Molecular characterisation of the effects of classical chemotherapy on Candida albicans and systemic candidiasis

Abstract

of C. albicans 1.3. Chemotherapy 1.3.1. Different classes of chemotherapy 1.3.2. Inhibition of ribonucleotide reductase (RNR) by HU 1.3.3. RNR in C. albicans 1.3.4. Side effects of chemotherapy on the human host 1.3.4.1. Effects of chemotherapy on the epithelial barrier 1.3.4.2. Effects of chemotherapy on innate immunity 1.3.4.3. Mutagenic effects of chemotherapeutic drugs in mammalian cells and C. albicans 1.3.4.4. Stress-induced mutagenesis 1.3.4.5. Effects of chemotherapeutic drugs on the phenotype of C. albicans 1.4. Summary and aims 2. Materials and Methods 2.1. Dose-response relationship of C. albicans and chemotherapeutic drugs 2.1.2. Overnight culture 2.1.2. Growth rate measurement 2.1.3. Viability measurement 2.1.4. Morphological changes 2.2. Generation of genomic changes in C. albicans by acute exposure to chemotherapeutic drugs 2.2.1. Analysis of ploidy changes in C. albicans using flow cytometry 2.2.2. Karyotyping of C. albicans by quantitative PCR (qPCR) 2.3. Generation of isogenic strains to assess requirement of trisomy 2 for acquired HU resistance 2.3.1. Spotting assays for screening HU resistance 2.4. Adaptive evolution experiments in C. albicans 2.5. Effect of HU treatment on C. albicans colonising the adult murine GI tract 2.6. Effect of HU treatment on the outcome of systemic candidiasis in the adult mouse i ii v vi vii ii 2.7. Generation of knockout mutants for RNR genes located on Chromosome 2 2.8. Overexpression of individual RNR genes in C. albicans 2.9. Quantification of RNR gene expression levels by quantitative PCR 3. Results 3.1. HU induces immunosuppression and increased mortality during systemic C. albicans infection 3.2. Effect of chemotherapeutic drugs on the growth, morphology and viability of C. albicans 3.3. In vitro and in vivo induction of aneuploidy in C. albicans by chemotherapeutic drugs 3.4. Adaptive advantage of trisomy 2 under HU-induced stress 3.5. Role of RNR genes in conferring HU resistance 3.5.1. Requirement of individual RNR genes on Chromosome 2 for trisomy 2-mediated HU resistance 3.5.2. Sufficiency of individual RNR genes on Chromosome 2 for trisomy 2-mediated HU resistance 3.6. Concomitant resistance of trisomic 2 strains to caspofungin in vitro and in vivo 4. Discussion 5. Conclusion 6. References 7. Appendix 8. Author's publications 9. Posters, awards, invited talks i Acknowledgements "I can no other answer make but thanks, and thanks; and ever thanks."-Twelfth Night, William Shakespeare It has been a long journey, with sunshine and rain in equal measure. I am glad I made the choice to take this path, in good part because I have met many wonderful people along the way, who helped me at many points on the road, and without whom I would not have made it. I would like to express my gratitude to: The past and present members of the NP Lab: Fabien Cottier, Giang Le, XiaoHui Sem, Alrina Tan, Gloria Tso, and José Reales. Thank you for the patience and generosity you showed every time I landed on your (metaphorical) doorstep (at occasionally less than opportune momen...