Crude gliadins were extracted from Canada Western Red Spring (CWRS) wheat flour with 70% (v/v) aqueous ethanol solutions and then lyophilized. Lyophilized crude gliadins were dissolved in 70% (v/v) aqueous ethanol (EtOH) or 4 mM acetic acid (HAc) and the density and ultrasound properties were measured at 20 °C. Good linear relationships of density, ultrasound velocity and ultrasound attenuation with solution concentrations were found. Solvent and sonication effects on the crude gliadins were discussed in terms of the values of the partial specific volume and the partial specific adiabatic compressibility coefficient for crude gliadins. The ethanol soluble crude gliadins had a larger partial specific volume and larger partial specific adiabatic compressibility coefficient than those for acidic soluble crude gliadins. These large values for the physical properties of ethanol soluble crude gliadins were thought to be evidence for the existence of complexes formed by some proteins (ethanol soluble LMW-glutenins and gliadins) and lipids in ethanol solutions and it was also found that the protein-lipid complexes were not destroyed by sonication treatment. Besides, there was no evidence showing that gliadins change with different wheat flours and cause different volume and compressibility properties of crude gliadins.

Chloroform methanol (CM) soluble extract of a cereal-based diet has been suggested to induce type 1 diabetes in an experimental animal model of type 1 diabetes. However, the individual constituent of this extract responsible for the disease induction and its disease pathogenesis mechanism remained unexplored. A previous study in our laboratory failed to show that the sphingolipid enriched fraction of CM soluble extract of wheat gluten triggers type 1 diabetes. Therefore, to study the involvement of CM soluble gliadin proteins in type 1 diabetes, we retrospectively analyzed proteins from sphingolipid enriched extract. SDS-PAGE analysis of CM soluble extract of wheat gluten and sphingolipid enriched fraction exhibited protein bands corresponding to the masses of the wheat gliadins, suggesting the presence of gliadin proteins in the CM soluble sphingolipid enriched extract. We studied the effect of five different dietary treatments on the histopathology of pancreatic tissue from BBdp rats includes insulitis scores i.e. lymphocytic infiltration in islet of Langerhans in order to test gliadin specific sphingolipid enriched extract (GSLEE) as possible a diabetogen. However, there were no significant differences in pancreatic insulitis scores and lymphoid tissue content due to addition of (GSLEE) to the hydrolyzed casein (HC) diet. We also investigated changes in jejunal histopathology and sera IFN-γ, TNF-α cytokine concentration in type 1 diabetes, induced by GSLEE. A decrease in jejunal CD4+ and γδ TCR + cell counts and inflammatory cell infiltrate were observed due to presence of CM soluble GSLEE in the HC diet, although this decrease was not statistically significant. A significant increase in sera IFN-γ cytokine concentration was found in BBdp rats fed the HC + GSLEE diet as compared to rats on HC diet. A numerical decrease in sera TNF-α concentration was also observed in BBdp rats fed the HC + GSLEE diet, when compared to BBdp rats on the HC diet. In contrast, a significant increase in serum IFN-γ concentrations in BBdp rats were observed after removing the CM soluble GSLEE from the wheat gluten based diet (WG) when compared to the WG diet alone. Removing GSLEE from WG diet resulted in insignificant increase in serum TNF-α concentration in BBdp rats when compared to WG dietary treatment group's BBdp rats. However, there were no significant differences in jejunal enteropathy parameters (i.e. lymphocytic infiltration, mucosal thickness, epithelial erosion, jejunal villi flattenings), jejunal CD4+ and jejunal γδ TCR+ cell counts; pancreatic insulitis scores, lymphoid tissue content after removing the CM soluble GSLEE from the WG diet when compared to the WG diet. Since overall findings regarding the CM soluble GSLEE's potential to induce type 1 diabetes by changing pancreatic and jejunal histopathology and elevating serum IFN-γ, TNF-α cytokine levels largely remained inconclusive, further investigations are warranted regarding immune suppression potential of the CM soluble sphingolipids in type 1 diabetes and the search of diabetogenic agents remaining in the residue after CM extraction. / Master of Science

Celiac disease is an autoimmune disease which occurs in susceptible individuals after ingestion of food containing gluten. Gluten and its monomeric fraction gliadin induce inflammatory damage of the small intestine by activating the immune cells that react strongly to gluten peptides. Gluten peptides have the ability to activate cells of adaptive as well as innate immune system. This work is focused on the production of interleukin (IL)-1 in antigen presenting cells stimulated with peptic gliadin digest. We found that monocytes and peripheral blood mononuclear cells (PBMC) isolated from blood of celiac patients secrete significantly more IL-1α and IL-1β than cells of healthy donors after stimulation with gliadin digest. The gliadin-induced IL-1β expression is controlled by a signaling cascade that includes MAPK kinase family molecules and transcription factor NF-κB. Moreover, we found that the adaptor proteins MyD88 and TRIF as well as Toll-like receptor (TLR) 2 and 4 play a role in the signaling cascade underlying gliadin-induced IL-1β expression by using murine bone marrow derived dendritic cells (BMDC). The precursor form of IL-1β in gliadin- stimulated PBMC and murine BMDC is maturated by caspase-1. In celiac PBMC the gliadin- induced maturation and secretion of IL-1β depends on the potassium...

Celiac disease is attributable to autoimmune diseases, where treatment is based on absolute respect for gluten-free diet. As the gluten used widely in all areas of food production, strict adherence to a gluten-free diet really can be difficult for people with celiac disease. Products suitable also for those suffering from the disease are usually expensive. Finally, patients that undergo exclusively on a gluten-free diet are robbing the important nutritional components. Therefore, interest in this type of products innovation enhance as to the nutritional, technological, as well as organoleptic properties. The aim of this final thesis was to draw up literature review of the celiac disease and gluten allergen, or his particular part of gliadin. In the experimental part, attention was focused on the first hydrolytic cleavage of starch, the substrate was sterilized wheat flour and water, where is incorporated spiral just gluten. The best ability of degrading the starch grains, the combination of enzymes and MT3K and GLUAMK concentration of 0, 1% over 4 hours of action, as indicated by a reduction in the starch content of the original amount of 72,65 % at a concentration of 29,00 %. The next item of work distribution used different proteases, to reduce the amount of gliadin to below 20 ppm per kilogram of final product. Low levels investigated allergen (10,79 ppm/kg) recorded by the enzyme combination exoprotease Flavourzyme (0,1 %) and endoprotease Neutral Protease (0,1 %) at 7 hours hydrolytic action. The conclusion of the thesis was prepared several alternatives gluten-free breads and through sensory evaluation assessed the characteristics of color, aroma and taste to predict the location of the products on the commercial market.

Gluten sensitive entheropathy - celiac disease is a lifelong, genetically predisposed, immunologically mediated susceptibility to dietary wheat gluten, most frequently demonstrated by small-bowel damage and malabsorption syndrome. Strict adherence to gluten-free diet is the sole rational therapy of the disease. One of the possible therapeutic strategy for the treating of celiac disease is to utilize the synthetic polymer P(HEMA-co-SS). This polymer is capable specifically bound gliadin in gastrointestinal tract and by this way to neutralize the damaging effect of this alimentary protein on mucosa of small intestine in celiac patients. The in vitro study on human PBMC and specimens of small intestinal biopsies of celiac patients in our laboratory demonstrated that putative therapeutic ability of P(HEMA-co-SS) is substantially influenced by degree of proteolytic processing of gliadin and P(HEMA- co-SS) and also by different timing of per os administration of both components in organism. Another putative adjuvant therapy of celiac disease is employing of the beneficial probiotic bacterial strains. Our experiments were based on the findings of Prof. Y Sánz and her group demonstrating the significant differences in the composition of bacterial microflora in patients with active form of celiac disease,...

Durum wheat (Triticum durum Desf.) is produced primarily in the Northern Great Plains and the Pacific Southwest of the United States. Current germplasm is predominantly of the spring growth habit. The objective of this study was to determine the feasibility of winter durum production in Virginia based upon both yield and quality parameters. Adaptation and yield potential of available winter durum lines were assessed from 1993-1998, in three physiographic regions in Virginia. The highest average durum yields were produced in the northern piedmont plateau at the Orange County location. Winter durum yields generally averaged 1600-2800 kg/ha less than soft red winter wheat, traditionally grown in the state. The price premium for high quality durum can compensate for this difference in yield. Based on average durum yields, and assuming the grain meets U.S. No. 2 Hard Amber Durum standards, durum production in Virginia would have been more profitable than soft red winter wheat production in 1994 and 1997. Physical and chemical quality analyses of the top 19 performing durum lines were performed to determine grain marketability, suitability for pasta, and potential consumer acceptance of the end product. Protein content and gluten strength of the Virginia grown durum were acceptable. Color, firmness, and cooking loss of pasta produced from Virginia grown durum were comparable to pasta produced from commercial semolina. Requirements for U.S. No. 2 Amber Durum were met by 21% of the lines in both 1996 and 1997. Overall, the wet, humid Virginia climate was the greatest hindrance to durum production and quality. The field trials and quality analyses showed that high quality durum production in Virginia is possible, but not consistent over all years. / Master of Science

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20430号 / 農博第2215号 / 新制||農||1048(附属図書館) / 学位論文||H29||N5051(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 谷 史人, 教授 安達 修二, 教授 松村 康生 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Master of Science / Department of Chemistry / Christopher T. Culbertson / Chemical separation involves selective movement of a component out of a region shared by multiple components into a region where it is the major occupant. The history of the field of chemical separations as a concept can be dated back to ancient times when people started improving the quality of life by separation of good materials from bad ones. Since then the field of chemical separation has become one of the most continually evolving branches of chemical science and encompasses numerous different techniques and principles. An analytical chemist’s quest for a better way of selective identification and quantification of a component by separating it from its mixture is the cause for these ever evolving techniques. As a result, today there are numerous varieties of analytical techniques for the separation of complex mixtures. High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Capillary Electrophoresis (CE) and Gel Electrophoresis are a few out of a long list. Each these techniques manipulates the different physical and chemical properties of an analyte to achieve a useful separation and thus certain techniques will be suited for certain molecules. This work primarily focuses on the use of Capillary Electrophoresis as a separation technique. The mechanism of separation in Capillary Zone Electrophoresis and principles of UV detection will discussed in chapter one. Chapter two contains a discussion about the application of Capillary Electrophoresis (CE) on microfluidc devices. This will include sections on: microfabrication techniques of PDMS and photosensitized PDMS (photoPDMS), a UV detector for microfluidic devices and its application for the detection of wheat proteins. In Chapter three we report the experimental part of this project which includes; investigations on the effect of UV exposure time and thermal curing time on feature dimensions of photoPDMS microfluidic device, investigations on the injection and separation performances of the device, characterization of a UV detector set up and its application for the separation and detection of wheat gliadin proteins. The results of these investigations are presented in chapter four.

A tese consistiu no estudo por técnicas reológicas da dinâmica de géis e dispersões da gliadina do glúten do trigo em meios de dimetilsulfóxido (DMSO), dimetilformamida (DMF) e formamida puros e em suas misturas com a água. Investigou-se o efeito da temperatura e das concentrações das espécies componentes (proteína e constituintes dos sistemas solventes) sobre o comportamento de fluxo e de deformação dos sistemas. Foram realizados testes em regime estacionário, ensaios transientes e ensaios dinâmicos (oscilatórios). A caracterização da proteína nos diversos meios foi realizada através de técnicas de espalhamento de luz dinâmico e estático e de espalhamento de raios X em baixos ângulos. Esses recursos permitiram descrever a distribuição de populações da proteína nos géis e dispersões, em suas formas livres ou associadas, assim como estimar aspectos de tamanho e mobilidade a partir da determinação dos raios hidrodinâmicos e coeficientes de difusão. As composições de aminoácidos da gliadina, na fração bruta e em uma fração isolada em meio etanólico, foram determinadas por técnicas de CLAE Cromatografia Líquida de Alta Eficiência. Um isolamento preliminar das sub-frações de gliadina foi obtido por outra técnica cromatogrática (FPLC Fast Polymer Liquid Chromatography). Foram ainda desenvolvidos estudos relativos ao efeito de gliadina sobre a bicamada lipídica de vesículas gigantes de fosfolipídio, através da técnica de aspiração por micropipetas, com monitoramento por vídeo-microscopia. O objetivo foi o de se verificar o eventual efeito da proteína sobre a constante de elasticidade, kc, da bicamada. Os resultados obtidos podem ser resumidos como se segue: a) o comportamento viscoelástico apresentado pela gliadina mostrou dependência com a natureza e a composição do meio de dispersão. Foi verificada a ocorrência de comportamento tixotrópico ou reopéxico como função da temperatura e um comportamento cíclico para a viscosidade em função do tempo. b) foi possível correlacionar alguns aspectos da estrutura molecular e polaridade do solvente com a maior ou menor capacidade de formação de gel de gliadina: solventes com regiões apolares mais expressivas, como DMSO e DMF (ε= 47,24 e 38,25, respectivamente) induzem a formação de géis rígidos, enquanto um composto como formamida, destituído de um domínio apolar minimamente significativo e ε maior que o da água (ε = 84), tem capacidade significativamente menor de promover a formação de géis com as mesmas características de rigidez. c) os géis são formados por unidades com raios de giro da ordem de 15Å, além de agregados de tamanhos bem maiores. Considerando-se a massa molar média de 32 350 g/mol para a gliadina em nossa amostra, é de se supor que a proteína encontre-se muito compactada no gel (considere-se que lisozima, com massa molar muito menor, igual a 14300 g/mol, possui raio de giro nesse mesmo valor). Um modelo de enovelamento interpenetrante, proposto em nosso grupo através de métodos de dinâmica molecular para a conformação da gliadina, seria assim compatível com essas observações experimentais. d) gliadina ω, seletivamente extraída por tratamento com etanol, mostrou-se capaz de reduzir a elasticidade de membranas de microvesículas de fosfolipídios, aumentando o valor de sua constante de curvatura kc. Um efeito de saturação foi observado, para razões de massa gliadina/DOPC ~2-3%. e) coeficientes de difusão para dispersões de gliadina são da ordem de 10-10 cm2.s-1, com raios hidrodinâmicos na faixa nm-µm. Duas populações dinamicamente distintas foram identificadas nos géis de gliadina. f) o grau de polidispersão e o tamanho dos agregados mostraram dependência com a concentração da proteína nos géis: são gerados agregados menores (diminuição da massa molar média) em tamanhos mais uniformes (menor polidispersão), com o aumento da concentração de gliadina. Essa mesma tendência de variação é observada como função do tempo, indicando a ocorrência de rearranjos estruturais na fase de envelhecimento dos géis. / The thesis consisted in a rheological study of the dynamics of gels and dispersions of gliadin from wheat gluten in pure dimethylsulfoxide, dimethylformamide or formamide and in their mixtures with water. The effect of temperature and that of the concentration of the component species (protein and solvent constituents) on flow behavior and deformation were evaluated. Steady-state, transient and dynamical (oscillatory) tests were performed. Protein characterization in the various media was performed through dynamic and static light scattering and small angle X ray scattering techniques. The application of techniques allowed to describe the distribution of protein populations in the gels and dispersions, in their free and associated forms, as well as to estimate their size and mobility, through the determination of the hydrodynamical radii and diffusion coefficients. The amino acid compositions of gliadin, both in the crude protein and in an isolated fraction in ethanol, were determined by HPLC technique. A preliminary isolation procedure of gliadin sub-fractions was achieved by another chromatographic technique (FPLC). The effect of gliadin on the phospholipid bilayer of giant vesicles was studied through the technique of micropipet aspiration coupled to videomicroscopy. The aim was to verify the possible effect of protein on the elasticity constant, kC of the bilayer. Results obtained can be summarized as follows: a) the viscoelastic behavior presented by gliadin was found to be dependent on the nature and composition of the dispersing media. Temperature-dependent tixotropic or rheopexic behavior were observed, as well as a cyclic behaviour for viscosity as a function of time. b) it was possible to correlate some aspects of solvent molecular structure and polarity with its greater or lesser capacity of gel formation: solvents with more expressive apolar regions, such as DMSO and DMF (ε = 47,24 and 38,25, respectively) induce formation of rigid gels, whereas a compound such as formamide, devoid of a minimally significant apolar domain and ε greater than that of water (ε = 84), is significantly less able to promote gel formation with the same characteristics of rigidity. c) gels are formed by units with gyration radius around 15Å and also by aggregates of greater size. Considering that the average molar mass in our sample for gliadin is 32350, one can assume that the protein is very densely packed in the gel (consider for instance that lysozyme, with a much lower molar mass, 14300 g/mol, has a gyration radius of this same size). A compact, interpenetrating folding model for gliadin, developed in our group through molecular dynamics, is compatible with such experimental observations. d) ω gliadin, selectively extracted in ethanol, was found to be able to reduce membrane elasticity of phospholipid microvesicles, by increasing its elastic curvature constant kC. A saturation effect was observed, for a mass ratio as low as gliadin/DOPC ~2-3%. e) diffusion coefficients for gliadin dispersions are circa 10-10 cm2.s-1, with hydrodynamical radii in the nm-µm range. Two dynamically distinct populations are identified in the gliadin gels. f) gel polydispersity and aggregate size depend on protein concentration: smaller aggregates (lower average molar mass) of more uniform sizes are produced for increasing gliadin concentration. Variation with time followed the same pattern, indicating that structural rearrangements take place during gel aging.

The aim of this study was to identify QTLs associated with the maintenance of grain quality following post-anthesis heat stress. A population of 64 F6Halberd X Cutter recombinant inbred lines (RILs) was exposed to either heat stress or control conditions in the greenhouse, starting ten days after anthesis. Grain quality was determined using the sodium dodecyl sulfate (SDS) sedimentation test, a significant predictor of bread baking quality. The percent change in SDS sedimentation test scores between the heat and control populations was used to identify QTLs associated with quality stability. Four QTLs were identified, located one each on chromosomes 1B, 1D, 4A, and 7A. Three of the QTLs, those on 1B, 1D, and 4A, were associated with variations in SDS sedimentation level. The QTL on chromosome 7A was associated with the percent change in SDS sedimentation scores between heat-stressed and control conditions. This indicated a relationship between the identified QTL and quality stability. To confirm the detected QTLs, eighty advanced lines grown at three Texas nurseries were genotyped and tested for relationships between QTL-associated markers, quality traits, and stability of the quality traits. Quality trait stability was estimated using the coefficient of variability (CV%) of quality traits between growing sites. Quality characters analyzed in the advanced lines included kernel hardness, mixograph peak time, kernel weight, flour yield, SDS sedimentation, and grain yield. The analysis showed support for the effect of the QTLs on chromosomes 1B, 1D, and 4A. Further analysis will be needed to confirm the QTL on 7A, in particular. The mapping of additional markers will be necessary. However, the potential importance of this QTL and the abundance of other QTLs detected in this region make it worth investigating.