An integral model for natural regeneration of Pinus pinea L. in the Northern Plateau (Spain) = Modelo integral de regeneración natural para Pinus pinea L. en la Meseta Norte (España)

Natural regeneration is an ecological key-process that makes plant persistence possible and, consequently, it constitutes an essential element of sustainable forest management. In this respect, natural regeneration in even-aged stands of Pinus pinea L. located in the Spanish Northern Plateau has not always been successfully achieved despite over a century of pine nut-based management. As a result, natural regeneration has recently become a major concern for forest managers when we are living a moment of rationalization of investment in silviculture. The present dissertation is addressed to provide answers to forest managers on this topic through the development of an integral regeneration multistage model for P. pinea stands in the region. From this model, recommendations for natural regeneration-based silviculture can be derived under present and future climate scenarios. Also, the model structure makes it possible to detect the likely bottlenecks affecting the process. The integral model consists of five submodels corresponding to each of the subprocesses linking the stages involved in natural regeneration (seed production, seed dispersal, seed germination, seed predation and seedling survival). The outputs of the submodels represent the transitional probabilities between these stages as a function of climatic and stand variables, which in turn are representative of the ecological factors driving regeneration. At subprocess level, the findings of this dissertation should be interpreted as follows. The scheduling of the shelterwood system currently conducted over low density stands leads to situations of dispersal limitation since the initial stages of the regeneration period. Concerning predation, predator activity appears to be only limited by the occurrence of severe summer droughts and masting events, the summer resulting in a favourable period for seed survival. Out of this time interval, predators were found to almost totally deplete seed crops. Given that P. pinea dissemination occurs in summer (i.e. the safe period against predation), the likelihood of a seed to not be destroyed is conditional to germination occurrence prior to the intensification of predator activity. However, the optimal conditions for germination seldom take place, restraining emergence to few days during the fall. Thus, the window to reach the seedling stage is narrow. In addition, the seedling survival submodel predicts extremely high seedling mortality rates and therefore only some individuals from large cohorts will be able to persist. These facts, along with the strong climate-mediated masting habit exhibited by P. pinea, reveal that viii the overall probability of establishment is low. Given this background, current management –low final stand densities resulting from intense thinning and strict felling schedules– conditions the occurrence of enough favourable events to achieve natural regeneration during the current rotation time. Stochastic simulation and optimisation computed through the integral model confirm this circumstance, suggesting that more flexible and progressive regeneration fellings should be conducted. From an ecological standpoint, these results inform a reproductive strategy leading to uneven-aged stand structures, in full accordance with the medium shade-tolerant behaviour of the species. As a final remark, stochastic simulations performed under a climate-change scenario show that regeneration in the species will not be strongly hampered in the future. This resilient behaviour highlights the fundamental ecological role played by P. pinea in demanding areas where other tree species fail to persist.

Towards measuring the complexity of introducing semantics into a company

The Semantics Difficulty Model (SDM) is a model that measures the difficult of introducing semantics technology into a company. SDM manages three descriptions of stages, which we will refer to as ?snapshots?: a company semantic snapshot, data snapshot and semantic application snapshot. Understanding a priory the complexity of introducing semantics into a company is important because it allows the organization to take early decisions, thus saving time and money, mitigating risks and improving innovation, time to market and productivity. SDM works by measuring the distance between each initial snapshot and its reference models (the company semantic snapshots reference model, data snapshots reference model, and the semantic application snapshots reference model) with Euclidian distances. The difficulty level will be "not at all difficult" when the distance is small, and becomes "extremely difficult" when the the distance is large. SDM has been tested experimentally with 2000 simulated companies with arrangements and several initial stages. The output is measured by five linguistic values: "not at all difficult, slightly difficult, averagely difficult, very difficult and extremely difficult". As the preliminary results of our SDM simulation model indicate, transforming a search application into integrated data from different sources with semantics is a "slightly difficult", in contrast with data and opinion extraction applications for which it is "very difficult".

Desarrollo de un modelo de predimensionado de costes de construcción en el proyecto arquitectónico

El objetivo de la presente investigación es el desarrollo de un modelo de cálculo rápido, eficiente y preciso, para la estimación de los costes finales de construcción, en las fases preliminares del proyecto arquitectónico. Se trata de una herramienta a utilizar durante el proceso de elaboración de estudios previos, anteproyecto y proyecto básico, no siendo por tanto preciso para calcular el “predimensionado de costes” disponer de la total definición grafica y literal del proyecto. Se parte de la hipótesis de que en la aplicación práctica del modelo no se producirán desviaciones superiores al 10 % sobre el coste final de la obra proyectada. Para ello se formulan en el modelo de predimensionado cinco niveles de estimación de costes, de menor a mayor definición conceptual y gráfica del proyecto arquitectónico. Los cinco niveles de cálculo son: dos que toman como referencia los valores “exógenos” de venta de las viviendas (promoción inicial y promoción básica) y tres basados en cálculos de costes “endógenos” de la obra proyectada (estudios previos, anteproyecto y proyecto básico). El primer nivel de estimación de carácter “exógeno” (nivel .1), se calcula en base a la valoración de mercado de la promoción inmobiliaria y a su porcentaje de repercusión de suelo sobre el valor de venta de las viviendas. El quinto nivel de valoración, también de carácter “exógeno” (nivel .5), se calcula a partir del contraste entre el valor externo básico de mercado, los costes de construcción y los gastos de promoción estimados de la obra proyectada. Este contraste entre la “repercusión del coste de construcción” y el valor de mercado, supone una innovación respecto a los modelos de predimensionado de costes existentes, como proceso metodológico de verificación y validación extrínseca, de la precisión y validez de las estimaciones resultantes de la aplicación práctica del modelo, que se denomina Pcr.5n (Predimensionado costes de referencia con .5niveles de cálculo según fase de definición proyectual / ideación arquitectónica). Los otros tres niveles de predimensionado de costes de construcción “endógenos”, se estiman mediante cálculos analíticos internos por unidades de obra y cálculos sintéticos por sistemas constructivos y espacios funcionales, lo que se lleva a cabo en las etapas iniciales del proyecto correspondientes a estudios previos (nivel .2), anteproyecto (nivel .3) y proyecto básico (nivel .4). Estos cálculos teóricos internos son finalmente evaluados y validados mediante la aplicación práctica del modelo en obras de edificación residencial, de las que se conocen sus costes reales de liquidación final de obra. Según va evolucionando y se incrementa el nivel de definición y desarrollo del proyecto, desde los estudios previos hasta el proyecto básico, el cálculo se va perfeccionando en su nivel de eficiencia y precisión de la estimación, según la metodología aplicada: [aproximaciones sucesivas en intervalos finitos], siendo la hipótesis básica como anteriormente se ha avanzado, lograr una desviación máxima de una décima parte en el cálculo estimativo del predimensionado del coste real de obra. El cálculo del coste de ejecución material de la obra, se desarrolla en base a parámetros cúbicos funcionales “tridimensionales” del espacio proyectado y parámetros métricos constructivos “bidimensionales” de la envolvente exterior de cubierta/fachada y de la huella del edificio sobre el terreno. Los costes funcionales y constructivos se ponderan en cada fase del proceso de cálculo con sus parámetros “temáticos/específicos” de gestión (Pg), proyecto (Pp) y ejecución (Pe) de la concreta obra presupuestada, para finalmente estimar el coste de construcción por contrata, como resultado de incrementar al coste de ejecución material el porcentaje correspondiente al parámetro temático/especifico de la obra proyectada. El modelo de predimensionado de costes de construcción Pcr.5n, será una herramienta de gran interés y utilidad en el ámbito profesional, para la estimación del coste correspondiente al Proyecto Básico previsto en el marco técnico y legal de aplicación. Según el Anejo I del Código Técnico de la Edificación (CTE), es de obligado cumplimiento que el proyecto básico contenga una “Valoración aproximada de la ejecución material de la obra proyectada por capítulos”, es decir , que el Proyecto Básico ha de contener al menos un “presupuesto aproximado”, por capítulos, oficios ó tecnologías. El referido cálculo aproximado del presupuesto en el Proyecto Básico, necesariamente se ha de realizar mediante la técnica del predimensionado de costes, dado que en esta fase del proyecto arquitectónico aún no se dispone de cálculos de estructura, planos de acondicionamiento e instalaciones, ni de la resolución constructiva de la envolvente, por cuanto no se han desarrollado las especificaciones propias del posterior proyecto de ejecución. Esta estimación aproximada del coste de la obra, es sencilla de calcular mediante la aplicación práctica del modelo desarrollado, y ello tanto para estudiantes como para profesionales del sector de la construcción. Como se contiene y justifica en el presente trabajo, la aplicación práctica del modelo para el cálculo de costes en las fases preliminares del proyecto, es rápida y certera, siendo de sencilla aplicación tanto en vivienda unifamiliar (aisladas y pareadas), como en viviendas colectivas (bloques y manzanas). También, el modelo es de aplicación en el ámbito de la valoración inmobiliaria, tasaciones, análisis de viabilidad económica de promociones inmobiliarias, estimación de costes de obras terminadas y en general, cuando no se dispone del proyecto de ejecución y sea preciso calcular los costes de construcción de las obras proyectadas. Además, el modelo puede ser de aplicación para el chequeo de presupuestos calculados por el método analítico tradicional (estado de mediciones pormenorizadas por sus precios unitarios y costes descompuestos), tanto en obras de iniciativa privada como en obras promovidas por las Administraciones Públicas. Por último, como líneas abiertas a futuras investigaciones, el modelo de “predimensionado costes de referencia 5 niveles de cálculo”, se podría adaptar y aplicar para otros usos y tipologías diferentes a la residencial, como edificios de equipamientos y dotaciones públicas, valoración de edificios históricos, obras de urbanización interior y exterior de parcela, proyectos de parques y jardines, etc….. Estas lineas de investigación suponen trabajos paralelos al aquí desarrollado, y que a modo de avance parcial se recogen en las comunicaciones presentadas en los Congresos internacionales Scieconf/Junio 2013, Rics‐Cobra/Septiembre 2013 y en el IV Congreso nacional de patología en la edificación‐Ucam/Abril 2014. ABSTRACT The aim of this research is to develop a fast, efficient and accurate calculation model to estimate the final costs of construction, during the preliminary stages of the architectural project. It is a tool to be used during the preliminary study process, drafting and basic project. It is not therefore necessary to have the exact, graphic definition of the project in order to be able to calculate the cost‐scaling. It is assumed that no deviation 10% higher than the final cost of the projected work will occur during the implementation. To that purpose five levels of cost estimation are formulated in the scaling model, from a lower to a higher conceptual and graphic definition of the architectural project. The five calculation levels are: two that take as point of reference the ”exogenous” values of house sales (initial development and basic development), and three based on calculation of endogenous costs (preliminary study, drafting and basic project). The first ”exogenous” estimation level (level.1) is calculated over the market valuation of real estate development and the proportion the cost of land has over the value of the houses. The fifth level of valuation, also an ”exogenous” one (level.5) is calculated from the contrast between the basic external market value, the construction costs, and the estimated development costs of the projected work. This contrast between the ”repercussions of construction costs” and the market value is an innovation regarding the existing cost‐scaling models, as a methodological process of extrinsic verification and validation, of the accuracy and validity of the estimations obtained from the implementation of the model, which is called Pcr.5n (reference cost‐scaling with .5calculation levels according to the stage of project definition/ architectural conceptualization) The other three levels of “endogenous” construction cost‐scaling are estimated from internal analytical calculations by project units and synthetic calculations by construction systems and functional spaces. This is performed during the initial stages of the project corresponding to preliminary study process (level.2), drafting (level.3) and basic project (level.4). These theoretical internal calculations are finally evaluated and validated via implementation of the model in residential buildings, whose real costs on final payment of the works are known. As the level of definition and development of the project evolves, from preliminary study to basic project, the calculation improves in its level of efficiency and estimation accuracy, following the applied methodology: [successive approximations at finite intervals]. The basic hypothesis as above has been made, achieving a maximum deviation of one tenth, in the estimated calculation of the true cost of predimensioning work. The cost calculation for material execution of the works is developed from functional “three‐dimensional” cubic parameters for the planned space and constructive “two dimensional” metric parameters for the surface that envelopes around the facade and the building’s footprint on the plot. The functional and building costs are analyzed at every stage of the process of calculation with “thematic/specific” parameters of management (Pg), project (Pp) and execution (Pe) of the estimated work in question, and finally the cost of contractual construction is estimated, as a consequence of increasing the cost of material execution with the percentage pertaining to the thematic/specific parameter of the projected work. The construction cost‐scaling Pcr.5n model will be a useful tool of great interest in the professional field to estimate the cost of the Basic Project as prescribed in the technical and legal framework of application. According to the appendix of the Technical Building Code (CTE), it is compulsory that the basic project contains an “approximate valuation of the material execution of the work, projected by chapters”, that is, that the basic project must contain at least an “approximate estimate” by chapter, trade or technology. This approximate estimate in the Basic Project is to be performed through the cost‐scaling technique, given that structural calculations, reconditioning plans and definitive contruction details of the envelope are still not available at this stage of the architectural project, insofar as specifications pertaining to the later project have not yet been developed. This approximate estimate of the cost of the works is easy to calculate through the implementation of the given model, both for students and professionals of the building sector. As explained and justified in this work, the implementation of the model for cost‐scaling during the preliminary stage is fast and accurate, as well as easy to apply both in single‐family houses (detached and semi‐detached) and collective housing (blocks). The model can also be applied in the field of the real‐estate valuation, official appraisal, analysis of the economic viability of real estate developments, estimate of the cost of finished projects and, generally, when an implementation project is not available and it is necessary to calculate the building costs of the projected works. The model can also be applied to check estimates calculated by the traditional analytical method (state of measurements broken down into price per unit cost details), both in private works and those promoted by Public Authorities. Finally, as potential lines for future research, the “five levels of calculation cost‐scaling model”, could be adapted and applied to purposes and typologies other than the residential one, such as service buildings and public facilities, valuation of historical buildings, interior and exterior development works, park and garden planning, etc… These lines of investigation are parallel to this one and, by way of a preview, can be found in the dissertations given in the International Congresses Scieconf/June 2013, Rics‐Cobra/September 2013 and in the IV Congress on building pathology ‐Ucam/April 2014.

Expresión de la resistencia en plantas de tomate a nematodos formadores de nódulos (Meloidogyne javanica)

La resistencia genética mediada por los genes R es uno de los sistemas de defensa de las plantas frente a patógenos y se activa una vez que los patógenos han superado la defensa basal que otorgan la cutícula y pared celular. Los mecanismos de resistencia genética se inician a su vez, por el reconocimiento de productos derivados de genes de avirulencia de los patógenos (avr) por parte de las proteínas R. Tanto la respuesta de defensa basal como la respuesta de defensa por genes R están influenciadas por patrones de regulación hormonal, que incluye a las principales hormonas vegetales ácido salicílico (SA), ácido jasmónico (JA) y etileno (ET). En tomate (Solanum lycopersicum) uno de los genes R es el gen MiG1, que confiere resistencia a nematodos formadores de nódulos (Meloidogyne javanica, M. incognita y M. arenaria). Uno de los eventos más importantes que caracterizan a la respuesta de resistencia es la reacción hipersensible (HR), que está mediada por la activación temprana de una serie de sistemas enzimáticos, entre los que destaca el de las peroxidasas (PRXs) Clase III. Su función es importante tanto para limitar el establecimiento y expansión del nematodo, al generar ambientes altamente tóxicos por su contribución en la producción masiva de ROS, como por su implicación en la síntesis y depósito de lignina generando barreras estructurales en el sitio de infección. Además de estos mecanismos de defensa asociados a la resistencia constitutiva, las plantas pueden desarrollar resistencia sistémica adquirida (SAR) que en la naturaleza ocurre, en ocasiones, en una fase posterior a que la planta haya sufrido el ataque de un patógeno. Así mismo hay diferentes productos de origen químico como el benzotiadiazol o BTH (ácido S-metil benzol-(1,2,3)-tiadiozole-7-carbónico ester) que pueden generar esta misma respuesta SAR. Como resultado, la planta adquiere resistencia sistémica frente a nuevos ataques de patógenos. En este contexto, el presente trabajo aborda en primer lugar el análisis comparativo, mediante microarrays de oligonucleótidos, de los transcriptomas de los sistemas radicales de plantas de tomate de 8 semanas de edad de dos variedades, una portadora del gen de resistencia MiG1 (Motelle) y otra carente del mismo y, por tanto, susceptible (Moneymaker), antes y después de la infección por M. javanica. Previo a la infección se observó que la expresión de un gran número de transcritos era más acusada en la variedad resistente que en la susceptible, entre ellos el propio gen MiG1 o los genes PrG1 (o P4), LEJA1 y ER24, lo que indica que, en ausencia de infección, las rutas hormonales del SA, JA y ET están más activas en la raíz de la variedad resistente. Por el contrario, un número mucho menor de transcritos presentaban su expresión más reducida en Motelle que en Moneymaker, destacando un gen de señalización para sintetizar la hormona giberelina (GA). La infección por M. javanica causa importantes cambios transcripcionales en todo el sistema radical que modifican sustancialmente las diferencias basales entre plantas Motelle y Moneymaker, incluida la sobreexpresión en la variedad resistente de los transcritos de MiG1, que se reduce parcialmente, mientras que las rutas hormonales del SA y el JA continuan más activas que en la susceptible (evidente por los genes PrG1 y LEJA1). Además, los cambios asociados a la infección del nematodo se evidencian por las grandes diferencias entre los dos tiempos post-infección considerados, de tal forma que en la fase temprana (2 dpi) de la interacción compatible predomina la sobreexpresión de genes de pared celular y en la tardía (12 dpi) los relacionados con el ARN. En el análisis de la interacción incompatible, aunque también hay muchas diferencias entre ambas fases, hay que destacar la expresión diferencial común de los genes loxA y mcpi (sobrexpresados) y del gen loxD (reprimido) por su implicación en defensa en otras interacciones planta-patógeno. Cabe destacar que entre las interacciones compatible e incompatible hubo muy pocos genes en común. En la etapa temprana de la interacción compatible destacó la activación de genes de pared celular y la represión de la señalización; en cambio, en la interacción incompatible hubo proteínas principalmente implicadas en defensa. A los 12 días, en la interacción compatible los genes relacionados con el ARN y la pared celular se sobreexpresaban principalmente, y se reprimían los de proteínas y transporte, mientras que en la incompatible se sobreexpresaron los relacionados con el estrés, el metabolismo secundario y el de hormonas y se reprimieron los de ARN, señalización, metabolismo de hormonas y proteínas. Por otra parte, la técnica de silenciamiento génico VIGS reveló que el gen TGA 1a está implicado en la resistencia mediada por el gen MiG1a M. javanica. Así mismo se evaluó el transcriptoma de todo el sistema radical de la variedad susceptible tras la aplicación del inductor BTH, y se comparó con el transcriptoma de la resistente. Los resultados obtenidos revelan que el tratamiento con BTH en hojas de Moneymaker ejerce notables cambios transcripcionales en la raíz; entre otros, la activación de factores de transcripción Myb (THM16 y THM 27) y del gen ACC oxidasa. Las respuestas inducidas por el BTH parecen ser de corta duración ya que no hubo transcritos diferenciales comunes a las dos fases temporales de la infección comparadas (2 y 12 dpi). El transcriptoma de Moneymaker tratada con BTH resultó ser muy diferente al de la variedad resistente Motelle, ambas sin infectar, destacando la mayor expresión en el primero del gen LeEXP2, una expansina relacionada con defensa frente a nematodos. Las respuestas inducidas por los nematodos en Moneymaker-BTH también fueron muy distintas a las observadas previamente en la interacción incompatible mediada por MiG1, pues sólo se detectaron 2 genes sobreexpresados comunes a ambos eventos. Finalmente, se abordó el estudio de la expresión diferencial de genes que codifican PRXs y su relación con la resistencia en la interacción tomate/M. javanica. Para ello, se realizó en primer lugar el estudio del análisis del transcriptoma de tomate de la interacción compatible, obtenido en un estudio previo a partir de tejido radical infectado en distintos tiempos de infección. Se han identificado 16 unigenes de PRXs con expresión diferencial de los cuales 15 se relacionan por primera vez con la respuesta a la infección de nematodos. La mayoría de los genes de PRXs identificados, 11, aparecen fuertemente reprimidos en el sitio de alimentación, en las células gigantes (CG). Dada la implicación directa de las PRXs en la activación del mecanismo de producción de ROS, la supresión de la expresión génica local de genes de PRXs en el sitio de establecimiento y alimentación pone de manifiesto la capacidad del nematodo para modular y superar la respuesta de defensa de la planta de tomate en la interacción compatible. Posteriormente, de estos genes identificados se han elegido 4: SGN-U143455, SGN-U143841 y SGN-U144042 reprimidos en el sitio de infección y SGN-U144671 inducido, cuyos cambios de expresión se han determinado mediante análisis por qRT-PCR y de hibridación in situ en dos tiempos de infección (2 dpi y 4 dpi) y en distintos tejidos radicales de tomate resistente y susceptible. Los patrones de expresión obtenidos demuestran que en la interacción incompatible la transcripción global de los 4 genes estudiados se dispara en la etapa más temprana en el sitio de infección, detectándose la localización in situ de transcritos en el citoplasma de las células corticales de la zona meristemática afectadas por el nematodo. A 4 dpi se observó que los niveles de expresión en el sitio de infección cambian de tendencia y los genes SGN-U144671 y SGN-U144042 se reprimen significativamente. Los diferentes perfiles de expresión de los genes PRXs en los dos tiempos de infección sugieren que su inducción en las primeras 48 horas es crucial para la respuesta de defensa relacionada con la resistencia frente a la invasión del nematodo. Por último, al analizar el tejido radical sistémico, se detectó una inducción significativa de la expresión en la fase más tardía de la infección del gen SGN-U144042 en el genotipo susceptible y del SGN-U143841 en ambos genotipos. En este estudio se describe por primera vez la inducción de la expresión sistémica de genes de PRXs en tomate durante la interacción compatible e incompatible con M. javanica lo que sugiere su posible implicación funcional en la respuesta de defensa SAR activada por la infección previa del nematodo. ABSTRACT Plants defend themselves from pathogens by constitutive and/or induced defenses. A common type of induced defense involves plant resistance genes (R), which are normally activated in response to attack by specific pathogen species. Typically, a specific plant R protein recognizes a specific pathogen avirulence (avr) compound. This initiates a complex biochemical cascade inside the plant that results in synthesis of antipathogen compounds. This response can involve chemical signaling, transcription, translation, enzymes and metabolism, and numerous plant hormones such as salicylic acid (SA), jasmonates (JA) and ethylene (ET). Induced plant defense can also activate Class III peroxidases (PRXs), which produce reactive oxygen species (ROS), regulate extracellular H2O2, and play additional roles in plant defense. R-gene activation and the resulting induced defense often remain localized in the specific tissues invaded by the plant pathogen. In other cases, the plant responds by signaling the entire plant to produce defense compounds (systemic induction). Plant defense can also be induced by the exogenous application of natural or synthetic elicitors, such as benzol-(1,2,3)-thiadiazole-7-carbothionic acid. There is much current scientific interest in R-genes and elicitors, because they might be manipulated to increase agricultural yield. Scientists also are interested in systemic induction, because this allows the entire plant to be defended. In this context, one of the aims of this investigation was the transcriptoma analysis of the root systems of two varieties of tomato, the resistant variety (Motelle) that carrier MiG1 and the susceptible (Moneymaker) without MiG1, before and after infection with M. javanica. The overexpression was more pronounced in the transcriptoma of the resistant variety compared with susceptible, before infection, including the MiG1 gene, PrG1 (or P4) genes, LEJA1 and ER24, indicating that hormone SA, JA and ET are active in the resistant variety. Moreover, GA hormone presents an opposite behavior. M. javanica infection causes significant transcriptional changes in both compatible (Moneymaker-M. javanica) and incompatible (Motelle-M. javanica) interaction. In the incompatible transcriptome root system, was notably reduced the expression of the MiG1 gene, and a continuity in the expression of the hormonal pathways of SA and JA. In other hand, transcriptional profile changes during compatible interaction were associated with nematode infection. The large differences between the two times point infection considered (2 dpi and 12 dpi) indicates an overexpression of cell wall related genes in the first phase, and conversely an overexpression of RNA genes in the late phase. Transcriptoma analysis of incompatible interaction, although there were differences between the two phases, should be highlighted the common differential gene expression: loxA and mcpi (overexpressed) and loxD gene (suppressed), as they are involved in defenses in other plant-pathogen interactions. The VIGS tool has provided evidence that TGA 1a is involved in MiG1 mediated resistance to M. javanica. Likewise, the systemic application of BTH was assessed and compared with susceptible and resistant variety. Root system transcriptoma of BTH treatment on leaves showed the activation of Myb transcription factors (THM16 and THM27), the ACC oxidase gene. and the LeEXP2 gene, encoding for an expansin enzyme, related with defense against nematodes. The activation appears to be reduced by subsequent infection and establishment of nematodes. To assist in elucidate the role of tomato PRXs in plant defence against M. javanica, the transcriptome obtained previously from isolated giant cells (GC) and galls at 3 and 7 dpi from the compatible interaction was analysed. A total of 18 different probes corresponding to 16 PRX encoding genes were differentially expressed in infection site compared to the control uninfected root tissues. Most part of them (11) was down-regulated. These results yielded a first insight on 15 of the PRX genes responding to tomato–Meloidogyne interaction and confirm that repression of PRX genes might be crucial for feeding site formation at the initial stages of infection. To study the involvement of PRX genes in resistance response, four genes have been selected: SGN-U143455, SGN-U143841 and SGN-U144042 consistently down-regulated and SGN-U144671 consistently up-regulated at infection site in compatible interaction. The expression changes were determined by qRT-PCR and in situ location at 2 dpi and 4 dpi, and in different root tissues of resistant and susceptible plants. Early upon infection (2 dpi), the transcripts levels of the four genes were strongly increased in infected tissue of resistant genotype. In situ hybridization showed transcript accumulation of them in meristem cortical cells, where the nematode made injury. The results obtained provide strong evidence that early induction of PRX genes is important for defence response of the resistance against nematode invasion. Moreover, the induction patterns of SGN-U144042 gene observed at 4 dpi in distal noninfected root tissue into the susceptible genotype and of SGN-U143841 gene in both genotypes suggest a potential involvement of PRX in the systemic defence response.

Functional genomics tools to decipher the pathogenicity mechanisms of the necrotrophic fungus Plectosphaerella cucumerina in Arabidopsis thaliana

The analysis of the interaction between Arabidopsis thaliana and adapted (PcBMM) and nonadapted (Pc2127) isolates of the necrotrophic fungus Plectosphaerella cucumerina has contributed to the identification of molecular mechanisms controlling plant resistance to necrotrophs.To characterize the pathogenicity bases of the virulence of necrotrophic fungi in Arabidopsis, we developed P. cucumerina functional genomics tools using Agrobacterium tumefaciens-mediated transformation.We generated PcBMM-GFP and Pc2127-GFP transformants constitutively expressing the green fluorescence protein (GFP), and a collection of random T-DNA insertional PcBMM transformants. Confocal microscopy analyses of the initial stages of PcBMM-GFP infection revealed that this pathogen, like other necrotrophic fungi, does not form an appressorium or penetrate into plant cells, but causes successive degradation of leaf cell layers

Aprendizaje integrado en arquitectura con modelos virtuales : implementación de metodología BIM en la docencia universitaria

Los procesos de diseño y construcción en Arquitectura han mostrado un desarrollo de optimización históricamente muy deficiente cuando se compara con las restantes actividades típicamente industriales. La aspiración constante a una industrialización efectiva, tanto en aras de alcanzar mayores cotas de calidad así como de ahorro de recursos, recibe hoy una oportunidad inmejorable desde el ámbito informático: el Building Information Modelling o BIM. Lo que en un inicio puede parecer meramente un determinado tipo de programa informático, en realidad supone un concepto de “proceso” que subvierte muchas rutinas hoy habituales en el desarrollo de proyectos y construcciones arquitectónicas. La inclusión y desarrollo de datos ligados al proyecto, desde su inicio hasta el fin de su ciclo de vida, conlleva la oportunidad de crear una realidad virtual dinámica y actualizable, que por añadidura posibilita su ensayo y optimización en todos sus aspectos: antes y durante su ejecución, así como vida útil. A ello se suma la oportunidad de transmitir eficientemente los datos completos de proyecto, sin apenas pérdidas o reelaboración, a la cadena de fabricación, lo que facilita el paso a una industrialización verdaderamente significativa en edificación. Ante una llamada mundial a la optimización de recursos y el interés indudable de aumentar beneficios económicos por medio de la reducción del factor de incertidumbre de los procesos, BIM supone un opción de mejora indudable, y así ha sido reconocido a través de la inminente implantación obligatoria por parte de los gobiernos (p. ej. Gran Bretaña en 2016 y España en 2018). La modificación de procesos y roles profesionales que conlleva la incorporación de BIM resulta muy significativa y marcará el ejercicio profesional de los futuros graduados en las disciplinas de Arquitectura, Ingeniería y Construcción (AEC por sus siglas en inglés). La universidad debe responder ágilmente a estas nuevas necesidades incorporando esta metodología en la enseñanza reglada y aportando una visión sinérgica que permita extraer los beneficios formativos subyacentes en el propio marco BIM. En este sentido BIM, al aglutinar el conjunto de datos sobre un único modelo virtual, ofrece un potencial singularmente interesante. La realidad tridimensional del modelo, desarrollada y actualizada continuamente, ofrece al estudiante una gestión radicalmente distinta de la representación gráfica, en la que las vistas parciales de secciones y plantas, tan complejas de asimilar en los inicios de la formación universitaria, resultan en una mera petición a posteriori, para ser extraída según necesidad del modelo virtual. El diseño se realiza siempre sobre el propio modelo único, independientemente de la vista de trabajo elegida en cada momento, permaneciendo los datos y sus relaciones constructivas siempre actualizados y plenamente coherentes. Esta descripción condensada de características de BIM preconfiguran gran parte de las beneficios formativos que ofrecen los procesos BIM, en especial, en referencia al desarrollo del diseño integrado y la gestión de la información (incluyendo TIC). Destacan a su vez las facilidades en comprensión visual de elementos arquitectónicos, sistemas técnicos, sus relaciones intrínsecas así como procesos constructivos. A ello se une el desarrollo experimental que la plataforma BIM ofrece a través de sus software colaborativos: la simulación del comportamiento estructural, energético, económico, entre otros muchos, del modelo virtual en base a los datos inherentes del proyecto. En la presente tesis se describe un estudio de conjunto para explicitar tanto las cualidades como posibles reservas en el uso de procesos BIM, en el marco de una disciplina concreta: la docencia de la Arquitectura. Para ello se ha realizado una revisión bibliográfica general sobre BIM y específica sobre docencia en Arquitectura, así como analizado las experiencias de distintos grupos de interés en el marco concreto de la enseñanza de la en Arquitectura en la Universidad Europea de Madrid. El análisis de beneficios o reservas respecto al uso de BIM se ha enfocado a través de la encuesta a estudiantes y la entrevista a profesionales AEC relacionados o no con BIM. Las conclusiones del estudio permiten sintetizar una implantación de metodología BIM que para mayor claridad y facilidad de comunicación y manejo, se ha volcado en un Marco de Implantación eminentemente gráfico. En él se orienta sobre las acciones docentes para el desarrollo de competencias concretas, valiéndose de la flexibilidad conceptual de los Planes de Estudio en el contexto del Espacio Europeo de Educación Superior (Declaración de Bolonia) para incorporar con naturalidad la nueva herramienta docente al servicio de los objetivos formativo legalmente establecidos. El enfoque global del Marco de Implementación propuesto facilita la planificación de acciones formativas con perspectiva de conjunto: combinar los formatos puntuales o vehiculares BIM, establecer sinergias transversales y armonizar recursos, de modo que la metodología pueda beneficiar tanto la asimilación de conocimientos y habilidades establecidas para el título, como el propio flujo de aprendizaje o learn flow BIM. Del mismo modo reserva, incluso visualmente, aquellas áreas de conocimiento en las que, al menos en la planificación actual, la inclusión de procesos BIM no se considera ventajosa respecto a otras metodologías, o incluso inadecuadas para los objetivos docentes establecidos. Y es esta última categorización la que caracteriza el conjunto de conclusiones de esta investigación, centrada en: 1. la incuestionable necesidad de formar en conceptos y procesos BIM desde etapas muy iniciales de la formación universitaria en Arquitectura, 2. los beneficios formativos adicionales que aporta BIM en el desarrollo de competencias muy diversas contempladas en el currículum académico y 3. la especificidad del rol profesional del arquitecto que exigirá una implantación cuidadosa y ponderada de BIM que respete las metodologías de desarrollo creativo tradicionalmente efectivas, y aporte valor en una reorientación simbiótica con el diseño paramétrico y fabricación digital que permita un diseño finalmente generativo. ABSTRACT The traditional architectural design and construction procedures have proven to be deficient where process optimization is concerned, particularly when compared to other common industrial activities. The ever‐growing strife to achieve effective industrialization, both in favor of reaching greater quality levels as well as sustainable management of resources, has a better chance today than ever through a mean out of the realm of information technology, the Building Information Modelling o BIM. What may initially seem to be merely another computer program, in reality turns out to be a “process” concept that subverts many of today’s routines in architectural design and construction. Including and working with project data from the very beginning to the end of its full life cycle allows for creating a dynamic and updatable virtual reality, enabling data testing and optimizing throughout: before and during execution, all the way to the end of its lifespan. In addition, there is an opportunity to transmit complete project data efficiently, with hardly any loss or redeveloping of the manufacture chain required, which facilitates attaining a truly significant industrialization within the construction industry. In the presence of a world‐wide call for optimizing resources, along with an undeniable interest in increasing economic benefits through reducing uncertainty factors in its processes, BIM undoubtedly offers a chance for improvement as acknowledged by its imminent and mandatory implementation on the part of governments (for example United Kingdom in 2016 and Spain in 2018). The changes involved in professional roles and procedures upon incorporating BIM are highly significant and will set the course for future graduates of Architecture, Engineering and Construction disciplines (AEC) within their professions. Higher Education must respond to such needs with swiftness by incorporating this methodology into their educational standards and providing a synergetic vision that focuses on the underlying educational benefits inherent in the BIM framework. In this respect, BIM, in gathering data set under one single virtual model, offers a uniquely interesting potential. The three‐dimensional reality of the model, under continuous development and updating, provides students with a radically different graphic environment, in which partial views of elevation, section or plan that tend characteristically to be difficult to assimilate at the beginning of their studies, become mere post hoc requests to be ordered when needed directly out the virtual model. The design is always carried out on the sole model itself, independently of the working view chosen at any particular moment, with all data and data relations within construction permanently updated and fully coherent. This condensed description of the features of BIM begin to shape an important part of the educational benefits posed by BIM processes, particularly in reference to integrated design development and information management (including ITC). At the same time, it highlights the ease with which visual understanding is achieved regarding architectural elements, technology systems, their intrinsic relationships, and construction processes. In addition to this, there is the experimental development the BIM platform grants through its collaborative software: simulation of structural, energetic, and economic behavior, among others, of the virtual model according to the data inherent to the project. This doctoral dissertation presents a broad study including a wide array of research methods and issues in order to specify both the virtues and possible reservations in the use of BIM processes within the framework of a specific discipline: teaching Architecture. To do so, a literature review on BIM has been carried out, specifically concerning teaching in the discipline of Architecture, as well as an analysis of the experience of different groups of interest delimited to Universidad Europea de Madrid. The analysis of the benefits and/or limitations of using BIM has been approached through student surveys and interviews with professionals from the AEC sector, associated or not, with BIM. Various diverse educational experiences are described and academic management for experimental implementation has been analyzed. The conclusions of this study offer a synthesis for a Framework of Implementation of BIM methodology, which in order to reach greater clarity, communication ease and user‐friendliness, have been posed in an eminently graphic manner. The proposed framework proffers guidance on teaching methods conducive to the development of specific skills, taking advantage of the conceptual flexibility of the European Higher Education Area guidelines based on competencies, which naturally facilitate for the incorporation of this new teaching tool to achieve the educational objectives established by law. The global approach of the Implementation Framework put forth in this study facilitates the planning of educational actions within a common perspective: combining exceptional or vehicular BIM formats, establishing cross‐disciplinary synergies, and sharing resources, so as to purport a methodology that contributes to the assimilation of knowledge and pre‐defined competencies within the degree program, and to the flow of learning itself. At the same time, it reserves, even visually, those areas of knowledge in which the use of BIM processes is not considered necessarily an advantage over other methodologies, or even inadequate for the learning outcomes established, at least where current planning is concerned. It is this last category which characterizes the research conclusions as a whole, centering on: 1. The unquestionable need for teaching BIM concepts and processes in Architecture very early on, in the initial stages of higher education; 2. The additional educational benefits that BIM offers in a varied array of competency development within the academic curriculum; and 3. The specific nature of the professional role of the Architect, which demands a careful and balanced implementation of BIM that respects the traditional teaching methodologies that have proven effective and creative, and adds value by a symbiotic reorientation merged with parametric design and digital manufacturing so to enable for a finally generative design.

Automated wordlength optimization framework for multi-source statistical interval-based analysis of nonlinear systems with control-flow structures

El uso de aritmética de punto fijo es una opción de diseño muy extendida en sistemas con fuertes restricciones de área, consumo o rendimiento. Para producir implementaciones donde los costes se minimicen sin impactar negativamente en la precisión de los resultados debemos llevar a cabo una asignación cuidadosa de anchuras de palabra. Encontrar la combinación óptima de anchuras de palabra en coma fija para un sistema dado es un problema combinatorio NP-hard al que los diseñadores dedican entre el 25 y el 50 % del ciclo de diseño. Las plataformas hardware reconfigurables, como son las FPGAs, también se benefician de las ventajas que ofrece la aritmética de coma fija, ya que éstas compensan las frecuencias de reloj más bajas y el uso más ineficiente del hardware que hacen estas plataformas respecto a los ASICs. A medida que las FPGAs se popularizan para su uso en computación científica los diseños aumentan de tamaño y complejidad hasta llegar al punto en que no pueden ser manejados eficientemente por las técnicas actuales de modelado de señal y ruido de cuantificación y de optimización de anchura de palabra. En esta Tesis Doctoral exploramos distintos aspectos del problema de la cuantificación y presentamos nuevas metodologías para cada uno de ellos: Las técnicas basadas en extensiones de intervalos han permitido obtener modelos de propagación de señal y ruido de cuantificación muy precisos en sistemas con operaciones no lineales. Nosotros llevamos esta aproximación un paso más allá introduciendo elementos de Multi-Element Generalized Polynomial Chaos (ME-gPC) y combinándolos con una técnica moderna basada en Modified Affine Arithmetic (MAA) estadístico para así modelar sistemas que contienen estructuras de control de flujo. Nuestra metodología genera los distintos caminos de ejecución automáticamente, determina las regiones del dominio de entrada que ejercitarán cada uno de ellos y extrae los momentos estadísticos del sistema a partir de dichas soluciones parciales. Utilizamos esta técnica para estimar tanto el rango dinámico como el ruido de redondeo en sistemas con las ya mencionadas estructuras de control de flujo y mostramos la precisión de nuestra aproximación, que en determinados casos de uso con operadores no lineales llega a tener tan solo una desviación del 0.04% con respecto a los valores de referencia obtenidos mediante simulación. Un inconveniente conocido de las técnicas basadas en extensiones de intervalos es la explosión combinacional de términos a medida que el tamaño de los sistemas a estudiar crece, lo cual conlleva problemas de escalabilidad. Para afrontar este problema presen tamos una técnica de inyección de ruidos agrupados que hace grupos con las señales del sistema, introduce las fuentes de ruido para cada uno de los grupos por separado y finalmente combina los resultados de cada uno de ellos. De esta forma, el número de fuentes de ruido queda controlado en cada momento y, debido a ello, la explosión combinatoria se minimiza. También presentamos un algoritmo de particionado multi-vía destinado a minimizar la desviación de los resultados a causa de la pérdida de correlación entre términos de ruido con el objetivo de mantener los resultados tan precisos como sea posible. La presente Tesis Doctoral también aborda el desarrollo de metodologías de optimización de anchura de palabra basadas en simulaciones de Monte-Cario que se ejecuten en tiempos razonables. Para ello presentamos dos nuevas técnicas que exploran la reducción del tiempo de ejecución desde distintos ángulos: En primer lugar, el método interpolativo aplica un interpolador sencillo pero preciso para estimar la sensibilidad de cada señal, y que es usado después durante la etapa de optimización. En segundo lugar, el método incremental gira en torno al hecho de que, aunque es estrictamente necesario mantener un intervalo de confianza dado para los resultados finales de nuestra búsqueda, podemos emplear niveles de confianza más relajados, lo cual deriva en un menor número de pruebas por simulación, en las etapas iniciales de la búsqueda, cuando todavía estamos lejos de las soluciones optimizadas. Mediante estas dos aproximaciones demostramos que podemos acelerar el tiempo de ejecución de los algoritmos clásicos de búsqueda voraz en factores de hasta x240 para problemas de tamaño pequeño/mediano. Finalmente, este libro presenta HOPLITE, una infraestructura de cuantificación automatizada, flexible y modular que incluye la implementación de las técnicas anteriores y se proporciona de forma pública. Su objetivo es ofrecer a desabolladores e investigadores un entorno común para prototipar y verificar nuevas metodologías de cuantificación de forma sencilla. Describimos el flujo de trabajo, justificamos las decisiones de diseño tomadas, explicamos su API pública y hacemos una demostración paso a paso de su funcionamiento. Además mostramos, a través de un ejemplo sencillo, la forma en que conectar nuevas extensiones a la herramienta con las interfaces ya existentes para poder así expandir y mejorar las capacidades de HOPLITE. ABSTRACT Using fixed-point arithmetic is one of the most common design choices for systems where area, power or throughput are heavily constrained. In order to produce implementations where the cost is minimized without negatively impacting the accuracy of the results, a careful assignment of word-lengths is required. The problem of finding the optimal combination of fixed-point word-lengths for a given system is a combinatorial NP-hard problem to which developers devote between 25 and 50% of the design-cycle time. Reconfigurable hardware platforms such as FPGAs also benefit of the advantages of fixed-point arithmetic, as it compensates for the slower clock frequencies and less efficient area utilization of the hardware platform with respect to ASICs. As FPGAs become commonly used for scientific computation, designs constantly grow larger and more complex, up to the point where they cannot be handled efficiently by current signal and quantization noise modelling and word-length optimization methodologies. In this Ph.D. Thesis we explore different aspects of the quantization problem and we present new methodologies for each of them: The techniques based on extensions of intervals have allowed to obtain accurate models of the signal and quantization noise propagation in systems with non-linear operations. We take this approach a step further by introducing elements of MultiElement Generalized Polynomial Chaos (ME-gPC) and combining them with an stateof- the-art Statistical Modified Affine Arithmetic (MAA) based methodology in order to model systems that contain control-flow structures. Our methodology produces the different execution paths automatically, determines the regions of the input domain that will exercise them, and extracts the system statistical moments from the partial results. We use this technique to estimate both the dynamic range and the round-off noise in systems with the aforementioned control-flow structures. We show the good accuracy of our approach, which in some case studies with non-linear operators shows a 0.04 % deviation respect to the simulation-based reference values. A known drawback of the techniques based on extensions of intervals is the combinatorial explosion of terms as the size of the targeted systems grows, which leads to scalability problems. To address this issue we present a clustered noise injection technique that groups the signals in the system, introduces the noise terms in each group independently and then combines the results at the end. In this way, the number of noise sources in the system at a given time is controlled and, because of this, the combinato rial explosion is minimized. We also present a multi-way partitioning algorithm aimed at minimizing the deviation of the results due to the loss of correlation between noise terms, in order to keep the results as accurate as possible. This Ph.D. Thesis also covers the development of methodologies for word-length optimization based on Monte-Carlo simulations in reasonable times. We do so by presenting two novel techniques that explore the reduction of the execution times approaching the problem in two different ways: First, the interpolative method applies a simple but precise interpolator to estimate the sensitivity of each signal, which is later used to guide the optimization effort. Second, the incremental method revolves on the fact that, although we strictly need to guarantee a certain confidence level in the simulations for the final results of the optimization process, we can do it with more relaxed levels, which in turn implies using a considerably smaller amount of samples, in the initial stages of the process, when we are still far from the optimized solution. Through these two approaches we demonstrate that the execution time of classical greedy techniques can be accelerated by factors of up to ×240 for small/medium sized problems. Finally, this book introduces HOPLITE, an automated, flexible and modular framework for quantization that includes the implementation of the previous techniques and is provided for public access. The aim is to offer a common ground for developers and researches for prototyping and verifying new techniques for system modelling and word-length optimization easily. We describe its work flow, justifying the taken design decisions, explain its public API and we do a step-by-step demonstration of its execution. We also show, through an example, the way new extensions to the flow should be connected to the existing interfaces in order to expand and improve the capabilities of HOPLITE.

Retinoic acid alters photoreceptor development in vivo

Application of exogenous retinoic acid (RA) to zebrafish during the initial stages of photoreceptor differentiation results in a precocious development of rod photoreceptors and an inhibition of cone photoreceptor maturation. The acceleration of rod differentiation is observed initially within the ventral retina 3 days after fertilization, following 24 hr of RA application, and within the dorsal retina 4 days after fertilization, following 48 hr of RA application. The differentiation of rods was impeded significantly when the synthesis of endogenous retinoic acid was inhibited by citral prior to the initial stage of rod differentiation. RA-treated embryos labeled for bromodeoxyuridine (BrdU) uptake revealed that RA exerts its effect on a postmitotic cell population within the developing retina. During normal development in zebrafish, rod differentiation is most robust within the ventral retina, a region previously shown to be rich in RA. Our data suggest that the RA signaling pathway is involved in the differentiation and maturation of both the rod and cone photoreceptors within the developing zebrafish retina.

Inactivation of the α C protein antigen gene, bca, by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus

The α C protein of group B Streptococcus (GBS) is a major surface-associated antigen. Although its role in the biology and virulence of GBS has not been defined, it is opsonic and capable of eliciting protective immunity. The α C protein is widely distributed among clinical isolates and is a potential protein carrier and antigen in conjugate vaccines to prevent GBS infections. The structural gene for the α C protein, bca, has been cloned and sequenced. The protein encoded by bca is related to a class of surface-associated proteins of Gram-positive cocci involved in virulence and immunity. To investigate the potential roles of the α C protein, bca null mutants were generated in which the bca gene was replaced with a kanamycin resistance cassette via homologous recombination using a novel shuttle/suicide vector. Studies of lethality in neonatal mice showed that the virulence of the bca null mutants was attenuated 5- to 7-fold when compared with the isogenic wild-type strain A909. Significant differences in mortality occurred in the first 24 h, suggesting that the role of the α antigen is important in the initial stages of the infection. In contrast to A909, bca mutants were no longer killed by polymorphonuclear leukocytes in the presence of α-specific antibodies in an in vitro opsonophagocytic assay. In contrast to previous studies, α antigen expression does not appear to play a role in resistance to opsonophagocytosis in the absence of α-specific antibodies. In addition, antibodies to the α C protein did not passively protect neonatal mice from lethal challenge with bca mutants, suggesting that these epitopes are uniquely present within the α antigen as expressed from the bca gene. Therefore, the α C protein is important in the pathogenesis of GBS infection and is a target for protective immunity in the development of GBS vaccines.

The Relationship between Photosynthesis and a Mastoparan-Induced Hypersensitive Response in Isolated Mesophyll Cells1

The G-protein activator mastoparan (MP) was found to elicit the hypersensitive response (HR) in isolated Asparagus sprengeri mesophyll cells at micromolar concentrations. The HR was characterized by cell death, extracellular alkalinization, and an oxidative burst, indicated by the reduction of molecular O2 to O2⋅−. To our knowledge, this study was the first to monitor photosynthesis during the HR. MP had rapid and dramatic effects on photosynthetic electron transport and excitation energy transfer as determined by variable chlorophyll a fluorescence measurements. A large increase in nonphotochemical quenching of chlorophyll a fluorescence accompanied the initial stages of the oxidative burst. The minimal level of fluorescence was also quenched, which suggests the origin of this nonphotochemical quenching to be a decrease in the antenna size of photosystem II. In contrast, photochemical quenching of fluorescence decreased dramatically during the latter stages of the oxidative burst, indicating a somewhat slower inhibition of photosystem II electron transport. The net consumption of O2 and the initial rate of O2 uptake, elicited by MP, were higher in the light than in the dark. These data indicate that light enhances the oxidative burst and suggest a complex relationship between photosynthesis and the HR.

Temporal Sequence of Cell Wall Disassembly in Rapidly Ripening Melon Fruit1

The Charentais variety of melon (Cucumis melo cv Reticulatus F1 Alpha) was observed to undergo very rapid ripening, with the transition from the preripe to overripe stage occurring within 24 to 48 h. During this time, the flesh first softened and then exhibited substantial disintegration, suggesting that Charentais may represent a useful model system to examine the temporal sequence of changes in cell wall composition that typically take place in softening fruit. The total amount of pectin in the cell wall showed little reduction during ripening but its solubility changed substantially. Initial changes in pectin solubility coincided with a loss of galactose from tightly bound pectins, but preceded the expression of polygalacturonase (PG) mRNAs, suggesting early, PG-independent modification of pectin structure. Depolymerization of polyuronides occurred predominantly in the later ripening stages, and after the appearance of PG mRNAs, suggesting the existence of PG-dependent pectin degradation in later stages. Depolymerization of hemicelluloses was observed throughout ripening, and degradation of a tightly bound xyloglucan fraction was detected at the early onset of softening. Thus, metabolism of xyloglucan that may be closely associated with cellulose microfibrils may contribute to the initial stages of fruit softening. A model is presented of the temporal sequence of cell wall changes during cell wall disassembly in ripening Charentais melon.

Epicuticular Wax Accumulation and Fatty Acid Elongation Activities Are Induced during Leaf Development of Leeks1

Epicuticular wax production was evaluated along the length of expanding leek (Allium porrum L.) leaves to gain insight into the regulation of wax production. Leaf segments from the bottom to the top were analyzed for (a) wax composition and load; (b) microsomal fatty acid elongase, plastidial fatty acid synthase, and acyl-acyl carrier protein (ACP) thioesterase activities; and (c) tissue and cellular morphological changes. The level of total wax, which was low at the bottom, increased 23-fold along the length of the leaf, whereas accumulation of the hentriacontan-16-one increased more than 1000-fold. The onset of wax accumulation was not linked to cell elongation but, rather, occurred several centimeters above the leaf base. Peak microsomal fatty acid elongation activity preceded the onset of wax accumulation, and the maximum fatty acid synthase activity was coincident with the onset. The C16:0- and C18:0-ACP-hydrolyzing activities changed relatively little along the leaf, whereas C18:1-ACP-hydrolyzing activity increased slightly prior to the peak elongase activity. Electron micrographic analyses revealed that wax crystal formation was asynchronous among cells in the initial stages of wax deposition, and morphological changes in the cuticle and cell wall preceded the appearance of wax crystals. These studies demonstrated that wax production and microsomal fatty acid elongation activities were induced within a defined and identifiable region of the expanding leek leaf and provide the foundation for future molecular studies.

Nuclear LIM interactor, a rhombotin and LIM homeodomain interacting protein, is expressed early in neuronal development.

LIM domain-containing transcription factors, including the LIM-only rhombotins and LIM-homeodomain proteins, are crucial for cell fate determination of erythroid and neuronal lineages. The zinc-binding LIM domains mediate protein-protein interactions, and interactions between nuclear LIM proteins and transcription factors with restricted expression patterns have been demonstrated. We have isolated a novel protein, nuclear LIM interactor (NLI), that specifically associates with a single LIM domain in all nuclear LIM proteins tested. NLI is expressed in the nuclei of diverse neuronal cell types and is coexpressed with a target interactor islet-1 (Isl1) during the initial stages of motor neuron differentiation, suggesting the mutual involvement of these proteins in the differentiation process. The broad range of interactions between NLI and LIM-containing transcription factors suggests the utilization of a common mechanism to impart unique cell fate instructions.

Escherichia coli trigger factor is a prolyl isomerase that associates with nascent polypeptide chains.

Correct folding of newly synthesized proteins is proposed to be assisted by molecular chaperones and folding catalysts. To identify cellular factors involved in the initial stages of this process we searched for proteins associated with nascent polypeptide chains. In an Escherichia coli transcription/translation system synthesizing beta-galactosidase we identified a 58-kDa protein which associated with translating ribosomes but dissociated from these ribosomes upon release of nascent beta-galactosidase. N-terminal sequencing identified it as trigger factor, previously implicated in protein secretion. Direct evidence for association of trigger factor with nascent polypeptide chains was obtained by crosslinking. In a wheat germ translation system complemented with E. coli lysates, epsilon-4-(3-trifluoromethyldiazirino)benzoic acid-lysine residues were incorporated into nascent secretory preprolactin and a nonsecretory preprolactin mutant. Trigger factor crosslinked to both types of nascent chains, provided they were ribosome bound. Trigger factor contains key residues of the substrate-binding pocket of FK506-binding protein-type peptidyl-prolyl-cis/trans-isomerases and has prolyl isomerase activity in vitro. We propose that trigger factor is a folding catalyst acting cotranslationally.

The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen.

Selective lodgement or homing of transplanted hemopoietic stem cells in the recipient's bone marrow (BM) is a critical step in the establishment of long-term hemopoiesis after BM transplantation. However, despite its biologic and clinical significance, little is understood about the process of homing. In the present study, we have concentrated on the initial stages of homing and explored the functional role in vivo of some of the adhesion pathways previously found to mediate in vitro adhesion of hemopoietic cells to cultured BM stroma. We have found that homing of murine hemopoietic progenitors of the BM of lethally irradiated recipients at 3 h after transplant was significantly reduced after pretreatment of the donor cells with an antibody to the integrin very late antigen 4 (VLA4). This inhibition of marrow homing was accompanied by an increase in hemopoietic progenitors circulating in the blood and an increased uptake of these progenitors by the spleen. Similar results were obtained by treatment of the recipients with an antibody to vascular cell adhesion molecule 1 (VCAM-1), a ligand for VLA4. Furthermore, we showed that administration of the same antibodies (anti-VLA4 or anti-VCAM-1) to normal animals causes mobilization of hemopoietic progenitors into blood. These data suggest that hemopoietic cell lodgement in the BM is a regulatable process and can be influenced by VLA4/VCAM-1 adhesion pathway. Although additional molecular pathways are not excluded and may be likely, our data establish VCAM-1 as a BM endothelial addressin, analogous to the role that mucosal addressin cell adhesion molecule (MAdCAM) plays in lymphocyte homing. Whether splenic uptake of hemopoietic progenitors is passive or controlled through different mechanisms remains to be clarified. In addition, we provide experimental evidence that homing and mobilization are related phenomena involving, at least partly, similar molecular pathways.