Made available in DSpace on 2018-08-01T21:58:51Z (GMT). No. of bitstreams: 1 tese_9982_Dissertação_Radigya (1).pdf: 1760976 bytes, checksum: 51ceba8c31d07e4fd2e5e4d40c305358 (MD5) Previous issue date: 2016-03-04 / O Brasil é o maior produtor e segundo consumidor mundial de café, sendo o Espírito Santo um dos maiores pólos de produção nacional deste produto. O café é uma fruta de grande importância para o PIB do país e a presença de adulterantes infringe o direito do consumidor, causando-lhe prejuízos e possíveis danos à saúde. Desta maneira, desenvolver um método analítico para determinar possíveis adulterações, agregaria valor de commodity, fortalecendo o crescimento econômico do país. Amostras de café torrados Conilon e Arábica foram analisadas através da técnica de Espectrometria de Massas por Ressonância Ciclotrônica de Íons com Transformada de Fourier (ESI(-)FT-ICR MS) e Espectroscopia na região do infravermelho médio com refletância total atenuada (ATR-FTIR) com o objetivo de desenvolver um método de quantificação de café Conilon em blendas de café Arábica, utilizando modelos de análise de dados uni e multivariados. Para construção do modelo de calibração univariada por ESI(-)FT-ICR MS, foram usadas 10 amostras de café Arábica (Coffea Arábica) adulteradas com diferentes proporções de café Conilon (Coffea Canephora), e 2 amostras puras (0% e 100 wt% de café Conilon) dopadas com um padrão interno de ácido esteárico de concentração 5.10-3 mol.L-1 (m/z 283 e 567). Para a análise de ATR-FTIR foram usadas uma maior variabilidade de adulterações, totalizando 21 amostras de blendas e 2 amostras puras (0% e 100 wt% de café Conilon), a partir dos resultados foi proposto um modelo de regressão por mínimos quadrados parciais (PLS). A partir dos resultados de ESI(-)FT-ICR foi possível identificar as principais espécies químicas presentes nas amostras de cafés, tais como: ácido quínico (m/z 191), ácido cafeoilquínico (m/z 353) e ácido feruloilquínico (m/z 367). O modelo de calibração univariada desenvolvido apresentou boa linearidade (R2 de 0,976) e limites de detecção e de quantificação de 0.2 e 0.3 wt%, respectivamente. O modelo PLS construído com dados ATR-FTIR resultou em um RMSECV de 5.7 wt% e RMSEP de 9.2 wt%, com coeficientes de determinação para validação cruzada e previsão de 0.9635 e 0.9295, respectivamente. Foram encontrados valores de repetibilidade e precisão intermediária de 4 wt% e 5 wt% para ESI(-)FT-ICR MS respectivamente, e 1,7 wt% para ambas as figuras de mérito nas análises de ATR-FTIR.

Orientador: Marcos Nogueira Eberlin / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-07T11:19:58Z (GMT). No. of bitstreams: 1 Marques_LygiadeAzevedo_M.pdf: 2432568 bytes, checksum: 7bf6003b0fa0df13e1ba0f91d7ebf00b (MD5) Previous issue date: 2006 / Resumo: Neste trabalho aplicamos técnicas avançadas de espectrometria de massas, (MALDI-TOF e ESI-MS) na análise de micotoxinas em alimentos e na tipificação e verificação de fraudes em perfumes. Aplicamos a técnica MALDI-TOF em análises de micotoxinas, e esta mostrou excelente desempenho nas análises de aflatoxinas e ocratoxina e vantagem sobre a técnica de escolha atual, o método ELISA. Esta vantagem é principalmente maior especificidade através de maior exatidão em medidas de massas e, portanto, maior confiabilidade. O Planejamento de experimento foi uma ferramenta valiosa para obtenção das melhores condições e estudo dos parâmetros de interferência. O limite de detecção encontrado para a técnica foi da ordem de 25 pg para aflatoxinas e de 1 ng para ocratoxina, com perspectiva de melhoria através de aumento da massa amostral em estudos futuros para adaptação da metodologia de extração na matriz de interesse à técnica MALDI-TOF. A técnica ESI-MS foi utilizada para a tipificação e detecção de perfumes proporcionando, através da análise de componentes principais (PCA), a diferenciação com segurança entre perfumes originais, falsos e inspirados, utilizando como indicadores componentes polares não majoritários característicos de cada categoria avaliada. Este estudo abre caminho para que esta técnica seja utilizada na avaliação de perfumes que estão sob suspeita de falsificação com auxilio de uma biblioteca de "fingerprint" de perfumes por ESI-MS. O emprego da técnica de MALDI-TOF também é uma opção vantajosa para o monitoramento da qualidade de grãos quanto a presença de toxinas indesejáveis, bem como ameaças de bioterrorismo. / Abstract: In this work we applied advanced mass spectrometry techniques (MALDI-TOF and ESI-MS) to micotoxin analysis in food and for the typification and detection of counterfeit perfumes. MALDI-TOF was applied to micotoxin analysis, which showed excellent performance for the analysis of aflatoxins and ochratoxin with advantage over the current technique of choice, the ELISA method. This advantage is mainly its greater specifity due the exactness of the measurements, therefore with higher reliability. The surface analysis was a valuable tool to attain the best conditions and study the interference of several parameters. The detection limit found for the technique was 25 pg for aflatoxins and 1 ng for ochratoxins, with perspective of improvement through increase of the sample mass in future studies for adaptation of the methodology of extration in the matrix of interest for the MALDI-TOF technique. The ESI-MS technique was used for typification and detection of counterfeit perfumes, providing, through principal component analysis (PCA), the characterization of original, counterfeit and inspired perfumes, using as minoritarian polar compounds as diagnostic ions of each perfume category evaluated. We envisage that the method can be used to establish a ESI-MS fingerprinting library of perfumes for comparison with those from samples under investigation, and that such a library could be updated constantly by the addition of ESI-MS of new perfumes even before they are commercially released. MALDI-TOF technique is also an advantageous option for the monitoring of crop quality relating to the presence of undesirable toxins, as well as bioterrorism threats by micotoxin poisoning. / Mestrado / Quimica Analitica / Mestre em Química

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

The possibility to orient a protein in space using an external electrical field was studied bymeans of molecular dynamics simulations. To model the possible conditions of an electrospray ionization (ESI) the protein Lysozyme in vacuum was considered under the influence ofdifferent field strengths. The simulations showed three distinct patterns: (1) the protein wasdenaturated when exposed to too strong electrical fields, above 1.5 V/nm; (2) the proteinoriented without being denaturated at field strengths between 0.5 V/nm and 1.5 V/nm (3) theprotein did not orient and did not denaturate if the strength of the field became to low, below0.5 V/nm. Our simulations show that the orientation of the protein in the fields correspondingto the second pattern takes place within time intervals from about 100 ps at 1.5 V/nm to about1 ns at 0.5 V/nm. We therefore predict, that there exists a window of field strengths, which issuitable for orientation of proteins in experimental studies without affecting their structure.The orientation of proteins potentially increases the amount of information that can beobtained from experiments such as single particle imaging. This study will therefore bebeneficial for the development of such modern techniques.

This study has been partly inspired by the fact that historical narratives on the evolution of the South African Electricity Supply Industry (ESI), have for the most part focused on the national vertically integrated utility, Eskom; with far less attention being paid to the role that the municipal electricity undertakings (MEU's) have, and continue to, play. Indeed, this is somewhat surprising if one considers that MEU's began operating more than 20 years before Eskom's 1923 formation; and perhaps this lack of focus on MEU's is compounded by Eskom's operational crisis from 2006 (threatening its ongoing viability), which has overshadowed the perilous situation that MEU's have found themselves in. The research thus has two objectives. The first is to provide a detailed historical account of the role of MEU's and their contribution to the country's ESI from their genesis; while demonstrating the linkages between Eskom, MEU's and the three tiers of government. The second then examines how from the formation of the Union (1910), two fundamental but diametrically opposing objectives continue to prevail: 1) An over-burdened, financially ‘self-sufficient', local government, whose limited scope to collect revenue means electricity surpluses must be maximised to cross-subsidise its operations; and, 2) A vertically integrated utility, mandated to generate electricity at the lowest unit price, so as to provide the energy intensive economy with a competitive advantage. These contradictions, which have endured for many decades, reached fever pitch in the last 20 years, contributing significantly to the demise of ESI reforms initiated in 2000 and abandoned in 2010. Simultaneously, they have worsened the crisis of local government, which is constitutionally mandated to deliver basic services to its constituents, whose failure to do so, in many instances now threatens national government legitimacy at the most fundamental level. Within this context, the research, (based on the premise that history and institutions matter), employs the theoretical framework of new institutionalism, as applied through the lens of historical institutionalism (HI). Here, application of HI's core tenets revolves around identifying and explaining the critical junctures which create path dependency and institutional lock-in, while also accounting for incremental change which undoubtedly exists over a 120-year period. However, the unjust social and economic history of the country, where political decisions (pre and post-apartheid) have had a disproportional impact on state entities, requires closer scrutiny. For this, a detailed conceptual framework is employed to disentangle the complex relationship that has developed between the three tiers of government and their respective interacting powers. Ultimately, in delivering a detailed historiography of municipal electricity supply, the research posits that the ESI requires deeper fundamental reform than envisaged in 2000; and that most importantly, this must take cognisance of the extent to which MEU's are embedded within local government. This, the research believes, will increase the likelihood of local government participation and acceptance; perhaps pointing to an as yet unexplored path forward out of the South African ESI's current quandary.

No description available.

No description available.

CE-ESI-MS has become a powerful analytical tool capable of simultaneous identification and quantification of many classes of biologically relevant molecules. For studies in metabolomics, CE-ESI-MS offers a unique platform which will allow for the systematic elucidation of unknown metabolites in complex matrices without the need for complex sample preparation steps required with other techniques. In this thesis, a novel theoretical prediction model which will allow the estimation of detector response in ESIMS is outlined. This response model will allow researchers to quantitatively predict relative ionization effiency of compounds based on proposed two-dimensional structures without the need for a purified standard. Another feature of this model is that it can be applied to complex biological samples without the need for off-line sample pretreatment. Also in this thesis, a novel neonatal screening method will be presented which will aid clinical chemists in determining the presence of inborn metabolic disorders. This screening method which aims to compliment current protocols will allow health care professionals to further assess dried blood spot samples by providing simultaneous separation, identification, and quantification of relevant metabolites. This method also offers an alternatives to other protocols in place which are necessary to measure acid labile compounds which cannot be assessed by standard screening techniques. / Thesis / Master of Science (MSc)

A película adquirida (PA) é um filme formado pela adsorção seletiva de proteínas, glicoproteínas e lipídeos à superfície dentária. A presença de proteínas na PA forma uma interface protetora sobre a superfície do dente, participando em todos os eventos interfaciais que ocorrem na cavidade bucal, tais como des- e remineralização, lubrificação das superfícies dos dentes, e aderência bacteriana. Com o advento da proteômica, tem havido um aumento considerável no conhecimento acerca do perfil proteico de PAs adquiridas formadas sobre o esmalte dentário, em diferentes situações, mas nenhum trabalho até o momento descreveu o perfil proteômico de PAs formadas sobre a dentina. Este estudo foi pioneiro em comparar o perfil proteico de PAs formadas in situ sobre o esmalte e a dentina, nos tempos de 10 minutos e 2 horas, utilizando análise proteômica quantitativa livre de marcadores. Os experimentos foram realizados por três dias consecutivos. Em cada dia, os 9 voluntários receberam profilaxia dentária e em seguida utilizaram um aparelho vestibular com 6 blocos de esmalte e 6 de dentina humanos por 10 minutos ou 2 horas. Após esses períodos, a PA formada era coletada com auxílio de um papel filtro de eletrodos embebido em ácido cítrico 3%. Para as análises foi realizado um pool com os papéis dos 9 voluntários de todos os dias, para cada substrato e tempo de formação. Após a extração e digestão das proteínas, a separação dos peptídeos foi realizada por nano-HPLC (nano-Cromatografia Líquida de Alta Performace), interligada a um espectrômetro de massa (nLC-ESI-MS/MS). Os dados MS/MS obtidos foram processados e pesquisados em bancos de dados de proteínas humanas (UniProt e TrEMBL), utilizando o algoritmo SEQUEST no software Proteome Discoverer 1.3. Para a PA formada sobre o esmalte, foram identificadas 160 e 64 proteínas, nos tempos de formação de 10 minutos e 2 horas, respectivamente. Os respectivos números de proteínas identificadas para a dentina foram 86 e 52, respectivamente. Nos tempos de 10 minutos e 2 horas, respectivamente, 25 e 11 proteínas foram comuns a ambos os substratos e foram submetidas à quantificação livre de marcadores (SIEVE), revelando que a maioria das proteínas com diferença de expressão entre os dois substratos teve sua expressão aumentada na dentina. Foram identificadas ainda, no tempo de 10 minutos de formação da PA, 135 e 61 proteínas exclusivas ao esmalte ou à dentina, respectivamente. O número correspondente de proteínas exclusivas para o tempo de 2 horas foi de 53 e 41 proteínas, para o esmalte e dentina, respectivamente. Dentre as proteínas exclusivas da dentina, foram identificadas várias proteínas relacionadas ao complexo cálcio/calmodulina, assim como proteínas associadas à tumorigênese e à fosforilação/desfosforilação de proteínas. Em adição, muitas das proteínas identificadas no presente estudo, tanto para o esmalte quanto para a dentina, ainda não foram caracterizadas e, portanto, não têm função conhecida na PA. Sua caracterização e estudos funcionais futuros poderão trazer novos horizontes no entendimento da importância da PA para a proteção da estrutura dentária, bem como do papel da PA como sítio de biomarcadores para doenças bucais e sistêmicas. / The acquired pellicle (AP) is a film that results from selective adsorption of proteins, glicoproteins and lipids on the tooth surface. The presence of proteins in the AP forms a protective interface on the tooth surface that participates in all the surface events occurring in the oral cavity, such as de- and remineralization, lubrification of the tooth surfaces and bacterial adherence. With the advent of Proteomics, considerable increase in the knowledge of the protein profile of the AP formed on tooth enamel, under different circunstances, has been observed. However, so far the proteomic profile of the AP formed on dentin has not been described. This is the first study to compare the proteomic profile of APs formed in situ for 10 minutes and 2 hours, on enamel and dentin, using quantitative label-free proteomics. The experiments were conducted for 3 consecutive days. Each day, 9 volunteers were submitted to dental prophylaxis and in sequence wore a vestibular device containing 6 human enamel and 6 human dentin blocks for 10 minutes or 2 hours. After these periods, the PA formed was collected with an electrode filter paper soaked in 3% citric acid. The papers from the 9 volunteers, for each substrate and time of pellicle formation were pooled and used for analysis. After protein extraction and digestion, peptides were separated by nano-HPLC (High-performance liquid chromatography) coupled to a mass spectrometer (nLC-ESI- MS/MS). The obtained MS/MS spectra were searched against human protein databases (UniProt and TrEMBL) using SEQUEST algorithm in Proteome Discoverer 1.3 software. For the AP formed on enamel, 160 and 64 proteins were identified for the times of pellicle formation of 10 minutes and 2 hours, respectively. The respective numbers of identified proteins for dentin were 86 and 52, respectively. For the times of 10 minutes and 2 hours, respectively, 25 and 11 proteins were common to both substrates. They were submitted to label-free quantification, which revealed that most of the proteins with differential expression were overexpressed in the dentin. For APs formed for 10 minutes, 135 and 61 proteins were identified exclusively for enamel or dentin, respectively. The corresponding number for the 2-hour APs was 53 and 41 proteins, respectively. Among the proteins identified exclusively in dentin, many proteins related with calcium/calmodulin complex, as well as proteins associated with tumorigenesis and protein phosphorylation/dephosphorylation were found. In addition, many of the identified proteins, both for enamel and dentin, remain uncharacterized and, therefore have no described function in the AP. In the future, their characterization and functional studies might open new avenues for the understanding of the importance of the AP for the protection of the dental structure, as well as for the use of the AP as a site for biomarkers of oral and systemic diseases.

O arsênio é um dos mais tóxicos elementos químicos e reconhecidamente carcinogênico. Ele pode ser encontrado em alimentos basicamente em 5 formas: arsenobetaína (AsB), dimetil-arsênio (DMA), monometil-arsênio (MMA), arsenato (As5+) e arsenito (As3+), sendo estas duas últimas (As-i) as mais tóxicas. Assim, é de suma importância a utilização da especiação química de As para avaliação dos reais riscos associados à ingestão de alimentos contaminados. Neste sentido, o presente trabalho teve como objetivos o desenvolvimento de um método para separação das espécies de As por LC e detecção por ICP-MS; extrações quantitativas das espécies de As de tecidos animais e em grãos de arroz; aplicação dos métodos em amostras de alimentos consumidos no Brasil; e estudo do metabolismo do As em diferentes cultivares de arroz. O método desenvolvido para a extração das espécies de As em tecidos animais (ovo, músculos de ave, peixe e boi, etc.) utilizou apenas metanol (10%v/v) e ácido nítrico (2%v/v) como extratores e 2 minutos de sonicação, mostrando recuperação quantitativa do analito (>88%, n=3) pela análise dos materiais de referência (CE278, DOLT-3, DORM-3 e SRM NIST 1577). No entanto, para a análise de arroz, apenas ácido nítrico 2%v/v foi utilizado como extrator, possibilitando uma recuperação quantitativa (>94%, n=6), quando da análise do material de referência (NIST Rice Flour 1568a). Para a separação cromatográfica foram avaliadas diversas colunas, das quais a troca aniônica (Hamilton PRP-X100®) foi utilizada em todos os experimentos. A aplicação dos métodos na análise de alimentos diversos consumidos no Brasil mostrou uma grande variação nas concentrações das diferentes espécies do arsênio em músculos, ovos e, especialmente, no arroz. Devido as altas concentrações de As encontradas em amostras de arroz comercializadas no Brasil (em vários casos com predominância das espécies mais tóxicas, As3+ e As5+) e ao grande consumo deste alimento no país e no mundo, foi também realizado um estudo de metabolismo do As em 6 diferentes cultivares. O foco deste estudo foi a síntese de fitoquelatinas (PCs), compostos não peptídicos produzidos por plantas expostas a elementos tóxicos, e sua influência no fator de transferência (TF) do As solo-grãos. O TF do As, assim como a concentração das PCs, variaram em relação aos cultivares (genótipos) estudados. Além disso, as PCs mostraram ter uma forte correlação positiva entre si e com a concentração de As-i nos grãos, bem como uma correlação negativa do TF do As raízes-grãos. Portanto, os métodos desenvolvidos demonstraram fácil aplicação em rotina para avaliação toxicológica dos alimentos em relação às espécies de As e, finalmente, o estudo de metabolismo do As pela planta do arroz pode contribuir para escolha de cultivares que o absorvam menos, reduzindo sua ingestão frente ao consumo de arroz. / Arsenic is one of the most toxic chemicals and known carcinogen. It can be found in food basically in 5 different forms: arsenobetaine (AsB), dimethyl arsenic (DMA), monomethyl arsenic (MMA), arsenate (As5+) and arsenite (As3+), the latter two forms (i-As) are the most toxic. Thus, it is of extreme importance the use of chemical speciation for the evaluation of the real risks associated to arsenic intake from contaminated food. In this sense, this study aimed develop a simple method for separation of arsenic species by LC and detection by ICP-MS, quantitative extraction of arsenic species from animal tissues and rice grains, the application of the proposed method in the analysis of food samples commercialized in Brazilian markets, and the study of arsenic metabolism in different rice cultivars. The method developed for the extraction of arsenic species in animal tissues (egg, muscle of chicken, fish and cattle, etc.) used only methanol (10%v/v) and nitric acid (2%v/v) as extractant and 2 minutes of sonication, showing quantitative recovery of the analyte (>88%, n=3) when analyzing reference materials (CE278, DOLT-3, DORM-3 e SRM NIST 1577). However, for the analysis of rice grains, only nitric acid (2%v/v) was used as extractant, allowing a quantitative recovery (>94%, n=6), when analyzing the reference material (NIST Rice Flour 1568a). For the chromatographic separation several columns were evaluated and an anion exchange column (Hamilton PRP X-100) was used in all experiments. The analysis of several foods consumed in Brazil, showed a wide variation in the concentrations of different arsenic species in muscle, eggs, and especially in rice. Due to the high concentrations of arsenic found in rice samples (in several cases with predominance of more toxic species, As3+ and As5+) and the large consumption of this food in the country and abroad, it was also carried out a study on arsenic metabolism in 6 different rice cultivars. The focus of this study was the synthesis of phytochelatins (PCs), non-peptide compounds produced by plants exposed to toxic elements, and its influence on the transfer factor (TF) soil-grains. The TF of arsenic as well as the concentration of PCs varied in relation to the cultivars (genotype) studied. Furthermore, the PCs have shown a strong positive correlation between themselves and with the concentration of i-As in the grains, and a negative correlation with TF roots-grains. Therefore, the developed method demonstrated feasibility for routine use in toxicological studies of arsenic species in food samples and, finally, the study of arsenic metabolism in rice plant can contribute to the selection of cultivars that absorb less arsenic, thereby reducing the intake of this toxic element by rice consumption.