Metabólitos secundários produzidos por Streptomyces com atividade antibiótica apresentam relevante importância biotecnológica para as indústrias farmacêuticas e agroquímicas. Dentre estes metabólitos, podemos destacar a nigericina, um antibiótico poliéter usado como aditivo em ração animal atuando como promotor de crescimento e no tratamento de algumas doenças, como a malária, em carcinoma nasofaríngeo, a vaccínia, entre outras. Neste trabalho foram avaliadas duas cepas de actinobactérias potenciais produtoras de nigericina, a EUCAL 26 e a EUCAL 74. As duas actinobatérias foram fermentadas em cinco meios de cultivo diferentes (BD, Czapek, ISP2, M29 e TSB). A cepa EUCAL 26 foi a mais promissora na produção de nigericina em meio Czapeck. A partir da EUCAL 26, foi feito um estudo da máxima produção de nigericina em meio Czapek variando o pH do meio, temperatura de fermentação, e período de fermentação. As melhores condições encontradas foram em pH 7,0 a 25 °C por 27 dias. Foi realizado também um estudo de otimização de aumento de escala de fermentação, de um volume de meio Czapeck de 50 mL, para um volume de 4 L. Também foram avaliados dois resíduos agroindustriais (Farmal e Melaço de Soja) para a produção de nigericina. O meio de Melaço de Soja aumento a produção em aproximadamente 300x quando comparado com o meio Czapeck padrão. Os efeitos dos nutrientes do meio Czapeck também foram avaliados. A retida do K2HPO4 do meio produziu um aumento de 50x na produção de nigericina, quando comparado com o meio Czapeck controle. Também foi avaliado o efeito da adição de -butirolactonas sintéticas, moléculas de sinalização hormonal, para a produção de nigericina. Das 15 -butirolactonas testadas, a DP21A foi a mais eficiente, pois além de aumentar a produção de nigericina em 23x, também diminui o período máximo de sua produção. Todas as analises realizadas neste trabalho para o monitoramento da produção de nigericina, foram feitas empregando a espectrometria de massas sequencial acoplada à cromatografia liquida de ultra eficiência. / Secondary metabolites produced by Streptomyces with antibiotic activity have significant biotechnological importance for the pharmaceutical and agrochemical industries. Among them, nigericin stands out as an antibiotic polyether used as growth promoter in animal feed and for treatment of some diseases such as malaria, nasopharyngeal carcinoma, and vaccinia. In this study, two actinobacteria strains considered potential producers of nigericin named EUCAL 26 and 74 were tested. The two actinobacteria were fermented in five different culture media (BD, Czapek, ISP2, M29 and TSB). EUCAL 26 strain was the most promising in producing nigericin in amid Czapeck media. For EUCAL 26, a study of maximum production of nigericin in Czapek medium at varying the pH, fermentation temperature and fermentation period have been performed. As a result, the best conditions were pH 7.0, at 25 °C for 27 days. In addition, an optimization study for scale-up fermentation have been done, where a volume of 50 mL Czapeck medium have been expanded to 4 L, in order to obtain the highest production of nigericin. Two agroindustrial residues (FARMAL and Honey Soy) have also been evaluated for nigericin production. The honey soy medium increased nigericin production in the rate of 300 when compared with standard Czapeck medium. The effects of nutrients from Czapeck medium have also been evaluated. Removal of K2HPO4 from culture medium resulted in an increase of 50 times when compared with the control Czapeck medium. The effect of adding synthetics -butyrolactones (hormone signaling molecules) for the production of nigericin have also been evaluated. From 15 tested -butyrolactones, DP21A was the most efficient. In addition to increase nigericin yield in 23x it also reduced the period for its maximum production. All analyzes performed in this study to monitor the nigericin production were performed using tandem mass spectrometry coupled to ultra high performance liquid chromatography.

Neste trabalho foi empregada uma metodologia rápida e eficiente para a identificação de metabólitos fitotóxicos produzidos por microrganismos. O isolamento do composto bioativo foi guiado através de bioensaio com Lemna minor. A espectrometria de massas, em especial o LC-MS, foi utilizada para acelerar o processo de identificação do composto ativo. As bactérias estudadas eram simbióticas do fungo fitopatogênico Sclerotium rolfsii. Seus respectivos extratos orgânicos obtidos de culturas em meio BD (batata dextrose) foram submetidos ao ensaio de fitotoxicidade com Lemna minor. Entre cinco bactérias foi selecionada a bactéria Burkholderia sp, a qual apresentou maior atividade no ensaio de fitotoxicidade. O fracionamento por cromatografia em coluna de sílica propiciou a identificação de uma fração ativa. A fitotoxina foi caracterizada como sendo um macropentólido de 20 membros. O composto pertence à classe dos polihidroxibutiratos (PHBs). Sua estrutura foi determinada por RMN 1H, RMN 13C, HMQC, HMBC, IV, ESI-MS/MS e também por comparação com dados da literatura. Esse composto nunca foi isolado de fontes naturais. Foi descrito na literatura uma rota sintética para sua obtenção, porém esta é a primeira vez que sua atividade fitotóxica é relatada. Este trabalho mostra uma nova perspectiva para o emprego de PHBs de baixo peso molecular e apresenta uma proposta de estrutura de composto fitotóxico que pode servir de modelo para a síntese de novos herbicidas. / In this work a quick and efficient methodology was employed for the identification of phytotoxic metabolites produced by microorganisms. The isolation of the bioactive compound was guided by Lemna minor bioassay. Mass spectrometry, especially LC-MS, was used to accelerate the process of identification of the phytotoxin. All bacteria were symbiotic to the phytopatogenic fungi Sclerotium rolfsii.. The bacterium Burkholderia sp was selected among the five bacteria analyzed, due to its greater phytotoxic activity in the bioassay. The phytotoxin was characterized as a 20 member macropentolide. This compound belongs to the polyhidroxybutirates (PHBs) chemical class. Its structure was determined by NMR1H, NMR 13C, HMQC, HMBC, IV, ESI-MS/MS and HRMS. It has never been isolated from natural sources before. Although a synthetic route has been proposed in the literature this is the first time that its phytotoxic activity is reported. This work leads to a new perspective for the application of low molecular weight PHBs and propose a phytotoxic structure that can be used as a model for the synthesis of new herbicide class.

Neste trabalho foi desenvolvida, otimizada, e validada, uma metodologia para análise de 15 pesticidas organoclorados em sedimento através de HS/SPME-GC/MS. Esta metodologia foi aplicada para análise de amostras reais visando à realização de um diagnóstico da contaminação por pesticidas organoclorados do Rio Atibaia, principalmente em sua região próxima a contaminação oriunda da empresa Shell Brasil S.A. unidade de Paulínia. Para realização deste diagnóstico, também foi desenvolvida uma metodologia padronizada para diagnóstico ambiental de áreas contaminadas, embasada principalmente em conceitos de Gestão de Qualidade e Meio Ambiente abordadas nas normas ISO 17025, ISO 14001 e GLP. Finalmente, aplicando a metodologia analítica validada, e a metodologia para realização de um diagnóstico ambiental, foi realizado o diagnóstico da presença de pesticidas organoclorados no Rio Atibaia, indicando a existência de diferentes pesticidas organoclorados em sedimento, com destaque para os compostos DDE e BHC que se encontraram em valores acima dos recomendados pela legislação canadense. Os resultados encontrados também demonstraram que as metodologias desenvolvidas foram adequadas para análise de uma contaminação real. / In this work a methodology was developed, optimized and validated for the analysis of 15 organochlorine pesticides in sediment through HS/SPME-GC/MS. This methodology was applied to the analysis of real samples for diagnosis of the organochlorine pesticides contamination of the Atibaia River, mainly in its region close the contamination from company Shell Brasil S.A. site of Paulínia. For accomplishment of this diagnosis, also a standard methodology for environmental diagnosis of contaminated areas was developed, mainly based in concepts of Quality Management and Environmental Management find in standards like ISO 17025, ISO 14001 and GLP. Finally, applying the validated analytical methodology and the methodology proposed for environmental diagnosis, the diagnosis of the organoclhorine pesticide presence in the Atibaia River was performed, indicating different organoclorine pesticides in sediment, mainly DDE and yBHC, which were found in values above of those recommended by the Canadian legislation. The results also demonstrated that the developed methodologies are adequate for analysis of a real contamination.

A poluição dos corpos d\'água é de grande preocupação mundial, pois a maioria da população utiliza a água doce de reservatórios, represas ou rios como principal fonte de água potável. A presença no Brasil de florações de cianobactérias capazes de produzir anatoxina-a, revela a necessidade de métodos simples e rápidos que permitam sua detecção e monitoramento. Neste trabalho foram desenvolvidos, otimizados e validados dois métodos analíticos por GC/MS para identificação e quantificação de anatoxina-a em amostras de água e florações, respectivamente. A norcocaína foi usada como padrão interno em ambos os métodos. Os íons escolhidos para serem monitorados foram (íons quantificadores sublinhados): anatoxina-a: 191,164, 293 e norcocaína: 195, 136, 168. As curvas de calibração dos métodos mostraram-se lineares nas faixas de 2.5-200 ng.mL-1 e 13-250 ng.mg-1. Os limites de detecção obtidos foram 2 ng.mL-1 e 10 ng.mg-1. Os métodos demonstraram sensibilidade e especificidade adequada para seu uso no monitoramento ambiental da anatoxina-a. / The water pollution is a big concern around the world, since the most of cities use freshwater reservoirs, dams or rivers as the main drinking water suppliers. Cyanobacterial blooms capable to produce anatoxin-a are regularly present in Brazilian waters. Therefore, there is a necessity of simple and rapid analytical methods to monitor this cyanotoxin. In the present work, two analytical methods by GC/MS for identification and quantification of anatoxin-a in water and algae bloom samples were developed, optimized and validated. Norcocaine was used as internal standard in both methods. The ions chosen to be monitorated were (quantification ions underlined): anatoxin-a 191, 164, 293 and norcocaine: 195, 136, 168. Both method calibration curves showed linearity in the ranges of: 2.5-200 ng.mL-1 and 13-250 ng.mg-1. The obtained limit of detection were: 2 ng.mL-1 and 10 ng.mg-1. The methods showed sensitivity and specificity enough to be used routinely as a tool for anatoxin-a monitoring.

Orientadores: Marco Aurélio Zezzi Arruda, Ljubica Tasic / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-23T02:17:28Z (GMT). No. of bitstreams: 1 Mataveli_LidianeRaquelVerola_D.pdf: 1935019 bytes, checksum: 059066ee53b01850e2858460ba23617e (MD5) Previous issue date: 2013 / Resumo: Este trabalho consistiu em estudos de metalômica comparativa de sementes de soja transgênica (T) e não-transgênica (NT). Para tanto, as sementes de soja T e NT foram comparadas, em um primeiro, momento levando-se em conta: concentração total dos elementos, comportamento dos elementos durante extração utilizando-se fracionamento sequencial, e bioacessibilidade dos elementos após procedimento de digestão gastrointestinal simulada (in vitro). As análises preliminares foram feitas utilizando-se ICP-MS com analisador de massas quadrupolar, e as análises posteriores utilizando espectrometria de massas de alta resolução com plasma acoplado indutivamente (HR-ICP-MS). Foram determinados 25 elementos em concentrações variando de ng g até %. Foi observado que as sementes de soja T e NT exibem diferenças estatisticamente significantes nas concentrações de Cu, Fe e Sr, sendo os dois primeiros apresentando maiores concentrações na semente T, e, o último, com maior concentração nas sementes de soja NT. Estes resultados também se refletiram nos conteúdos desses elementos em extratos aquosos e resíduos obtidos por meio de fracionamento sequencial. Ainda, os experimentos de bioacessibilidade realizados mostraram que as frações bioacessíveis de Cu, Fe, e outros elementos (Mn, S, Zn) contribuíram em maior porcentagem para a concentração total dos elementos nas sementes de soja T do que para as sementes de soja NT. Posteriormente, foi dada continuidade aos estudos utilizando cromatografia líquida bidimensional off line para as amostras de sementes de soja, sendo a primeira dimensão constituída de cromatografia de exclusão por tamanho (SEC,) e a segunda dimensão constituída de cromatografia de troca aniônica (AEX). Na primeira dimensão cromatográfica foram identificadas três frações contendo metais por meio da hifenação SEC-ICP-MS: a primeira correspondendo a massas molares entre 38,1 e 181,1 kDa, a segunda correspondendo a massas molares entre 8,2 e 17,2 kDa, e a terceira fração correspondendo a massas molares entre 0,4 e 3,8 kDa. As três frações identificadas foram separadas, liofilizadas, e separadas novamente utilizando a cromatografia de troca aniônica. Foram detectados metais em todas as frações separadas por SEC: três sub-frações da primeira fração, uma sub-fração na segunda fração e três sub-frações na terceira fração. Os eluatos foram coletados, liofilizados, digeridos e levados ao espectrômetro de massas com fonte de ionização por electrospray (ESI-MS) para identificação de proteínas. Foram identificadas 33 e, entre elas, duas proteinas previamente relacionadas a metais foram encontradas: seed lipoxygenase 1 e b-conglycinin. Após a separação na segunda dimensão cromatográfica, as sub-frações resultantes foram liofilizadas e submetidas a uma terceira dimensao de separacao, utilizando a eletroforese em gel de poliacrilamida unidimensional (SDS-PAGE). As bandas obtidas por SDS-PAGE foram recortadas e digeridas a fim de analisar os metais presentes nas mesmas, destacando os resultados obtidos para Fe, onde o mesmo foi quantificado nas bandas da sub-fração onde o pico deste elemento foi encontrado nas análises por AEX-ICP-MS. Ainda, as bandas foram digeridas tripticamente a fim de identificar as proteínas presentes, e, novamente, proteínas associadas a metais foram identificadas: chain A lipoxygenase-3 (Soybean) complex with 13(S)-hydroperoxy- 9(Z),11(E)-octadecadienoic acid; beta-amylase [Glycine max]; seed lipoxygenase-1, lipoxygenase [Glycine max], seed lipoxygenase-2 (PISUM SATIVUM) e beta-conglycinin / Abstract: This work consisted of comparative metallomics studies of transgenic (T) and nontransgenic (NT) soybean seeds. To that end, T and NT seeds were compared at first taking into account: the total concentration of the elements, the elements behavior during extraction using sequential fractionation, and bioacessibility of the elements after simulated gastrointestinal digestion procedure (in vitro). Preliminary analyzes were done using ICP-MS with quadrupole mass analyzer, and the subsequent analysis using high-resolution inductively coupled plasma mass spectrometry, (HR-ICP-MS). 25 elements were determined at concentrations ranging from 1 ng g up to %. It was observed that T and NT soybean seeds exhibit statistically significant differences in the concentrations of Cu, Fe and Sr, the first two having higher concentrations in the T seeds, and the last with the highest concentration in soybeans NT. These results were also reflected in the contents of these elements in aqueous extracts and residues obtained through sequential fractionation. Also, the contributions of bioaccessible fractions of Cu, Fe and other elements (Mn, S, Zn) to the total content of the elements in T soybean seeds were higher than those found in NT soybean seeds. Subsequently, studies were continued using bidimensional liquid chromatography, the first dimension consisting of size exclusion chromatography (SEC) and the second dimension of anion exchange chromatography (AEX). In the first chromatographic dimension three fractions containing metals were identified using hyphenation SEC-ICP-MS, the first corresponding to molar masses between 38.1 and 181.1 kDa, the second corresponding to molar masses between 8.2 and 17.2 kDa and the third fraction corresponding to molar masses between 0.4 and 3.8 kDa. The three identified fractions were separated and lyophilized, and again separated using anion exchange chromatography (AEX). Metals were found in all the separated fractions by SEC: three sub-fractions of the first fraction, a subfraction in the second fraction and three sub-fractions in the third fraction. These peaks were collected, lyophilized, digested and taken to the mass spectrometer for protein identification. 33 proteins were identified, and, among them, two proteins previously related to metals were found: seed lipoxygenase 1 and b-conglycinin. After chromatographic separation in the second dimension, the resultant subfractions were lyophilized and subjected to a third separation dimension using onedimensional polyacrylamide gel electrophoresis (SDS-PAGE). After the separation, the bands were cut out and digested to examine the metals contained therein, highlighting the results obtained for Fe, which was quantified in the bands of the sub-fraction where the peak of the element is found in the analysis by ICP-AEX - MS. Also, the bands were digested triptically to identify the proteins, and once again proteins associated to metals were identified: 3-lipoxygenase A chain (Soybean) complex with 13 (S)-Hydroperoxy-9 (Z), 11 ( E)-octadecadienoic acid, beta-amylase [Glycine max]; seed lipoxygenase-1, lipoxygenase [Glycine max] seed lipoxygenase-2 (Pisum sativum) and beta-conglycinin / Doutorado / Quimica Analitica / Doutora em Ciências

Electrospray ionization mass spectrometry (ESI-MS) is a fast and sensitive technique that is ideal for detecting low concentration species of interest within complex mixtures. Because ESI-MS simply transfers charged species to the gas phase, only ions pre-formed in solution are visible. Accordingly, the charged tag, 3-(4-(bromomethyl)benzyl)-1-methylimidazolium hexafluorophosphate, was designed and synthesized to allow selective detection of phenols in petroleum fractions. Pressurized sample infusion (PSI) was optimized and used for time dependent analyses. PSI ESI-MS was applied to measure O-alkylation of the phenol species leading to rate information about the overall reaction along with dynamic information about reaction progress. The relative intensity of the charged tag was used to determine semi-quantitatively the presence of phenols in different petroleum fractions. Other derivatization methods in petroleomics were also explored. 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide methiodide (EDT) derivatization followed by PSI ESI-MS analysis was applied for the selective measurement and identification of naphthenic acids in petroleum fractions. The reactions of standard naphthenic acids and EDT were studied by PSI ESI-MS to assess the efficiency of EDT and the rate of reactions. The same petroleum fractions were analysed by cold Electron ionization (Cold EI) gas chromatography-mass spectrometry (GC/MS) and classical EI GC/MS. The combination spectra from the subtraction from cold EI spectra to classical EI spectra provide us a new dimension to cold EI analysis of complex matrices. Meanwhile, a Python program was written to rapidly screen cold EI GC/MS data for routine tasks, such as retention time comparison on different instrument parameters for single petroleum sample and spectrum comparison on the same retention time for different petroleum samples. / Graduate

Ces travaux ont pour but d’identifier des métabolites dans des teintures-mères (TMs) utilisées en homéopathie et en phytothérapie pour mieux contrôler la qualité des TMs et de mieux comprendre leur mode d’action sur la santé humaine. Nous avons étudié, par une approche de métabolomique globale, 19 TMs et un produit leader : le L52, fabriqués par les Laboratoires Lehning. Premièrement, nous avons utilisé des approches non-ciblées en construisant des banques de données GC-MS et UPLC-MS/MS, afin d’identifier un maximum de molécules dans chaque extrait. Puis, nous avons fractionné certaines TMs et purifié des métabolites inconnus pour une identification fine en HRMS. Nous avons identifié de nombreuses molécules dans chaque TM, montrant que ces dernières sont très riches en molécules pouvant être utilisées pour le contrôle-qualité des TMs et valorisées pour la santé humaine. / Tinctures defined as hydro-alcoholic extracts have been used from centuries in homeopathy and phytotherapy, but their chemical compositions remain still unknown. During my PhD, metabolomics analyses of nineteen tinctures and one leader product, L52, made by Laboratoires Lehning, were conducted using untargeted metabolomic approach. We build GC-MS and UPLC-MS/MS databases to identify a large amount of metabolites. Then, we used semi-preparative HPLC with both UV and mass detection to isolate some compounds from tinctures. We used UPLC-HRMS to obtain chemical formula, a prerequisite for metabolites identification. Finally, we identified a broad range of different metabolites in each tincture, highlighting the metabolic complexity of the TMs. These molecules can now be used for quality-control and valued for a better understanding of these products on human health.

La consommation excessive d’alcool présente des risques élevés pour l’individu et pour la société ; elle est fréquemment associée à une augmentation du risque d’accidents, d’actes de violence, et peut également conduire à court et/ou à long terme à de graves maladies et à des problèmes sociaux. Dès lors, l’utilisation de marqueurs fiables permettant de détecter une consommation excessive d’alcool, ponctuelle ou chronique, s’avère nécessaire pour prévenir des conséquences néfastes de l’abus d’alcool. L’éthylglucuronide (EtG) est un marqueur d’alcoolisation utilisé en toxicologie clinique (alcoologie) et médicolégale. Par rapport aux marqueurs indirects d’alcoolisation (CDT, &#947;-GT), ce métabolite mineur de l’éthanol est très spécifique et est quantifiable dans diverses matrices biologiques. La production d’EtG est catalysée par des enzymes de la famille des UDP-glucuronosyl-transférases (UGT). Cependant, les UGT impliquées dans la glucuronoconjugaison de l'éthanol, ainsi que les sources potentielles de variabilité interindividuelle de la production d'EtG, sont encore mal connues. Nos travaux ont ainsi consisté à (1) développer et valider une méthode de dosage de l’EtG dans différentes matrices biologiques par chromatographie en phase gazeuse couplée à la spectrométrie de masse en tandem, (2) identifier les UGT humaines impliquées dans la glucuronoconjugaison de l’éthanol et étudier la contribution relative de chaque isoforme active au niveau hépatique, (3) étudier l’impact de substances fréquemment utilisées par les consommateurs d’alcool sur la production d’EtG in vitro, (4) étudier l’impact de polymorphismes génétiques fonctionnels des UGT sur la production hépatique d’EtG, et enfin (5) évaluer l’impact de la consommation de cannabis et d’autres drogues sur la production d’EtG à l’aide de prélèvements post-mortem. Ces travaux ont notamment permis de montrer que (1) l'éthanol est glucuronoconjugué principalement par le foie, puis dans une moindre mesure par les reins et par l'intestin, (2) les UGT1A9 et 2B7 sont les deux enzymes majoritairement impliquées dans la glucuronoconjugaison de l’éthanol, quel que soit l’organe considéré, (3) la morphine, la codéine, la nicotine et la cotinine n’entraînent aucune modification des taux de production d’EtG in vitro ; le lorazépam et l'oxazépam augmentent légèrement cette production (p = 0,2 et 0,065, respectivement) ; le cannabidiol inhibe la glucuronoconjugaison de l’éthanol par un mécanisme non-compétitif (CI50 = 1,17 mg/L; Ki = 3,1 mg/L), alors que le cannabinol augmente cette glucuroconjugaison de manière concentration-dépendante (p <0,05), (4) les SNP c.-900G>A affectant l’UGT2B7 et IVS1+399T>C affectant l’UGT1A9 augmentent légèrement la production d’EtG in vitro. Enfin (5) le rapport des concentrations sanguines EtG/éthanol apparaît significativement plus élevé chez des co-consommateurs de cannabis et/ou d’autres drogues que chez des consommateurs d’alcool seul. L’ensemble de ces résultats démontre l’existence de plusieurs facteurs pouvant potentiellement influencer la production d’EtG et devraient donc être pris en considération lors de l’interprétation de sa concentration in vivo. / Alcohol abuse is frequently associated with an increased risk of road accidents and violence, and can also lead to serious social and health problems. Therefore, the use of reliable markers to detect excessive punctual and/or chronic consumption of alcohol is necessary to prevent the harmful consequences of alcohol abuse. Ethylglucuronide (EtG) has been proposed as a marker of alcohol consumption in a variety of clinical and forensic contexts. Compared with the indirect markers (e.g. CDT, &#947;-GT), this minor metabolite of ethanol is very sensitive and specific, and is quantifiable in various biological matrices. It is formed by conjugation of ethanol with uridine 5’-diphosphate glucuronic acid (UDP-GA) via the action of UDP-glucuronosyltransferase (UGT) enzymes. However, the knowledge of the UGTs involved in the glucuronidation of ethanol, and the potential sources of the interindividual variability of EtG production are still not clearly established. The aims of our work were (1) to develop and validate a method for the determination of EtG in different biological matrices by gas chromatography coupled with tandem mass spectrometry, (2) to identify the human UGT isoforms involved in the glucuronidation of ethanol, and then to evaluate qualitatively and quantitatively their specific contribution in the formation of EtG, (3) to study the impact of the co-administration of drugs frequently used by ethanol consumers on the in vitro production of EtG, (4) to study the impact of functional genetic polymorphisms of two UGTs on the hepatic production of EtG, and finally (5) to study the impact of the consumption of cannabis and other drugs on the production of EtG using post-mortem samples. The main results of our study showed that (1) ethanol is primarily glucuronidated by the liver and, to a lesser extent, by kidneys, (2) UGT1A9 and 2B7 were identified as the main human UGTs involved in ethanol glucuronidation, (3) morphine, codeine, nicotine, and cotinine did not modify EtG in vitro formation rate; lorazepam and oxazepam produced a minor, but not significant, increase of EtG formation. Only cannabinol and cannabidiol significantly affected ethanol glucuronidation; cannabinol significantly increased the glucuronidation of ethanol in a concentration-dependent manner, whereas cannabidiol inhibited the glucuronoconjugaison of ethanol by a non-competitive mechanism (CI50 = 1.17 mg / L; Ki = 3.1 mg / L), (4) the SNP c.-900G>A and IVS1+399T>C affecting UGT2B7 and UGT1A9, respectively, seem to increase the in vitro production of EtG, and (5) cannabis and/or drugs consumption (mainly opioids, benzodiazepines, and paracetamol) seem to be associated with ratios of blood concentrations of EtG/ethanol significantly higher than those observed among only alcohol consumers. Taken together, these results show the existence of several factors that could potentially influence the production of EtG, and that should be taken into account when interpreting its concentration in vivo.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22487号 / 農博第2391号 / 新制||農||1076(附属図書館) / 学位論文||R2||N5267(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 梅澤 俊明, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Genus Phytophthora represents a world-wide spread pathogen with more than hundred recognized species and its devastating effect on plants has a serious economic and ecological impact. This diploma thesis entitled „Proteome and metabolome of genus Phytophthora” briefly summarizes knowledge about this pathogen, including its life cycle and interactions with its host. Twelve species representing six Phytophthora clades that were selected for experimental work are then discussed in details. Phytophthora isolates were characterized on proteome and metabolome level employing an LC-MS untargeted proteome profiling and a GC-MS analysis of volatiles. The results were then processed to identify candidate molecules for a targeted identification of Phytophthora and these results were validated in an independent experiment with P. palmivora and Hordeum vulgare. We found that a proteome profiling can be employed as a tool to differentiate individual Phytophthora species and that the marker peptides can be employed for a targeted monitoring of Phytophthora presence in plants.