Title Page
Contents
ABSTRACT 10
PART Ⅰ. Candidacidal and antibiofilm activity of PS1-3 peptide against drug-resistant Candida albicans on contact lenses 12
ABSTRACT 13
Ⅰ-1. INTRODUCTION 14
Ⅰ-2. MATERIALS AND METHODS 17
Ⅰ-2-1. Materials 17
Ⅰ-2-2. Synthesis of PS1 analog peptides 17
Ⅰ-2-3. Antifungicidal activity 18
Ⅰ-2-4. FAM-PS1-1 and FAM-PS1-3 peptide binding affinity 19
Ⅰ-2-5. Assessment of time kill-kinetic activity using fluorescence assisted cell sorting (FACS) analysis and fluorescence microscopy 19
Ⅰ-2-6. Analysis of morphological changes using SEM 20
Ⅰ-2-7. Biofilm inhibition assay 21
Ⅰ-2-8. Biofilm reduction assay 22
Ⅰ-2-9. CLSM analysis 23
Ⅰ-2-10. Biofilm activity assays on coverslips 23
Ⅰ-2-11. Biofilm activity assay on soft CLs (SCLs) 24
Ⅰ-3. RESULTS 26
Ⅰ-3-1. Candidacidal activity of PS1 analog peptides 26
Ⅰ-3-2. 5'-Fluorescein phosphoramidite (FAM)-labeled Peptide binding activity 28
Ⅰ-3-3. Time kill-kinetic activity 30
Ⅰ-3-4. Analysis of morphological changes through scanning electron microscopy (SEM) 32
Ⅰ-3-5. Antibiofilm activity of PS1 analog peptides 34
Ⅰ-3-6. Ex-vivo antibiofilm activities of PS1-1 and PS1-3 peptides 37
Ⅰ-3-7. Anti-biofilm activity on the CLs 40
Ⅰ-4. DISCUSSION 42
Ⅰ-5. CONCLUSIONS 46
Ⅰ-6. REFERENCES 47
PART Ⅱ. Antifungal and anti-inflammatory activities of PS1-2 peptide against fluconazole-resistant Candida albicans 51
ABSTRACT 52
Ⅱ-1. INTRODUCTION 53
Ⅱ-2. MATERIALS AND METHODS 57
Ⅱ-2-1. Materials 57
Ⅱ-2-2. Synthesis of the PS1 peptides 57
Ⅱ-2-3. Anti-fungicidal activity 58
Ⅱ-2-4. Binding affinity of FAM-labeled peptide 59
Ⅱ-2-5. Time-killing kinetic assay 59
Ⅱ-2-6. Drug-resistance assay 60
Ⅱ-2-7. Trypsin hydrolysis assay 60
Ⅱ-2-8. Anti-inflammatory effects 61
Ⅱ-2-9. In vivo study 62
Ⅱ-2-10. Statistical data analysis 64
Ⅱ-3. RESULTS AND DISCUSSION 65
Ⅱ-3-1. In vitro antifungal activity of PS1-2, PS1-5, and PS1-6 against drug-resistant C. albicans 65
Ⅱ-3-2. Fungicidal kinetics of PS1-2, PS1-5, and PS1-6 68
Ⅱ-3-3. Binding affinity of FAM-labeled PS1-2 peptide to C. albicans cells 71
Ⅱ-3-4. Peptides did not induce the drug-resistance 73
Ⅱ-3-5. Inhibition of LPS binding to RAW 264.7 cells 75
Ⅱ-3-6. In vitro anti-inflammatory activity of PS1-2 peptide in co-culture of RAW 264.7 and C. albicans 77
Ⅱ-3-7. In vivo anti-inflammatory effects of PS1-2 in acute lung injury 80
Ⅱ-4. CONCLUSIONS 84
Ⅱ-5. REFERENCES 85
KOREAN ABSTRACT 91
CURRICULUM VITAE 95
LIST of PUBLICATIONS 96
PART Ⅰ. Candidacidal and antibiofilm activity of PS1-3 peptide against drug-resistant Candida albicans on contact lenses 27
Table 1. Sequence, molecular weight, and Candidacidal activity of PS1-1, PS1-3, and Fluconazole. 27
PART Ⅱ. Antifungal and anti-inflammatory activities of PS-1 peptide against fluconazole-resistant Candida albicans 67
Table 1. Candidacidal activity of PS1-2, PS1-5, PS1-6 peptide, and fluconazole against Candida albicans CCARM 14007 (fluconazole-resistant... 67
PART Ⅰ. Candidacidal and antibiofilm activity of PS1-3 peptide against drug-resistant Candida albicans on contact lenses 29
Figure 1. Binding affinity effects of FAM-labeled PS1-1 and PS1-3 peptides against C. albicans. PS1-3 peptide structure was modeled using the PEP-... 29
Figure 2. Inhibitory effects of PS1-1 and PS1-3 peptides against C. albicans cells. Time-kill kinetics of PS1-1 and PS1-3 peptides with C.... 31
Figure 3. Morphological characterization of cell surface of the peptide-treated C. albicans CCARM 14007. After incubation of C. albicans CCARM... 33
Figure 4. Anti-biofilm activity of PS1-1 and PS1-3. Biofilm inhibition (A) and reduction (B) in C. albicans CCARM 14007 cells strain by treating PS1-... 36
Figure 5. Image analysis of C. albicans CCARM 14007 strain using confocal laser scanning microscopy (CLSM). The biofilm inhibition and reduction on C.... 38
Figure 6. Image analysis of C. albicans CCARM 14007 strain using scanning electron microscopy (SEM). The biofilm inhibition and reduction on C.... 39
Figure 7. The visual effects of PS1-3 peptide against biofilm on the soft contact lenses (SCL). Biofilm inhibition (A) and reduction (B) in C.... 41
PART Ⅱ. Antifungal and anti-inflammatory activities of PS1-2 peptide against fluconazole-resistant Candida albicans 70
Figure 1. Time-kill kinetic activity of PS1-2, PS1-5, PS1-6, and fluconazole against drug-resistant C. albicans. (a and b) After incubation of each peptide... 70
Figure 2. Binding affinity of 5-carboxyfluorescein (FAM)-labeled PS1-2 peptide. After incubation of FAM-labeled PS1-2 peptide for 1 min, C. albicans... 72
Figure 3. Induction of drug-resistance of peptides and fluconazole in C. albicans CCARM 14007 cells. Development of resistance in C. albicans cells... 74
Figure 4. Protease protection effects of PS1-2, PS1-5, and PS1-6 peptide. After treatment of 1 μg/mL trypsin to peptide solutions of MFCs at 37 ℃ for... 76
Figure 5. Anti-inflammatory effects of fluconazole and PS1-2. (a) Pre-cultured RAW 264.7 cells were stimulated with C. albicans CCARM 14007... 79
Figure 6. In vivo anti-inflammatory effects of peptides in lung injury. C. albicans CCARM 14007 (1 x 10⁵ cells) was intratracheally instilled in mice... 82