O amendoim é freqüentemente invadido por fungos toxigênicos antes da colheita. Este trabalho teve como objetivos: determinar a micoflora do solo, ar, sementes plantadas, flores, ginóforo e grãos e cascas de amendoim em diferentes fases de maturação e após a secagem; analisar aflatoxinas B1, B2, G1 e G2, ácido ciclopiazônico e fumonisinas B1 e B2 nas sementes plantadas, grãos e cascas de amendoim em diferentes fases de maturação e após a secagem. Os fungos mais freqüentes nos grãos e cascas de amendoim foram Fusarium spp. e Aspergillus flavus. A espécie A. flavus também foi encontrada em amostras do solo e do ar. Aflatoxinas e ácido ciclopiazônico foram detectados em 32 % das amostras de grãos de amendoim analisadas em concentrações que variaram de 4,20<font face=\"symbol\">mg/kg a 198,84 <font face=\"symbol\">mg/kg e de 260 <font face=\"symbol\">mg/kg a 600 <font face=\"symbol\">mg/kg, respectivamente. Nas cascas somente aflatoxinas foram detectadas em concentrações que variaram de 5,76 <font face=\"symbol\">mg/kg a 218,52 <font face=\"symbol\">mg/kg. Fumonisinas não foram detectadas. Boas práticas agrícolas são indicadas para região. / Peanuts are contaminated frequently by toxigenic fungi before harvest. the present study aimed to: identify the mycoflora of the soil, air, flower, pegs, peanut kernels and hulls in the different maturation stages and after drying; determinate the occurrence of aflatoxins B1, B2, G1 and G2, cyclopiazonic acid (CPA) and fumonisins B1 and B2 in peanut kernels and hulls in different maturation stages and after drying. Fusarium spp. Aspergullus flavus were the most frequent fungi isolated in peanuts hulls and kernels. A. flavus was isolated, also, in air and soil samples. Aflatoxins and cyclopiazonic acid were detected in 32% of the kernels samples in concentration from 4.20 <font face=\"symbol\">mg/kg to 198.84 <font face=\"symbol\">mg/kg and from 260 <font face=\"symbol\">mg/kg to 600 <font face=\"symbol\">mg/kg, respectively. In the peanut hulls only aflatoxins were detected in 24 % of the samples in concentration from 5.76 <font face=\"symbol\">mg/kg to 218.52 <font face=\"symbol\">mg/kg. Fumonisins were not detected. Good agriculture practices are indicated to region.

Thesis (Ph.D. (Medical Science)) -- University of Limpopo, 2012

Subtilase cytotoxin (SubAB) is the prototype of a novel class of AB₅ cytotoxins produced by Shiga-toxigenic Escherichia coli (STEC). The A subunit (SubA) is a serine protease that cleaves the ER chaperone BiP causing cell death by a previouslyundetermined mechanism. The B subunits of AB₅toxins typically recognise host cell glycan receptors and direct the subcellular transport of the A subunit. Although the function of SubA and its intracellular substrate have been elucidated, the B subunit (SubB) is relatively uncharacterised. The subcellular trafficking pathway of SubAB was initially examined. SubAB conjugated to Oregon Green 488 (SubAB-OG) was internalised by Vero cells by 5 min, and co-localised with its ER target BiP within 30 min. When Vero cells were incubated with SubAB-OG and either Alexa Fluor 594-conjugated Cholera toxin B subunit (CtxBAF594) or Texas Red-conjugated Shiga toxin B subunit (StxB-TR), individual cells exhibited differential toxin uptake. This was shown to be cell cycle-dependent, in which, SubAB-OG was preferentially internalised by cells migrating through G1 and early S phases. In contrast, CtxB-AF594 was taken up by cells in S through M phases and by a majority of cells in G1, while StxB-TR endocytosis occurred in cells traversing G1. Fluorescent SubAB co-localised with the clathrin marker transferrin, but not with Caveolin-1 (a marker for cholesterol-associated caveolae) and was subsequently trafficked via a retrograde pathway to the TGN, Golgi and ER. The clathrin inhibitor phenylarsine oxide prevented SubAB entry and BiP cleavage in SubAB-treated Vero, HeLa and N2A cells, while cholesterol depletion did not, demonstrating that, unlike either Stx or Ctx, SubAB internalisation is exclusively clathrin-dependent. Identification of the SubB receptor was initially approached using toxin overlay assays in which Vero cell glycolipid extracts were separated by thin-layer chromatography and overlaid with SubAB. SubAB exhibited a high affinity for particular acidic species in the ganglioside fraction. However, none co-migrated with commercial glycolipid standards. SubAB-OG also exhibited an affinity for the oligosaccharide structures of chimeric LPS from GM₂ and GM₃ bacterial receptor mimic constructs in an LPS toxin overlay assay. Glycan array analysis revealed that SubB possessed a unique affinity for carbohydrate receptors with a terminal Neu5Gcα(2→3)Galβ disaccharide. Monovalent receptor analogues with distal Neu5Gc or Neu5Gcα(2→3)Galβ and highly-sialylated α₁-AGP did not prevent endocytosis of SubAB-OG, BiP cleavage or cytotoxicity in Vero cells. This indicated that SubAB has a greater affinity for the host cell receptors than the receptor analogues and may engage multiple receptors displayed on a lipid bilayer. In addition to mediating toxin binding and subcellular trafficking, CtxB and StxB can also potentiate the immune response to co-administered antigen. Accordingly, the systemic immunomodulatory properties of SubB administered by the i.p. route were assessed in mice. Using SubAA₂₇₂ as a bystander antigen, SubB significantly increased mouse anti-SubAA₂₇₂ titres to levels that were comparable to those obtained using Alum adjuvant. However, when admixed with structurally-unrelated OVA, SubB did not significantly affect anti-OVA titres whereas Alum and CtxB did. This indicated that SubB may function as a systemic carrier protein (rather than an adjuvant) for particular antigens. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1363363 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2009

Thesis (Ph. D.)--University of Nevada, Reno, 2008. / Includes bibliographical references. Online version available on the World Wide Web.

Thesis (Ph.D.) - Carleton University, 2007. / Includes bibliographical references (p. 129-156). Also available in electronic format on the Internet.

Significantes quantidades de maçãs produzidas no Rio Grande do Sul são usadas para a produção de suco destinado à exportação. As frutas que são utilizadas pela indústria são aquelas que não atingem o padrão exigido para o consumo in natura. Estas maçãs constituem fator de risco quanto à contaminação por fungos, que podem produzir micotoxinas, como a patulina. O Brasil não tem legislação que estabeleça níveis permitidos de patulina em alimentos. Mas muitos países têm adotado a recomendação do Codex Alimentarius que indica um nível máximo permitido de 50 μg/L de patulina em suco de maçã. Esse fato estimula países exportadores, como o Brasil, a pesquisar a qualidade micotoxicológica dos seus produtos, a fim de se tornarem competitivos no mercado internacional e de fornecerem alimentos seguros. Nesse contexto, os objetivos desse trabalho foram: (1) avaliar a contaminação fúngica de maçãs destinadas à produção de suco, bem como a possível presença de patulina nos sucos produzidos a partir destas frutas; (2) adaptar metodologicamente a técnica de cromatografia em camada delgada à quantificação de patulina em maçãs e sucos de maçã; (3) analisar os níveis de patulina nas fases do processamento de suco de maçã; e (4) pesquisar fungos termorresistentes nos sucos em estudo. As amostras de maçãs e de sucos foram fornecidas pela Golden Sucos Ltda de Farroupilha, RS. Penicillium expansum foi a espécie isolada predominantemente, sendo que 94,44% dos isolados pertencentes a esta espécie se mostraram produtores de patulina nas condições testadas. A prevalência de fungos produtores de patulina nas maçãs processadas resultou em altas concentrações da micotoxina nessas maçãs, variando entre 254,62 e 653,37 μg/kg e também nos sucos concentrados produzidos, com níveis de patulina nestes entre 56,04 e 231,44 μg/L. O método desenvolvido para extração e quantificação da patulina foi eficaz. O detector de carga acoplada forneceu boa sensibilidade, precisão, linearidade e recuperação para a determinação quantitativa de patulina. Todos os estágios do processamento do suco contribuíram para a redução dos níveis de patulina. Desde a maçã até o suco a redução foi de 75,2%. A presença de Byssochlamys nivea em uma das amostras de suco indica que a patulina poderia ser produzida por este fungo durante o armazenamento. / Significant amounts of apples produced in the state of Rio Grande do Sul, Brazil, are used to juice production destined to exportation. Fruits used by the industry show some defects and are not accepted for fresh consumption. These apples are a risk factor considering the contamination by fungus, which can produce mycotoxins, such as patulin. Brazil does not have legislation for patulin in food. But many countries have adopted the recommendation of the Codex Alimentarius, which indicates a maximum permitted level of 50 μg/L of patulin in apple juice. This concern stimulates exporting countries, like Brazil, in the research of mycotoxigenic quality of apples and by-products, to become competitive and provide safe foods. In this context, the objectives of this work were (1) evaluate mould contamination of apples used to juice production and possible presence of patulin in apples; (2) methodologically adapt the thin-layer chromatographic technique for quantification of patulin in apples and apple juice; (3) analyze patulin levels in some processing stages of apple juice production; and (4) investigate heat-resistant moulds in juices. The apples and juice samples were obtained from Golden Sucos Ltda, located in Farroupilha city, in Rio Grande do Sul state, Brazil. Penicillium expansum was the most frequently isolated fungus, among these isolates, 94.44% were able to produce patulin under the tested conditions. The prevalence of patulin-producing fungi in apples resulted in high patulin levels in these apples, ranging from 254.62 to 653.37 μg/Kg and in juices ranging from 56.04 to 231.44 μg/L. The method developed to patulin extraction and quantification was effective. The charged coupled device imaging system provides good sensitivity, precision and linearity for the quantitative determination of patulin. All stages of apple juice production contributed to patulin reduction. The overall loss of patulin through processing from apple to apple juice was 75.2%. The presence of Byssochlamys nivea in a juice sample indicates that this fungus could produce patulin during storage.

Significantes quantidades de maçãs produzidas no Rio Grande do Sul são usadas para a produção de suco destinado à exportação. As frutas que são utilizadas pela indústria são aquelas que não atingem o padrão exigido para o consumo in natura. Estas maçãs constituem fator de risco quanto à contaminação por fungos, que podem produzir micotoxinas, como a patulina. O Brasil não tem legislação que estabeleça níveis permitidos de patulina em alimentos. Mas muitos países têm adotado a recomendação do Codex Alimentarius que indica um nível máximo permitido de 50 μg/L de patulina em suco de maçã. Esse fato estimula países exportadores, como o Brasil, a pesquisar a qualidade micotoxicológica dos seus produtos, a fim de se tornarem competitivos no mercado internacional e de fornecerem alimentos seguros. Nesse contexto, os objetivos desse trabalho foram: (1) avaliar a contaminação fúngica de maçãs destinadas à produção de suco, bem como a possível presença de patulina nos sucos produzidos a partir destas frutas; (2) adaptar metodologicamente a técnica de cromatografia em camada delgada à quantificação de patulina em maçãs e sucos de maçã; (3) analisar os níveis de patulina nas fases do processamento de suco de maçã; e (4) pesquisar fungos termorresistentes nos sucos em estudo. As amostras de maçãs e de sucos foram fornecidas pela Golden Sucos Ltda de Farroupilha, RS. Penicillium expansum foi a espécie isolada predominantemente, sendo que 94,44% dos isolados pertencentes a esta espécie se mostraram produtores de patulina nas condições testadas. A prevalência de fungos produtores de patulina nas maçãs processadas resultou em altas concentrações da micotoxina nessas maçãs, variando entre 254,62 e 653,37 μg/kg e também nos sucos concentrados produzidos, com níveis de patulina nestes entre 56,04 e 231,44 μg/L. O método desenvolvido para extração e quantificação da patulina foi eficaz. O detector de carga acoplada forneceu boa sensibilidade, precisão, linearidade e recuperação para a determinação quantitativa de patulina. Todos os estágios do processamento do suco contribuíram para a redução dos níveis de patulina. Desde a maçã até o suco a redução foi de 75,2%. A presença de Byssochlamys nivea em uma das amostras de suco indica que a patulina poderia ser produzida por este fungo durante o armazenamento. / Significant amounts of apples produced in the state of Rio Grande do Sul, Brazil, are used to juice production destined to exportation. Fruits used by the industry show some defects and are not accepted for fresh consumption. These apples are a risk factor considering the contamination by fungus, which can produce mycotoxins, such as patulin. Brazil does not have legislation for patulin in food. But many countries have adopted the recommendation of the Codex Alimentarius, which indicates a maximum permitted level of 50 μg/L of patulin in apple juice. This concern stimulates exporting countries, like Brazil, in the research of mycotoxigenic quality of apples and by-products, to become competitive and provide safe foods. In this context, the objectives of this work were (1) evaluate mould contamination of apples used to juice production and possible presence of patulin in apples; (2) methodologically adapt the thin-layer chromatographic technique for quantification of patulin in apples and apple juice; (3) analyze patulin levels in some processing stages of apple juice production; and (4) investigate heat-resistant moulds in juices. The apples and juice samples were obtained from Golden Sucos Ltda, located in Farroupilha city, in Rio Grande do Sul state, Brazil. Penicillium expansum was the most frequently isolated fungus, among these isolates, 94.44% were able to produce patulin under the tested conditions. The prevalence of patulin-producing fungi in apples resulted in high patulin levels in these apples, ranging from 254.62 to 653.37 μg/Kg and in juices ranging from 56.04 to 231.44 μg/L. The method developed to patulin extraction and quantification was effective. The charged coupled device imaging system provides good sensitivity, precision and linearity for the quantitative determination of patulin. All stages of apple juice production contributed to patulin reduction. The overall loss of patulin through processing from apple to apple juice was 75.2%. The presence of Byssochlamys nivea in a juice sample indicates that this fungus could produce patulin during storage.

Master of Science / Entomology / Ludek Zurek / House flies (Musca domestica L.) and stable flies (Stomoxys calcitrans L.) are insects of medical and veterinary importance. House flies are recognized as mechanical vectors of human foodborne pathogens and stable files are known for their painful bites resulting in reduction of body weight gain and milk production in cattle. The larval development of both fly species takes place in decaying organic materials (primarily animal manure), resulting in large fly populations in confined cattle environments. Shiga toxin-producing Escherichia coli (STEC) are a major foodborne pathogen. Cattle are the asymptomatic reservoir of STEC with bacteria being released to the environment via their feces. STEC O157 is the main serogroup causing human illness. However, infections with non-O157 STEC are increasing: more than 70% of non-O157 infections are caused by six serogroups of non-O157, referred as "Big six" (O26, O45, O103, O111, O121, and O145). In addition, there was a large 2011 outbreak in Europe caused by STEC O104. The objectives of my thesis were: 1) To assess the prevalence of seven serogroups of non-O157 STEC (O26, O45, O103, O104, O111, O121, and O145) (STEC-7) in house flies and stable flies collected from confined cattle environments; 2) To investigate the vector competence of house flies for non-O157 STEC-7. A total of 463 house flies from feedlots and dairies from six states, and 180 stable flies collected from a feedlot in Nebraska were processed for the isolation and identification of STEC-7 using a culture-based approach followed by PCR for the confirmation of serogroups, and virulence genes. A total of 34.3% of house flies and 1.1% of stable flies tested positive for at least one serogroup of E. coli of interest, and 1.5% of house flies harbored STEC with the Shiga-toxin gene (stx1). No STEC were detected in stable flies. Vector competence bioassays for non-O157 STEC revealed that house flies can carry non-O157 STEC for at least six days with the exception STEC O145. Overall, the findings of this research demonstrate that house flies, but not stable flies, likely play an important role in the ecology and transmission of non-O157 STEC in confined cattle environments.

O amendoim é freqüentemente invadido por fungos toxigênicos antes da colheita. Este trabalho teve como objetivos: determinar a micoflora do solo, ar, sementes plantadas, flores, ginóforo e grãos e cascas de amendoim em diferentes fases de maturação e após a secagem; analisar aflatoxinas B1, B2, G1 e G2, ácido ciclopiazônico e fumonisinas B1 e B2 nas sementes plantadas, grãos e cascas de amendoim em diferentes fases de maturação e após a secagem. Os fungos mais freqüentes nos grãos e cascas de amendoim foram Fusarium spp. e Aspergillus flavus. A espécie A. flavus também foi encontrada em amostras do solo e do ar. Aflatoxinas e ácido ciclopiazônico foram detectados em 32 % das amostras de grãos de amendoim analisadas em concentrações que variaram de 4,20<font face=\"symbol\">mg/kg a 198,84 <font face=\"symbol\">mg/kg e de 260 <font face=\"symbol\">mg/kg a 600 <font face=\"symbol\">mg/kg, respectivamente. Nas cascas somente aflatoxinas foram detectadas em concentrações que variaram de 5,76 <font face=\"symbol\">mg/kg a 218,52 <font face=\"symbol\">mg/kg. Fumonisinas não foram detectadas. Boas práticas agrícolas são indicadas para região. / Peanuts are contaminated frequently by toxigenic fungi before harvest. the present study aimed to: identify the mycoflora of the soil, air, flower, pegs, peanut kernels and hulls in the different maturation stages and after drying; determinate the occurrence of aflatoxins B1, B2, G1 and G2, cyclopiazonic acid (CPA) and fumonisins B1 and B2 in peanut kernels and hulls in different maturation stages and after drying. Fusarium spp. Aspergullus flavus were the most frequent fungi isolated in peanuts hulls and kernels. A. flavus was isolated, also, in air and soil samples. Aflatoxins and cyclopiazonic acid were detected in 32% of the kernels samples in concentration from 4.20 <font face=\"symbol\">mg/kg to 198.84 <font face=\"symbol\">mg/kg and from 260 <font face=\"symbol\">mg/kg to 600 <font face=\"symbol\">mg/kg, respectively. In the peanut hulls only aflatoxins were detected in 24 % of the samples in concentration from 5.76 <font face=\"symbol\">mg/kg to 218.52 <font face=\"symbol\">mg/kg. Fumonisins were not detected. Good agriculture practices are indicated to region.

Significantes quantidades de maçãs produzidas no Rio Grande do Sul são usadas para a produção de suco destinado à exportação. As frutas que são utilizadas pela indústria são aquelas que não atingem o padrão exigido para o consumo in natura. Estas maçãs constituem fator de risco quanto à contaminação por fungos, que podem produzir micotoxinas, como a patulina. O Brasil não tem legislação que estabeleça níveis permitidos de patulina em alimentos. Mas muitos países têm adotado a recomendação do Codex Alimentarius que indica um nível máximo permitido de 50 μg/L de patulina em suco de maçã. Esse fato estimula países exportadores, como o Brasil, a pesquisar a qualidade micotoxicológica dos seus produtos, a fim de se tornarem competitivos no mercado internacional e de fornecerem alimentos seguros. Nesse contexto, os objetivos desse trabalho foram: (1) avaliar a contaminação fúngica de maçãs destinadas à produção de suco, bem como a possível presença de patulina nos sucos produzidos a partir destas frutas; (2) adaptar metodologicamente a técnica de cromatografia em camada delgada à quantificação de patulina em maçãs e sucos de maçã; (3) analisar os níveis de patulina nas fases do processamento de suco de maçã; e (4) pesquisar fungos termorresistentes nos sucos em estudo. As amostras de maçãs e de sucos foram fornecidas pela Golden Sucos Ltda de Farroupilha, RS. Penicillium expansum foi a espécie isolada predominantemente, sendo que 94,44% dos isolados pertencentes a esta espécie se mostraram produtores de patulina nas condições testadas. A prevalência de fungos produtores de patulina nas maçãs processadas resultou em altas concentrações da micotoxina nessas maçãs, variando entre 254,62 e 653,37 μg/kg e também nos sucos concentrados produzidos, com níveis de patulina nestes entre 56,04 e 231,44 μg/L. O método desenvolvido para extração e quantificação da patulina foi eficaz. O detector de carga acoplada forneceu boa sensibilidade, precisão, linearidade e recuperação para a determinação quantitativa de patulina. Todos os estágios do processamento do suco contribuíram para a redução dos níveis de patulina. Desde a maçã até o suco a redução foi de 75,2%. A presença de Byssochlamys nivea em uma das amostras de suco indica que a patulina poderia ser produzida por este fungo durante o armazenamento. / Significant amounts of apples produced in the state of Rio Grande do Sul, Brazil, are used to juice production destined to exportation. Fruits used by the industry show some defects and are not accepted for fresh consumption. These apples are a risk factor considering the contamination by fungus, which can produce mycotoxins, such as patulin. Brazil does not have legislation for patulin in food. But many countries have adopted the recommendation of the Codex Alimentarius, which indicates a maximum permitted level of 50 μg/L of patulin in apple juice. This concern stimulates exporting countries, like Brazil, in the research of mycotoxigenic quality of apples and by-products, to become competitive and provide safe foods. In this context, the objectives of this work were (1) evaluate mould contamination of apples used to juice production and possible presence of patulin in apples; (2) methodologically adapt the thin-layer chromatographic technique for quantification of patulin in apples and apple juice; (3) analyze patulin levels in some processing stages of apple juice production; and (4) investigate heat-resistant moulds in juices. The apples and juice samples were obtained from Golden Sucos Ltda, located in Farroupilha city, in Rio Grande do Sul state, Brazil. Penicillium expansum was the most frequently isolated fungus, among these isolates, 94.44% were able to produce patulin under the tested conditions. The prevalence of patulin-producing fungi in apples resulted in high patulin levels in these apples, ranging from 254.62 to 653.37 μg/Kg and in juices ranging from 56.04 to 231.44 μg/L. The method developed to patulin extraction and quantification was effective. The charged coupled device imaging system provides good sensitivity, precision and linearity for the quantitative determination of patulin. All stages of apple juice production contributed to patulin reduction. The overall loss of patulin through processing from apple to apple juice was 75.2%. The presence of Byssochlamys nivea in a juice sample indicates that this fungus could produce patulin during storage.