Made available in DSpace on 2014-06-12T18:05:12Z (GMT). No. of bitstreams: 2 arquivo6215_1.pdf: 1677963 bytes, checksum: e550537f637e0f99ab5e08d9174c5447 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2007 / O gênero Aspergillus pertence ao grupo de fungos filamentosos de grande dispersão no meio ambiente. Algumas espécies estão associadas a doenças, principalmente em pessoas imunocomprometidas. Outras são importantes por razões econômicas, devido à aplicação biotecnológica ou à produção de aflatoxinas. A identificação das espécies por métodos tradicionais, aliados ao uso de marcadores moleculares, estão somando conhecimentos e estabelecendo maior eficiência na caracterização de isolados. Foram utilizados os marcadores moleculares ISSR, utilizando os primers (GACA)4 e (GTG)5, e os marcadores ITS e RAPD, com o objetivo de caracterizar geneticamente linhagens de Aspergillus flavus e linhagens de outras espécies pertencentes ao grupo A. flavus. Alta diversidade genética foi obtida com os marcadores RAPD e ISSR, sendo que o primer (GTG)5 gerou menor diversidade que o (GACA)4, mas apresentou perfis característicos para cada espécie. A partir destes dados foi construída uma matriz de similaridade e confeccionados os dendrogramas, através do método de agrupamento UPGMA. Os perfis dos marcadores ISSR e RAPD mostraram que entre as linhagens estudadas de A. flavus, quatro apresentaram perfis diferenciados, tendo sido reclassificadas como A. oryzae, A. parasiticus e duas como A. tamarii. No entanto, uma das linhagens previamente identificadas como A. parasiticus, devido ao seu perfil de marcadores ser idêntico ao de A. flavus, foi revisada como sendo desta espécie. Esses resultados reforçam a importância do uso de marcadores moleculares para a diferenciação de espécies e linhagens fúngicas

Submitted by Danielle Karla Martins Silva (danielle.martins@ufpe.br) on 2015-03-04T12:56:34Z No. of bitstreams: 2 Andre Ferraz Goiana Leal Tese Doutorado_2012.pdf: 1567423 bytes, checksum: 51bb64ce5a0aada499a3747efab44594 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-04T12:56:34Z (GMT). No. of bitstreams: 2 Andre Ferraz Goiana Leal Tese Doutorado_2012.pdf: 1567423 bytes, checksum: 51bb64ce5a0aada499a3747efab44594 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012 / Aspergillus é um fungo ubíquo que pode causar uma variedade de síndromes clínicas, especialmente em pacientes imunossuprimidos. Esta pesquisa teve como objetivos caracterizar o perfil de carboidratos da parede celular de espécies de Aspergillus, avaliar a susceptibilidade antifúngica in vitro ao itraconazol e o efeito antifúngico de formulação lipossomal de itraconazol no tratamento de ceratite experimental por A. flavus. As lectinas usadas no ensaio foram wheat germ agglutinin (WGA), Ulex europeus agglutinin I (UEA I), peanut agglutinin (PNA) e concanavalina A (Con A) todas conjugadas a peroxidase. A metodologia utilizada na susceptibilidade antifúngica in vitro seguiu as condições descritas no protocolo M38-A2 do Clinical and Laboratory Standard Institute. Os lipossomas foram obtidos pelo método de hidratação do filme lipídico seguido de sonicação. Fêmeas adultas de ratos Wistar (pesando 200-220g) foram imunossuprimidas com apenas uma aplicação intraperitoneal de 150mg/kg de ciclofosfamida três dias antes da infecção com A. flavus na concentração de 107 esporos/mL. Após 48h da infecção, os animais foram tratados com a formulação lipossomal. Para efeito de comparação um grupo de animais (n=6) foi tratado com o fármaco não encapsulado. Ao fim do experimento os animais foram avaliados quanto a: manifestações clínicas, unidade formadora de colônias (UFC/g) e exame direto/histopatológico. Nossos resultados mostraram que houve expressão de N-acetil-D-glicosamina na superfície da parede celular das espécies de Aspergillus analisadas em espécimes histopatológicas (cérebro e pulmão) e crescidos em meio de cultura batata dextrose ágar (BDA). Todas as amostras sensíveis ao itraconazol expressaram de moderada a alta concentração de N-acetil-D-glicosamina na parede celular. No estudo in vivo o grupo de animais tratados com o fármaco livre, um apresentou opacificação total da córnea, três apresentaram edema de córnea em dois quadrantes e dois apresentaram um pequeno edema. No grupo de animais tratados com a formulação lipossomal dois animais apresentaram edema de córnea moderada, um apresentou um pequeno edema e três animais não apresentaram qualquer lesão. No grupo de animais tratados com o fármaco livre foi possível quantificar de 2 a 13 UFC/g e visualizar estruturas fúngicas ao exame direto/histopatológico em todas as amostras de globo ocular. No grupo tratado com a formulação lipossomal metade das amostras avaliadas (três animais) não foi visualizado nenhum crescimento fúngico (cultura) e nem filamentos micelianos ao exame direto/histopatológico. Os resultados obtidos neste estudo indicam a formulação lipossomal do itraconazol apresenta uma atividade antifúngica significativamente maior do que o fármaco livre no tratamento de ceratite fúngica experimental por A. flavus em ratos Wistar.

This dissertation consists of three manuscripts for publication: Appendix A presents a genomic comparison of Aspergillus flavus isolates with different morphologies, and Appendices B and C present the identification and systematics of an emerging snake pathogen, Ophidiomyces ophiodiicola. The comparative genomics project of A. flavus tests the hypothesis that isolates with different morphologies within the species are adapted to different niches. Our results reveal differences in genome structure and protein content that are implicated in niche adaptation to the soil and phyllosphere. The systematics project of O. ophiodiicola was initiated to resolve the frequent misidentification of emerging reptilian diseases that is occuring in the literature. One of these emerging pathogens, O. ophiodiicola, was incorrectly described in the genus Chrysosporium due to its resemblance in spore morphology; therefore, the taxonomy of the genus was revised. We hope the review will aid in accurate identification and tracking of emerging reptilian diseases to better understand their epidemiology.

My research focuses on the development of a novel method for the fast detection of aflatoxin-producing fungi from the volatile organic compounds that they produce. Aflatoxins have received great attention because of their demonstrated potent carcinogenic effect in susceptible laboratory animals and their acute toxicological effects in humans. Traditional detection and quantification techniques are considered time-consuming, high cost, and require technical professionals. The `odor' or so called volatile metabolites released by a fungus is the key for fast detection. Several researchers have reported that diverse fungi species have unique volatile metabolite patterns. This study focuses on answering several questions: Is it possible to discriminate aflatoxins-producing fungi from other fungi based on volatile metabolites? What are the key discriminating biomarkers related to each fungus? Does the growth environment have an effect on the production of volatile metabolites? What chemicals are consistently emitted by a fungus under varied conditions? To answer these questions, one toxigenic and one nontoxigenic A. flavus isolate were studied to evaluate the microbial volatile organic compound (MVOC) profiles. The results described in chapter two of this dissertation indicate that MVOC production is time-dependent and that aflatoxigenic and non-aflatoxigenic strains have different MVOC expression patterns. Chapter three describes the effects of experimental parameters on fungal volatile metabolites. The identity and quantity of MVOCs can be affected by many factors including SPME fiber type, fungal growth media, and growth temperature. A CAR/PDMS coated fiber performed better than the other SPME fibers by collecting a larger variety and quantity of MVOCs. Fungi grown on the chemical defined liquid media produced much larger quantities of MVOCs compared to the other media. The highest MVOC production results were found at 30 degrees Celsius. The fungi discrimination study was extended in chapter four by including 3 toxigenic and 3 non-toxigenic isolates using multivariate analysis. The results indicate that volatile patterns vary even at the fungal isolate level and that discrimination of aflatoxin-producing fungi from non-toxigenic fungi is possible.

Maize (Zea mays L.) is a global food staple and is at risk from infection by the pathogenic fungus Aspergillus flavus L. The ubiquitous, soil-borne fungus causes ear rot of maize and produces the carcinogenic secondary metabolite known as aflatoxin. Aflatoxin B1 is the most potent carcinogenic mycotoxin known, causing hepatocellular carcinoma, along with many other serious health problems such as immunosuppression. Previous studies have shown that maize cob tissue plays an essential role in both facilitating and limiting the spread of the fungal pathogen A. flavus, however, little attention in the literature has been given to the cob. To date, there have not been any studies published describing the metabolome of maize cob tissue. This study assessed three different methods for disruption of maize cob tissue and investigated the global metabolome of maize cob of two resistant (Mp313E and Mp420) and two susceptible (B73 and SC212m) genotypes via liquid chromatography-mass spectrometry. Three treatments (control, water-inoculated, and fungus-inoculated) and three-time points (3, 9, 15 days after inoculation) were included in the experimental design. For the first time in maize cob, 69 metabolites were identified via the mzCloud online database. Out of them, 28 metabolites showed statistically significant differences in abundance across the treatments. Twenty-two metabolites were identified via Fragment Ion Search, of which 14 were statistically significant differences in abundance across the treatments. The majority of the metabolites identified where from the phenylpropanoid, linoleic acid, and terpenoid biosynthesis pathways. Thousands of unknown ions were detected, and for 521 compounds the formula could be derived, based on accurate monoisotopic masses. In the targeted metabolomics analysis, the MS3 spectral tree was obtained for zealexin B1 for the first time via a highly induced sample and was subsequently used to identify zealexin B1 in maize cobs (Va35). To date, this work is the sole metabolomic profiling study of maize cob tissue, and it provides insight into constitutive and induced molecular antifungal defenses of resistant and susceptible genotypes. A list of significant fungal-induced metabolites related to the maize-A. flavus defense response was compiled for further targeted metabolomic identification.

Aspergillus flavus exemplifies a pathogenic fungus that remains a significant contributor to the loss of corn (Zea mays) crops. The production of carcinogenic aflatoxins renders the crop hazardous for consumption and causes significant loss to farmers. Therefore, the prevention of A. flavus contamination continues to persist as a topic for research intervention. Host resistance to this pathogen provides a promising source of defense for the corn plant. Corn inbred line Mp313E was previously identified to exhibit significant resistance against the A. flavus fungal infection and aflatoxin contamination. Quantitative trait loci (QTL) mapping has previously established four major QTL locations associated with aflatoxin resistance in the corn inbred line Mp313E. Near-isogenic lines were developed containing these previously identified QTLs from backcrossing of inbred lines Mp313E (resistant donor parent) and Va35 (susceptible recurrent parent). Quantitative RT-PCR (qRT-PCR) was used to study gene expression patterns of 17 genes selected from plant-pathogen interaction pathways. Furthermore, genomic primer analysis was used for establishment of 15 candidate genes for marker- assisted breeding.

Aflatoxin is a carcinogen produced by the fungus Aspergillus flavus that causes millions of dollars in agriculture losses in the southeastern US. This thesis examines the dispersal of A. flavus on two corn inbred lines, resistant (Mp313E) and susceptible (B73), which differ in total aflatoxin accumulation after infection with A. flavus. After inoculating corn kernels with the fungus an RNA analysis was used to determine the location (number of kernels away from inoculation site) and abundance of A. flavus at weekly intervals. A. flavus started its spread at 7 days after inoculation (DAI) on both corn lines. The B73 corn line showed a constant spread of 3.4mm per day until the entire ear was infected at 21 DAI. The spread on Mp313E did not proceed beyond 3 kernels away from the inoculation site following 7 DAI. The results are significant because they show a faster rate of spread than previously reported and they help quantify the ability of Mp313E to mitigate infection.

No description available.

La contamination des aliments par les mycotoxines est une source très importante de gaspillage de nourriture. Depuis des années, la stratégie classique pour lutter contre ces contaminants est l’utilisation de pesticides. Cependant, il a été montré que ces produits pouvaient avoir des effets nocifs sur la biodiversité et la santé humaine et animale. Il est donc nécessaire de développer de nouvelles stratégies, plus écologiques, pour lutter contre les mycotoxines. L’utilisation de produits naturels pourrait représenter une alternative intéressante et plus respectueuse de l’environnement. En effet, certains produits naturels sont capables d’inhiber la production de mycotoxines. Cependant, le mécanisme d’action mis en jeu est souvent mal compris. Un des objectifs principaux de ce travail a consisté à identifier des produits naturels capables d’inhiber la production de mycotoxines et d’élucider leur mécanisme d’action moléculaire. Pour ce faire, un outil moléculaire a été conçu pour comprendre les mécanismes permettant à des produits naturels d’inhiber la production d’Aflatoxine B1 (AFB1) chez Aspergillus flavus. L’étude de cette mycotoxine est importante puisque c’est un cancérigène puissant chez l’homme et les animaux. Un outil moléculaire permettant l’analyse simultanée de l’expression de 60 des principaux gènes impliqués dans la synthèse de l’aflatoxine B1 par q-*-PCR a été développé. Il permet d’étudier l’ensemble des gènes du cluster d’AFB1 mais également 33 autres gènes codant pour des facteurs de régulation liés à l’environnement dans lequel se trouve la moisissure. Par cette approche, le mécanisme d'action moléculaire de l’eugénol et la pipérine, ainsi que celui de 3 extraits naturels de plantes a été identifié et l'impact de ces composés sur les gènes impliqués dans la production d'AFB1 a été élucidé. L'un des principaux résultats de cette étude a été de montrer que les différents produits naturels modulent systématiquement plusieurs gènes au cours de l'inhibition de la production d’AFB1. Notre étude montre que les produits naturels représentent une alternative possible pour limiter la contamination des aliments par l’AFB1. L'élucidation du mécanisme d'action des produits naturels ouvre de nouvelles perspectives sur les méthodes à employer pour inhiber la production de la toxine. / Mycotoxin’s contamination represents an important source of food spoilage that has to be taken in consideration. For years pesticides, have been used as a common strategy to combat mycotoxin contamination. However, such products were also demonstrated to be harmful to humans and animals’ health. Therefore, it is necessary to find new strategies in order to avoid mycotoxin contamination and the use of natural products could be a promising alternative. Indeed, these compounds may be eco-friendly and several of them are demonstrated aseffective agents against toxin production. Nevertheless, their precise mechanism of action is poorly documented. One of the principal aim of this work consisted in the identification of natural sources capable to inhibit mycotoxin production and in the elucidation of their molecular mechanism of action. For that, we developed a molecular tool aiming the analysis of the impact of natural extracts on the expression of several genes related to Aflatoxin B1 synthesis in Aspergillus flavus. The study of this mycotoxin is an important issue since it is one of the most dangerous compounds inducing cancer in humans and animals. Taking advantage of the well-studied genome of Aspergillus flavus and considering that AFB1 production involves a great number of genetic elements, a q-PCR approach including 60 of the principal genes involved in toxin biosynthesis was developed. This tool simultaneously studies the entire AFB1 gene cluster but also 33 regulatory factors coding for external stimuli to which fungus is exposed. Using this molecular approach, the study of already known but also, new sources of anti-aflatoxigenic compounds was performed. The molecular mechanism of action of 2 isolated molecules and 3 whole plant extracts were determined and the impact of these compounds on the genes involved in AFB1 production was analysed. One of the innovative findings consisted in the demonstration that natural products systematically modulate several genes within AFB1’s inhibition. Taken together, our approach demonstrated that the use of natural products against mycotoxin production can represent an alternative strategy to inhibit food contamination. The elucidation of the mechanism of action of natural products allowed a better understanding of the fungal machinery through which toxin can be inhibited.

Orientador: Ely Nahas / Banca: José da Cruz Machado / Banca: Antônio Carlos Monteiro / Resumo: O conhecimento da atividade das enzimas hidrolíticas é essencial para compreender as transformações dos nutrientes no solo. O milho é uma das principais culturas sob o ponto de vista econômico. O objetivo deste estudo foi avaliar a influência do crescimento dos fungos em dois tipos de solos, na presença e ausência de glicose, no desenvolvimento das plântulas de milho, no padrão isoenzimático dos coleóptilos, nos teores de carboidratos totais residuais, nas atividades da desidrogenase e amilase. Utilizou-se delineamento inteiramente casualizado com esquema fatorial. A adição de glicose no meio de cultura aumentou a velocidade de crescimento de A. flavus e Penicillium sp., mas não de F. verticillioides. Na presença de glicose, a produção de esporos aumentou de 1,2 (F. verticillioides) e 8,2 vezes (A. flavus), e reduzido de 3,5 vezes com Penicillium sp. A redução dos teores de carboidratos totais ajustou-se significativamente às equações de 1º e 2º grau. A.flavus e Penicillium sp., apresentaram maior consumo de carboidratos totais que o solo sem inoculação ou inoculado com F. verticillioides. A adição de glicose no solo favoreceu o consumo de carboidratos residuais, provavelmente devido ao estímulo do crescimento dos fungos. Exceto com F. verticillioides, a atividade da desidrogenase aumentou em média de 1,5 a 1,8 vez (p<0,05) nos solos com glicose em relação aos solos sem glicose. À atividade da amilase, aumentou 1,3 a 1,5 vez por efeito da adição de glicose no solo. Maior atividade da amilase foi observada no solo Latossolo Vermelho distrófico (LVd) com glicose e inoculado com A.flavus e Penicillium sp. em relação ao controle. As dimensões das plântulas como altura dos coleóptilos, comprimento das raízes e massa seca das plântulas de milho dos solos adicionados de glicose e/ou inoculados com os fungos foram diminuídas. A expressão genética... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The knowledge about hydrolytic enzymes activity is essential to understand the nutrients transformations in soil. Corn is one of the main crops from the economic view in the whole world. The objective of this study was evaluate the fungi growth in two soil types, in the presence and absence of glucose, coleoptiles development, roots and weight of corn seedlings, coleoptiles isoenzyme pattern, content of total residual carbohydrates, enzymes dehydrogenase and amylase activity. Was used a completely randomized design with factorial arrangement. The glucose addition in culture medium increased the growth rate of A. flavus and Penicillium sp., but not F. verticillioides. In the presence of glucose, the spores number was increased from 1.2 (F. verticillioides) and 8.2 times (A. flavus), but was reduced 3.5 times with Penicillium sp. Reducing the levels of total carbohydrates, significantly adjusted to the equations of 1st and 2nd grade. A.flavus and Penicillium sp., showed higher carbohydrates consumption that the soil control or inoculated with F. verticillioides. The glucose addition in soil, favored the use of residual carbohydrates, probably due fungi growth stimulation. Except F. verticillioides, dehydrogenase activity increased in the range 1.5 to 1.8 times (p<0.05) in soils with glucose compared to glucose free soil. Amylase activity increased 1.3 to 1.5 times by glucose addition effect in soil. Increased amylase activity was observed in the Distrophic Red Latosol (DRL) with glucose and inoculated with A.flavus and Penicillium sp., compared to control. The seedlings dimensions as coleoptiles height, root length and dry weight of maize seedlings of the soils added with glucose and inoculated with fungi were decreased. Gene expression of maize seed was changed due probable by fungi inoculated infection in soil. The coleoptiles isoenzymes pattern was amended as early as 5 incubation... (Complete abstract click electronic access below) / Mestre