Background: The psychology of behavior change and addictive substance users' motivation to get treatment is a topical issue in addictology. The reasons why patients enter treatment vary; they have different goals, and they differ in the degree to which they are able to reflect these goals. To understand addictive behavior, it is necessary to pay attention to all aspects that determine it, i.e. to recognize the abilities, opportunities and motivations of their behavior and their interaction. The topic of patient motivation at the very beginning of the institutional treatment process of addiction, i.e. when entering detox, was not given enough attention in the Czech Republic, despite the fact that detoxification is in many cases the first, indispensable step in starting medium-term and long-term residential treatment of addiction, and work with motivation is a method of direct work with patients. Aims: To map the motivation for treatment of addiction to addictive substances in patients voluntarily hospitalized at the Detox K20department at the Kosmonosy Psychiatric Hospital. To describe the characteristics of these patients, the differences in their motivation and compare their motivation in terms of socio-demographic data, preferential addictive substances, experience with addiction treatment and...

Bifenilas policloradas (PCBs) foram produzidas comercialmente entre 1929 e meados da década de 1980 para propósitos industriais. As mesmas propriedades que despertaram o interesse industrial, tais como: inércia química, alta constante dielétrica, resistência à queima; foram responsáveis pelo espalhamento dos PCBs em todos os compartimentos ambientais, de tal forma que são encontrados em amostras de tecidos adiposos de animais e humanos, leite, sedimentos dentre outras matrizes. Enormes quantidades de PCBs continuam em uso ou estão estocadas a espera de uma destinação final. No presente estudo demonstramos o uso da reação de hidrodescloração catalítica como forma de destruição/destoxificação de bifenilas policloradas. Para tanto, a reação foi estudada em amostras reais de PCBs (óleo dielétrico - Ascarel®), amostras comerciais (Aroclor® 1242 e 1254) e amostra sintética (2,4-diclorobifenila). O estudo se baseia no uso de solventes orgânicos como meio reacional e paládio suportado em carvão ativado como catalisador, devido à sua seletividade para a reação desejada, bem como sua baixa capacidade de hidrogenar compostos aromáticos. xii A condição experimental ótima para a hidrodescloração foi determinada a partir da aplicação de planejamento experimental do tipo Doehlert. Esta condição ótima foi aplicada com sucesso a PCBs contidos em outras matrizes. A cinética da reação é apresentada para o 2,4-diclorobifenila como estudo de caso e uma proposta de mecanismo da reação de hidrodescloração de PCBs é apresentada baseada nos resultados experimentais. / Polychlorinated biphenyls (PCBs) were produced between 1929 and the 1980s for industrial applications. The same properties that make it a chemical of interest for industrial applications, such as: chemical inertness, high dielectric constant, fire resistance; were responsible for the widespreading of PCBs over all enviornmental compartments. They can be found in samples of fat tissues of humans and animals, milk, sediments, among other matrices. Enormous quantities of PCBs are still in use or stocked waiting for a final destination. In the present study, we have shown the use of catalytic hydrodechlorination as an alternative for the destruction/detoxification of polychlorinated biphenyls. For this, the reaction was studied in real samples of PCBs (dielectric oil - Ascarel®), commercial samples (Aroclor® 1242 e 1254) and pure chlorinated biphenyls (2,4-dichlorobiphenyl). The study is based in the use of organic solvents as reactional media and palladium supported in activated carbon as catalyst, due to its selectivity for the desired reaction and to its low capacity to hydrogenate aromatic compounds. xiv The optimal hydrodechlorination condition was determined through the application of a Doehlert experimental planning. This optimal condition was applied with success to PCBs contained in other matrices. The reaction kinetics for 2,4-dichlorobiphenyl was presented as a case study and a mechanistic proposal was presented for the hydrodechlorination of PCBs based on these experimental conditions.

A exposição humana a microrganismos patogênicos e suas toxinas em alimentos constitui um grave problema de saúde pública. O uso indiscriminado de antimicrobianos convencionais promove o aumento da resistência dos microrganismos às principais moléculas existentes no mercado. Além disso, as limitações do uso de substâncias químicas em matérias primas alimentares têm estimulado pesquisas para o desenvolvimento de metodologias eco-amigáveis para evitar a multiplicação de fungos e/ou eliminar suas toxinas. O Staphylococcus aureus é uma das principais bactérias patogênicas que apresenta taxas elevadas de resistência aos antimicrobianos, e por este motivo tem sido objeto de estudo de pesquisas que tentam identificar novos compostos bioativos para o combate de infecções. O Aspergillus parasiticus é um dos principais fungos produtores de aflatoxinas, substâncias carcinogênicas que contaminam diversos tipos de cereais antes e após o processamento. Estudos recentes demonstraram que extratos de plantas possuem atividade antimicrobiana e antifúngica, além de potencial para degradação de micotoxinas. Neste contexto, o presente estudo teve por objetivos avaliar a atividade antimicrobiana in vitro de extratos brutos e liofilizados de folhas de maracujá, araçá, alecrim e orégano sobre células planctônicas de S. aureus e A. parasiticus, bem como verificar a capacidade dos referidos extratos em degradar in vitro a aflatoxina B1 (AFB1), ocratoxina A (OTA) e zearalenona (ZEA). Os efeitos antibacterianos e antifúngicos dos extratos foram avaliados através da determinação da concentração inibitória mínima (CIM) e concentração bactericida / fungicida mínima (CBM/CFM). Os ensaios de degradação da micotoxinas foram realizados em diferentes tempos de incubação (12-48 h) a 37º C, utilizando-se cromatografia líquida de alta eficiência (HPLC) para determinação das concentrações das micotoxinas. O maracujá não demonstrou atividade antimicrobiana tanto para S. aureus quanto para A. parasiticus. Dos quatro extratos estudados, o araçá demonstrou o maior efeito antibacteriano, cujos valores de CIM para extratos bruto e liofilizado foram 0.39 mg/mL e 0.45 mg/mL, respectivamente. Os menores valores de CIM para A. parasiticus foram obtidos com o orégano liofilizado (8.33 mg/mL) e o araçá bruto (3.215 mg/mL). Contudo, não foi possível identificar valores de CFM para o fungo analisado. Não houve efeito de degradação de OTA e ZEA por nenhum dos extratos avaliados. Todos os extratos reduziram a concentração de AFB1 após 48 h de incubação. A maior porcentagem (60.3%) de redução de AFB1 foi obtida com o extrato de alecrim após 48 h de incubação. A atividade antimicrobiana demonstrada pelos extratos das plantas avaliadas indica um potencial para sua aplicação no combate a bactérias patogênicas e fungos toxigênicos. Este é o primeiro estudo realizado com extratos dessas quatro espécies de plantas que evidenciou a capacidade de redução in vitro de AFB1. / Human exposure to pathogenic microorganisms and their toxins in food is a serious public health problem. The indiscriminate use of conventional antimicrobials increases the resistance of microorganisms to the main molecules available in the market. In addition, limitations on the use of chemical substances in food raw materials have stimulated researches for the development of eco-friendly methodologies to avoid the multiplication of fungi and/or eliminate their toxins. Staphylococcus aureus is one of the major pathogenic bacteria with high rates of antimicrobial resistance, and for this reason it has been the subject of research studies aiming to identify new bioactive compounds to combat infections. Aspergillus parasiticus is one of the main aflatoxin-producing fungi, which are carcinogenic substances that contaminate many types of cereals before and after processing. Recent studies have demonstrated that plant extracts have antimicrobial and antifungal activity, as well as potential for degradation of mycotoxins. In this context, the objective of the present study was to evaluate the in vitro antimicrobial effects of crude and lyophilized extracts of leaves from sweet passion fruit, araçá, rosemary and oregano on planktonic cells of S. aureus and A. parasiticus. The in vitro degradation of aflatoxin B1 (AFB1), ochratoxin A (OTA) and zearalenone (ZEN) by the crude extracts was also investigated. The antimicrobial and antifungal effects were evaluated by determining the minimum inhibitory concentration (MIC) and minimum bactericidal / fungicidal concentration (MBC / MFC). Mycotoxin detoxification assays were conducted at different incubation times (12-48h) at 37 °C. The concentrations of mycotoxins were determined by high performance liquid chromatography (HPLC). Sweet passion fruit had no antimicrobial activity on S. aureus or A. parasiticus. Out of the four extracts evaluated, araçá showed the highest antimicrobial effect with MIC of 0.39 mg/mL and 0.45 mg/mL for crude and lyophilized extracts, respectively. The lowest MIC values for A. parasiticus were obtained with lyophilized oregano (8.33 mg/mL) and crude araçá (3.215 mg/mL). However, no MFC values were obtained for the analyzed fungi. Although OTA e ZEN were not degraded by any extract evaluated, all extracts reduced the concentration of AFB1 after 48 h of incubation. The highest percentage of AFB1 reduction (60.3%) was obtained with rosemary extract after 48h of incubation. The antimicrobial activity demonstrated by extracts of the evaluated plants indicates a potential for application against pathogenic bacteria and toxigenic fungi. This is the first study carried out with extracts of these four plants species that demonstrated the in vitro ability for AFB1 reduction.

Este trabalho objetivou o estudo de rotas de formação de metabólitos secundários utilizando precursores isotopicamente marcados com 13C. Os experimentos de crescimento com adição de [1-13C]acetato, [1,2-13C2]acetato e [U-13C315N1]-L-cisteína para o fungo do ambiente marinho Penicillium sp. DRF2 mostrou que as ciclotiocurvularinas são provenientes da rota de formação de policetídeos e pela incorporação de L-cisteína, depois da transformação desta em 3-mercaptopiruvato. Os resultados sugerem que a formação das ciclotiocurvularinas provém de um processo de detoxificação da α,β-desidrocurvularina. O estudo do metabolismo secundário de Aspergillus sp. DLM3-8, também do ambiente marinho, mostrou que o seu perfil metabólico produzido em experimentos de crescimento sob diferentes condições é constante. Os experimentos de incorporação de precursores isotopicamente marcados com 13C na naftoquinonaimina, produzida por Aspergillus sp. DLM3-8 foram inconclusivos, indicando que outras abordagens experimentais devem ser realizadas para se investigar a biossíntese deste metabólito. / This investigation aimed investigated the formation routes of secondary metabolites using 13C-labelled precursors. Feeding experiments with [1-13C]acetate, [1,2-13C2]acetate and [U-13C315N1]-L-cysteine within the growth medium of the marine-derived fungi Penicillium sp. DRF2 showed that cyclothiocurvularins are derived from polyketides and from the incorporation of a L-cysteine residue, after its transformation into 3-mercaptopyruvate. The results suggest that the formation of cyclothiocurvularins is derived from a detoxification process ofα,β-dehydrocurvularin. Investigation of the secondary metabolism of a marine-derived Aspergillus sp. DLM3-8 indicated a stable metabolic profile under a variety of growth conditions. Feeding experiments with 13C-labelled precursors for the biosynthesis investigation of naphthoquinoneimine were inconclusive, indicating that other methodologies should be envisaged in order to investigate the biosynthesis of this metabolite.

Thesis (Ph. D.)--Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), 2008. / Page 362 blank. / Includes bibliographical references. / Understanding how organisms deal with potentially toxic or fitness-reducing allelochemicals is important for understanding patterns of predation and herbivory in the marine environment. The ability of marine consumers to tolerate dietary toxins may involve biochemical resistance mechanisms, which increase the hydrophilicity of compounds and facilitate their active efflux out of sensitive cells and tissues. While several allelochemical-responsive detoxification enzymes have been sequenced and functionally characterized in terrestrial invertebrates feeding on chemically defended host plants, there is virtually no information concerning the role of these biotransformation enzymes that may mediate feeding tolerance in marine invertebrates. The objective of this research was to assess the diversity and dietary regulation of cytochrome P450s (CYP), glutathione S-transferases (GST) and ABC transporters in the generalist marine gastropod Cyphoma gibbosum feeding on a variety of chemically defended gorgonian corals, and to identify those dietary natural products that act as substrates for these proteins. Molecular and proteomic techniques identified both allelochemically-responsive CYPs, and constitutively expressed GSTs and transporters in Cyphoma digestive glands. Inhibition of Cyphoma GST activity by gorgonian extracts and selected allelochemicals (i.e., prostaglandins) indicated that gorgonian diets are likely to contain substrates for molluscan detoxification enzymes. In vitro metabolism studies with recombinant CYPs suggested those Cyphoma enzymes most closely related to vertebrate fatty acid hydroxylating enzymes may contribute to the detoxification ofichthyodeterrent cyclopentenone prostaglandins found in abundance in selected gorgonian species. / (cont.) Finally, the presence and activity of multixenobiotic resistance transporters in Cyphoma and the co-occurring specialist nudibranch, Tritonia hamnerorum, suggests these efflux transporters could function as a first line of defense against dietary intoxication. Together, these results suggest marine consumers that regularly exploit allelochemical-rich prey have evolved both general (GST and ABC transporters) and allelochemical-specific (CYP) detoxification mechanisms to tolerate prey chemical defenses. / by Kristen Elizabeth Whalen. / Ph.D.

As bases genéticas e moleculares da resistência de Spodoptera frugiperda (J.E. Smith) a lufenuron foram exploradas no presente estudo. Inicialmente, uma linhagem de S. frugiperda resistente a lufenuron foi selecionada a partir de uma população coletada na cultura do milho na região de Montevidiu-GO com intenso uso desse inseticida. As curvas de concentração-resposta a lufenuron para as linhagens de S. frugiperda suscetível (SUS) e resistente (LUF-R) a lufenuron foram caracterizadas pelo método de bioensaio com tratamento superficial da dieta artificial. As CL50 (I.C. 95%) estimadas para as linhagens SUS e LUF-R foram de 0,23 (0,18 - 0,28) e 210,6 (175,90 - 258,10) ?g de lufenuron.mL-1 respectivamente, com razão de resistência de ? 915 vezes. A partir dos resultados de cruzamentos recíprocos entre as linhagens SUS e LUF-R, concluiu-se que a herança da resistência de S. frugiperda a lufenuron é autossômica e incompletamente recessiva. Os testes de retrocruzamentos da progênie F1 de cruzamentos recíprocos com o parental LUF-R demonstraram um efeito poligênico para a resistência, com a estimativa do número mínimo de segregações independentes entre 1,54 e 1,71, indicando que o número de loci associado à resistência é baixo. Para conhecer o perfil de transcritos de lagartas de S. frugiperda e avaliar o padrão de expressão gênica diferencial entre lagartas da linhagem LUF-R em comparação ao de lagartas da linhagem SUS, buscando identificar o(s) mecanismo(s) de resistência a lufenuron, foram utilizadas novas tecnologias de sequenciamento em larga escala. Para isso, foram utilizados sequenciamentos de quatro bibliotecas de cDNA (plataforma HiScan 1000, Illumina©) obtidas de lagartas de 4º ínstar de S. frugiperda das linhagens LUF-R e SUS, induzidas ou não com lufenuron. O transcritoma foi construído utilizando aproximadamente 19,6 milhões de leituras single-end, o que gerou 18.506 transcritos, com N50 de 996 pb. A pesquisa contra o banco de dados nr (NCBI) proporcionou anotação funcional de 51,1% (9.457) dos transcritos obtidos, grande parte dos alinhamentos apresentaram homologia a insetos, com o maior número deles (45%) se assemelhando aos de Bombyx mori (Lepidoptera: Bombycidae), enquanto 10% se assemelharam a sequências de diversas espécies do gênero Spodoptera (Lepidoptera: Noctuidae), sendo 3% dos alinhamentos obtidos contra sequências de Spodoptera frugiperda. A análise comparativa da expressão gênica entre lagartas de S. frugiperda resistente e suscetível a lufenuron identificou 1.224 transcritos expressos diferencialmente (p <= 0,05, teste t; expressão relativa > 2). Sete destes transcritos foram associados ao metabolismo da cutícula, sendo cinco deles superexpressos na linhagem LUFR. O metabolismo de detoxificação apresentou 48 transcritos expressos diferencialmente, dos quais foram identificados 40 transcritos associados às monooxigenases P450, cinco a glutationa-S-transferase, dois às carboxilesterases e um a esterase. Foi observado que 39 dos 48 transcritos associados ao metabolismo de detoxificação foram superexpressos na linhagem resistente. Este padrão foi confirmado a partir da expressão relativa utilizando \"PCR quantitativa em Tempo Real - qPCR\". Estes resultados representam um importante passo para o entendimento dos mecanismos moleculares da resistência de S. frugiperda a lufenuron, proporcionando, ainda, uma visão mais ampla do perfil de expressão gênica de insetos a inseticidas. / The genetic and molecular basis of resistance to lufenuron in Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae) were exploited in this study. The resistant population of S. frugiperda was selected from a population collected in Montevidiu, Goiás. Initially, a luferunon-resistant strain of S. frugiperda was selected from a population collected in cornfields located in Montevidiu, Goiás State, Brazil, with intense use of this insecticide. The diet surface treatment bioassay was used to characterize the concentration-response to lufenuron in the susceptible (SUS) and resistant (LUF-R) strains of S. frugiperda. The estimated LC50s (95% C.I.) for the SUS and LUF-R strains were 0.23 (0.18 - 0.28) and 210.6 (175.90 - 258.10) ?g of lufenuron.mL-1 respectively, with resistance ratio of ? 915-fold. Based on reciprocal crosses between SUS and LUF-R strains, the inheritance of S. frugiperda resistance to lufenuron was incomplete autosomal recessive. Backcrosses between F1 of the reciprocal crosses and the parental LUF-R revealed a polygenic resistance, with an estimation of the minimum number of resistance genes from 1.54 to 1.71, indicating that the number of loci associated to resistance is low. Then, a new high-throughput cDNA sequencing technologies was explored to characterize the transcriptional profile of larvae of Spodoptera frugiperda, and to compare the differential gene expression between resistant and susceptible strains of S. frugiperda to lufenuron in order to identify the resistance mechanism(s) involved. Four cDNA libraries obtained from fourth instars of the resistant (LUF-R) and the susceptible (SUS) S. frugiperda strains, exposed or not to lufenuron, were sequenced in a HiScan1000® platform (Illumina©). The transcriptome was de novo assembled using nearly 19.6 million single-end reads, leading to 18,506 transcripts with a N50 of 996 bp in length. A Blast search against the non-redundant database available in NCBI allowed the functional annotation of 51.1% (9,457) of the obtained transcripts. Most of these transcripts aligned with insect sequences, and a majority of them (45%) with Bombyx mori (Lepidoptera: Bombycidae). Nearly 10% of the transcripts aligned with species belonging to Spodoptera (Lepidoptera: Noctuidae), with 3% of the alignments matching sequences from Spodoptera frugiperda. Differential gene expression analysis between the resistant and the susceptible strains identified 1,224 differentially expressed transcripts (p <= 0.05, t-test; fold change > 2). Seven of them were associated with the cuticle metabolism, and five out seven were up-regulated in the resistant strain (LUF-R). A large set of transcripts (48) associated with the detoxification metabolism was differentially expressed; 40 P450 monooxygenases, five glutathione-Stransferases, two carboxylesterase and one esterase were identified. Thirty-nine out of these 48 transcripts were up-regulated in the resistant strain. Gene expression data obtained by RNA-Seq analysis was validated by quantitative real time PCR (qPCR) of several selected target transcripts. These results represent an important step toward the understanding of the molecular mechanisms of resistance of S. frugiperda to lufenuron, and provide a broader view on the gene expression profile of insects to insecticides.

Les polluants atmosphériques de type oxydes d’azote (NOx), principalement constitués du NO, NO2 et leurs dérivés, représentent une énorme menace d’un point de vue environnemental et sanitaire. Leurs propriétés chimiques sont largement exploitées à l’échelle du vivant pour leur rôle dans divers processus de signalisation (systèmes nerveux et cardiovasculaire) ou l’élimination de pathogènes (système immunitaire). Néanmoins, des dérégulations dans la production cellulaire ou l’apport exogène de ces composés est à l’origine de nombreuses pathologies humaines (e.g. pulmonaires), généralement attribuées à la pollution. Toutefois, un grand nombre de microorganismes aéroportés sont continuellement exposés à ces composés délétères, intimement connectés aux espèces réactives de l’oxygène (ROS). Ainsi l’hypothèse de l’ensemble de ce travail a porté sur l’impact du NO2, NOx majoritairement retrouvés dans l’atmosphère, sur une souche aéroportée de P. fluorescens, espèce désormais associée aux voies aériennes et potentiellement pathogène. A l’issue d’une exposition à 45 ppm de NO2, la survie de P. fluorescens MFAF76a est significativement impactée suggérant un effet bactériostatique, conforté par l’impact observé sur le métabolisme énergétique. De plus, le NO2 induit un stress d’enveloppe via la perte d’un glycérophospholipide (UGP) et le remaniement de divers composants membranaires (LPS, peptidoglycane, acides gras). La pompe à efflux MexEF-OprN semblent participer à la stabilisation de la membrane et pourraient être également impliquée dans l’efflux des oxydes d’azotes, mécanismes confortés par l’étude d’un mutant MFAF76a-oprN. La porine majoritaire OprF semble également contribuer à la stabilisation de la membrane externe, néanmoins son implication reste à confirmer. De plus, une interconnexion entre ROS et NOx dans la signalisation (OxyR, IscR), et les mécanismes de détoxification, a été observée. La flavohémoprotéine Hmp semble être un élément crucial dans la détoxification des NOx chez P. fluorescens comme l’illustre un mutant MFAF76a-hmp. Les similitudes importantes entre les effets connus du NO et ceux observés lors d’une exposition au NO2 suggèrent une conversion non enzymatique du NO2, une fois pénétré dans la cellule, en NO. Désormais, une étude plus approfondie est nécessaire afin de décrypter (i) les mécanismes impliqués dans la régulation de la pompe à efflux RND MexEF-OprN et de la flavohémoprotéine Hmp, (ii) d’autres acteurs intervenant dans la réponse au stress d’enveloppe et la détoxification ainsi que (iii) le devenir de NO2 dans la cellule. / Nitrogen oxides (NOx) atmospheric pollutants, mainly constituted of NO, NO2 and derived compounds, are a big threat to the environment and health. Their chemical properties are largely exploited at the cellular scale for their role in diverse physiological processes such as signalisation (nervous and cardiovascular systems) or in pathogens eradication (immunity system).However, dysregulation in production pathways or exogenous input of these compounds lead to several pathologies (e.g. respiratory diseases), usually attributed to atmospheric pollution. However, a wide range of airborne microorganisms are constantly exposed to these deleterious compounds, intimately connected to reactive oxygen species (ROS). Thus, the hypothesis of this work deals with the impact of NO2, the main atmospheric NOx, on an airborne P. fluorescens, a strain usually neglected but yet associated with human airways, and potentially pathogenic. Following an exposure to 45 ppm of NO2, the survival of P. fluorescens MFAF76a is severely impaired, suggesting a bacteriostatic effect, as comforted by NO2 impact on energetic metabolism. Moreover, an exposure to NO2 induces an envelope stress through the loss of an Unknown Glycerophospholipid (UGP) and the reorganisation of membrane constituents (LPS, peptidoglycan, fatty acids). The efflux pump MexEF-OprN is involved in membrane stabilization and could also efflux NOx, as highlighted by a MFAF76a-oprN mutant. The major porin OprF could also contribute in external membrane stabilisation, however its implication is still under investigation. Moreover, ROS and NOx are interconnected as illustrated by their shared signalisation (OxyR, IscR) and detoxification pathways. The flavohemoprotein Hmp is a crucial element in the detoxification of NOx in P. fluorescens as illustrated in an MFAF76a-hmp mutant. The similarities between the known effects of NO and those observed in the case of an exposure to NO2, suggest a non-enzymatic conversion of NO2, following cell penetration, into NO. Henceforth, deeper studies are required to decode (i) the mechanisms involved in the regulation of the RND efflux pump MexEF-OprN and the flavohemoprotein Hmp, (ii) other relevant actor implicated in the envelope stress response and in detoxification pathways as well as (iii) the fate of NO2 within the cell.

A exposição humana a microrganismos patogênicos e suas toxinas em alimentos constitui um grave problema de saúde pública. O uso indiscriminado de antimicrobianos convencionais promove o aumento da resistência dos microrganismos às principais moléculas existentes no mercado. Além disso, as limitações do uso de substâncias químicas em matérias primas alimentares têm estimulado pesquisas para o desenvolvimento de metodologias eco-amigáveis para evitar a multiplicação de fungos e/ou eliminar suas toxinas. O Staphylococcus aureus é uma das principais bactérias patogênicas que apresenta taxas elevadas de resistência aos antimicrobianos, e por este motivo tem sido objeto de estudo de pesquisas que tentam identificar novos compostos bioativos para o combate de infecções. O Aspergillus parasiticus é um dos principais fungos produtores de aflatoxinas, substâncias carcinogênicas que contaminam diversos tipos de cereais antes e após o processamento. Estudos recentes demonstraram que extratos de plantas possuem atividade antimicrobiana e antifúngica, além de potencial para degradação de micotoxinas. Neste contexto, o presente estudo teve por objetivos avaliar a atividade antimicrobiana in vitro de extratos brutos e liofilizados de folhas de maracujá, araçá, alecrim e orégano sobre células planctônicas de S. aureus e A. parasiticus, bem como verificar a capacidade dos referidos extratos em degradar in vitro a aflatoxina B1 (AFB1), ocratoxina A (OTA) e zearalenona (ZEA). Os efeitos antibacterianos e antifúngicos dos extratos foram avaliados através da determinação da concentração inibitória mínima (CIM) e concentração bactericida / fungicida mínima (CBM/CFM). Os ensaios de degradação da micotoxinas foram realizados em diferentes tempos de incubação (12-48 h) a 37º C, utilizando-se cromatografia líquida de alta eficiência (HPLC) para determinação das concentrações das micotoxinas. O maracujá não demonstrou atividade antimicrobiana tanto para S. aureus quanto para A. parasiticus. Dos quatro extratos estudados, o araçá demonstrou o maior efeito antibacteriano, cujos valores de CIM para extratos bruto e liofilizado foram 0.39 mg/mL e 0.45 mg/mL, respectivamente. Os menores valores de CIM para A. parasiticus foram obtidos com o orégano liofilizado (8.33 mg/mL) e o araçá bruto (3.215 mg/mL). Contudo, não foi possível identificar valores de CFM para o fungo analisado. Não houve efeito de degradação de OTA e ZEA por nenhum dos extratos avaliados. Todos os extratos reduziram a concentração de AFB1 após 48 h de incubação. A maior porcentagem (60.3%) de redução de AFB1 foi obtida com o extrato de alecrim após 48 h de incubação. A atividade antimicrobiana demonstrada pelos extratos das plantas avaliadas indica um potencial para sua aplicação no combate a bactérias patogênicas e fungos toxigênicos. Este é o primeiro estudo realizado com extratos dessas quatro espécies de plantas que evidenciou a capacidade de redução in vitro de AFB1. / Human exposure to pathogenic microorganisms and their toxins in food is a serious public health problem. The indiscriminate use of conventional antimicrobials increases the resistance of microorganisms to the main molecules available in the market. In addition, limitations on the use of chemical substances in food raw materials have stimulated researches for the development of eco-friendly methodologies to avoid the multiplication of fungi and/or eliminate their toxins. Staphylococcus aureus is one of the major pathogenic bacteria with high rates of antimicrobial resistance, and for this reason it has been the subject of research studies aiming to identify new bioactive compounds to combat infections. Aspergillus parasiticus is one of the main aflatoxin-producing fungi, which are carcinogenic substances that contaminate many types of cereals before and after processing. Recent studies have demonstrated that plant extracts have antimicrobial and antifungal activity, as well as potential for degradation of mycotoxins. In this context, the objective of the present study was to evaluate the in vitro antimicrobial effects of crude and lyophilized extracts of leaves from sweet passion fruit, araçá, rosemary and oregano on planktonic cells of S. aureus and A. parasiticus. The in vitro degradation of aflatoxin B1 (AFB1), ochratoxin A (OTA) and zearalenone (ZEN) by the crude extracts was also investigated. The antimicrobial and antifungal effects were evaluated by determining the minimum inhibitory concentration (MIC) and minimum bactericidal / fungicidal concentration (MBC / MFC). Mycotoxin detoxification assays were conducted at different incubation times (12-48h) at 37 °C. The concentrations of mycotoxins were determined by high performance liquid chromatography (HPLC). Sweet passion fruit had no antimicrobial activity on S. aureus or A. parasiticus. Out of the four extracts evaluated, araçá showed the highest antimicrobial effect with MIC of 0.39 mg/mL and 0.45 mg/mL for crude and lyophilized extracts, respectively. The lowest MIC values for A. parasiticus were obtained with lyophilized oregano (8.33 mg/mL) and crude araçá (3.215 mg/mL). However, no MFC values were obtained for the analyzed fungi. Although OTA e ZEN were not degraded by any extract evaluated, all extracts reduced the concentration of AFB1 after 48 h of incubation. The highest percentage of AFB1 reduction (60.3%) was obtained with rosemary extract after 48h of incubation. The antimicrobial activity demonstrated by extracts of the evaluated plants indicates a potential for application against pathogenic bacteria and toxigenic fungi. This is the first study carried out with extracts of these four plants species that demonstrated the in vitro ability for AFB1 reduction.

Les maladies du blé causées par Fusarium, comme la brulure de l’épi (FHB) et la pourriture de la tige (FCR), entrainent une réduction de production, de la qualité du blé et des problèmes de sécurité alimentaire liés à la présence de mycotoxines affectant la santé de l’Homme et des animaux: la plus représentée étant le déoxynivalénol (DON). Le DON est un inhibiteur de la synthèse protéique qui agit durant l’infection comme un facteur de virulence. La glycosylation du DON en D3G (DON-3-O-glicoside) catalysée par des UDP-glycosyltransférases (UGTs) est le principal mécanisme de protection des plantes contre sa toxicité. Dans ce travail, nous avons démontré que la détoxification du DON par l’UGT confère une résistance à large spectre contre les champignons produisant DON F.graminearum et F.culmorum. Nous avons produit des plants de blé dur exprimant de manière constitutive le gène HvUGT13248 (Ubi-UGT) et des plants de blé panifiables exprimant ce gène au niveau du tissu floral (Lem-UGT). Les plants Ubi-UGT ont montré une réduction significative des symptômes de FHB durant les stades précoces et médians de l’infection, et de FCR à tous les stades de l’infection. De plus, les plants Lem-UGT ont montré une corrélation entre les niveaux d’expression de l’UGT et de protection observée. Finalement, nous avons démontré que la pyramidation des gènes associés à des mécanismes de résistance différents peut renforcer la résistance de l’hôte à l’infection. Des plants de blé ont été générés exprimant à la fois l’enzyme HvUGT13248, et des inhibiteurs de glycosidases: AcPMEI ou PvPGIP2, impliqués dans la dégradation de la paroi cellulosique, et qui ont montré une résistance accrue à la FHB. / Fusarium diseases, including Fusarium head blight (FHB) and Fusarium crown rot (FCR) represent major agricultural problems worldwide, causing reduction of grain yield and quality and food safety. In particular, grain contamination by Fusarium mycotoxins, mainly deoxynivalenol (DON), is responsible for health problems in humans and animals. DON is a protein synthesis inhibitor, acting as a virulence factor during pathogenesis. The principal mechanism involved in enhancing plant tolerance to DON is glycosylation, forming DON-3-β-D-glucoside (D3G), performed by specific UDP-glucosyltransferases (UGTs). In this work, we demonstrated that DON-detoxification by UGT confers a broad-spectrum resistance against the DON-producing fungi F. graminearum and F. culmorum, characterized by different time of infection and target organs. We produced transgenic durum wheat plants (Ubi-UGT) constitutively expressing the barley HvUGT13248 and bread wheat plants (Lem-UGT) expressing HvUGT13248 in flower tissues. Ubi-UGT plants revealed significant reduction of FHB symptom, during early-mid stages of infection, and of FCR symptom, throughout the infection timing. The floral-specific expression highlighted a dose-dependent efficacy of the UGT detoxification mechanism. In addition, we demonstrated that pyramiding of genes controlling different resistance mechanisms can further reinforce the host response by stacking transgenes controlling the DON-to-D3G conversion and the inhibition of cell wall degrading enzymes by glycosidase inhibitors in the same wheat genotype. We obtained plants expressing HvUGT13248 and AcPMEI or HvUGT13248 and PvPGIP2, which exhibited increased FHB resistance.

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