In this study, the adaptation and response of Aspergillus nidulans and Aspergillus fumigatus wild type and cpcA strains to antifungal compounds were studied using cultural, genetic and proteomic methods. CpcA is the fungal cross pathway control protein which may also have a role in the development of resistance to antifungal that has become a major problem in human and plant fungal diseases and many studies are devoted to address the drug resistance mechanisms. Cell adapts itself to stress when it is subjected to a stress repeatedly. The ancestor of CpcA, ATF4 (CREB2) has recently been found to be important in the survival of tumor cells after starvation and nutrient limitation and these findings are expected to open new insights into the future antifungal therapy. Fungal cross pathway control system conserves similar mechanism with the stress response pathway in humans as a response to amino acid starvation. Fungal adaptation to antifungal agents was studied using the genetic model A. nidulans with the experimentally induced adaptation setup. It was concluded that A. nidulans cells are able to adapt to antifungal. In order to understand how cell becomes resistant to a previously susceptible agent, it is important to investigate the process when the cell encounters the agent for the first time. Fungal cellular response to antifungal drugs was studied using the human opportunistic pathogen A. fumigatus at the protein level. This is the first proteomic study directed to investigate the A. fumigatus response to voriconazole (VRC). The recently developed two dimensional gel electrophoresis approach, Fluorescence 2-D Differential Gel Electrophoresis (DIGE) method was applied to visualize differentially expressed proteins. It was concluded that, about 150 proteins were differentially regulated as a response to stress exerted by azole group antifungal drugs. cpcA strains of A. nidulans and A. fumigatus were compared to wild type strains in terms of susceptibility to various stresses, adaptation potential also at the proteome level. The results obtained in this study showed that CpcA was important in the response of Aspergillus to oxidative, heat stress and in the adaptation of cells to VRC and that its absence drastically changed the cellular response to VRC at the protein level by changing the expression of about 80 proteins. Thus, this protein is a good candidate in future as a potential drug resistance mediator and further characterization is needed to elucidate its mechanism of action on drug resistance.

Contamination of peanuts by secondary metabolites of certain fungi, namely aflatoxins present a great health hazard when exposed either at low levels for prolonged times (carcinogenic) or at high levels at once (poisonous). It is important to develop an accurate and rapid measurement technique to trace the aflatoxin and/or source fungi presence in peanuts. Thus, current research focused on development of vibrational spectroscopy based methods for detection and separation of contaminated peanut samples. Aflatoxin incidence, as a chemical contaminant in peanut paste samples, was investigated, in terms of spectral characteristics using FTIR-ATR. The effects of spectral pre-processing steps such as mean-centering, smoothing the 1st derivative and normalizing were studied. Logarithmic method was the best normalization technique describing the exponentially distributed spectral data. Spectral windows giving the best correlation with respect to increasing aflatoxin amount led to selection of fat associated spectral bands. Using the multivariate analysis tools, structural contributions of aflatoxins in peanut matrix were detected. The best region was decided as 3028-2752, 1800-1707, 1584-1424, and 1408-1127 cm-1 giving correlation coefficient for calibration (R2C), root mean square error for calibration (RMSEC) and root mean square error for prediction (RMSEP) of 98.6%, 7.66ppb and 19.5ppb, respectively. Applying the constructed partial least squares model, 95% of the samples were correctly classified while the percentage of false negative and false positive identifications were 16% and 0%, respectively. Aspergillus species of section Flavi and the black fungi, A. niger are the most common colonists of peanuts in nature and the majority of the aflatoxin producing strains are from section Flavi. Seed colonization by selected Aspergillus spp. was investigated by following the chemical alterations as a function of fungal growth by means of spectral readouts. FTIR-ATR was utilized to correlate spectral characteristics to mold density, and to separate Aspergillus at section, species and strain levels, threshold mold density values were established. Even far before the organoleptic quality changes became visually observable (~10,000 mold counts), FTIR distinguished the species of same section. Besides, the analogous secondary metabolites produced increased the similarity within the spectra even their spectral contributions were mostly masked by bulk peanut medium; and led to grouping of species producing the same mycotoxins together. Aflatoxigenic and non-aflatoxigenic strains of A. flavus and A. parasiticus were further studied for measurement capability of FTIR-ATR system in discriminating the toxic streams from just moldy and clean samples. Owing to increased similarity within the collected spectral data due to aflatoxin presence, clean samples (having aflatoxin level lower than 20 ppb, n=44), only moldy samples (having aflatoxin level lower than 300 ppb, n=28) and toxic samples (having aflatoxin level between 300-1200 ppb, n=23) were separated into appropriate classes (with a 100% classification accuracy). Photoacoustic spectroscopy (PAS) is a non-invasive technique and offers many advantages over more traditional ATR system, specifically, for in-field measurements. Even though the sample throughput time is longer compared to ATR measurements, intact seeds can be directly loaded into sample compartment for analysis. Compared to ATR, PAS is more sensitive to high moisture in samples, which in our case was not a problem since peanuts have water content less than 10%. The spectral ranges between: 3600-2750, 1800-1480, 1200-900 cm-1 were assigned as the key bands and full separation between Aspergillus spp. infected and healthy peanuts was obtained. However, PAS was not sensitive as ATR either in species level classification of Aspergillus invasion or toxic-moldy level separation. When run for separation of aflatoxigenic versus non-aflatoxigenic batches of samples, 7 out of 54 contaminated samples were misclassified but all healthy peanuts were correctly identified (15 healthy/ 69 total peanut pods). This study explored the possibility of using vibrational spectroscopy as a tool to understand chemical changes in peanuts and peanut products to Aspergillus invasion or aflatoxin contamination. The overall results of current study proved the potential of FTIR, equipped with either ATR or PAS, in identification, quantification and classification at varying levels of mold density and aflatoxin concentration. These results can be used to develop quality control laboratory methods or in field sorting devices. / Ph. D.

Mycotoxins are toxic secondary metabolites produced by filamentous fungi. Aflatoxins and fumonisins are among the most toxic mycotoxins. They are a significant risk factor for a cocktail of chronic health conditions including cancer of the liver, oesophagus and kidney, teratogenicity, neural tube defects, interference with lipid metabolism, a weakened immune system and a negative impact on micronutrient absorption in both man and animals. This study compared natural contamination of peanuts, peanut butter and sorghum from Gaborone, Botswana and Bulawayo, Zimbabwe with aflatoxins and fumonisins. In total 34 peanut samples, 34 sorghum samples and 11 peanut butter samples were collected randomly from retail shops and informal markets in the two cities. Fungal contamination was determined using standard mycology methods. Aflatoxin and fumonisin contamination was determined using HPLC-FLD. A. flavus/parasiticus species were detected in 66% and 100% of randomly analysed peanut samples from Bulawayo and Gaborone respectively and 27% (3/11) of peanut butter samples from Bulawayo. 67% of randomly analysed sorghum samples from Bulawayo showed A. flavus/parasiticus and Fusarium species contamination while none of the randomly analysed sorghum samples from Gaborone showed any fungal contamination. Furthermore aflatoxins were not detected in any of the sorghum samples; however 61% (11/18) of the Bulawayo sorghum samples showed fumonisin contamination (Range: 8 – 187 ng/g). Three of the peanut samples from Bulawayo were contaminated with aflatoxins (range: 6.6 – 622 ng/g) and no aflatoxins were detected in Gaborone peanuts. All 11 peanut butter samples from Bulawayo were contaminated with aflatoxins (Mean: 73.5 ng/g, Range: 6.8-250 ng/g) and AFB1 was the most prevalent. These preliminary results indicate that peanut butter and peanuts from Bulawayo are contaminated with high levels of aflatoxins. Stricter policing of regulations should be implemented to ensure compliance by manufacturers and public health interventions implemented in vulnerable communities. / Life & Consumer Sciences / M. Sc. (Life Sciences)

Mycotoxins are toxic secondary metabolites produced by filamentous fungi. Aflatoxins and fumonisins are among the most toxic mycotoxins. They are a significant risk factor for a cocktail of chronic health conditions including cancer of the liver, oesophagus and kidney, teratogenicity, neural tube defects, interference with lipid metabolism, a weakened immune system and a negative impact on micronutrient absorption in both man and animals. This study compared natural contamination of peanuts, peanut butter and sorghum from Gaborone, Botswana and Bulawayo, Zimbabwe with aflatoxins and fumonisins. In total 34 peanut samples, 34 sorghum samples and 11 peanut butter samples were collected randomly from retail shops and informal markets in the two cities. Fungal contamination was determined using standard mycology methods. Aflatoxin and fumonisin contamination was determined using HPLC-FLD. A. flavus/parasiticus species were detected in 66% and 100% of randomly analysed peanut samples from Bulawayo and Gaborone respectively and 27% (3/11) of peanut butter samples from Bulawayo. 67% of randomly analysed sorghum samples from Bulawayo showed A. flavus/parasiticus and Fusarium species contamination while none of the randomly analysed sorghum samples from Gaborone showed any fungal contamination. Furthermore aflatoxins were not detected in any of the sorghum samples; however 61% (11/18) of the Bulawayo sorghum samples showed fumonisin contamination (Range: 8 – 187 ng/g). Three of the peanut samples from Bulawayo were contaminated with aflatoxins (range: 6.6 – 622 ng/g) and no aflatoxins were detected in Gaborone peanuts. All 11 peanut butter samples from Bulawayo were contaminated with aflatoxins (Mean: 73.5 ng/g, Range: 6.8-250 ng/g) and AFB1 was the most prevalent. These preliminary results indicate that peanut butter and peanuts from Bulawayo are contaminated with high levels of aflatoxins. Stricter policing of regulations should be implemented to ensure compliance by manufacturers and public health interventions implemented in vulnerable communities. / Life and Consumer Sciences / M. Sc. (Life Sciences)