Análisis comparativo de procedimientos para la medición de las gemas y evaluación de su influencia en la determinación de las propiedades mecánicas de la madera de Pinus sylvestris L. de gran escuadría. Comparison of wanes measurement systems to evaluate their influence on the mechanical properties of large cross section timber of Pinus sylvestris L.

Las gemas se evalúan mediante la norma de clasificación visual (UNE 56544), pero su aplicación en estructuras existentes y grandes escuadrías resulta poco eficaz y conduce a estimaciones demasiado conservadoras. Este trabajo analiza la influencia de las gemas comparando la resistencia de piezas con gemas y piezas correctamente escuadradas. Se han analizado 218 piezas de pino silvestre con dimensiones nominales 150 x 200 x 4.200 mm, de las que 102 presentaban una gema completa a lo largo de toda su longitud y el resto estaban correctamente escuadradas. En las piezas con gema se ha medido la altura de la sección cada 30 cm (altura en cada cara y altura máxima). Para determinar la resistencia se han ensayado todas las piezas de acuerdo a la norma EN 408. Se ha comparado la resistencia obtenida para las piezas con gema, diferenciando si la gema se encuentra en el borde comprimido o en el borde traccionado, con las piezas escuadradas. Puede concluirse que la presencia de gemas disminuye la resistencia excepto si la gema se encuentra en el borde traccionado, en cuyo caso los resultados obtenidos han sido similares a los de las piezas escuadradas. The wanes on structural timber are evaluated according to the visual grading standard (UNE 56544), but its application on existing structures and large cross sections is ineffective and leads to conservative estimations. This paper analyzes the influence of the wanes by comparing the resistance of pieces with wanes and square pieces. 218 pieces of Scotch pine with nominal dimensions 150 x 200 x 4200 mm have been analyzed, 102 of them had a complete wane along its length and the rest were properly squared. The height of the cross section was measured every 30 cm (the height on each side and the maximum height) for the pieces with wane. The bending strength of all the pieces was obtained according to the EN 408 standard. The bending strength of the pieces with wane has been compared with the strength of the squared pieces, taking into account if the wane is positioned on the compressed edge or on the tensioned edge. It can be concluded that the bending strength of the pieces with wanes is lower than the one of squared pieces, except if the wanes are on the tensioned edge of the beam.

Lupinus mariae-josphi, a new lupin endemic of soils with active lime and high pH in South Eastern Spain, is nodulated by a new bacterial lineage within Bradyrhizobium genus

Lupinus mariae-josephi is a recently described species (Pascual, 2004) able to grow in soils with high pH and active lime content in the Valencia province (Spain). L. mariae-josephi endosymbionts are extremely slowgrowing bacteria with genetic and symbiotic characteristics that differentiate them from Bradyrhizobium strains nodulating Lupinus spp. native of the Iberian Peninsula and adapted to grow in acid soils. Cross-inoculation experiments revealed that all the endosymbiotic isolates from L. mariae-josephi tested are legume-host selective and are unable to nodulate species such as L. angustifolius, and L. luteus. In contrast, Bradyrhizobium strains from Lupinus spp. tested were able to nodulate L. mariae-josephi, although the nodules fixed nitrogen inefficiently. Phylogenetic analysis was performed with housekeeping genes (rrn, glnII, recA, atpD) and nodulation gene nodC. Housekeeping gene phylogeny revealed that L. mariae-josephi rhizobia form a strongly supported monophyletic group within Bradyrhizobium genus. This cluster also includes B. jicamae and certain strains of B. elkanii. Contrarily, isolates from other Lupinus spp. native of the Iberian Peninsula were grouped mainly within B. canariense and two B. japonicum lineages. Phylogenetic analysis of L. mariae-josephi isolates based on the nodC symbiotic gene defined a solid clade close to isolates from Algerian Retama spp. and to fast-growing rhizobia.

Trajes espaciales: la vestimenta como proyecto arquitectónico

¿Qué son los TRAJES ESPACIALES? El término Trajes Espaciales da nombre al “equipo de inmersión” 1 necesario para garantizar las condiciones de habitabilidad en determinados entornos o circunstancias. En diferentes coyunturas sociales, políticas o ambientales el formato convencional de la arquitectura se vuelve limitado o poco eficaz. Determinadas configuraciones de “vida a la intemperie” 2, asociadas a necesidades de protección, energía, comunicación, socialización…, en situaciones de alta emancipación e itinerancia, solicitan una reflexión sobre la posible portabilidad, o más concretamente, sobre la idoneidad del uso 3 de las condiciones arquitectónicas. La tesis pretende acotar o definir un no reconocido ámbito de los recursos proyectuales del arquitecto dirigido a la confección de las más cercanas envolventes corporales entendidas como elementos arquitectónicos. Casa y vestimenta, citando a Marshall McLuhan, se identifican en un discurso que entiende ambas esferas como “extensiones de la piel” 4 que garantizan el control energético y la definición social del sujeto que las habita. En determinados momentos el arquitecto ha tenido que desestimar como principal herramienta de trabajo la construcción de edificios, para abordar ciertas demandas sociales. La tesis pretende demostrar cómo el diseño de trajes ha sido en ocasiones una estrategia proyectual del repertorio de la arquitectura. Su aportación es la de abordar el mundo de la vestimenta más allá del simple ejercicio estilístico, defendiendo estas construcciones como un elemento capaz de hacer habitable un determinado entorno. Este acercamiento ha permitido a los arquitectos ensayar en los trajes lo que posteriormente desarrollarían en sus edificios. Temas tratados por la arquitectura como higienismo, ornamento, estandarización, pliegue… han encontrado en la escala de la vestimenta su expresión más pura. El diseño de estos trajes, su pertinencia y su confección como parte del proyecto arquitectónico, conformará el hilo conductor de esta tesis. ABSTRACT The term spacesuits tries to define the “diving equipment” 5 necessary to ensure the living conditions in certain environments or circumstances. In specific social, political or environmental circumstances, the architectural standard format becomes limited or ineffective. Some configurations of “life outdoor” 6, associated with protection, energy, communication and socialization needs, in situations of high emancipation and roaming, seek a reflection on the possible portability, or more specifically, on the appropriateness of wearing the architectural features. The thesis aims to limit or define an unrecognized field from the resources of the architect focused on body emvelopes as architectural elements. House and clothing, quoting Marshall McLuhan, are matched in a speech that understand both spheres as “extensions of the skins” 7, to ensure energy control and social definition of the subject who inhabit them. At times, the architect has had to dismiss the construction of buildings as the main tool for working, to address certain social demands. The thesis aims to demonstrate how the costume design is sometimes a projectual strategy in the repertoire of architecture. Its contribution splits with Fashion as a mere stylistic exercise, defending the dress as an artefact capable of making livable a certain environment. This approach has allowed architects to explore at costumes what they develope in their buildings later. Topics covered by the architecture as hygienism, ornament, standardization, folding... have found in the scale of the dress its purest expression. The design of these suits, their techniques and their relevance as part of the architectural project, will form the core of this thesis.

Pru p 3 mutants exhibit low IgE-binding capacity: a good strategy for specific peach immunotherapy.

Treatment of food allergy consists of the avoidance of the specific allergenic food. However, the possibility of cross-reactivity with other food sources makes this practice sometimes ineffective. The use of hypoallergenic molecules with the ability to stimulate T cells may be a promising tool for specific immunotherapy.

Probe-based end-to-end overload control for networks of SIP servers

The Session Initiation Protocol (SIP) has been adopted by the IETF as the control protocol for creating, modifying and terminating multimedia sessions. Overload occurs in SIP networks when SIP servers have insufficient resources to handle received messages. Under overload, SIP networks may suffer from congestion collapse due to current ineffective SIP overload control mechanisms. This paper introduces a probe-based end-to-end overload control (PEOC) mechanism, which is deployed at the edge servers of SIP networks and is easy to implement. By probing the SIP network with SIP messages, PEOC estimates the network load and controls the traffic admitted to the network according to the estimated load. Theoretic analysis and extensive simulations verify that PEOC can keep high throughput for SIP networks even when the offered load exceeds the capacity of the network. Besides, it can respond quickly to the sudden variations of the offered load and achieve good fairness.

Análisis molecular de sistemas génicos implicados en la homeostasis de níquel y eficiencia simbiótica en Rhizobium leguminosarum

Los suelos ultramáficos, que poseen elevadas concentraciones de níquel, cobalto y cromo de manera natural, son fuente de bacterias resistentes a altas concentraciones de metales. Se realizó la caracterización físico-química de seis suelos ultramáficos del suroeste europeo, seleccionándose un suelo de la región de Gorro, Italia, como el más adecuado para aislar bacterias endosimbióticas resistentes a metales. A partir de plantas-trampa de guisante y lenteja inoculados con suspensiones de ese suelo, se obtuvieron 58 aislados de Rhizobium leguminosarum bv. viciae (Rlv) que fueron clasificados en 13 grupos según análisis de PCR-RAPDs. Se determinó la resistencia a cationes metálicos [Ni(II), Co(II), Cu(II), Zn(II)] de una cepa representante de cada grupo, así como la secuencia de los genomas de las cepas que mostraron altos niveles (UPM1137 y UPM1280) y bajos niveles (UPM1131 y UPM1136) de tolerancia a metales. Para identificar mecanismos de resistencia a metales se realizó una mutagénesis al azar en dicha cepa mediante la inserción de un minitransposón. El análisis de 4313 transconjugantes permitió identificar 14 mutantes que mostraron una mayor sensibilidad a Ni(II) que la cepa silvestre. Se determinó el punto de inserción del minitransposón en todos ellos y se analizaron en más detalle dos de los mutantes (D2250 y D4239). En uno de los mutantes (D2250), el gen afectado codifica para una proteína que presenta un 44% de identidad con dmeF (divalent efflux protein) de Cupriavidus metallidurans. Cadena arriba de dmeF se identificó un gen que codifica una proteína con un 39% de identidad con el regulador RcnR de Escherichia coli. Se decidió nombrar a este sistema dmeRF, y se generó un mutante en ambos genes en la cepa Rlv SPF25 (Rlv D15). A partir de experimentos de análisis fenotípico y de regulación se pudo demostrar que el sistema dmeRF tiene un papel relevante en la resistencia a Ni(II) y sobre todo a Co(II) en células en vida libre y en simbiosis con plantas de guisante. Ambos genes forman un operón cuya expresión se induce en respuesta a la presencia de Ni(II) y Co(II). Este sistema se encuentra conservado en distintas especies del género Rhizobium como un mecanismo general de resistencia a níquel y cobalto. Otro de los mutantes identificados (D4239), tiene interrumpido un gen que codifica para un regulador transcripcional de la familia AraC. Aunque inicialmente fue identificado por su sensibilidad a níquel, experimentos posteriores demostraron que su elevada sensibilidad a metales era debida a su sensibilidad al medio TY, y más concretamente a la triptona presente en el medio. En otros medios de cultivo el mutante no está afectado específicamente en su tolerancia a metales. Este mutante presenta un fenotipo simbiótico inusual, siendo inefectivo en guisantes y efectivo en lentejas. Análisis de complementación y de mutagénesis dirigida sugieren que el fenotipo de la mutación podría depender de otros factores distintos del gen portador de la inserción del minitransposón. ABSTRACT Ultramafic soils, having naturally high concentrations of nickel, cobalt and chrome, are potential sources of highly metal-resistant bacteria. A physico-chemical characterization of six ultramafic soils from the European southwest was made. A soil from Gorro, Italy, was chosen as the most appropriated for the isolation of heavy-metal-resistant endosymbiotic bacteria. From pea and lentil trap plants inoculated with soil suspensions, 58 isolates of Rhizobium leguminosarum bv. viciae (Rlv) were obtained and classified into 13 groups based on PCR-RAPDs analysis. The resistance to metallic cations [Ni(II), Co(II), Cu(II), Zn(II)] was analyzed in a representative strain of each group. From the results obtained in the resistance assays, the Rlv UPM1137 strain was selected to identify metal resistance mechanism. A random mutagenesis was made in UPM1137 by using minitransposon insertion. Analysis of 4313 transconjugants allowed to identify 14 mutants with higher sensitivity to Ni(II) than the wild type strain. The insertion point of the minitransposon was determined in all of them, and two mutants (D2250 and D4239) were studied in more detail. In one of the mutants (D2250), the affected gene encodes a protein with 44% identity in compared with DmeF (divalent efflux protein) from Cupriavidus metallidurans. Upstream R. leguminosarum dmeF, a gene encoding a protein with 39% identity with RcnR regulator from E. coli was identified. This protein was named DmeR. A mutant with both genes in the dmeRF deleted was generated and characterized in Rlv SPF25 (Rlv D15). From phenotypic and regulation analysis it was concluded that the dmeRF system is relevant for Ni(II) and specially Co(II) tolerance in both free living and symbiotic forms of the bacteria. This system is conserved in different Rhizobium species like a general mechanism for nickel and cobalt resistance. Other of the identified mutants (D4239) contains the transposon insert on a gene that encodes for an AraC-like transcriptional regulator. Although initially this mutant was identified for its nickel sensitivity, futher experiments demonstrated that its high metal sensitivity is due to its sensitivity to the TY medium, specifically for the tryptone. In other media the mutant is not affected specifically in their tolerance to metals. This mutant showed an unusual symbiotic phenotype, being ineffective in pea and effective in lentil. Complementation analysis and directed mutagenesis suggest that the mutation phenotype could depend of other factors different from the insertion minitransposon gene.

Integrated studies for the evaluation of conservation treatments on buildings materials from archaeological sites: application to the case of Merida (Spain)

The application of conservation treatments, such as consolidation and protection ones, has been demonstrated ineffective in many cases, and even harmful. Evaluation studies should be a mandatory task, ideally before and after the intervention, but both tasks are complex and unusual in the case of archaeological heritage. This study is mainly focused on analyzing changes in petrophysical properties of stone material from archaeological sites of Merida (Spain), evaluating, both on site and in laboratory, effects derived from different conservation treatments applied in past interventions, throughout the integration of different non-destructive techniques (NDT) and portable devices of analysis available at the Institute of Geosciences (CSIC,UCM). These techniques allow, not only assessment of effectiveness and alteration processes, but also monitoring durability of treatments, focused mainly on 1996 intervention in the case of Roman Theater, as well as different punctual interventions from the 90?s until date in the House of Mitreo. Studies carried out on archaeological sites of Merida permit us to compare outcomes and also check limitations in the use of those equipments. In this paper we discuss about the use of some techniques, their integration and limits, for the assessment of conservation treatments, showing some examples of Merida?s case study.

Experimental and numerical study of cw green laser crystallization of a-Si:H thin films

Crystallization and grain growth technique of thin film silicon are among the most promising methods for improving efficiency and lowering cost of solar cells. A major advantage of laser crystallization and annealing over conventional heating methods is its ability to limit rapid heating and cooling to thin surface layers. Laser energy is used to heat the amorphous silicon thin film, melting it and changing the microstructure to polycrystalline silicon (poly-Si) as it cools. Depending on the laser density, the vaporization temperature can be reached at the center of the irradiated area. In these cases ablation effects are expected and the annealing process becomes ineffective. The heating process in the a-Si thin film is governed by the general heat transfer equation. The two dimensional non-linear heat transfer equation with a moving heat source is solve numerically using the finite element method (FEM), particularly COMSOL Multiphysics. The numerical model help to establish the density and the process speed range needed to assure the melting and crystallization without damage or ablation of the silicon surface. The samples of a-Si obtained by physical vapour deposition were irradiated with a cw-green laser source (Millennia Prime from Newport-Spectra) that delivers up to 15 W of average power. The morphology of the irradiated area was characterized by confocal laser scanning microscopy (Leica DCM3D) and Scanning Electron Microscopy (SEM Hitachi 3000N). The structural properties were studied by micro-Raman spectroscopy (Renishaw, inVia Raman microscope).

Identification and analysis of a nickel-inducible cation diffusion facilitator-NiCoT combined system in Rhizobium leguminosarum bv. viciae

Nickel, like other transition metals, can be toxic to cells even at moderate concentration (low microM range) by displacing essential metals from their native binding sites or by generating reactive oxygen species that cause oxidative DNA damage. For this reason, cells have evolved mechanisms to deal with excess nickel. Efflux systems include members of the Resistance-Nodulation-cell Division (RND) protein family, P-type ATPases, cation diffusion facilitators (CDF) and other resistance factors. Nickel-specific exporters have been characterized in Cupravidus metallidurans, Helicobacter pylori, Achromobacter xylosoxidans, Serratia marcenses and Escherichia coli.

Categorization of Anti-Patterns in Software Project Management

In the last decades, software systems have become an intrinsic element in our daily lives. Software exists in our computers, in our cars, and even in our refrigerators. Today’s world has become heavily dependent on software and yet, we still struggle to deliver quality software products, on-time and within budget. When searching for the causes of such alarming scenario, we find concurrent voices pointing to the role of the project manager. But what is project management and what makes it so challenging? Part of the answer to this question requires a deeper analysis of why software project managers have been largely ineffective. Answering this question might assist current and future software project managers in avoiding, or at least effectively mitigating, problematic scenarios that, if unresolved, will eventually lead to additional failures. This is where anti-patterns come into play and where they can be a useful tool in identifying and addressing software project management failure. Unfortunately, anti-patterns are still a fairly recent concept, and thus, available information is still scarce and loosely organized. This thesis will attempt to help remedy this scenario. The objective of this work is to help organize existing, documented software project management anti-patterns by answering our two research questions: · What are the different anti-patterns in software project management? · How can these anti-patterns be categorized?

Los residuos de envases y embalajes en edificación residencial. Patrones de generación y estrategias de mejora de su gestión

Uno de los retos a los que se enfrenta la humanidad es la demanda de una sociedad con casi cero residuos: la búsqueda de una economía más eficiente en el uso de los recursos teniendo en cuenta el cambio climático y un abastecimiento sostenible de materias primas que permitan satisfacer las necesidades de una población mundial cada vez mayor, dentro de los límites sostenibles de los recursos naturales del planeta. Abordar este desafío implica la recuperación y reciclaje de materiales para reducir consumos de materias primas y, para ello, debe promoverse la innovación en la prevención y gestión de residuos, como estrategia hacia un desarrollo urbano sostenible. Los envases y embalajes forman parte de la cultura actual, relacionándose con el grado de desarrollo de países y regiones; el sector de la construcción no es ajeno a la problemática y genera un importante volumen de embalajes en las obras que no se gestionan de forma adecuada. El modelo de gestión de Residuos de Construcción Demolición (RCD) actual es poco eficaz, ya que los vertederos siguen recibiendo grandes cantidades de residuos valorizables. La normativa ha conseguido minimizar las prácticas ilegales, pero se comprueba que la gestión actual de los RCD en obra deja en manos de las plantas de tratamiento la misión de separar los residuos según su naturaleza y los datos aportados por las distintas plantas no permiten afirmar el grado de alcance de dicho objetivo, ni el destino de los mismos. Los envases y embalajes son un tipo de residuos que por su naturaleza suponen una fuente de recursos valorizables y por ello están presentes en directivas y leyes, dirigidas fundamentalmente al sector de la alimentación. En el ámbito de la construcción, hasta la fecha, no se habían tratado de forma específica, motivo que justifica este trabajo. Esta Tesis Doctoral tiene como objetivo principal mejorar la gestión de residuos de embalajes procedentes de materiales de construcción. Para ello se estudia un importante conjunto de obras de edificación residencial colectiva de 1173 viviendas, análisis que llevará a conocer los patrones de producción de los Residuos de Envases y Embalajes en Construcción (REEC) e identificar los productos o materiales responsables de su generación. La monitorización realizada, permitirá diseñar estrategias específicas que minimicen la generación de estos residuos y optimicen su valorización. Se ha podido comprobar que el tratamiento particularizado de los REEC puede mejorar los resultados de la gestión del conjunto de RCD, debido a que, al no mezclarse, se elimina un importante agente contaminador de los residuos inertes. Las obras analizadas alcanzan un nivel de segregación in situ muy bajo, y los capítulos de cerramientos e instalaciones suman más del 50% del global de REEC. Particularizando en cada tipo de material, el cartón predomina en los trabajos de electricidad, el plástico en las particiones y la madera en fachadas. Se evalúa la opción del eco-­‐rediseño para uno de los embalajes predominantes, y se identifican estrategias para minimizar su impacto. Se comprueba que el ahorro en la gestión de RCD en caso de impulsar la segregación in situ no es muy significativo en las obras estudiadas, si bien se demuestra que la viabilidad económica es el factor más importante para motivar a las empresas constructoras. Las Administraciones Públicas deberían incentivar la gestión responsable, estableciendo medidas de control en las plantas de tratamiento e implementando en sus obras medidas de gestión de RCD ejemplares para el mercado. Una adecuada planificación de la obra, y formación específica para el personal son medidas que facilitan la segregación in situ y mejoran los resultados. Los promotores, mediante su relación contractual con el resto de agentes, tienen la capacidad de establecer objetivos de reducción, planificación y gestión sostenible de los REEC. Por último, se propone un sistema de gestión de REEC, integrable dentro del Sistema de Gestión de la empresa constructora, que permite evitar que residuos valorizables tales como los envases y embalajes se envíen a vertedero, guiando al sector hacia un futuro más respetuoso, alineado con el desarrollo sostenible. ABSTRACT One of the challenges humanity faces is the demand of an almost zero waste society: the search for a more efficient economy in the use of resources, taking into account climate change and a sustainable supply of raw materials, that meet the needs of an increasing world population within the sustainable limits of the planet's natural resources. Addressing this challenge involves the recovery and recycling of materials to reduce consumption of raw materials, so innovation must therefore be promoted in the prevention and management of waste, as a strategy towards a sustainable urban development. Packaging is part of our current culture and is related to the degree of development of countries and regions; the construction sector is no stranger to this problem and generates a significant amount of packaging waste in the site works, which nowadays is not managed properly. The current Construction Demolition Waste (CDW) management model is ineffective, since landfills continue to receive large amounts of recoverable waste. The legislation has managed to minimize illegal practices, but it has been observed that the current management of CDW in the analysed works leaves the mission of separating waste according to their nature to the plants, and data provided by the latter does not allow for checking the scope of this objective, nor the destiny of the waste. Packaging is a type of waste which, by its nature, represents a source of recoverable resources and is therefore present in directives and laws, focused primarily on the food industry. It has not been specifically treated to date in the construction field, hence the reason that justifies this work. This Thesis aims to improve the management of packaging waste from construction materials. An important set of residential building works with 1173 dwellings is analysed, which leads to knowledge on the production patterns of packaging waste (PW) in the construction industry, and the identification of the products responsible for its generation. The monitorization also allows for the design of specific strategies which minimise the generation of waste and optimize recovery. It has been found that the individualized treatment of PW can improve the results of the management of the whole CDW since, when not mixed, a major pollutant of inert waste is removed. The analysed works reached a very low segregation level and the façades and building services phases account for more than 50% of the overall PW. Focusing on each type of material, cardboard predominates electricity works, plastic on partitions and wood on façades. Eco-­‐redesign is evaluated for one of the predominant packages, and strategies are identified to minimise their impact are . There is evidence that the savings in the CDW management in case of promoting segregation on site are not very significant at the studied works, although economic feasibility is the most important factor to motivate the construction companies. Public Administrations should encourage responsible management, establishing control measures in treatment plants and implementing CDW management exemplary measures on their work sites for the market. A proper planning of the works and specific training for the personnel are measures that facilitate in situ segregation and improve outcomes. The developers, through their contractual relationship with the other agents, have the ability to set reduction targets, planning and sustainable management of PW. Finally, a management system for PW is proposed, integrated within the Management System of the construction company, which aims to avoid that recoverable waste such as packaging is sent to landfill, leading the industry towards a more environmentally friendly future, aligned with sustainable development.

Integrated Studies for the Evaluation of Conservation Treatments on Stone Material from Archaeological Sites. Application to the Case of Merida (Spain)

The application of conservation treatments, such as consolidation and protection ones, has been demonstrated ineffective in many cases, and even harmful. Evaluation studies should be a mandatory task, ideally before and after the intervention, but both tasks are complex and unusual in the case of archaeological heritage. This study is mainly focused on analyzing changes in petrophysical properties of stone material from archaeological sites of Merida (Spain), evaluating, both on site and in laboratory, effects derived from different conservation treatments applied in past interventions, throughout the integration of different non-destructive techniques (NDT) and portable devices of analysis available at the Institute of Geosciences (CSIC,UCM). These techniques allow, not only assessment of effectiveness and alteration processes, but also monitoring durability of treatments, focused mainly on 1996 intervention in the case of Roman Theater, as well as different punctual interventions from the 90’s until date in the House of Mitreo. Studies carried out on archaeological sites of Merida permit us to compare outcomes and also check limitations in the use of those equipments. In this paper we discuss about the use of some techniques, their integration and limits, for the assessment of conservation treatments, showing some examples of Merida’s case study.

Native bradyrhizobial symbionts of Lupinus mariae-josephae, a unique endemism thriving in alkaline soils in Eastern Spain

Lupinus mariae-josephae (Lmj) es una especie de lupino endémica de una pequeña y específica área de Comunidad Valenciana (Este de España), donde prospera en suelos alcalinoscalcáreos, un hábitat singular para los altramuces, que crecen preferentemente en suelos ácidos o neutros. Esto hace de Lmj una especie de lupino única. Cuando se inició este trabajo, la extensión conocida de este endemismo abarcaba unos 700 kilómetros cuadrados, confinados en la provincia de Valencia. En esta área, Lmj prospera en pequeñas poblaciones aisladas que contienen un número reducido de plantas por lo que se la consideró una especie en peligro de extinción. Todos los esfuerzos, utilizando estrategias clásicas dirigidas a ampliar el área de crecimiento de Lmj y garantizar su conservación, han tenido un éxito limitado. El trabajo que se presenta está dirigido a mejorar el conocimiento de la ecología de Lmj, en particular la interacción simbiótica que establece con bacterias del suelo denominadas rizobios y se centra en la caracterización fenotípica, filogenética y genómica de esos rizobios. También se investiga la posible contribución de la simbiosis en mejorar la conservación de Lmj. Para este fin, se han estudiado diferentes aspectos que se describen a continuación. El primero objetivo se centró en aislar y estudiar de la diversidad genética de las bacterias endosimbióticas de Lmj. . Se realizó un análisis filogenético de genes esenciales que mostró que las cepas de Lmj pertenecen al género Bradyrhizobium y que presentan una gran diversidad con características fenotípicas y simbióticas diferentes de cepas de Bradyrhizobium que nodulan otras especies de lupinos nativos de España (cepas ISLU). Las cepas estudiadas se dividieron en dos grupos (Clado I y Clado II). El Clado I, incluye a las cepas Lmj, definiendo un nuevo linaje, filogenéticamente relacionado con otras especies de Bradyrhizobium, como B. jicamae y B. elkanii. El Clado II contiene cepas ISLU relacionadas con cepas de B. canariense y B. japonicum que establecen simbiosis con lupinos de suelos ácidos. Otro análisis filogenético basado en genes simbióticos, distribuyó las cepas de Lmj en sólo dos grupos diferentes. La singularidad y gran diversidad de estas cepas en una pequeña área geográfica, hacen de este, un atractivo sistema para el estudio de la evolución y adaptación de las bacterias simbióticas a su respectiva planta huésped. Adicionalmente, se estudio la presencia de bacterias capaces de nodular Lmj en suelos básicos de Chiapas, México. Sorprendentemente, estos suelos contienen bacterias capaces establecer interacciones simbióticas eficientes con Lmj en ensayos de invernadero. A continuación se investigó la taxonomía de los endosimbiontes de Lmj analizando la secuencia de cuatro genes esenciales (16S rRNA, recA, glnII y atpD) y el promedio de identidad de nucleótidos de genomas completos de algunas cepas representativas de la diversidad (ANIm). Se identificaron nuevas especies de Bradyrhizobium dentro del Clado I y se definió una de ellas: 'Bradyrhizobium valentinum' sp. nov (cepa tipo LmjM3T = CECT 8364T, LMG 2761T). También se abordó cómo conservar Lmj en su hábitat natural mediante inoculación con alguna de las cepas aisladas. Se demostró la ausencia de bacterias capaces de nodular Lmj en suelos rojos alcalinos o ‘‘terra rossa’’ de la Península Ibérica y Baleares. Dos cepas, altamente eficientes en cuanto a la fijación de nitrógeno, LmjC y LmjM3T, fueron seleccionadas para ser empleadas como inoculantes. Dos experimentos de campo llevados a cabo en años consecutivos en áreas con características edafoclimáticas similares a las que presentan las poblaciones de Lmj, lograron la reproducción exitosa de la planta. Se concluyó que un ciclo reproductivo exitoso de Lmj es absolutamente dependiente de la inoculación con sus simbiontes naturales y que la simbiosis debe ser considerada un factor esencial en estrategias de conservación de leguminosas en peligro. La obtención de varias secuencias genómicas de cepas aisladas de Lmj y de otras cepas de Bradyrhizobium reveló una alta similitud entre los genomas de las cepas del Clado I, y permitió la identificación de cinco posibles nuevas especies. Además, se estudiaron tres agrupaciones de genes relacionados con la simbiosis (nod, nif y fix) definiendo un nuevo linaje para las cepas de Lmj, diferente del symbiovar “genistearum” de B. canariense y B. japonicum. La baja diversidad encontrada en el análisis filogenético de los genes simbióticos contrasta con la gran diversidad asociada a genes esenciales. La presencia de plásmidos en cepas del género Bradyrhizobium ha sido descrita en muy pocas ocasiones, sin embargo el análisis de la secuencia genómica de la cepa ISLU101, aislada de Lupinus angustifolius, reveló la presencia de un origen de replicación extracromosómico homólogo al operón repABC, presente en el plásmido de Bradyrhizobium sp BTAi1. Gracias a esta secuencia se identificaron genes homólogos en 19 de 72 cepas ISLU. Filogenéticamente, las secuencias de repABC se agruparon en un grupo monofilético con las de pBTAi1 y separadas de los rizobios de crecimiento rápido. Finalmente, se identificaron sistemas de secreción de proteínas de tipo III (T3SS) en nueve genomas de cepas de Lmj. Los T3SS pueden inyectar proteínas efectoras al interior de células vegetales. Su presencia en rizobios se ha relacionado con la gama de hospedador que pueden nodular y puede tener un efecto beneficioso, neutro o perjudicial en la simbiosis. Los T3SS de las cepas de Lmj codifican para una proteína efectora similar a NopE, un efector dependiente de T3SS descrito en B. diazoefficiens USDA 110T. La proteína NopE de la cepa LmjC se ha caracterizado bioquímicamente. ABSTRACT Lupinus mariae-josephae (Lmj) is a lupine species endemic of a unique small area in Valencia region (Eastern Spain) where the lupine plants thrive in alkaline-limed soils, which preferentially grow in acid or neutral soils. This is the type of soils native lupines of Spain. When this work was initiated, the extension of the endemic area of Lmj was of about 700 squared kilometers confined to the Valencia province. In this area, Lmj thrives in small, isolated patches containing a reduced number of plants, and points to an endemism that can easily became endangered or extinct. Consequently, the Valencia Community authorities gave a ‘‘microreserve” status for conservation of the species. All efforts, using classical strategies directed to extend the area of Lmj growth and ensure its conservation have been so far unsuccessful. The work presented here is directed to improve our knowledge of Lmj ecology and it is centered in the characterization of the rhizobial symbiosis by phenotypic, phylogenetic and genomic analysis as well as in investigate the potential contribution of the symbiosis to improve its conservation. To this end, five different topics have been studied, and results are briefly described here. Extensive details can be followed en the attached, published articles. The first topic deals with the indigenous rhizobial symbionts of the Lmj endemism, and its genetic diversity was investigated. The Lmj root symbionts belong to the Bradyrhizobium genus, and phylogenetic analysis based on core genes identified a large diversity of Bradyrhizobium strains with phenotypic and symbiotic characteristics different from rhizobia nodulating other Lupinus spp. native of Spain. The strains were split in two clades. Clade II contained strains close to classical B. canariense and B. japonicum lineages that establish symbioses with lupines in acid soils of the Mediterranean area. Clade I included Lmj strains that define a new lineage, close to other Bradyrhizobium species as B. jicamae and B. elkanii. The phylogenetic analysis based on symbiotic genes identified only two distinct clusters. The singularity and large diversity of these strains in such a small geographical area makes this an attractive system for studying the evolution and adaptation of the rhizobial symbiont to the plant host. Additionally, the presence of bacteria able to nodulate Lmj in basic soils from Chiapas, Mexico was investigated. Surprisingly, these soils contain bacteria able to effectively nodulate and fix nitrogen with Lmj plants in greenhouse assays. In the second topic, the taxonomic status of the endosymbiotic bacteria of Lmj from Valencia endemism and Chiapas was investigated. Results from phylogenetic analysis of core genes and Average Nucleotide Identity (ANIm) using draft genomic sequences identified new Bradyrhizobium species within strains of Clade I of Lmj endosymbiotic bacteria. Only one of these potentially new species has been defined, meanwhile the others are under process of characterization. The name ‘Bradyrhizobium valentinum’ sp. nov. was proposed for the defined species (type strain LmjM3T= CECT 8364T, LMG 2761T). The third topic was directed to conservation of endangered Lmj in its natural habitat. The relevant conclusion of this experimentation is that the symbiosis should be considered as a relevant factor in the conservation strategies for endangered legumes. First, we showed absence of bacteria able to nodulate Lmj in all the inspected ‘‘terra rossa’’ or alkaline red soils of the Iberian Peninsula and Balearic Islands. Then, two efficient nitrogen fixing strains with Lmj plants, LmjC and LmjM3T, were selected as inoculum for seed coating. Two planting experiments were carried out in consecutive years under natural conditions in areas with edapho-climatic characteristics identical to those sustaining natural Lmj populations, and successful reproduction of the plant was achieved. The relevant conclusion from these assays was that the successful reproductive cycle was absolutely dependent on seedling inoculation with effective bradyrhizobia The forth topic deep into the analysis of the genomic of Lmj representative strains. To this end, draft genomic sequences of selected Lmj strains and type strains of Bradyrhizobium spp. were assembled. The comparison analysis of the draft genomic sequences of Lmj strains and related Bradyrhizobium species grouped in Clade I, revealed a high genomic homology among them, and allowed the definition of five potentially new species of Lmj nodulating bacteria. Also, based on the available draft genomic sequences, only three clusters of nod, fix and nif genes from Lmj strains were identified and showed to define a new symbiotic lineage, distant from that of B. canariense and B. japonicum bv. genistearum. The low diversity exhibited by the phylogenetic analysis of symbiotic genes contrast with the large diversity of strains as regards the housekeeping genes analyzed. Besides, the genomic analysis of a Lupinus angustifolius strain ISLU101, revealed the presence of an extrachromosomal replication origin homologous to repABC cluster from plasmid present in Bradyrhizobium spp BTAi1. This repABC cluster gene sequence allowed the identification of extrachromosomic replication origin in 19 out of 72 Bradyrhizobium strains from Lupinus spp., a highly significant result since the absence of plasmids in the Bradyrhizobium genus was traditionally assumed. The repABC gene sequences of these strains grouped them in a unique monophyletic group, related to B. sp. BTAi1 plasmid, but differentiated from the repABC gene cluster of plasmids in fast growing rhizobium strains. The last topic was focused on characterization of type III secreted effectors present in Lmj endosymbiotic bacteria. Type III secretion systems (T3SS) are specialized protein export machineries which can deliver effector proteins into plant cells. The presence of T3SS in rhizobia has frequently been related to the symbiotic nodulation host-range and may have a beneficial or detrimental effect on the symbiosis with legumes. In this context, the presence of T3SS in genomes of nine Lmj strains was investigated, and it was shown the presence of clusters encoding NopE type III-secreted protein similar to the NopE1 and NopE2 of B. diazoefficiens USDA 110T. The putative NopE protein of LmjC strain is at present being characterized regarding its structure and function.

Analysis and optimization for a hexapod walking robot for planetary missions

La tecnología de las máquinas móviles autónomas ha sido objeto de una gran investigación y desarrollo en las últimas décadas. En muchas actividades y entornos, los robots pueden realizar operaciones que son duras, peligrosas o simplemente imposibles para los humanos. La exploración planetaria es un buen ejemplo de un entorno donde los robots son necesarios para realizar las tareas requeridas por los científicos. La reciente exploración de Marte con robots autónomos nos ha mostrado la capacidad de las nuevas tecnologías. Desde la invención de la rueda, que esta acertadamente considerado como el mayor invento en la historia del transporte humano, casi todos los vehículos para exploración planetaria han empleado las ruedas para su desplazamiento. Las nuevas misiones planetarias demandan maquinas cada vez mas complejas. En esta Tesis se propone un nuevo diseño de un robot con patas o maquina andante que ofrecerá claras ventajas en entornos extremos. Se demostrara que puede desplazarse en los terrenos donde los robots con ruedas son ineficientes, convirtiéndolo en una elección perfecta para misiones planetarias. Se presenta una reseña histórica de los principales misiones espaciales, en particular aquellos dirigidos a la exploración planetaria. A través de este estudio será posible analizar las desventajas de los robots con ruedas utilizados en misiones anteriores. El diseño propuesto de robot con patas será presentado como una alternativa para aquellas misiones donde los robots con ruedas puedan no ser la mejor opción. En esta tesis se presenta el diseño mecánico de un robot de seis patas capaz de soportar las grandes fuerzas y momentos derivadas del movimiento de avance. Una vez concluido el diseño mecánico es necesario realizar un análisis que permita entender el movimiento y comportamiento de una maquina de esta complejidad. Las ecuaciones de movimiento del robot serán validadas por dos métodos: cinemático y dinámico. Dos códigos Matlab® han sido desarrollados para resolver dichos sistemas de ecuaciones y han sido verificados por un tercer método, un modelo de elementos finitos, que también verifica el diseño mecánico. El robot con patas presentado, ha sido diseñado para la exploración planetaria en Marte. El comportamiento del robot durante sus desplazamientos será probado mediante un código de Matlab®, desarrollado para esta tesis, que permite modificar las trayectorias, el tipo de terreno, y el número y altura de los obstáculos. Estos terrenos y requisitos iniciales no han sido elegidos de forma aleatoria, si no que están basados en mi experiencia como miembro del equipo de MSL-NASA que opera un instrumento a bordo del rover Curiosity en Marte. El robot con patas desarrollado y fabricado por el Centro de Astrobiología (INTA-CSIC), esta basado en el diseño mecánico y análisis presentados en esta tesis. ABSTRACT The autonomous machines technology has undergone a major research and development during the last decades. In many activities and environments, robots can perform operations that are tought, dangerous or simply imposible to humans. Planetary exploration is a good example of such environment where robots are needed to perform the tasks required by the scientits. Recent Mars exploration based on autonomous vehicles has shown us the capacity of the new technologies. From the invention of the wheel, which is rightly regarded as the greatest invention in the history of human transportation, nearly all-planetary vehicles are based in wheeled locomotion, but new missions demand new types of machines due to the complex tasks needed to be performed. It will be proposed in this thesis a new design of a legged robot or walking machine, which may offer clear advantages in tough environments. This Thesis will show that the proposed walking machine can travel, were terrain difficulties make wheeled vehicles ineffective, making it a perfect choice for planetary mission. A historical background of the main space missions, in particular those aimed at planetary exploration will be presented. From this study the disadvantages found in the existing wheel rovers will be analysed. The legged robot designed will be introduced as an alternative were wheeled rovers could be no longer the best option for planetary exploration. This thesis introduces the mechanical design of a six-leg robot capable of withstanding high forces and moments due to the walking motion. Once the mechanical design is concluded, and in order to analyse a machine of this complexity an understanding of its movement and behaviour is mandatory. This movement equation will be validated by two methods: kinematics and dynamics. Two Matlab® codes have been developed to solve the systems of equations and validated by a third method, a finite element model, which also verifies the mechanical design. The legged robot presented has been designed for a Mars planetary exploration. The movement behaviour of the robot will be tested in a Matlab® code developed that allows to modify the trajectories, the type of terrain, number and height of obstacles. These terrains and initial requirements have not been chosen randomly, those are based on my experience as a member of the MSL NASA team, which operates an instrument on-board of the Curiosity rover in Mars. The walking robot developed and manufactured by the Center of Astrobiology (CAB) is based in the mechanical design and analysis that will be presented in this thesis.

EFICÁCIA ADAPTATIVA DE MULHERES E HOMENS INSERIDOS NUM PROGRAMA DE FERTILIZAÇÃO IN VITRO

Este estudo teve como objetivos: caracterizar uma amostra de pacientes inseridos num programa de fertilização in vitro; avaliar a eficácia adaptativa de homens e mulheres inseridos neste programa; analisar dois casos clínicos representativos da amostra, quanto aos micro e macro fatores indicadores de adaptação psicológica. Os dados foram coletados num Centro de Reprodução Humana, tendo sido selecionados 65 homens e 65 mulheres, num período de março a outubro de 2002 e caracterizados segundo dados sócio-econômico-culturais. Foram extraídos 57 pares, sendo 57 homens e 57 mulheres diagnosticados pela Escala Diagnóstica Adaptativa Operacionalizada representando a maior concentração de casos com diagnóstico médico de mulheres inférteis e homens férteis, dos quais foram realizados os diagnósticos adaptativos. Depois, foram selecionados dois casos um homem e uma mulher, representativos deste grupo, que foram estudados em análise qualitativa, clínica, seguindo-se os pressupostos da teoria da adaptação. Os homens encontravam-se em faixa etária de 23 a 56 anos e as mulheres de 23 a 45 anos. Em relação ao diagnóstico, houve as seguintes concentrações: os homens (64,9 %) foram classificados no Grupo 2 Adaptação Ineficaz Leve e as mulheres (52,6 %) no Grupo 4 Adaptação Ineficaz Severa. Durante o diagnóstico, verificou-se que 20,2 % dos sujeitos estavam em crise, sendo 8,8 % dos homens e 31,6 % das mulheres. No estudo de caso representativo sendo H = Grupo 2 em condição adaptativa melhor do que a sua parceira que se encontrava no Grupo 4, verificou-se, que neste homem sua adequação nos setores era: Afetivo-Relacional - resposta pouco adequada; Produtividade - resposta adequada; Sócio-Cultural - resposta adequada, e Orgânico - resposta adequada. Sua parceira, entretanto, com diagnóstico do Grupo 4 Adaptação Ineficaz Severa sem crise, apresentou resposta pouquíssima adequada no setor Afetivo-Relacional, resposta pouco adequada no setor Produtividade, resposta pouco adequada no Sócio-Cultural e resposta pouco adequada no Orgânico. Concluímos que os homens desta amostra possuíam melhor eficácia adaptativa em relação às mulheres durante o processo de fertilização in vitro. Levantamos a necessidade de acompanhamento psicológico preventivo ante possível entrada em crise e de intervenções específicas para o momento de crise.