2. MATERIALS AND METHODS

CONTENTS

1. INTRODUCTION

1.1 The Candida genus 1.2 Candida glabrata

1 4 1.2.1 Candida glabrata epidemiology

1.2.2 Vulvovaginal candidiasis 1.2.3 Candida glabrata virulence

5 6 8

1.3 Antifungal drugs 10

1.3.1 Polyenes 1.3.2 Flucytosine 1.3.3 Echinocandins 1.3.4 Azoles

1.3.5 Candida glabrata azole resistance

11 12 12 13 14

1.4 Antimicrobial peptides 16

1.4.1 Human antimicrobial peptides

1.4.2 Antimicrobial peptide mode of action 1.4.3 Antimicrobial peptide role in immunity

1.4.4 Advantages and disavantages in the use of antimicrobial peptides 1.4.5 Hepcidins

17 20 22 24 25

1.5 Aims of the study 27

2. MATERIALS AND METHODS

2.1 Yeast strains and growth media 2.2 Antimicrobial peptide and antifungals

2.3 Killing kinetics of Hep-20 in sodium phosphate buffer (SPB)

2.4 Evaluation of antifungal activity of Hep-20 in combination with antifungal drugs in SPB

2.5 Fungicidal activity of Hep-20 in an artificial vaginal fluid simulant (VFS) 2.6 Evaluation of antifungal activity of Hep-20 in combination with antifungal

drugs in VFS

2.7 Human vaginal fluid (HVF) extraction

28 29 29 30

30 32

32

2.8 Fungicidal activity of Hep-20 in HVF 2.9 Haemolytic assay

2.10 Peripheral blood mononuclear cell (PBMC) isolation and A549 culturing 2.11 Propidium iodide staining of PBMCs and A549 cell line incubated with

Hep-20

2.12 XTT reduction assay on PBMCs and A549 cell line incubated with Hep-20 2.13 Evaluation of Hep-20 degradation in HVF

2.14 Statistical analysis

34 34 35 35

36 37 38

3. RESULTS

3.1 Antifungal Activity of Hepcidin 20 in sodium phosphate buffer (SPB) 3.2 Killing kinetics of Hep-20 in SPB

3.3 Activity of Hep-20 in combination with antifungal drugs in SPB

3.4 Evaluation of antifungal activity of Hep-20 in combination with fluconazole in SPB

3.5 Fungicidal activity of Hep-20 in an artificial vaginal fluid stimulant (VFS) 3.6 Evaluation of antifungal activity of Hep-20 in combination with common

antifungal drugs in VFS

3.7 Antifungal activity of Hep-20 in human vaginal fluid (HVF)

39 41 43 44

45 47

48 3.8 Cytotoxicity studies

3.8.1 Hemolytic assay 3.8.2 XTT reduction assay 3.8.3 Propidium iodide staining

3.9 Evaluation of Hep-20 degradation in HVF

54 54 55 57 59

4. DISCUSSION

5. REFERENCES