Dissertations / Theses on the topic '2D-Fluorescence'

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica, Especialidade em Engenharia Bioquímica
Os biorreactores de membranas (MBRs, ‘membrane bioreactors’) para o tratamento de águas residuais combinam o processo de lamas activadas com um passo de filtração para obtenção de um efluente limpo, livre de sólidos. Os MBRs representam uma tecnologia em expansão no tratamento de águas residuais sobretudo devido ao reduzido espaço que requerem e à elevada qualidade do efluente obtido. No entanto, a colmatação das membranas pode reduzir o desempenho do MBR. Por este motivo, no presente trabalho, pretendeu-se estudar a monitorização dos MBRs, com o objectivo de minimizar o número de parâmetros de monitorização necessários para descrever o desempenho do processo e obter uma monitorização em tempo real com recurso mínimo a técnicas laboratoriais demoradas. Para este fim, estudou-se a aplicabilidade da fluorescência bidimensional em meios biológicos complexos, tais como as lamas activadas utilizadas para o tratamento de águas residuais. A fluorescência bidimensional mostrou ser uma técnica abrangente, capaz de recolher informação relevante sobre o estado do sistema em tempo real. Devido à complexidade da informação contida nos espectros de fluorescência, usaram-se técnicas de estatística multivariada, tais como análise de componentes principais e projecção de estruturas latentes (PLS, ‘projection to latent structures’), para extrair a informação dos espectros e correlacioná-la com parâmetros de operação e de desempenho do MBR. O uso de modelos estatísticos permitiu a previsão de parâmetros chave para o desempenho do MBR usando somente dados de processo impostos ou facilmente adquiríveis em tempo real. Adicionalmente, a modelação estatística foi combinada com um modelo mecanístico, numa estrutura híbrida, de forma a melhorar a previsão mecanística. Este estudo demonstrou ser possível usar modelos PLS para incorporar dados de fluorescência obtidos em tempo real, de modo a melhorar a previsão mecanística sem requerer análises laboratoriais adicionais.

Biological systems present many challenges to researchers attempting to study them using spectroscopy. Low specificity, low sensitivity, and broad and overlapping lineshapes limit the amount of information that can be obtained in experiments. Two-dimensional fluorescence spectroscopy (2D FS) is a highly sensitive and information-rich spectroscopic technique that was developed to study the conformations and excited state dynamics of systems exhibiting exciton coupling. In this dissertation, I describe a variety of extensions of 2D FS that further increase its utility for the study of biological systems. I describe experiments on a dimer of zinc tetraphenylporphyrin embedded in a membrane, in which the signals from two conformational subpopulations were separated in order to study the thermodynamics of their interconversion. I present proof-of-principle experiments on nucleic acids that utilize fluorescence resonance energy transfer to separate signals from different subpopulations. I also describe experiments in which 2D FS was performed using ultraviolet excitation to determine the conformation of a dinucleotide of a fluorescent analogue of the nucleic acid base adenine. I discuss experiments on porphyrin dimers in which 2D FS was used as a probe of excited state dynamics. Finally, I present model calculations for a proposed variation of 2D FS in which entangled photons would be used as the excitation source. These calculations suggest that this approach has the potential to yield significantly narrower spectral lineshapes than conventional 2D FS. These experiments and calculations yield new insight into the systems investigated and establish a `toolbox' of variations of 2D FS that can be used to gain as much information as possible from experiments on challenging systems such as protein-DNA complexes. This dissertation contains previously published and unpublished co-authored material.

A dispersão de óxidos sulfurados no meio ambiente, proveniente da queima de combustíveis fosseis, contribui significativamente para a poluição atmosférica. A presença de compostos sulfurados em combustíveis também apresenta desvantagens práticas na operação das unidades, corroendo tubulações e unidades de armazenamento, além de causar danos aos motores. De acordo com a Resolução nº 50, de 23 de dezembro de 2013 da ANP, todo o diesel metropolitano vendido no Brasil deve conter no máximo 10 ppm de enxofre, forçando uma adaptação dos processos produtivos, que, por sua vez, forçam as industrias a investirem em sistemas de controle e consequentemente metodologias de análise on-line de correntes de processo. As análises atuais certificadoras de enxofre em diesel requerem preparação amostral, equipamentos de alto custo e possuem alto tempo morto associado. Surge a necessidade de estudo de uma metodologia capaz de ser aplicada em analisadores de linha para predição de enxofre em tempo real. Com este intuito, este trabalho visa estudar a viabilidade do uso de espectroscopia por fluorescência 2D para predição de enxofre em óleo diesel. A espectroscopia por fluorescência é uma técnica rápida, que dispensa preparação de amostra e possui alta sensibilidade para compostos naturalmente fluorescentes. Um estudo envolvendo quatro componentes sulfurados presentes em diesel foi realizado por meio de metodologias não supervisionadas - PCA, e supervisionadas - PSCM. Não foi possível segmentar as quatro soluções-padrão por meio de gráfico de escores da PCA, mas com PSCM foi factível o ajuste de modelos multilineares para predição de enxofre nas soluções-padrão, com coeficientes de determinação superiores a 0,97. Ainda, é possível definir a região de fluorescência mais significativa para cada um dos padrões, constatando que todos possuem regiões de emissão de fluorescência similares. Com relação à amostras de diesel, foram analisados dois grupos distintos de óleo diesel, (i) diesel HDT com média de 100 ppm de enxofre, e (ii) diesel S10 com média de 6,5 ppm de enxofre. Avaliando os resultados da PCA, foi possível segmentar os dois conjuntos de diesel utilizando os dados de escores. Por PSCM, foi possível ajustar modelos baseados em pares de fluorescência capazes de predizer satisfatoriamente concentrações de enxofre em amostras de diesel S10. Os modelos ajustados para diesel HDT apresentaram resultados menos significativos. Assim, a viabilidade do uso de espectroscopia por fluorescência 2D para a caracterização de enxofre em correntes de diesel foi confirmada, viabilizando a construção de sensores de processos baseados nesta técnica analítica.
The dispersion of sulfur oxides in the environment from the burning of fossil fuels contributes significantly to air pollution. The presence of sulfur compounds in fuel also presents disadvantages in operation units, corroding pipes and storage units and may even damage the engine. According to ANP Resolution No. 50, dated December 23, 2013 the entire metropolitan diesel sold in Brazil must contain no more than 10 ppm sulfur, forcing an adaptation of production processes. The current analysis to certify sulfur content in diesel requires sample preparation, expensive equipment and have high dead time associated. Therefore, it is important the study of a method capable of being applied to online analyzers for predicting sulfur in real time. This work aims to study the feasibility of using 2D fluorescence spectroscopy for prediction of sulfur in diesel fuel. Fluorescence spectroscopy is a rapid technique that, usually, does not require sample preparation and has high sensitivity to naturally fluorescent compounds. Initially, a study involving four sulfur containing compounds present in diesel was carried out by means of unsupervised methods - PCA and supervised - PSCM. It was not possible to segregate the four standard solutions by score plot from the PCA, but PSCM was able to fit general multilinear models for the prediction of sulfur in standard solutions with coefficients of determination greater than 0.97. It was possible to define the region of most significant fluorescence for each standard, noting that all standards present similar fluorescence emission regions. After, two groups of different diesel, diesel HDT averaging 100 ppm sulfur and diesel S10 averaged 6.5 ppm sulfur were analyzed. Evaluating the results of PCA was possible to segregate both sets of diesel data using score plot. With PSCM was possible to find models based on fluorescence able to satisfactorily predict concentrations of sulfur in diesel samples S10. The models adjusted for diesel HDT showed less significant results. Thus, the feasibility of prediction of sulfur in diesel using 2D fluorescence spectroscopy was confirmed.

O desenvolvimento e aperfeiçoamento de métodos de otimização são pontos de profundo interesse em todas as áreas de pesquisa. Tais técnicas muitas vezes envolvem a aquisição de métodos de controle novos ou melhores, o que está diretamente ligado a duas tarefas importantes: a escolha de formas eficientes de monitoramento do processo e a obtenção de modelos confiáveis para a variável de interesse a partir de dados experimentais. Graças às suas diversas vantagens, os sensores óticos vêm sendo amplamente aplicados na primeira tarefa. Uma vez que é possível a utilização de vários tipos de espectroscopia através deste tipo de sensor, modelos capazes de lidar com dados espectrais estão se tornando cada vez mais atraentes. A segunda tarefa, por sua vez, depende não só de quais preditores são utilizados na construção do modelo, mas também de quantos. Como a qualidade do modelo depende também do número de variáveis selecionadas, é importante desenvolver métodos que identifiquem aqueles que explicam o máximo possível da variabilidade dos dados. O método de otimização Colônia de Formigas (ACO) aparece como uma ferramenta bastante útil na seleção de variáveis, podendo-se encontrar muitas variações desse algoritmo na literatura. O propósito deste trabalho é desenvolver métodos de seleção de variáveis com base no algoritmo ACO, conceitos estatísticos e testes de hipóteses. Para isso, diversos critérios de decisão foram implementados nas etapas do algoritmo referentes à atualização de trilha de feromônios (C1) e à seleção de modelos (C2). A fim de estudar estas modificações, foram realizados dois estudos de caso: o primeiro na área de bioprocessos e o segundo na área de caracterização de alimentos. Ambos os estudos mostraram que, em geral, os modelos com menores erros são obtidos utilizando-se métricas dos componentes do modelo, tal como o tamanho do intervalo de confiança de cada parâmetro e o teste-t de hipóteses. Além disso, a modificação do critério de seleção de modelos parece não interferir significativamente no resultado final do algoritmo. Por último, foi feito um estudo da aplicação dessas versões do ACO no campo de caracterização de combustíveis, mais especificamente diesel, associando-se duas análises espectroscópicas para predição do conteúdo de enxofre. Algumas das versões desenvolvidas mostraram-se superior ao algoritmo ACO utilizado como base para este trabalho, proposto por Ranzan (2014), e todas os versões forneceram melhores resultados na quantificação de enxofre que aqueles obtidos por PCR. Dessa forma, comprova-se a potencialidade de métricas implementadas no algoritmo ACO, associadas à espectroscopia, na seleção de preditores significativos.
The development and improvement of optimization methods are points of deep interest in all areas of research. These techniques are often related to the acquisition of new or better control methods, which are directly attached to two importante tasks: choosing efficient forms of process monitoring and obtaining reliable models for the monitored variable from experimental data. Due to their several advantagens, optical sensors are being widely applied in the first task. Since several types of spectroscopy are possible through this type of sensor, models capable of dealing with spectral data are becoming increasingly attractive. The second task depends not only on which predictors are used in the model, but also on how many. Since the quality of the model depends on the number of selected variables, it is important to develop methods that identify those that explain the greater amount of data variability as possible, without compromising the reliability of the model. The Ant Colony Optimization is an important tool for variable selection, being possible to find a lot of variations of this method in literature. The purpose of this work is to develop a method of variable selection based on the Ant Colony Optimization (ACO) algorithm, statistical concepts and hypothesis testing. For this purpose, several decision criteria for trail update (C1) and model selection (C2) were implemented within the routine. In order to study these modifications, two case study was conducted: one related to bioprocess monitoring and another one envolving the characterization of food products. Both studies showed that, in general, the models with the lowest errors were obtained through the use of model component metrics, such as the length of the confidence interval associated with each parameter and the t hypothesis test. Besides, the modification of the model selection criterion doesn’t seem to affect the algorithm final result. Finally, the aplicattion of these methods in the field of fuels characterization, specifically diesel fuel, was studied, associating two spectroscopical analyses in order to predict the sulfur content. Some of the new developed methods appeared to be better than the ACO algorithm used as basis in this work, proposed by Ranzan (2014), and all methods showed better results than those from the models constructed by PCR. Thus, it is proved the high potencial of using different metrics within ACO algorithm, associated with spectroscopy, in order to select significative predictors.

We present the synthesis and characterisation of novel single organic molecules known as phenoxylene borazines and borazatruxenes. Using temperature-dependant and concentration-dependant 1H NMR, we probe the supramolecular aggregation of these molecules in solution. Finally, we synthesise 2D hybrid material comprised of electron delocalised benzene rings and electron localised borazine rings. Using a combination of solid-state 11B and 13C NMR techniques, Raman spectroscopy and XPS, we confirm the presence of benzene and borazine regions in these novel materials.

Up-conversion (UC) in rare-earth co-doped fluorides to convert diode laser light in the near infrared to red, green and blue visible light is applied to make possible high performance emissive displays. The infrared-to-visible UC in the materials we study is a sequential form of non-linear two photon absorption in which a strong absorbing constituent absorbs two low energy photons and transfers this energy to another constituent which emits visible light. Some of the UC emitters' most appealing characteristics for displays are: a wide color gamut with very saturated colors, very high brightness operation without damage to the emitters, long lifetimes and efficiencies comparable to those of existing technologies. Other advantages include simplicity of fabrication, versatility of operating modes, and the potential for greatly reduced display weight and depth. Thanks to recent advances in material science and diode laser technology at the excitation wavelength, UC selected materials can be very efficient visible emitters. However, optimal UC efficiencies strongly depend on chosing proper operating conditions. In this thesis, we studied the conditions required for optimization. We demonstrated that high efficiency UC depends on high pump irradiance, low temperature and low scattering. With this understanding we can predict how to optimally use UC emitters in a wide range of applications. In particular, we showed how our very efficient UC emitters can be applied to make full color displays and very efficient white light sources.
Ph.D.
Other
Optics and Photonics
Optics

Le travail de cette thèse porte sur l'amélioration des techniques de microscopies optiques 2D et 3D. Dans une première partie des travaux, j'ai proposé une technique d'illumination structurée focalisée et des raitements numériques spécifiques permettant un gain de la résolution latérale d'un facteur deux en microscopie confocale de fluorescence. La deuxième partie des travaux a consisté à réaliser un montage expérimental de microscopie tomographique optique diffractive qui permet, après une étape de reconstruction numérique, l'imagerie de spécimen transparents en trois imensions. La caractérisation du montage expérimental a montré que cette technique pennettait d'obtenir une information jusqu'alors inaccessible : la cartographie des indices optiques au sein du spécimen observé. En outre un gain en résolution vis à vis des techniques de microscopie plus classiques a été obtenu
This work concerns the improvement of 2D and 3D optical microscopy techniques. In a first part, I propose a focused structured illumination technique with specific numérical processing allowing an improvement of a factor two of the lateral resolution in confocal fluorescence microscopy. The second part of work consisted in carrying an experimental diffractive optical tomographic microscopy set-up. This one allows, after a stage of numerical processing of the images, to image 3D transparent specimens. The characterization of the experimental set-up showed that this technique allowed to obtain information not accessible before : the cartography of the optical indices within the specimen observed. Moreover a improvement of the resolution with respect to the more traditional techniques of microscopy was obtained

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Universidade Estadual Paulista (UNESP)
O presente trabalho teve como objetivo investigar a presença de manganês e zinco em “spots” protéicos de amostras de plasma de tilápia do Nilo (Oreochromis niloticus) obtidos após separação das proteínas por eletroforese em gel de poliacrilamida em segunda dimensão (2D-PAGE) para posterior avaliação qualitativa e quantitativa utilizando fluorescência de raios-X com radiação síncrotron (SR-XRF) e espectrometria de absorção atômica em chama e em forno de grafite (FAAS/GFAAS). As análises dos espectros de fluorescência indicaram a presença de manganês e zinco em quatro e seis “spots” protéicos de plasma, respectivamente. Observou-se que os íons metálicos estão ligados em proteínas com massa molar na faixa de 19 a 70 kDa e com pI na faixa de 4,7 a 6,30. A concentração de manganês e zinco ligados a essas proteínas foi determinada por GFAAS após a mineralização ácida dos spots protéicos, encontrandose concentrações na faixa de 3,40 a 4,20 mg g-1 e 2,30 a 13,90 mg g-1, respectivamente
The aim of the present study was to investigate the presence of manganese and zinc in protein spots in samples of Nile tilapia (Oreochromis niloticus) plasma obtained after protein separation by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and subsequent qualitative and quantitative determination by synchrotron radiation X-ray fluorescence (SRXRF) and graphite furnace atomic absorption spectrometry (GFAAS). An analysis of the fluorescence spectra indicated the presence of manganese and zinc in four and six plasma protein spots, respectively. It was observed that the metal ions are bound in proteins with molecular weight ranging from 19 to 70 kDa and pI ranging from 4.7 to 6.30. The manganese and zinc concentrations bound to these proteins were determined by GFAAS after acid digestion of protein spots, finding concentrations ranging from 3.40 to 4.20 mg g-1 and 2.30 to 13.90 mg g - 1, respectively

Orientador: Pedro de Magalhães Padilha
Banca: Gustavo Rocha de Castro
Banca: Eduardo José de Arruda
Resumo: O presente trabalho teve como objetivo investigar a presença de manganês e zinco em "spots" protéicos de amostras de plasma de tilápia do Nilo (Oreochromis niloticus) obtidos após separação das proteínas por eletroforese em gel de poliacrilamida em segunda dimensão (2D-PAGE) para posterior avaliação qualitativa e quantitativa utilizando fluorescência de raios-X com radiação síncrotron (SR-XRF) e espectrometria de absorção atômica em chama e em forno de grafite (FAAS/GFAAS). As análises dos espectros de fluorescência indicaram a presença de manganês e zinco em quatro e seis "spots" protéicos de plasma, respectivamente. Observou-se que os íons metálicos estão ligados em proteínas com massa molar na faixa de 19 a 70 kDa e com pI na faixa de 4,7 a 6,30. A concentração de manganês e zinco ligados a essas proteínas foi determinada por GFAAS após a mineralização ácida dos spots protéicos, encontrandose concentrações na faixa de 3,40 a 4,20 mg g-1 e 2,30 a 13,90 mg g-1, respectivamente
Abstract: The aim of the present study was to investigate the presence of manganese and zinc in protein spots in samples of Nile tilapia (Oreochromis niloticus) plasma obtained after protein separation by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and subsequent qualitative and quantitative determination by synchrotron radiation X-ray fluorescence (SRXRF) and graphite furnace atomic absorption spectrometry (GFAAS). An analysis of the fluorescence spectra indicated the presence of manganese and zinc in four and six plasma protein spots, respectively. It was observed that the metal ions are bound in proteins with molecular weight ranging from 19 to 70 kDa and pI ranging from 4.7 to 6.30. The manganese and zinc concentrations bound to these proteins were determined by GFAAS after acid digestion of protein spots, finding concentrations ranging from 3.40 to 4.20 mg g-1 and 2.30 to 13.90 mg g - 1, respectively
Mestre

碩士
國立臺灣大學
生化科學研究所
106
Alzheimer’s disease (AD) is a slowly progressing neurodegenerative disease and the most common type of dementia. The accumulation of amyloid-β (Aβ) in brain is generally considered to be the major culprit of AD. Aβ is a catabolic product of amyloid precursor protein (APP), generated in the amyloidogenic pathway by β- and γ-secretase cleavage. After γ-secretase cleavage, Aβ peptides will be released into extracellular spaces and aggregate into oligomers and fibrils. Despite numerous researches of fibrillogenesis mechanism, there are very few studies exploring how Aβ peptides are released from membrane and associate together after the γ-secretase cleavage. In addition, it has been reported that membrane fluidity, which is affected by lipid composition, can influence Aβ generation and aggregation. Whether the dynamic behavior of Aβ peptides in the membrane is affected by lipid composition or not is rarely discussed. In this study, we aimed to develop a method to observe the dynamic behavior of Aβ peptide in membrane with fluorescence correlation spectroscopy (FCS). We have successfully synthesized the fluorophore-labeled peptide corresponding to APP transmembrane region, as well as the one with a photolabile linker to mimic the γ-secretase cleavage on APP. We have also built a method to prepare peptide-inserted liposomes and lipid bilayers, and preliminarily set up a suitable condition for FCS measurements. This system, combined with the fluorescent photolabile linker introduced peptide we produced, can allow us to further investigate the movement of N-terminus of APP after photolysis in membrane with different lipid composition.

Part of the definition given to the new emerging science discipline of tissue engineering is the understanding of the structure-function relationships at the cellular level. In this context it is important for vascular tissue engineering to understand the mechanisms involved in the vascular cell responses to their mechanically active environment. This work has elucidated some aspects of the complicated puzzle of mechanotransduction in vascular smooth muscle cells (SMC) and endothelial cells (EC). Two dimensional timelapse fluorescence microscopy revealed rapid alkalinization occurring in cultured human aortic SMC exposed to well defined fluid flow profiles. The response was reversible and persisted for at least 20 min after flow initiation. The magnitude of the alkalinization (0.14 pH units) was enough to increase the nitric oxide synthase activity and account at least in part for the flow-induced increases in NO production by SMC. Use of specific inhibitors demonstrated the involvement of the Na$\sp+$/H$\sp+$ exchanger in the flow-induced response, whereas the Cl$\sp-$/HCO$\sb3\sp-$ exchanger was active even under stationary conditions. The involvement of calcium as a second messenger in the EC flow-induced mechanotransduction and the localization of possible signals within the cell was addressed by a three dimensional fluorescence microscopy technique. After 5 min of flow initiation there was a significant calcium increase in the nuclear region. The response was cytoskeleton independent. The same technique revealed early flow-induced changes in the three dimensional EC architecture. Nuclear and whole cell heights were reduced by about 1 $\mu$m with a corresponding increase in the cross-sectional area at lower optical sections. Using specific cytoskeleton disrupters we demonstrated that the whole cell height response was cytoskeleton independent and the nuclear height response was microtubule dependent. Thus, it has been shown that force imposed on the EC membrane is rapidly transmitted by microtubules to the endothelial nucleus. A mechanical equivalent model is presented to explain the cytoskeleton involvement in flow-induced structural changes based on tensegrity arguments. The early responses in the nuclear calcium and structure demonstrated in this study may be important for the shear-induced gene regulation.

My Ph.D. work was aimed at developing new methodologies based on combination of traditional spectroscopic techniques with multivariate analysis tools to analyze binding media used in traditional easel European paintings. My dissertation explored the possibility of fully characterizing traditional neat binding media and complex systems such as oil-paint models, applying Principal Component Analysis (PCA) on combined first derivative Fourier Transform Near Infrared (FT-NIR) and micro-Raman spectra. Furthermore, I also carried out a preliminary study to evaluate potentialities and limits of the 2D-Fluorescence spectroscopy, applied in combination with PCA to study and characterize neat binding media. This work was performed within the framework of the national project “SICH PRIN 2010-11 - Sustainability in Cultural Heritage from diagnosis to the development of innovative systems for consolidation, cleaning and protection” that was funded by the Italian Ministry of University and Research.

Die Demenz mit Lewy-Körperchen (DLB) ist eine progrediente neurodegenerative Erkrankung und stellt nach der Alzheimer-Erkrankung eine der häufigsten Ursachen einer Demenz dar. Betroffene leiden neben dem zentralen Merkmal Demenz an Fluktuationen der Kognition, Parkinsonismus und visuellen Halluzinationen. Charakteristische neuropatholgische Kennzeichen der DLB sind α-Synuklein-enthaltende Lewy-Körperchen und -Neuriten, die sich in kortikalen und subkortikalen Hirnregionen finden. Bei der klinischen Diagnostik dieser Erkrankung sind neben der Beurteilung klinischer Befunde laborchemische, psychometrische, apparative und bildgebende Verfahren von Bedeutung, jedoch ist eine sichere Diagnose nur bioptisch zu stellen. Gegenstand dieser Arbeit war die Untersuchung des Liquorproteomprofils von DLB-Patienten im Vergleich zu neurologisch gesunden Kontrollen und die Identifikation von regulierten Proteinen im Liquor bei der DLB durch Verwendung klassischer Methoden der Proteomik. Nach initialer Depletion von zwölf häufigen Proteinen wurden die Liquorproben mittels zweidimensionaler Gelektrophorese aufgetrennt, die Proteinexpressionsmuster quantitativ verglichen und anschließend insgesamt 23 verschiedene Proteine aus 44 regulierten Gelspots massenspektrometrisch identifiziert. Es fanden sich Proteine involviert in die Immunantwort, den Lipidstoffwechsel, den Glukosestoffwechsel, die Signaltransduktion und die Zellstruktur sowie einige, die sich keiner dieser funktionellen Gruppen zuordnen ließen. Von vier ausgewählten Proteinen - Complement C4a, Transthyretin, Contactin-1 und Chromogranin A - wurden Western Blots angefertigt, wofür Liquor sowohl von DLB-Patienten und gesunden Kontrollen als auch zum weiterführenden Vergleich von Parkinson- und Alzheimer-Patienten verwendet wurde. Die Ergebnisse dieser Arbeit zeigen auf Proteinebene die Vielfalt der biologischen Prozesse, die bei der DLB gestört ist. Zum Teil lassen sich Parallelen zu anderen neurogenerativen Erkrankungen erkennen, einige Proteine konnten jedoch erstmalig und einzig als bei der DLB reguliert nachgewiesen werden.

The Energy Dispersive X-ray Fluorescence (EDXRF) analysis technique presents unique features for elemental content evaluation of varied samples, such as its non-destructive nature, high sensitivity, or reduced costs. Many EDXRF studies have been focused on the determination of elemental concentration variations between normal and tumour tissues, to compare and correlate findings with factors such as the type and the stage of tumours, or the patients’ age and sex. Mapping elemental distributions may also provide insightful information on these types of samples, namely the identification of regions of elemental accumulation or depletion, and the demarcation of tumour margins, thus contributing to the unbiased differentiation between normal and abnormal tissues. The main goals of this work are to quantitatively determine the elemental content in paired samples of normal and tumour human tissues, and to map the corresponding elemental distributions. An improved methodology for the quantitative analysis of biological tissues is introduced, to surpass existing limitations (e.g., reduced number of tissue samples) that have lead to low-accuracy quantifications and statistically irrelevant conclusions. Moreover, a Full-field Energy Dispersive X-ray Fluorescence (FF-XRF) imaging spectrometer based on the 2D Thick-COBRA (2D-THCOBRA) micropattern gas detector is presented as a novel, cost-effective tool of simple instrumentation, for detection and mapping of elements in biological tissues. Different sample sets of normal and tumour human tissues were analysed, and it was possible to quantify concentration variations of light (e.g., K) and heavy elements (e.g., Fe and Zn). However, the contribution of these results to cancer studies is minimal due to the reduced number of analysed samples. To overcome this limitation, mirror samples of "fresh" and paraffin embedded tissues were analysed, and it was verified that the embedding process alters the elemental content of tissues. As such, the monitoring of the elemental content of tissues throughout the process is suggested. The FF-XRF imaging spectrometer based on the 2D-THCOBRA detector was used to map paired samples of normal and tumour human tissues. Elements present in small amounts (a few μg/g) were detected and the corresponding distributions were mapped. Overall, the detected elements are evenly distributed on the samples’ surface, with the exception of Ca in the lung tumour tissue. Throughout the analysed sample, calcium deposits, a possible malignancy indicator, were identified. The imaging spectrometer was also applied in the study of Zebrafish exposed to Mn, Se, and Pb. Heavy metal accumulation in the intestinal region was identified in all the analysed fish. Despite the suitability of the system for these studies, the need to improve detection efficiency remains. As such, the iFluX imaging spectrometer, with a large area X-ray panel based on a redesigned 2D-THCOBRA structure, is introduced.
As técnicas de análise por Fluorescência de Raios-X Dispersiva em Energia (EDXRF) apresentam características únicas para a avaliação do conteúdo elemental de diversas amostras, tais como o seu carácter não destrutivo, elevada sensibilidade ou custo reduzido. Muitos estudos têm-se focado na determinação de variações da concentração elemental entre tecidos normais e tumorais, para comparar e correlacionar resultados com diversos factores, como o tipo e estádio de tumores ou a idade e sexo dos doentes. O mapeamento da distribuição elemental poderá também providenciar informações importantes sobre este tipo de amostras, que contribuam para a diferenciação entre tecido com e sem patologia, através da identificação de regiões de acumulação ou depleção de elementos e da demarcação de margens tumorais. Os principais objectivos deste trabalho são a determinação da concentração elemental em amostras pareadas de tecidos com e sem patologia tumoral, e o mapeamento da distribuição elemental correspondente. Para tal, é introduzida uma metodologia melhorada para a quantificação elemental em tecidos biológicos, que permite ultrapassar as limitações (e.g., o número reduzido de amostras estudadas) que têm levado à obtenção de resultados pouco precisos e estatisticamente irrelevantes. Apresenta-se também um sistema de imagem full-field EDXRF (FF-XRF) baseado no detector gasoso micro-estruturado 2D Thick-COBRA (2D-THCOBRA), como uma alternativa inovadora, de instrumentação simples e custo reduzido, para detecção e mapeamento elemental em amostras biológicas. Foram analisados diferentes conjuntos de amostras de tecidos com e sem patologia tumoral, tendo sido possível quantificar a variação de elementos leves (e.g., K) e pesados (e.g., Fe e Zn). No entanto, a contribuição destes resultados para estudos de patologias tumorais é mínima devido ao número reduzido de amostras. De modo a ultrapassar esta limitação, foram analisadas amostras espelhadas de tecidos "frescos" e preservados em parafina. Verificou-se que este processo de preservação altera o contéudo elemental dos tecidos, propondo-se a sua monitorização em cada estágio. O sistema de imagem FF-XRF baseado no detector 2D-THCOBRA foi utilizado no mapeamento de amostras pareadas de tecido normal e tumoral. Foram detectados elementos presentes em quantidades reduzidas (alguns μg/g) e a sua distribuição foi mapeada. Verificou-se a distribuição homogénea de todos os elementos, à excepção de Ca em tecido tumoral do pulmão; e foram identificados aglomerados de cálcio que poderão ser uma característica de tumores malignos. O sistema FF-XRF foi também aplicado no mapeamento de peixes Zebra expostos a metais pesados, tendo sido verificada a acumulação de Mn, Se e Pb na zona intestinal dos peixes analisados. Apesar da adequação do sistema para este tipo de aplicações, permanece a necessidade de aumentar a eficiência de detecção. Neste sentido, é introduzido o sistema de imagem full-field iFluX, com um painel detector de raios-X de grande área baseado numa estrutura 2D-THCOBRA redesenhada.