Several countries have enacted regulations on tolerance limits for common mycotoxins because of the hazardous nature and widespread occurrence of these fungal secondary metabolites in agricultural commodities. Screening of agricultural commodities destined for animal consumption for the presence of mycotoxins is now becoming a prerequisite in several countries as a means of minimizing ingestion of these toxins. Silage samples were analyzed for pH, % dry matter (DM) content, and the presence of total fungi, yeasts and the types of lactic acid bacteria present. The samples were also analyzed for mycotoxins that have been reported to commonly occur in silage. The pH of the samples was found to be acidic ranging from pH 3.4 to 4.7, with few samples having pH values above 6. There was a significant difference in the % DM content amongst the sampling regions. There was no significant difference in the extent of fungal contamination amongst the different regions. Aspergillus fumigatus was the predominant species from all the samples. Most of the yeast species were isolated from the Bergville region. The yeast species isolated from all samples were Trichosporon, Cryptococcus and Candida species, which are all regarded as nonlactate fermenters. Lactobacillus plantarum and Lactobacillus buchneri were the only two lactic acid producers isolated from the samples. Aflatoxins, citrinin and patulin were the most predominant toxins in the samples. Ochratoxin A and deoxynivalenol was not detected in all samples using thin layer chromatography, while the latter two toxins were only detected in two samples using VICAM fluorometry. The level of fumonisins that was found in the forage crops used for silage production was fairly low with the highest level being 9.36 ppb. Most of the mycotoxin extracts were found to reduce the % cell viability of human lymphocytes after 18 hours of incubation as determined by the MTT assay. / Professor Mike Dutton Mr. F. E. Van Zyl

Thesis (MSc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Mycotoxins have assumed worldwide importance due to the ubiquitous occurrence of toxigenic fungi, their infestation of plant-based foods and feeds and the subsequent economical and health impact it because of their contamination of commercial products. Ochratoxin A (OA) is a nephrotoxic mycotoxin produced by isolates of Aspergillus ochraceus and Penicillium verrucosum and occurs frequently in nature. The major target for toxicity of OA in mammalian species is the kidneys and it has been the major cause of Danish Porcine Nephropathy. OA has also been extensively implicated in the aetiology of Balkan Endemic Nephropathy and Chronic Interstitial Nephropathy in Northern-Africa. Furthermore, OA has been identified as a carcinogen, an immunosuppressant and a teratogen with respect to the foetal central nervous system. Although a large amount of research has been conducted into the chemical nature of the toxicity of OA, the exact molecular mechanism of action of OA is not yet conclusive. Numerous structure activity relationship studies have suggested that the toxicity of OA may be assigned to three major processes: (i) inhibition of ATP production; (ii) inhibition of protein synthesis; and (iii) the disruption of hepatic microsomal calcium homeostasis through the promotion of membrane lipid peroxidation. It is the aim of this thesis to gain a better understanding, through the synthesis ofOA analogues, of the chemical structure responsible for the toxic function of the ochratoxins. The halogen-group has extensively been implicated in the toxicity of the ochratoxins. This is evident in ochratoxin B (OB), the dechloro analogue of OA, which is approximately ten times less toxic than OA. Preliminary tests have indicated that bromo-ochratoxin B(BrOB), the bromo analogue of OA, is more toxic than ochratoxin A to renal cells. Fluoro-ochratoxin B and other analogues of OA, where other amino acids are incorporated, should provide invaluable information on the structure-activity relationships and the mode of action of the ochratoxins. Our research effort addresses both these aspects (i) fluorination of the dihydroisocoumarin moiety and (ii) the coupling of different amino acids and dipeptides to the non-toxic hydrolysed product of OA, ochratoxin a. Chapter one includes a review of the important biological aspects of OA that has served as a guideline to the synthesis of effective OA analogues. An overview of the relevant chemistry involved in the modification of OA will conclude the chapter. Chapter two entails a discussion of fluorine in bio-organic chemistry. This includes an overview of the impact that fluorine substitution has on the biological reactivity of molecules. A review on the synthesis of organofluorine compounds, which forms the emphasis of this study, concludes the chapter. Chapter three elaborates on the different methodologies used in our attempts to synthesise fluoro-ochratoxin B and other analogues. These included the direct electrophilic fluorination of OB and different analogous aromatic model compounds by xenon difluoride, N-fluorobenzenesulfonimides and Selectfluor™ as fluorinating agents. Also involved is an investigation into an alternative route for the synthesis of fluoro aromatic compounds from bromo and chloro analogues by means of palladium catalysed trimethyl- and tributylstannyl and trimethylsilylation which in tum may be substituted with fluorine by means of xenon difluoride. Efforts towards the direct catalytic fluorosubstitution of aryl halides are also investigated. The synthesis of a key intermediate, fluoroacetoacetaldehyde, in a de nova synthetic route to fluoroochratoxin B is also discussed. Furthermore, the synthesis of novel OA analogues with respect to the replacement of the L-phenylalanine moiety is addressed. This includes the conversion of OA to Oa, by acid hydrolysis, followed by the coupling of ortho-, meta- and para- substituted DL-fluorophenylalanine to the lactone acid. This is followed by the synthesis of histidylhistidine methyl ester and attempted coupling to Oa. The coupling of halosalicylic acids and salicylic acid to L-phenylalanine, for use as model aromatic substrates for fluorination, IS discussed. Peptide coupling by dicyclohexylcarbodiimide carboxyl activation, with reference to the protection of the phenolic hydroxyl group in 5-chlorosalicylic acid for application to Oa, concludes this work. / AFRIKAANSE OPSOMMING: Mikotoksiene is van wêreld-wye belang as gevolg van die alomteenwoordige voorkoms van toksigeniese fungi, hul besmetting van plantaardige kossoorte en voerstowwe en die gevolglike ekonomiese en gesondheidsimpak deur die besoedeling van kommersiële produkte. Ochratoksien A (OA) is 'n nefrotoksiese mikotoksien wat geproduseer word deur isolate van Aspergillus ochraceus en Penicillium verrucosum en kom algemeen in die natuur voor. Die niere is die hoof teiken vir vergifiting deur OA in soogdierspesies en is as die vername oorsaak van "Danish Porcine Nephropathy" aangewys. OA word verder aangedui as die oorsaak vir "Balkan Endemic Nephropathy" en "Chronic Interstitial Nephropathy" in Noord- Afrika. OA is verder geïdentifiseer as 'n karsinogeen, immuno-onderdrukker en is teratogenies ten opsigte van die sentrale senuweestelsel van fetusse. Alhoewel aansienlike navorsing alreeds gewei is aan die chemiese natuur van die toksisiteit van OA, is die presiese molekulêre meganisme van OA reaktiwiteit onbeslis. Verskeie struktuur-aktiwitweit verwantskaps studies dui daarop dat die toksisiteit van OA hoofsaaklik toegeskryf kan word aan drie hoof prosesse: (i) inhibisie van ATP produksie; (ii) inhibisie van proteïen sintese; en (iii) die ontwrigting van hepatiese mikrosomale kalsiumhomeostase deur die bevordering van membraanlipiedperoksidasie. Hierdie tesis het ten doel, deur die sintese van OA analoë, om 'n beter insig oor die chemiese struktuur wat verantwoordelik is vir die toksiese funksionaliteit van ochratoksiene te verkry. Die halogeen substituent is grootliks geïmpliseer in die toksisiteit van OA. 'n Bewys hiervan is ochratoksien B (OB), die dechlooranaloog van OA, wat ongeveer tien maal minder toksies is as OA. Voorlopige ondersoeke het aangetoon dat bromoochratoksien B (BrOB), die broomanaloog van OA, meer toksies is vir nierselle as OA. Fluoorochratoksien B en ander analoë van OA, waar ander aminosure geïnkorporeer word, behoort waardevolle inligting te voorsien met betrekking tot die struktuur-aktiwiteitsverwantskappe en die wyse waarop ochratoksiene funksioneer. Hierdie navorsingspoging spreek beide aspekte aan; (i) die fluorering van die dihidroïsokumarien gedeelte en, (ii) die koppeling van verskillende armnosure en dipeptiede aan die nie-toksiese hidrolieseproduk van OA, nl. ochratoksien a. Hoofstuk een vervat 'n oorsig van die belangrike biologiese aspekte van OA wat dien as riglyn vir die sintese van doeltreffende OA analoë. Die hoofstuk word afgesluit met 'n oorsig van die relevante chemie betrokke by die modifisering van die struktuur van OA. Hoofstuk twee bevat 'n bespreking van die aanwending van fluoor in bio-organiese chemie. Dit bevat 'n oorsig van die impak wat fluoorsubstitusie het op die biologiese reaktiwiteit van molekules. 'n Opsomming oor die sintese van organofluoorverbindings, wat die essensie van hierdie studie is, beëindig die hoofstuk. Hoofstuk drie handeloor die veskillende metodes wat toegepas is in pogings om fluoorochratoksien B en ander analoë te sintetiseer. Dit sluit in die direkte elektrofiliese fluorering van OB en ander verwante aromatiese modelverbindings deur gebruik te maak van xenondifluoried, N-fluoorbenseensulfonimied en Selectfluor™ as fluoreringsreagense. Dit behels verder ook 'n ondersoek na 'n alternatiewe roete tot die sintese van fluooraromatiese verbindings vanaf broom- en chlooranaloë. Vir die doel word palladiumgekataliseerde trimetiel- en tributielstannilering, en trimetielsililering wat vervolgens deur middel van xenondifluoried met fluoor gesubstitueer kan word, aangewend. Pogings tot die direkte katalitiese fluoorsubstitusie van arielhaliede word ook bespreek. Die sintese van 'n sleutelintermediêr, fluoroasetoasetaldehied, in 'n de nova sintese roete tot fluoorochratoksien B word bespreek. Die sintese van nuwe OA analoë, met betrekking to die vervangmg van die Lfenielalanien (L-Phe) groep word ondersoek. Dit bevat die omsetting van OA na Oa, deur suurhidrolise, gevolg deur die koppeling van orto-, meta- en paragesubstitueerde DL-fluoorfenielalanien aan die laktoonsuur, Oa. Daarna word die sintese van histidielhistidienmetielester en die verdere pogings aangaande koppeling met Oa bespreek. Die koppeling van halosalisielsure en salisielsuur aan L-Phe wat dien as model aromatiese verbindings vir fluorering, word behandel. Peptiedkoppeling met behulp van disikloheksielkarbodiimied-karboksielaktivering, met inbegrip van die beskerming van die fenoliese hidroksiel groep m 5-chloorsalisielsuur Vir die toepassing op Oa, beëindig hierdie werk.

Tan spot of wheat is an economically significant disease caused by the fungal pathogen, Pyrenophora tritici-repentis. Certain races of the fungus secrete Ptr ToxA (ToxA), a 13.2 kDa proteinaceous host-selective toxin that is responsible and sufficient to cause disease in susceptible wheat varieties. Disease symptoms develop only when the ToxA gene in the fungus and a single gene in the wheat host are expressed. The understanding of this gene-for-gene interaction could be instrumental towards control of the disease and is also being developed as a model system for understanding host-pathogen interactions. Here, this effort is given a solid structural foundation through crystallographic analysis of the ToxA structure. The ToxA structure was solved at 1.65 Å resolution using the anomalous signal from inherently present sulfur atoms. The monomeric toxin adopts a β-sandwich fold of two anti-parallel β-sheets composed of four strands each. The mapping of existing mutation data onto the structure reveals that a sequence of Arg- Gly-Asp(RGD) and surrounding residues required for activity are present on a solvent-exposed loop thereby making them potential candidates for recognition events that are required for ToxA activity. Unexpectedly, after a simple circular permutation, the ToxA structure is topologically identical to the classic mammalian RGD containing fibronectin type III (FnIII) domain, and furthermore the RGD residues are topologically equivalent. These results support the hypothesis that ToxA, like FnIII, interacts with an integrin-like receptor on the host plant cell surface. There has been a renewed interest in the method of using the anomalous signal from sulfur atoms to solve protein structures. As a spin-off of the structure solution work, the data were systematically analyzed to study the effects of crystal decay, resolution and data redundancy on the ability to locate the sulfur positions and subsequent phasing of the protein. The analyses show that the choices made about data redundancy and resolution limits may be crucial for the structure determination and that anomalous correlation coefficients are helpful indicators in making these choices. / Graduation date: 2006

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 90-104). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / "May 2000." Typescript. Vita. Includes bibliographical references (leaves 90-104). Also available on the Internet.

Thesis (M. Sc.)--Carleton University, 2004. / Includes bibliographical references (p. 56-69). Also available in electronic format on the Internet.

Thesis (D. Tech.) -- Central University of Technology, Free State, 2008 / The purpose of this study was to evaluate brewing and consumption practices and to screen for micro-organisms and mycotoxins associated with traditional beer produced and consumed in the marginal urban settlements of the city of Kimberley in the Northern Cape Province of South Africa. The survey study revealed that traditional beer is no longer being brewed for traditional purposes only, as was the case in the past, but rather for commercial gain. Both brewers and consumers, however, appeared to be largely unaware of disease-causing micro-organisms present on the hands or bodies of handlers that can be transferred to the beverage during the handling process, and were seemingly not conversant with regard to the effects of hazardous ingredients sometimes incorporated during the brewing process. Unemployment and a lack of education emerged as pivotal factors related to the production of traditional beer and the ignorance of the associated safety thereof. The survey further indicated that although facilities such as the availability of potable water (taps in yards) and flushing toilets were sometimes in place, other facilities such as basins with hot running water were often not available. In commercially produced and homebrewed traditional beer the mean counts for total coliforms and Staphylococcus spp. were circa 105 cfu.ml-1 whereas the TVC (Total Viable Counts) and total fungi counts were 106 and 107 cfu.ml-1 respectively. The total coliforms and Staphylococcus spp. counts for homebrewed traditional beer were approximately one log-phase higher than the commercial version. The counts in the homebrewed beer probably originated from contamination during handling, while in the commercial product contamination originated either in the raw ingredients or during postprocessing and consumption. Apart from staphylococci, considerable numbers of total coliforms indicating faecal contamination were noted. A rapid, easy, reliable and accurate technique that could be used to quantify the level of mycotoxins (deoxynivalenol and citrinin) in the beer was developed through validation of the ELISA Ridascreen methodology. Using this method, the deoxynivalenol (DON) level in the beer samples was found to exceed the recommended levels suggested by the European Union, while citrinin levels in the samples varied between 35.6 ppb and 942.2 ppb. In the case of citrinin there were statistically significant differences between spring, summer and winter samples, confirming the seasonal impact on fungal growth and consequent mycotoxin production. An R2-value of 0.409 was noted between DON and citrinin, indicating a weak positive association. Finally, an awareness programme in the format of a poster with accompanying subscripts was developed to address issues of safety and hygiene of traditional beer in the study area. The poster utilises animatedstyle colour images of selected practices that need to be addressed, accompanied by slogans summarising the particular image in English, Afrikaans and Setswana. It is envisaged that, as part of a comprehensive awareness programme, the poster will contribute greatly to the quality, safety and promotion of traditional beer in the area.