O presente trabalho visou utilizar o perfil polifásico, envolvendo características fenotípicas e genotípicas, na identificação de Fusarium spp. isolados de grãos de trigo como também investigar a presença das principais toxinas: desoxinivalenol e zearalenona nos grãos de trigo de três regiões produtoras de trigo (São Paulo, Paraná e Rio Grande do Sul). Nossos resultados indicaram que os gêneros mais frequentes no trigo foram Alternaria, Fusarium e Epicoccum. A determinação do genótipo por qPCR demonstrou predomínio de 15-ADON seguido por NIV e 3-ADON. A quantificação de DNA demostrou que o perfil 15-ADON foi responsável por 96% de todo DNA quantificado, seguido por NIV com 3.84% e 3-ADON, com somente 0.06%, indicando que 15-ADON, é o principal genótipo de tricoteceno nos grãos de trigo nacional. A toxina desoxinivalenol foi detectada em todas amostras analisadas, com mediana de 323, 466 e 783 µg/kg em SP, PR e RS, respectivamente. A determinação de zearalenona demonstrou contaminação em 100%, 80% e 42% dos grãos dos Estados do RS, PR e SP e medianas de 843, 100 e 14 µg/kg, respectivamente. / The present work aimed to use a polyphasic approach, by phenotypic and genotypic characteristics for the identification of Fusarium strains from wheat grains as well to investigate the presence of deoxynivalenol and zearalenone on wheat grains from three wheat producers States (Parana, Rio Grande do Sul and Sao Paulo). Our results showed that the main genera found were Alternaria, Fusarium and Epicoccum. Genotype detection from qPCR revealed predominance of 15-ADON, followed by NIV and 3-ADON. The qPCR demonstrated that 15-ADON genotype was responsible for 96% of all DNA quantified, followed by NIV with 3.84% and 3-ADON with 0.06%, indicating that 15-ADON is the main trichothecene genotype from Brazilian wheat grains. The toxin deoxynivalenol was detected in all 150 samples analyzed from wheat grains, with medians of 323, 466 and 783 µg/kg in SP, PR and RS, respectively. The zearalenone determination showed contamination on 100%, 80% and 42% of wheat grains from RS, PR and SP State and medians of 843, 100 and 14 µg/kg, respectively.

Les mycotoxines sont des métabolites secondaires de moisissures contaminant de façon naturelle de nombreuses denrées alimentaires, notamment les céréales. Le déoxynivalénol (DON), produit par Fusarium sp., est la mycotoxine la plus répandue dans le monde. Du fait de sa grande stabilité chimique, le DON est difficile à éliminer, et se retrouve dans les céréales et les produits finis ou il induit des effets toxiques pour l'homme et l'animal. De nouvelles stratégies de lutte sont mises en places, telle la transformation biologique utilisant des bactéries ou des plantes. En effet certaines bactéries possèdent des enzymes capables de transformer le DON en de nouveaux composés, le déepoxy-déoxynivalénol (DOM-1) et le 3-épi-déoxynivalénol (3-epi-DON). De plus, certaines plantes sont naturellement capables de transformer le DON dans le but de l'éliminer et de le détoxifier, formant ainsi le deoxynivalénol-3-ß-D-glucoside (D3G). L'objectif de cette thèse était d'évaluer la toxicité de ces dérivés du DON au niveau de l'intestin et du système immunitaire par le biais d'analyses in silico, in vitro, ex vivo et in vivo. Les tests de toxicité in vitro sur la lignée humaine intestinale cellulaire Caco-2 montrent que le DOM-1, le 3-epi-DON et le D3G n'étaient pas cytotoxiques, ils ne modifiaient ni la viabilité, ni la fonction de barrière des cellules, mesurée par la résistance électrique transépithéliale. Les tests de toxicité ex vivo sur des explants jéjunum porcin ont montré que le DOM-1, le 3-epi-DON ou le D3G n'induisaient pas de modifications histomorphologiques. En revanche, les explants exposés au DON montraient des lésions morphologiques et une régulation positive de l'expression des cytokines pro-inflammatoires. L'impact de ces trois dérivés a été également analysé sur l'expression de l'ensemble des gènes du tissu, avec une analyse microarray. Ceci a montré que ces dérivés du DON n'induisaient aucun changement dans l'expression des gènes par rapport au groupe contrôle. Le DON quand a lui exprimait différentiellement 747 sondes, correspondantes à 333 gènes impliqués dans l'immunité, la réponse inflammatoire, le stress oxydatif, la mort cellulaire, le transport moléculaire et la fonction mitochondriale. L'analyse in silico a montré que le D3G, contrairement au DON était incapable de se lier au site-A du ribosome, principale cible de la toxicité pour le DON. Les deux dérivés microbiens eux, étaient capables de se fixer au site-A au sein du ribosome, mais contrairement au DON ils ne formaient que deux liaisons hydrogènes au lieu de trois. De plus, ces trois dérivés n'induisaient pas de stress ribotoxique, d'activation des MAPKs (mitogen-activated protein kinases), et de réponse pro-inflammatoire. Une étude complémentaire a été menée in vivo pour évaluer la toxicité du DOM-1 chez le porc (gavage pendant 21 jours avec .0.14mg / kg de poids vif). Les résultats ont montré que le DOM-1, contrairement au DON n'induisait pas les effets toxiques du DON au niveau des paramètres zootechniques (pas de vomissements, aucune diminution de la consommation alimentaire ou de perte de poids), sur l'intestin et le foie (pas de dommages tissulaires), ou sur la réponse immunitaire (pas de réponse inflammatoire induite). En conclusion, nos résultats montrent l'efficacité de ces transformations enzymatiques. La déepoxydation et l'épimérisation bactérienne, ainsi que la glycosylation par les plantes permettent de sensiblement diminuer la toxicité du DON, passant par une absence de toxicité sur le ribosome avec une absence d'activation des MAPKs et de réponses inflammatoires. Dans ce contexte de contamination par les mycotoxines, ces méthodes de luttes alternatives semblent être des approches prometteuses. / The Fusarium sp. mycotoxin deoxynivalenol (DON) is one of the most frequently widespread mycotoxin worldwide. Due to its high structural stability, the elimination of DON, once present in cereals or feed materials, becomes difficult. Thereby, it is present in many cereals and final feed products, inducing several toxic effects on human and animals, and causing big economic losses. New strategies of to fight against mycotoxins were developed, as biological transformation, either by the use of bacteria or plants. Indeed, some microorganisms are able to transform DON in new products, by enzymatic reaction, forming the deepoxy-deoxynivalenol (DOM-1) and the 3-epi-deoxynivalenol (3-epi-DON). Moreover, some plants naturally own the capacity to glycosylate DON in the aim to detoxify it, forming the deoxynivalenol-3-ß-D-glucoside (D3G). The aim of this thesis was to assess the toxicity of these DON derivatives, on the intestine and immune response, using several approaches such as in silico, in vitro, ex vivo and in vivo models. On the human intestinal Caco-2 cell line, DOM-1, 3-epi-DON and D3G were not cytotoxic; they did not alter its viability and barrier function, as measured by the trans epithelial electrical resistance. The expression profile of DOM-1, 3-epi-DON and D3G-treated jejunal explants was similar to that of controls and these explants did not show any histomorphology alteration. On the other hand, the treatment of intestinal explants with DON, induced morphological lesions and upregulated the expression of proinflammatory cytokines. The impact of these three derivatives was also studied on intestinal explants with a pan-genomic transcriptomic analysis. Results show that the derivatives of DON did not induce any change on the gene expression in comparison to the control-treated explants. In contrary, DON-treated explants differentially expressed 747 probes, representing 323 genes involved in immune and inflammatory responses, oxidative stress, cell death, molecular transport and mitochondrial function. In silico analysis revealed that D3G, opposing to DON, was unable to bind to the A site of the ribosome, which is the main target for DON toxicity. Both DOM-1 and 3-epi-DON were able to fit into the pockets of the A site of the ribosome but only by forming two hydrogen bonds, while in this position, DON forms three hydrogen bonds. Moreover, the three derivatives do not elicit a ribotoxic stress, MAPKinase activation, and inflammatory response. Then, an in vivo study was carried out to assess the toxicity of DOM-1 on pig (feed forced during 21 days at 0.14 mg/Kg BW). The results showed that DOM-1 does not have as much toxic effects as DON on zootechnical parameters (no emesis induced, no decrease of food consumption or weight loss observed), on intestine and liver (no tissues damages), or on the immune response (no inflammatory response induced). Our data demonstrate that bacterial de-epoxidation or epimerization of deepoxy-DON modified its interaction with the ribosome, leading to an absence of MAPKinase activation and toxicity; and that the glycosylation of DON suppresses its ability to bind to the ribosome and decreases its intestinal toxicity. The mycotoxin deoxynivalenol (DON) remains an important challenge in many regions in the world. Thus, these biological detoxifications of DON seem to represent a new promising approach helping manage the problem of its contamination.

Deoxynivalenol (DON) contamination of grain destined for animal feeds is a major toxicological risk to monogastrics and is suspected of restricting productivity in ruminants. Whereas bacterial additives have been developed that can detoxify DON in the rumen and lower intestine, there are currently no commercial inoculants able to perform this task in crimped grain (CG) silage, a regionally important method of moist grain preservation based on homo- and heterofermentative lactic acid bacteria or chemical additives. Determining whether this ensiling process alongside the action of detoxifying bacteria has the potential to remove DON in CG prior to ingestion, was explored in mini-silo ensiling experiments. CG was heat treated (100 °C, 60 min) or ensiled fresh in triplicate 50 g silos, spiked with 5 mg/kg DON and inoculated with lactic acid bacteria derived from wild birds, natural epiphytic inoculants and commercially sourced silage additives (21 d). DON recovery was only significantly reduced (31.2 ± 14.4% recovery, p < 0.001, n= 30) by heat treatment, as determined by IAC-RP-HPLC-UV. Bacterial assemblage analysis by 16S rRNA PCR-DGGE-SEQ identified Weissella cibaria, Pantoea agglomerans, Bacillus subtilis, B. licheniformis and Hafnia alvei as candidate detoxification agents, of which W. cibaria and H. alvei decreased DON recovery in vitro (11.3 and 6.2% recovery respectively, p < 0.05, n = 18), which translated to inoculated W. cibaria yielding a decrease in DON recovery (67.2± 14.4%, 28 d) in naturally contaminated crimped wheat (13.5 ± 1.0 mg/kg, 35-40% moisture, p < 0.05, n =15). As W. cibaria is a lactic acid bacteria already associated with fermented CG by default it has promise as a novel DON detoxification agent in CG silage. DON is however just one of many hepatotoxic co-contaminants. Retrorsine, a DNA-crosslinking pyrrolizidine alkaloid derived from Ragwort (Senecio sp.) was investigated for interactive toxicity with DON in an in vitro co-exposure experiment. HepG2 cells were exposed to Log10 multifactorial binary exposures for 48 h followed by a suite of assays to elucidate mechanisms of interactive cytotoxicity, genotoxicity and modulation of the proteome. Retrorsine was tentatively confirmed to form DNA/protein crosslinks in the comet, micronucleus and crosslinking assays, whilst DON was found to potently induce cytotoxicity and apoptosis. Co-exposure yielded a complex toxicity response, with low doses yielding antagonistic effects and high doses trending towards additive effects, although DON dose was generally the principle component. The difficulties associated with undertaking an interactive toxicity study where both toxins have multiple metabolic and cellular targets are highlighted.

A presença de desoxinivalenol (DON) em produtos da moagem de trigo tem sido estudada, evidenciando que a contaminação ocorre em todas as frações. No Brasil, os limites máximos toleráveis (LMT) para DON em cereais e produtos de cereais são regulamentados pela Agência Nacional de Vigilância Sanitária - ANVISA. O entendimento da distribuição de DON nas frações da moagem do trigo é importante, pois pode fornecer base técnica para o gerenciamento desta contaminação e subsídios para as agências regulamentadoras no estabelecimento e ou revisão dos LMT. Este estudo teve como objetivo principal avaliar a distribuição de DON entre as frações obtidas da moagem experimental de grãos de trigo naturalmente contaminados. Trinta amostras de grãos de trigo (3-4 kg), naturalmente contaminadas com DON em concentrações que variaram de 350 a 4.150 ?g.kg-1, passaram por processo de limpeza (Labofix Brabender), seguido de condicionamento e moagem experimental (Brabender Quadrumat Senior). Quatro frações foram obtidas, de acordo com o tamanho das partículas: farelo (>=530 ?m), farelinho (>=195 e <=529 ?m), farinha de quebra (<=154 ?m) e farinha de redução (>=155 e <=194 ?m). As frações e o resíduo de limpeza foram avaliados quanto ao percentual obtido e concentração de DON. O DON foi extraído com H2O destilada, seguido de purificação em coluna de imunoafinidade e detecção/quantificação empregando cromatografia líquida de alta eficiência e detector de arranjo de diodos (UV-DAD). O percentual médio de resíduo de limpeza foi de 15,9% (6,9 a 23,2%). A redução média de DON nos grãos limpos foi 22,5% de (5,5 a 37,3%). A moagem experimental do trigo produziu em média 28,5% de farelo, 5,8% de farelinho, 27,9% de farinha de quebra, 37,8% de farinha de redução, totalizando 65,7% de farinha. As concentrações de DON verificadas no farelo e farelinho foram significativamente maiores quando comparadas com as farinhas (p<=0,05), as quais não diferiram entre si. A concentração relativa (CRel) de DON (relação entre a concentração da fração e do grão) indicou que a concentração de DON foi, em média, 73% maior no farelo e 35% maior no farelinho, comparada à concentração inicial nos grãos limpos. Na farinha de quebra, farinha de redução e farinha total a CRel foi em média 24%, 38% e 33% menor que nos grãos limpos, respectivamente. De acordo com os LMT para DON em farelo (2.000 ?g.kg-1) e farinha de trigo (1.750 ?g.kg-1), em vigência no Brasil, das 30 amostras avaliadas, 15 amostras de farelo (50%) e 1 amostra de farinha (3%) encontravam-se acima dos LMT. Considerando os LMT previstos pela legislação para 2017 (1.000 ?g.kg-1 para farelo e 750 ?g.kg-1 para farinha), 22 amostras de farelo (73%) e 16 amostras de farinha (53%) estariam acima dos LMT. Os resultados observados neste estudo demonstraram que a moagem experimental de grãos de trigo com contaminação igual a 3.000 ?g.kg-1, que será o LMT para grãos para posterior processamento em 2017, poderá acarretar a produção de farelos e farinhas com contaminação acima dos LMT previstos nesta mesma legislação. / The presence of deoxynivalenol (DON) in wheat milling products has been studied, showing that the contamination occurs in all streams. In Brazil, the maximum tolerable limits (MLT) for DON in cereals and cereal products are regulated by Agência Nacional de Vigilância Sanitária - ANVISA. The knowledge of DON distribution in wheat milling streams is important to provide the technical basis for the management of this contamination and could assist regulatory agencies to establish or review MTL. The main purpose of this study was to evaluate the fate of DON among experimental milling streams of naturally DON contaminated wheat kernels. Thirty wheat samples (3-4 kg), naturally DON contaminated (350-4.150 ?g.kg-1) were cleaned (Labofix Brabender), conditioned and milled (Brabender Senior Quadrumat mill). Four milled streams were obtained, according to the particle size: bran (>=530 ?m), shorts (>=195 e <=529 ?m), break flour (<=154 ?m) and reduction flour (>=155 e <=194 ?m). The streams and the screenings were evaluated for percentage and DON contents. DON analysis was performed with distilled H2O as extraction solvent, immunoaffinity column for cleanup and HPLC/diode array detector. The mean screening percentage obtained was 15,9% (6,9 to 23,2%). The mean reduction of DON in the cleaned wheat was 22,5% (5,5 to 37,3%). In the experimental milling of wheat it was obtained 28,5% of bran, 5,8% of shorts, 27,9% of break flour, and 37,8% of reduction flour, totaling 65,7% of flour. DON concentration obtained in bran and shorts were significantly higher when compared with the flours (p<=0,05), which did not differ from each other. The Relative Concentration (RConc) of DON (ratio between stream and cleaned wheat concentration) indicated that DON concentration was, on average, 73% higher in bran and 35% higher in shorts, compared to the initial concentration of the cleaned wheat, whereas in the break flour, reduction flour and total flour it was 24%, 38% and 33%, respectively, lower than the cleaned wheat. According to MTL for DON in bran (2.000 ?g.kg-1) and wheat flour (1.750 ?g.kg-1), in force in Brazil, the 30 samples evaluated, 15 samples of bran (50%) and 1 sample of flour (3%) were above the MLT. Considering the MTL provided in the legislation for 2017 (1.000 ?g.kg-1 for bran and 750 ?g.kg-1 for flour), 22 samples of bran (73%) and 16 samples of flour (53%) were above the MTL. The results of this study demonstrate that the experimental milling of wheat kernel contaminated at level of 3.000 ?g.kg-1 of DON, which will be the MTL for wheat kernels for further processing in 2017, may result in bran and flour contamination above the MTL provided in the same legislation.

Mycotoxins are secondary metabolites of moulds, which attack cereals, for example barley, from which mycotoxins then get to beer. This submitted work is focused on ochratoxin A, deoxynivalenol and zearalenone, which can occur in beer. The first part of this master’s thesis consists of literary research, which describes mycotoxins in general, points out their occurrence, prevention of their formation and delivers information about their physical and chemical properties and toxicity. Furthermore, the research contains basis of malt and beer technology, the occurrence of mycotoxins in beer and raw materials for its production. The research describes changes in concentration of mycotoxins across malt and beer production. The next part deals with possibilities of determination of mycotoxins in barley, malt and beer, compares individual methods of their determination and points out many difficulties of some analyses. The experimental part of this work pursues determination of ochratoxin A, deoxynivalenol and zearalenone in different types of beer with the help of UPLC-FLR, HPLC-MS and ELISA. Instrumental techniques are validated and gathered results are compared with the results in literature. The goal of this master’s thesis is to assess the stability of ochratoxin A and deoxynivalenol in beer over time. The gained results show that there are changes in the concentration of ochratoxin A over time, nevertheless those changes show no pattern. Overall, there was a decrease in concentration in 47 % of the samples and an increase in 28 % of them. In the rest of the samples the concentration did not change. The concentration of deoxynivalenol does not change over time. One of the other goals of this thesis is monitoring of selected mycotoxins in beer. The average concentration of ochratoxin A in the samples was 39 ng/l and deoxynivalenol 9,9 g/l. Zearalenone did not occur in any of the samples when determined by liquid chromatography. All results agree with literature. Next, the thesis compares different analytical methods for determination of ochratoxin A, deoxynivalenol and zearalenone. The screening method ELISA is compared to UPLC-FLR and HPLC-MS. The determination of ochratoxin A by ELISA has shown to be time consuming, nevertheless the results responded to instrumental technique. ELISA overestimated the results of determination of deoxynivalenol in beer by 363–697 % and with zearalenone there were found false positive results.

This diploma thesis is focused on the analysis of toxins and their extraction from sandyloamy soil. Particularly, saxitoxin and trichothecene mycotoxins deoxynivalenol, HT-2 and T-2 toxins are in the centre of interest of this work. Their occurrence, toxic properties and influence on living organisms are described in theoretical part of this thesis. In next chapters, currently published extraction methods for individual toxins and analytical approaches for their quantitative evaluation are summarized. In experimental part of this thesis, optimized process of sample pretreatment based on extraction of toxins from soil using 1mM HCOONH4 in 84% acetonitrile was proposed as the best option. Simultaneous determination of toxins was performed by liquid chromatography on a CN column (3.0 x 150 mm, 3 Îm, 100 ) in gradient elution mode. Mass spectrometer with electrospray as ion source and linear ion trap as analyzer was used as detector. Recovery of designed method was over 80% for trichothecene mycotoxins and 51% for saxitoxin.

No description available.

<p>In sum, these studies described in this dissertation showed that both mycotoxin deoxynivalenol exposure and nutrient starvation can increase endocytosis and degradation of tight junction proteins in the lysosome. Therefore, manipulation of endocytic pathway as well as the signaling pathway involved in this process can be used to prevent intestinal barrier dysfunction in animals.</p> <p><br></p>

Phenotypic and genotypic characterization of hulled and hulless winter barley (Hordeum vulgare L.) is necessary for improvement using traditional and molecular breeding techniques.  Identification of genomic regions conferring resistance to Fusarium head blight (caused by Fusarium graminearum), leaf rust (caused by Puccinia hordei G. Otth), powdery mildew [caused by Blumeria graminis (DC.) E.O. Speer f. sp. hordei Em. Marchal], net blotch (caused by Pyrenophora teres) and spot blotch [caused by Cochliobolus sativus (Ito & Kuribayashi) Drechsler ex Dastur] will greatly aid in breeding for improved resistance.  Determining factors that contribute to yield differences between hulled and hulless genotypes, and identification of markers associated with yield and yield related traits will greatly aid in improvement of hulled and hulless genotypes.  The hulled cultivar Nomini, hulless cultivar Eve, and hulless line VA06H-48 were consistently resistant to Fusarium head blight (FHB) and had low deoxynivalenol (DON) accumulation.  Screening with molecular markers on chromosomes 2H and 6H for FHB and DON identified quantitative trait loci (QTL) which may confer resistance in Virginia Tech germplasm.  Evaluation of hulled and hulless full-sibs from four populations indicated  that  grain volume weight and protein concentration were significantly (P d 0.05) higher for hulless genotypes, while seedling emergence and grain ash concentration were significantly (P d 0.05) higher for hulled genotypes.  In linear regression analysis, none of the assessed traits explained yield variation in all populations and environments.  Identification of hulless genotypes having yield potentials similar to those of their hulled sibs should be possible after adjusting for hull weight.  A genome wide association study was used to identify chromosome regions governing traits of importance in six-rowed winter barley germplasm and to identify single nucleotide polymorphisms (SNPs) markers for use in a marker-assisted breeding program. Significant SNPs associated with previously described QTL or genes were identified for heading date, test weight, yield, grain protein, polyphenol oxidase activity, and resistance to leaf rust, powdery mildew, net blotch, and spot blotch.  Novel QTL also were identified for agronomic, quality, and disease resistance traits.  These SNP-trait associations provide the opportunity to directly select for QTL contributing to multiple traits in breeding programs. / Ph. D.

Doctor of Philosophy / The digital imaging technology, geographical analyses tool, and computer vision (a technique that enables computers and systems to get meaningful information from images) methods can be used to extract traits-related branching pattern, canopy cover, and pod location in edamame for many plant populations in short time using less labor and resources. Using genome-wide association study, we identified several genetic markers that were associated with those traits. These markers can be used in marker-assisted selection to develop the edamame varieties that are more adaptable to mechanical harvesting and give more yield, along with understanding the physiological mechanisms for better shoot architecture traits and better yield. We used spectral signatures of different edamame at several harvesting time along with machine learning methods to identify the optimal harvest time of edamame. Hyperspectral imaging (a technique that analyzes a wide spectrum of light instead of just assigning primary colors (red, green, blue) to each pixel) when combined with computer vision and machine learning methods can be used to quantify the levels of vomitoxin (chemical that causes vomiting and feed refusal in animal and humans) for larger wheat kernel samples in a cheaper and faster way.