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En la actualidad existe una gran expectación ante la introducción de nuevas herramientas y métodos para el desarrollo de productos software, que permitirán en un futuro próximo un planteamiento de ingeniería del proceso de producción software. Las nuevas metodologías que empiezan a esbozarse suponen un enfoque integral del problema abarcando todas las fases del esquema productivo. Sin embargo el grado de automatización conseguido en el proceso de construcción de sistemas es muy bajo y éste está centrado en las últimas fases del ciclo de vida del software, consiguiéndose así una reducción poco significativa de sus costes y, lo que es aún más importante, sin garantizar la calidad de los productos software obtenidos. Esta tesis define una metodología de desarrollo software estructurada que se puede automatizar, es decir una metodología CASE. La metodología que se presenta se ajusta al modelo de ciclo de desarrollo CASE, que consta de las fases de análisis, diseño y pruebas; siendo su ámbito de aplicación los sistemas de información. Se establecen inicialmente los principios básicos sobre los que la metodología CASE se asienta. Posteriormente, y puesto que la metodología se inicia con la fijación de los objetivos de la empresa que demanda un sistema informático, se emplean técnicas que sirvan de recogida y validación de la información, que proporcionan a la vez un lenguaje de comunicación fácil entre usuarios finales e informáticos. Además, estas mismas técnicas detallarán de una manera completa, consistente y sin ambigüedad todos los requisitos del sistema. Asimismo, se presentan un conjunto de técnicas y algoritmos para conseguir que desde la especificación de requisitos del sistema se logre una automatización tanto del diseño lógico del Modelo de Procesos como del Modelo de Datos, validados ambos conforme a la especificación de requisitos previa. Por último se definen unos procedimientos formales que indican el conjunto de actividades a realizar en el proceso de construcción y cómo llevarlas a cabo, consiguiendo de esta manera una integridad en las distintas etapas del proceso de desarrollo.---ABSTRACT---Nowdays there is a great expectation with regard to the introduction of new tools and methods for the software products development that, in the very near future will allow, an engineering approach in the software development process. New methodologies, just emerging, imply an integral approach to the problem, including all the productive scheme stages. However, the automatization degree obtained in the systems construction process is very low and focused on the last phases of the software lifecycle, which means that the costs reduction obtained is irrelevant and, which is more important, the quality of the software products is not guaranteed. This thesis defines an structured software development methodology that can be automated, that is a CASE methodology. Such a methodology is adapted to the CASE development cycle-model, which consists in analysis, design and testing phases, being the information systems its field of application. Firstly, we present the basic principies on which CASE methodology is based. Secondly, since the methodology starts from fixing the objectives of the company demanding the automatization system, we use some techniques that are useful for gathering and validating the information, being at the same time an easy communication language between end-users and developers. Indeed, these same techniques will detail completely, consistently and non ambiguously all the system requirements. Likewise, a set of techniques and algorithms are shown in order to obtain, from the system requirements specification, an automatization of the Process Model logical design, and of the Data Model logical design. Those two models are validated according to the previous requirement specification. Finally, we define several formal procedures that suggest which set of activities to be accomplished in the construction process, and how to carry them out, getting in this way integrity and completness for the different stages of the development process.

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Esta tesis se centra en el estudio y desarrollo de algoritmos de guerra electrónica {electronic warfare, EW) y radar para su implementación en sistemas de tiempo real. La llegada de los sistemas de radio, radar y navegación al terreno militar llevó al desarrollo de tecnologías para combatirlos. Así, el objetivo de los sistemas de guerra electrónica es el control del espectro electomagnético. Una de la funciones de la guerra electrónica es la inteligencia de señales {signals intelligence, SIGINT), cuya labor es detectar, almacenar, analizar, clasificar y localizar la procedencia de todo tipo de señales presentes en el espectro. El subsistema de inteligencia de señales dedicado a las señales radar es la inteligencia electrónica {electronic intelligence, ELINT). Un sistema de tiempo real es aquel cuyo factor de mérito depende tanto del resultado proporcionado como del tiempo en que se da dicho resultado. Los sistemas radar y de guerra electrónica tienen que proporcionar información lo más rápido posible y de forma continua, por lo que pueden encuadrarse dentro de los sistemas de tiempo real. La introducción de restricciones de tiempo real implica un proceso de realimentación entre el diseño del algoritmo y su implementación en plataformas “hardware”. Las restricciones de tiempo real son dos: latencia y área de la implementación. En esta tesis, todos los algoritmos presentados se han implementado en plataformas del tipo field programmable gate array (FPGA), ya que presentan un buen compromiso entre velocidad, coste total, consumo y reconfigurabilidad. La primera parte de la tesis está centrada en el estudio de diferentes subsistemas de un equipo ELINT: detección de señales mediante un detector canalizado, extracción de los parámetros de pulsos radar, clasificación de modulaciones y localization pasiva. La transformada discreta de Fourier {discrete Fourier transform, DFT) es un detector y estimador de frecuencia quasi-óptimo para señales de banda estrecha en presencia de ruido blanco. El desarrollo de algoritmos eficientes para el cálculo de la DFT, conocidos como fast Fourier transform (FFT), han situado a la FFT como el algoritmo más utilizado para la detección de señales de banda estrecha con requisitos de tiempo real. Así, se ha diseñado e implementado un algoritmo de detección y análisis espectral para su implementación en tiempo real. Los parámetros más característicos de un pulso radar son su tiempo de llegada y anchura de pulso. Se ha diseñado e implementado un algoritmo capaz de extraer dichos parámetros. Este algoritmo se puede utilizar con varios propósitos: realizar un reconocimiento genérico del radar que transmite dicha señal, localizar la posición de dicho radar o bien puede utilizarse como la parte de preprocesado de un clasificador automático de modulaciones. La clasificación automática de modulaciones es extremadamente complicada en entornos no cooperativos. Un clasificador automático de modulaciones se divide en dos partes: preprocesado y el algoritmo de clasificación. Los algoritmos de clasificación basados en parámetros representativos calculan diferentes estadísticos de la señal de entrada y la clasifican procesando dichos estadísticos. Los algoritmos de localization pueden dividirse en dos tipos: triangulación y sistemas cuadráticos. En los algoritmos basados en triangulación, la posición se estima mediante la intersección de las rectas proporcionadas por la dirección de llegada de la señal. En cambio, en los sistemas cuadráticos, la posición se estima mediante la intersección de superficies con igual diferencia en el tiempo de llegada (time difference of arrival, TDOA) o diferencia en la frecuencia de llegada (frequency difference of arrival, FDOA). Aunque sólo se ha implementado la estimación del TDOA y FDOA mediante la diferencia de tiempos de llegada y diferencia de frecuencias, se presentan estudios exhaustivos sobre los diferentes algoritmos para la estimación del TDOA, FDOA y localización pasiva mediante TDOA-FDOA. La segunda parte de la tesis está dedicada al diseño e implementación filtros discretos de respuesta finita (finite impulse response, FIR) para dos aplicaciones radar: phased array de banda ancha mediante filtros retardadores (true-time delay, TTD) y la mejora del alcance de un radar sin modificar el “hardware” existente para que la solución sea de bajo coste. La operación de un phased array de banda ancha mediante desfasadores no es factible ya que el retardo temporal no puede aproximarse mediante un desfase. La solución adoptada e implementada consiste en sustituir los desfasadores por filtros digitales con retardo programable. El máximo alcance de un radar depende de la relación señal a ruido promedio en el receptor. La relación señal a ruido depende a su vez de la energía de señal transmitida, potencia multiplicado por la anchura de pulso. Cualquier cambio hardware que se realice conlleva un alto coste. La solución que se propone es utilizar una técnica de compresión de pulsos, consistente en introducir una modulación interna a la señal, desacoplando alcance y resolución. ABSTRACT This thesis is focused on the study and development of electronic warfare (EW) and radar algorithms for real-time implementation. The arrival of radar, radio and navigation systems to the military sphere led to the development of technologies to fight them. Therefore, the objective of EW systems is the control of the electromagnetic spectrum. Signals Intelligence (SIGINT) is one of the EW functions, whose mission is to detect, collect, analyze, classify and locate all kind of electromagnetic emissions. Electronic intelligence (ELINT) is the SIGINT subsystem that is devoted to radar signals. A real-time system is the one whose correctness depends not only on the provided result but also on the time in which this result is obtained. Radar and EW systems must provide information as fast as possible on a continuous basis and they can be defined as real-time systems. The introduction of real-time constraints implies a feedback process between the design of the algorithms and their hardware implementation. Moreover, a real-time constraint consists of two parameters: Latency and area of the implementation. All the algorithms in this thesis have been implemented on field programmable gate array (FPGAs) platforms, presenting a trade-off among performance, cost, power consumption and reconfigurability. The first part of the thesis is related to the study of different key subsystems of an ELINT equipment: Signal detection with channelized receivers, pulse parameter extraction, modulation classification for radar signals and passive location algorithms. The discrete Fourier transform (DFT) is a nearly optimal detector and frequency estimator for narrow-band signals buried in white noise. The introduction of fast algorithms to calculate the DFT, known as FFT, reduces the complexity and the processing time of the DFT computation. These properties have placed the FFT as one the most conventional methods for narrow-band signal detection for real-time applications. An algorithm for real-time spectral analysis for user-defined bandwidth, instantaneous dynamic range and resolution is presented. The most characteristic parameters of a pulsed signal are its time of arrival (TOA) and the pulse width (PW). The estimation of these basic parameters is a fundamental task in an ELINT equipment. A basic pulse parameter extractor (PPE) that is able to estimate all these parameters is designed and implemented. The PPE may be useful to perform a generic radar recognition process, perform an emitter location technique and can be used as the preprocessing part of an automatic modulation classifier (AMC). Modulation classification is a difficult task in a non-cooperative environment. An AMC consists of two parts: Signal preprocessing and the classification algorithm itself. Featurebased algorithms obtain different characteristics or features of the input signals. Once these features are extracted, the classification is carried out by processing these features. A feature based-AMC for pulsed radar signals with real-time requirements is studied, designed and implemented. Emitter passive location techniques can be divided into two classes: Triangulation systems, in which the emitter location is estimated with the intersection of the different lines of bearing created from the estimated directions of arrival, and quadratic position-fixing systems, in which the position is estimated through the intersection of iso-time difference of arrival (TDOA) or iso-frequency difference of arrival (FDOA) quadratic surfaces. Although TDOA and FDOA are only implemented with time of arrival and frequency differences, different algorithms for TDOA, FDOA and position estimation are studied and analyzed. The second part is dedicated to FIR filter design and implementation for two different radar applications: Wideband phased arrays with true-time delay (TTD) filters and the range improvement of an operative radar with no hardware changes to minimize costs. Wideband operation of phased arrays is unfeasible because time delays cannot be approximated by phase shifts. The presented solution is based on the substitution of the phase shifters by FIR discrete delay filters. The maximum range of a radar depends on the averaged signal to noise ratio (SNR) at the receiver. Among other factors, the SNR depends on the transmitted signal energy that is power times pulse width. Any possible hardware change implies high costs. The proposed solution lies in the use of a signal processing technique known as pulse compression, which consists of introducing an internal modulation within the pulse width, decoupling range and resolution.

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Context. This thesis is framed in experimental software engineering. More concretely, it addresses the problems arisen when assessing process conformance in test-driven development experiments conducted by UPM's Experimental Software Engineering group. Process conformance was studied using the Eclipse's plug-in tool Besouro. It has been observed that Besouro does not work correctly in some circumstances. It creates doubts about the correction of the existing experimental data which render it useless. Aim. The main objective of this work is the identification and correction of Besouro's faults. A secondary goal is fixing the datasets already obtained in past experiments to the maximum possible extent. This way, existing experimental results could be used with confidence. Method. (1) Testing Besouro using different sequences of events (creation methods, assertions etc..) to identify the underlying faults. (2) Fix the code and (3) fix the datasets using code specially created for this purpose. Results. (1) We confirmed the existence of several fault in Besouro's code that affected to Test-First and Test-Last episode identification. These faults caused the incorrect identification of 20% of episodes. (2) We were able to fix Besouro's code. (3) The correction of existing datasets was possible, subjected to some restrictions (such us the impossibility of tracing code size increase to programming time. Conclusion. The results of past experiments dependent upon Besouro's data could no be trustable. We have the suspicion that more faults remain in Besouro's code, whose identification requires further analysis.

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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.

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The mobile apps market is a tremendous success, with millions of apps downloaded and used every day by users spread all around the world. For apps’ developers, having their apps published on one of the major app stores (e.g. Google Play market) is just the beginning of the apps lifecycle. Indeed, in order to successfully compete with the other apps in the market, an app has to be updated frequently by adding new attractive features and by fixing existing bugs. Clearly, any developer interested in increasing the success of her app should try to implement features desired by the app’s users and to fix bugs affecting the user experience of many of them. A precious source of information to decide how to collect users’ opinions and wishes is represented by the reviews left by users on the store from which they downloaded the app. However, to exploit such information the app’s developer should manually read each user review and verify if it contains useful information (e.g. suggestions for new features). This is something not doable if the app receives hundreds of reviews per day, as happens for the very popular apps on the market. In this work, our aim is to provide support to mobile apps developers by proposing a novel approach exploiting data mining, natural language processing, machine learning, and clustering techniques in order to classify the user reviews on the basis of the information they contain (e.g. useless, suggestion for new features, bugs reporting). Such an approach has been empirically evaluated and made available in a web-­‐based tool publicly available to all apps’ developers. The achieved results showed that the developed tool: (i) is able to correctly categorise user reviews on the basis of their content (e.g. isolating those reporting bugs) with 78% of accuracy, (ii) produces clusters of reviews (e.g. groups together reviews indicating exactly the same bug to be fixed) that are meaningful from a developer’s point-­‐of-­‐view, and (iii) is considered useful by a software company working in the mobile apps’ development market.

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Rhizobium leguminosarum establishes highly specific nitrogen-fixing symbioses. We have applied a Pool-Seq approach to study plant host selection of genotypes. Our results confirm, at the genomic level, previous observations regarding plant selection of specific genotypes

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Rhizobium leguminosarum bv.viciae is able to establish nitrogen-fixing symbioses with legumes of the genera Pisum, Lens, Lathyrus and Vicia. Classic studies using trap plants (Laguerre et al., Young et al.) provided evidence that different plant hosts are able to select different rhizobial genotypes among those available in a given soil. However, these studies were necessarily limited by the paucity of relevant biodiversity markers. We have now reappraised this problem with the help of genomic tools. A well-characterized agricultural soil (INRA Bretennieres) was used as source of rhizobia. Plants of Pisum sativum, Lens culinaris, Vicia sativa and V. faba were used as traps. Isolates from 100 nodules were pooled, and DNA from each pool was sequenced (BGI-Hong Kong; Illumina Hiseq 2000, 500 bp PE libraries, 100 bp reads, 12 Mreads). Reads were quality filtered (FastQC, Trimmomatic), mapped against reference R. leguminosarum genomes (Bowtie2, Samtools), and visualized (IGV). An important fraction of the filtered reads were not recruited by reference genomes, suggesting that plant isolates contain genes that are not present in the reference genomes. For this study, we focused on three conserved genomic regions: 16S-23S rDNA, atpD and nodDABC, and a Single Nucleotide Polymorphism (SNP) analysis was carried out with meta / multigenomes from each plant. Although the level of polymorphism varied (lowest in the rRNA region), polymorphic sites could be identified that define the specific soil population vs. reference genomes. More importantly, a plant-specific SNP distribution was observed. This could be confirmed with many other regions extracted from the reference genomes (data not shown). Our results confirm at the genomic level previous observations regarding plant selection of specific genotypes. We expect that further, ongoing comparative studies on differential meta / multigenomic sequences will identify specific gene components of the plant-selected genotypes

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(SPA) Introducción: los medios de fijación a la piel de un catéter venoso periférico, de un catéter venoso central o de un catéter arterial central presentan los siguientes graves inconvenientes: ser la sede de infecciones a nivel del lugar de la punción, provocar una saturación de la zona anatómica, la cual resulta muy difícil de soportar e incómodo para el paciente portador, y suponer un riesgo para el personal de enfermería cuando se usan hilos de sutura para fijar el soporte del catéter. Objetivo: por lo anteriormente expuesto, se ha detecta- do la necesidad de diseñar un dispositivo que reduzca la complejidad y aparatosidad de los sistemas conocidos y empleados en la actualidad, favoreciendo la asepsia y la movilidad del paciente. Método: el desarrollo presentado en este artículo se refie- re a un sistema mecánico de fijación, cierre y acoplamiento para catéter de perfusión intravenosa. El sistema dispone de una pieza circular de pequeño espesor con al menos dos orificios para su fijación subcutánea, centralmente de uno o más conductos para acoplar lúmenes, y de una parte cilín- drica hueca que sobresale a uno de los lados, exteriormente roscada e interiormente lisa con un tetón de posición. Para uso domiciliario se dispone de un tapón de estanqueidad con uno o más tubos de pequeño diámetro que se acoplan en los orificios para lúmenes, una ranura de posición, un tirador y un tapón roscado ciego para el cierre hermético con la parte roscada. Para uso hospitalario se dispone de un tapón intermedio con una o más entradas para lúmenes, así como de un tapón roscado hueco que permite su acopla- miento hermético con la parte roscada. Resultados: el desarrollo descrito en este artículo va a ser utilizado en dos ambientes: ambiente domiciliario y ambiente hospitalario. Además, reduce el riesgo de infec- ción y saturación de la zona anatómica de los pacientes en los que se realiza la punción cutánea en la que se fijan catéteres venosos periféricos, catéteres venosos centrales o catéteres arteriales centrales. (ENG) Introduction: skin fixing devices in peripheral, central or arterial catheters have several important drawbacks: site infection, stacking of material in the anatomical area which is very annoying for the patient and medical staff risk when fixating stitches are used. Objective: to develop a fixing device that simplifies presently used systems, favoring asepsis and motility. Methods: the device herein described is composed by a mechanical fixation, a closing system and coupling for intravenous catheters. The system has a thin circular pie- ce with at least two holes for subcutaneous fixation, one or several conducts for lumina and a hollow cylindrical part in one side, screwed exteriorly and flat inside, with an oriented protuberance. A watertight plug with one or several thin tubes that adapt to the lumina, a positional slot, a handle and a solid screwed tap for perfect closure are available for at home use. An intermediate plug with one or several lumina and a screwed hollow plug are pro- vided for in hospital use. Results: the above described device is intended to be used in two settings: in hospital and at home. It is su- pposed to reduce the risk of infection and stacking of the anatomical site where cutaneo us puncture with fixation of peripheral, central or arterial catheters is performed.

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Esta dissertação apresenta um estudo exploratório que tem como objetivo a avaliação da reação do mercado frente aos problemas de agência e assimetria informacional entre os acionistas majoritários-controladores e os acionistas minoritários de uma empresa brasileira de capital aberto, no tocante ao valor das suas ações negociadas na Bolsa de Valores de São Paulo. O estudo se propõe a investigar o impacto de precificação dessas ações promovendo a análise do seu comportamento frente à divulgação de notícias de crise e fraude da empresa por meio da mídia especializada. A metodologia adotada consiste na aplicação de estudo de eventos para identificação de retornos anormais da empresa desde a divulgação da primeira notícia selecionada, datada de maio de 2001, até a última notícia em outubro de 2005, utilizando-se de procedimentos estatísticos como a regressão linear e aplicação do teste t de student para estimar e comparar os resultados. Os dados foram obtidos por meio do banco de dados da Economática Ltda, conforme acesso realizado na Universidade de São Paulo. Para objeto de pesquisa foi selecionada a empresa Bombril S/A, por estar em evidências quanto a problemas de agência no âmbito do mercado nacional. Os resultados obtidos apontaram que o mercado reagiu significantemente aos anúncios dos conflitos selecionados, apresentando um valor de p-value <0,05 para os blocos de eventos, o que significa a rejeição da hipótese nula, constatando que a evidencia estatística dos dados testados comprova retornos anormais acumulados diferentes de zero. Entretanto, sugere-se novas pesquisas com outros parâmetros de eventos na busca de mais evidências sobre o efeito das informações no preço das ações.

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Esta dissertação apresenta um estudo exploratório que tem como objetivo a avaliação da reação do mercado frente aos problemas de agência e assimetria informacional entre os acionistas majoritários-controladores e os acionistas minoritários de uma empresa brasileira de capital aberto, no tocante ao valor das suas ações negociadas na Bolsa de Valores de São Paulo. O estudo se propõe a investigar o impacto de precificação dessas ações promovendo a análise do seu comportamento frente à divulgação de notícias de crise e fraude da empresa por meio da mídia especializada. A metodologia adotada consiste na aplicação de estudo de eventos para identificação de retornos anormais da empresa desde a divulgação da primeira notícia selecionada, datada de maio de 2001, até a última notícia em outubro de 2005, utilizando-se de procedimentos estatísticos como a regressão linear e aplicação do teste t de student para estimar e comparar os resultados. Os dados foram obtidos por meio do banco de dados da Economática Ltda, conforme acesso realizado na Universidade de São Paulo. Para objeto de pesquisa foi selecionada a empresa Bombril S/A, por estar em evidências quanto a problemas de agência no âmbito do mercado nacional. Os resultados obtidos apontaram que o mercado reagiu significantemente aos anúncios dos conflitos selecionados, apresentando um valor de p-value <0,05 para os blocos de eventos, o que significa a rejeição da hipótese nula, constatando que a evidencia estatística dos dados testados comprova retornos anormais acumulados diferentes de zero. Entretanto, sugere-se novas pesquisas com outros parâmetros de eventos na busca de mais evidências sobre o efeito das informações no preço das ações.

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Under nitrogen-limiting conditions Rhizobium meliloti can establish symbiosis with Medicago plants to form nitrogen-fixing root nodules. Nodule organogenesis starts with the dedifferentiation and division of root cortical cells. In these cells the early nodulin gene enod40, which encodes an unusually small peptide (12 or 13 amino acids), is induced from the beginning of this process. Herein we show that enod40 expression evokes root nodule initiation. (i) Nitrogen-deprived transgenic Medicago truncatula plants overexpressing enod40 exhibit extensive cortical cell division in their roots in the absence of Rhizobium. (ii) Bombardment of Medicago roots with an enod40-expressing DNA cassette induces dedifferentiation and division of cortical cells and the expression of another early nodulin gene, Msenod12A. Moreover, transient expression of either the enod40 region spanning the oligopeptide sequence or only the downstream region without this sequence induces these responses. Our results suggest that the cell-specific growth response elicited by enod40 is involved in the initiation of root nodule organogenesis.

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Transcripts for two genes expressed early in alfalfa nodule development (MsENOD40 and MsENOD2) are found in mycorrhizal roots, but not in noncolonized roots or in roots infected with the fungal pathogen Rhizoctonia solani. These same two early nodulin genes are expressed in uninoculated roots upon application of the cytokinin 6-benzylaminopurine. Correlated with the expression of the two early nodulin genes, we found that mycorrhizal roots contain higher levels of trans-zeatin riboside than nonmycorrhizal roots. These data suggest that there may be conservation of signal transduction pathways between the two symbioses—nitrogen-fixing nodules and phosphate-acquiring mycorrhizae.

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Lateral transfer of bacterial plasmids is thought to play an important role in microbial evolution and population dynamics. However, this assumption is based primarily on investigations of medically or agriculturally important bacterial species. To explore the role of lateral transfer in the evolution of bacterial systems not under intensive, human-mediated selection, we examined the association of genotypes at plasmid-encoded and chromosomal loci of native Rhizobium, the nitrogen-fixing symbiont of legumes. To this end, Rhizobium leguminosarum strains nodulating sympatric species of native Trifolium were characterized genetically at plasmid-encoded symbiotic (sym) regions (nodulation AB and nodulation CIJT loci) and a repeated chromosomal locus not involved in the symbiosis with legumes. Restriction fragment length polymorphism analysis was used to distinguish genetic groups at plasmid and chromosomal loci. The correlation between major sym and chromosomal genotypes and the distribution of genotypes across host plant species and sampling location were determined using χ2 analysis. In contrast to findings of previous studies, a strict association existed between major sym plasmid and chromosomal genetic groups, suggesting a lack of successful sym plasmid transfer between major Rhizobium chromosomal types. These data indicate that previous observations of sym plasmid transfer in agricultural settings may seriously overestimate the rates of successful conjugation in systems not impacted by human activities. In addition, a nonrandom distribution of Rhizobium genotypes across host plant species and sampling site demonstrates the importance of both factors in shaping Rhizobium population dynamics.

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Photosynthesis, biological nitrogen fixation, and carbon dioxide assimilation are three fundamental biological processes catalyzed by photosynthetic bacteria. In the present study, it is shown that mutant strains of the nonsulfur purple photosynthetic bacteria Rhodospirillum rubrum and Rhodobacter sphaeroides, containing a blockage in the primary CO2 assimilatory pathway, derepress the synthesis of components of the nitrogen fixation enzyme complex and abrogate normal control mechanisms. The absence of the Calvin–Benson–Bassham (CBB) reductive pentose phosphate CO2 fixation pathway removes an important route for the dissipation of excess reducing power. Thus, the mutant strains develop alternative means to remove these reducing equivalents, resulting in the synthesis of large amounts of nitrogenase even in the presence of ammonia. This response is under the control of a global two-component signal transduction system previously found to regulate photosystem biosynthesis and the transcription of genes required for CO2 fixation through the CBB pathway and alternative routes. In addition, this two-component system directly controls the ability of these bacteria to grow under nitrogen-fixing conditions. These results indicate that there is a molecular link between the CBB and nitrogen fixation process, allowing the cell to overcome powerful control mechanisms to remove excess reducing power generated by photosynthesis and carbon metabolism. Furthermore, these results suggest that the two-component system integrates the expression of genes required for the three processes of photosynthesis, nitrogen fixation, and carbon dioxide fixation.

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The symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti results in the formation of nitrogen-fixing nodules on the roots of the host plant. The early stages of nodule formation are induced by bacteria via lipochitooligosaccharide signals known as Nod factors (NFs). These NFs are structurally specific for bacterium–host pairs and are sufficient to cause a range of early responses involved in the host developmental program. Early events in the signal transduction of NFs are not well defined. We have previously reported that Medicago sativa root hairs exposed to NF display sharp oscillations of cytoplasmic calcium ion concentration (calcium spiking). To assess the possible role of calcium spiking in the nodulation response, we analyzed M. truncatula mutants in five complementation groups. Each of the plant mutants is completely Nod− and is blocked at early stages of the symbiosis. We defined two genes, DMI1 and DMI2, required in common for early steps of infection and nodulation and for calcium spiking. Another mutant, altered in the DMI3 gene, has a similar mutant phenotype to dmi1 and dmi2 mutants but displays normal calcium spiking. The calcium behavior thus implies that the DMI3 gene acts either downstream of calcium spiking or downstream of a common branch point for the calcium response and the later nodulation responses. Two additional mutants, altered in the NSP and HCL genes, which show root hair branching in response to NF, are normal for calcium spiking. This system provides an opportunity to use genetics to study ligand-stimulated calcium spiking as a signal transduction event.