method 19
2.15 Lipid peroxidation assay 19
2.16 Scanning Electron Microscopy (SEM) 19
2.17 Assay for C. albicans killing by macrophage 20
2.18 Statistical analysis 21
3. Results 22
3.1 DNA microarray analysis revealed the potential role of Aro1 in regulation of oxidative stress response. 22
3.2 Knockdown of ARO1 led to cell resistance to oxidative stress 23
3.3 The ARO1-knockdown strain had a lower ROS level and exhibited a higher total antioxidant capacity 23
3.4 ARO1 influenced multiple antioxidative mechanisms in C. albicans 24
3.4.1 Antioxidants to detoxify hydrogen peroxide 24
3.4.2 Antioxidants to detoxify superoxide 27
3.5 Measurement of the oxidative damage 28
3.6 The ARO1-knockdown strain showed a higher ability to survive in macrophages 28
4. Discussion 29
5.Future Perspectives 32
6.References 33






List of Tables
Table 1. Strains used in this study. 42
Table 2. Oligonucleotides used in this study. 43
Table 3. Fold changes of antioxidant related genes from DNA microarray analysis. 44
















List of Figures
Fig 1. The ARO1-knockdown strain exhibits higher viability with the treatment of H2O2. 45
Fig 2. The ARO1-knockdown strain demonstrated lower intracellular ROS level and higher antioxidative capacity with and without H2O2 treatment. 46
Fig 3. The ARO1 knockdown strain demonstrated higher catalase gene expression and enzyme activity. 47
Fig 4. The ARO1-knockdown strain showed higher expression level in glutathione-related gene expression level. 48
Fig 5. The ARO1-knockdown strain exhibits a difference in the glutathione content compared to the wild type strain. 49
Fig 6. The ARO1-knockdown strain had lower glutathione content and GSH/GSSG ratio compared to the wild type strain. 50
Fig 7. The ARO1-knockdown strain exhibited lower glutathione peroxidase and glutathione S-transferase activity. 51
Fig 8. The ARO1-knockdown strain demonstrated higher superoxide dismutase gene expression and enzyme activity. 52
Fig 9. The ARO1-knockdown strain demonstrated higher superoxide dismutase gene expression and enzyme activity. 53
Fig 10. The ARO1-knockdown strain demonstrated lower oxidative damage and higher viability in macrophage killing assay. 54

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