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One of the major aims of this thesis was to identify novel regulators that drive C. albicans adaptation during growth under different nutrient and temperature conditions. The classical stress response cascades have been previously characterised under standardized, but physiologically irrelevant growth conditions (YPD at 30°C). In this study these pathways and other regulators were examined under more physiologically relevant inputs because metabolic plasticity and thermo-tolerance have been shown to affect stress adaptation (Arguelles et al., 1999; Brown et al., 2014; Cowen, 2009; Diezmann et al., 2014). In this study, we characterized 18.5% of the functional C. albicans ORFeome under 144 different stress conditions by employing a standardized system of robotic screening (Chapter 3). These screens highlighted extensive carbon and temperature-conditional regulators in C. albicans. We identified carbon-conditional contributions of the transcriptional regulators Sfp1 and Rtg3 to stress adaptation in this pathogenic fungus (Chapter 4). Sfp1 was found to regulate the expression of key stress regulators during growth on glucose, whereas Rtg3 induced the expression of these stress genes during growth on lactate. Our screens also revealed a distinct set of transcription factors, Hap43, Swi4, Sfp1, Cap1 and Zcf31, that control regulators of cell wall integrity and that promote antifungal drug resistance in a temperature dependent and yet Hsp90- independent manner. The screens also provided new information about a relatively obscure group of transcriptional regulators in C. albicans; the zinc cluster proteins with focus on Zcf3 and Zcf18 which we further pursued with RNA-sequencing to establish them as modulators of cell cycle, stress resistance and virulence in C. albicans. Lastly, our screens reveal a network of regulators that are homologous to human oncogenes and control fungal growth via modulation of TOR signaling. In conclusion, this thesis has revealed many novel targets for possible antifungal drug development and highlighted the extensive and intricate cross-talk between stress response modules facilitated by physiologically relevant nutrient sources and ambient temperatures.

Os pesticidas organofosforados são amplamente utilizados na agricultura, pois são muito eficazes no controle de pragas, promovendo um aumento na produtividade dos alimentos. Contudo, sua utilização indiscriminada provoca graves problemas ambientais e para a saúde humana, uma vez que são tóxicos também para as espécies que não são alvos e acumulam grandes quantidades de metabólitos tóxicos, como por exemplo, fenois. Os compostos fenólicos enquadram-se nos resíduos resultantes da degradação de compostos naturais e xenobióticos da atividade antrópica. Este trabalho teve por objetivo estudar as reações de conjugação de fase II em compostos fenólicos derivados da hidrólise de pesticidas organosfosforados (clorpirifós, metil paration e profenofós) e a biotransformação de outros fenois por enzimas provenientes de fungos de ambiente marinho. Primeiramente foi realizado um screening com os fungos de ambiente marinho Aspergillus sydowii CBMAI 934, A. sydowii CBMAI 935, A. sydowii CBMAI 1241, Penicillium decaturense CBMAI 1234, P. raistrickii CBMAI 931, P. raistrickii CBMAI 1235 e Trichoderma sp. CBMAI 932 para avaliar a resistência destes microrganismos frente à toxicidade dos pesticidas organofosforados para posterior escolha da cepa mais resistente e melhor adaptada aos pesticidas testados nesse trabalho. O fungo selecionado para as reações em meio líquido de malte 2%, que melhor adaptou-se na presença dos pesticidas testados foi a cepa do fungo A. sydowii CBMAI 935. Foram realizadas curvas analíticas com o objetivo de estimar a extensão da biodegradação dos pesticidas clorpirifós, metil paration, profenofós e seus respectivos produtos de hidrólise, os fenois 3,5,6-tricloro-2-piridinol, 4-nitrofenol e 4-bromo-2-clorofenol, respectivamente. As reações de biotransformação em meio líquido de malte 2% foram avaliadas com 10, 20, 30 d de reação com concentração inicial dos pesticidas organofosforados de 50 mg.L-1. Todos os metabólitos encontrados nas reações de biotransformação dos pesticidas organofosforados com o fungo A. sydowii CBMAI 935 foram comparados com os seus padrões analíticos e sintéticos (metilação) com o objetivo de corroborar as reações de bioconjugação. Através deste estudo foi possível sugerir a presença de enzimas fosfotriesterases e enzimas metiltransferases provenientes do fungo A. sydowii CBMAI 935. Enzimas que promoveram a hidrólise e metilação dos pesticidas e compostos fenólicos testados nesse trabalho. Segundo a literatura, as reações de biotransforrmação e bioconjugação dos pesticidas orgafosforados, diminuem consideravelmente a toxicidade desses compostos recalcitrantes. / Organophosphate pesticides are widely used in agriculture, as they are very effective in pest control, promoting an increase in productivity of food. However, indiscriminate use causes serious problems environmental and for human health, since they are also toxic to non-target species and accumulate large amounts of toxic metabolites, such as phenols. Phenolic compounds are part of the waste resulting from the degradation of natural compounds and xenobiotics of human activity. This work aimed to study the phase II conjugation reactions in phenolic compounds derived from hydrolysis of pesticides organophosphates (chlorpyrifos, methyl parathion and profenofos) and the biotransformation of other phenols for enzymes from marine environment fungi. First was conducted a screening with the marine environment fungi. Aspergillus sydowii CBMAI 934, A. sydowii CBMAI 935, A. sydowii CBMAI 1241, Penicillium decaturense CBMAI 1234, P. raistrickii CBMAI 931, P. raistrickii CBMAI 1235 and Trichoderma sp. CBMAI 932 to evaluate the resistance of these microorganisms front the toxicity of organophosphate pesticides to later choose the most resistant strain and better adapted to pesticides tested in this work. The fungus selected to the reactions in liquid medium 2% malt, which best adapted in the presence of the pesticide tested was the fungal strain of A. sydowii CBMAI 935. Standard curves were performed in order to estimate the extent of biodegradation of pesticides chlorpyrifos, methyl parathion, profenofos and their hydrolysis products, phenols 3,5,6-trichloro-2-pyridinol, 4-nitrophenol and 4-bromo- 2-chlorophenol, respectively. The biotransformation reactions in liquid medium 2% malt were evaluated in 10, 20, 30 days reaction of with initial concentration of organophosphate pesticides of 50 mg.L-1. All metabolites found in the biotransformation reactions of organophosphate pesticides with the fungus A. sydowii CBMAI 935 were compared with their synthetic and analytical standards (methylation) in order to corroborate the bioconjugation reactions. Through this study was possible suggest the presence of enzymes phosphotriestesterases and methyltransferases from fungus A. sydowii CBMAI 935. Enzymes that promote hydrolysis and methylation of pesticides and phenolic compounds tested in this work. According to the literature, the reactions of biotransformation and biodegradation of organophosphate pesticides, greatly reduce the toxicity of recalcitrant compounds.

Natural products continue to be a valuable source of compounds in research involving chemical ecology and drug discovery. Secondary metabolites are biosynthesized to benefit the host organism in its environment (feeding deterrence from predators, antibiotic properties to avoid infection, etc.) but these compounds also serve as useful scaffolds in drug discovery applications. The research herein describes both aspects of these two branches of natural products chemistry. The Antarctic sponge Dendrilla membranosa produces diterpenes, of which membranolide A, deters feeding of the predatory amphipod Gondogenia antarctica. A metabolomic study of several sponges was undertaken to determine environmental factors that govern the metabolism of D. membranosa. Habitat specificity, above or below the algal canopy, was a significant factor for the chemical clustering of sponges as well as the abundance of potential amphipod predators that are prevalent within the canopy. Another D. membranosa diterpene, aplysulphurin, undergoes degradation upon methanolic treatment to form the methoxy membranolides B-H. An investigation of these artifacts reveals potent activity against the leishmaniasis-causing parasite Leishmania donovani. Microorganisms also generate a significant number of bioactive natural products. Biotic and abiotic culture stressors such as co-culturing and epigenetic modification, respectively, will be explored to turn on cryptic biosynthetic pathways. These techniques are shown to produce unique secondary metabolites from cultures and further reinforce the one strain many compounds approach to the versatile and formidable microbial domain.

1. Fusarium roseum �Graminearum� was the predominant fungus associated with stalk rot of maize in eastern Australia in the 1972, 1973 and 1974 growing seasons. All isolates of this pathogen were of the Group 2 type. Thus Group 2 contrasts with Group I which is normally isolated :Erora crown rot of wheat and grasses. Other fungi isolated in order of frequency were Diplodia maydis, F. rnoniliforme �Subglutinans�, Bipolaris sorokiniana, Nigrospora oryzac, F. roseum �Semitectum�, F. moniliforme, F. roseum �Equiseti�, F. roseum �Concolor�, Macrophomina phaseolina, Rhizoctonia sp., F. roseum �Acuminatum�, F. oxysporum, F. solani, F. tricinctum and F. roseum �Heterosporum�. The relative isolation frequencies of the fungi varied according to the seasonal conditions. Stalk rots were not of major importance in 1973, a relatively dry growing season. However, in 1974, a wet growing season, stalk rot diseases were common in all areas investigated. 2. Isolates of F. roseum �Graminearum�,derived mainly from wheat and maize but also from other sources and from various regions of eastern Australia, were examined for perithecia formation, colony characteristics, fertility, colony growth, conidia production and conidia size. The distribution of the fungus in field colonized maize and wheat plants was also studied. The Group 1 isolates did not produce perithecia, were heterothallic and very infertile, had a mean colony growth of 4.4 cm per 3 days (range, 3.9- 5.1) and produced relatively large numbers of conidia. In contrast, Group 2 isolates were homothallic and produced perithecia readily, had a mean colony growth of 5.4 cm per 3 days (range, 4.7�6.1) and produced relatively low numbers of conidia. Group 1 isolates were found to be commonly associated with crowns and roots of plants and Group 2 isolates were commonly associated with aerial plant parts. 3. The ability of a number of Group 1 and Group 2 isolates to produce the fungal hormone, zearalenone was assessed. Group I isolates produced three to four times more zearalenone than Group 2 isolates. In addition, a. culture which had previously produced perithecia but had lost that ability following numerous transfers, produced no detectable zearalenone. The results provided good evidence that the observed difference in perithecia formation was directly related to the ability to produce zearalenone. 4. The pathogenicity to wheat, maize and carnations of Group 1 isolates from crown rot affected wheat plants and Group 2 isolates from stalk rot affected maize plants was tested. Pathogenicity of 11 other isolates from teosinte, carnations, pearl millet, wheat and barley scab, banana, ginger and common wheat grass was also assessed. The results indicated that pathogenic specialization exists within F. roseum �Graminearum�. Wheat isolates were the most pathogenic to wheat, carnation isolates were the most pathogenic to carnations and all maize isolates were pathogenic to maize while those from wheat and common wheat grass were not as pathogenic to maize. Moreover, Group 2 isolates were more pathogenic when inoculated in aerial plant parts, and the Group I isolates were more pathogenic when inoculated in plant parts in soil. Inoculations on wheat seedlings in sterile field soil demonstrated that the inherent pathogenicity to wheat seedlings of isolates from wheat and maize were similar. 5. Some factors which could contribute to the observed pathogenic differences between isolates from wheat and maize to wheat seedlings in field soil were examined. Conidia volume, germination rate and inherent germinability in the soil were studied. The Group I isolates had the largest volume, the most rapid germination and the highest inherent germinability. Pathogenicity was positively correlated with conidium volume and inherent germinability. In addition, the inherent germinability and conidium volume were positively correlated. Thus, it was established that pathogenic behaviour of conidia of Group 1 and Group 2 reflected differences in conidia morphology.

Fusarium verticillioides is a pathogen of maize causing ear rot and stalk rot. The fungus also produces fumonisins, a group of mycotoxins linked to disorders in animals and humans. A cluster of genes, designated FUM genes, plays a key role in the synthesis of fumonisins. However, our understanding of the regulatory mechanism of fumonisin biosynthesis is limited. It was previously demonstrated that Cpp1, a protein phosphatase type 2A (PP2A) catalytic subunit, negatively regulates fumonisin production and is involved in cell shape maintenance. Typically, a structural A subunit, a catalytic C subunit, and a regulatory B subunit form PP2A heterotrimer complex. Significantly, there are two PP2A regulatory subunits in F. verticillioides genome, Ppr1 and Ppr2, which are homologous to Saccharomyces cerevisiae Cdc55 and Rts1, respectively. Based on preliminary data, I hypothesized that Ppr1 and Ppr2 are independently involved in the regulation of fumonisin biosynthesis and/or cell development, and to test this hypothesis I generated gene-deletion mutants of PPR1 and PPR2. The ppr1 deletion strain (Δppr1) resulted in drastic growth defect, but with increased microconidia production. The ppr2 deletion mutant strain (Δppr2) showed elevated fumonisin production similar to the Δcpp1 strain. Germinating Δppr1 conidia formed abnormally swollen cell with central septation. Δppr2 showed early hyphal branching during conidia germination. Results from this study suggest that two PP2A regulatory subunits in F. verticillioides carry out unique roles in regulating fumonisin biosynthesis and fungal development.

This research is a two year study on the effects of endomycorrhizae on vegetable production using conventional vs. organic practices. Objective of this study was initiated to determine if mycorrhizae improve yield, available soil nutrients and soil quality from two different fertilizer sources. Measurements were taken on yield, available soil nutrients, and soil quality in comparison of glomalin production and soil loss percentage. Two plant species were chosen, Tomatoes (‘Big Beef’) and Bush Beans (‘Tenderette’). A randomized split block 2 x 3 factorial treatment arrangement was used with two crops and three different inputs: Mo- 0 mycorrhizae, M1- recommended rate, and M2- 2x recommended rate of mycorrhizae. Each mycorrhizal input was replicated three times in both the conventional and organic system. Results show there was no difference in yield based on mycorrhizae additions at any rate. There was a significant yield difference based on conventional production over organic production in tomatoes and snap beans in 2010 and tomatoes in 2011. Possible explanations for yield difference in the organic production system include: different insect controls and a slower release of nutrients from poultry litter. Available soil nutrients were not influenced in the study based on mycorrhizal inputs in inorganic or organic tomato production. Soil available nutrients were significantly influenced in organic tomato when compared to inorganic tomato production at selected sampling dates. Mycorrhizae did not influence soil fertility in inorganic snap bean or organic snap bean production. Soil available nutrients were significantly influenced in organic snap bean when compared to inorganic snap bean production at selected sampling dates. Glomalin production and soil loss percentage were not shown to be significantly different within organic or inorganic treatments based on mycorrhizae inputs. However, glomalin production was shown to be significantly greater in organic production compared to inorganic in 2011. An explanation of this could be due to the use of leaf mulch as organic weed control. Although a numerical decrease was observed in soil loss percentage in organic production compared to inorganic production from the first year to the second, it was not shown to be a significant amount.