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.

<p>U radu je ispitivan uticaj karakteristika glutenina i<br />glijadina uzoraka p&scaron;enice domaćeg sortimenta kako<br />bi se na osnovu tumačenja njihove strukture<br />omogućilo dono&scaron;enje egzaktnih zaključaka o<br />uzročno-posledičnom odnosu vezanom za stanje<br />gluteninsko-glijadinskog kompleksa zrna različitih<br />sorti p&scaron;enice i njihovog ispoljenog tehnolo&scaron;kog<br />kvaliteta posmatrano i u zavisnosti od uticaja<br />spoljne sredine. Za ispitivanje su kori&scaron;ćene A-PAGE<br />i SDS-PAGE elektroforetske tehnike ispitivanja<br />proteinskih frakcija, uz pomoć profesionalnog<br />softverskog paketa za obradu elektroforegrama.<br />Takođe, kori&scaron;ćene su i standardne metode za<br />ispitivanje tehnolo&scaron;kog kvaliteta p&scaron;enice i njihove<br />modifikacije. Na osnovu dobijenih rezultata<br />utvrđeno je da je tehnolo&scaron;ki kvalitet p&scaron;enice<br />neophodno povezivati i sa gluteninskim i sa<br />glijadinskim frakcijama proteina i da su osobine<br />otpornosti prema proteolitičkim procesima i<br />temperaturnom stresu sortno specifične. Takođe je<br />utvrđeno da su elektroforetske tehnike primenljive<br />u tumačenju tehnolo&scaron;kog kvaliteta p&scaron;enice, a da su<br />modifikacije standardnih metoda pokazale prednost<br />u objektivnijem i bržem predviđanju tehnolo&scaron;kog<br />kvaliteta domaćeg sortimenta hlebnih p&scaron;enica u<br />odnosu na standardne metode ispitivanja<br />tehnolo&scaron;kog kvaliteta p&scaron;enice.</p> / <p>The influence of glutenin and gliadin characteristics of<br />samples of domestic wheat varieties was investigated<br />with the aim to obtain on the basis of their structure,<br />conclusions about the interactions of condition of<br />glutenin and gliadin complex of the kernel of different<br />wheat varieties and their expressed technological<br />quality in the dependence of agro ecological growing<br />conditions. A-PAGE and SDS-PAGE electrophoretic<br />techniques were used for the investigation, with<br />consequent use of professional software for<br />electrophoregrams analysis. Standard and modified<br />methods were used for determination of wheat<br />technological quality. On the basis of obtained results<br />it was concluded that the technological quality of<br />wheat should be analyzed based structure of both<br />glutenin and gliadin fractions of wheat proteins, and<br />that the resistance to both, temperature stress and<br />proteolitic degradation is variety specific.<br />Electrophoretic techniques are applicable in analysis<br />of wheat technological quality. Modification of<br />standard methods expressed advantages in more<br />objective and more rapid determination and<br />foreseeing of technological quality of domestic wheat<br />varieties in comparison to standard methods.</p>

The aim of thesis was monitoring of changes in the content of gluten proteins in the biotechnological process of beer production. During the production process of wheat and barley beer, the samples were collected and analysed using the electrophoresis and immunoassay method. The results of the analyses were compared with commercial Czech beers. The theoretical part contains description and composition of gluten proteins, malt and beer technology, the changes that occur in this process, and methods of gluten proteins analysis. The experimental part contains procedures for laboratory production of barley and wheat beer and analyses of gluten proteins. To identify the individual gluteal protein fraction acid and SDS electrophoresis methods were used. For quantification, enzyme immunoassay was used and evaluated spectrophotometrically. The identification of the gluten‘s fractions by electrophoretic methods has been shown to be less specific for samples with lower content of gluten proteins and for barley specimens. A decrease in the concentration of gliadins and glutenins in the beer production process was demonstrated. A significant change was found during wort production with 98% decrease of gluten content compared to the feedstock and during the fermentation, when the gluten concentration dropped below 10 mg/kg. This value is acceptable from the legislation for products labelled „gluten-free“.

A doença celíaca (DC) é uma doença inflamatória induzida pela ingestão de glúten em indivíduos geneticamente predispostos e seu tratamento é baseado em uma dieta sem glúten por toda a vida. A doença celíaca refratária é um problema comum que afeta de 10% a 19% dos pacientes célicos tratados. Provavelmente, a contaminação da dieta por glúten é uma das razões principais para a persistência de sintomas em pacientes celíacos tratados, assim como a ingestão inadvertida de glúten, devido a rotulagem incorreta. Assim, o objetivo deste estudo foi avaliar a confiabilidade dos rótulos dos alimentos brasileiros processados, através de testes de contaminação de glúten nos seguintes grupos (a) produtos \"livres de glúten\" - preparados especificamente para a população celíaca; (b) produtos \"naturalmente sem glúten\" feitos com arroz, milho, soja e mandioca, utilizados por toda a população e (c) produtos rotulados com \"contém glúten\", mas que não apresentam glúten em sua composição no rótulo. Foram analisados 213 produtos alimentícios agrupados em: 115 produtos do grupo \"sem glúten\"; 86 produtos do grupo \"naturalmente sem glúten\" e 12 produtos do grupo rotulados com \"contém glúten\". O teor de glúten foi detectado e quantificado por ELISA-R5 (Ridascreen®gliadin) e os resultados foram expressos em ppm e mg/100 g de alimento. A linha de corte foi estabelecida em 20 ppm para a contaminação de glúten. Todas as contaminações por glúten foram confirmadas por Western-blotting. Resultados: (a) alimentos livres de glúten 15 das 115 (13%) apresentaram contaminação por glúten (20 ppm), (b) grupo de alimentos naturalmente sem glúten - 8 de 86 (9,3%) apresentaram contaminação por glúten (20 ppm); (c) grupo de alimentos rotulados com contem glúten - somente 2 de 12 (16,7%) apresentaram contaminação por glúten (20 ppm). A análise de Western-blotting confirmou 36 das 38 (95%) contaminações encontradas no ELISA-R5. CONCLUSÕES: Ambos os grupos de alimentos \"sem glúten\" e \"naturalmente sem glúten\" comercializados no Brasil apresentaram razoável porcentagem de contaminação por glúten, o que dificulta a realização de uma dieta adequada ao paciente celíaco. O grupo de alimentos rotulado \"com glúten\" não apresentou 100% de contaminação, o que revela que a rotulagem desses produtos deve ser feita como uma medida preventiva. Uma maior chance de contaminação pelo glúten foi observada para os produtos a base de arroz (13,6x), soja (13,3x) e milho (9,3x), mas não naqueles à base de mandioca. Em média, encontramos 10,8% (23 de 213) de contaminação de glúten para os alimentos analisados, um panorama positivo para a população brasileira celíaca, principalmente devido ao uso da mandioca, uma alternativa para a farinha de trigo. No entanto, a contaminação de glúten encontrada mostra a importância da quantificação de glúten em todos os alimentos industrializados. / Celiac disease (CD) is an inflammatory disorder induced by ingestion of gluten in genetically predisposed individuals and its treatment is based on a life-time gluten-free diet. Nonresponsive celiac disease is a common problem affecting from 10% to 19% of treated celiac patients. Probably a gluten contamination in diet is one of the major reasons for symptoms persistence in celiac patients as well as an inadvertent gluten intake due to a misleading nutritional label. The aim of this study was to evaluate the reliability of Brazilian processed food labels by testing gluten contamination in (a) gluten-free products - prepared specifically for the celiac population; (b) in naturally gluten-free products made with rice, corn, soy bean and cassava and used by all population and (c) in not gluten-free products labeled to contain gluten but not having it in their composition. We analyzed 213 food samples grouped accordingly to its type: 115 samples of \"gluten-free food, 86 samples of \"naturally gluten-free food and 12 samples of not-gluten free labeled products. The gluten content was detected and quantified by ELISA-R5 (Ridascreen® Gliadin) and the results were expressed in ppm and mg/100 g of food. A cut-off line was established in 20 ppm for gluten contamination. All gluten contaminations were confirmed by Western-blotting. Results: (a) Gluten-free foods - we found 100 of 115 samples (87%) with no contamination (< 20 ppm) and 15 of 115 (13%) showed gluten contamination 20 ppm; (b) Naturally Gluten-free foods - we found 78 of 86 samples (90,7%) showing no contamination (< 20 ppm) and 8 of 86 (9,3%) with gluten levels 20 ppm; (c) Not gluten-free foods - we found 10 of 12 samples (83,3%) showing no contamination (< 20 ppm) and 2 of 12 (16,7%) with gluten contamination 20 ppm. The Western-blotting analysis confirmed 36 of 38 (95%) contaminations found in the ELISA-R5. CONCLUSIONS: Both \"gluten-free and \"naturally gluten-free foods commercialized in Brazil have presented some gluten contamination making a restricted gluten-free diet hard to be achieved by the celiac population. Unexpectedly the not gluten-free group was not entirely contaminated showing a preventive measure in labeling by food companies. A higher odds ratio for gluten contamination was observed for products made with rice (13.6), soy bean (13.3) and corn (9.3) but not to cassava products (not significant). In general, we found a 10.8% (23 of 213) of gluten contamination for all food products analyzed, a positive panorama for the Brazilian celiac population mainly due to cassava products, an alternative for wheat starch. Nevertheless the gluten contamination found here leads us to the importance for a gluten quantification in all industrialized food to guarantee an appropriated diet to the Brazilian celiac group

The increasing environmental concern has reawakened an interest in materials based on renewable resources as replacement for petroleum-based materials. The main objective of this thesis was to explore plant proteins, more specifically wheat gluten, as a binder in wood adhesives intended for typical solid wood applications such as furniture and flooring. Alkaline and acidic dispersions of wheat gluten were used as wood adhesives to bond together beech wood substrates. Soy protein isolate was used as a reference. The tensile shear strengths of the substrates were measured for comparison of bond strength and resistance to cold water. AFM in colloidal probe mode was used to investigate nanoscale adhesion between cellulose and protein films. Wheat gluten was divided into the two protein classes; glutenins and gliadins, and their adhesive performance was compared with that of wheat gluten. Heat treatment and mild hydrolysis were investigated as means for improving bonding performance of wheat gluten. The treated wheat gluten samples were analysed by SE-HPLC and 13C-NMR to correlate molecular size distribution and structural changes with bonding performance. Soy protein isolate is superior to wheat gluten, especially in regards to water resistance. However, the bond strength of wheat gluten is improved when starved bond lines are avoided. The AFM analysis reveals higher interfacial adhesion between soy protein isolate and cellulose than between wheat gluten and cellulose. These results partly explain some of the differences in bonding performance between the plant proteins. Soy protein isolate contains more polar amino acid residues than wheat gluten and possibly interacts more strongly with cellulose. Furthermore, the bond performances of wheat gluten and glutenin are similar, while that of gliadin is inferior to the others, especially regarding water resistance. The extent of penetration of the dispersions into the wood material has a large impact on the results. The bonding performance of gliadin is similar to the others when over-penetration of the dispersion into the wood material is avoided. Moreover, the bond strength of the wheat gluten samples heated at 90°C was in general improved compared to that of wheat gluten. A small improvement was also obtained for some of the hydrolyzed wheat gluten samples (degree of hydrolysis: 0-0.6 %). The improvements in bonding performance for the heat treated samples are due to polymerization, while the improvements for the hydrolyzed samples are due to denaturation. The 13C-NMR analysis of the treated samples confirms some degree of denaturation. / QC 20120514

Zöliakie ist eine immunologisch vermittelte Erkrankung bei der die Dünndarm-schleimhaut durch das in zahlreichen Getreidesorten vorkommende Klebereiweiß Gliadin geschädigt wird. Dabei wird die typische Architektur der Mukosa zerstört und imponiert histologisch als Zottenatrophie. In Folge dessen zeigen Betroffene Mangelerscheinungen und Verdauungsbeschwerden sowie zahlreiche extra-intestinale, atypische Symptome. Bei Kindern können zusätzlich gravierende Wachstums- und Entwicklungsstörungen auftreten. Die Therapie besteht in einer lebenslangen glutenfreien Diät. Die Diagnostik der Erkrankung basiert auf vier Säulen: Neben der Beurteilung der klinischen Symptomatik werden zöliakie-typische Antikörper nachgewiesen, welche bei hoher Konzentration die Indikation zur Biopsie darstellen. Die bioptische Untersuchung mit anschließendem histolo-gischem Nachweis der Zottenatrophie stellt den Goldstandard der Diagnostik dar und wird durch die Besserung der klinischen Symptomatik unter glutenfreier Diät gestützt. Bei der serologischen Untersuchung haben Antikörper gegen natives Gliadin auf Grund niedriger diagnostischer Genauigkeit an Bedeutung verloren. Sie wurden durch die Bestimmung von Autoantikörpern gegen die Gewebstransglutaminase abgelöst, die eine höhere Sensitivität und Spezifität aufweisen. Im Jahr 2000 konn-te jedoch gezeigt werden, dass sich Antikörper von Zöliakiepatienten an Gliadin-peptide besser nach selektiver Deamidierung (Austausch der Aminosäure Glutamin durch Glutaminsäure) binden. Darauf aufbauend entwickelte die Firma INOVA Diagnostics im Jahr 2006 erst-mals einen ELISA zur Zöliakiediagnostik mit synthetisch hergestellten, deamidier-ten Gliadinpeptiden (DGP) als Antigen. Zu Beginn unserer Arbeit existierten keine Veröffentlichungen zur diagnostischen Genauigkeit dieser Tests bei Kindern und nur eine Veröffentlichung, die zwei der ELISA an Seren von erwachsenen Patienten untersuchte. Bei Kindern ist es jedoch besonders wichtig, durch die Antikörperbestimmung eine hohe Diagnosesicher-heit zu erlangen, weil für sie die Biopsie eine große Belastung darstellt und eine nicht erkannte Zöliakieerkrankung zu schwerwiegenden Entwicklungsstörungen führen kann. Aus diesem Grund soll die vorliegende Studie den Nutzen dieser neuen ELISA in der Diagnostik der Zöliakie im Kindesalter evaluieren. Es wurden dazu insgesamt 340 Seren von bioptisch bestätigten Zöliakiepatienten und Kontrollen, bei denen die Erkrankung histologisch ausgeschlossen wurde, gesammelt, verblindet und retrospektiv analysiert. Dabei wurden vier verschiede-ne ELISA der Firma INOVA Diagnostics eingesetzt: drei ELISA mit DGP als An-tigen sowie ein weiterer ELISA, in dem DGP mit Gewebstransglutaminase kombiniert war. Es wurden folgende Erkenntnisse gewonnen: 1. Alle vier ELISA eignen sich zur Diagnostik von Zöliakie bei Kindern und weisen eine hohe Trennschärfe auf. Die Fläche unter der Receiver Operating Characteristic (ROC)-Kurve ist für alle vier Tests größer als 0,96. Mit den Tests kann somit die Indikation zur bioptischen Untersuchung mit großer Sicherheit gestellt werden. 2. Die Bestimmung der Antikörper gegen DGP ist der Antikörperbestimmung gegen natives Gliadin überlegen. Die DGP-Tests weisen eine signifikant größe-re Fläche unter der ROC-Kurve als die Tests auf Antikörper gegen natives Gli-adin auf. Die Bestimmung der Antikörper gegen DGP ist der Bestimmung von Antikörpern gegen Gewebstransglutaminase nicht unterlegen. Die Flächen un-ter den ROC-Kurven unterscheiden sich nicht signifikant voneinander. 3. Überraschenderweise zeigt die IgG-Klasse der DGP eine signifikant höhere diagnostische Genauigkeit als die IgA-Klasse. Somit ist auch bei Patienten mit IgA-Mangel eine sichere Diagnosestellung gegeben und es kann auf die gene-relle Bestimmung des Gesamt-IgA verzichtet werden. 4. Der DGP-IgG-Test weist von den vier validierten Antikörpertests bei einer Sensitivität von 100 % die höchste Spezifität (90 %) und bei einer Spezifität von 100 % die höchste Sensitivität (64 %) auf. 5. Durch den kombinierten Test mit zwei Antigenen und den gleichzeitigen Nachweis von IgA und IgG in einem ELISA (tTG/DGP-Screen-Test) lassen sich Kosten und Zeit sparen. Dieser kombinierte Test weist die höchste diagnosti-sche Genauigkeit der untersuchten DGP-Tests auf. 6. Durch Angabe von Likelihood Ratios und mittels grafischer Darstellung der Posttest-Wahrscheinlichkeit in Abhängigkeit der Antikörperkonzentration und Prävalenz können den behandelnden Ärzten wertvolle Informationen zur Di-agnosesicherheit eines einzelnen Testergebnisses vermittelt werden. Mit den evaluierten DGP-Tests stehen neue und zuverlässige ELISA zur Diagnos-tik der Zöliakie im Kindesalter zur Verfügung. Sie weisen eine hohe diagnostische Sicherheit auf und unterscheiden sicher zwischen Gesunden und Zöliakiepatien-ten. Die Indikation für eine Dünndarmbiopsie kann mit Hilfe der DGP-Tests sehr sicher gestellt werden. Ob darüber hinaus auf der Basis sehr hoher Antikörper-konzentrationen ohne histologische Untersuchung die Diagnose Zöliakie ausrei-chend sicher gestellt werden kann oder bei sehr niedrigen Konzentrationen auf einen bioptischen Ausschluss verzichtet werden kann, soll in einer weiteren be-reits in Planung befindlichen prospektiven Studie geklärt werden. Zusätzlich sollte die Diagnosestellung durch DGP-Tests bei besonders jungen Kindern in prospektiven Studien untersucht werden. Die vorliegende Arbeit legt gemeinsam mit anderen Untersuchungen den Grundstein dafür.

A doença celíaca (DC) é uma doença inflamatória induzida pela ingestão de glúten em indivíduos geneticamente predispostos e seu tratamento é baseado em uma dieta sem glúten por toda a vida. A doença celíaca refratária é um problema comum que afeta de 10% a 19% dos pacientes célicos tratados. Provavelmente, a contaminação da dieta por glúten é uma das razões principais para a persistência de sintomas em pacientes celíacos tratados, assim como a ingestão inadvertida de glúten, devido a rotulagem incorreta. Assim, o objetivo deste estudo foi avaliar a confiabilidade dos rótulos dos alimentos brasileiros processados, através de testes de contaminação de glúten nos seguintes grupos (a) produtos \"livres de glúten\" - preparados especificamente para a população celíaca; (b) produtos \"naturalmente sem glúten\" feitos com arroz, milho, soja e mandioca, utilizados por toda a população e (c) produtos rotulados com \"contém glúten\", mas que não apresentam glúten em sua composição no rótulo. Foram analisados 213 produtos alimentícios agrupados em: 115 produtos do grupo \"sem glúten\"; 86 produtos do grupo \"naturalmente sem glúten\" e 12 produtos do grupo rotulados com \"contém glúten\". O teor de glúten foi detectado e quantificado por ELISA-R5 (Ridascreen®gliadin) e os resultados foram expressos em ppm e mg/100 g de alimento. A linha de corte foi estabelecida em 20 ppm para a contaminação de glúten. Todas as contaminações por glúten foram confirmadas por Western-blotting. Resultados: (a) alimentos livres de glúten 15 das 115 (13%) apresentaram contaminação por glúten (20 ppm), (b) grupo de alimentos naturalmente sem glúten - 8 de 86 (9,3%) apresentaram contaminação por glúten (20 ppm); (c) grupo de alimentos rotulados com contem glúten - somente 2 de 12 (16,7%) apresentaram contaminação por glúten (20 ppm). A análise de Western-blotting confirmou 36 das 38 (95%) contaminações encontradas no ELISA-R5. CONCLUSÕES: Ambos os grupos de alimentos \"sem glúten\" e \"naturalmente sem glúten\" comercializados no Brasil apresentaram razoável porcentagem de contaminação por glúten, o que dificulta a realização de uma dieta adequada ao paciente celíaco. O grupo de alimentos rotulado \"com glúten\" não apresentou 100% de contaminação, o que revela que a rotulagem desses produtos deve ser feita como uma medida preventiva. Uma maior chance de contaminação pelo glúten foi observada para os produtos a base de arroz (13,6x), soja (13,3x) e milho (9,3x), mas não naqueles à base de mandioca. Em média, encontramos 10,8% (23 de 213) de contaminação de glúten para os alimentos analisados, um panorama positivo para a população brasileira celíaca, principalmente devido ao uso da mandioca, uma alternativa para a farinha de trigo. No entanto, a contaminação de glúten encontrada mostra a importância da quantificação de glúten em todos os alimentos industrializados. / Celiac disease (CD) is an inflammatory disorder induced by ingestion of gluten in genetically predisposed individuals and its treatment is based on a life-time gluten-free diet. Nonresponsive celiac disease is a common problem affecting from 10% to 19% of treated celiac patients. Probably a gluten contamination in diet is one of the major reasons for symptoms persistence in celiac patients as well as an inadvertent gluten intake due to a misleading nutritional label. The aim of this study was to evaluate the reliability of Brazilian processed food labels by testing gluten contamination in (a) gluten-free products - prepared specifically for the celiac population; (b) in naturally gluten-free products made with rice, corn, soy bean and cassava and used by all population and (c) in not gluten-free products labeled to contain gluten but not having it in their composition. We analyzed 213 food samples grouped accordingly to its type: 115 samples of \"gluten-free food, 86 samples of \"naturally gluten-free food and 12 samples of not-gluten free labeled products. The gluten content was detected and quantified by ELISA-R5 (Ridascreen® Gliadin) and the results were expressed in ppm and mg/100 g of food. A cut-off line was established in 20 ppm for gluten contamination. All gluten contaminations were confirmed by Western-blotting. Results: (a) Gluten-free foods - we found 100 of 115 samples (87%) with no contamination (< 20 ppm) and 15 of 115 (13%) showed gluten contamination 20 ppm; (b) Naturally Gluten-free foods - we found 78 of 86 samples (90,7%) showing no contamination (< 20 ppm) and 8 of 86 (9,3%) with gluten levels 20 ppm; (c) Not gluten-free foods - we found 10 of 12 samples (83,3%) showing no contamination (< 20 ppm) and 2 of 12 (16,7%) with gluten contamination 20 ppm. The Western-blotting analysis confirmed 36 of 38 (95%) contaminations found in the ELISA-R5. CONCLUSIONS: Both \"gluten-free and \"naturally gluten-free foods commercialized in Brazil have presented some gluten contamination making a restricted gluten-free diet hard to be achieved by the celiac population. Unexpectedly the not gluten-free group was not entirely contaminated showing a preventive measure in labeling by food companies. A higher odds ratio for gluten contamination was observed for products made with rice (13.6), soy bean (13.3) and corn (9.3) but not to cassava products (not significant). In general, we found a 10.8% (23 of 213) of gluten contamination for all food products analyzed, a positive panorama for the Brazilian celiac population mainly due to cassava products, an alternative for wheat starch. Nevertheless the gluten contamination found here leads us to the importance for a gluten quantification in all industrialized food to guarantee an appropriated diet to the Brazilian celiac group

La maladie cœliaque ou sprue cœliaque est une intolérance au gluten. Il s’agit d’une maladie inflammatoire de l’intestin liée à l’ingestion de gluten chez des personnes génétiquement susceptibles. Ce désordre présente une forte prévalence puisqu’il touche 1 % de la population mondiale. En l’état actuel des choses, il n’existe aucun outil pharmacologique pour traiter ou pallier à cette maladie. Cependant, grâce aux avancées dans la compréhension de sa pathogenèse, de nouvelles cibles thérapeutiques ont été identifiées. À l’heure actuelle, le seul traitement efficace consiste à suspendre la consommation de l’agent pathogène, à savoir le gluten. Le gluten est un ensemble de protéines de stockage des céréales contenu dans le blé, l’orge et le seigle. Le gluten du blé se subdivise en gluténines et gliadines. Ce sont ces dernières qui semblent les plus impliquées dans la maladie cœliaque. Les gliadines et ses protéines apparentées (i.e. sécalines et hordéines, respectivement dans le seigle et l’orge) sont riches en prolines et en glutamines, les rendant résistantes à la dégradation par les enzymes digestives et celles de la bordure en brosse. Les peptides résultant de cette digestion incomplète peuvent induire des réponses immunitaires acquises et innées. L’objectif principal de cette thèse était de tester un nouveau traitement d’appoint de la maladie cœliaque utile lors de voyages ou d’évènements ponctuels. Dans les années 80, une observation italienne montra l’inhibition de certains effets induits par des gliadines digérées sur des cultures cellulaires grâce à la co-incubation en présence de mannane: un polyoside naturel composé de mannoses. Malheureusement, ce traitement n’était pas applicable in vivo à cause de la dégradation par les enzymes du tractus gastro-intestinales du polymère, de par sa nature osidique. Les polymères de synthèse, grâce à la diversité et au contrôle de leurs propriétés physico-chimiques, se révèlent être une alternative attrayante à ce polymère naturel. L’objectif de cette recherche était d’obtenir un polymère liant la gliadine, capable d’interférer dans la genèse de la maladie au niveau du tube digestif, afin d’abolir les effets délétères induits par la protéine. Tout d’abord, des copolymères de type poly (hydroxyéthylméthacrylate)-co-(styrène sulfonate) (P(HEMA-co-SS)) ont été synthétisés par polymérisation radicalaire contrôlée par transfert d’atome (ATRP). Une petite bibliothèque de polymères a été préparée en faisant varier la masse molaire, ainsi que les proportions de chacun des monomères. Ces polymères ont ensuite été testés quant à leur capacité de complexer la gliadine aux pH stomacal et intestinal et les meilleurs candidats ont été retenus pour des essais cellulaires. Les travaux ont permis de montrer que le copolymère P(HEMA-co-SS) (45:55 mol%, 40 kDa) permettait une séquestration sélective de la gliadine et qu’il abolissait les effets induits par la gliadine sur différents types cellulaires. De plus, ce composé interférait avec la digestion de la gliadine, suggérant une diminution de peptides immunogènes impliqués dans la maladie. Ce candidat a été testé in vivo, sur un modèle murin sensible au gluten, quant à son efficacité vis-à-vis de la gliadine pure et d’un mélange contenant du gluten avec d’autres composants alimentaires. Le P(HEMA-co-SS) a permis de diminuer les effets sur les paramètres de perméabilité et d’inflammation, ainsi que de moduler la réponse immunitaire engendrée par l’administration de gliadine et celle du gluten. Des études de toxicité et de biodistribution en administration aigüe et chronique ont été réalisées afin de démontrer que ce dernier était bien toléré et peu absorbé suite à son administration par la voie orale. Enfin des études sur des échantillons de tissus de patients souffrants de maladie cœliaque ont montré un bénéfice therapeutique du polymère. L’ensemble des travaux présentés dans cette thèse a permis de mettre en évidence le potentiel thérapeutique du P(HEMA-co-SS) pour prévenir les désordres reliés à l’ingestion de gluten, indiquant que ce type de polymère pourrait être exploité dans un avenir proche. / Celiac Disease or celiac sprue is identified as a gluten intolerance. It is an inflammatory disorder of the intestine triggered by the ingestion of gluten in genetically susceptible individuals. This condition is highly prevalent because it affects up to 1% of the worldwide population. Nowadays, there is no pharmacological treatment available to treat or off set to the disease. Due to the huge progress in the understanding of the pathogenesis, new therapeutic targets have been discovered. At the present time, the only effective treatment remains the strict lifelong abandonment of the pathogen agent, gluten. Gluten encompasses the storage proteins in wheat, rye and barley. The wheat gluten is divided into glutenins and gliadins. The latter seem to be the most important trigger in the celiac disease. Gliadins and the related proteins (i.e. secalins and hordeins, respectively in rye and barley) are rich in prolin and glutamin residues, conferring them to be resistant by enzymatic digestion. The resulting peptides of the incomplete process may set off the inflammatory reaction. The main objective of this thesis was to test a new supportive therapy in treating celiac disease, useful in punctual occasion (i.e. during travel or social event when the gluten-free property cannot be ascertained). In the 1980’s, inhibition of some gliadin-induced effects on cell cultures were observed owing to mannan co-incubation. However, this compound, due to his osidic nature, may be cleaved by digestive enzymes in vivo. Synthetic polymers prove to be an attracting alternative owing to the tunability of their physical and chemical properties. The goal of this study was to obtain a polymeric gliadin-binder, interferring with the pathogenesis of the celiac disease in the gastro-intestinal tract, to abrogate the gliadin induced effects. Atom transfer radical polymerization was used to synthesize copolymers of the type poly (hydroxyethylméthacrylate)-co-(styren sulfonate) (P(HEMA-co-SS)). A small library of polymers varying in their molecular weight and in their constituting monomers ratio was prepared. The ability of these polymers to sequester gliadin was assessed at stomacal and intestinal pH. The best candidates were further evaluated in cell cultures. Our results revealed that a selective binding was obtained with the P(HEMA-co-SS) (45:55 mol%, 40 kDa). This compound abolished gliadin-induced effects on various cell lines. In addition, gliadin digestion was hindered, suggesting a decrease in the formation of immunogenic peptides known to trigger the diseases. In vivo experiments were additionally carried out on a murine model of gluten-sensitivity with this polymeric candidate. Its efficacy towards pure gliadin and gluten containing food was tested. P(HEMA-co-SS decreased gliadin–induced effect on permeability and inflammatory parameters and modulated the immune response due to gliadin/gluten gavage. Toxicity and biodistribution studies following acute and chronic administration were performed on murine to demonstrate that the polymer was well tolerated and not absorbed after oral administration. Finally, biopsies of patients suffering from CD disease exhibited therapeutic benefit of the polymer. Altogether, the results presented in this thesis evidenced the potential of P(HEMA-co-SS) to prevent the gluten-induced disorder, indicating that this type of polymer may be useful in a near future.

La maladie cœliaque ou sprue cœliaque est une intolérance au gluten. Il s’agit d’une maladie inflammatoire de l’intestin liée à l’ingestion de gluten chez des personnes génétiquement susceptibles. Ce désordre présente une forte prévalence puisqu’il touche 1 % de la population mondiale. En l’état actuel des choses, il n’existe aucun outil pharmacologique pour traiter ou pallier à cette maladie. Cependant, grâce aux avancées dans la compréhension de sa pathogenèse, de nouvelles cibles thérapeutiques ont été identifiées. À l’heure actuelle, le seul traitement efficace consiste à suspendre la consommation de l’agent pathogène, à savoir le gluten. Le gluten est un ensemble de protéines de stockage des céréales contenu dans le blé, l’orge et le seigle. Le gluten du blé se subdivise en gluténines et gliadines. Ce sont ces dernières qui semblent les plus impliquées dans la maladie cœliaque. Les gliadines et ses protéines apparentées (i.e. sécalines et hordéines, respectivement dans le seigle et l’orge) sont riches en prolines et en glutamines, les rendant résistantes à la dégradation par les enzymes digestives et celles de la bordure en brosse. Les peptides résultant de cette digestion incomplète peuvent induire des réponses immunitaires acquises et innées. L’objectif principal de cette thèse était de tester un nouveau traitement d’appoint de la maladie cœliaque utile lors de voyages ou d’évènements ponctuels. Dans les années 80, une observation italienne montra l’inhibition de certains effets induits par des gliadines digérées sur des cultures cellulaires grâce à la co-incubation en présence de mannane: un polyoside naturel composé de mannoses. Malheureusement, ce traitement n’était pas applicable in vivo à cause de la dégradation par les enzymes du tractus gastro-intestinales du polymère, de par sa nature osidique. Les polymères de synthèse, grâce à la diversité et au contrôle de leurs propriétés physico-chimiques, se révèlent être une alternative attrayante à ce polymère naturel. L’objectif de cette recherche était d’obtenir un polymère liant la gliadine, capable d’interférer dans la genèse de la maladie au niveau du tube digestif, afin d’abolir les effets délétères induits par la protéine. Tout d’abord, des copolymères de type poly (hydroxyéthylméthacrylate)-co-(styrène sulfonate) (P(HEMA-co-SS)) ont été synthétisés par polymérisation radicalaire contrôlée par transfert d’atome (ATRP). Une petite bibliothèque de polymères a été préparée en faisant varier la masse molaire, ainsi que les proportions de chacun des monomères. Ces polymères ont ensuite été testés quant à leur capacité de complexer la gliadine aux pH stomacal et intestinal et les meilleurs candidats ont été retenus pour des essais cellulaires. Les travaux ont permis de montrer que le copolymère P(HEMA-co-SS) (45:55 mol%, 40 kDa) permettait une séquestration sélective de la gliadine et qu’il abolissait les effets induits par la gliadine sur différents types cellulaires. De plus, ce composé interférait avec la digestion de la gliadine, suggérant une diminution de peptides immunogènes impliqués dans la maladie. Ce candidat a été testé in vivo, sur un modèle murin sensible au gluten, quant à son efficacité vis-à-vis de la gliadine pure et d’un mélange contenant du gluten avec d’autres composants alimentaires. Le P(HEMA-co-SS) a permis de diminuer les effets sur les paramètres de perméabilité et d’inflammation, ainsi que de moduler la réponse immunitaire engendrée par l’administration de gliadine et celle du gluten. Des études de toxicité et de biodistribution en administration aigüe et chronique ont été réalisées afin de démontrer que ce dernier était bien toléré et peu absorbé suite à son administration par la voie orale. Enfin des études sur des échantillons de tissus de patients souffrants de maladie cœliaque ont montré un bénéfice therapeutique du polymère. L’ensemble des travaux présentés dans cette thèse a permis de mettre en évidence le potentiel thérapeutique du P(HEMA-co-SS) pour prévenir les désordres reliés à l’ingestion de gluten, indiquant que ce type de polymère pourrait être exploité dans un avenir proche. / Celiac Disease or celiac sprue is identified as a gluten intolerance. It is an inflammatory disorder of the intestine triggered by the ingestion of gluten in genetically susceptible individuals. This condition is highly prevalent because it affects up to 1% of the worldwide population. Nowadays, there is no pharmacological treatment available to treat or off set to the disease. Due to the huge progress in the understanding of the pathogenesis, new therapeutic targets have been discovered. At the present time, the only effective treatment remains the strict lifelong abandonment of the pathogen agent, gluten. Gluten encompasses the storage proteins in wheat, rye and barley. The wheat gluten is divided into glutenins and gliadins. The latter seem to be the most important trigger in the celiac disease. Gliadins and the related proteins (i.e. secalins and hordeins, respectively in rye and barley) are rich in prolin and glutamin residues, conferring them to be resistant by enzymatic digestion. The resulting peptides of the incomplete process may set off the inflammatory reaction. The main objective of this thesis was to test a new supportive therapy in treating celiac disease, useful in punctual occasion (i.e. during travel or social event when the gluten-free property cannot be ascertained). In the 1980’s, inhibition of some gliadin-induced effects on cell cultures were observed owing to mannan co-incubation. However, this compound, due to his osidic nature, may be cleaved by digestive enzymes in vivo. Synthetic polymers prove to be an attracting alternative owing to the tunability of their physical and chemical properties. The goal of this study was to obtain a polymeric gliadin-binder, interferring with the pathogenesis of the celiac disease in the gastro-intestinal tract, to abrogate the gliadin induced effects. Atom transfer radical polymerization was used to synthesize copolymers of the type poly (hydroxyethylméthacrylate)-co-(styren sulfonate) (P(HEMA-co-SS)). A small library of polymers varying in their molecular weight and in their constituting monomers ratio was prepared. The ability of these polymers to sequester gliadin was assessed at stomacal and intestinal pH. The best candidates were further evaluated in cell cultures. Our results revealed that a selective binding was obtained with the P(HEMA-co-SS) (45:55 mol%, 40 kDa). This compound abolished gliadin-induced effects on various cell lines. In addition, gliadin digestion was hindered, suggesting a decrease in the formation of immunogenic peptides known to trigger the diseases. In vivo experiments were additionally carried out on a murine model of gluten-sensitivity with this polymeric candidate. Its efficacy towards pure gliadin and gluten containing food was tested. P(HEMA-co-SS decreased gliadin–induced effect on permeability and inflammatory parameters and modulated the immune response due to gliadin/gluten gavage. Toxicity and biodistribution studies following acute and chronic administration were performed on murine to demonstrate that the polymer was well tolerated and not absorbed after oral administration. Finally, biopsies of patients suffering from CD disease exhibited therapeutic benefit of the polymer. Altogether, the results presented in this thesis evidenced the potential of P(HEMA-co-SS) to prevent the gluten-induced disorder, indicating that this type of polymer may be useful in a near future.

<p>Zein is a major storage protein of corn, with unique amphiphilic film forming properties. It is insoluble in water, but soluble in 70% ethanol and acetic acid, and has been declared ‘generally recognized as safe’ (GRAS) by the FDA. Due to new advances in food nanotechnology, zein is being investigated for various applications such as biodegradable packaging, oral delivery of proteins and peptides, scaffold for tissue engineering, as well as biodegradable sensor platforms. The time consuming and highly complicated methods for toxin and allergen analysis in the food industry necessitates the need for a rapid, selective, compact and easy-to-use method of detection for analytes. In the scope of this dissertation, we investigated the feasibility of functional zein nanocomposite films and formation of a zein nanocomposite sensor assembly for rapid and highly selective electrochemical measurements of food toxins and allergens. Fabrication of a zein based electrochemical amperometric sensor assembly was studied, first through the comparison of various zein film characteristics changes with the application of Laponite®, graphene oxide and carbon nanotube nanoparticles, followed by a proof-of-concept study by detecting the gluten allergen protein gliadin. </p> <p>To mechanistically study the functional zein nanocomposite films, Laponite®, a silica nanoparticle, was added in the presence of 70% ethanol solvent and oleic acid plasticizer. The films were studied using various characterization techniques like transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), water contact angle measurements etc. Through Si-N bond formation between Laponite® and zein, fabricated zein nanocomposite films showed increase in surface hydrophobicity, water vapor barrier properties, tensile strength and Young’s modulus. Graphene oxide (GO), a carbon nanoparticle, was also incorporated into zein through the solvent casting process. Uniform dispersion of GO nanoparticles within zein matrix were confirmed up to 1% GO loading, and covalent and hydrogen bonding mechanisms were proposed. Similar to zein-Laponite® (Z-LAP) nanocomposites, zein-GO (Z-GO) showed increase in hydrophobic tendencies, rougher surface and a 300% improvement in Young’s modulus and 180% improvement in tensile strength at only 3% GO loading. Both nanoparticles increased tensile strength, thermal stability and water vapor barrier property of the films, indicating a potential for food packaging as an alternative application for the nanocomposite films.</p> Finally, the research focused on the fabrication of an electrochemical amperometric sensor, capable of detecting the protein gliadin, which is responsible for the allergic reaction with people having celiac disease. Novel biodegradable coatings made from zein nanocomposites: zein-graphene oxide, zein-Laponite® and zein-multiwalled carbon nanotubes (Z-CNT) using drop casting technique were tested for fabricating the electrochemical sensors using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV) techniques. As Z-CNT produced the strongest signals compared to other nanomaterials, the active tip of the electrochemical sensor was functionalized through a sequence of layer by layer deposition of Z-CNT nanocomposite, antibody and target analyte. Here, Z-CNT acts as a natural linker molecule with large number of functional groups, that causes immobilization of capture antibody and target, to ensure high sensor performance. Both CV curves and SWV curves indicated successful sequential immobilization of gliadin antibody onto the Z-CNT coated electrode. The Z-CNT biosensor was successfully able to give CV signals for gliadin toxins for as low as 0.5 ppm and was highly specific for gliadin in the presence of other interfering molecules, and remained stable over a 30-day period. The low-cost, thin, conductive zein films offered a promising alternative for protein immobilization platforms used in sensors and can be extended to other matrices in biosensors as well as other functional film applications