O trabalho apresenta procedimentos e resultados referentes à análise de solos, rejeitos ferrosos de origem mínero-metalúrgica, ligas ferrosas e minérios ferrosos por espectroscopia de fluorescência de raios X com dispersão de energia (EDXRF), com vistas especialmente à identificação e quantificação de metais pesados em ensaios realizados com equipamento portátil. Para solos e lamas, foram realizados estudos com e sem o recurso de adições de padrões internos. Em ambos os casos, foram ignorados os teores de elementos leves importantes como alumínio e silício. Sem a adição de padrões internos, não se obtiveram resultados absolutos, e os teores dos elementos claramente identificados eram expressos de modo relativo. Com a adição de padrões internos, foi possível expressar os teores daqueles elementos como valores absolutos. Para a análise de ligas ferrosas e minérios, utilizou-se o recurso da fluorescência secundária com alvo de ferro, para aumentar a excitação dos elementos mais leves que o ferro e diminuir a intensidade dos picos de escape da família Fe-K. Com este recurso, obteve-se uma elevada correlação entre as áreas dos picos destes elementos e seus teores nominais. Para determinação dos elementos mais pesados que o ferro, utilizou-se o feixe direto. Foi elaborada metodologia para conciliar os resultados obtidos com feixe direto e os obtidos com fluorescência secundária, obtendo-se uma boa aderência aos valores dos teores nominais. Complementarmente o trabalho apresenta uma forma alternativa de estimar erros quantitativos, baseando-se em simulações de espectros, e traz uma forma de visualizar o espectro sob o domínio da frequência para encontrar e remover ruído. / The work presents procedures and results concerning the analysis of soils, ferrous wastes of mineral-metallurgical origin, ferrous alloys and ferrous minerals by Energy-Dispersive X-ray Fluorescence spectroscopy (EDXRF), with a special focus on the identification and quantification of heavy metals in tests performed with portable equipment. For soils and sludges, studies were carried out with and without the use of additions of internal standards. In both cases, the contents of important light elements such as aluminum and silicon were ignored. Without the addition of internal standards, no absolute results were obtained, and the contents of the clearly identified elements were expressed in relative terms. With the addition of internal standards, it was possible to express the contents of these elements as absolute values. For the analysis of ferrous alloys and ores, the secondary fluorescence with iron target was used to increase the excitation of lighter elements than iron and to reduce the intensity of the escape peaks of the Fe-K family. With this feature, a high correlation was obtained between the areas of the peaks of these elements and their nominal contents. To determine the elements heavier than iron, the direct beam was used. A methodology was developed to reconcile the results obtained with direct beam and those obtained with secondary fluorescence, obtaining a good adhesion to the values of the nominal values. In addition, the work presents an alternative way of estimating quantitative errors, based on spectra simulations, and provides a way to visualize the spectrum under the frequency domain to find and remove noise.

Diabetes Mellitus (DM) é uma síndrome metabólica complexa, causada pela secreção diminuída ou ausente de insulina pelas células beta pancreáticas, levando a hiperglicemia. A hiperglicemia promove a glicação de proteínas e, conseqüentemente, o aparecimento de produtos finais da glicação avançada (AGEs). Atualmente, os pacientes diabéticos são monitorados pela determinação dos níveis de glicemia e hemoglobina glicada (HbA1c). As complicações geradas pela hiperglicemia podem ser divididas em micro e macrovasculares, representadas por retinopatias, nefropatias, neuropatias e doenças cardiovasculares. A albumina (HSA) é a proteína sérica mais abundante no organismo humano e está sujeita à glicação. A protoporfirina XI (PpIX) é a molécula precursora da síntese do heme, componente estrutural da hemoglobina. Ensaios in vitro e em animais indicaram que a hiperglicemia promove uma diminuição de sua concentração em eritrócitos. A espectroscopia de fluorescência é uma técnica bastante utilizada na área biomédica. A autofluorescência corresponde à fluorescência intrínseca presente em algumas moléculas, estando esta associada à estrutura das mesmas. O objetivo deste trabalho foi utilizar a técnica de espectroscopia de fluorescência para mensurar os níveis de autofluorescência da PpIX eritrocitária e AGE-HSA em pacientes diabéticos e indivíduos saudáveis e compará-los com os níveis de glicemia e HbA1c. Este estudo foi realizado com 151 indivíduos (58 controles e 93 diabéticos). Os dados epidemiológicos de pacientes e controles foram obtidos nos prontuários médicos. Para os indivíduos controle, os valores de glicemia foram adquiridos dos prontuários médicos e os níveis de Hb1Ac obtidos pela utilização de kits comerciais. A determinação da autofluorescência da PpIX foi realizada com excitação de 405 nm e emissão de 632 nm. Para a determinação do AGE-HSA foi realizada excitação de 370 nm e emissão de 455 nm. Aproximadamente 50% dos diabéticos apresentaram lesões micro ou macrovasculares decorrentes da hiperglicemia. Não foram observadas diferenças significativas nos valores de intensidade de emissão de PpIX entre os grupos estudados (P=0,89). Na análise do AGE-HSA observou-se diferenças significativas dos valores de intensidade de emissão entre os dois grupos, sendo este valor 1,45 vezes maior para o grupo de indivíduos diabéticos (P<0,0001). Os pacientes com complicações diabéticas apresentavam intensidade de emissão de fluorescência 1,19 vezes maior que os indivíduos sem complicações decorrentes da doença (P= 0,01), mesmo não havendo diferenças significativas nos valores de HbA1c entre os dois grupos. Concluímos que a espectroscopia de fluorescência foi uma técnica eficaz na identificação da autofluorescência da PpIX e do AGE-HSA. A PpIX não foi um biomarcador eficiente para o acompanhamento do DM. A determinação dos níveis de autofluorescência do AGE-HSA foi eficiente para a discriminação entre os grupos e para o monitoramento da progressão da doença, podendo ser mais eficiente que a dosagem de HbA1c. A espectroscopia de fluorescência é uma técnica simples, rápida e de baixo custo para o acompanhamento de indivíduos diabéticos. / Diabetes Mellitus (DM) comprises a complex metabolic syndrome, caused by reduced or absent secretion of insulin by pancreatic beta cells, leading to hyperglycemia. Hyperglycemia promotes glycation of proteins and, consequently, the appearance of advanced glycation end products (AGEs). Currently, diabetic patients are monitored by determining levels of glucose and glycated hemoglobin (HbA1c). The complications caused by hyperglycemia may be divided into micro and macrovascular complications, represented by retinopathy, nephropathy, neuropathy and cardiovascular disease. Albumin (HSA) is the most abundant serum protein in the human body and is subject to glycation. The Protoporphyrin IX (PpIX) is the precursor molecule of heme synthesis, structural component of hemoglobin. The in vitro and animals studies have indicated that hyperglycemia promotes a decrease in its concentration in erythrocytes. The fluorescence spectroscopy is a technique widely used in biomedical field. The autofluorescence corresponds to the intrinsic fluorescence present in some molecules, this being associated with the same structure. The aim of this study was to use fluorescence spectroscopy to measure levels of erythrocyte PpIX autofluorescence and AGE-HSA in diabetic and healthy subjects and compare them with levels of blood glucose and HbA1c. This study was conducted with 151 subjects (58 controls and 93 diabetics). Epidemiological data of patients and controls were obtained from medical records. For control subjects, blood glucose levels were obtained from medical records and levels of Hb1Ac obtained by using commercial kits. The determination of the PpIX autofluorescence was performed with excitation at 405 nm and emission at 632 nm. Determination of AGE-HSA was performed with excitation at 370 nm and emission at 455 nm. Approximately 50% of diabetic had micro and macrovascular lesions resulting from hyperglycemia. There were no significant differences in the PpIX emission intensity values between groups (P = 0.89). In the analysis of AGE-HSA was observed significant differences in the values of emission intensity between the two groups, and this value was 1.45-fold greater for the group of diabetic (P <0.0001). Patients with diabetic complications had fluorescence emission intensity of 1.19-fold higher than individuals without disease complications (P = 0.01), even with no significant differences in HbA1c values between the two groups. We conclude that fluorescence spectroscopy was an effective technique in the identification of the PpIX autofluorescence and AGE-HSA. The PpIX was not an effective biomarker for the monitoring of diabetes. The determination of AGE-HSA autofluorecência was efficient for the discrimination between groups and monitoring disease progression, may be more effective than HbA1c dosage. The fluorescence spectroscopy is a simple, fast and low cost for the monitoring of diabetic patients.

A espectroscopia fotoacústica e de fluorescência foram empregadas no estudo de reação da transferência de elétrons entre um doador (Octaetil-porfirina) dois tipos de aceitadores [Diclorodiciano-benzoquinona (DDQ) e Duroquinona (DQ)] em um meio rígido [poli (metil-metacrilato)] . Foram preparados filmes nos quais a concentração de doador foi mantida constante e a de aceitador foi variada. Observou-se um aumento exponencial da amplitude do sinal fotoacústico do doador em 620 nm, com o aumento da concentração de aceitadores nos filmes. O modelo de Perrin para a supressão de fluorescência foi aplicado aos dados de fotoacústica e o raio da esfera de ação, que representa a distância crítica para a ocorrência da transferência de elétrons, foi determinado. Os resultados obtidos foram: para DDQ37 angstron e para aDQ 32 a 34angstron. Era esperado um raio maior para a DDQ devido a sua maior elétron afinidade. Também foi aplicado a estes dados o modelo de Kaneko, desenvolvido para a supressão de fluorescência. Dele se obtém a distribuição de moléculas aceitadoras incorporadas na esfera de ação. Para uma mesma concentração de aceitadores (2,8mmoldm-3) , a probabilidade de encontrar uma molécula de DDQ na esfera de ação foi 27%, enquanto que a probabilidade de encontrar uma molécula de DQ na esferade ação foi de 20%. Este resultado é, na verdade, equivalente ao encontrado no modelo de Perrin pois a probabilidade maior encontrada para a DDQ vem do fato do raio da sua esfera de ação ser maior. O valor encontrado para o raio crítico com os dados de fluorescência foi de 34 A para a DQ. Isto demonstra que as duas técnicas são complementares e que a espectroscopia fotoacústica pode ser usada para monitorar o processo de transferência de elétrons / Steady-state photoacoustic and fluorescence spectroscopy were employed to study the electron-transfer reaction from donor molecules (Octaethyl-porphirin) to two types of acceptor molecules [Dichloro-dicyano-benzoquinone (DDQ) and Duroquinone (DQ)I in a rigid medium [poly (methyl -metacrylate)] . Films were prepared with a fixed donor concentration and severa1 acceptor concentrations. It was observed an exponential growth in the donor photoacoustic signal amplitude at 620 nm, with the increase in the acceptor concentration. Perrin model, used in fluorescence quenching, was applied to the photoacoustic data and the radius o£ an action sphere, which represents a critica1 distance for the electron transfer process, was determined. The results obtained were: for DDQ 37 angstron and for DQ from 32 to 34 angstron. It was expected a larger radius for DDQ than for DQ due to its stronger electron affinity. Kaneko\'s model, was also applied to these data. It provides the distribution o£ incorporated acceptor molecules in the action sphere. For the same acceptor concentration (2.8 mmol , the probability of finding one DDQ molecule in the action sphere was 27%, while for the DQ molecule this value was 20%. Actually, this result is equivalent to that found using Perrin model, because the larger probability obtained for DDQ comes frm the fact that its critical radius is bigger . The value obtained for the critica1 radius from the fluorescence data was 34 angstron for DQ. This shows that these two techniques are complementary and that photoacoustic spectroscopy can be used to monitor the electron transfer process

Sem resumo / Sem abstract

Este trabalho teve como objetivo estudar a transferência de elétrons em um meio rígido através de técnicas espectroscópicas convencionais, tais como a fluorescência e a fotoacústica, e verificar a possibilidade de se determinar a taxa de transferência através destas técnicas. Foram utilizados como doador de elétrons a tetrafenilporfirina (TPP), como aceitador de elétrons a duroquinona (DQ), e como meio rígido o polimetilmetacrilato (PMMA) e o poliestireno (PS). As amostras foram estudadas na forma de filmes, com uma distribuição aleatória de doadores e aceitadores de elétrons. Com a espectroscopia de fotoacústica não foi possível se detectar a ocorrência da transferência de elétrons. Com a fluorescência, usando o modelo de Perrin, foi possível determinar o raio crítico de transferência, 72k para o par TPP/DQ em poliestireno. No entanto, verificou-se a impossibilidade de se determinar univocamente a taxa de transferência de elétrons. Nos filmes preparados com polimetilmetacrilato foi observada uma fotodegradação irreversível, o que inviabilizou seus estudos / The objective of this work was to study the electron transfer process in a rigid medium using conventional spectroscopic techniques, such as fluorescence and photoacoustic, and verify the possibility of determining the electron transfer rate through these techniques. The molecules used were: tetraphenylporphyrin (TPP) as electron donor, duroquinone (DQ) as the electron acceptor, and polystyrene (PS) and polymethylmetacrilate (PMMA) as the rigid medium. The samples were studied in the form of films, with a random distribution of electron donor and acceptor molecules. With the photoacoustic spectroscopy it was not possible to detect the occurrence of the electron transfer process. With the fluorescence technique, using Perrin model, it was possible to determine the criticai radius for electron transfer, 7 À, for the par TPP/DQ in polystyrene. However, it was verified the impossibility of determining univocally the electron transfer rate. In the films made with polymethylmetacrilate, an irreversible photodegradation was observed, what made the study of these films non viable

A adição de corantes fluorescentes a adesivos odontológicos possibilita a investigação da distribuição espacial desses materiais na interface dente-restauração, utilizando-se a microscopia confocal de varredura a laser (MCVL). A literatura indica falta de padronização na aplicação de agentes fluorescentes com tal finalidade. Esse estudo sistematizou estratégias para a adição de rodamina B (RB) e fluoresceína sódica (FS) a um sistema adesivo convencional de três passos, Adper Scotchbond Multi-Purpose (MP), e um autocondicionante de dois passos, Clearfil SE Bond (SE), considerados padrão-ouro na Odontologia. Os objetivos principais foram (a) determinar a menor faixa de concentrações de RB e FS necessária para produzir imagens satisfatórias da interface dentina-adesivo e (b) avaliar o efeito da adição desses corantes sobre algumas propriedades das resinas. Os adesivos foram marcados com RB ou FS em concentrações decrescentes (0,5, 0,1, 0,02 e 0,004 mg/mL) por meio de um método de dispersão semidireto. O comportamento fotofísico/ fluorescente dos adesivos marcados foi investigado por espectroscopia de fotoluminescência e MCVL. Paralelamente, avaliaram-se os adesivos quanto ao grau de conversão (GC) e ao ângulo de contato (AC). Tanto os resultados de GC como os de AC foram submetidos à análise de variância com dois fatores (adesivo e tratamento) com = 0,05, seguida de teste post-hoc de Tukey. Os máximos comprimentos de onda de emissão e de excitação da RB e da FS foram influenciados pelo meio polimérico e pela concentração de corante de modo geral. A MCVL preliminar de amostras de adesivo polimerizado, realizada sob condições experimentais padronizadas, mostrou que o comportamento fluorescente da RB em MP e SE foi muito semelhante na mesma concentração de corante, mas o mesmo não pôde ser dito do comportamento da FS, que foi notavelmente inferior no adesivo autocondicionante, SE, na concentração mais alta. Em dentina, os adesivos preparados com RB nas concentrações-alvo de 0,1 e 0,02 mg/mL apresentaram fluorescência ótima; já aqueles preparados com 0,004 mg/mL produziram fraco sinal. Adesivos preparados com FS a 0,5 mg/mL apresentaram ótima fluorescência na interface de adesão, enquanto que concentração menor desse corante não produziu sinal suficiente. Padrões morfológicos aparentemente atípicos foram observados na interface de adesão, quando da associação do adesivo SE com o corante FS. A adição de RB e FS nas quatro concentrações indicadas aos adesivos MP e SE não afetou o GC nem o AC em comparação com os grupos de controle correspondentes. Em suma, a RB mostra-se um corante mais versátil que a FS na avaliação morfológica das interfaces dentina-MP e dentina-SE via MCVL. A menor faixa de concentrações de RB nos adesivos MP e SE, na qual é possível produzir imagens satisfatórias das interfaces, situa-se entre 0,10,02 mg/mL. Já o corante FS deve ser adicionado a esses adesivos a pelo menos 0,5 mg/mL para produzir níveis de fluorescência satisfatórios na interface de adesão. A não ocorrência de efeitos deletérios sobre a polimerização e a molhabilidade das resinas estabelece uma margem de segurança para a incorporação desses agentes fluorescentes (em concentração 0,5 mg/mL) nesses sistemas monoméricos. / The addition of fluorescent dyes to dental adhesives makes it possible to investigate the spatial distribution of such resin-based materials in the tooth-restoration interface, using confocal laser scanning microscopy (CLSM). Literature indicates a lack of standardization on the application of fluorescent agents for this purpose. This work presents strategies for adding rhodamine B (RB) and fluorescein sodium salt (FS) to a three-step etch-and-rinse adhesive system, Adper Scotchbond Multi-Purpose (MP), and a two-step self-etching one, Clearfil SE Bond (SE), both regarded as \"gold standard\" in restorative dentistry. The main objectives were (a) to determine the lowest range of RB and FS concentrations required to produce suitable images of the dentin-adhesive interface via CLSM and (b) to investigate potential effects of addition of these dyes on some resin properties. The adhesives were labeled with RB or FS at decreasing concentrations (0.5, 0.1, 0.02 and 0.004 mg/mL) by means of a semi-direct dispersion method. The photophysical/fluorescent behavior of the labeled resins was investigated by photoluminescence spectroscopy and by CLSM. The adhesives were also investigated with regards to the degree of conversion (DC) and contact angle (CA). A two-way ANOVA of adhesive and treatment was conducted on DC and CA separately, followed by Tukeys test. The maximum emission and excitation wavelengths of RB and FS were influenced by the host polymer and the dye concentration in general. The preliminary CLSM of cured adhesive samples, performed with standardized settings, showed that the fluorescent behavior of RB in MP and SE was very similar in the same dye concentration, unlike the behavior of FS, which was lower in the self-etching adhesive for the highest dye concentration. In dentin, the adhesives prepared with RB at the target concentrations of 0.1 and 0.02 mg/mL presented optimal fluorescence; those with 0.004 mg/mL produced poor signal. Adhesives prepared with FS at 0.5 mg/mL presented optimal fluorescence at the bonding interface, whereas lower concentrations of FS did not produce sufficient signal. Atypical morphological features were observed at the bonding interface, when adhesive SE was used with FS. The addition of RB and FS at the four decreasing concentrations to adhesives MP and SE did not affect DC or CA compared to the corresponding controls. In short, RB is more versatile than FS for the morphological characterization of dentin-MP and dentin-SE interfaces via MCVL. The lowest range of RB concentrations in adhesives MP and SE that can produce suitable images of the bonding interface lies between 0.10.02 mg/mL. The dye FS should be added to these adhesives at 0.5 mg/mL at least to produce satisfactory fluorescence levels at the bonding interface. Since negative effects on polymerization and wettability of the resins were not observed, the use of RB and FS (in concentration 0.5 mg/mL) together with MP and SE should be reliable in terms of resin properties.

Single step co-sintering is proposed as a method to minimise the time and cost of fabricating solid oxide fuel cells (SOFCs). Such a methodology is attractive but challenging due to the differing sintering behaviours and thermal mismatch of the constituent materials of the anode, cathode and electrolyte in solid oxide fuel cells. As a result it is likely that compromises are made for one layer with respect to optimising another. The single chamber solid oxide fuel cell (SC-SOFC) has not seen widespread adoption due to poor selectivity and fuel utilisation, but relaxed some of the stringent SOFC requirements such as sealing, and the need for a dense electrolyte layer. Thus, to initiate the study into single step co-sintering, the single chamber SOFC is earmarked as the first candidate. The effect of single step co-sintering on cell performance is also an attractive area to investigate. Therefore, in this study, a new co-sintering process (single step co-sintering) was applied to fabricate three different types (in terms of the supporting structure) of planar SC-SOFCSs (the anode, cathode and electrolyte supported planar cells) and anode supported wavy types of SC-SOFC in order to reduce fabrication cost and time owing to effective fabrication process. In addition, their performances were tested to establish functionality of the sintered specimens as working electrochemical cells as well as to investigate the maximum performance possible with these cells under single chamber conditions. Moreover, it is also aimed to improve the performance of SC-SOFCs by extending TPB (Triple phase boundary) via wavy type. This study presents a single step co-sintering manufacturing process of planar and wavy single chamber solid oxide fuel cells with porous multilayer structures, consisting of NiO-CGO, CGO and CGO-LSCF as anode, electrolyte and cathode respectively. Pressure of 2 MPa, with the temperature at 60˚C for 5 minutes, was deemed optimal for the hot pressing of these layers. The best result of sintering profile was obtained with heating rate of 1˚C min-1 to 500˚C, 2˚C min-1 to 900˚C and 1˚C min-1 to 1200˚C with 1 hour dwelling; the cooling rate was 3˚C min-1. Hence anode supported SC-SOFC (thickness: 200:40:40 µm, thickness ratio: 10:2:2, anode (A): electrolyte (E): cathode (C)) was fabricated via a single co-sintering process, albeit with curvature formation at edges. Its performance was investigated in methane-oxygen mixtures at a temperature of 600˚C. Maximum open circuit voltage (OCV) and power density of the anode supported planar cell were obtained as 0.69 V and 2.83 mW cm-2, respectively, at a fuel-oxygen ratio of 1. Subsequently, anode thickness was increased to 800 µm and electrolyte thickness was reduced 20 µm (thickness ratio of cell 40:1:2) to obtain curvature-free anode-supported SOFCs with the help of a porous alumina cover plate placed on the top of the cell. The highest power density and OCV obtained from this cell was 30.69 mW cm-2 and 0.71 V, respectively, at the same mix ratio. In addition, the maximum residual stresses between cathode end electrolyte layers of anode supported cells after sintering were investigated using the fluorescence spectroscopy technique. The total mean residual stresses along the x-direction of the final anode supported planar cell after sintering were measured to range from -488.688 MPa to -270.781 MPa. Determination of optimum thickness and thickness ratio of the cell with the defined ideal hot pressing and sintering conditions for single step co-sintering were carried out for cathode and electrolyte supported planar cells using similar fabrication processes. Their performance changes with thickness ratio were examined. The results show that the cathode and electrolyte supported planar cells can be obtained successfully via single step co-sintering technique with the help of alumina cover plates, as with the anode supported cell. In addition, an anode supported wavy SC-SOFC was fabricated via single step co-sintering and its performance was also investigated. The maximum power density and OCV from the final curvature free cathode supported planar cell (thickness: 60:20:800 µm, thickness ratio: 3:1:20, A:E:C) was measured to be 1.71 mW cm-2 and 0.20 V, respectively, at a fuel-oxygen ratio of 1.6. Likewise, the maximum OCV and power density were found to be 0.55 V and 29.39 mW cm-2, respectively, at a fuel-oxygen ratio of 2.6, for the final electrolyte supported curvature free planar cell (thickness: 60:300:40 µm, thickness ratio: 3:15:2, A:E:C). Furthermore, a maximum OCV of 0.43 V and power density of 29.7 mW cm-2 were found from the final anode supported wavy cell (thickness: 800:20:40 µm, thickness ratio: 40:1:2, A:E:C) at a fuel-oxygen ratio of 1. In essence, this study can be divided into five chapters. The first chapter addresses the overview of the research background, problem statement, aims and objective of this study as well as that of novelty and impact. In the second chapter, fundamental information is provided regarding SOFCs and SC-SOFCs in terms of working principles, main components including electrodes electrolytes, advantages and disadvantages, types, material used for each cell components, losses in the system, and so forth. Moreover, the second chapter also contains essential sintering information in order to understand how to approach sintering of ceramics or cermet to fabricate SC-SOFCs. The overall methodology of this study is explained in detail in the third chapter while experimental works are described in the chapter 4, chapter 5, chapter 6, chapter 7 and chapter 8. Chapter 5 also contains background for the fluorescence spectroscopy and a modelling technique for residual stress measurement between ceramic layers. The results of experiments with discussion session are also in the same chapter. The last chapter presents conclusions and the possible routes for future works of the study.

Acute lymphoblastic leukemia, one of the most malignant cancer forms in children is commonly treated with the thiopurine 6-mercaptopurine (6-MP) in combination with a high dose of methotrexate (MTX). 6-Mercaptopurine is in the body metabolized by the enzyme thiopurine S-methyltransferase (TPMT). Polymorphic variants of TPMT express different catalytic activities, and for this reason the dosage of 6-MP needs to be individualized. In order to better optimize the treatment it is important to understand how mutations in TPMT affect its enzymatic activity. In this thesis we have investigated how the wild type and two variants of TPMT interact with different ligands using fluorescence and isothermal titration calorimetry. Experiments with MTX, ANS and furosemide resulted in a similar binding strength for the wild type and the variant TPMT*8, while the other variant TPMT*6 showed a slightly weaker binding. A binding affinity for polyglutamated MTX to TPMTwt was also determined which resulted in an almost twice as strong binding compared to MTX. Today’s methods to determine enzymatic activity are either based on radioactivity, time consuming or expensive. As an alternative the use of a spectrophotometric assay using 5-thio-2-nitrobenzoic acid (TNB) was investigated. The method showed positive results and could hopefully be adapted to plate readers in future experiments. Using 5.5’-dithiobis-(2-nitrobenzoic acid) (DTNB, also known as Ellman’s reagent) the amount of accessible thiol groups on the protein was estimated. This revealed a similar relationship between TPMTwt and TPMT*6, while the result for TPMT*8 was inconclusive.

Waterborne disease is a significant contributor to the global burden of disease, in particular among high-risk populations in developing nations. State-of-the-art methods for the enumeration of microbial pathogens in drinking water sources have important limitations, including high initial cost, 24-48 hour delays in results, high staffing and facility requirements, and training requirements which all become especially problematic in the developing nation context. A number of alternative approaches to microbial water quality testing have been proposed, with the goal of decreasing the required testing time, decreasing overall costs, leveraging appropriate technology approaches, or improving sensitivity or specificity of the water quality testing method. One approach that may offer solutions to some of these limitations involves the deployment of sensor networks using fluorescent spectroscopy to detect intrinsic protein fluorescence in water samples as a proxy for microbial activity. In recent years, a number of researchers have found significant and meaningful correlations between indicator bacteria species and the protein fluorescence of drinking water samples. Additionally, advances in the semiconductor industry could be used to drive down the cost of such sensors. This technology may also be extensible to other water quality parameters, including dissolved organic matter or the presence of fluorescent pollutants. In this thesis, a literature review describes the fundamentals of fluorescence spectroscopy, historical and recent work regarding the fluorescence of the amino acid tryptophan and associated bacterial fluorescence, possible mechanisms for this association, and potential applications of this technology for drinking water quality monitoring and waste water process control. Extensibility of the technology is also discussed. Next, experimental methodology in reproduction of similar results is described. Samples were taken from seven (7) surface water sources and tested using membrane filtration and an off-the-shelf fluorescence spectrometer to help examine the association between the presence of indicator bacteria and the tryptophan fluorescence of the water sample. The results, showing an association of R2 = 0.560, are compared to the results of recent similar experiments. Finally, two prototypes are described, including their design requirements and data from prototype testing. The results of the testing are briefly discussed, and next steps are outlined with the goal of developing a low-cost, in-situ microbial water quality sensor using fluorescence spectroscopy principles.

Dissolved organic carbon (DOC) plays an important role in the global carbon (C) cycle because increases in aqueous C potentially contribute to rising atmospheric CO2 levels. Over the past few decades, headwater streams of the northern hemisphere have shown increased amounts of DOC coinciding with decreased acid deposition. Although the issue is widely discussed in the literature, a mechanistic link between precipitation composition and stream water DOC has not yet been proposed. In this study, the breakup of soil aggregates is hypothesized as the mechanistic link between reduced acid deposition and DOC increases in surface waters. Specific hypotheses state that soil aggregate dispersion (and the ensuing release of DOC from these aggregates) is driven by a decrease in soil solution ionic strength (IS, decreasing the tendency of flocculation) as well as a shift from divalent to monovalent cations (reducing the propensity for cation bridging) in soil solution. These hypotheses were tested on soil samples collected from several riparian zone and hillslope positions along three flagged transects in the acid-impacted Sleepers River Research Watershed in northeastern Vermont. To determine soil C content by landscape position, samples from transects spanning hilltop to hillslope and riparian area, as well as replicated hillslope and riparian samples (n=40) were analyzed. Aqueous soil extracts simulate the flushing of soils during hydrologic events (e.g. rain or snowmelt) and were used to test the effect of soil solution chemistry on DOC release. Extracts were prepared with solutions of varying IS (0-0.005M) and composition (CaCl2 and NaCl) on replicated soil samples (n=54) and changes in DOC release and aggregate size were monitored. As IS of the extraction solution increased, the amount of DOC in solution decreased, and aggregate size increased. This was presumably due to cations bridging and diffuse double layer effects. This effect was reversed in low ionic strength solutions where DOC release was significantly higher and average aggregate size was smaller. While extraction solution controlled the amount of C liberated, landscape position impacted the quality, but not quantity, of released DOC. This study is the first to propose a mechanistic link observed changes in DOC in surface waters and recovery from acidification and provides initial experimental evidence that soil aggregates indeed play a role in the generation of DOC.