Made available in DSpace on 2017-07-10T17:37:45Z (GMT). No. of bitstreams: 1 Patricia_Paro.pdf: 402899 bytes, checksum: cb9972fdc35b447bcb1020e13a07782b (MD5) Previous issue date: 2011-04-08 / Fundação Araucária / The wheat is a species of great importance in human nutrition, wheat grain is extracted from wheat flour used in the preparation of various food products. The rheological properties of wheat flour is a feature that determines the fate of the flour, being of great importance to this assessment. In breeding programs for wheat determining the quality of flour is essential for the development of cultivars, and it is necessary that the reading of the quality parameters are carried out early, with the use of small amounts of sample, reliable and the level of genotype. The development of this work aimed at the validation of molecular markers for selection of genotypes from high molecular weight glutenin, given their great influence on baking quality of wheat flour and check the correlation between parameters of quality of flour and the high molecular weight glutenin. Analyses were performed in the laboratory of biotechnology COODETEC using 77 samples of wheat (cultivars and lines) from program for wheat breeding of the institution, with data quality technology of wheat flour has characterized. The set of primers called Glu1-DX2-DX5 was used to select individuals that contained subunits Glu1-DX5 + Dy10 by PCR, nevertheless was necessary to make adjustments so that the marker amplified a fragment corresponding to allele Glu1-DX5. To check the reliability of molecular marker, all samples were characterized by electrophoresis of proteins by SDS-PAGE method and scores as a function of the subunits of glutenin high molecular weight they have. The results of the analysis of protein and molecular differ, indicating that the use of molecular markers should be used with any control in the PCR reaction. From the data collected and the quality of flour, the results were submitted to Pearson correlation analysis. It appears that the score of the protein is positively correlated with W, P and P / L, indicating that the composition of glutenin high molecular weight have significant influence on quality of wheat flour. Alleles Glu1-DX5 and the subunits of the A genome are positively correlated with protein scores and consequently with the wheat breadmaking. The allelic variants of the A genome were positively correlated with W and P. From the results it is concluded that the selection of genotypes with superior quality of flour can be made taking into account the high molecular weight glutenin encoded by the A and D genomes of wheat / O trigo é uma espécie de grande importância na alimentação humana, de seus grãos é extraída a farinha de trigo utilizada no preparo de diversos produtos alimentícios. As propriedades reológicas da farinha de trigo são características que determinam o destino final da farinha, sendo de grande importância a sua avaliação. Em programas de melhoramento genético do trigo a determinação da qualidade de farinha é indispensável para o desenvolvimento de cultivares, e é necessário que a leitura dos parâmetros de qualidade sejam realizados precocemente, com a utilização de pouca quantidade de amostra, confiáveis e a nível de genótipo. Este trabalho teve por objetivo a validação de marcadores moleculares para a seleção de genótipos a partir de gluteninas de alta massa molecular, visto que apresentam grande influencia na qualidade de panificação da farinha de trigo e verificar a correlação existente entre parâmetros de qualidade de farinha e as gluteninas de alta massa molecular. As análises foram realizadas no laboratório de biotecnologia da COODETEC utilizando 77 amostras de trigo (linhagens e cultivares) provenientes do programa de melhoramento genético de trigo da instituição, com dados de qualidade tecnológica da farinha de trigo já caracterizados. O conjunto de primers denominados Glu1-Dx5-Dx2 foi utilizado para selecionar indivíduos que continham o conjunto de subunidades Glu1-Dx5+Dy10 através da técnica de PCR, porém foram necessário ajustes para que o marcador amplificasse o fragmento correspondente ao alelo Glu1-Dx5. Para verificar a confiabilidade do marcador molecular todas as amostras foram caracterizadas através de eletroforese de proteínas pelo método SDS-PAGE, e escoreadas em função das subunidades de gluteninas de alta massa molecular que apresentam. Os resultados entre a análise de proteína e molecular apontam divergências, indicando que para a utilização do marcador molecular deve ser utilizada com algum controle na reação de PCR. A partir dos dados coletados e os de qualidade de farinha os resultados foram submetidos a análise de correlação de Pearson. Verifica-se que o escore de proteína é positivamente correlacionado com W, P e P/L, indicando que a composição de gluteninas de alta massa molecular apresentam influencia significativa na qualidade de farinha de trigo. Os alelos Glu1-Dx5 e as subunidades do genoma A estão positivamente correlacionados com escore de proteína e conseqüentemente com a qualidade e panificação do trigo. Os variantes alélicos do genoma A foram positivamente correlacionados com W e P. A partir dos resultados conclui-se que a seleção de genótipos de trigo com qualidade superior de farinha podem ser realizados levando em consideração as gluteninas de alta massa molecular codificadas pelos genomas A e D do trigo

The main focus of the PhD study was to develop new gel electrophoresis and ICP-MS based methods to analyze a wide variety of the bio-molecules such as proteins, phosphoproteins and metalloproteins etc. The tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine-SDS-PAGE) method is commonly used to resolve low molecular mass proteins, however, it requires a high percentage gel and a very complicated procedure to achieve this separation. This study describes a modification to tricine-SDS-PAGE to make it more effective for the separation of smaller proteins and for coupling to ICP-MS. The modified method employs low percentage PAGE gels and low reagent concentrations that provide efficient separations, good quantitation and low matrix levels that are compatible with ICP-MS. This modified method was applied to analyze phosphopeptides. Phosphopeptides are very small in size and difficult to separate using the other techniques such as Laemmli SDS-PAGE, original tricine-SDS-PAGE, immobilized metal affinity chromatography (IMAC), size exclusion chromatography (SEC) etc. In this study a simplified procedure is described based on modifying the original tricine-SDS-PAGE method. A comparative study showed that this modified method successfully resolved a digest mixture of very low to high molecular mass phosphopeptides/peptides. In off-line coupling of this method with ICP-MS, much better recoveries of the peptides from the gel were obtained as compared to traditional methods which indicate the compatibility of this modified method for quantitative studies. An on-line coupling of the modified system with ICP-MS was also demonstrated and it was applied for the separation, detection and quantification of phosphopeptides. Another application of this modified system was the separation of serum proteins. Blood serum contains five major protein groups i.e., albumin, alpha-1 globulin, alpha-2 globulin, beta globulin and gamma globulin. The separation of these five major proteins in a single gel is difficult to achieve using traditional methods. The modified system was shown to be superior for the separation of these serum proteins in a 7% (m/v) native-PAGE gel and a cellulose acetate membrane. A further study was carried out into controlling the factors that cause metal loss and protein fragmentation in SDS-PAGE. Using a reducing sample buffer, and heating to high temperatures (90-100ºC) in alkaline or acidic conditions may cause protein fragmentation and decrease the metal binding affinity. 70ºC was found suitable to prepare the sample at neutral, alkaline or acidic pH as no fragmentation observed. To prevent metal loss, the binding constant (log K) values of metal-amino acids, play the major role. Those metals which have high binding affinities with the amino acids in proteins can also be affected by the variation of the pH so prior information about pH to maintain the binding constant values is essential to minimize metal loss. This was observed in the loss of zinc, and to a lesser extent copper from human serum albumin (HSA) as measured by inductively coupled plasma mass spectrometry (ICP-MS). The method described above was applied for the separation and quantification of the serum proteins obtained from age-related macular degeneration (AMD) patients (where the AMD patients were from Moorfields Eye Hospital, London). Zn and Cu were quantified employing external calibration. Zn concentration showed variation whilst Cu did not show any significant variations in samples from AMD patients. A brief study of the interaction of cisplatin and oxaliplatin with HSA and transferrin was also performed. Cisplatin bound much faster than oxaliplatin with HSA. After 24 hours incubation, cisplatin showed a decrease in signal intensity which indicates that cisplatin binding decreases with time. Cisplatin binding with transferrin as compared to HSA was not significant, which could be the result of unstable Pt-transferrin complex formation. Oxaliplatin did not show high binding to either protein, perhaps due to the presence of the bulky, non polar DACH ligand.