Made available in DSpace on 2014-06-11T19:29:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-27Bitstream added on 2014-06-13T20:18:59Z : No. of bitstreams: 1 leandro_fz_me_araiq_prot.pdf: 1135475 bytes, checksum: f2d8e4f49663bf83eb7c7e55cb4d5a0c (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Atualmente há um grande interesse no estudo dos desreguladores endócrinos (substâncias estranhas aos organismos, que mimetizam ou bloqueiam a ação natural dos hormônios naturais), tanto em relação ao seu destino ambiental quanto à toxicologia dos mesmos. Neste trabalho, avaliou-se o Bisfenol A (BPA), um monômero do qual se produz o plástico policarbonato. Recentemente foi proposto que o BPA exibe atividade estrogênica em concentrações de 1 ng mL-1. Neste trabalho, desenvolveu-se um método para a determinação de BPA em águas naturais, fazendo-se uso de cartuchos de poliestireno-divinilbenzeno (PSDVB) para a extração em fase sólida (SPE) e cromatografia líquida de alta eficiência com um detector de fluorescência (HPLC/FLU) para a quantificação do composto. A recuperação do método foi avaliada utilizando-se amostras de água potável fortificada em 3 níveis de concentração: 0,06; 0,2 e 0,6 ng mL-1. A extração do analito (500 mL; n=4) resultou valores de recuperações entre 99 e 100% e CV entre 0,30 e 3,9%. Os limites de detecção e quantificação do método foram 0,02 e 0,06 ng mL-1, respectivamente. O mesmo estudo foi efetuado para água superficial sendo obtidos valores de recuperação entre 85,8 e 87% para níveis de fortificação similares aos utilizados para água potável, com CV entre 1,1 e 2,3%. O método validado foi aplicado com bom desempenho às amostras de água de entrada e saída da Estação de Tratamento de Água, onde constatou-se a presença de BPA entre 11,7 e 16,8 ng mL-1 na água bruta e 6,2 e 7,3 ng mL-1 na água potável. Desta forma, o monitoramento ambiental do BPA torna-se extremamente relevante e necessário. / Nowadays it has been increased the concern about endocrine disrupters (unknown substances for organisms that mimic or block the natural action of endogenous hormones), so much at relation of your environmental fate as toxicology about them. In this study, it had been evaluated Bisphenol A (BPA), a monomer, wich produces polycarbonate. Recently, it has been proposed that BPA exhibits estrogenic activity at 1 ng mL-1. At this work, it had been developed a method for determination of BPA at natural water with polistyrene-divinylbenzene (PSDVB) cartridge on phase solid extraction (SPE) and high performance chromatography liquid with fluorescence detection (HPLC/FLU) for the analyte's quantification. The method's recovery was evaluated with spiked drink water sample at three concentrations levels: 0,06; 0,2 and 0,6 ng mL-1. The analyte extraction (500 mL; n=4) gave recoveries between 99 and 100% with CV between 0,30 and 3,9%. The method detection and quantification limits were 0,02 and 0,06 ng mL-1, respectively. The same study was developed for superficial water with recoveries between 85,8% and 87% for similar spiked levels at the drink water with CV between 1,1 and 2,3%. The method developed was applied with good performance at water's samples of Water Treatment Plant, verifying the appearance of BPA between 11,7 and 16,8 ng mL-1 on sewage water and 6,2 until 7,3 ng mL-1 on drink water. Therefore the environmental monitoring of BPA is very important and indispensable.

Specific amino acids (i.e. arginine, citrulline, aspartic acid, histamine, glutamic acid and taurine) play significant roles in a number of physiological processes such as neurotransmission, inflammation and cell proliferation. Analytical methods that are capable of measuring these compounds in small volumes are important for in vivo sampling strategies and single cell analysis. Electrophoresis-based methods (e.g. capillary (CE) and microchip (ME) electrophoresis) with fluorescence detection have been applied to determine various biological compounds due to the high separation efficiency, simplicity, very low sample and solvent volume consumption and short analysis time. In this study, selected amino acids were off-line derivatized with naphthalene-2,3-dicarboxaldehyde/cyanide (NDA/CN-). NDA itself is not fluorescent, but can react with primary amines in the presence of cyanide to produce N-substituted 1-cyanobenz[f]isoindole (CBI) derivatives, which are fluorescent. Run conditions (e.g. buffer additives, organic solvent and separation voltage) were optimized for both methods (CE and ME) to obtain baseline separation of the six analytes. Excitation was accomplished using a diode laser (λex = 445 nm and λem = 490 m). Based on five-point calibration curves, both methods showed good linearity in the range of 10 to 0.1 µmolL-1 for each amino acid. Precision with %RSDs of less than 6.9 and 13.2% was obtained from CE and ME, respectively. For CE, the number of plates (N) was greater than 150 000/m, resolution values were more than 3.4 and migration time was between 11.2 to 18.2 min. ME offered a better efficiency (N > 300 000/m) and resolution (Rs > 3.9) with a much shorter analysis time (3.8 min) than the CE method. Limits of detection and quantification were significantly lower than the concentrations measured in microdialysis sample (MD) for both methods. According to the results with MD, it was possible to identify and quantify all analytes using CE and ME method. A portable fluorescence detection system for use with microchip electrophoresis was developed. Using the portable system with ME, limits of detection for the analytes of interest were 250 nmolL-1 – 1.3 μmolL-1, which were adequate for most analyte detection in brain microdialysis samples. (P.S.: some expressions without training) / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Aminoácidos específicos (arginina, citrulina, ácido aspártico, histamina, ácido glutâmico e a taurina) apresentam funções importantes em vários processos fisiológicos, tais como neurotransmissão, inflamação e proliferação celular. Métodos analíticos que são capazes de quantificar estes compostos utilizando baixo volume de amostra são importantes para estratégias de amostragem in vivo e análises de célula única. Métodos baseados em eletroforese, como eletroforese capilar (EC) e microchip eletroforético (ME), com detecção por fluorescência têm sido utilizados para determinar vários compostos biológicos, devido à elevada eficiência de separação, simplicidade, baixo consumo de amostra, volume do solvente e o curto tempo de análise. Neste estudo, os aminoácidos selecionados foram derivatizados off-line com naftaleno-2,3-dicarboxaldeído/cianeto (NDA / CN-). NDA não é por si só fluorescente, mas pode reagir com aminas primárias na presença de cianeto para produzir 1-cianobenz[f]isoindoline N-substituídos (CBI), que são derivados fluorescentes. As condições de execução, incluindo aditivos no tampão, solvente orgânico e voltagem de separação foram otimizados para ambos os métodos (EC e ME) visando obter adequada resolução de separação para seis analitos. A excitação foi conseguida utilizando um laser de diodo (λex = 445 nm e λem = 490 m). Com base nas curvas analíticas com cinco pontos, os dois métodos demonstraram uma boa linearidade no intervalo de 10 a 0,1 µmolL-1 para cada aminoácido. Precisão com RSD% menor do que 6,9 e 13,2% foram obtidos a partir da técnica de EC e ME, respectivamente. Para EC, o número de pratos teóricos (N) foi maior do que 150 000 / m, com valores de resolução maiores que 3,4; sendo que o tempo de migração variou entre 11,2-18,2 min. ME ofereceu uma melhor eficiência (N> 300 000 / m) e resolução (Rs> 3,9) com um tempo de análise muito mais curto (3,8 minutos) do que o método aplicando EC. Os limites de detecção e quantificação para ambos os métodos foram significativamente menores do que as concentrações medidas nas amostras de microdiálise (MD). De acordo com os resultados com MD, foi possível identificar e quantificar todos os analitos usando o método de EC e ME. Um sistema portátil de detecção de fluorescência para ser utilizado com microchip eletroforético foi desenvolvido. Usando o sistema portátil com o ME, os limites de detecção para os analitos de interesse foram 250 nmolL-1 – 1,3 μmolL-1, que são adequados para a detecção da maioria dos analitos nas amostras de microdiálise cerebral. (OBS.: algumas expressões sem formação)

G protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that perform transmembrane signal transduction. Due to their pivotal role in a wide range of essential physiological functions GPCRs represent a high proportion of all drug targets. High resolution X-ray structures of GPCRs are however underrepresented in the Protein Data Bank. This is due to their instability in detergent, low expression levels and the presence of misfolded receptors in many heterologous expression systems. The objective of this project was to engineer the angiotensin II type 1 receptor (AT1R), a human GPCR, to make it suitable for structural studies. It was determined that detergentsolubilised AT1R was thermostable with antagonist bound with an apparent Tm of ~45°C, which was sufficiently stable for purification without further thermostabilisation by rational mutagenesis. Two expression systems were then evaluated for large-scale production of AT1R, namely baculovirus-mediated expression in insect cells and mammalian expression in HEK293 cells. Radioligand binding assays showed that only the mammalian system produced sufficient quantities of active AT1R for structural studies. Expression in the mammalian system was further optimised to approximately 6 mg/L. An AT1R-GFP fusion was created to examine membrane localisation using confocal laser scanning microscopy, to assay expression levels, to select highly expressing monoclonal cell lines using fluorescence activated flow cytometry and to develop a fluorescence size-exclusion chromatographybased assay to examine the suitability of 12 different ligands for co-crystallization. AT1R was also engineered to facilitate crystallisation, including C-terminal truncations to remove predicted disordered regions and bacteriophage T4-lysozyme being added to the third intracellular loop to provide additional points of contact for crystallisation, which increased the apparent Tm by approximately 10°C. All modified versions of AT1R were assessed for expression, stability and monodispersity. Additionally a rapid western blotting based assay was developed for the detection of unfolded membrane proteins, which will have wide applicability in the field.

Fluorescence lifetime determination and allied studies find application in spectroscopy in general and fiber optic biosensors in particular. Instruments and sensors cited in literature however use open loop, intensity based techniques with sophisticated detectors and components. We propose phase sensitive signal processing schemes to estimate the fluorescence lifetime using simple detectors and components, without compromising on accuracy. The performance of the schemes proposed is analysed and contrasted from a communications (signals and systems) point of view. The resolution and sensitivity limits imposed in processing the signal, by systematic errors and additive noise, are derived for the schemes suggested. It is found that systematic errors impose a phase resolution limit of about 2°. We then study the suitability of different detectors and channels for application in phase sensitive fluorescence biosensors we analyse the effect of systematic limitations as well as additive noise, in the detection/transmission process, from the point of view of the components used. Certain fundamental limits of operation in terms of excitation intensities are derived for different detector-channel combinations, with a view to obtain a given resolution. A photodiode used with a fiber bundle is found to be sufficient for accurate phase read outs with 10"4 radians resolution. A PMT used in conjunction with a multimode fiber serves as a very good device for microsensing applications Lastly, the biosensor for oxygen sensing, the ruthenium complex, is studied for standardisation of the sensor. We examine the quenching of fluorescence, the repeatability and reusability of the sensor, the stability of the instrument and such.

Orientador: Mary Rosa Rodrigues de Marchi / Banca: Manoel Lima de Menezes / Banca: Eny Maria Vieira / Resumo: Atualmente há um grande interesse no estudo dos desreguladores endócrinos (substâncias estranhas aos organismos, que mimetizam ou bloqueiam a ação natural dos hormônios naturais), tanto em relação ao seu destino ambiental quanto à toxicologia dos mesmos. Neste trabalho, avaliou-se o Bisfenol A (BPA), um monômero do qual se produz o plástico policarbonato. Recentemente foi proposto que o BPA exibe atividade estrogênica em concentrações de 1 ng mL-1. Neste trabalho, desenvolveu-se um método para a determinação de BPA em águas naturais, fazendo-se uso de cartuchos de poliestireno-divinilbenzeno (PSDVB) para a extração em fase sólida (SPE) e cromatografia líquida de alta eficiência com um detector de fluorescência (HPLC/FLU) para a quantificação do composto. A recuperação do método foi avaliada utilizando-se amostras de água potável fortificada em 3 níveis de concentração: 0,06; 0,2 e 0,6 ng mL-1. A extração do analito (500 mL; n=4) resultou valores de recuperações entre 99 e 100% e CV entre 0,30 e 3,9%. Os limites de detecção e quantificação do método foram 0,02 e 0,06 ng mL-1, respectivamente. O mesmo estudo foi efetuado para água superficial sendo obtidos valores de recuperação entre 85,8 e 87% para níveis de fortificação similares aos utilizados para água potável, com CV entre 1,1 e 2,3%. O método validado foi aplicado com bom desempenho às amostras de água de entrada e saída da Estação de Tratamento de Água, onde constatou-se a presença de BPA entre 11,7 e 16,8 ng mL-1 na água bruta e 6,2 e 7,3 ng mL-1 na água potável. Desta forma, o monitoramento ambiental do BPA torna-se extremamente relevante e necessário. / Abstract: Nowadays it has been increased the concern about endocrine disrupters (unknown substances for organisms that mimic or block the natural action of endogenous hormones), so much at relation of your environmental fate as toxicology about them. In this study, it had been evaluated Bisphenol A (BPA), a monomer, wich produces polycarbonate. Recently, it has been proposed that BPA exhibits estrogenic activity at 1 ng mL-1. At this work, it had been developed a method for determination of BPA at natural water with polistyrene-divinylbenzene (PSDVB) cartridge on phase solid extraction (SPE) and high performance chromatography liquid with fluorescence detection (HPLC/FLU) for the analyte's quantification. The method's recovery was evaluated with spiked drink water sample at three concentrations levels: 0,06; 0,2 and 0,6 ng mL-1. The analyte extraction (500 mL; n=4) gave recoveries between 99 and 100% with CV between 0,30 and 3,9%. The method detection and quantification limits were 0,02 and 0,06 ng mL-1, respectively. The same study was developed for superficial water with recoveries between 85,8% and 87% for similar spiked levels at the drink water with CV between 1,1 and 2,3%. The method developed was applied with good performance at water's samples of Water Treatment Plant, verifying the appearance of BPA between 11,7 and 16,8 ng mL-1 on sewage water and 6,2 until 7,3 ng mL-1 on drink water. Therefore the environmental monitoring of BPA is very important and indispensable. / Mestre

Rtuť je v přírodě přirozeně se vyskytujícím toxickým prvkem, jehož globální emise jsou ovlivňovány zejména antropogenními zdroji znečištění. Obrovský globální nárůst v usazování rtuti, zejména ve vodných ekosystémech, byl zaznamenán současně s počátkem průmyslové revoluce. Sedimenty jsou posledním místem úložiště nejrůznějších komplexů rtuti. Rtuť však zde může být přeměněna na toxičtější organickou formu, methylrtuť, pomocí transformačních procesů kontrolovaných různými fyzikálními, chemickými, ale i biologickými faktory. Navíc mohou být specie rtuti remobilizovány ze sedimentů pomocí difuze a resuspenzace a tak se sedimenty mohou stát i potenciálním zdrojem rtuti. Proces bioakumulace a bioobohacování tak pokračuje v potravním řetězci, ve kterém se člověk, i další zvířata, stává konzumentem methylrtuti. Stanovení celkové koncentrace rtuti není dostačující k porozumění osudu rtuti v přírodním prostředí a tak stanovení MeHg poskytuje nezbytnou doplňující informaci. Dostatečně citlivá a přesná analytická metoda pro stanovení specií rtuti je nezbytným nástrojem environmentální chemie. Metody vhodné pro stanovení specií rtuti v sedimentech jsou popsány v části metodologie disertační práce. Metoda stanovení methylrtuti v sedimentech pomocí automatické Headspace vybavené pastí („trap“) a spojené s plynovou chromatografií a fluorescenční detekcí je zde také popsána. Zvláštní pozornost je také věnována potřebám zásad čistého vzorkování, skladování vzorků a přípravě vzorků před samotou analýzou, jakož i samostatné části věnující se terénní studii rtuti a methylrtuti v sedimentech vytipovaných lokalit. Sedimenty jižní Moravy a severní Francie jsou srovnány z hlediska znečištění rtutí. Specie rtuti a další ukazatele (Fe, Mn, S) byly analyzovány v sedimentech, pórové vodě a povrchové vodě řek Dele a Lys (Francie) a Jihlava a Morava (Česká republika). Z hlediska posouzení vodních ekosystémů a jejich znečištění rtutí, je vhodné znát koncentraci rtuti v pórové vodě a posoudit dostupnost rtuti ze sedimentů. Technika difuzního gradientu v tenkém filmu je vhodným způsobem jak stanovit koncentraci rtuti v pórové vodě sedimentů. Do roku 2005 bylo použití této techniky pro měření rtuti značně limitováno. Ale nedávný pokrok především v dostupnosti možných sorpčních gelů vhodných pro stanovení rtuti umožnilo využití této techniky i pro stanovení rtuti. Byly použity různé sorpční gely: Spheron.Thiol, Duolite GT-73 a TiO2. Řeka Dele představuje past enormního množství antropogenní rtuti pocházející z průmyslových zdrojů a je považována za potenciální významný zdroj methylrtuti pro okolní prostředí a živé organismy především. Poslední část dizertační práce se zabývá aplikací dobře zavedeného experimentu využívajícím stabilní isotopy ke studiu metylačních a demethylačních procesů v sedimentech řeky Dele. Obohacené stabilní značkovače rtuti v anorganické formě (199Hg) and methylované formě (201MeHg) byly přidány do sedimentů. Tyto označené specie rtuti tak pomohly sledovat osud specií rtuti a vypočítat rozsah jejich přeměny v průběhu experimentu.

No description available.

L'assainissement non collectif concerne 12 à 15 millions de personnes en France. La filière classique de ce mode d’assainissement se compose généralement d'un prétraitement anaérobie par une fosse septique recevant l’ensemble des eaux usées domestiques suivi d’un système d’infiltration dans le sol ou d’un filtre à sable. Le filtre à sable vertical drainé met à profit le pourvoir épuratoire qui est principalement lié à la présence d’une biomasse sous forme d’un biofilm. Cette dynamique de la croissance de la biomasse ou du biofilm est soumise à l’impact de la nature de matériaux filtrants. L’écoulement insaturé dans ces systèmes conditionne également cette croissance du biofilm. Dans ce contexte, l'objectif du travail de la thèse est d’appréhender les mécanismes mis en jeu et particulièrement l’impact des matériaux dans le fonctionnement des filtres en comparant notamment deux types de matériaux: les sables de rivière et les agrégats concassés. Pour cela, une étude expérimental sur une unité pilote composé des réacteurs de filtration du diamètre de 30cm et différents épaisseurs de garnissage (15, 30 et 70cm) a été construite. Les réacteurs garnis de deux sables roulés et deux agrégats concassés, sont alimentés en effluent septique avec une charge volumique 12cm/jour par 10 bâchés par jour. Suite des matériaux, une étude de la performance épuratoire avec le suivi des composants biochimiques de la biomasse totale et de la matrice extracellulaire du biofilm est réalisée en comparant notamment les deux types de matériaux filtrants. / The onsite wastewater treatment systems concern 12 to 15 million of people in France. The treatment plant is generally composed by a septic tank as pretreatment, followed by soil infiltration field or sand filtration bed. The vertical drained sand filter provides the purification capacity thanks to the presence of a biomass in form of the biofilm. The dynamic of the biomass growth or the biofilm development is under the impact of filter materials’ nature. In this context, the objective of this work is to understand the mechanisms involved and especially the impact of medium in the functioning of the filtration reactor by comparing two types of packing materials: river sands and crushed aggregates. For this purpose, an experimental study is conducted with pilot unity composed by filtration reactors of 30cm of diameter and different packing thicknesses (15, 30 and 70cm). The reactors packed with two river sands and two crushed aggregates are fed with septic effluent with a volumic hydraulic charge of 12cm/day by 10 batches per day. Based on a characterization of materials, a study of purification performance with biochemical components monitoring of the total biomass and the extracellular matrix of the biofilm is realized by comparing two types of filter materials. The purification performance has presented similar efficiencies of particulates and organic matters removals by fine river sand and fine crushed aggregate. The nitrogen pollutants removals are more effective in the fine river sand which presents the finest granulometry with an alternative of aerobic and anoxic phases along the reactor depth and with a biomass more abundant. The distribution and the quality of the total biomass and the extracellular matrix differentiate between the river sand and the crushed aggregates. As the reference material, the fine river sand presented an earlier stabilization of total biomass growth with a less important production of extracellular exudates compared to the crushed aggregates. The origin of impacts brought by the crushed aggregates might be due to the higher fine particles content which created microenvironments poor in substrates or in oxygen and also due to a more heterogeneous mineralogy. The extracellular components of highest percentage in the biofilm of crushed aggregate are polysaccharides type substances.