La séparation de sources en aveugle (SSA) vise à rechercher des signaux sources inconnus et mélangés de manière inconnue au sein de plusieurs observations. Cette approche très générique et non-supervisée ne fournit cependant pas nécessairement des résultats exploitables. Il est alors nécessaire d’ajouter des contraintes, notamment physiques, afin de privilégier la recherche de sources ayant une structure particulière. La factorisation en matrices positives (non-negative matrix factorization, NMF) qui fait plus précisément l’objet de cette thèse recherche ainsi des sources positives observées au travers de mélanges linéaires positifs.L’ajout de davantage d’information reste cependant souvent nécessaire afin de pouvoir séparer les sources. Nous nous intéressons ainsi au concept de parcimonie qui permet d’améliorer le contraste entre celles-ci tout en produisant des approches très robustes, en particulier au bruit. Nous montrons qu’afin d’obtenir des solutions stables, les contraintes de positivité et la régularisation parcimonieuse doivent être appliqués de manière adéquate. Aussi, l’utilisation de la parcimonie dans un espace transformé potentiellement redondant, permettant de capturer la structure de la plu- part des signaux naturels, se révèle difficile à appliquer au côté de la contrainte de positivité dans l’espace direct. Nous proposons ainsi un nouvel algorithme de NMF parcimonieuse, appelé nGMCA (non-negative Generalized Morphological Component Analysis), qui surmonte ces difficultés via l’utilisation de techniques de calcul proximal. Des expérimentations sur des données simulées montrent que cet algorithme est robuste à une contamination par du bruit additif Gaussien, à l’aide d’une gestion automatique du paramètre de parcimonie. Des comparaisons avec des algorithmes de l’état-de-l’art en NMF sur des données réalistes montrent l’efficacité ainsi que la robustesse de l’approche proposée.Finalement, nous appliquerons nGMCA sur des données de chromatographie en phase liquide - spectrométrie de masse (liquid chromatography - mass spectrometry, LC-MS). L’observation de ces données montre qu’elles sont contaminées par du bruit multiplicatif, lequel détériore grandement les résultats des algorithmes de NMF. Une extension de nGMCA conçue pour prendre en compte ce type de bruit à l’aide d’un a priori non-stationnaire permet alors d’obtenir d’excellents résultats sur des données réelles annotées. / Blind source separation aims at extracting unknown source signals from observations where these sources are mixed together by an unknown process. However, this very generic and non-supervised approach does not always provide exploitable results. Therefore, it is often necessary to add more constraints, generally arising from physical considerations, in order to favor the recovery of sources with a particular sought-after structure. Non-negative matrix factorization (NMF), which is the main focus of this thesis, aims at searching for non-negative sources which are observed through non-negative linear mixtures.In some cases, further information still remains necessary in order to correctly separate the sources. Here, we focus on the sparsity concept, which helps improving the contrast between the sources, while providing very robust approaches, even when the data are contaminated by noise. We show that in order to obtain stable solutions, the non-negativity and sparse constraints must be applied adequately. In addition, using sparsity in a potentially redundant transformed domain could allow to capture the structure of most of natural image, but this kind of regularization proves difficult to apply together with the non-negativity constraint in the direct domain. We therefore propose a sparse NMF algorithm, named nGMCA (non-negative Generalized Morphological Component Analysis), which overcomes these difficulties by making use of proximal calculus techniques. Experiments on simulated data show that this algorithm is robust to additive Gaussian noise contamination, with an automatic control of the sparsity parameter. This novel algorithm also proves to be more efficient and robust than other state-of-the-art NMF algorithms on realistic data.Finally, we apply nGMCA on liquid chromatography - mass spectrometry data. Observation of these data show that they are contaminated by multiplicative noise, which greatly deteriorates the results of the NMF algorithms. An extension of nGMCA was designed to take into account this type of noise, thanks to the use of a non-stationary prior. This extension is then able to obtain excellent results on annotated real data.

Parmi les nombreux pathogènes fongiques susceptibles d’infecter les épis de maïs, les espèces appartenant au genre Fusarium sont particulièrement préoccupantes pour la filière maïsicole. La fusariose est susceptible d’induire des pertes de rendement considérables et est fréquemment associée à une contamination des épis par des mycotoxines. Un des leviers prometteur repose sur la sélection génétique de plantes résistantes à Fusarium et à l’accumulation de mycotoxines. Plusieurs Quantitative Trait Loci (QTL) ont été identifiés d’après la caractérisation moléculaire de la résistance à la fusariose chez le maïs. Cependant malgré les progrès des approches génétiques, les mécanismes moléculaires impliqués restent en grande partie inconnus. L’identification de métabolites majeurs associés à la résistance reste donc indispensable pour la création d’un outil d’aide à la sélection variétale. Une approche métabolomique combinant de la spectrométrie de masse et de la 1H-RMN a été mise au point pour identifier un ensemble de métabolites de défense, constitutifs ou induits par l’infection, susceptibles d’intervenir dans la résistance à Fusarium. Cette approche a été appliquée aux grains à deux stades de développement sur 20 variétés présentant des degrés de résistance contrastés inoculés ou non avec une souche de Fusarium graminearum toxinogène par le canal des soies. Les résultats obtenus mettent en évidence un panel de métabolites liés à la résistance ou la sensibilité des variétés de maïs. / Fusarium graminearum is the main causal agent of maize ear rot or Gibberella ear rot (GER), an important fungal disease affecting maize. GER leads to significant economic loss and serious health issues due to the ability of F. graminearum to produce mycotoxins such as type B trichothecenes. One promising approach to control Giberella Ear Rot and reduce mycotoxins contamination is to promote host-genetic resistance. Several Quantitative Trait Loci (QTLs) have been identified in maize. However molecular basis to resistance to Fusarium infection remains largely unknown and the success of selection for GER resistance is still challenging. Biochemical approaches can provide valuable insights in the mechanisms crops employ against F. graminearum and its production of mycotoxins. A biochemical profiling could actually be an efficient way to decipher plant-pathogen interactions and progress in screening resistant maize lines. This study aims to elucidate the metabolic profiling of F. graminearum resistance and toxin accumulation in kernels toward the combination of high resolution mass spectrometry and 1H NMR to identify a large set of metabolites, preformed, constitutive as well as inducible defense metabolites that could play a key role in GER resistance. This approach was applied to kernels harvested at two developmental stages. Twenty genotypes with contrasting levels of resistance were inoculated, or not, with a toxigenic Fusarium graminearum strain through the silk channel. The obtained data allowed highlighting a set of biochemical compounds linked to the resistance or susceptibility of maize genotypes

O presente trabalho apresenta os dados obtidos no estudo para o desenvolvimento de método multirresíduo de pesticidas dos grupos de benzimidazol (carbendazim, tiabendazol e tiofanato metílico), carbamatos (aldicarbe, aldicarbe sulfona, aldicarbe sulfóxido, carbaril, carbofurano, metomil, metiocarbe, pirimicarbe, propoxur) e triazinas (atrazina e simazina) em amostras de milho verde. Os pesticidas foram extraídos em acetona, sob agitação, diluídos em água e injetados no sistema LC/MS/MS. A técnica analítica de quantificação e confirmação utilizada foi a Cromatografia Líquida acoplada à Espectrometria de Massas em Tandem com ionização por Electrospray no modo positivo. A validação do método multirresíduo foi submetida em conformidade com a norma EC/2002/657 da Comunidade Européia. Para quantificação dos analitos utillizouse as curvas analíticas dos princípios ativos, nas concentrações que variaram de 0,04 a 8,0 ng/ml, correspondentes a 2,0 a 400 ?g.kg-1 na amostra. Para o estudo da recuperação, os pesticidas foram avaliados em cinco níveis, de ½ Limite de quantificação (LOQ) a 5 LOQ, correspondentes a níveis de fortificação de 4,0 a 200 ?g.kg-1 . Os resultados da recuperação apresentaram, em níveis aceitáveis, na faixa de 80 a 110% e com precisão satisfatórias, CV <=20%, com exceção de aldicarbe e de aldicarbe sulfona. Os limites de quantificação do método variaram de 8 a 40 ?g.kg-1 e os limites de detecção do método, de 0,2 a 2,9 ?g.kg-1. Os limites de quantificação do método atendem aos limites máximos de resíduos da legislação brasileira em vigor. Nas amostras estudadas, não foram encontrados resíduos de pesticidas acima do limite de quantificação do método. Por outro lado, todos os pesticidas, exceto carbaril e pirimicarbe, foram detectados em todas as amostras e em níveis acima dos limites de detecção do método. A alta incidência da presença de resíduos de pesticidas se deve possivelmente ao uso inadequado de agrotóxico, quando não autorizado o seu uso para a cultura de milho no Brasil ou proveniente de alguma contaminação, como o tratamento de agrotóxicos aplicados em outras culturas plantadas no mesmo solo. / The present work presents the results of a study of development about a multiresidue method for determining pesticides of the following groups in corn: benzimidazolic compounds (carbendazim, thiabendazol and methyl thiofanate), carbamates (aldicarb, aldicarb sulfone, aldicarb sulfoxide, carbaryl, carbofuran, methomyl, methiocarb, pirimicarb, propoxur), and triazines (atrazine and simazine). The pesticides were extracted in acetone under constant stirring, diluted in water and injected in the LC/MS/MS system. The analytical technique for quantification and confirmation was Liquid Chromatography coupled to tandem Mass Spectrometry with Electrospray ionization in the positive mode The validation of the multiresidue method underwent the European Community EC/2002/657 standard. For the quantification of the analytes, calibration curves of the substances in concentrations ranging from 0.04 to 8.0 ng/mL were used, corresponding to 2.0 to 400 ?g.kg-1 in the sample. For recovery tests, the pesticides were assessed on five levels, from ½ Limit of Quantification (LOQ) to 5 LOQ, corresponding to the fortification levels of 4.0 to 200 ?g.kg-1. The results of the tests showed a recovery of 80-110% with satisfactory precision, and CV <=20%, except for aldicarb and aldicarb sulfone. The limits of quantification of the method ranged from 8 to 40 ?g.kg-1 and the limits of detection ranged from 0.2 to 2.9 ?g.kg-1. The limits of quantification of the method meet the maximum residue limit allowed by the Brazilian law. In the studied samples, pesticide residues above the limit of quantification of the method were not found. On the other hand, all pesticides, except for carbaryl and pirimicarb, in all samples, were detected and level above the limits of detection of the method. The high incidence of residues of these pesticides is probably due to their inadequate use, since it is not allowed on corn cultures in Brazil, or any kind of contamination like the use of such pesticides on other cultures over the same soil.

A identificação de intermediários formados durante os processos de tratamentos de efluentes tem se tornado alvo de estudos. Entretanto, uma das questões mais importantes nos estudos de intermediários formados durante a fotólise ou degradação de fármacos por processos oxidativos avançados publicados atualmente é a concentração inicial utilizada. Altas concentrações dificultam a compreensão da real situação que ocorre na natureza, onde esses compostos são encontrados em baixas concentrações. Dentro deste contexto, o presente estudo, avaliou a pré-concentração de intermediários formados durante a fotólise em solução aquosa e em esgoto sintético da sulfametazina (SMZ), do ciprofloxacino (CIP) e do enrofloxacino (ENR) em diferentes concentrações (25 mg L-1 e 250 &mu;g L-1 para SMZ; 10 mg L-1 e 100 &mu;g L-1 para CIP e ENR) utilizando espectromatria de massas sequencial de alta resolução (Q-ToF e LIT-Orbitrap MS). Foram identificados oito intermediários da fotodegradação da SMZ, dentre estes os de m/z nominal 140 e 229 ainda não relatados na literatura. A metodologia desenvolvida para a SMZ foi aplicada a análises de produtos de transformação vindos de reator anaeróbio. As análises da biodegradação da SMZ permitiram a identificação de três produtos de transformação. O m/z 295 foi comum aos dois processos, fotólise e biodegradação. O m/z 227 foi descrito pela primeira vez como produto de biodegradação anaeróbia da SMZ. Tanto para o CIP quanto para o ENR foram identificados 10 intermediários, apesar da similaridade entre os dois fármacos, apenas um produto de degradação foi comum aos dois, o m/z 346, além do próprio CIP que é intermediário do ENR. Dentre as fases extratoras avaliadas, a Oasis HLB se mostrou mais eficiente na recuperação dos intermediários. A pré-concentração de intermediários formados durante a fotodegradação dos antimicrobianos estudados permitiu a avaliação da degradação destes em concentrações menores do que aquelas atingidas pelos trabalhos já publicados que abordaram rotas de degradação. Os perfis de degradação em diferentes concentrações e em diferentes matrizes apresentam indícios de serem diferente, tanto no perfil quanto na composição proporcional entre os produtos formados. Esses resultados são promissores, visto que permitem uma aproximação às concentrações ambientalmente relevantes. / Identification of intermediate products formed during effluent treatment has become the focus of some studies. However, one of the most important issues during the photolysis of drugs or degradation by advanced oxidation processes is the high and invariable initial concentration of the treated drug. High concentrations, used in currently publications, potentially hinder the understanding of the real situation that occurs in nature, where low concentrations of these drugs are usually found. Here we focused in the identification of the degradation products of sulfamethazine (SMZ), ciprofloxacin (CIP) and enrofloxacin (ENR) obtained by photolysis in aqueous and synthetic wastewater medium. Solid-phase pre-concentration and chromatographic separation of the products obtained during the degradation process at different concentrations (25 mg L-1 and 250 &mu;g L-1 for SMZ ; 10 mg L-1 and 100 &mu;g L-1 for CIP and ENR) was developed in association with high-resolution tandem mass spectrometry (Q-ToF and LIT-Orbitrap MS). Eight photodegradation products of SMZ, among them those of nominal m/z 140 and 229 not reported yet in the literature, were identified. The methodology developed for SMZ was applied to transformation products analysis coming from anaerobic reactor. The biodegradation analysis of SMZ allowed the identification of three transformation products. The m/z 295 was common to processes, photolysis and biodegradation. The m/z 227 was first described as a product of anaerobic biodegradation of SMZ. Both for the CIP as well as for the ENR, ten intermediates have been identified, despite the similarity between the two drugs, only one degradation product is common to both, the one with nominal m/z 346, besides the CIP itself which is an intermediate of ENR. Among the evaluated extracting phases, the Oasis HLB was more efficient in recovering the intermediaries. The pre-concentration of intermediates formed during the photochemical degradation of antimicrobials allowed the evaluation of the degradation at lower concentration than the previously published papers that addressed routes for degradation. The degradation profile at different concentrations and matrices showed evidences of being different, both profile and regarding proportional composition of the products formed. These results are promising, as they provide an approach for application at environmentally relevant concentrations.

Algumas espécies de cianobactérias podem produzir diferentes tipos de peptídeos bioativos (cianopeptídeos), como aeruginosinas, cianopeptolinas, microgininas, microviridinas, anabaenopeptinas e microcistinas. As microcistinas, que compõem a classe mais conhecida na literatura científica, são hepatotoxinas e inibem fosfatases do tipo 1 e 2A. Já as microgininas, moléculas inibidoras da enzima conversora de angiotensina (ECA) e de outras proteases, são importantes candidatas ao desenvolvimento de fármacos anti-hipertensivos. No entanto, as funções fisiológicas desses compostos ainda não foram completamente elucidadas. O objetivo desse estudo é identificar e isolar cianopeptídeos, especialmente da classe das microgininas, produzidos por cepas de cianobactérias brasileiras. O fracionamento dos extratos das cianobactérias foi biomonitorado por meio de ensaios de inibição da ECA. Para tanto, foram produzidos extratos de culturas de 59 cepas de cianobactérias mantidas em laboratório e, quando pertinente, seguiu-se o pré-fracionamento por extração em fase sólida em coluna de fase reversa. As frações obtidas foram testadas para a inibição da ECA e analisadas por cromatografia líquida acoplada à espectrometria de massas (LC-MS). Nos casos em que houve inibição, o fracionamento foi continuado por cromatografia líquida de alta eficiência semipreparativa para o isolamento dos cianopeptídeos. As espécies analisadas pertencem aos gêneros: Microcystis, Oscillatoria, Radiocystis, Sphaerocavum, Sphaerospermopsis, Cylindrospermopsis, Leptolyngbya, Pleurocapsa, Chrooccocidiopsis, Nostoc, Brasilonema, Desmonostoc, Oculatella e Limnothrix. Além de outras espécies das famílias Pseudanabaenaceae, Nostocaceae e Michochaetaceae que não tiveram suas identificações concluídas. Nas análises por HPLC-DAD não foram encontrados picos com espectros de UV característicos de microgininas nas 59 cepas avaliadas. Esses resultados foram corroborados por análises por LC-MS, nas quais também não foi possível a detecção desses compostos. Como método de triagem, as análises por LC-MS triplo quadrupolo foram conduzidas via precursor ion scan para o monitoramento das perdas m/z 128 e 142, que são fragmentos oriundos do aminoáciodo Ahda, exclusivamente presente em microgininas. Algumas linhagens se revelaram produtoras de variantes do cianopeptídeo microcistina, possuindo absorção UV máxima em 238 nm. Amostras de Israel, doadas pelo Prof. Shmuel Carmeli, da Universidade de Tel Aviv, também foram analisadas por espectrometria de massas no modo precursor ion para a investigação da produção de microgininas. Foram encontrados íons de m/z 128 e 142 em uma das amostras de Israel. Ensaios enzimáticos iniciais da ECA foram realizados com frações das cepas controle LTPNA 08 e 09, sabidamente produtoras de microgininas. Realizou-se a otimização do método inicial de detecção dos cianopeptídeos para que fosse procedido o isolamento dos compostos de interesse da cultura LTPNA 08. As frações obtidas foram analisadas por cromatografia líquida acoplada à espectrometria de massas (LC-MS) e isoladas por um coletor de frações. 4,8 mg de microgininas foram obtidos com o isolamento das moléculas a partir de 3 g de biomassa seca da linhagem. Ensaios enzimáticos de inibição da ECA com as frações isoladas de microgininas e microcistina-LR comercial também foram realiazados. Os resultados mostram que tanto as microgininas quanto a microcistina-LR comercial apresentam potencial de inibição da enzima ECA. As microgininas e a microcistina-LR podem ser consideradas como novas moléculas a serem estudadas para o desenvolvimento de fármacos inibidores da ECA. Continuar a investigação da produção desses compostos é relevante tanto por seu potencial ecotoxicológico quanto por seu potencial farmacológico. / Some cyanobacterial species can produce different types of bioactive peptides (cyanopeptides), such as aeruginosins, cyanopeptolins, microginins, microviridins, anabaenopeptins and microcystins. Microcystins are well known in the scientific literature as hepatotoxins that inhibit phosphatases type 1 and 2A. Microginins are reported as inhibitors of the angiotensin converting enzyme (ACE) and other proteases, being important candidates for the development of anti-hypertensive compounds. Nevertheless, the physiological functions of these compounds have not been fully elucidated. The aim of this study is to isolate and identify cyanopeptides, namely microginins, produced by Brazilian cyanobacterial strains. The fractionation of cyanobacterial extracts was screened by ACE inhibition assays. Extracts of 59 cyanobacterial strains were produced from cultures cultivated under laboratory conditions and, in cases where microginins were found, prefractionation were carried out by solid phase extraction on a reverse phase column. The fractions obtained were tested for inhibition of ACE and analyzed by liquid chromatographymass spectrometry (LC-MS) by using precursor ion scan to monitor the loss of m/z 128 and 142, typical fragments detected from the amino acid Ahda, which is exclusively found in microginins. In cases where fractions demonstrated inhibitory activity, the fractionation was continued by semi-preparative high performance liquid chromatography for the isolation of cyanopeptides. The analyzed species belong to the genere: Microcystis, Oscillatoria, Radiocystis, Sphaerocavum, Sphaerospermopsis, Cylindrospermopsis, Leptolyngbya, Pleurocapsa, Chrooccocidiopsis, Nostoc, Brasilonema, Desmonostoc, Oculatella and Limnothrix. And other species of Pseudanabaenaceae, Nostocaceae and Michochaetaceae families did not have their peptides completely characterized. Analyses of HPLC-DAD did not revealed characteristic features by UV spectra of microginins in the 59 strains. These results were corroborated by analyses of LC-MS triple quadrupole, microginins were not detected as well. Some strains were positive for microcystin variants, showing maximum UV absorption at 238 nm and typical MS/MS spectra. Initial enzyme assays for the inhibition of ACE were performed with fractions of the control strains LTPNA 08 and 09, known as microginins\' producers. Samples fromIsrael, kindly donated by Prof. Shmuel Carmeli, of Tel Aviv University, were analyzed by mass spectrometry in the precursor ion mode for the investigation of microginins. Ions m/z 128 and 142 were founded on one sample of Israel. A HPLC method was optimized for the detection and isolation of cyanopeptides produced by culture LTPNA 08. Fractions isolated by semi-preparative HPLC were analyzed by LC-MS and collected when UV and MS spectra were characteristically similar to microginins . 4.8 mg of microginins were obtained by semi-preparative HPLC from 3 g of dried strains. The inhibition assays of ECA were performed with fractions of microginins and microcystin-LR analytical standard. The results showed that microginins as well as microcystin-LR have the potential of inhibiting ACE. Microginins and microcystin-LR can be considered as target molecules for the study of ACE inhibitors. Further research on the production of these classes of peptides is relevant because of its ecotoxicological and pharmacological potential.

A presen&ccedil;a de antibi&oacute;ticos em &aacute;guas superficiais e subterr&acirc;neas &eacute; motivo de preocupa&ccedil;&atilde;o, devido ao surgimento de bact&eacute;rias resistentes. Contudo, a maioria dos trabalhos publicados na literatura cient&iacute;fica mant&eacute;m o foco nos antibi&oacute;ticos inalterados, sendo que as mol&eacute;culas degradadas tamb&eacute;m est&atilde;o no ambiente. Esses produtos de degrada&ccedil;&atilde;o podem ser t&atilde;o ou mais prejudiciais do que as mol&eacute;culas que lhes deram origem. Esse trabalho teve por objetivo desenvolver m&eacute;todos de extra&ccedil;&atilde;o utilizando a microextra&ccedil;&atilde;o por sorvente empacotado (MEPS) para pr&eacute;-concentrar e recuperar produtos de fotodegrada&ccedil;&atilde;o de representantes das sulfonamidas (sulfametazina) e fluoroquinlonas (ciprofloxacino), duas das mais importantes classes de antibi&oacute;ticos. MEPS &eacute; considerada uma t&eacute;cnica promissora utilizando pequenos volumes de amostra e de solventes, al&eacute;m de empregar pequenas quantidades de fase extratora, que ainda pode ser reutilizada. Foram comparadas as fases Oasis&reg; HLB e nanotubos de carbono, sendo que a primeira apresentou melhores resultados. Seis produtos de degrada&ccedil;&atilde;o da sulfametazina (SMZ) e oito produtos do ciprofloxacino (CIP), al&eacute;m das mol&eacute;culas inalteradas, foram pr&eacute;-concentrados e analisados por cromatografia l&iacute;quida acoplada &agrave; espectrometria de massas de alta resolu&ccedil;&atilde;o (LC-ESI-ToF). Inicialmente, as concentra&ccedil;&otilde;es de f&aacute;rmaco inalterado utilizadas na fotodegrada&ccedil;&atilde;o foram de 25 mg L-1 (SMZ) e 10mg L-1 (CIP), para que os produtos fossem identificados. As taxas de recupera&ccedil;&atilde;o por MEPS ficaram acima de 50%, o que &eacute; um resultado promissor, considerando-se as diferen&ccedil;as estruturais dos produtos de degrada&ccedil;&atilde;o. Em concentra&ccedil;&otilde;es 100 vezes menores, MEPS conseguiu pr&eacute;-concentrar todos os produtos da SMZ e do CIP, facilitando sua detec&ccedil;&atilde;o. Ap&oacute;s desenvolver a pr&eacute;-concentra&ccedil;&atilde;o por MEPS em &aacute;gua purificada, foram realizados estudos de efeito de matriz em esgoto sint&eacute;tico. Enquanto somente um produto de degrada&ccedil;&atilde;o da SMZ sofreu supress&atilde;o de ioniza&ccedil;&atilde;o, todos os outros (inclusive os de CIP) experimentaram um aumento de sinal devido &agrave; presen&ccedil;a dos interferentes de matriz. O m&eacute;todo desenvolvido para MEPS tamb&eacute;m foi testado para pr&eacute;-concentrar produtos de degrada&ccedil;&atilde;o anaer&oacute;bica da SMZ em reator biol&oacute;gico, obtendo-se &ecirc;xito. Dessa forma, MEPS desponta como uma t&eacute;cnica promissora para pr&eacute;-concentrar produtos de degrada&ccedil;&atilde;o e para que pesquisadores possam acompanhar a degrada&ccedil;&atilde;o de reatores biol&oacute;gicos, visto que requer pequenas quantidades de amostra, n&atilde;o alterando significativamente o volume do meio reacional. / Antibiotics are present both surface water and groundwater and this is motive of concern, due to their ability to cause bacterial resistance. Nevertheless, most of publications in scientific area focuses in unchanged compounds, even knowing the presence of degraded molecules in the environment. These degradation products might be so or more dangerous than unchanged compounds. In this work, extraction methods for degradation products were developed using microextraction by packed sorbent (MEPS). MEPS is an eco-friendly technique due to little consumption of sample, solvents and sorbent. The degradation products of sulfamethazine (SMZ) and ciprofloxacin (CIP) were generated by photodegradation at 25 mg L-1 and <br clear=\"all\" /> 10 mg L-1 of unchanged drug, respectively. The number of degradation products was six for SMZ and eight for CIP. Oasis&reg; HLB and carbon nanotubes were tested as sorbents and the first got better results in preconcentration. The chromatographic analysis was performed by liquid chromatography coupled to high resolution mass spectrometry (LC-ESI-ToF). Recovery rates obtained by MEPS were greater than 50%, which is a significant result, considering structural differences among degradation products. After setting extraction conditions, MEPS was able to recover degradation products at concentrations 100 times lower and more akin to those find in the environment. Using synthetic sewage as medium, matrix effect studies were performed. The prevalent effect was an increase of ionization in degradation products (both SMZ and CIP), just one SMZ product experienced suppression of ionization. At last, SMZ was degraded in an anaerobic reactor and the degradation products were preconcentrated by MEPS. Although the biological degradation products were not the same of photodegradation (except by one), MEPS was capable to preconcentrate them. Thereby, MEPS starts to dawn as a promising technique to preconcentrate degradation products, specially for researchers using biological reactors, since MEPS requires low volumes of sample and almost do not change the final bulk of reactor.

As elevadas taxas de colesterol plasmático representam um grande risco à saúde, uma vez que podem causar doenças cardiovasculares. Para o tratamento e prevenção da dislipidemia são utilizados medicamentos reguladores do colesterol, como as estatinas. Embora eficazes e extensamente utilizados, esses fármacos apresentam efeitos adversos se administrados na dosagem errada. Assim, faz-se necessário o desenvolvimento de um método de monitorização terapêutica a fim de se ajustar a concentração desses compostos no sangue. Este trabalho visa o desenvolvimento de um método para análise de pravastatina (PRA), atorvastatina (AT), fluvastatina (FLV) e sinvastatina (SV) em plasma humano usando cromatografia líquida acoplada à espectrometria de massas (LC-MS). Na etapa de preparo de amostras, de forma inédita, utilizou-se a técnica microextração por sorvente empacotado (MEPS) para a análise de plasma humano contendo quatro estatinas. Para a otimização das condições de extração avaliaram-se, por experimentos univariados, parâmetros como fase extratora, composição do solvente de eluição e de lavagem. Outros fatores como volume de amostra, ciclos de amostragem, ciclos de eluição e etapas de eluição foram avaliados empregando-se planejamento experimental multivariado. A extração foi realizada utilizando-se uma fase estacionária C18 Chromabond como sorvente. O método MEPS-LC-MS/MS desenvolvido foi validado baseando-se nas recomendações da agência nacional de vigilância sanitária (ANVISA) e apresentou linearidade, seletividade, precisão, exatidão e recuperação adequadas para as estatinas, excetuando-se para a sinvastatina. A faixa de linearidade obtida foi de 10-200 ng mL-1 (FLV e AT) e 20-200 ng mL-1 (PRA). Os limites de quantificação obtidos foram da ordem de 10 ng mL-1 (AT e FLV) e 20 ng mL-1 (PRA). Desta forma o método desenvolvido poderá ser utilizado para a determinação dos níveis de pravastatina, fluvastatina e atorvastatina em amostras de plasma humano. / Elevated plasma cholesterol level is a risk factor for coronary diseases, which are the most deadly sickness according to the World Health Organization (WHO). In order to fight the hypercholesterolemia in patients, statins are a well-established class of drugs to be prescribed. Even though they are efficient, some side effects can be associated with statin therapy, especially when interactions with other drugs occur. In these cases, monitoring the concentration can optimize the drug dosage to therapeutic effectiveness whilst minimizing the adverse effects. The aim of this work was to develop a method for analysis of pravastatin (PRA), atorvastatin (AT), fluvastatin (FLV) and simvastatin (SV) in human plasma. The experimental means chosen to attain the goal was liquid chromatography-tandem mass spectrometry (LC-MS/MS) and for the sample preparation, microextraction by packed sorbent (MEPS). To optimize the extraction conditions, parameters such as sorbent, elution and washing solution were evaluated. Other parameters such as sampling, elution cycles, sample volume and elution steps were evaluated using multivariate experimental design. The extraction was performed using C18 Chromabond as sorbent. The method was validated based on ANVISA recommendations and featured appropriated linearity, selectivity, accuracy, precision, and recovery, except for simvastatin. The calibration curve in plasma was obtained in the concentration range 10-200 ng mL-1 (FLV and AT) and 20-200 ng mL-1 (PRA) and the limit of quantification (LOQ) was 10 ng mL-1 (FLV and AT) and 20 ng mL-1 (PRA). The method developed proved to be suitable for the analysis of pravastatin, fluvastatin and atorvastatin in human plasma sample, but not simvastatin, and it can contribute to a more efficient usage of the statins in the treatment of hypercholesterolemia.

En milieu marin, toute surface immergée est soumise à une colonisation par de nombreux organismes (biofouling). Le développement de biofilms est une étape clé du phénomène. Les systèmes de communication y sont contrôlés par le biais de signaux chimiques. Dans ce travail, l’étude de la signature métabolique de biofilms naturels formé in situ a été réalisée selon un gradient de pollution en contaminants métalliques dans la rade de Toulon et selon la nature du revêtement de la surface immergée. De nettes variations chimiques des biofilms prélevés sont observées et sont corrélées avec des variations en termes de communauté microbiennes. L’étude in vitro de 4 souches bactériennes issues de biofilms naturels a permis, après optimisation des méthodologies d’analyse, une discrimination selon leur profil métabolique. Des biomarqueurs ont été mis en évidence, avec notamment la production de lipides ornithine par la souche Pseudoalteromonas lipolytica. La réponse biologique de cette souche en fonction de son phénotype et face à un stress cuprique a été étudiée par métabolomique et protéomique révélant d’importantes modulations de certaines voies biosynthétiques. / In the marine environment, any immersed surface is subjected to colonization by many organisms (biofouling). The biofilms development is a key stage of this phenomenon. Communication systems are controlled in these structures by chemical signals. In this work, the study of the chemical signature of natural biofilms formed in situ was carried out among a gradient of contamination of metal contaminants in the bay of Toulon and according to the nature of the coating on the immersed surface. Clear chemical variations of the biofilms collected were observed and were correlated with variations in microbial community. The in vitro study of 4 bacterial strains harvested from natural biofilms allowed, after optimization of the analysis methodologies, their discrimination according to their metabolic profile. Biomarkers were highlited, particularly ornithine lipids production by the Pseudoalteromonas lipolytica strain. The biological response of this strain depending on its phenotype and face to copper stres was studied by metabolomics and proteomics revealing important modulations of certain biosynthetic patways.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O cajueiro (Anarcadium occidentale) à uma planta tÃpica da regiÃo do nordeste que responde por mais de 95% da produÃÃo nacional, sendo os estados do CearÃ, PiauÃ, Rio Grande do Norte e Bahia os principais produtores. Com o incremento de Ãreas plantadas com o cajueiro anÃo precoce, verificou-se que esta cultura era susceptÃvel ao ataque de pragas, causando prejuÃzos no desenvolvimento, produÃÃo e qualidade do fruto. Para evitar esses prejuÃzos, estudos foram realizados no sentido de controlar as pragas atravÃs do uso de agrotÃxicos. Estes compostos representam uma classe de compostos quÃmicos orgÃnicos que sÃo usados para aumentar a produtividade, no armazenamento e beneficiamento de produtos agrÃcolas. Os agrotÃxicos mesmo favorecendo a produÃÃo e conservaÃÃo de alimentos, sÃo nocivos ao homem e ao meio ambiente, e a determinaÃÃo e detecÃÃo de seus resÃduos tÃm exigido rigorosos mÃtodos de anÃlises. O avanÃo de tecnologias analÃticas sensÃveis e seletivas tem permitido a detecÃÃo destas substÃncias em nÃveis traÃos. Este trabalho descreve uma metodologia analÃtica empregando o mÃtodo de extraÃÃo QuEChERS (Quick, Easy, Cheap, Effective, Robustand Safe) e Cromatografia LÃquida acoplada à Espectrometria de Massas em sÃrie, utilizando fonte de ionizaÃÃo por eletrospray. Esta metodologia foi desenvolvida e validada para determinaÃÃo de 20 agrotÃxicos multiclasses em amostras de caju. Avaliou-se a seletividade, faixa de linearidade (6 nÃveis de concentraÃÃo), limites de detecÃÃo (LD) e quantificaÃÃo (LQ), efeito matriz, bem como a precisÃo e exatidÃo, em termos de percentual de recuperaÃÃo. Para isso, efetuou-se fortificaÃÃo dos 20 agrotÃxicos em 3 nÃveis de contraÃÃo em 10 g de caju recÃm processado. Todos agrotÃxicos estudados apresentaram boa linearidade com coeficiente de correlaÃÃo maior que 0,99. Devido à complexidade da amostra foi efetuado o estudo do efeito matriz, onde foi necessÃrio fazer a calibraÃÃo externa com superposiÃÃo na matriz. Os limites de detecÃÃo variaram entre 0,1 e 0,25Âg.L-1 e os limite de quantificaÃÃo entre 0,3 e 0,75Âg.L-1. A validaÃÃo da metodologia mostrou bons resultados de recuperaÃÃes, com taxas dentro dos limites aceitÃveis de 50% a 120%, e com RSD > 20%, estando de acordo com a faixa recomendada pela norma brasileira ABNT-NBR 14029/2005. AtravÃs do monitoramento de mÃltiplas reaÃÃes (MRM), duas transiÃÃes diferentes (Ãon precursor â Ãon produto) foram selecionadas para cada composto, uma para quantificaÃÃo e outra para confirmaÃÃo, o que aumentou a seletividade. O mÃtodo foi aplicado em amostras coletadas no comÃrcio de Fortaleza-CE e nÃo apresentaram contaminaÃÃo pelos agrotÃxicos em estudo / The cashew tree is a plant typical of the region which accounts for over 95% of national production, and the states of CearÃ, PiauÃ, Rio Grande do Norte and Bahia major producers. With the increase in areas planted with war of cashew, this culture was sensitive to pests, causing damage in the development, production and fruit quality. To avoid these losses, studies were made to control pests through the use of pesticides. These compounds represent a class of chemical compounds that are used to increase productivity, storage and processing f agricultural products. Pesticides despite favoring the production and preservation of food are harmful to humans and the environment, and the determination and detection of its residues have required rigorous analysis methods. The advancement of analytical technologies has allowed sensitive and selective detection of these substances in trace levels. This paper describes an analytical methodology employing the extraction method QuEChERS (Quick, Easy, Cheap, Effective, Robust and Safe) and Liquid Chromatography coupled to Mass Spectrometry in series, using electrospray ionization source was developed and validated for the determination of twenty multiclass pesticides in samples of cashew apple. It was evaluated the selectivity, linearity range (six concentration levels), limits of detection (LOD) and quantification (LOQ), matrix effect, as well as the precision and accuracy in terms of percentage of recovery. For this, we performed fortification of 20 pesticides in three contraction levels in 10g cashew newly processed. All pesticides studied showed good linearity with correlation coefficient greater than 0,99. Because of the complexity of the sample was performed to study the effect matrix where it was necessary to overlap with the external calibration matrix. Detection limits ranged from 0,1 to 0,25Âg.L-1 and the limit of quantification of 0,3 to 0,75Âg.L-1. The validation of the methodology showed good results of recoveries with rates within acceptable limits of 50% to 120%, and% RSD>20% which is consistent with that recommended by the Brazilian standard ABNT-NBR 14029/2005. By monitoring multiple reactions (MRM) two different transitions (precursor ion -product ion) were selected for each compound, one for quantitation and confirmation which increased the selectivity. The method was applied to samples collected in trade in Fortaleza-CE and not contaminated by pesticides under study

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-10-29T14:34:23Z No. of bitstreams: 2 Dissertação - Iury Valentim Jorge Zoghaib - 2013.pdf: 1607539 bytes, checksum: d92b3313edee2773935cb667be5b908d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-10-30T10:48:45Z (GMT) No. of bitstreams: 2 Dissertação - Iury Valentim Jorge Zoghaib - 2013.pdf: 1607539 bytes, checksum: d92b3313edee2773935cb667be5b908d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-30T10:48:45Z (GMT). No. of bitstreams: 2 Dissertação - Iury Valentim Jorge Zoghaib - 2013.pdf: 1607539 bytes, checksum: d92b3313edee2773935cb667be5b908d (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-10-18 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The LQFM030 was obtained by molecular simplification of nutlins (inhibitors of p53-MDM2 interaction) and, having demonstrated excellent antineoplastic activity in vitro (cytotoxicity against K-562 cell line with IC50 = 23 M) and in vivo against Ehrlich ascitic tumor with increased survival in treated animals developed the pharmacokinetic study of p.o. in rats. It was used for LC-MS/MS analytical method (Applied Biosystems MDS Sciex API 3200) previously validated together. O LQFM030 was administered to 3 rats (mean weight 186g) in predetermined dose of 100 mg/kg by gavage. After administration, samples were collected from 1.0 mL of blood by cannulation of the left jugular vein with heparinized syringe, the intervals 0-8 h. The blood samples were identified and centrifuged to obtain plasma which was frozen at -20°C until analysis. The kinetic parameters were calculated in the software WinNonlin 5.0 (Pharsight™). Results (mean ± SD) half-life (t1/2) 3.61 ± 0.68 h, total clearance (CLT) 36.49 ± 2.23 mL/min/kg, volume of distribution (Vd) 11,40 ± 1.58 L/kg. The LQFM030 had low value of t1/2, Vd high and high value of CLT. These values allow us to understand that the prototype study demonstrated a good safety profile of tissue distribution and/or has been extensively eliminated. When compared with the kinetic parameters obtained in other studies, it was observed difference in results is justified by the high interspecies variability, mainly in basal metabolic rate and body weight, once the route of administration, orally and intraperitoneally, are kinetically similar. / O LQFM030 foi obtido por simplificação molecular dos nutlins (inibidores da interação MDM2-p53) e, tendo demonstrado excelente atividade antineoplásica in vitro (citotoxicidade contra a linhagem celular K-562 com IC50 = 23 M) e in vivo contra tumor ascítico de Ehrlich, com aumento de sobrevida em animais tratados, desenvolveu-se o estudo do perfil farmacocinético, p.o., em ratos. Empregou-se método analítico em LC-MS/MS (Applied Biosystems MDS Sciex API 3200) previamente validado em colaboração. O LQFM030 foi administrado a 3 ratos (peso médio de 186g), em dose preestabelecida de 100 mg/kg, por gavagem. Após a administração, foram coletadas amostras de 1,0 mL de sangue, por canulação da veia jugular esquerda, com seringa heparinizada, nos intervalos de tempo de 0 a 8 h. As amostras sanguíneas foram identificadas e centrifugadas para obtenção do plasma, que foi congelado a -20ºC até o momento da análise. Os parâmetros cinéticos foram calculados no software Winnonlin 5.0 (Pharsight™). Resultados (média ± DP): meia-vida (t1/2) 3,61 ± 0,68 h; clearance total (CLT) 36,49 ± 2,23 mL/min/kg; volume de distribuição (Vd) 11,40 ± 1,58 L/kg. O LQFM030 apresentou baixo valor de t1/2, elevado Vd e elevado valor de CLT. Estes valores permitem entender que o protótipo estudado demonstrou um bom perfil de distribuição tecidual e/ou foi extensivamente eliminado. Quando comparados com parâmetros cinéticos obtidos em outros estudos, observou-se diferença nos resultados, justificada pela elevada variabilidade interespécies, principalmente na taxa de metabolismo basal e peso corporal, uma vez que as vias de administração, oral e intraperitoneal, são cineticamente semelhantes.