No abstract available. / Thesis (M. Sc.)-University of Durban-Westville, 1994.

The Canadian Council of Ministers of the Environment (CCME) developed guidelines for petroleum hydrocarbon (PHC) impacted field soils based on the “worst case” scenario of a fresh petroleum spill (CCME, 2001b; CCME, 2008b). Therefore, when these guidelines are applied as remedial benchmarks, they may be too conservative to be used as realistic targets as they do not account for weathering, which has been shown to decrease the toxicity of PHCs in soil. Chronic toxicity tests were performed using weathered PHC-impacted field soil from three different field sites (ON1, AB1 and BC1) and Folsomia candida. The highest PHC concentration of soil obtained from ON1 (635 mg/kg F2 and 12,000 mg/kg F3) and AB1 (610 mg/kg F2 and 2,900 mg/kg F3) did not affect F. candida survival and reproduction. However, when F. candida were exposed to PHC-impacted soil obtained from the BC1 site, a LC25 of 2,809 mg F2 + F3/kg was calculated for adult survival while an IC25 of 1,030 mg F2 + F3/kg was calculated for juvenile production. The toxicity at BC1 was postulated to be caused by the F2 concentration (it was the only site with high F2). Heat extraction and floatation methods were compared using the soil obtained from the ON1 field site. The number of adults obtained using the floatation method was always higher than the total number of adults obtained from the heat extraction method; however, only two of these results were statistically significant. This suggests that the floatation method is the best method to use to extract Folsomia candida and also indicates either method can be used with no significant effect on the conclusions. Chronic toxicity tests usually focus on measuring sub-lethal endpoints; however, only juvenile production was included in the Environment Canada protocol (Environment Canada, 2005; Environment Canada, 2007a). The endpoints of weight, length and width were added to chronic toxicity tests on AB1 and BC1 soils to determine if they were suitable endpoints. The highest concentration tested for AB1 (610 mg F2/kg and 2,900 mg F3/kg) had no effect on the weight, length or width of the adults. However, the toxicity data obtained for the BC1soils provided an EC25 of 421 mg F2 + F3/kg, 13,750 mg F2 + F3/kg and 17,425 mg F2 + F3/kg for weight, length and width, respectively. The EC25 of 421 mg F2 + F3/kg obtained for the weight of adults is lower than the IC25 of 1,030 mg F2 + F3/kg obtained for juvenile production which indicating that weight is a more sensitive endpoint than juvenile production. Avoidance-response tests involved placing a control and test soil on either side of a cylindrical container and adding 20 Folsomia candida to the midline (Environment Canada, 2007a; Liu et al., 2010). The results using soil obtained from AB1 showed no trend between soil avoidance and increasing PHC concentration. However, the avoidance-response test, using soil obtained from BC1, indicated that F. candida avoidance increased with increasing petroleum concentration. These results show that avoidance-response tests were able to predict the outcome of the chronic toxicity tests. Overall, the above results indicate that the CCME guidelines are too conservative to apply to weathered PHC-impacted field soil when the impacts are primarily F3. Results also indicate that F2 and F3 concentrations of 250 mg/kg and 2,900 mg/kg, respectively would not adversely affect F. candida adult survival, juvenile production or adult weight.

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.

Thesis (Ph. D.)--University of Sydney, 1997. / Includes tables. Title from title screen (viewed Apr. 27, 2009) Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Discipline of Removable Prosthodontics, Faculty of Dentistry. Degree awarded 1997; thesis submitted 1996. Includes bibliography. Also available in print form.

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.