1H nuclear magnetic resonance (NMR) metabolomics was used to determine the response of earthworm exposure to polycyclic aromatic hydrocarbons (PAHs) in contact and soil tests. Eisenia fetida is recommended for toxicology testing, but to date this species has not frequently been used in environmental metabolomic studies. The metabolic profile of E. fetida was characterized with the goal of using this species in metabolomic studies. Testing several individual solvents for earthworm tissue extraction indicated that D2O buffer extracted the highest concentration of the widest variety of earthworm metabolites. Sample preparation methods were evaluated to reduce variability and achieve reproducible control groups for use in metabolomic studies. 96h depuration and intact lyophilization of earthworms before homogenization resulted in the least variation between sample extracts. This sample preparation method was used to compare E. fetida and two other earthworm species (Lumbricus rubellus and Lumbricus terrestris) and E. fetida had the most reproducible 1H NMR spectra. E. fetida was then used to identify the metabolic response after exposure to several concentrations of the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene and pyrene, individually and in mixtures. With exposure to individual PAHs in contact tests, earthworm responses were both PAH- and concentration- dependent. In earthworms exposed to PAH mixtures in contact tests, an increase in amino acids was measured. Furthermore, an increase in specific amino acids and a decrease in maltose were identified as potential indicators of sub-lethal phenanthrene exposure in soil. Lastly, the relationship between earthworm response and contaminant bioavailability in soil was tested. Contaminant bioavailability is typically assessed using indirect methods [e.g., ‘soft’ extraction techniques like hydroxypropyl cyclodextrin (HPCD) extraction]. However, it was found that the directly measured response of earthworm exposure to sub-lethal concentrations of phenanthrene in soil is related to both the total and bioavailable phenanthrene. This suggests there is potential for the use of 1H NMR metabolomics for the assessment of contaminant bioavailability. This thesis has demonstrated that E. fetida are suitable for metabolomic studies and has indicated that 1H NMR metabolomics may have potential for measuring and monitoring earthworm exposure to sub-lethal concentrations of PAHs.

1H nuclear magnetic resonance (NMR) metabolomics was used to determine the response of earthworm exposure to polycyclic aromatic hydrocarbons (PAHs) in contact and soil tests. Eisenia fetida is recommended for toxicology testing, but to date this species has not frequently been used in environmental metabolomic studies. The metabolic profile of E. fetida was characterized with the goal of using this species in metabolomic studies. Testing several individual solvents for earthworm tissue extraction indicated that D2O buffer extracted the highest concentration of the widest variety of earthworm metabolites. Sample preparation methods were evaluated to reduce variability and achieve reproducible control groups for use in metabolomic studies. 96h depuration and intact lyophilization of earthworms before homogenization resulted in the least variation between sample extracts. This sample preparation method was used to compare E. fetida and two other earthworm species (Lumbricus rubellus and Lumbricus terrestris) and E. fetida had the most reproducible 1H NMR spectra. E. fetida was then used to identify the metabolic response after exposure to several concentrations of the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene and pyrene, individually and in mixtures. With exposure to individual PAHs in contact tests, earthworm responses were both PAH- and concentration- dependent. In earthworms exposed to PAH mixtures in contact tests, an increase in amino acids was measured. Furthermore, an increase in specific amino acids and a decrease in maltose were identified as potential indicators of sub-lethal phenanthrene exposure in soil. Lastly, the relationship between earthworm response and contaminant bioavailability in soil was tested. Contaminant bioavailability is typically assessed using indirect methods [e.g., ‘soft’ extraction techniques like hydroxypropyl cyclodextrin (HPCD) extraction]. However, it was found that the directly measured response of earthworm exposure to sub-lethal concentrations of phenanthrene in soil is related to both the total and bioavailable phenanthrene. This suggests there is potential for the use of 1H NMR metabolomics for the assessment of contaminant bioavailability. This thesis has demonstrated that E. fetida are suitable for metabolomic studies and has indicated that 1H NMR metabolomics may have potential for measuring and monitoring earthworm exposure to sub-lethal concentrations of PAHs.

Orientador: Anita Jocelyne Marsaioli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-21T04:07:12Z (GMT). No. of bitstreams: 1 Lopes_ThiagoInacioBarros_M.pdf: 3472847 bytes, checksum: 6ec44b5e5d2ad869887d1d1a884a0682 (MD5) Previous issue date: 2012 / Resumo: A organização mundial de saúde (OMS) estima que existam atualmente mais de 1,5 bilhões de adultos com sobrepeso, número que é esperado dobrar até 2015. Obesidade e sobrepeso são enfermidades caracterizadas pelo acúmulo excessivo de gordura corporal e têm sido associadas a vários problemas de saúde. Vários fármacos têm sido utilizados no tratamento desta enfermidade, entre os mais utilizados se encontra o Orlistate, um inibidor de lipases gástricas utilizado na redução da absorção de gordura da dieta, auxiliando na perda e manutenção do peso. Esta dissertação teve como objetivo principal avaliar as alterações metabólicas sofridas por pacientes com sobrepeso tratados com Orlistate por 120 dias. Para tornar mais didático, o trabalho foi divido em duas Partes: Parte I, implementação da metodologia analítica para análise de ácidos graxos por CG-EM e avaliação do perfil de ácidos graxos em indivíduos com sobrepeso tratados com Orlistate; e Parte II, avaliação do perfil metabólico de plasma por RMN de ¹H de indivíduos com sobrepeso tratados com Orlistate. Na primeira etapa do trabalho, análise de componentes principais foi aplicada na seleção íons (m/z) para quantificação de ácidos graxos, após preparação de ésteres metílicos correspondentes, por CG-EM usando monitoramento de íons selecionados. Quatro íons foram então selecionados de forma a aumentar a detectividade sem perda completa de informação qualitativa. Os íons de m/z 74, 79, 81 e 87 foram selecionados e permitiram a quantificação de vários ácidos graxos, além da determinação do número de insaturações dos mesmos relacionando a abundância relativa dos íons à presença de insaturações. A metodologia analítica implementada permitiu quantificar ácidos graxos esterificados em vários lipídios presentes no sangue, após transesterificação para produção dos ésteres metílicos de ácidos graxos, com adequada precisão, repetitividade e baixos limites de detecção e quantificação. Na segunda etapa, a metodologia analítica foi aplicada no estudo do perfil de ácidos graxos de 20 mulheres com sobrepeso tratadas com Orlistate durante 120 dias. O tratamento não reduziu o índice de massa corpórea contribuiu para diminuição significativa dos níveis de colesterol-HDL no plasma e do conteúdo de colesterol na membrana de eritrócitos, além de alterar as proporções relativas de vários ácidos graxos essenciais e exógenos em vários lipídios estudados. Adicionalmente foi observado um perfil de ácido graxo significativamente diferente para os indivíduos magros (controles) em comparação com indivíduos com sobrepeso. Na última etapa, o perfil metabólico de plasma por RMN de ¹H foi estudado por uma abordagem metabolômica. A análise discriminatória por quadrados mínimos parciais (PLS-DA) revelou que alterações nos níveis de lactato e magnésio são importantes na diferenciação entre indivíduos com sobrepeso tratados e não-tratados com Orlistate, sinalizando diminuição destes metabólitos relacionada ao tratamento. Não há relatos anteriores da alteração dos níveis de lactato devido ao tratamento. Adicionalmente, os níveis de triacilglicerídios, alanina e lactato contribuíram significativamente na distinção entre indivíduos magros e com sobrepeso / Abstract: The World Health Organization (WHO) estimates that there are currently more than 1.5 billion overweight adults, a number that is expected to double until 2015. Obesity and overweight are diseases characterized by excessive accumulation of body fat and linked to various health problems. Several drugs have been used to treat such diseases. Orlistat is a gastric lipase inhibitor largely used to reduce the absorption of dietary fat, helping weight loss. Thus this thesis aimed at the metabolic variation of overweight subjects treated with Orlistat for 120 days. This work is divided into two parts: Part I, implementation of the analytical methodology for analysis of fatty acids by GC-MS and evaluation of the fatty acid profile in overweight subjects treated with Orlistat; and Part II, evaluation of the metabolic profile of plasma of overweight subjects treated with Orlistat using ¹H NMR. In the Part I of this work, principal component analysis was applied to selected ions (m/z) for determination of fatty acids, after preparation of the corresponding methyl esters, by GC-MS using selected ion monitoring. Four ions were selected. The ions of m/z 74, 79, 81 and 87 allowed the quantification of various fatty acids and determination of the number of double bounds in theo fatty acids through the relative abundance of these selected ions. The analytical methodology implemented permitted quantify esterifing fatty acids in various lipids in blood, after transesterification for production of methyl esters of fatty acids, with adequate accuracy, repeatability and low limits of detection and quantification. In the second step, the analytical methodology was applied to study the fatty acid profile of 20 overweight women treated with Orlistat for 120 days. Although there was no significant reduction in body mass index, the treatment contributed to significant reduction of HDL-cholesterol levels in plasma and the cholesterol content in erythrocyte membranes. The Orlistat also alters the relative proportions of various fatty acids in the several lipids studied. Additionally, a significantly different fatty acid profile was observed for lean subjects (controls) compared to overweight subjects. In Part II, the metabolic profile of plasma obtained by ¹H NMR was studied by a metabolomics approach. The partial least squares discriminant analysis (PLS-DA) revealed changes in lactate and magnesium level, these metabolites were important to differentiating between overweight subjects treated and not-treated with Orlistat, suggesting that the level of these metabolites decreased with treatment. There are no previous reports of changes in lactate levels. Additionally, levels of triglycerides, alanine and lactate were highlighted by PLS-DA into distinguishing between lean and overweight individuals / Mestrado / Quimica Organica / Mestre em Química

Ce travail étudie les matériaux composites zéolithes surfactifs, principes actifs pour des applications pharmaceutiques. D'abord sont identifiées les principales propriétés physiques, chimiques et biologiques des matériaux, requises pour des applications en tant que supports médicamenteux ciblant certaines formes de cancer. Par la suite sont décrits les différentes étapes de formulation des supports médicamenteux à base des zéolithes naturelles, les caractéristiques des matériaux intermédiaires ainsi obtenus, ainsi que les mécanismes des phénomènes interfaciaux à l'origine de l'action de ces matériaux. La dernière partie est consacrée à l'étude d'un relargage contrôlé des principes actifs de l'interface jusque dans un milieu aqueux modèle. / This work studies the composite zeolites surfactants, active principles for pharmaceutical applications. First identified the main physical, chemical and biological materials, required for applications as drug carriers targeting some cancers. Subsequently described the various stages of formulation of drug carriers based on natural zeolites, the characteristics of intermediate materials obtained, and the mechanisms of interfacial phenomena at the origin of the action of these materials. The last part is devoted to the study of controlled release of active ingredients of the interface into a model aqueous medium.

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The aim of this study was to develop an analytical method for fluorinated silanes. Furthermore, as a secondary aim, to explore if there would be possible to detect 1H,1H,2H,2H-perfluorooctyl triethoxysilane (6:2 PTrEtSi) and 1H,1H,2H,2H-perfluorodecyl triethoxysilane (8:2 PTrEtSi) in two different matrices, sludge and cosmetic extract. The method development included experiments using LC-MS, LC-MS/MS, UPC2, GC-MS and APGC-MS/MS and was carried out using standards containing 6:2 PTrEtSi and 8:2 PTrEtSi. The analytical method that worked best for the compounds was GC-MS/MS and an analytical method using APGC-MS/MS was developed for fluorinated silanes. The IDL for 6:2 PTrEtSi was 0.0012 μg/mL and 1.32 μg/mL for 8:2 PTrEtSi. This makes the developed method suitable for high contaminated samples, such as extracts from cosmetic products. It was concluded that a method using LC as the analytical instrument would not work for the two target compounds since they were too reactive with the mobile phase. However, LC could be a good choice for siloxanes, compounds that are formed from hydrolysis and condensation of fluorinated silanes. The samples analyzed in this study were three sludge extracts and one extract from a cosmetic product. 6:2 PTrEtSi was expected to be detected in the cosmetic sample since the compound was stated on the table of contents of the cosmetic product. No detection of 6:2 TrEtSi or 8:2 TrEtSi could be made in either of the samples. The reason for this was suspected to be transformation or degradation of the compounds into other compounds. Therefore, a full scan of the cosmetic sample using LC-MS/MS was included in the experiment as an addition to verify the suspicions that compounds such as siloxanes could have been formed. An interesting peak was discovered with m/z 947 which could be a disiloxane of 6:2 PTrEtSi.

Made available in DSpace on 2014-06-12T15:50:11Z (GMT). No. of bitstreams: 2 arquivo2029_1.pdf: 3912167 bytes, checksum: 4f659ba400462a69544db2ffbff56bf5 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / A metabonômica constitui nova metodologia que vem em ascensão pelo seu importante papel na análise de materiais biológicos para o diagnóstico e o prognóstico de certas doenças, principalmente as crônico-degenerativas e as alterações genéticas, além do seu lugar frente à toxicidade por drogas. Refere-se à detecção, identificação, quantificação e classificação das mudanças metabólicas, tempo-relacionadas, integrada ao sistema biológico, numa visão global ao invés da avaliação da célula individualmente. Essa revisão visa introduzir o conceito da metabonômica baseada na utilização da espectroscopia de ressonância magnética nuclear de hidrogênio-1 (RMN 1H), uma das ferramentas analíticas preferidas por apresentar a vantagem de ser técnica não destrutiva, rápida e requerer pouco ou nenhum pré-tratamento das amostras. Além disso, dispensa o uso de amostras padrões para a co-injeção e identificação dos compostos, necessitando, entretanto, de métodos estatísticos multivariados para a análise dos complexos espectros obtidos. Buscaram-se, no banco de dados MEDLINE e SciELO, os artigos publicados no período de janeiro de 1998 a dezembro de 2007, escritos nos idiomas inglês ou português, que avaliassem seres humanos e utilizassem biofluidos para análise em RMN de 1H, sendo empregados os seguintes termos: metabonomics and 1H NMR and nuclear magnetic resonance spectroscopy and disease. Os artigos publicados inicialmente estavam mais relacionados à investigação dos erros inatos do metabolismo e à toxicidade de drogas, sendo bastante aplicados à indústria farmacêutica. Recentemente, os estudos estão mais voltados ao diagnóstico e ao prognóstico das doenças crônico-degenerativas e infecciosas, um dos motivos para a elaboração desta revisão

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