published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

This thesis is an investigation of the exopolysaccharides produced by Lactococcus lactis subsp. Cremoris JFR1 and the hyphal cell wall glucans of Candida albicans. L. lactis is an important organism in the dairy industry for the production of fermented dairy products and the exopolysaccharides have been shown to add textural qualities to the foods. C. albicans is a fungal pathogen responsible for the common yeast infection and many post-surgery complications in hospitals and can grow in both the yeast and hyphae form. Through a series of GC-MS, NMR and chemical degradation experiments three unique polysaccharides are discovered in the L. lactis samples giving a molecular basis to the textural qualities provided by these molecules. Additionally, several unique structural features are discovered on the C. albicans hyphal glucan providing possible explanations for the differing immune responses elicited by the hyphae form of the fungus.

Thesis (M.Tech.: Homoeopathy) - Dept. of Homoeopathy, Durban Institute of Technology, 2004 xxvii, 155 leaves / The aim of this in vitro study was to determine the effect of Commiphora molmol tincture prepared in 86% v/v ethanol, Hydrastis canadensis tincture prepared in 62% v/v ethanol and Warburgia salutaris tincture prepared in 62% v/v ethanol against Candida albicans (C. albicans) with 62% v/v ethanol, 86% v/v ethanol and fluconazole as the control agents, and to determine the Minimum Inhibitory Concentration/s (MIC/s) of the effective tincture/s using the disc diffusion assay.

Fungal pathogens are a leading cause of human mortality, at least in part due to their ability to thwart therapeutic regimens by rapidly evolving resistance to antifungal drugs, and as a consequence of the increasing frequency of immunocompromised individuals most vulnerable to fungal infection. Candida albicans, the leading human fungal pathogen, has evolved an elegant repertoire of mechanisms to survive the cellular stress exerted by the azoles, which are the most widely deployed class of antifungals and inhibit ergosterol biosynthesis, inducing cell membrane stress. The evolution and maintenance of diverse resistance phenotypes is contingent upon cellular stress response circuitry, including that regulated by the molecular chaperone Hsp90 and its client protein calcineurin. My doctoral research focuses on three aspects of the role of fungal stress responses in regulation of azole resistance. First, I establish a novel role for nutrients and nutrient signalling in azole resistance of C. albicans and the model yeast Saccharomyces cerevisiae. Compromising a global regulator that couples growth to environmental cues, Tor kinase, provides a powerful strategy to abrogate fungal drug resistance with broad therapeutic potential. Second, I implicate the molecular chaperone Hsp90 as a key regulator of biofilm drug resistance in C. albicans. Compromising Hsp90 function transforms the azoles from ineffective to highly efficacious at eradicating biofilms in vitro and in vivo. Depletion of Hsp90 leads to reduction of client proteins’ calcineurin and Mkc1 in planktonic but not biofilm conditions, suggesting that Hsp90 regulates drug resistance through different mechanisms in these distinct cellular states. Third, I establish that inhibition of lysine deacetylases (KDACs) blocks the emergence and maintenance of Hsp90-dependent azole resistance in C. albicans and S. cerevisiae. S. cerevisiae Hsp90 is acetylated on lysine 27 and 270, and key KDACs for drug resistance are Hda1 and Rpd3. Compromising KDACs alters stability and function of Hsp90 client proteins, including drug resistance regulator calcineurin. Overall, this work provides novel insight into the mechanisms by which cellular stress responses mediate azole resistance, and establishes acetylation as a novel mechanism of post-translational control of Hsp90 function in fungi; ultimately, this unveils numerous targets that could be exploited for therapeutic benefit in the treatment of fungal disease.

Fungal pathogens are a leading cause of human mortality, at least in part due to their ability to thwart therapeutic regimens by rapidly evolving resistance to antifungal drugs, and as a consequence of the increasing frequency of immunocompromised individuals most vulnerable to fungal infection. Candida albicans, the leading human fungal pathogen, has evolved an elegant repertoire of mechanisms to survive the cellular stress exerted by the azoles, which are the most widely deployed class of antifungals and inhibit ergosterol biosynthesis, inducing cell membrane stress. The evolution and maintenance of diverse resistance phenotypes is contingent upon cellular stress response circuitry, including that regulated by the molecular chaperone Hsp90 and its client protein calcineurin. My doctoral research focuses on three aspects of the role of fungal stress responses in regulation of azole resistance. First, I establish a novel role for nutrients and nutrient signalling in azole resistance of C. albicans and the model yeast Saccharomyces cerevisiae. Compromising a global regulator that couples growth to environmental cues, Tor kinase, provides a powerful strategy to abrogate fungal drug resistance with broad therapeutic potential. Second, I implicate the molecular chaperone Hsp90 as a key regulator of biofilm drug resistance in C. albicans. Compromising Hsp90 function transforms the azoles from ineffective to highly efficacious at eradicating biofilms in vitro and in vivo. Depletion of Hsp90 leads to reduction of client proteins’ calcineurin and Mkc1 in planktonic but not biofilm conditions, suggesting that Hsp90 regulates drug resistance through different mechanisms in these distinct cellular states. Third, I establish that inhibition of lysine deacetylases (KDACs) blocks the emergence and maintenance of Hsp90-dependent azole resistance in C. albicans and S. cerevisiae. S. cerevisiae Hsp90 is acetylated on lysine 27 and 270, and key KDACs for drug resistance are Hda1 and Rpd3. Compromising KDACs alters stability and function of Hsp90 client proteins, including drug resistance regulator calcineurin. Overall, this work provides novel insight into the mechanisms by which cellular stress responses mediate azole resistance, and establishes acetylation as a novel mechanism of post-translational control of Hsp90 function in fungi; ultimately, this unveils numerous targets that could be exploited for therapeutic benefit in the treatment of fungal disease.

Many drug-resistant mechanisms in Candida albicans (C. albicans), a clinical important fungal pathogen, have been well characterized. However, few studies investigated the emergence of drug resistance from the evolutionary perspective and little is known about the evolutionary trajectories during the adaptation to the drug. Here, we examined the evolutionary dynamics of C. albicans both in the presence and absence of fluconazole, a first line drug, using the visualizing evolution in real-time (VERT) method. Evolutionary dynamics of replicate C. albicans populations, either in the presence or absence of fluconazole, were determined and adaptive mutants arose in the populations were systematically isolated using the VERT method. Drug susceptibility assays were performed to measure the fluconazole minimum inhibitory concentration (MIC) for the adaptive isolates from drug-exposed populations. Analysis of the evolutionary dynamics revealed that mutations arose more frequently in the presence of the drug compared to the absence of the drug and the drug-resistant mutations occurred in independent lineages, suggesting a heterogeneous nature of the populations during the adaptation. In addition, fitness effects were evaluated for each adaptive mutant both in the presence and absence of drug and we found most of them gained significant increase in the drug resistance without a fitness cost in the absence of the drug. Interestingly, the aneuploidy and gross chromosomal rearrangements, common drug-resistant mechanisms, were not responsible for the increased resistance to fluconazole of most adaptive isolates, suggesting single-nucleotide polymorphisms (SNPs) or other stable unknown chromosomal rearrangements may contribute to the increased drug resistance.

Thesis (M.S.)---University of Nevada, Reno, 2001. / Includes bibliographical references. Online version available on the World Wide Web.

Zugl.: Giessen, Universiẗat, Diss., 2008.

Würzburg, Univ., Diss., 2008

Thèse (M.Sc.)--Université Laval, 2008. / Bibliogr.: f. 99-105. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.