[pt] A presença de metais-traço nos cigarros oferece uma fonte de informação potencial do ponto de vista forense e criminológico, na determinação e classificação de marcas de tabaco, com o intuito de diferenciar as marcas legalizadas das contrabandeadas. Por isso, neste trabalho, almejou-se desenvolver e validar métodos para determinação metais e semimetais presentes nos cigarros, com o intuito de avaliar a viabilidade de discriminação entre as diferentes marcas, analisando as amostras após o processo de calcinação e dissolução ácida. Estes métodos englobam as análises químicas por técnicas espectrométricas (CV-AAS, ICP-OES e DRC-ICP-MS) e posterior interpretação de dados por ACP (Análise dos Componentes Principais) e AAH (Análise de Agrupamento Hierárquico). As concentrações de metais e semimetais (As, Ba, B, Cd, Co, Cu, Fe, Mn, Hg, Ni, Sr e Zn) foram estudadas para dez diferentes marcas de cigarros, obtidas em tabacarias e lojas locais, representando as marcas mais consumidas no país. A utilização da ACP indicou a presença de quatro componentes principais que, em conjunto, explicavam mais de 80 por cento da variabilidade dos dados amostrais. Esses resultados comprovaram a existência de diferenças significativas entre as marcas de tabaco em seus conteúdos elementares, possibilitando a diferenciação entre marcas ilegais e marcas legalizadas e o agrupamento das marcas legalizadas de acordo com as empresas de tabaco as quais pertencem, sendo corroborado pela AAH, feita pelo método da distância média de ligação (UPGMA). Os teores de umidade, cinzas totais e pH de cada marca também foram determinados, no entanto não contribuíram efetivamente para a diferenciação entre as marcas estudadas. A AAH foi utilizada para a interpretação de possíveis similaridades entre os elementos, e em conjunto com o teste de correlação linear de Pearson, indicou similaridades entre os elementos Ba e Sr . Agrupamentos entre Fe e Zn com o Sr e Co com o Mn também foram detectados. / [en] The presence of trace metals in cigarettes offers potential source of information from a forensic and criminological point of view, which deals with the determination and classification of tobacco brands, with the aim of differentiating legal brands from smuggled brands. Therefore, this work aimed to develop and validate methods for determining the metallic content present in cigarettes, in order to assess the feasibility of discrimination between different brands, analyzing cigarettes after the process of calcination and acid digestion. These methods include chemical analysis by spectrometric techniques (CV-AAS, ICP-OES and DRC-ICPMS) and subsequent data interpretation by PCA (Principal Component Analysis) and HCA (Hierarchical Cluster Analysis). The concentration of metals and semimetals (As, Ba, B, Cd, Co, Cu, Fe, Mn, Hg Ni, Sr and Zn) were studied for ten different brands of cigarettes, obtained in tobacconists and local stores, representing the brands most consumed in the country. The analysis of PCA indicated the presence of four principal components that together explained more than 80 percent of the variability of the sample data. These results proved the existence of significant differences between tobacco brands in their elemental content, allowing the differentiation between illegal brands and legalized brands and the grouping of legal brands according to the tobacco companies to which they belong, being corroborated by the HCA performed by the average link distance method (UPGMA). The moisture content, total ash and pH of each brand were also determined, however they did not contribute effectively to the differentiation between the brands studied. The HCA used for the interpretation of possible similarities between the elements, and interpreted together with Pearson s linear correlation test indicated similarities between the elements Ba, Sr, in addition to indicating the proximity between the Mn and the Cd. Clusters between Fe and Zn with Sr and Co with Mn were also detected.

Les autotransporteurs monomériques, appartenant au système de sécrétion de type V, correspondent à une famille importante de facteurs de virulence bactériens. Plusieurs fonctions, souvent essentielles pour le développement d’une infection ou pour le maintien et la survie des bactéries dans l’organisme hôte, ont été décrites pour cette famille de protéines. Malgré l’importance de ces protéines, notre connaissance de leur biogenèse et de leur mécanisme d’action demeure relativement limitée. L’autotransporteur AIDA-I, retrouvé chez diverses souches d’Escherichia coli, est un autotransporter multifonctionnel typique impliqué dans l’adhésion et l’invasion cellulaire ainsi que dans la formation de biofilm et d’agrégats bactériens. Les domaines extracellulaires d’autotransporteurs monomériques sont responsables de la fonctionnalité et possèdent pratiquement tous une structure caractéristique d’hélice β. Nous avons mené une étude de mutagenèse aléatoire avec AIDA-I afin de comprendre la base de la multifonctionnalité de cette protéine. Par cette approche, nous avons démontré que les domaines passagers de certains autotransporteurs possèdent une organisation modulaire, ce qui signifie qu’ils sont construits sous la forme de modules fonctionnels. Les domaines passagers d’autotransporteurs peuvent être clivés et relâchés dans le milieu extracellulaire. Toutefois, malgré la diversité des mécanismes de clivage existants, plusieurs protéines, telles qu’AIDA-I, sont clivées par un mécanisme qui demeure inconnu. En effectuant une renaturation in vitro d’AIDA-I, couplée avec une approche de mutagenèse dirigée, nous avons démontré que cette protéine se clive par un mécanisme autocatalytique qui implique deux acides aminés possédant un groupement carboxyle. Ces résultats ont permis la description d’un nouveau mécanisme de clivage pour la famille des autotransporteurs monomériques. Une des particularités d’AIDA-I est sa glycosylation par une heptosyltransférase spécifique nommée Aah. La glycosylation est un concept plutôt récent chez les bactéries et pour l’instant, très peu de protéines ont été décrites comme glycosylées chez E. coli. Nous avons démontré que Aah est le prototype pour une nouvelle famille de glycosyltransférases bactériennes retrouvées chez diverses espèces de protéobactéries. La glycosylation d’AIDA-I est une modification cytoplasmique et post-traductionnelle. De plus, Aah ne reconnaît pas une séquence primaire, mais plutôt un motif structural. Ces observations sont uniques chez les bactéries et permettent d’élargir nos connaissances sur la glycosylation chez les procaryotes. La glycosylation par Aah est essentielle pour la conformation d’AIDA-I et par conséquent pour sa capacité de permettre l’adhésion. Puisque plusieurs homologues d’Aah sont retrouvés à proximité d’autotransporteurs monomériques putatifs, cette famille de glycosyltranférases pourrait être importante, sinon essentielle, pour la biogenèse et/ou la fonction de nombreux autotransporteurs. En conclusion, les résultats présentés dans cette thèse apportent de nouvelles informations et permettent une meilleure compréhension de la biogenèse d’une des plus importantes familles de protéines sécrétées chez les bactéries Gram négatif. / Monomeric autotransporters, a family of proteins that use the type V secretion pathway, are important mediators of virulence for many bacterial pathogens. Many functions important for host colonization and survival have been described for these proteins. Despite the recognized importance of this family of proteins, the mechanisms that are required for the biogenesis and functionality of monomeric autotransporters still remain poorly understood. The Escherichia coli adhesin involved in diffuse adherence (AIDA-I) is a classical multifunctional autotransporter protein that mediates bacterial aggregation and biofilm formation, as well as adhesion and invasion of cultured epithelial cells. Extracellular domains of autotransporters are responsible for the protein function and fold into a characteristic β-helical structure. We performed a random mutagenesis of the AIDA-I passenger domain in order to identify regions involved in the various phenotypes associated with the expression of this protein. Our study suggests that the passenger domain of AIDA-I possesses a modular organization, which means that AIDA-I is built with individual functional modules. Autotransporter passenger domains can be cleaved from the β-domain and released into the extracellular milieu. However, despite the fact that diverse cleavage mechanisms have been previously described, many autotransporters, like AIDA-I, are cleaved by an unknown mechanism. By monitoring the in vitro refolding and cleavage following by site-directed mutagenesis, we showed that AIDA-I processing is an autocatalytic event that involves two acidic residues. Our results unveil a new mechanism of auto-processing in the autotransporter family. AIDA-I is one of the few glycosylated proteins found in Escherichia coli. Glycosylation is mediated by a specific heptosyltransferase encoded by the aah gene, but little is known about the role of this modification and the mechanism involved. Our findings suggest that Aah represents the prototype of a new large family of bacterial protein O-glycosyltransferases that modify various substrates recognized through a structural motif. Furthermore, we showed that glycosylation occurs in the cytoplasm by a cotranslational mechanism. These observations are unique in bacteria and represent a significant advance in our comprehension of prokaryotic glycosylation. We also showed that glycosylation is required to ensure a normal conformation of AIDA-I and, as a consequence, is necessary for its cell-binding function. The finding that other autotransporters or large adhesin-encoding genes are linked to Aah homologue-encoding genes suggests that glycosylation may be important, if not essential, for the function of these proteins, as for AIDA-I. In conclusion, the results presented in this thesis bring new information about the autotransporter family and also give new insight into the mechanisms that are important for different aspects of the biogenesis of monomeric autotransporters.

Les autotransporteurs monomériques, appartenant au système de sécrétion de type V, correspondent à une famille importante de facteurs de virulence bactériens. Plusieurs fonctions, souvent essentielles pour le développement d’une infection ou pour le maintien et la survie des bactéries dans l’organisme hôte, ont été décrites pour cette famille de protéines. Malgré l’importance de ces protéines, notre connaissance de leur biogenèse et de leur mécanisme d’action demeure relativement limitée. L’autotransporteur AIDA-I, retrouvé chez diverses souches d’Escherichia coli, est un autotransporter multifonctionnel typique impliqué dans l’adhésion et l’invasion cellulaire ainsi que dans la formation de biofilm et d’agrégats bactériens. Les domaines extracellulaires d’autotransporteurs monomériques sont responsables de la fonctionnalité et possèdent pratiquement tous une structure caractéristique d’hélice β. Nous avons mené une étude de mutagenèse aléatoire avec AIDA-I afin de comprendre la base de la multifonctionnalité de cette protéine. Par cette approche, nous avons démontré que les domaines passagers de certains autotransporteurs possèdent une organisation modulaire, ce qui signifie qu’ils sont construits sous la forme de modules fonctionnels. Les domaines passagers d’autotransporteurs peuvent être clivés et relâchés dans le milieu extracellulaire. Toutefois, malgré la diversité des mécanismes de clivage existants, plusieurs protéines, telles qu’AIDA-I, sont clivées par un mécanisme qui demeure inconnu. En effectuant une renaturation in vitro d’AIDA-I, couplée avec une approche de mutagenèse dirigée, nous avons démontré que cette protéine se clive par un mécanisme autocatalytique qui implique deux acides aminés possédant un groupement carboxyle. Ces résultats ont permis la description d’un nouveau mécanisme de clivage pour la famille des autotransporteurs monomériques. Une des particularités d’AIDA-I est sa glycosylation par une heptosyltransférase spécifique nommée Aah. La glycosylation est un concept plutôt récent chez les bactéries et pour l’instant, très peu de protéines ont été décrites comme glycosylées chez E. coli. Nous avons démontré que Aah est le prototype pour une nouvelle famille de glycosyltransférases bactériennes retrouvées chez diverses espèces de protéobactéries. La glycosylation d’AIDA-I est une modification cytoplasmique et post-traductionnelle. De plus, Aah ne reconnaît pas une séquence primaire, mais plutôt un motif structural. Ces observations sont uniques chez les bactéries et permettent d’élargir nos connaissances sur la glycosylation chez les procaryotes. La glycosylation par Aah est essentielle pour la conformation d’AIDA-I et par conséquent pour sa capacité de permettre l’adhésion. Puisque plusieurs homologues d’Aah sont retrouvés à proximité d’autotransporteurs monomériques putatifs, cette famille de glycosyltranférases pourrait être importante, sinon essentielle, pour la biogenèse et/ou la fonction de nombreux autotransporteurs. En conclusion, les résultats présentés dans cette thèse apportent de nouvelles informations et permettent une meilleure compréhension de la biogenèse d’une des plus importantes familles de protéines sécrétées chez les bactéries Gram négatif. / Monomeric autotransporters, a family of proteins that use the type V secretion pathway, are important mediators of virulence for many bacterial pathogens. Many functions important for host colonization and survival have been described for these proteins. Despite the recognized importance of this family of proteins, the mechanisms that are required for the biogenesis and functionality of monomeric autotransporters still remain poorly understood. The Escherichia coli adhesin involved in diffuse adherence (AIDA-I) is a classical multifunctional autotransporter protein that mediates bacterial aggregation and biofilm formation, as well as adhesion and invasion of cultured epithelial cells. Extracellular domains of autotransporters are responsible for the protein function and fold into a characteristic β-helical structure. We performed a random mutagenesis of the AIDA-I passenger domain in order to identify regions involved in the various phenotypes associated with the expression of this protein. Our study suggests that the passenger domain of AIDA-I possesses a modular organization, which means that AIDA-I is built with individual functional modules. Autotransporter passenger domains can be cleaved from the β-domain and released into the extracellular milieu. However, despite the fact that diverse cleavage mechanisms have been previously described, many autotransporters, like AIDA-I, are cleaved by an unknown mechanism. By monitoring the in vitro refolding and cleavage following by site-directed mutagenesis, we showed that AIDA-I processing is an autocatalytic event that involves two acidic residues. Our results unveil a new mechanism of auto-processing in the autotransporter family. AIDA-I is one of the few glycosylated proteins found in Escherichia coli. Glycosylation is mediated by a specific heptosyltransferase encoded by the aah gene, but little is known about the role of this modification and the mechanism involved. Our findings suggest that Aah represents the prototype of a new large family of bacterial protein O-glycosyltransferases that modify various substrates recognized through a structural motif. Furthermore, we showed that glycosylation occurs in the cytoplasm by a cotranslational mechanism. These observations are unique in bacteria and represent a significant advance in our comprehension of prokaryotic glycosylation. We also showed that glycosylation is required to ensure a normal conformation of AIDA-I and, as a consequence, is necessary for its cell-binding function. The finding that other autotransporters or large adhesin-encoding genes are linked to Aah homologue-encoding genes suggests that glycosylation may be important, if not essential, for the function of these proteins, as for AIDA-I. In conclusion, the results presented in this thesis bring new information about the autotransporter family and also give new insight into the mechanisms that are important for different aspects of the biogenesis of monomeric autotransporters.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / This work aimed to isolate and identify endophytic fungi associated to three chemotypes of Lippia alba (Mill.) NE Brown found in Brazilian Northeast and investigate the potential of these fungi in the production of volatile organic compounds (VOCs) and in front biotransformation to organic substrates. In addition, there was a comparative study of the chemical composition of the leaves three chemotypes of L. alba detected both in essential oils obtained by hydrodistillation as the volatile constituents obtained by solid phase microextraction (SPME). The analysis and identification of volatile constituents was performed by gas chromatography coupled to mass spectrometry (GC-MS). The GC-MS data of the samples obtained by hydrodistillation and SPME were subjected to multivariate data analysis, which provided principal component analysis charts (PCA) and hierarchical cluster analysis (HCA), where it was observed that the methodology used obtaining volatile compounds can influence the classification of chemotypes made based on volatile chemically L. alba reported in the literature. The essential oils obtained from the leaves of the three chemotypes of L. alba were tested against nematicide activity, where the essential oil chemotype II had a higher potential nematicide (LD50 = 71.30 ppm). Of the six endophytic fungi isolated from L. alba leaves, only the fungi Colletotrichum sp. (chemotypes I and II) and Scopulariopsis fusca were able to produce VOCs considerably; and wherein the linalool and caryophyllene compounds produced by S. fusca have also been detected in the chemical composition leaves from L. alba chemotype I, where the fungus was isolated. The study of VOCs also pointed a biocatalytic potential for endophytic fungi from L. alba. This potential was investigated using (R)-carvona and (R)-limonene as substrates. Variables such as time and culture medium were investigated in both experiments, and the biotransformation of (R)-carvona by strains of L. alba isolated endophytes resulted in the production of three derivatives, among which one of them has not yet been reported in the literature as a product of biotransformation of carvone. Since the biotransformation of (R) led to the production of limonene nine derivatives among which four have not been reported in the literature as the limonene biotransformation products. / Este trabalho teve como objetivo principal isolar e identificar fungos endofÃticos associados a trÃs quimiotipos de Lippia alba (Mill.) N. E. Brown encontrados no Nordeste brasileiro e investigar o potencial desses fungos na produÃÃo de compostos orgÃnicos volÃteis (COVs) e na biotransformaÃÃo frente a substratos orgÃnicos. AlÃm disso, realizou-se um estudo comparativo entre a composiÃÃo quÃmica das folhas de trÃs quimiotipos de L. alba detectada tanto nos Ãleos essenciais obtidos por hidrodestilaÃÃo quanto nos constituintes volÃteis obtidos por microextraÃÃo em fase sÃlida (MEFS). A anÃlise e identificaÃÃo dos constituintes volÃteis foi realizada por cromatografia gasosa acoplada à espectrometria de massa (CG-EM). Os dados de CG-EM das amostras obtidas por hidrodestilaÃÃo e por MEFS foram submetidos à anÃlise multivariada de dados, que forneceu grÃficos de anÃlise de componente principal (ACP) e anÃlise de agrupamento hierÃrquico (AAH), onde foi possÃvel observar que a metodologia utilizada na obtenÃÃo dos compostos volÃteis pode influenciar a classificaÃÃo dos quimiotipos feita com base na constituiÃÃo quÃmica volÃtil de L. alba relatada na literatura. Os Ãleos essenciais obtidos das folhas dos trÃs quimiotipos de L. alba foram testados frente a atividade nematicida, onde o Ãleo essencial do quimiotipo II apresentou maior potencial nematicida (DL50 = 71,30 ppm). Dos seis fungos endofÃticos isolados das folhas de L. alba, somente os fungos Colletotrichum sp. (quimiotipos I e II) e Scopulariopsis fusca foram capazes de produzir COVs consideravelmente; sendo que os compostos linalol e cariofileno, produzidos por S. fusca tambÃm foram detectados na constituiÃÃo quÃmica das folhas de L. alba quimiotipo I, de onde o fungo foi isolado. O estudo dos COVs produzidos por fungos tambÃm apontou um potencial biocatalÃtico para os endÃfitos de L. alba. Este potencial foi investigado utilizando-se (R)-carvona e (R)-limoneno como substratos. VariÃveis como meio de cultivo e tempo foram investigadas em ambos experimentos, sendo que a biotransformaÃÃo da (R)-carvona por cepas de endÃfitos isolados de L. alba resultou na produÃÃo de trÃs derivados, dentre os quais um deles ainda nÃo foi citado na literatura como produto de biotransformaÃÃo da carvona. Jà a biotransformaÃÃo do (R)-limoneno levou à produÃÃo de 9 derivados, dentre os quais, 4 ainda nÃo foram relatados na literatura, como produtos de biotransformaÃÃo do limoneno.

Several animals, both vertebrate and invertebrate, communicate using sound and they do so in a non-ideal medium, the habitat in which they live. As acoustic signals pass through the habitat, they suffer loss of information due to signal degradation, which is often determined by the acoustic properties of the habitat. Understanding the influence of habitat-induced constraints on signaling is vital to the understanding of evolution of signal structure. Over time signals can evolve their temporal and/or spectral characteristics so as to resist or reduce degradation. Conversely, signalers may modify their behaviour so as to improve transmission for effective communication. The Acoustic Adaptation Hypothesis (AAH) predicts that given the properties of the habitat in which animals communicate, signals should evolve so as to maximize their broadcast range and the number of potential receivers. The prediction of the hypothesis is that signal transmission is best in the native habitat as compared to non-native habitats. Since its inception, the idea of acoustic adaptation has been tested in several vertebrate species including birds, mammals and frogs but rarely in invertebrates. Moreover, most of these studies have been carried out at the macrohabitat level, such as transmission in forests versus grasslands. For animals with limited mobility such as invertebrates, a finer level of investigation at the microhabitat level is more relevant. Only one study on cicadas has investigated the predictions of the AAH at the microhabitat level. Besides, investigations done at the community level are largely missing in the literature. The effect of height on signal transmission is well documented, however, only one study on birds has investigated the AAH with respect to vertical stratification of coexisting species. Among invertebrates, crickets are well known for their calling songs which males use to attract conspecific females over long distances. No study so far has tested the prediction of the AAH in these acoustically communicating invertebrates. The central focus of this study was to test the predictions of the AAH in a natural assemblage of ensiferan (cricket) species. I examined the prediction of the hypothesis at the microhabitat level with respect to the vertical stratification of co-existing ensiferan species. The study was carried out on an assemblage of crickets in the wet evergreen forests of Kudremukh National Park in the Western Ghats in Southern India. For this purpose, it was important to examine calling height and microhabitat selection in these animals because if the use of calling height and microhabitat was random, then there would not be any native height/microhabitat and the question of acoustic adaption would not arise. Therefore, I first standardized methods to characterize the habitat of the crickets. Using resource selection functions, I then quantified microhabitat selection in 13 ensiferan species. I also examined the calling heights of these species. My results suggest that these species are microhabitat specialists and also distribute vertically within the forest with respect to calling height. Based on the knowledge of the vertical distribution of these animals in the forest I then carried out playback experiments using natural calls of 12 species of Ensifera in their natural habitat. The transmission experiments were carried out at five heights in the forest, including the ground, different parts of the understorey as well as in the canopy. The study aimed to examine whether vertical stratification in the ensiferan species of Kudremukh is driven by selection for maximizing transmission range. I examined the effect of different heights on signal degradation. The investigation was carried out with respect to three different measures of signal degradation, namely, total attenuation, signal to noise ratio as well as envelope distortion. The results indicate a lack of overall support for the AAH with respect to vertical stratification of crickets in Kudremukh. However, a strong, independent effect of height of calling on signal degradation was found, with the ground being the worst layer for transmission and the mid-understorey (2 m) emerging to be good for all species with respect to all three measures of signal degradation. I then analysed the transmission data from a different point of view, exploring the possibility of evolution of signal structures that may confer some advantages in terms of signal transmission, given the habitat-induced constrains on signal propagation. The idea was to examine why certain species perform better than others in terms of signal transmission. This investigation was aimed at characterizing the effect of call features on signal attenuation. I found that temporal features of calls did not have a strong effect on height-specific signal attenuation. While call duration had no effect on signal attenuation, duty cycle did influence attenuation profiles of the calls, with high duty cycle calls performing better than low duty cycle calls. However, there was no interaction of height with the temporal features of calls, eliminating the possibility of these features being shaped by microhabitat or height dependent transmission characteristics. Spectral features of calls, on the other hand, affected signal attenuation very strongly. As expected, low frequency calls performed better than high frequency calls and pure tone calls fared much better than the broadband calls, especially on the ground and the canopy. To the best of my knowledge, this is the first study to carry out a rigorous quantification of microhabitat selection in Ensifera. This is also the first detailed examination of the Acoustic Adaptation Hypothesis at the microhabitat level, tested in a natural assemblage of coexisting invertebrate species.