Stability and kinetics of nucleic acid triplexes with chimaeric DNA/RNA third strands

A series of chimaeric DNA/RNA triplex-forming oligonucleotides (TFOs) with identical base-sequence but varying sequential composition of the sugar residues were prepared. The structural, kinetic and thermodynamic properties of triplex formation with their corresponding double-helical DNA target were investigated by spectroscopic methods. Kinetic and thermodynamic data were obtained from analysis of non-equilibrium UV-melting- and annealing curves in the range of pH 5.1 to 6.7 in a 10 mM citrate/phosphate buffer containing 0.1M NaCl and 1 mM EDTA. It was found that already single substitutions of ribo- for deoxyribonucleotides in the TFOs greatly affect stability and kinetics of triplex formation in a strongly sequence dependent manner. Within the sequence context investigated, triplex stability was found to increase when deoxyribonucleotides were present at the 5'-side and ribonucleotides in the center of the TFO. Especially the substitution of thymidines for uridines in the TFO was found to accelerate both, the association and dissociation process, in a strongly position-dependent way. Differential structural information on triplexes and TFO single-strands was obtained from CD-spectroscopy and gel mobility experiments. Only minor changes were observed in the CD spectra of the triplexes at all pH values investigated, and the electrophoretic mobility was nearly identical in all cases, indicating a high degree of structural similarity. In contrast, the single-stranded TFOs showed high structural variability as determined in the same way. The results are discussed in the context of the design of TFOs for therapeutic or biochemical applications.

Conformational diversity versus nucleic acid triplex stability, a combinatorial study

The stability of a triple helix formed between a DNA duplex and an incoming oligonucleotide strand strongly depends on the solvent conditions and on intrinsic chemical and conformational factors. Attempts to increase triple helix stability in the past included chemical modification of the backbone, sugar ring, and bases in the third strand. However, the predictive power of such modifications is still rather poor. We therefore developed a method that allows for rapid screening of conformationally diverse third strand oligonucleotides for triplex stability in the parallel pairing motif to a given DNA double helix sequence. Combinatorial libraries of oligonucleotides of the requisite (fixed) base composition and length that vary in their sugar unit (ribose or deoxyribose) at each position were generated. After affinity chromatography against their corresponding immobilized DNA target duplex, utilizing a temperature gradient as the selection criterion, the oligonucleotides forming the most stable triple helices were selected and characterized by physicochemical methods. Thus, a series of oligonucleotides were identified that allowed us to define basic rules for triple helix stability in this conformationally diverse system. It was found that ribocytidines in the third strand increase triplex stability relative to deoxyribocytidines independently of the neighboring bases and position along the strand. However, remarkable sequence-dependent differences in stability were found for (deoxy)thymidines and uridines

Nucleic-Acid Analogs with Restricted Conformational Flexibility in the Sugar-Phosphate Backbone (‘Bicyclo-DNA’). Part 5. Synthesis, Characterization, and Pairing Properties of Oligo-αlpha-D-(bicyclodeoxynucleotides) of the Bases Adenine and Thymine (αlpha-Bicyclo-DNA)

10.1002/hlca.19950780816.abs A conformational analysis of the (3′S,5′R)-2′-deoxy-3′,5′-ethano-α-D-ribonucleosides (a-D-bicyclodeoxynucleosides) based on the X-ray analysis of N4-benzoyl-α-D-(bicyclodeoxycytidine) 6 and on 1H-NMR analysis of the α-D-bicyclodeoxynucleoside derivatives 1-7 reveals a rigid sugar structure with the furanose units in the l′-exo/2′-endo conformation and the secondary OH groups on the carbocyclic ring in the pseudoequatorial orientation. Oligonucleotides consisting of α-D-bicyclothymidine and α-D-bicyclodeoxyadenosine were successfully synthesized from the corresponding nucleosides by phosphoramidite methodology on a DNA synthesizer. An evaluation of their pairing properties with complementary natural RNA and DNA by means of UV/melting curves and CD spectroscopy show the following characteristics: i) α-bcd(A10) and α-bcd(T10) (α = short form of α-D)efficiently form complexes with complementary natural DNA and RNA. The stability of these hybrids is comparable or slightly lower as those with natural β-d(A10) or β-d(T10)( β = short form ofβ-D). ii) The strand orientation in α-bicyclo-DNA/β-DNA duplexes is parallel as was deduced from UV/melting curves of decamers with nonsymmetric base sequences. iii) CD Spectroscopy shows significant structural differences between α-bicyclo-DNA/β-DNA duplexes compared to α-DNA/β-DNA duplexes. Furthermore, α-bicyclo-DNA is ca. 100-fold more resistant to the enzyme snake-venom phosphodiesterase with respect to β-DNA and about equally resistant as α-DNA.

Folic acid fortification, maternal obesity and neural tube defects: Policy implications

In 1996, the Food and Drug Administration (FDA) mandated that beginning in January 1998, flour and other enriched grain products be fortified with 140 μg of folic acid per 100 g of grain to prevent neural tube defects (NTDs) that occur in approximately 1 in 1,000 pregnancies in the United States (U.S.). Although this program has demonstrated important public health effects, it is argued that current fortification levels may not be enough to prevent all folic acid-preventable NTD cases. This study reviews published literature, on folic acid fortification in the U.S. and countries with mandatory folic acid fortification programs reported after 1992 and through January 2008. Published studies are evaluated to determine if the current level of folic acid fortification in the U.S. is adequate to prevent the most common forms of NTDs (spina bifida and anencephaly), particularly among overweight and obese women. ^ Although consistent improvement in blood folate levels of child bearing age women is reported in almost all studies, the RBC folate concentration has not reached the level associated with the most significant reduction of risk for NTDs (906 nmol/L); approximately half of the potentially preventable NTDs are prevented by fortification at the current U.S. level. Furthermore, the blood folate status of women in higher BMI categories (obese or overweight) has not improved as much as among women in lower BMI categories. Therefore, women classified as overweight or obese have not benefited from the preventive effects of folic acid fortification as much as normal or underweight women. ^ To reduce risk of folate preventable NTDs, especially in overweight and obese women, it may be necessary to increase the current level of folic acid fortification. However, further research is required to determine the optimal levels of fortification to achieve this goal without causing adverse health effects in the general population. ^

HEALTH STATUS - HEALTH SERVICE RESOURCES - ECONOSOCIOCULTURAL DEVELOPMENT: IMPLICATIONS FOR HEALTH CARE POLICY IN AFRICA

Rational health services planning requires an examination of the effects of various factors on the health status of a population within a given set of socioeconomic circumstances. The commonly accepted explanations for improved health in the less developed countries (LDCs) are: Health Service Resources available to a population, Environmental and Life conditions, and the Econosociocultural Characteristics of the population.^ In the context of the low economic base from which many LDCs initiate development activities, a strong imperative exists for identifying in which of these major areas public health policy would be most effective in terms of improving health. A new conceptual model is proposed that would be used for future policy analyses to assess what changes in health status of populations in LDCs can be expected as direct functions of increased health service resources, and of improved environmental and econosociocultural conditions.^ While direct policy analysis is ill-advised at this time due to data inadequacy, the model is illustrated using data presently available for twenty-five relatively homogeneous Sub-Sahara African countries. Within the limitations of available data, study findings indicate that while econosociocultural conditions were the most important explanatory factors of the three major independent variables in 1970, health service resources became the most important in 1975. Study findings are inconclusive at this time with regards to the relative contributions of physicians and medical assistants in explaining variances in mortality in these countries.^ Because of the deficient nature of available data, study findings should be interpreted very cautiously. Tests of statistical significance of study findings were by-passed because of their situational technical inappropriateness. This study is significant in being the first of its kind and scope to focus on the Sub-Sahara African region of the World Health Organization, using the Wroclaw Taxonomic Method in conjunction with a stepwise regression technique. It is desirable, therefore, to examine the observed magnitude and directional consistency of all hypothesized relationships, even if evidence is inconclusive. ^

Factors associated with HPV16 status and survival among patients with squamous cell carcinoma of the oropharynx

Background: Squamous cell carcinoma of the oropharynx (SCCOP) is characterized by local tumor aggressiveness, high recurrence rates, a high incidence of second primary tumors, and medical comorbidities. Significant trends in demographic and clinical characteristics as well as survival among SCCOP patients have been observed over time, likely owing to the changing etiology of the disease. Human papillomavirus type 16 (HPV16) infection is associated with a significant proportion of these cancers. Biomarkers that may aid in identifying patients that are at higher risk of recurrence and death are important so that these patients may be followed more closely to improve their quality of life. ^ Study population and methods: The retrospective review (Specific Aim 2) included 3891 newly diagnosed, previously untreated patients presenting to our institution between 1955 and 2004. A total of 2299 patients treated at our institution were included in survival and recursive partitioning analysis. The prospective cohort study (Specific Aim 3) included 266 patients presenting to our institution between January 2006 and September 2009. ^ Results: The results from the retrospective review showed that over time, patients presented at younger ages and were more likely to have base of tongue/tonsil tumors and to be never/former smokers and moreover survival improved significantly over time. In survival and recursive partitioning analyses, the TNM staging system was efficient in prognosticating patients prior to 1995. However, in the recent decade, the TNM staging system was completely inadequate. The factors having the greatest positive effect on overall survival since 1995 were those common to HPV-associated SCCOP. The results from the prospective cohort study indicate that patients with high nodal stage and those with late stage disease have increased levels of pretreatment serum HPV DNA. ^ Conclusions: We saw a distinct improvement in survival among SCCOP patients over the past 50 years at our institution. The main factors contributing to this were changes in clinical characteristics, in particular surrogates for HPV status. The current TNM staging system for SCCOP is inadequate and incorporation of HPV status (and perhaps smoking status) is encouraged. Furthermore, although pretreatment circulating levels of HPV DNA was associated with higher N category and overall disease stage, it has limited utility as a marker for recurrence among SCCOP patients.^

Lugar que ocupa la evaluación en los proyectos de investigación. Una opinión construida en base a la experiencia

El presente trabajo surge ante una invitación por parte de las autoridades del Departamento de Educación Física para participar de las 3ras. Jornadas de Investigación en Educación Corporal. Tiene como intención dar respuesta a la convocatoria en cuanto a plantear opinión y experiencia acerca del lugar que ocupa la evaluación en los Proyectos de Investigación. Se parte de contextualizar tres núcleos temáticos polísémicos y de variabilidad interpretativa como son: opinión, experiencia y evaluación. El trabajo se centra en el planteo de cuestiones e interrogantes a partir de un breve análisis de las políticas sobre evaluación con inicio en la década del 90 y realiza un recorte en el análisis de las normas vigentes y que tienen como cometido por una lado impulsar la generación de conocimientos a partir de la elaboración de proyectos de investigación y por otro pautar aspectos a considerar al momento de acreditar los proyectos de referencia. En este sentido se analiza en forma sucinta el Decreto 2427/93 y con especial énfasis los Artículos 31 a 34 de la Resolución nº 811/03- Manual de Procedimientos del MECyT. El texto concluye abogando por la necesidad de construir una cultura evaluativa en el ámbito académico- universitario.

Lugar que ocupa la evaluación en los proyectos de investigación. Una opinión construida en base a la experiencia

El presente trabajo surge ante una invitación por parte de las autoridades del Departamento de Educación Física para participar de las 3ras. Jornadas de Investigación en Educación Corporal. Tiene como intención dar respuesta a la convocatoria en cuanto a plantear opinión y experiencia acerca del lugar que ocupa la evaluación en los Proyectos de Investigación. Se parte de contextualizar tres núcleos temáticos polísémicos y de variabilidad interpretativa como son: opinión, experiencia y evaluación. El trabajo se centra en el planteo de cuestiones e interrogantes a partir de un breve análisis de las políticas sobre evaluación con inicio en la década del 90 y realiza un recorte en el análisis de las normas vigentes y que tienen como cometido por una lado impulsar la generación de conocimientos a partir de la elaboración de proyectos de investigación y por otro pautar aspectos a considerar al momento de acreditar los proyectos de referencia. En este sentido se analiza en forma sucinta el Decreto 2427/93 y con especial énfasis los Artículos 31 a 34 de la Resolución nº 811/03- Manual de Procedimientos del MECyT. El texto concluye abogando por la necesidad de construir una cultura evaluativa en el ámbito académico- universitario.

Lugar que ocupa la evaluación en los proyectos de investigación. Una opinión construida en base a la experiencia

El presente trabajo surge ante una invitación por parte de las autoridades del Departamento de Educación Física para participar de las 3ras. Jornadas de Investigación en Educación Corporal. Tiene como intención dar respuesta a la convocatoria en cuanto a plantear opinión y experiencia acerca del lugar que ocupa la evaluación en los Proyectos de Investigación. Se parte de contextualizar tres núcleos temáticos polísémicos y de variabilidad interpretativa como son: opinión, experiencia y evaluación. El trabajo se centra en el planteo de cuestiones e interrogantes a partir de un breve análisis de las políticas sobre evaluación con inicio en la década del 90 y realiza un recorte en el análisis de las normas vigentes y que tienen como cometido por una lado impulsar la generación de conocimientos a partir de la elaboración de proyectos de investigación y por otro pautar aspectos a considerar al momento de acreditar los proyectos de referencia. En este sentido se analiza en forma sucinta el Decreto 2427/93 y con especial énfasis los Artículos 31 a 34 de la Resolución nº 811/03- Manual de Procedimientos del MECyT. El texto concluye abogando por la necesidad de construir una cultura evaluativa en el ámbito académico- universitario.

Absorption and mobility of foliar-applied boron in soybean as affected by plant boron status and application as a polyol complex

In the present study (i) the impact of plant Boron (B) status on foliar B absorption and (ii) the effect of B complexation with polyols (sorbitol or mannitol) on B absorption and translocation was investigated. Soybean (Glycine max (L.) Meer.) plants grown in nutrient solution containing 0 μM, 10 μM, 30 μM or 100 μM 11B labelled boric acid (BA) were treated with 50 mM 10B labelled BA applied to the basal parts of two leaflets of one leaf, either pure or in combination with 500 mM sorbitol or mannitol. After one week, 10B concentrations in different plant parts were determined. In B deficient leaves (0 μM 11B), 10B absorption was significantly lower than in all other treatments (9.7% of the applied dose vs. 26%–32%). The application of BA in combination with polyols increased absorption by 18–25% as compared to pure BA. The absolute amount of applied 10B moving out of the application zone was lowest in plants with 0 μM 11B supply (1.1% of the applied dose) and highest in those grown in 100 μM 11B (2.8%). The presence of sorbitol significantly decreased the share of mobile 10B in relation to the amount absorbed. The results suggest that 11B deficiency reduces the permeability of the leaf surface for BA. The addition of polyols may increase 10B absorption, but did not improve 10B distribution within the plant, which was even hindered when applied a sorbitol complex.

Distribution of indole-3-acetic acid in Petunia hybrida shoot tip cuttings and relationship between auxin transport, carbohydrate metabolism and adventitious root formation.

To determine the contribution of polar auxin transport (PAT) to auxin accumulation and to adventitious root (AR) formation in the stem base of Petunia hybrida shoot tip cuttings, the level of indole-3-acetic acid (IAA) was monitored in non-treated cuttings and cuttings treated with the auxin transport blocker naphthylphthalamic acid (NPA) and was complemented with precise anatomical studies. The temporal course of carbohydrates, amino acids and activities of controlling enzymes was also investigated. Analysis of initial spatial IAA distribution in the cuttings revealed that approximately 40 and 10% of the total IAA pool was present in the leaves and the stem base as rooting zone, respectively. A negative correlation existed between leaf size and IAA concentration. After excision of cuttings, IAA showed an early increase in the stem base with two peaks at 2 and 24h post excision and, thereafter, a decline to low levels. This was mirrored by the expression pattern of the auxin-responsive GH3 gene. NPA treatment completely suppressed the 24-h peak of IAA and severely inhibited root formation. It also reduced activities of cell wall and vacuolar invertases in the early phase of AR formation and inhibited the rise of activities of glucose-6-phosphate dehydrogenase and phosphofructokinase during later stages. We propose a model in which spontaneous AR formation in Petunia cuttings is dependent on PAT and on the resulting 24-h peak of IAA in the rooting zone, where it induces early cellular events and also stimulates sink establishment. Subsequent root development stimulates glycolysis and the pentosephosphate pathway

Potencial de la producción de bioelectricidad de las Comarcas Agrarias de la Comunidad de Madrid en base a centeno y triticale

En el presente trabajo se estudia la producción potencial de biomasa procedente de los cultivos de centeno y triticale en las seis comarcas agrarias de la Comunidad de Madrid (CM) y la posibilidad de su aplicación a la producción de bioelectricidad en cada una de ellas. En primer lugar se realiza un estudio bibliográfico de la situación actual de la bioelectricidad. Uno de los principales datos a tener en cuenta es que en el PER 2011- 2020 se estima que el total de potencia eléctrica instalada a partir de biomasa en España en el año 2020 sea de 1.350 MW, unas dos veces y media la existente a finales de 2010. Además, se comenta el estado de la incentivación del uso de biomasa de cultivos energéticos para producción de electricidad, la cual se regula actualmente según el Real Decreto-ley 9/2013, de 12 de Julio, por el que se adoptaron medidas urgentes para garantizar la estabilidad financiera del sistema eléctrico, y se consideran los criterios de sostenibilidad en el uso de biocombustibles sólidos. Se realiza una caracterización de las seis comarcas agrarias que forman la Comunidad Autónoma de Madrid: Área Metropolitana, Campiña, Guadarrama, Lozoya- Somosierra, Sur-Occidental y Vegas, la cual consta de dos partes: una descripción de la climatología y otra de la distribución de la superficie dedicada a barbecho y cultivos herbáceos. Se hace una recopilación bibliográfica de los modelos de simulación más representativos de crecimiento de los cultivos (CERES y Cereal YES), así como de ensayos realizados con los cultivos de centeno y triticale para la producción de biomasa y de estudios efectuados mediante herramientas GIS y técnicas de análisis multicriterio para la ubicación de centrales de bioelectricidad y el estudio de la logística de la biomasa. Se propone un modelo de simulación de la productividad de biomasa de centeno y de triticale para la CM, que resulta de la combinación de un modelo de producción de grano en base a datos climatológicos y a la relación biomasa/grano media de ambos cultivos obtenida en una experiencia previa. Los modelos obtenidos responden a las siguientes ecuaciones (siendo TN = temperatura media normalizada a 9,9 ºC y PN = precipitación acumulada normalizada a 496,7 mm): - Producción biomasa centeno (t m.s./ha) = 2,785 * [1,078 * ln(TN + 2*PN) + 2,3256] - Producción biomasa triticale (t m.s./ha) = 2,595 * [2,4495 * ln(TN + 2*PN) + 2,6103] Posteriormente, aplicando los modelos desarrollados, se cuantifica el potencial de producción de biomasa de centeno y triticale en las distintas comarcas agrarias de la CM en cada uno de los escenarios establecidos, que se consideran según el uso de la superficie de barbecho de secano disponible (25%, 50%, 75% y 100%). Las producciones potenciales de biomasa, que se podrían alcanzar en la CM utilizando el 100% de la superficie de barbecho de secano, en base a los cultivos de centeno y triticale, se estimaron en 169.710,72 - 149.811,59 - 140.217,54 - 101.583,01 - 26.961,88 y 1.886,40 t anuales para las comarcas de Campiña - Vegas, Sur - Occidental - Área Metropolitana - Lozoya-Somosierra y Guadarrama, respectivamente. Se realiza un análisis multicriterio basado en la programación de compromiso para definir las comarcas agrarias con mejores características para la ubicación de centrales de bioelectricidad en base a los criterios de potencial de biomasa, infraestructura eléctrica, red de carreteras, espacios protegidos y superficie de núcleos urbanos. Al efectuar el análisis multicriterio, se obtiene la siguiente ordenación jerárquica en base a los criterios establecidos: Campiña, Sur Occidental, Vegas, Área Metropolitana, Lozoya-Somosierra y Guadarrama. Mediante la utilización de técnicas GIS se estudia la localización más conveniente de una central de bioelectricidad de 2,2 MW en cada una de las comarcas agrarias y según el uso de la superficie de barbecho de secano disponible (25%, 50%, 75% y 100%), siempre que exista potencial suficiente. Para el caso de la biomasa de centeno y de triticale en base seca se considera un PCI de 3500 kcal/kg, por lo que se necesitarán como mínimo 17.298,28 toneladas para satisfacer las necesidades de cada una de las centrales de 2,2 MW. Se analiza el potencial máximo de bioelectricidad en cada una de las comarcas agrarias en base a los cultivos de centeno y triticale como productores de biomasa. Según se considere el 25% o el 100% del barbecho de secano para producción de biomasa, la potencia máxima de bioelectricidad que se podría instalar en cada una de las comarcas agrarias variaría entre 5,4 y 21,58 MW en la comarca Campiña, entre 4,76 y 19,05 MW en la comarca Vegas, entre 4,46 y 17,83 MW en la comarca Sur Occidental, entre 3,23 y 12,92 MW en la comarca Área Metropolitana, entre 0,86 y 3,43 MW en la comarca Lozoya Somosierra y entre 0,06 y 0,24 MW en la comarca Guadarrama. La potencia total que se podría instalar en la CM a partir de la biomasa de centeno y triticale podría variar entre 18,76 y 75,06 MW según que se utilice el 25% o el 100% de las tierras de barbecho de secano para su cultivo. ABSTRACT In this work is studied the potential biomass production from rye and triticale crops in the six Madrid Community (MC) agricultural regions and the possibility of its application to the bioelectricity production in each of them. First is performed a bibliographical study of the current situation of bioelectricity. One of the main elements to be considered is that in the PER 2011-2020 is estimated that the total installed electric power from biomass in Spain in 2020 was 1.350 MW, about two and a half times as at end 2010. Also is discussed the status of enhancing the use of biomass energy crops for electricity production, which is currently regulated according to the Real Decreto-ley 9/2013, of July 12, by which urgent measures were adopted to ensure financial stability of the electrical system, and there are considered the sustainability criteria in the use of solid biofuels. A characterization of the six Madrid Community agricultural regions is carried out: Area Metropolitana, Campiña, Guadarrama, Lozoya-Somosierra, Sur-Occidental and Vegas, which consists of two parts: a description of the climatology and another about the distribution of the area under fallow and arable crops. It makes a bibliographic compilation of the most representative crop growth simulation models (CERES and Cereal YES), as well as trials carried out with rye and triticale crops for biomass production and studies conducted by GIS tools and techniques multicriteria analysis for the location of bioelectricity centrals and the study of the logistics of biomass. Is proposed a biomass productivity simulation model for rye and triticale for MC that results from the combination of grain production model based on climatological data and the average relative biomass/grain of both crops obtained in a prior experience. The models obtained correspond to the following equations (where TN = normalized average temperature and PN = normalized accumulated precipitation): - Production rye biomass (t d.m./ha) = 2.785 * [1.078 * ln (TN + 2*PN) + 2.3256] - Production triticale biomass (t d.m./ha) = 2,595 * [2.4495 * ln (TN + 2*PN) + 2.6103] Subsequently, applying the developed models, the biomass potential of the MC agricultural regions is quantified in each of the scenarios established, which are considered as the use of dry fallow area available (25%, 50%, 75 % and 100%). The potential biomass production that can be achieved within the MC using 100% of the rainfed fallow area based on rye and triticale crops, were estimated at 169.710,72 - 149.811,59 - 140.217,54 - 101.583,01 - 26.961,88 and 1.886,40 t annual for the regions of Campiña, Vegas, Sur Occidental, Area Metropolitana, Lozoya- Somosierra and Guadarrama, respectively. A multicriteria analysis is performed, based on compromise programming to define the agricultural regions with better features for the location of bioelectricity centrals, on the basis of biomass potential, electrical infrastructure, road network, protected areas and urban area criteria. Upon multicriteria analysis, is obtained the following hierarchical order based on criteria: Campiña, Sur Occidental, Vegas, Area Metropolitana, Lozoya-Somosierra and Guadarrama. Likewise, through the use of GIS techniques, the most suitable location for a 2,2 MW bioelectricity plant is studied in each of the agricultural regions and according to the use of dry fallow area available (25%, 50% , 75% and 100%), if there is sufficient potential. In the case of biomass rye and triticale dry basis is considered a PCI of 3500 kcal/kg, so it will take at least 17,298.28 t to satisfy the needs of each plant. Is analyzed the maximum bioelectricity potential on each of the agricultural regions on the basis of the rye and triticale crops as biomass producers. As deemed 25% or 100% dry fallow for biomass, the maximum bioelectricity potential varies between 5,4 and 21,58 MW in the Campiña region, between 4,76 and 19,05 MW in the Vegas region, between 4,46 and 17,83 MW in the Sur Occidental region, between 3,23 and 12,92 MW in the Area Metropolitana region, between 0,86 and 3,43 MW in the Lozoya-Somosierra region and between 0,06 and 0,24 MW in the Guadarrama region. The total power that could be installed in the CM from rye and triticale biomass could vary between 18.76 and 75.06 MW if is used the 25% or 100% of fallow land for rainfed crop.

Respuesta fisiológica y agronómica del olivar en seto (cv. Arbequina) a la orientación de filas y al ancho de calle: relación entre la producción y calidad del aceite y la radiación interceptada

En las últimas dos décadas, los productores han plantado olivares en seto para lograr la mecanización de la poda y en especial de la cosecha, reducir los costes de mano de obra y permitir intervenciones de manejo rápidas y oportunas. Los olivares se desarrollaron en ausencia del conocimiento científico, sobre el diseño óptimo de la estructura de la copa, necesario para incrementar la producción y calidad del aceite. En contraste, con los árboles muy espaciados y distribuidos uniformemente de las plantaciones tradicionales, en el olivar en seto hay una marcada variabilidad espacial y temporal de la radiación disponible en función del diseño de la plantación. Así, conocer la respuesta fisiológica y productiva del olivo a la radiación resulta fundamental en el olivar en seto. La orientación de las filas y el ancho de calle son aspectos que se deciden en el diseño de las plantaciones en seto. Ambos aspectos modifican la radiación interceptada por la canopia y, por lo tanto, pueden incidir en la productividad y calidad del aceite. Una vez realizada la plantación no pueden ser modificados, y así las ventajas o desventajas permanecerán fijas durante toda la vida productiva del olivar. A pesar de esto, el impacto de la orientación de las filas y el ancho de calle han recibido poca atención en olivos y en la mayoría de los frutales conducidos en seto. Por todo ello, los objetivos principales de esta tesis fueron, (i) evaluar el efecto de la orientación del seto y del ancho de calle, sobre la productividad y calidad del aceite, (ii) evaluar un modelo que estime la radiación dentro de la canopia. Este modelo permitirá cuantificar las relaciones entre la radiación y los componentes del rendimiento y calidad del aceite de olivares en setos con un amplio rango de estructuras y (iii) conocer la variabilidad en las características de las hojas (morfológicas y fisiológicas) y de los tejidos del fruto (tamaño y composición) en diferentes posiciones de la copa de los setos. Para ello, se dispuso de 3 ensayos de olivar en seto (cv. Arbequina) implantados en 2008 en el municipio de La Puebla de Montalbán, Toledo. La primera cosecha fue en 2010 y a partir del 2012 los setos formaron una copa continua. A partir de ese año, los setos se mantuvieron mediante poda, con similar ancho (~1 m) y altura (~2,5 m), acordes a las dimensiones de la cosechadora vendimiadora. En los años 2012 y 2013 se estudió en profundidad la respuesta de las plantas de estos ensayos. En el ensayo 1, los setos fueron plantados con cuatro orientaciones de filas: N–S, NE–SO, NO–SE y E–O y el mismo ancho de calle (4 m). En los otros dos ensayos, los setos fueron plantados con tres anchos de calle (5,0, 4,0 y 2,5 m), y con filas orientadas N–S (ensayo 2) y E–O (ensayo 3). La respuesta de la orientación de las filas se evaluó a nivel de seto y de estratos del seto (alturas y caras), a través de mediciones del crecimiento de brotes, componentes reproductivos, características y temperatura del fruto, estado hídrico del suelo y de las plantas, fotosíntesis neta de las hojas y contenido de ácidos grasos. Los setos orientados NE–SO (2,7 t/ha) lograron la mayor producción de aceite, que fue significativamente más alta que la de los setos E–O (2,3 t/ha). La producción de aceite de los setos E–O no se diferenció estadísticamente de los setos N–S (2,5 t/ha). Las diferencias productivas entre orientaciones fueron explicadas por el número de frutos en cosecha, a su vez la variación en el número de frutos estuvo asociada al efecto de la orientación de las filas sobre el número de yemas desarrolladas y el porcentaje de inflorescencias fértiles. Las hojas en las caras iluminadas de los setos NE–SO y N–S presentaron mayor tasa fotosintética a la mañana (~10.0 h) que los setos E–O, en el año 2012, pero no en 2013. La orientación de las filas no tuvo un efecto significativo en el contenido de ácidos grasos de los aceites extraídos, esto ocurrió a pesar de variaciones en la temperatura interna de los frutos (3 °C) y de la radiación (40%) entre las distintas caras de los setos. La orientación del seto afectó significativamente al contenido relativo de agua del suelo, donde setos E–O presentaron valores más altos (12%) que setos N–S durante el verano y otoño. Sin embargo, el potencial hídrico de tallo fue similar entre orientaciones. En los ensayos 2 y 3, se evaluó el efecto que produce, a nivel de seto y de estratos (caras y alturas), reducir el ancho de calle de 5,0 a 4,0 y 2,5 m, en un seto orientado N–S y otro E–O, respectivamente. La relación entre altura/ancho de calle libre aumentó 0,6 a 0,8 y 1,6, al reducir 5,0, 4,0 y 2,5 m el ancho de calle, mientras la longitud de seto y el volumen de copa por hectárea incrementó 100% al reducir de 5,0 a 2,5 m, el ancho de calle. En los setos orientados N–S, la producción de aceite por ha acumulada en 4 campañas, incrementó significativamente un 52 %, al reducir de 5,0 a 2,5 m el ancho de calle. Los setos N–S con calle más estrecha (2,5 m) tuvieron un 19% menos frutos que los setos con calle más ancha (5,0 m) y a su vez el 60% de los mismos se localizaron los estratos altos de la canopia de los setos con calles estrecha en comparación al 40% en setos con calle de 5,0 m. En los estratos más bajos de los setos con calles de 2,5m hubo menor crecimiento de los brotes y los frutos tuvieron menor peso seco, contenido de aceite y madurez, que los frutos en los estratos bajos de los setos a 5,0 m. Los componentes del rendimiento y características de los frutos (agua y madurez) fueron similares entre la caras E y O, independientemente del ancho de calle. En los setos orientados E–O, la producción de aceite por ha acumulada en 4 campañas, no respondió significativamente al ancho de calle, debido a una disminución significativa en el número de frutos y producción de aceite por m de seto, al reducir de 5,0 a 2,5 m, el ancho de calle. En los setos orientados E–O, con calles de 5,0 m, los frutos presentaron similar peso seco, contenido de aceite y agua, en las caras S y N, sin embargo, cuando la calle fue reducida a 2,5, los frutos de la cara S fueron más pesado y maduros que en la cara N. Independientemente del ancho de calle y de la orientación del seto, el aceite presentó mayor contenido de ácidos palmitoleico, palmítico, esteárico y linoleico en los frutos del estrato más alto de la canopia disminuyendo hacia la base. En contraste, el contenido de ácido oleico aumentó desde el estrato más alto hacia la base de los setos. Las diferencias en el contenido de ácidos grasos entre la parte alta y baja de los setos, incrementó al reducir el ancho de calle en los setos N–S, pero no en los E-O. En conclusión, en olivares en seto, reducir el ancho de calle permite incrementar la producción de aceite, en setos orientados N–S, pero no en E–O. Un modelo que estima la cantidad y distribución de la radiación en toda la copa del seto, fue utilizado para estimar la radiación interceptada en distintos estratos del seto. El modelo requiere un valor del coeficiente de extinción (k) para estimar la transmisión de radiación a través de la copa, el cual fue obtenido experimentalmente (k=1,2). Utilizando los datos del ensayo 1, un único modelo lineal relacionó el peso seco y el rendimiento graso de setos con la radiación interceptada por los distintos estratos de setos con cuatro orientaciones de filas. La densidad de frutos fue también relacionada con la radiación, pero más débilmente. En los setos orientados N–S, plantados con tres anchos de calles, (ensayo 2) el contenido de ácidos palmitoleico y linoleico del aceite incrementó linealmente con el incremento de la radiación interceptada, mientras el contenido ácido oleico disminuyó linealmente con el incremento de la radiación. El contenido de ácidos grasos del aceite no estuvo relacionado con la radiación interceptada en setos orientados E–O (Ensayo 3). En los setos N–S y E–O, plantados con anchos de calle de 2,5 m, se estudiaron las interacciones entre la radiación y características de las hojas, número de fruto, tamaño y composición de los frutos a nivel de órgano, tejido y células. Independientemente de la orientación del seto, el área y el contenido de clorofila de las hojas incrementaron significativamente en los estratos más bajos de los setos. Mientras, las hojas de los estratos medios del seto presentaron mayor capacidad fotosintética que en los estratos bajos y alto de los setos. Los estratos del seto que interceptaron más radiación produjeron frutos con mayor tamaño y contenido de aceite en el mesocarpo, sin efectos sobre el tamaño y composición del endocarpo. A nivel celular, los frutos expuestos a mayor nivel de radiación desarrollaron en el mesocarpo células de mayor tamaño en comparación a frutos menos expuestos, mientras el número de células no fue afectado. Adicionalmente, el número y tamaño de las células estuvo relacionado con la composición del mesocarpo en términos de aceite, agua y peso seco menos aceite. Esta tesis, contribuye, desde una perspectiva integral del cultivo del olivo, a cuantificar el impacto de la orientación y ancho de calle sobre la producción y calidad del aceite en olivares conducidos en setos. El análisis y discusión de la relación entre la radiación y los componentes del rendimiento y calidad del aceite, puede ayudar a diseñar plantaciones en seto con dimensiones óptimas para la intercepción de la radiación. ABSTRACT In the last two decades, olive hedgerow system has been established by commercial growers to allow continuous mechanized pruning and especially harvest, reduce costs of manual labour and allow more rapid and timely management interventions. The adoption of hedgerow was done in the absence of adequate scientific knowledge of the impact of this orchard structure and associated mechanization on tree response, yield and quality, after centuries in low-density orchards and open-formed trees. The row orientation and width alley are fundamental aspects in the hedgerow design and have been scarcely studied in olive. Both aspects modify the radiation intercepted by the canopy, and consequently the productivity and oil quality, and once defined in orchard planting cannot be changed, so advantages and disadvantages remain fixed for the lifespan of the orchard. The main objectives of this thesis were to (i) evaluate the impact of the row orientation and width alley on productivity and oil quality by the measurements of profile of the determining processes of shoot growth, fruit temperature, yield components and fruit and oil characteristics on opposite sides of olive hedgerows. Additionally, the effect of row orientation on the plant water status was also evaluated; (ii) evaluate a mathematical model for estimating the radiation within the canopy and quantify the relationships between the radiation estimated and yield components and oil quality in olive hedgerows under wide range of structures and; (iii) determine the variability in the characteristics of the leaves (morphological and physiological) and fruit tissues (size and composition) in different positions of the hedgerows canopy. Three plots of olive hedgerows (cv. Arbequina) planted in 2008 in La Puebla de Montalbán, Toledo were evaluated during the 2012 and 2013 seasons. The hedgerows were maintained by lateral pruning and topping with the same width (1 m) and height (2.5 m) compatible with the intended harvester. In a plot (experiment 1), the hedgerows were planted with the same width alley (4 m) and four row orientations: N–S, NE–SW, NW–SE and E–W. Other two plots (Experiments 2 and 3) separated by approximately 100 m were planted with N–S and E–O oriented rows and three alley widths in each orientation: 5.0, 4.0 and 2.5 m. In the exp. 1, maximum fruit yield were achieved by NE–SW and NW–SW (15.7 t/ha). Of these, NE–SW achieved the highest oil yield (2.7 t/ha). There were no differences in fruit or oil yield between N–S (2.5 t oil/ha) and E–W (2.3 t oil/ha) orientations. Fruit number was the most important component to explain these differences, by previous influence on number of bud developed and percentage of fertile inflorescences. Fruit maturity and oil quality on both sides of the hedgerows were not affected by row orientation. This occurred despite significant variations in the internal fruit temperature, which was closely related to the irradiance received by the canopy and the time of day. Additionally, row orientation significantly affected the relative water content of the soil, where E–W oriented hedgerows showed consistently higher values than N–S during summer-autumn season. The stem water potential at midday, however, was similar between orientations, revealing possible lower water consumption of E–W than N–S oriented hedgerows. In the exp. 2, regardless of row orientation, reduction of row spacing from 5.0 to 4.0 and 2.5 m increases the ratio of canopy depth to free alley width (Al/An) from 0.6 to 0.8 and 1.6, respectively, and ads 25 and 100 % more hedgerow length per ha. In N–S oriented hedgerows, oil production per ha increased significantly by 14 and 52 % in 4.0 m and 2.5 m relative to 5.0 m row spacing, the effect being proportionally less than the increase in hedgerow length per ha. Hedgerows spaced 2.5 m with Al/An = 1.6 produced relatively fewer fruits per unit length than did wider spacings and were preferentially distributed in upper layers. Fruits located at the bottom of the canopy were smaller, with lower oil content and were less mature. In E–W oriented hedgerows, oil production per ha did not respond significantly to row spacing, despite the doubling of row length from the 5.0 to the 2.5 m row spacing. The explanation was found in fewer fruit per unit length of hedgerow and smaller oil content at 2.5 m than 5.0 m row spacing, averaged over the experimental period. In E–W hedgerows spaced at 5.0 m with Al/An = 0.6, the vertical profiles of fruit characteristics (mass, oil and water contents, and maturity) were similar between opposing sides, but at 4.0 m (Al/An= 0.8) and 2.5 m (Al/An=1.6) spacings, fruits on the S side were heavier and more mature than on N side. The oil extracted from fruits harvested at different heights of N–S and E–W oriented hedgerows showed higher palmitoleic, palmitic, stearic and linoleic contents at the canopy top decreasing toward base. The oleic content was reverse, increased from top to base. In N–S hedgerows, vertical gradients increased by reducing the alley width, but not in the E–W oriented hedgerows. The simulation of internal canopy irradiance was related in a single relationship (R2 = 0.63) to the vertical profiles of fruit weight and oil content of olive hedgerows with wide range of structures. The density of fruits was also associated with the irradiance but more weakly (R2 = 0.27), and revealed a more complex response involving changes in the vegetative structure by canopy management (topping) and the effect of radiation on the previous sequence that defines the number of fruits. The vertical profiles of oil quality traits were closely associated to canopy irradiance, but only when the N–S oriented hedgerows were considered. The contents of palmitoleic and linoleic acid in the oil increased linearly when intercepted irradiance increased from 9 to 19 mol PAR/m2. In contrast, oleic content decreased linearly in this irradiance range. Additionally, we advanced knowledge regarding the interactions among irradiance and leaf, fruit number, size and composition at organ-, tissue- and cellular- levels. The irradiance received at different positions in the canopy strongly affected the leaf area and chlorophyll content, and mesocarp size and composition (water and oil), without effects on endocarp size and composition. At the cellular level, light-exposed fruit developed larger mesocarp cells than shaded fruits, but cell number was not affected. Our results indicate that cell number and size are related to mesocarp composition in term of oil, water, and dry weight menus oil, although the specific manner in which they interact remains to be determined. This research contributes from an integral perspective of olive growing to quantify the impact of row orientation and width alley on productivity and oil quality in hedgerows systems. The analysis and discussion of the relationships between radiation and yield components and oil quality can help understand the impact of design olive hedgerows in general and in a wide range of environmental conditions.

Palaeoenvironmental reconstruction of Las Conchas Mire (Asturias, N. Spain) from the ketone and fatty acid record= reconstrucción paleoambiental de la Turbera de las Conchas (Asturias, N. España)a partir del registro de cetonas y ácidos grasos

Se han estudiado los biomarcadores, principalmente cetonas y ácidos, preservados en el registro de 3.2 m de la Turbera de Las Conchas. Las cetonas reflejan cierta actividad bacteriana desde 94 cm hasta la base del registro, Los ácidos grasos reflejan una buena preservación de la materia orgánica, salvo en los 20 cm superiores en los que hay indicios de oxidación microbiana de alcanos .The biomarkers, mainly ketones and fally aclds, preserved In 3.2 m deep Las Conchas Mire have been studied, Kelones reflect certain bacterial activity from 94 cm to the bottom of the record. Falty aclds indlcate a good preservation of the organlc matter, wlth the exception of the uppermost 20 cm In whlch mlcroblal oxldation of alkanes are likely to occur

A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly

Resistance to organophosphorus (OP) insecticides is associated with decreased carboxylesterase activity in several insect species. It has been proposed that the resistance may be the result of a mutation in a carboxylesterase that simultaneously reduces its carboxylesterase activity and confers an OP hydrolase activity (the “mutant ali-esterase hypothesis”). In the sheep blowfly, Lucilia cuprina, the association is due to a change in a specific esterase isozyme, E3, which, in resistant flies, has a null phenotype on gels stained using standard carboxylesterase substrates. Here we show that an OP-resistant allele of the gene that encodes E3 differs at five amino acid replacement sites from a previously described OP-susceptible allele. Knowledge of the structure of a related enzyme (acetylcholinesterase) suggests that one of these substitutions (Gly137 → Asp) lies within the active site of the enzyme. The occurrence of this substitution is completely correlated with resistance across 15 isogenic strains. In vitro expression of two natural and two synthetic chimeric alleles shows that the Asp137 substitution alone is responsible for both the loss of E3’s carboxylesterase activity and the acquisition of a novel OP hydrolase activity. Modeling of Asp137 in the homologous position in acetylcholinesterase suggests that Asp137 may act as a base to orientate a water molecule in the appropriate position for hydrolysis of the phosphorylated enzyme intermediate.