Laser Ablation (imaging) For Mapping And Determining Se And S In Sunflower Leaves.

Qualitative and quantitative methods were developed for selenium and sulphur mapping/quantification in sunflower (Helianthus annuus L.) leaves. The plants were grown for 50 days in a greenhouse, being divided into two groups, one irrigated with only deionized water and the other treated with a daily dose of 7.58 mg of Na(2)SeO(3). Leaves were collected during the growth period for directly evaluating the distribution of Se and S in these structures. For quantification analysis, pellets were produced from both CRM (100 and 1575a for Se and S, respectively) and the sunflower materials. The pellets were doped with 25 and 1200 μg g(-1) Se and 5 and 20 mg g(-1) S and analyzed by LA-ICP-MS. For accuracy purposes all the samples were also decomposed via microwave and analyzed by ICP-MS. To avoid polyatomic interferences Se and S were monitored as SeO(+) and SO(+) at m/z 96 and 48, respectively, (12)C(+) was used as an internal standard, and the ratios between SeO(+)/C(+) and SO(+)/C(+) were used for measurements. Statistic tests (t test at 95% confidence level) confirming good agreement between LA-ICP-MS and ICP-MS indicated the accuracy of this technique.562-

Evaluation and characterization of cloud point based systems for albumin removal from blood plasma

Orientador: Marco Aurelio Zezzi ArrudaDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de QuimicaResumo: Nesta dissertação foi estudado o comportamento de partição da albumina, proteína presente em elevada concentração no plasma sangüíneo e que interfere na determinação em diversas técnicas analíticas. No estudo efetuado foram avaliados diferentes sistemas aquosos de duas fases, explorando um fenômeno denominado ponto nuvem. Tais sistemas empregam surfactantes, sob condições experimentais específicas, obtendo-se a formação de duas fases imiscíveis: uma rica e outra pobre em tensoativo. Observou-se que devido às características hidrofílicas da albumina, bem como à dimensão dos agregados formados, sua extração para fase rica em tensoativo apresenta valores de coeficiente de partição que não ultrapassam 0,66 o que representa uma eficiência de extração de ca.40%. A extração da proteína apresentou-se viável mediante a utilização de uma mistura composta por um tensoativo não-iônico (Triton® X-114), que possibilita a separação de fases em temperatura biocompatível, na presença de outro tensoativo iônico (dodecil sulfato de sódio - SDS), que atua favorecendo as interações eletrostáticas entre os agregados e as moléculas protéicas, desde que se trabalhe em meio com pH abaixo de 5,0. Análises de dicroísmo circular complementaram o estudo e fornecerem evidências de que a aplicação do sistema baseado na mistura composta por Triton® X-114 e SDS causava desnaturação parcial da proteína, o que não inviabilizou sua aplicação para extração da mesma em uma amostra real. A eficiência deste sistema foi, então, avaliada para a remoção de albumina em plasma sangüíneo. Um coeficiente de partição de 1,1 foi obtido, indicando que ca.51% das proteínas encontravam-se na fase rica em tensoativo. As amostras submetidas ao procedimento de extração também foram avaliadas frente à técnica de eletroforese em gel, sendo que a fase pobre em sufactante apresentou um perfil eletroforético mais detalhado quando comparada a uma amostra que não foi submetida ao procedimento proposto. Já na fase rica em surfactante, foi observada a presença majoritária de albumina e, em menor concentração, outras proteínas de grande abundância no plasma tais como imunoglobulina G e transferrina. Por fim, o método apresentou desempenho semelhante ao de sistemas disponíveis comercialmente para remoção de albumina, tal como o sistema da Millipore®, apresentado neste trabalhoAbstract: In this work the partition behavior of albumin was studied, which is found at high concentrations in blood plasma, which interferes in many analytical techniques determinations. The present study evaluated different aqueous two-phase systems, exploiting a phenomenon called of cloud point. These systems employ surfactants under specific experimental conditions, enabling formation of two immiscible phases: one rich and another poor in surfactant. Due to the hydrophilic characteristcs of albumin, as well as its aggregate dimensions, its extration to the surfactant rich phase presented partition coefficients lower than 0.66, representing and extraction efficiency of ca. 40%. Protein extraction was feasible by applying a mixture comprised of a nonionic surfactant (Triton® X-114), which allowed the phase separation at biocompatible temperatures, and an ionic one (sodium dodecylsufate - SDS), wich promotes eletrostatic interactions between aggregates and protein molecules, since the extraction procedure is carried out at pH 5.0. Circular dichroism analysis complemented the study and it showed that a system based on a Triton® X-114 and SDS mixture causes partial protein denaturation, but its application for a real sample is feasible. The efficiency of this was evaluated for albumin removal from blood plasma. A partition coefficient of 1.1 was obtained, indicating that ca. 51% of proteins were contained in the surfactant rich phase. Albumin depleted samples were submitted to gel eletrophoresis and the surfactant poor phase presented a more detailed gel electrophoresis profile, when compared with a crude sample. The surfactant rich phase reveled that albumin is the predominant protein present, but it is possible to find other highly concentrated plasmatic proteins including immunoglobulin G and transferrin. Finally, the method presented similar performance when compared with commercially available systems for albumin removal, such as the Millipore® system, wich was also evaluated in this workMestradoQuimica AnaliticaMestre em Químic

Evaluation of the effect of selenium and zinc in the metabolism of sunflowers through a metallomic study

Orientador: Marco Aurélio Zezzi ArrudaTese (doutorado) - Universidade Estadual de Campinas, Instituto de QuímicaResumo: Inúmeros trabalhos envolvendo uma nova área de estudos denominada metalômica destacam a necessidade de melhor compreender a interação entre íons metálicos e proteínas. Esta Tese empregou o girassol (Helianthus annuus L.), uma planta da família das oleaginosas que apresenta uma relevante importância econômica na produção de óleo vegetal, bem como em processos de fitorremediação, em um estudo metalômico para avaliação de possíveis alterações na expressão de proteínas mediante o desenvolvimento das plantas irrigadas com íons selenito ou zinco. Inicialmente, o estudo avaliou quatro diferentes métodos para a adequada para extração e separação de proteínas das folhas de girassol empregando a técnica de eletroforese bidimensional em gel de poliacrilamida (2DPAGE), sendo que o melhor procedimento consistiu na extração empregando tampão na presença de fenol. A posterior caracterização das proteínas, nos spots proteicos, empregando espectrometria de massas também foi conduzida evidenciando, pela primeira vez para o organismo estudado, a expressão de 12 novas proteínas. Um estudo ionômico foi desenvolvido para avaliar a distribuição destes elementos nas diferentes estruturas das plantas, buscando correlação com os processos biológicos que estariam ocorrendo nos girassóis. O estudo revelou uma correlação entre o nível de selênio e enxofre nas plantas sugerindo a incorporação do selênio em aminoácidos utilizados para síntese de proteínas. O emprego da técnica de ablação a laser acoplada ao espectrômetro de massas com plasma acoplado indutivamente (LA-ICP-MS) também apresenta destaque na pesquisa, complementando os dados obtidos no estudo ionômico, por meio da identificação de selênio nos spots proteicos extraídos das folhas dos girassóis e separadas por 2D-PAGE. Desenvolveu-se também desenvolver um método para análise quantitativa direta e simultânea de selênio e enxofre nas folhas das plantas empregando a hifenação LA-ICP-MS, buscando correlação entre os dois elementosAbstract: Several papers highlight a new research area called metallomic, and points out the necessity to better understand the relationship between metallic ions and proteins. This Thesis has employed the sunflower (Helianthus annuus L.), which is considered an oilseed plant with significant economical importance for the production of vegetal oil, and also used in fitoremediation processes, to carry out a metallomic study focused on the evaluation of possible changes in proteins expression due to the irrigation of the plants with selenite or zinc ions. Initially, the study evaluated four different methods for the adequate extraction and separation of sunflower leaf proteins using the two-dimensional gel electrophoresis (2DPAGE) technique and the best result was obtained using a phenol based extraction procedure. Further protein characterization in protein spots were carried out using mass spectrometry and revealed for the first time for the studied organism, the expression of 12 new proteins. An ionomic study was also developed to investigate the distribution of these elements in the different structures of the plants, looking for the correlation of obtained data with biological processes that could be occurring in the sunflowers. The study showed a correlation between selenium and sulfur levels, suggesting the incorporation of selenium into amino acids used for the synthesis of proteins. The application of laser ablation technique coupled to the inductively coupled plasma mass spectrometer (LA-ICP-MS) has also been highlighted in this research, complementing the results obtained in the ionomic study through the identification of selenium in sunflower leaves protein spots. In addition, it was evaluated an analytical procedure for the direct and simultaneous quantitative determination of selenium and sulfur in the leaves of the plants using LA-ICP-MS, searching for a correlation between both elementsDoutoradoQuimica AnaliticaDoutor em Ciência

Cloud point method applied to the Apolipoprotein A-I extraction from human plasma and its identification by tandem mass spectrometry

This work describes the extraction of the apolipoprotein A-I (apoA-I) from human plasma using the cloud point extraction (CPE). The CPE was carried out with a nonionic surfactant (5 % w/v Triton® X-114), and the presence of a salting-out effect (10 % w/v NaCl) promoted biocompatible separation conditions at room temperature and pH 6.8. The apoA-I present in the surfactant-rich phase was identified by tandem mass spectrometry after two-dimensional gel electrophoresis

Taxonomic indexes for differentiating malignancy of lung nodules on CT images

Abstract Introduction Lung cancer remains the leading cause of cancer mortality worldwide, with one of the lowest survival rates after diagnosis. Therefore, early detection greatly increases the chances of improving patient survival. Methods This study proposes a method for diagnosis of lung nodules in benign and malignant tumors based on image processing and pattern recognition techniques. Taxonomic indexes and phylogenetic trees were used as texture descriptors, and a Support Vector Machine was used for classification. Results The proposed method shows promising results for accurate diagnosis of benign and malignant lung tumors, achieving an accuracy of 88.44%, sensitivity of 84.22%, specificity of 90.06% and area under the ROC curve of 0.8714. Conclusion The results demonstrate the promising performance of texture extraction techniques by means of taxonomic indexes combined with phylogenetic trees. The proposed method achieves results comparable to those previously published

Evaluation Of Sample Preparation Protocols For Proteomic Analysis Of Sunflower Leaves.

Six protocols for extraction of proteins from sunflower (Helianthus annuus L.) leaves were evaluated for their abilities in both removing interferents and attaining the best resolution in two-dimensional gel electrophoresis. Classical phenol extraction followed by precipitation with ammonium acetate in methanol displayed the most efficient protocol, which allowed the detection of 244 protein spots with ca. 485mug of protein in gel electrophoresis. Tandem mass spectrometry was performed to identify proteins in 61 spots, and cross species identification was used for this task. Proteins from twenty two spots were identified, and 12 of these proteins are up to now not included into the ExPASy sunflower protein databank.801545-5