Cette étude porte sur l'identification et la dispersion des bioaérosols générés sur les plates-formes de compostage et plus précisément lors du retournement des andains en cours de fermentation. L'analyse des bioaérosols émis sur cinq plates-formes par des inventaires moléculaires (ADNr 16S et ADNr 18S) a permis de montrer la dominance de deux phyla bactériens Firmicutes et Actinobacteria et du phylum fongique Ascomycota. En comparant la structure de la population microbienne des cinq bioaérosols, une signature a été identifiée. Dix phylotypes microbiens sont communs à au moins quatre bioaérosols. Deux sont présents dans les cinq bioaérosols : NA07 appartenant à l'espèce Saccharopolyspora rectivirgula et représentant 7% des séquences bactériennes totales et EQ07 affiliée à Thermomyces (49% des séquences fongiques). Un second phylotype bactérien, NC38, affilié à la famille des Thermoactinomycetaceae a été sélectionné du fait q u'il n'ait été identifié que dans le compost. Des systèmes de PCRq ont été développés pour quantifier ces trois indicateurs potentiels. Ces derniers ont été validés expérimentalement par des mesures sur sites industriels. La dispersion des bioaérosols a ensuite été caractérisée en utilisant plusieurs méthodologies, dont les trois indicateurs conçus et une technique d'empreinte moléculaire, la SSCP. Lors d'une activité de retournement, la concentration des indicateurs est supérieure à leur niveau basal dans l'air (bruit de fond). Les indicateurs présentent des profils de dispersion différents d'où l'intérêt de les coupler afin d'obtenir une meilleure vision de la dispersion des bioaérosols de compostage. / The aim of this work was to analyze the diversity and the dispersal of composting bioaerosol emitted during the turning of compost windrows in thermophilic phase on composting platforms. The study of the microbial diversity of aerosols emitted on five composting plants was realized by 16S and 18S rDNA molecular inventories. Two bacterial phyla Firmicutes and Actinobacteria and one fungal phylum Ascomycota dominated. A common microbial signature emerged from the five composting bioaerosols: ten microbial phylotypes (seven bacterial and three fungal) were common to at least four bioaerosols. Two have been identified in five bioaerosols: NA07 belonging to the species Saccharopolyspora rectivirgula, and representing 7% of total number of bacterial sequences, and EQ05,affiliated to Thermomyces (49% of total number of fungal sequences). A second bacterial phylotype, NC38, affiliated to the Thermoactinomycetaceae family, was selected because it was id entified only in the environmental source compost'. qPCR systems were then designed for each phylotype. Measurements performed on industrial composting sites validated the use of these microorganisms as indicators of composting bioaerosols. The dispersal of composting bioaerosols was then characterized using the three indicators developed and a fingerprint technique, the SSCP.

A obesidade é considerada uma doença multifatorial e pode envolver, em sua gênese, aspectos genéticos, metabólicos, ambientais, psicológicos e socioeconômicos. O crescimento expressivo da incidência mundial da obesidade desencadeia o surgimento contínuo de novas pesquisas em relação a esse tema e, nesse contexto, estudos recentes sugerem que a microbiota intestinal é um fator que pode contribuir com o desenvolvimento desta doença. Assim, existe a premissa que a alteração da composição da microbiota intestinal pode ser influenciada pelo estado nutricional (obesidade x eutrofia) e pela qualidade da alimentação. O presente estudo teve como objetivos: i. comparar a proporção dos filos predominantes da microbiota intestinal Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Verrucomicrobia e a razão Firmicutes/Bacteroidetes entre mulheres com obesidade grau III e mulheres eutróficas, e ii. investigar o impacto de uma dieta hipocalórica para perda de peso na composição da microbiota intestinal. Estudo prospectivo longitudinal, no qual foram selecionadas 20 mulheres com média de idade 33±3,1 anos, as quais foram divididas em dois grupos: Grupo Intervenção (GI): 10 mulheres com obesidade grau III (Índice de Massa Corporal (IMC) >40 kg/m2) que foram submetidas à intervenção nutricional (dieta hipocalórica) durante 8 semanas e Grupo Controle (GC):10 mulheres eutróficas (IMC entre 18,5 a 24,9 kg/m2). No GI, as coletas foram realizadas antes e após oito semanas da intervenção (GI) e, no GC em um único momento. Em cada momento, foram aferidos o peso e estatura; realizado o cálculo do IMC, análise composição corporal, taxa metabólica de repouso, consumo alimentar, glicemia e lipidograma, e coleta de amostra fecal. A análise da composição da microbiota intestinal em relação à abundância relativa dos Filos foi realizada por reação em cadeia da polimerase em tempo real (qPCR). Após oito semanas de dieta hipocalórica, houve redução do peso (119,5±10,3 para 114,9±10,2 kg), IMC (43,6±2,4 para 41,9±2,6 kg/m2), massa corporal gorda (MG) (62,4±7,5 para 58,9±7,7 kg), triglicérides (TG) (143,2±60,9 para 117,94±48,3 mg/dL). Evidenciou-se que mulheres com obesidade apresentam menor abundância dos filos pesquisados em relação às eutróficas. Ainda, a dieta hipocalórica promoveu diminuição da abundância relativa do filo Proteobacteria, o qual apresentou correlações positivas com a ingestão de lipídio total (%), ômega 6 (g). Conclui-se que a intervenção com dieta hipocalórica foi eficaz na redução de peso, IMC, MG e TG e na modulação da microbiota intestinal, com a diminuição do filo Proteobacteria / Obesity is considered a multifactorial disease and it may involve, in its genesis, genetic, metabolic, environmental, psychological and socioeconomic aspects. The expressive growth of a worldwide incidence of obesity triggers the continual emergence of new researches on this subject and, in this context, recent studies have suggested that the intestinal microbiota is a factor that may contribute to the development of this disease. Thus, there is the premise that the changes in the composition of the intestinal microbiota can be influenced by the nutritional status (obesity x eutrophy) and by the diet quality.The present study aimed at: i. comparing the proportion of the predominant phyla of the intestinal microbiota Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Verrucomicrobia and ratio Firmicutes/Bacteroidetes among degree III obese women and eutrophic women, and ii. investigating the impact of a low-calorie diet for weight loss on the intestinal microbiota composition. This was a longitudinal prospective study in which 20 women at the average age of 33 ± 3.1 years old were selected and divided into two groups: Intervention Group (IG): 10 women with grade III obesity (Body Mass Index (BMI) > 40 kg / m2) who were submitted to a nutritional intervention (a low-calorie diet) for 8 weeks, and the Control Group (CG): 10 eutrophic women (BMI from 18.5 to 24.9 kg / m2) who were not submitted to any intervention. In the IG, sampling was carried out before and after the eight-week intervention (IG) and in the CG it was carried out only in a single moment. At each sampling, weight and height were checked; BMI was calculated, body composition was analyzed, resting metabolic rate, food intake, fasting blood glucose, and lipidogram tests were performed, and fecal sample collected. The analysis of the intestinal microbiota composition in relation to a relative abundance of every phila was performed by real-time polymerase chain reaction (qPCR). After eight weeks of a low-calorie diet, some of the observed results were weight reduction (119.5 ± 10.3 to 114.9 ± 10.2 kg), BMI (43.6 ± 2.4 to 41.9 ± 2.6 kg / m2), fat body mass (FM) (62.4 ± 7.5 to 58.9 ± 7.7 kg), triglycerides (TG) (143.2 ± 60.9 to 117.94 ± 48.3 mg / dL). It was evidenced that obese women present a lower abundance of the studied phyla in relation to the eutrophic ones. Moreover, the lowcalorie diet promoted a decrease in the relative abundance of Proteobacteria phylum, which presented positive correlations with the intake of total lipid (%) and omega 6 (g). It was concluded that the intervention with a low-calorie diet was effective in reducing BMI, FM and TG and in modulating the intestinal microbiota, by reducing Proteobacteria phylum

The diploma thesis is focussed on understanding relationships between soil chemical characteristics, actinobacterial communities of agricultural field soils and occurrence of potato common scab, a disease caused by members of the genus Streptomyces. The aim of monitoring study, on thirty-three sites covering main potato- growing regions in the Czech Republic, was to find relationships suitable for prediction of common scab severity. The second part of the thesis compared actinobacterial communities and incidence of Streptomyces harboring a pathogenic determinant, gene txtA (gene of biosynthetic pathway of phytotoxin thaxtomin A), in soils differing in occurrence of common scab. In the screening study, analysis of terminal restriction fragment length polymorphism (T-RFLP) was employed to compare composition of soil actinobacterial communities. Real-time PCR was used to quantify total actinobacteria and streptomycetes harboring txtA gene in soils differing in scab incidence. The screening study revealed negative correlations between the scab severity and (i) available phosphorus in soil and (ii) diversity of actinobaterial community. The results were used to design a model for scab prediction. A qPCR analysis showed difference in numbers of total actinobacteria and the strains harboring txtA gene in...

As actinobactérias são uma reconhecida fonte de metabólitos secundários bioativos com grande diversidade estrutural e funcional. Dentre elas, o gênero Streptomyces é sem dúvida o mais explorado, correspondendo a cerca de 75% dos metabólitos secundários estudados, e por dois terços dos antibióticos conhecidos. Dessa forma, ainda é crescente a necessidade de novos herbicidas com perfil de baixa toxicidade ambiental e novos mecanismos de ação, para a substituição de produtos que são utilizados extensivamente há anos. Grandes avanços tecnológicos têm sido alcançados em termos de instrumentação analítica nas últimas décadas, tornando a espectrometria de massas a técnica mais eficiente e robusta na identificação de metabólitos secundários, principalmente quando acoplada a técnicas cromatográficas de ultra eficiência (UHPLC-MS). Nessa dissertação foi explorada a diversidade metabólica de actinobactérias isoladas do bioma Caatinga para a identificação de metabólitos secundários com atividade fitotóxica. Foram produzidos 166 extratos brutos de actinobactérias, das quais, 27 apresentaram algum nível de atividade fitotóxica contra Lemna minor. Devido pronunciada bioatividade (MIC = 6,25 ?g) o extrato bruto da actinobactéria, CAAT 7-52, foi selecionado para desreplicação, caracterização e fracionamento guiado por bioensaios, e, ao mesmo tempo, um estudo taxonômico foi realizado para determinar o posicionamento filogenético do novo isolado. A análise da sequência do gene 16S rRNA suportou a classificação da linhagem no gênero Streptomyces e mostrou que CAAT 7-52 formou uma linha filética distinta junto com a linhagem tipo de Streptomyces actinomycinicus, com um valor de identidade da sequência de 99,0%. No extrato bruto de CAAT 7-52 foi identificada a Albociclina como responsável pela atividade fitotóxica (MIC = 3,12 ?g), assim como alguns compostos análogos. O monitoramento da massa micelial seca e produção de Albociclina mostrou uma maior produção do composto ativo aos 21 dias de crescimento. Esse extrato foi submetido a ensaios de inibição de germinação, onde apresentou atividade contra sementes de buva (Conyza canadensis) (MIC = 12,5 ?g), alface (Lactuca sativa) e rúcula (Eruca sativa). Variações no meio de cultivo mostraram uma produção seletiva de Albociclina nos meios de cultivo PMB e Czapeck-GL. Ensaios de fitotoxicidade contra Lemna minor, utilizando o fermentado do meio PMB sem extração com solventes orgânicos, mostrou atividade em uma diluição de 20%. Portanto, foi possível demostrar através desse estudo o grande potencial das actinobactérias na produção de metabólitos secundários bioativos e da espectrometria de massas como técnica analítica na identificação dos compostos ativos. / Actinobacterias are a well know source of bioactive secondary metabolites with great structural and functional diversity. Among them, the Streptomyces genus is certainly the most explored, corresponding to about 75% of studied secondary metabolites, and for two thirds of known antibiotics. Thus, there is still a growing need for new herbicides with low environmental toxicity e new modes of action, for the replacement of products that have been broadly used for years. Great technological progress has been achieved in analytical instrumentation in the last few decades, making mass spectrometry the most efficient and robust technique to identify secondary metabolites, mainly when accoupled to ultra-performance chromatographic techniques (UHPLC-MS). In this essay, was explored the metabolic diversity of the actinobacterias isolated from the Caatinga biome to identification of secondary metabolites with phytotoxic activity. Were produced 166 crude extracts from actinobacterias, from this, 27 showed some phytotoxic activity against Lemna minor. Due to noticeable bioactivity (MIC = 6,25 ?g) the crude extract from actinobacteria CAAT 7-52, was selected to dereplication, characterization and bioassay guided fractionation, and, at the same time, a taxonomic study was carried out to determinate the phylogenetic location of the new isolated. The analysis of 16S rRNA gene sequence supported the lineage classification on the genus Streptomyces and established that CAAT 7-52 assembled a distinct phyletic line together with the lineage type of Streptomyces actinomycinicus, with an identity sequence value of 99,0%. In the crude extract CAAT 7-52, Albocycline was identified as the responsible for the phytotoxic activity (MIC = 3,12 ?g), so as some analogous compounds. The mycelial dry mass and Albocycline production monitoring revealed a major production of the active compound at 21 days of growth. This extract was subjected to germination inhibition assays, where revealed activity against horseweed (Conyza canadensis) (MIC = 12,5 ?g), lettuce (Lactuca sativa) and arugula (Eruca sativa) seeds. Variations in the culture media showed a selective production of Albocycline in the PMB and Czapeck-GL culture media. Phytotoxicity assays against Lemna minor, applying the fermented PMB media without organic solvents extractions, revealed activity with a 20% dilution. Therefore, through this study was able to demonstrate the great potential of the actinobacterias in the production of bioactive secondary metabolites and the mass spectrometry as analytical technique to identification of the active compounds.

A crescente resistência dos micro-organismos patogênicos aos fármacos já existentes gera intensa demanda por novos agentes terapêuticos. Em contrapartida, a eficiência na descoberta de compostos com novas estruturas químicas diminuiu nos últimos anos. Tendo em vista a vasta biodiversidade existente de micro-organismos, a redução da eficiência na descoberta de novos produtos naturais não indica que todos compostos existentes já foram descritos, mas sim que as metodologias para isolamento dos mesmos devem ser aperfeiçoadas e diversificadas, e novos nichos devem ser explorados. Esta tese compreende três capítulos, que trazem abordagens que podem ser utilizadas na busca por produtos naturais. O capítulo 1 aborda a aplicação de conhecimentos de biologia molecular na pesquisa de produtos naturais, através da metagenômica. O capítulo 2 aborda o conceito de química ecológica para estimular o metabolismo secundário, através da utilização de interações microbianas. O capítulo 3 aborda o uso de genome mining para entender a capacidade metabólica de uma linhagem bacteriana, bem como o uso de variações nos parâmetros da cultura para alterar o metabolismo desta. Metagenômica: a triagem anti-parasitária das bibliotecas metagenômicas detectou clones bioativos contra Leishmania major. As análises químicas e biológicas das culturas destes clones não permitiram a identificação dos compostos responsáveis pelas atividades observadas. Esta abordagem apresenta grandes desafios técnicos e tem passado por ajustes envolvendo sequenciamento e bioinformática para aumentar a taxa de sucesso em seu uso. Interações microbianas: esta metodologia mostrou-se promissora para a busca por compostos bioativos, tendo em vista que diversos pares exibiram nova atividade antibiótica, corroborando a hipótese que interações microbianas podem levar à expressão diferenciada do metabolismo secundário. Foi escolhida para caracterização química e biológica a interação entre uma actinobactéria rara (Krasilnikovia sp. T082) e uma actinobactéria endossimbionte de besouro (Streptomyces sp. SPB78). Esta interação é robusta e estimula a biossíntese de um antibiótico polar capaz de inibir o crescimento de uma bactéria multirresistente. Diversas técnicas foram testadas para o isolamento do composto indutor e do antibiótico induzido. Este processo foi desafiador devido ao caráter polar de ambos compostos e pelo fato da atividade antibiótica ser instável. Foi demonstrado que o antibiótico induzido é capaz de inibir o crescimento do micro-organismo indutor, sugerindo importância ecológica deste composto. A utilização desta abordagem metodológica permitiu a identificação de uma linhagem pertencente a um gênero de actinobactéria rara que nunca teve seu metabolismo secundário estudado (Krasilnikovia). Isto foi realizado através da investigação de seu genoma e também através do isolamento de compostos produzidos por esta linhagem. Esta linhagem demonstrou potencial para a produção de metabólitos secundários, apresentando pelo menos 21 potenciais clusters gênicos biossintéticos detectados pelo antiSMASH em seu genoma. Esta linhagem foi cultivada em dois meios de cultura (ISP2 e TSB) e diferentes metodologias de extração foram empregadas. O metabolismo secundário desta linhagem é expresso de maneira diferente de acordo com a metodologia de cultivo, evidenciando a importância da variação da composição do meio de cultura para o acesso da real capacidade metabólica de microorganismos. Foram identificadas algumas dicetopiperazinas, que são conhecidas por seu amplo espectro de atividades biológicas, e três compostos pertencentes a uma classe de peptídeos nãoribossomais não usuais com atividade antibiótica, que possuem estrutura química complexa e incomum para produtos naturais / Increasing drug resistance among microbial pathogens is a public health threat; therefore, new antibiotics are needed. On the other hand, the rate of discovering new compounds has diminished. Considering the wide biodiversity of microorganisms, reduced efficiency on the discovery of new natural products does not indicate that all existing compounds have been described, but that methods for isolation should be improved and diversified and new niches should be explored. This thesis comprises three chapters that demonstrate approaches that can be used in the search for natural products. Chapter 1 demonstrates the application of molecular biology tools on the search for natural products through metagenomics. Chapter 2 discusses the concept of chemical ecology for the stimulation of secondary metabolism by the use of microbial interactions. Chapter 3 discusses the use of genome mining to understand the metabolic capacity of a bacterial strain as well as the use of different culture parameters to alter bacterial metabolism. The anti-parasitic metagenomic screening on metagenomic libraries detected bioactive clones against L. major. Chemical and biological analysis of cultures of these clones did not permit identifying the compounds responsible for the observed activities. This approach faces diverse technical challenges and is currently being improved by the use of sequencing and bioinformatics analysis in order to increase its hit rate. Microbial interactions: this approach has shown to be promising in the search for bioactive compounds, considering that several pairs exhibited new antibiotic activity, supporting the hypothesis that microbial interactions can stimulate differential expression of secondary metabolism. It was chosen for chemical and biological characterization the interaction between a rare actinobacteria (Krasilnikovia sp. T082), which belongs to a genus that its secondary metabolism has not yet been studied in the literature, and an endosymbiont actinobacteria of beetle (Streptomyces sp. SPB78). This interaction is robust and stimulates the biosynthesis of a polar antibiotic capable of inhibiting the growth of multi-resistant bacteria. Several techniques have been tested for the isolation process of the inducer compound and the induced antibiotic. This process has been particularly challenging due to the polar character of both compounds, and because the antibiotic activity is unstable. It has been shown that the induced antibiotic is capable of inhibiting the growth of the inducer microorganism, suggesting an ecological importance of this compound. The use of this co-culture approach led to the identification of a strain belonging to a rare actinobacteria genus whose secondary metabolism has never been studied before (Krasilnikovia). This was accomplished through sequencing and analysis of its genome and also by isolating compounds produced by this strain (chapter 3). This strain has shown great potential for the production of secondary metabolites, exhibiting at least 21 biosynthetic gene clusters detected by antiSMASH in its genome, and only four of them showed high similarity to any known gene cluster. This strain was cultured in two different culture media (ISP2 and TSB), and different methods of extraction were used. The secondary metabolism of this strain is expressed differently according to the method of cultivation, showing the importance of variation in the composition of the culture medium to access the actual metabolic capacity of microorganisms. Some diketopiperazine were isolated, which are known for their wide spectrum of biological activities, and also three compounds belonging to a class of non-ribosomal peptides known for their high bioactivity, which have complex and unusual chemical structures for natural products

A biodiversidade microbiana de diferentes ecossistemas tem incentivado estudos químicos e biológicos com micro-organismos dos mais variados habitats, os quais têm conduzido à obtenção de moléculas bioativas com aplicações na medicina, indústria química e agricultura, proporcionando melhorias na qualidade de vida ao homem. O presente trabalho teve como objetivos a bioprospecção por actinobactérias endofíticas e seus metabólitos, além do estudo da via biossintética dos sesquiterpenos aristoloquenos produzidos pelo fungo fitopatogênico Botrytis cinerea. No estudo de bioprospecção foram isoladas 41 linhagens de actinobactérias endofíticas de duas espécies de Asteraceae (Thitonia diversifolia e Lychnophora ericoides). A identificação através do sequenciamento de DNAr indicou predominância do gênero Streptomyces. As linhagens foram cultivadas em meio de arroz e os extratos etanólicos submetidos aos ensaios de citotoxicidade frente a células tumorais e antimicrobiano. Um total de 58,5% dos extratos apresentou atividade em pelo menos um dos ensaios realizados. Foram selecionadas as linhagens Streptomyces cattleya RLe 4 e Streptomyces sp. RLe 8 para cultivo em escala ampliada, isolamento e identificação de metabólitos bioativos. O isolamento dos compostos foi realizado através de diferentes técnicas cromatográficas e a identificação estrutural foi baseada em dados de ressonância magnética nuclear de 1H e 13C e espectrometria de massas. De S. cattleya RLe 4 foram isolados quatro compostos: 2-hidroxibenzamida, desferrioxamina E, 1-(3\',4\'-dimetoxifenil)-1-propanona e 1-(3\',4\'-dimetoxifenil)-1-etanona. Dos extratos de Streptomyces sp. RLe 8 foram isolados dez compostos: benzamida, 3- hidroxibenzamida, 3-hidróxi-4-metoxibenzamida, 4-hidróxi-3-metoxibenzamida, 3,4- dimetoxibenzamida, 2-fenilacetamida, dois isômeros de 3,4-diidro-3,4,6,8-tetraidróxi-1(2H)- naftalenona, 2,3-diidro-2,2-dimetil-4(1H)-quinazolinona e desferrioxamina B. O composto 2,3-diidro-2,2-dimetil-4(1H)-quinazolinona apresentou elevada atividade frente as células de câncer de cólon (HCT-8) e glioblastoma (SF295), com 93,9 % e 87.0 % de inibição, respectivamente. O outro enfoque da tese envolveu a otimização da produção de sesquiterpenos aristoloquenos por linhagens de B. cinerea, seguido de estudo biossintético destes produtos naturais através de experimentos de incorporação de precursores isotopicamente enriquecidos com 2H (deutério) e 13C (carbono treze). As análises dos dados obtidos de RMN de 2H e de 13C do sesquiterpeno majoritário indicaram que a biossíntese desta substância ocorre pela via do mevalonato (MVA). Os resultados também sugeriram o possível envolvimento da via do metil-eritritolfosfato ou 1-desoxi-D-xilulose-fosfato (MEP/DPX) na biossíntese deste sequiterpeno. Estes resultados podem contribuir para o planejamento racional de fungicidas seletivos com aplicação na agricultura. O trabalho desenvolvido mostrou o grande potencial de actinobactérias endofíticas para a obtenção de moléculas bioativas e que estudos usando precursores isotopicamente marcados fornecem informações precisas acerca da origem biossintética de produtos naturais. / The microbial biodiversity from different ecosystems has incited chemical and biological studies with microorganisms from several habitats, leading to the isolation of bioactive natural products with applications in medicine, chemical industry and agriculture, and thus contributing to a better quality of life. This thesis aimed the biopropecting on endophytic actinobacteria and their natural products, and also the biosynthetic study of aristolochene sesquiterpenes in the phytopathogenic fungus Botrytis cinerea. A total of 41 actinobacterial strains were isolated of two Asteraceae species (Thitonia diversifolia and Lychnophora ericoides) for the bioprospecting study. The rDNA sequencing showed predominancy of Streptomyces genus. All the strains were cultured on rice medium, and the ethanolic extracts were screened in cytotoxity and antimicrobial assays. As a result, 58.5% of the extracts showed activity in al least one bioassay. The strains Streptomyces cattleya RLe 4 and Streptomyces sp. RLe 8 were selected for scale up cultures, isolation and identification of bioactive compounds. Different chromatographic methods were applied for the isolation of compounds, and structural analysis were based on 1H and 13C nuclear magnetic resonance and mass spectrometry data. Four compounds were isolated from S. cattleya RLe 4: 2- hydroxybenzamide, desferrioxamine E, 1-(3\',4\'-dimethoxyphenyl)-1-propanone, and 1-(3\',4\'- dimethoxyphenyl)-1-etanone. Ten compounds were isolated from Streptomyces sp. Rle 8: benzamide, 3-hydroxybenzamide, 3-hydroxy-4-methoxybenzamide, 4-hydroxy-3- methoxybenzamide, 2-phenylacetamide, two isomers of 3,4-dihydro-3,4,6,8-tetrahydroxy- 1(2H)-naphthalenone, 2,3-dihydro-2,2-dimethyl-4(1H)-quinazolinone, and desferrioxamine B. Compound 2,3-dihydro-2,2-dimethyl-4(1H)-quinazolinone showed high antiproliferative activity against colon cancer cells (HCT-8) and glioblastoma cells (SF295), with 93.9 and 87.0% of inhibition, respectively. The second focus of the thesis involved the optimization of aristolochene sesquiterpenes production by two strains of B. cinerea, followed by the biosynthetic study through feeding experiments with 2H (deuterium) and 13C isotopically labeled precursors. The 2H and 13C NMR obtained data showed that the biosynthesis of the sesquiterpene proceeds by the mevalonate pathway (MVA). The results also suggested the possible participation of the non mevalonate pathway, methylerytritol phosphate ou 1-deoxy- D-xylulose phosphate (MEP/DXP), in the biosynthesis. These results might contribute to the rational design of selective fungides with application in agriculture. This thesis showed the endophytic actinobacteria as promising sources of bioactive natural products, and also showed that the isotopically labeled feeding experiments give reliable information about the natural products biosynthetic pathways.

Wheat is the most economically important crop forming one quarter of Australian farm production. The wheat industry is severely affected by diseases, with fungal pathogens causing the most important economic losses in Australia. The application of fungicides and chemicals can control crop diseases to a certain extent, however, it is expensive and public concern for the environment has led to alternative methods of disease control to be sought, including the use of microorganisms as biological control agents. Microorganisms are abundant in the soil adjacent to plant roots (rhizosphere) and within healthy plant tissue (endophytic) and a proportion possess plant growth promotion and disease resistance properties. Actinobacteria are gram-positive, filamentous bacteria capable of secondary metabolite production such as antibiotics and antifungal compounds. A number of the biologically active endophytes belonging to the Actinobacteria phylum were isolated in our laboratory. A number of these isolates were capable of suppressing the wheat fungal pathogens Rhizoctonia solani, Pythium sp. and Gaeumannomyces graminis var. tritici, both in vitro and in planta indicating the potential for the actinobacteria to be used as biocontrol agents. The aim of this research was to investigate the molecular mechanisms underlying this plant-microbe interaction. The indigenous microbial populations present in the rhizosphere and endophytic environment are critical to plant health and disruptions of these populations are detrimental. The culture-independent technique Terminal Restriction Fragment Length Polymorphism (T-RFLP) was used to characterise the endophytic actinobacteria population of wheat roots under different conditions. Soils which support a higher number of indigenous microorganisms result in wheat roots with higher endophytic actinobacterial diversity and level of colonisation. Sequencing of 16S rRNA gene clones, obtained using the same actinobacteria-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity, and identified a number of Mycobacterium and Streptomyces species. It was found that the endophytic actinobacterial population of the wheat plants contained a higher diversity of endophytic actinobacteria than reported previously, and that this diversity varied significantly among different field soils. The endophytic actinobacteria have previously been shown to protect wheat from disease and enhance growth when coated onto the seed before sowing. As the endophytes isolated were recognised as potential biocontrol agents, the impact on the indigenous endophytic microbial population was investigated. Utilising the T-RFLP technique it was established that the use of a commercial microbial inoculant, containing a large number of soil bacterial and fungal strains applied to the soil, disrupts the indigenous endophyte population present in the wheat roots. The hypothesis is that non-indigenous microbes proliferate and dominate in the soil preventing a number of endophytic-competent actinobacterial genera from access to the seed and ultimately endophytic colonisation of the wheat roots. This dramatically reduces diversity of endophytes and level of colonisation. In contrast the use of a single endophytic actinobacteria endophyte inoculant results in a 3-fold increase in colonisation by the added inoculant, but does not significantly affect this indigenous population. Colonisation of healthy plant tissues with fungal endophytes has been shown to improve the competitive fitness with enhanced tolerance to abiotic and biotic stress and improved resistance to pathogens and herbivores. In this study the fungal endophyte population of wheat plants grown in four different soils was analysed using partial sequencing of 18S rRNA gene sequences. Sequence anlaysis of clones revealed a diverse range of fungal endophytes. In this diverse range of fungal endophytes a number sequences were highly similar to those of previously known fungal phytopathogens. A number of sequences detected were similar to fungal species previously identified in soil or plant material but not as endophytes. The remaining sequences were similar to fungal species without a known relationship with plants. Plants have developed an inducible mechanism of defence against pathogens. In addition to local responses plants have developed a mechanism to protect uninfected tissue through a signal that spreads systemically inducing changes in gene expression. In the model plant Arabidopsis thaliana activation of the Systemic Acquired Resistance (SAR) pathway and the Jasmonate (JA)/Ethylene (ET) pathway is characterised by the production of pathogenesis-related (PR) and antimicrobial proteins resulting in systemic pathogen resistance. Endophytic actinobacteria, isolated from healthy wheat roots in our laboratory, have been shown to enhance disease resistance to multiple pathogens in wheat when coated onto the seed before sowing. Real Time RT-PCR was used to determine if key genes in the SAR and JA/ET pathways were induced in response to inoculation with endophytic actinobacteria. Inoculation of wild-type Arabidopsis thaliana with selected strains of endophytic actinobacteria was able to �prime� the defence pathways by inducing low level expression of SAR and JA/ET genes. Upon pathogen infection the defence-genes are strongly up-regulated and the endophyte coated plants had significantly higher expression of these genes compared to un-inoculated plants. Resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora was mediated by the JA/ET pathway whereas the fungal pathogen Fusarium oxysporum triggered primarily the SAR pathway. Further analysis of the endophytic actinobacteria-mediated resistance was performed using the Streptomyces sp. EN27 and Arabidopsis defence-compromised mutants. It was found that resistance to E. carotovora subsp. carotovora mediated by Streptomyces sp. EN27 occurred via a NPR1-independent pathway and required salicylic acid whereas the jasmonic acid and ethylene signalling molecules were not essential. In contrast resistance to F. oxysporum mediated by Streptomyces sp. EN27 occurred via a NPR1-dependent pathway but also required salicylic acid and was JA- and ET-independent. This research demonstrated that inoculating wheat with endophytic actinobacteria does not disrupt the indigenous endophytic population and may be inducing systemic resistance by activating defence pathways which lead to the expression of antimicrobial genes and resistance to a broad range of pathogens.

Descrição: As doenças causadas por actinomicetos patógenos, aeróbios e anaeróbios facultativos, diferem consideravelmente no que diz respeito à sua etiologia, patogênese, apresentação clínica e epidemiologia. Objetivos: Analisar a distribuição etária, manifestações clínicas, doenças de base e condições associadas, achados radiológicos, microbiológicos, tratamento e evolução, nos pacientes com actinomicetoses (actinomicose, nocardiose, rodococose). Delineamento: Foram analisados, retrospectivamente, prontuários de pacientes com achados microbiológicos positivos para infecções por actinomicetos. Local do estudo: Um hospital universitário de atendimento terciário em Porto Alegre, Rio Grande do Sul, Brasil. Pacientes e métodos: Foram incluídos neste estudo, pacientes com diagnóstico de actinomicose, nocardiose e rodococose, num período de 1978 a 2009. Os critérios microscópicos para o diagnóstico de actinomicetose foram os seguintes: actinomicose - composto por grânulos actinomicóticos, filamentos Gram-positivos, não ácido-resistentes; nocardiose - bactérias filamentosas ramificadas, Gram-positivas, e ácido-resistentes; rodococose - cocobacilos Gram-positivos, ácido resistentes. Resultados: Foram incluídos 59 pacientes com actinomicetose. Actinomicose foi obervada em 27 pacientes entre 8 e 65 anos (idade média de 39,9 anos), 22 do sexo masculino (81,5%). Doença oral (cárie dentária, a doença periodontal) esteve frequentemente associada, sendo procedimento odontológico o fator de risco mais importante. A apresentação clínica foi actinomicose torácica em 24 casos, em dois facial e em um cérvico-facial e mediastinal. O diagnóstico microscópico foi positivo em 25, com o isolamento do organismo em cultivo anaeróbico em um, e, pelo teste de imunofluorescência direta em um. Estes dois últimos casos foi identificado como A. israelii. O tratamento mais utilizado consiste na administração prolongada de penicilina e esteve associado a boa evolução na maioria dos casos. Nocardiose foi observada em 27 pacientes, a idade variou entre 21 e 84 anos, idade média de 51,8 anos. A manifestação mais comum foi pneumonia cavitária, apresentado no paciente imunossuprimido, especialmente recebendo altas doses de corticoterapia. Todos os casos foram positivos para filamentos bacterianos ramificados Gram-positivos, ácido resistentes, sugestivos de espécies de Nocardia. Nocardia sp foi isolada em 14 casos, ―N. asteroides” em 7, N. farcinica em 2, N. brasiliensis em 1, N. pseudobrasiliensis em 1, N. abscessus em 1 e N. cyriacigeorgica em 1. Doze pacientes foram a óbito e os restantes tiveram melhora clínica. A rodococose foi diagnosticada em 5 pacientes, com idade, no momento do diagnóstico, de 22-69 anos (média de 45,6). Rhodococcus foi isolado em todos os 5 casos, três pacientes imunodeprimidos apresentaram infecção pulmonar pelo R. equi. O caso do paciente com HIV/AIDS foi fatal. Conclusões: Esta experiência, indica que a informação clínica associada ao Gram e a ácido-resistência em amostras clínicas é útil no reconhecimento da infecção por actinomicetos. A actinomicetose deve ser sempre considerada em pacientes apresentando doença febril supurativa ou radiografia de tórax anormal, em paciente com estado imune alterado causado por determinadas drogas (corticoterapia) e condições associadas (HIV/AIDS). / Background: Diseases caused by pathogenic aerobic and facultative anaerobic actinomycetes differ considerably with respect to their etiology, pathogenesis, clinical appearence and epidemiology. Objectives: To analyse the age distribution, clinical manifestations, underlying diseases and associated medical conditions, radiographic findings, microbiology, treatment and outcome, in patients with actinomycetosis (actinomycosis, nocardiosis, rhodococcosis). Design: The medical records of patients with positive microbiology findings to actinomycetes infections were retrospectively analysed. Settings: A university-based tertiary care hospital in Porto Alegre, Rio Grande do Sul, Brazil. Patients and methods: From 1978 through 2009 patients diagnosed with actinomycosis, nocardiosis, and rhodococcosis were included in this study. The microscopic criteria for diagnosis of actinomycetosis were as follow: actinomycosis –granules composed by branching Gram-positive organisms non acid-fast stained; nocardiosis - branched filamentous, Gram-positive, and acid-fast bacteria; rhodococcosis - coccobacilli Gram-positive, and acid-fast organism. Results: Sixty-five patients with actinomycetosis were included. Actinomycosis was oberved in 27 patients between 8 and 65 years old (mean age, 39,9 years), 22 were male (81,5%). Oral disease (poor dentition, periodontal disease) frequently associated with dental procedure was the most important risk factor. The clinical presentation was thoracic actinomycosis in 24 cases, facial in two, and cervico-facial and mediastinal one. Microcopic diagnosis were positive in 25, recovery of organism in anaerobic culture in one, and by fluorescent antibody test in one. These last two cases was identified as A. israelii. Treatment most commonly consisted of prolonged administration of penicillin and was associated with good outcome in the majority of cases. Nocardiosis was observed in 27 patients, aged 21 to 84 years old, with a mean age of 51,8 years. Cavitary pneumonia was the most common manifestation, presented in immunosuppresed patient, especially receiving high-dose corticotherapy. All cases were positive for branching Gram-positive, acid-fast bacterial filaments, suggestive of a Nocardia species. Nocardia sp was isolated in 14 cases, ―N. asteroides” in 7, N. farcinica in 2, N. brasiliensis in 1, N. pseudobrasiliensis in 1, N. abscessus in 1 and N. cyriacigeorgica in 1. Twelve patients died and the remaining cases were well improved. The diagnosis of rhodococcosis was made in five patients, ranged in age at time of diagnosis from 22 to 69 years, with a mean age of 45,6 years. Rhodococcus was isolated in all 5 cases, three immunocompromised patients showed pulmonary infection by R. equi. The case with HIV/AIDS was fatal. Conclusions: This experience, indicates that clinical information with Gram and acid-fast stains on clinical specimens is helpful in recognizing the possibility os actinomycetes should always be considered as a cause os suppurative febrile illness or abnormal chest roentgenograms in patient who may have an altered immune status caused by certain drugs (corticotherapy) and underlying conditions (HIV/AIDS).

L’ochratoxine A (OTA) est une mycotoxine issue du métabolisme secondaire des champignons filamenteux appartenant aux genres Penicillium et Aspergillus. Cependant, Aspergillus carbonarius est le majeur producteur de l’OTA sur les raisins. L’OTA a été retrouvée dans différents types de denrées alimentaires ainsi que lesurs produits dérivés. Le profil toxicologique de l’OTA due aux effets néfastes qu’elle présente sur la santé humaine et animale (effets hépatotoxiques, immunotoxiques, génotoxiques, tératogènes et cancérogènes) a conféré à cette mycotoxine une attention majeure auprès des instances internationales afin de limiter son occurrence. Ce projet est dédié pour trouver un moyen de lutte biologique, pouvant réduire l’OTA produite par A. carbonarius d’une part, et détoxifier les matrices alimentaires non conformes aux normes d’une autre part. La première stratégie était d’employer des huiles essentielles (cardamome, céleri, cannelle, taramira, origan, feuille de laurier, cumin, fenugrec, mélisse, menthe, sauge, anis, camomille, fenouil, romarin, romarin et thym) ainsi que des composés phénoliques extraits de plantes médicinales (feuille de laurier, cumin, fenugrec, mélisse, menthe, sauge, anis, camomille, fenouil, romarin et thym) afin d’évaluer leur effet sur la production de l’OTA dans le milieu SGM. Cette approche a été complétée par une étude moléculaire dans le but d’évaluer l’expression des gènes de biosynthèse de l’OTA (acpks, acOTApks et acOTAnrps) ainsi que les gènes de régulation (veA et laeA) chez A. carbonarius. Les résultats ont décelé que les huiles essentielles ont une activité fongicide plus élevée que celle des extraits phénoliques. Effectivement, les huiles essentielles du thym, de l’origan, du taramira, et de la cannelle ont bloqué complètement la croissance d’A. carbonarius. Cependant, les huiles essentielles du fenouil, de la cardamome, de l’anise, de la camomille, du céleri et du romarin ont réduit l’OTA sans autant affecter la croissance fongique. Le mode d’action de ces dernières a été mis en évidence en suivant l’expression des gènes acpks, acOTApks, acOTAnrps, veA et laeA, impliqués dans la biosynthèse de l’OTA chez A. carbonarius. Le gène acpks a été réprimée le plus (99.2%) quand A. carbonarius a été mis en culture avec 5 µL/mL du fenouil, entrainant ainsi une réduction de 88.9% de l’OTA. La deuxième stratégie était de développer un moyen de lutte biologique pouvant détoxifier les matrices alimentaires contaminées. Cette méthodologie a été développée suite à l’utilisation de sept souches d’actinobactéries (AT10, AT8, SN7, MS1, ML5, G10 et PT1), en évaluant leur capacité à métaboliser l’OTA, adhérer cette toxine à leur paroi membranaire ainsi que leur effet sur l’expression des gènes impliqués dans la biosynthèse de l’OTA chez A. carbonarius (acpks, acOTApks, acOTAnrps, veA et laeA). Les résultats ont montré que toutes les souches possèdent la capacité d’adhérer l’OTA à leur surface, notamment la souche SN7 qui a réduit 33% de l’OTA après 60 minutes d’incubation dans une solution PBS (Phosphate Buffer Solution) non nutritive. Les souches AT10 et SN7 ont métabolisé 51.94 et 52.68% de l’OTA ajoutée au milieu ISP2 (International Streptomyces Project-2) après 5 jours de culture à 28 °C. Cependant, les souches MS1, ML5 et G10 étaient les seules à avoir un effet sur l’expression des gènes de biosynthèse de l’OTA chez A. carbonarius. Effectivement les gènes acpks, acOTApks et acOTAnrps ont été réprimé respectivement de 37.1, 23.9 et 21% par MS1, de 39, 23 et 11.1% par ML5 et de 39, 18.3 et 11.1% par la souche G10. Ce projet a mis en valeur la capacité des extraits naturels (composés phénoliques et huiles essentielles) et des actinobactéries à prévenir d’une part la production de l’OTA et d’autre part réduire ses taux, sans pourtant affecter l’équilibre naturel ni engendrer l’apparition des débris toxiques dans les aliments traités. / Ochratoxin A (OTA) is a mycotoxin derived from the secondary metabolism of filamentous fungi belonging to the Penicillium and Aspergillus genera. However, Aspergillus carbonarius is the major producer of OTA on grapes. OTA has been detected in different types of foodstuffs as well as several products derived from these commodities. The toxicological profile of OTA and its adverse effects on human and animal health (hepatotoxic, immunotoxic, genotoxic, teratogenic and carcinogenic effects) has given this mycotoxin a major attention of international committees in order to limit its occurrence. The aim of this project was to develop biological techniques that can reduce OTA produced by A. carbonarius and detoxify non-compliant food matrices. The first strategy was achieved by using essential oils (cardamom, celery, cinnamon, taramira, oregano, bay leaf, cumin, fenugreek, melissa, mint, sage, anise, chamomile, fennel, rosemary and thyme) and phenolic compounds extracted from medicinal plants (bay leaf, cumin, fenugreek, melissa, mint, sage, anise, chamomile, fennel, rosemary, and thyme) to evaluate their effect on OTA production in SGM medium. This approach was complemented by a molecular study to evaluate the expression of the OTA biosynthesis genes (acpks, acOTApks and acOTAnrps) as well as the regulatory genes (veA and laeA) in A. carbonarius. The results revealed that essential oils had more significant fungicidal activity than phenolic extracts. Indeed, the essential oils of thyme, oregano, taramira, and cinnamon completely blocked the growth of A. carbonarius. However, essential oils of fennel, cardamom, anise, chamomile, celery and rosemary reduced OTA without affecting fungal growth. The mode of action of these essential oils has been demonstrated by evaluating the expression of acpks, acOTApks, acOTAnrps, veA and laeA genes in A. carbonarius. The expression of acpks was repressed the most (up to 99.2%) when A. carbonarius was cultured with 5 L / mL of fennel essential oil, resulting in a 88.9% of reduction in the OTA produced by this fungus. The second strategy was developed in order to detoxify contaminated food matrices. This methodology was achieved by using seven strains of actinobacteria (AT10, AT8, SN7, MS1, ML5, G10 and PT1), and evaluating their ability to metabolize OTA, adhere this toxin to their membrane walls and their effect on the expression of the genes involved in the biosynthesis of OTA in A. carbonarius (acpks, acOTApks, acOTAnrps, veA and laeA). The results showed that all strains were able to bind OTA to their surfaces, specially the SN7 strain which reduced 33% of OTA after incubation for 60 minutes in PBS (Phosphate Buffer Solution). The strains AT10 and SN7 metabolized 51.94 and 52.68% of the OTA added to the ISP2 medium (International Streptomyces Project-2) after 5 days of culture at 28° C. However, MS1, ML5 and G10 were the only strains to have an effect on the expression of the OTA biosynthesis genes in A. carbonarius. Indeed, acpks, acOTApks and acOTAnrps genes were repressed respectively by 37.1, 23.9 and 21% by MS1, 39, 23 and 11.1% by ML5 and 39, 18.3 and 11.1% by the strain G10. This project highlighted the power of natural extracts (phenolic compounds and essential oils) as well as strains of actinobacteria to prevent OTA production on one hand and to detoxify contaminated commodities on the other hand without altering the natural microbial balance.

This work is aimed for Actinobacteria and describes their importance in Arctic soil. The members of Actinobacteria are known for their ability to decompose complex natural biopolymers and because they are able to live in harsh arctic environment they could play there an important role in organic matter decomposition. The work compares their abundance in different soil horizons with the focus on cryoturbations and determines the influence of temperature on their amount. This work also represents the results of testing pure Actinobacterial isolates for the production of cellulolytic enzymes.