From the Sea to Paris and back: On the interdependence of economic warfare, weather conditions and the submarine campaign 1916-1917

Starting of from Avner Offer’s comment that the First World War was not only a war of steel and gold, but also of bread and potatoes (1989: 1) and my own research on British as well as Australian preparations for economic warfare and based on sources from the entente as well as the central powers but also from the United States, Canada and Australia, may presentation will focus on the interdependence of the measures taken by entente as well as central power authorities in the second half of 1916. Already a year before both sides had become aware that this war would not only be decided on the battlefield, but that the issues of primary as well as secondary resources would be decisive. Accordingly measures that could strike the enemy in this field were discussed and put into place more and more and this at time, when weather conditions caused a reduction of harvest all over Europe, Northern America and Argentina.

Tef – Cultivating a healthy gluten free lifestyle

Originally domesticated in Ethiopia, tef is a wholegrain cereal that has become a lifestyle food alternative in the West. Its appeal is due to its gluten free qualities and its light and soft texture which can easily be combined with other cuisines. Tef products including flour, bread, cookies and the flattened bread injera can be found in organic/health food stores in Europe and the USA or can be purchased online. It is estimated that there are more than 90 restaurants in Europe providing Ethiopian cuisine, at the heart of which is injera.

"Cruel sacrifices of popish preests" : Teología y cuestión eucarística en The Discoverie of Witchcraft de Reginald Scot

Las siguientes páginas intentarán echar luz sobre la teología desarrollada por Reginald Scot a partir de una de las temáticas más propias del siglo XVI: la cuestión eucarística. Uno de los objetivos es demostrar que la idea que el inglés tiene de la divinidad no sólo es la causa de su desmantelamiento de la demonología positiva, sino también de la aproximación en igual sentido al dogma católico de la transubstanciación. Lo que se plantea aquí es que Scot rechazaba las bases intelectuales de la persecución de brujas por el mismo motivo por el que negaba la transformación del pan y el vino en el cuerpo y sangre de Cristo. Producto del contexto histórico de su elaboración intelectual, el autor utiliza su teología como una herramienta de proselitismo anticatólico, relacionando al papismo con todas las creencias equivocadas en materia religiosa. A su vez, se abre la posibilidad de rastrear si el inglés propone una postura eucarística positiva, y -en caso de hacerlo- cómo se vincula con la posición oficial de la Iglesia de Inglaterra y la de las corrientes predominantes de la Reforma continental

"Cruel sacrifices of popish preests" : Teología y cuestión eucarística en The Discoverie of Witchcraft de Reginald Scot

Las siguientes páginas intentarán echar luz sobre la teología desarrollada por Reginald Scot a partir de una de las temáticas más propias del siglo XVI: la cuestión eucarística. Uno de los objetivos es demostrar que la idea que el inglés tiene de la divinidad no sólo es la causa de su desmantelamiento de la demonología positiva, sino también de la aproximación en igual sentido al dogma católico de la transubstanciación. Lo que se plantea aquí es que Scot rechazaba las bases intelectuales de la persecución de brujas por el mismo motivo por el que negaba la transformación del pan y el vino en el cuerpo y sangre de Cristo. Producto del contexto histórico de su elaboración intelectual, el autor utiliza su teología como una herramienta de proselitismo anticatólico, relacionando al papismo con todas las creencias equivocadas en materia religiosa. A su vez, se abre la posibilidad de rastrear si el inglés propone una postura eucarística positiva, y -en caso de hacerlo- cómo se vincula con la posición oficial de la Iglesia de Inglaterra y la de las corrientes predominantes de la Reforma continental

"Cruel sacrifices of popish preests" : Teología y cuestión eucarística en The Discoverie of Witchcraft de Reginald Scot

Las siguientes páginas intentarán echar luz sobre la teología desarrollada por Reginald Scot a partir de una de las temáticas más propias del siglo XVI: la cuestión eucarística. Uno de los objetivos es demostrar que la idea que el inglés tiene de la divinidad no sólo es la causa de su desmantelamiento de la demonología positiva, sino también de la aproximación en igual sentido al dogma católico de la transubstanciación. Lo que se plantea aquí es que Scot rechazaba las bases intelectuales de la persecución de brujas por el mismo motivo por el que negaba la transformación del pan y el vino en el cuerpo y sangre de Cristo. Producto del contexto histórico de su elaboración intelectual, el autor utiliza su teología como una herramienta de proselitismo anticatólico, relacionando al papismo con todas las creencias equivocadas en materia religiosa. A su vez, se abre la posibilidad de rastrear si el inglés propone una postura eucarística positiva, y -en caso de hacerlo- cómo se vincula con la posición oficial de la Iglesia de Inglaterra y la de las corrientes predominantes de la Reforma continental

A PCR-based method for discriminating between high molecular weight glutenin subunits Bx7 and Bx7* in Triticum aestivum L

The correct assignment of high molecular weight glutenin subunit variants is a key task in wheat breeding. However, the traditional analysis by protein electrophoresis is sometimes difficult and not very precise. This work describes a novel DNA marker for the accurate discrimination between the Glu-B1 locus subunits Bx7 and Bx7*. The analysis of one hundred and forty two bread wheat cultivars from different countries has highlighted a great number of misclassifications in the literature that could lead to wrong conclusions in studies of the relationship between glutenin composition and wheat quality.

Estudio molecular de gluteninas de alto y bajo peso molecular en Triticum aestivum ssp. vulgare L. y su relación con la calidad panadera

El trigo blando (Triticum aestivum ssp vulgare L., AABBDD, 2n=6x=42) presenta propiedades viscoélasticas únicas debidas a la presencia en la harina de las prolaminas: gluteninas y gliadinas. Ambos tipos de proteínas forman parte de la red de gluten. Basándose en la movilidad en SDS-PAGE, las gluteninas se clasifican en dos grupos: gluteninas de alto peso molecular (HMW-GS) y gluteninas de bajo peso molecular (LMW-GS). Los genes que codifican para las HMW-GS se encuentran en tres loci del grupo 1 de cromosomas: Glu-A1, Glu-B1 y Glu-D1. Cada locus codifica para uno o dos polipéptidos o subunidades. La variación alélica de las HMW-GS es el principal determinante de de la calidad harino-panadera y ha sido ampliamente estudiado tanto a nivel de proteína como de ADN. El conocimiento de estas proteínas ha contribuido sustancialmente al progreso de los programas de mejora para la calidad del trigo. Comparadas con las HMW-GS, las LMW-GS forman una familia proteica mucho más compleja. La mayoría de los genes LMW se localizan en el grupo 1 de cromosomas en tres loci: Glu-A3, Glu-B3 y Glu-D3 que se encuentran estrechamente ligados a los loci que codifican para gliadinas. El número de copias de estos genes ha sido estimado entre 10-40 en trigo hexaploide, pero el número exacto aún se desconoce debido a la ausencia de un método eficiente para diferenciar los miembros de esta familia multigénica. La nomenclatura de los alelos LMW-GS por electroforesis convencional es complicada, y diferentes autores asignan distintos alelos a la misma variedad lo que dificulta aún más el estudio de esta compleja familia. El uso de marcadores moleculares para la discriminación de genes LMW, aunque es una tarea dificil, puede ser muy útil para los programas de mejora. El objetivo de este trabajo ha sido profundizar en la relación entre las gluteninas y la calidad panadera y desarrollar marcadores moleculares que permitan ayudar en la correcta clasificación de HMW-GS y LMW-GS. Se han obtenido dos poblaciones de líneas avanzadas F4:6 a partir de los cruzamientos entre las variedades ‘Tigre’ x ‘Gazul’ y ‘Fiel’ x ‘Taber’, seleccionándose para los análisis de calidad las líneas homogéneas para HMW-GS, LMW-GS y gliadinas. La determinación alélica de HMW-GS se llevó a cabo por SDS-PAGE, y se complementó con análisis moleculares, desarrollándose un nuevo marcador de PCR para diferenciar entre las subunidades Bx7 y Bx7*del locus Glu-B1. Resumen 2 La determinación alélica para LMW-GS se llevó a cabo mediante SDS-PAGE siguiendo distintas nomenclaturas y utilizando variedades testigo para cada alelo. El resultado no fue concluyente para el locus Glu-B3, así que se recurrió a marcadores moleculares. El ADN de los parentales y de los testigos se amplificó usando cebadores diseñados en regiones conservadas de los genes LMW y fue posteriormente analizado mediante electroforesis capilar. Los patrones de amplificación obtenidos fueron comparados entre las distintas muestras y permitieron establecer una relación con los alelos de LMW-GS. Con este método se pudo aclarar la determinación alélica de este locus para los cuatro parentales La calidad de la harina fue testada mediante porcentaje de contenido en proteína, prueba de sedimentación (SDSS) y alveógrafo de Chopin (parámetros P, L, P/L y W). Los valores fueron analizados en relación a la composición en gluteninas. Las líneas del cruzamiento ‘Fiel’ x ‘Taber’ mostraron una clara influencia del locus Glu-A3 en la variación de los valores de SDSS. Las líneas que llevaban el nuevo alelo Glu-A3b’ presentaron valores significativamente mayores que los de las líneas con el alelo Glu-A3f. En las líneas procedentes del cruzamiento ‘Tigre ’x ‘Gazul’, los loci Glu-B1 y Glu-B3 loci mostraron ambos influencia en los parámetros de calidad. Los resultados indicaron que: para los valores de SDSS y P, las líneas con las HMW-GS Bx7OE+By8 fueron significativamente mejores que las líneas con Bx17+By18; y las líneas que llevaban el alelo Glu-B3ac presentaban valores de P significativamente superiores que las líneas con el alelo Glu-B3ad y significativamente menores para los valores de L . El análisis de los valores de calidad en relación a los fragmentos LMW amplificados, reveló un efecto significativo entre dos fragmentos (2-616 y 2-636) con los valores de P. La presencia del fragmento 2-636 estaba asociada a valores de P mayores. Estos fragmentos fueron clonados y secuenciados, confirmándose que correspondían a genes del locus Glu-B3. El estudio de la secuencia reveló que la diferencia entre ambos se hallaba en algunos SNPs y en una deleción de 21 nucleótidos que en la proteína correspondería a un InDel de un heptapéptido en la región repetida de la proteína. En este trabajo, la utilización de líneas que difieren en el locus Glu-B3 ha permitido el análisis de la influencia de este locus (el peor caracterizado hasta la fecha) en la calidad panadera. Además, se ha validado el uso de marcadores moleculares en la determinación alélica de las LMW-GS y su relación con la calidad panadera. Summary 3 Bread wheat (Triticum aestivum ssp vulgare L., AABBDD, 2n=6x=42) flour has unique dough viscoelastic properties conferred by prolamins: glutenins and gliadins. Both types of proteins are cross-linked to form gluten polymers. On the basis of their mobility in SDS-PAGE, glutenins can be classified in two groups: high molecular weight glutenins (HMW-GS) and low molecular weight glutenins (LMW-GS). Genes encoding HMW-GS are located on group 1 chromosomes in three loci: Glu-A1, Glu-B1 and Glu-D1, each one encoding two polypeptides, named subunits. Allelic variation of HMW-GS is the most important determinant for bread making quality, and has been exhaustively studied at protein and DNA level. The knowledge of these proteins has substantially contributed to genetic improvement of bread quality in breeding programs. Compared to HMW-GS, LMW-GS are a much more complex family. Most genes encoded LMW-GS are located on group 1 chromosomes. Glu-A3, Glu-B3 and Glu-D3 loci are closely linked to the gliadin loci. The total gene copy number has been estimated to vary from 10–40 in hexaploid wheat. However, the exact copy number of LMW-GS genes is still unknown, mostly due to lack of efficient methods to distinguish members of this multigene family. Nomenclature of LMW-GS alleles is also unclear, and different authors can assign different alleles to the same variety increasing confusion in the study of this complex family. The use of molecular markers for the discrimination of LMW-GS genes might be very useful in breeding programs, but their wide application is not easy. The objective of this work is to gain insight into the relationship between glutenins and bread quality, and the developing of molecular markers that help in the allele classification of HMW-GS and LMW-GS. Two populations of advanced lines F4:6 were obtained from the cross ‘Tigre’ x ‘Gazul’ and ‘Fiel’ x ‘Taber’. Lines homogeneous for HMW-GS, LMW-GS and gliadins pattern were selected for quality analysis. The allele classification of HMW-GS was performed by SDS-PAGE, and then complemented by PCR analysis. A new PCR marker was developed to undoubtedly differentiate between two similar subunits from Glu-B1 locus, Bx7 and Bx7*. The allele classification of LMW-GS was initially performed by SDS-PAGE following different established nomenclatures and using standard varieties. The results were not completely concluding for Glu-B3 locus, so a molecular marker system was applied. DNA from parental lines and standard varieties was amplified using primers designed in conserved domains of LMW genes and analyzed by capillary electrophoresis. The pattern of amplification products obtained was compared among samples and related to the protein allele classification. It was possible to establish a correspondence between specific amplification products and almost all LMW alleles analyzed. With this method, the allele classification of the four parental lines was clarified. Flour quality of F4:6 advanced lines were tested by protein content, sedimentation test (SDSS) and alveograph (P, L, P/L and W). The values were analyzed in relation to the lines prolamin composition. In the ‘Fiel’ x ‘Taber’ population, Glu-A3 locus showed an influence in SDSS values. Lines carrying new allele Glu-A3b’, presented a significantly higher SDSS value than lines with Glu-A3f allele. In the ‘Tigre ’x ‘Gazul’ population, the Glu-B1 and Glu-B3 loci also showed an effect in quality parameters, in SDSS, and P and L values. Results indicated that: for SDSS and P, lines with Bx7OE+By8 were significantly better than lines with Bx17+By18; lines carrying Glu-B3ac allele had a significantly higher P values than Glu-B3ad allele values. lines with and lower L The analysis of quality parameters and amplified LMW fragments revealed a significant influence of two peaks (2-616 y 2-636) in P values. The presence of 2-636 peak gave higher P values than 2-616. These fragments had been cloned and sequenced and identified as Glu-B3 genes. The sequence analysis revealed that the molecular difference between them was some SNPs and a small deletion of 21 nucleotides that in the protein would produce an InDel of a heptapeptide in the repetitive region. In this work, the analysis of two crosses with differences in Glu-3 composition has made possible to study the influence of LMG-GS in quality parameters. Specifically, the influence of Glu-B3, the most interesting and less studied loci has been possible. The results have shown that Glu-B3 allele composition influences the alveograph parameter P (tenacity). The existence of different molecular variants of Glu-B3 alleles have been assessed by using a molecular marker method. This work supports the use of molecular approaches in the study of the very complex LMW-GS family, and validates their application in the analysis of advanced recombinant lines for quality studies.

Some minor problems with B.E.M.

As it is well known B.E.M. works efficiently in the treatment of a bread class of potential and elasticity problems. In this paper we present the results of several runs established with linear elements in plane potential theory and treating the importance of singularities and the pattern and size of elements used in the boundary discretization.

Molecular characterization of Glu-B3 locus in wheat cultivars and segregating populations

Bread wheat quality constitutes a key trait for the demands of the baking industry as well as the broad consumer preferences. The role of the low molecular weight glutenin subunits (LMW-GS) with regard to bread quality is so far not well understood owing to their genetic complexity and to the use of different nomenclatures and standards for the LMW-GS assignment by different research groups, which has made difficult the undertaking of association studies between genotypes and bread quality. The development of molecular markers to carry out genetic characterization and allele determination is demanding. Nowadays, the most promising LMW gene marker system is based on PCR and high resolution capillary electrophoresis for the simultaneous analysis of the complete multigene family. The molecular analysis of the bread wheat Glu-B3 locus in F2 and F4:6 populations expressed the expected one-locus Mendelian segregation pattern, thus validating the suitability of this marker system for the characterization of LMW-GS genes in segregating populations, allowing for the successful undertaking of studies related to bread-making quality. Moreover, the Glu-B3 allele characterization of standard cultivars with the molecular marker system has revealed its potential as a complementary tool for the allelic determination of this complex multigene family.

Evaluación de la calidad funcional y sensorial en cultivares de Triticum aestivum ssp. vulgare y ssp. spelta en cultivo ecológico

En esta Tesis Doctoral se ha estudiado la influencia del cultivar sobre el comportamiento reológico y panadero de cinco cultivares de trigo sembrados en el mismo año y en el mismo ambiente, en condiciones de cultivo ecológico. Tres de ellos eran de trigo panadero (Triticum aestivum ssp. vulgare), ‘Bonpain’, ‘Craklin’ y ‘Sensas’ y los otros dos de trigo espelta (Triticum aestivum ssp. spelta), ‘Espelta Álava’ y ‘Espelta Navarra’. Actualmente, el alohexaploide trigo panadero (2n=6x=42 genomio AABBDD) supone en torno al 90% del trigo cultivado en el mundo. En cambio, el cultivo del trigo alohexaploide espelta (2n=6x=42 genomio AABBDD) se limita a pequeñas regiones de Europa y de América del Norte. En España, el cultivo de trigo espelta se ha mantenido durante años ligado a la región de Asturias, aunque en la actualidad su cultivo está empezando a diversificarse hacia otras regiones. Esto se debe, fundamentalmente, a su potencial nutricional y a su adaptabilidad a condiciones de agricultura sostenible. El reciente resurgimiento de la espelta en productos de panificación, se debe, en gran parte, a la percepción del consumidor de que se trata de un producto ”más saludable” y “más natural” y con menor requerimiento de insumos que los trigos modernos. A medida que el consumo de alimentos a base de harina de espelta aumenta, se plantea la necesidad de evaluar su calidad harino-panadera, nutricional y sensorial en comparación con los productos elaborados con variedades de trigo común. Se caracterizaron las gluteninas de alto peso molecular (HMW) y las puroindolinas de los cinco cultivares. Se evaluó la calidad del grano, la reología de sus masas y se analizó la calidad instrumental y sensorial de sus panes. Para tal fin se ha puesto a punto un protocolo de panificación adecuado a las características particulares de los trigos espelta y se ha propuesto para el análisis sensorial de los panes un protocolo de selección, entrenamiento y validación de jueces. Teniendo en cuenta la composición en gluteninas HMW de los cultivares, se comprobó su influencia en el volumen de sedimentación y en la fuerza panadera. La composición en puroindolinas se vió reflejada en el parámetro dureza del endospermo. Los resultados indicaron que hay diferencias entre trigo panadero y trigo espelta en parámetros como, la tenacidad y el equilibrio de sus masas, la capacidad de absorción de agua de la harina y el comportamiento de la masa durante el amasado. Los trigos espeltas mostraron menor valor en el tiempo en alcanzar la presión máxima y la tolerancia al amasado, mientras que presentaron valores superiores en el decaimiento a los 250 y 450 segundos respectivamente. Respecto a la calidad de los panes elaborados, los trigos espeltas tenían mayor elasticidad en la miga y mayores valores en el área y en el diámetro de sus alveolos. Estas diferencias en la estructura y textura de la miga fueron también detectadas a nivel sensorial por el panel de jueces. Mediante el perfil sensorial descriptivo, se determinó que uno de los dos panes elaborado con trigo espelta (‘Espelta Navarra’) fue el pan más complejo considerando conjuntamente los atributos de aroma y flavor. En este trabajo no se apreciaron diferencias entre ambos tipos de trigo ni en el contenido en proteína, ni en minerales, ni en la viscosidad de su almidón. ABSTRACT In this Doctoral Thesis, the influence of various cultivars on rheological and baking behavior was studied. Five wheat cultivars were used, all planted in the same year and same organic farming environment. Three were bread wheat (Triticum aestivum ssp. vulgare), 'Bonpain', 'Craklin' and 'Sensas' and the other two were spelt wheat (Triticum aestivum ssp. spelta) , 'Espelta Álava' and 'Espelta Navarra' . Currently, the allohexaploid bread wheat (2n=6x=42 genome AABBDD) represents about 90% of global wheat production. On the other hand, allohexaploid spelt wheat (2n=6x=42 genome AABBDD) is merely produced in small areas of Europe and North America. For many years, the cultivation of spelt wheat in Spain was limited to the region of Asturias, although nowadays its production has begun to spread into other regions. This is owing to its nutritional potential and adaptability to conditions of sustainable agriculture. The recent resurgence of spelt in baking products is mainly due to consumers perception of it, as "healthier" and "more natural", and to the fewer agricultural input requirements compared to modern wheat products. As the consumption of foods made from spelt flour increases, there is a need to assess its baking, nutritional and sensory quality, compared to products made with common varieties of wheat. High molecular weight glutenins and puroindolines from the five cultivars were characterized. The quality of the grain and the rheology of the dough were evaluated and the instrumental and sensory quality of its breads were analyzed. To this end it a baking protocol was appropriately developed to the particular characteristics of spelt wheat and a selection protocol was proposed for the sensory analysis of breads, after proper training and validation of judges. Considering the HMW glutenin composition of the cultivars, the influence on the sedimentation volume and the baking strength was proven. The composition of puroindolines was reflected in the endosperm hardness parameter. The results show that there are differences between bread wheat and spelt wheat on parameters such as the tenacity and tenacity/elasticity ratio of their masses, the water absorption capacity of the flour and the behavior of the dough during kneading. The values for total time to reach maximum pressure and tolerance to mixing were lower for spelt wheat, and higher values were found for the drop at 250 s and 450 s. Regarding the quality of manufactured bread, spelt wheat had the greatest elasticity of the crumb and higher values in the area and diameter of the cells. These differences in the structure and texture of the crumb were also noticed at a sensory level by the panel of judges. It was determined by a descriptive sensory profile that one of the two loaves of bread made with spelt ('Espelta Navarra') was the most complex in the sense of its attributes of scents and flavors altogether. In this study, no differences were appreciated between the two types of wheat or the protein composition, or minerals or viscosity of the starch.

Subgenome chromosome walking in wheat: A 450-kb physical contig in Triticum monococcum L. spans the Lr10 resistance locus in hexaploid wheat (Triticum aestivum L.)

For many agronomically important plant genes, only their position on a genetic map is known. In the absence of an efficient transposon tagging system, such genes have to be isolated by map-based cloning. In bread wheat Triticum aestivum, the genome is hexaploid, has a size of 1.6 × 1010 bp, and contains more than 80% of repetitive sequences. So far, this genome complexity has not allowed chromosome walking and positional cloning. Here, we demonstrate that chromosome walking using bacterial artificial chromosome (BAC) clones is possible in the diploid wheat Triticum monococcum (Am genome). BAC end sequences were mostly repetitive and could not be used for the first walking step. New probes corresponding to rare low-copy sequences were efficiently identified by low-pass DNA sequencing of the BACs. Two walking steps resulted in a physical contig of 450 kb on chromosome 1AmS. Genetic mapping of the probes derived from the BAC contig demonstrated perfect colinearity between the physical map of T. monococcum and the genetic map of bread wheat on chromosome 1AS. The contig genetically spans the Lr10 leaf rust disease resistance locus in bread wheat, with 0.13 centimorgans corresponding to 300 kb between the closest flanking markers. Comparison of the genetic to physical distances has shown large variations within 350 kb of the contig. The physical contig can now be used for the isolation of the orthologous regions in bread wheat. Thus, subgenome chromosome walking in wheat can produce large physical contigs and saturate genomic regions to support positional cloning.

Sequence-tagged microsatellite profiling (STMP): a rapid technique for developing SSR markers

We describe a technique, sequence-tagged microsatellite profiling (STMP), to rapidly generate large numbers of simple sequence repeat (SSR) markers from genomic or cDNA. This technique eliminates the need for library screening to identify SSR-containing clones and provides an ∼25-fold increase in sequencing throughput compared to traditional methods. STMP generates short but characteristic nucleotide sequence tags for fragments that are present within a pool of SSR amplicons. These tags are then ligated together to form concatemers for cloning and sequencing. The analysis of thousands of tags gives rise to a representational profile of the abundance and frequency of SSRs within the DNA pool, from which low copy sequences can be identified. As each tag contains sufficient nucleotide sequence for primer design, their conversion into PCR primers allows the amplification of corresponding full-length fragments from the pool of SSR amplicons. These fragments permit the full characterisation of a SSR locus and provide flanking sequence for the development of a microsatellite marker. Alternatively, sequence tag primers can be used to directly amplify corresponding SSR loci from genomic DNA, thereby reducing the cost of developing a microsatellite marker to the synthesis of just one sequence-specific primer. We demonstrate the utility of STMP by the development of SSR markers in bread wheat.

Targeted development of informative microsatellite (SSR) markers

We describe a novel approach, selectively amplified microsatellite (SAM) analysis, for the targeted development of informative simple sequence repeat (SSR) markers. A modified selectively amplified microsatellite polymorphic loci assay is used to generate multi-locus SSR fingerprints that provide a source of polymorphic DNA markers (SAMs) for use in genetic studies. These polymorphisms capture the repeat length variation associated with SSRs and allow their chromosomal location to be determined prior to the expense of isolating and characterising individual loci. SAMs can then be converted to locus-specific SSR markers with the design and synthesis of a single primer specific to the conserved region flanking the repeat. This approach offers a cost-efficient and rapid method for developing SSR markers for predetermined chromosomal locations and of potential informativeness. The high recovery rate of useful SSR markers makes this strategy a valuable tool for population and genetic mapping studies. The utility of SAM analysis was demonstrated by the development of SSR markers in bread wheat.

Innovative analytical strategies for the development of sensor devices and mass spectrometry methods

Il lavoro presentato in questa tesi di Dottorato è incentrato sullo sviluppo di strategie analitiche innovative basate sulla sensoristica e su tecniche di spettrometria di massa in ambito biologico e della sicurezza alimentare. Il primo capitolo tratta lo studio di aspetti metodologici ed applicativi di procedure sensoristiche per l’identificazione e la determinazione di biomarkers associati alla malattia celiaca. In tale ambito, sono stati sviluppati due immunosensori, uno a trasduzione piezoelettrica e uno a trasduzione amperometrica, per la rivelazione di anticorpi anti-transglutaminasi tissutale associati a questa malattia. L’innovazione di questi dispositivi riguarda l’immobilizzazione dell’enzima tTG nella conformazione aperta (Open-tTG), che è stato dimostrato essere quella principalmente coinvolta nella patogenesi. Sulla base dei risultati ottenuti, entrambi i sistemi sviluppati si sono dimostrati una valida alternativa ai test di screening attualmente in uso per la diagnosi della celiachia. Rimanendo sempre nel contesto della malattia celiaca, ulteriore ricerca oggetto di questa tesi di Dottorato, ha riguardato lo sviluppo di metodi affidabili per il controllo di prodotti “gluten-free”. Il secondo capitolo tratta lo sviluppo di un metodo di spettrometria di massa e di un immunosensore competitivo per la rivelazione di prolammine in alimenti “gluten-free”. E’ stato sviluppato un metodo LC-ESI-MS/MS basato su un’analisi target con modalità di acquisizione del segnale selected reaction monitoring per l’identificazione di glutine in diversi cereali potenzialmente tossici per i celiaci. Inoltre ci si è focalizzati su un immunosensore competitivo per la rivelazione di gliadina, come metodo di screening rapido di farine. Entrambi i sistemi sono stati ottimizzati impiegando miscele di farina di riso addizionata di gliadina, avenine, ordeine e secaline nel caso del sistema LC-MS/MS e con sola gliadina nel caso del sensore. Infine i sistemi analitici sono stati validati analizzando sia materie prime (farine) che alimenti (biscotti, pasta, pane, etc.). L’approccio sviluppato in spettrometria di massa apre la strada alla possibilità di sviluppare un test di screening multiplo per la valutazione della sicurezza di prodotti dichiarati “gluten-free”, mentre ulteriori studi dovranno essere svolti per ricercare condizioni di estrazione compatibili con l’immunosaggio competitivo, per ora applicabile solo all’analisi di farine estratte con etanolo. Terzo capitolo di questa tesi riguarda lo sviluppo di nuovi metodi per la rivelazione di HPV, Chlamydia e Gonorrhoeae in fluidi biologici. Si è scelto un substrato costituito da strips di carta in quanto possono costituire una valida piattaforma di rivelazione, offrendo vantaggi grazie al basso costo, alla possibilità di generare dispositivi portatili e di poter visualizzare il risultato visivamente senza la necessità di strumentazioni. La metodologia sviluppata è molto semplice, non prevede l’uso di strumentazione complessa e si basa sull’uso della isothermal rolling-circle amplification per l’amplificazione del target. Inoltre, di fondamentale importanza, è l’utilizzo di nanoparticelle colorate che, essendo state funzionalizzate con una sequenza di DNA complementare al target amplificato derivante dalla RCA, ne permettono la rivelazione a occhio nudo mediante l’uso di filtri di carta. Queste strips sono state testate su campioni reali permettendo una discriminazione tra campioni positivi e negativi in tempi rapidi (10-15 minuti), aprendo una nuova via verso nuovi test altamente competitivi con quelli attualmente sul mercato.

Base de dados de informação nutricional, relacionados com doenças crônicas não transmissíveis, de alimentos comercializados no Brasil

A recente evolução das doenças crônicas não transmissíveis (DCNT), possivelmente associadas às mudanças de hábitos alimentares, tem sido um desafio para a promoção da alimentação saudável em vários países, inclusive no Brasil, onde o sódio tem sido o foco de atenção, pela possibilidade da redução da sua elevada ingestão ser uma das medidas com melhor custo benefício para saúde pública. É necessário conhecer o conteúdo de sódio e de componentes específicos, relacionados às DCNT nos alimentos comercializados no país, para orientar o consumidor na seleção adequada dos alimentos e mesmo para modificar sua composição; no entanto, nas bases de dados, o conteúdo desse mineral está presente em um número reduzido de alimentos. O objetivo da presente pesquisa foi a elaboração de uma base de dados de alimentos processados com componentes específicos associados às DCNT, dando ênfase ao sódio, e avaliar o uso dessa base de dados para estimar a sua ingestão. Informações nutricionais de rótulos e de websites de indústrias de alimentos foram coletadas para inclusão na base de dados, elaborada de acordo com as diretrizes do International collaborative project to compare and monitor the nutritional composition of processed foods, coordenado pelo The George Institute for Global Health (Austrália). A avaliação da variação do conteúdo de sódio foi realizada para alguns alimentos processados presentes na base de dados, considerando os de maior consumo nacional. O conteúdo de sódio em refeições de restaurantes populares de São Paulo foi analisado por espectrofotometria de absorção atômica de chama e estimado pela base de dados elaborada para comparação. Na base de dados estão incluídas informações de 2.319 alimentos distribuídos em 14 grupos. Foi observada grande variação no conteúdo de sódio entre diferentes tipos de pães de forma industrializados, de salsichas, de linguiças, de queijos e iogurtes. O conteúdo de sódio analisado nas refeições (base integral) variou de 215,9 mg a 427,9 mg por 100 kcal, enquanto que o conteúdo de sódio estimado variou de 204,2 mg a 486,8 mg por 100 kcal (418 kJ). A análise do coeficiente de correlação entre valores analíticos e estimados do conteúdo de sódio em refeições mostrou forte correlação entre esses dados para dois restaurantes (r=0,703 e 0,897) e moderada correlação para outros dois (r=0,513 e 0,622) dos cinco restaurantes estudados, indicando que através da base de dados elaborada é possível obter uma estimativa da ingestão de sódio. A importância de se conhecer o conteúdo de sódio de refeições e/ou alimentos está na possibilidade de uso dessas informações para orientar a redução do sal empregado no preparo da refeição, e ampliar para o consumidor informações que permitam identificar alternativas para redução do consumo de sal.