Instability and topology bifurcations on a hemisphere-cylinder at high angle of attack

La configuración de un cilindro acoplado a una semi-esfera, conocida como ’hemispherecylinder’, se considera como un modelo simplificado para numerosas aplicaciones industriales tales como fuselaje de aviones o submarinos. Por tanto, el estudio y entendimiento de los fenómenos fluidos que ocurren alrededor de dicha geometría presenta gran interés. En esta tesis se muestra la investigación del origen y evolución de los, ya conocidos, patrones de flujo (burbuja de separación, vórtices ’horn’ y vórtices ’leeward’) que se dan en esta geometría bajo condiciones de flujo separado. Para ello se han llevado a cabo simulaciones numéricas (DNS) y ensayos experimentales usando la técnica de Particle Image Velocimetry (PIV), para una variedad de números de Reynolds (Re) y ángulos de ataque (AoA). Se ha aplicado sobre los resultados numéricos la teoría de puntos críticos obteniendo, por primera vez para esta geometría, un diagrama de bifurcaciones que clasifica los diferentes regímenes topológicos en función del número de Reynolds y del ángulo de ataque. Se ha llevado a cabo una caracterización completa sobre el origen y la evolución de los patrones estructurales característicos del cuerpo estudiado. Puntos críticos de superficie y líneas de corriente tridimensionales han ayudado a describir el origen y la evolución de las principales estructuras presentes en el flujo hasta alcanzar un estado de estabilidad desde el punto de vista topológico. Este estado se asocia con el patrón de los vórtices ’horn’, definido por una topología característica que se encuentra en un rango de números de Reynolds muy amplio y en regímenes compresibles e incompresibles. Por otro lado, con el objeto de determinar las estructuras presentes en el flujo y sus frecuencias asociadas, se han usado distintas técnicas de análisis: Proper Orthogonal Decomposition (POD), Dynamic Mode Decomposition (DMD) y análisis de Fourier. Dichas técnicas se han aplicado sobre los datos experimentales y numéricos, demostrándose la buena concordancia entre ambos resultados. Finalmente, se ha encontrado en ambos casos, una frecuencia dominante asociada con una inestabilidad de los vórtices ’leeward’. ABSTRACT The hemisphere-cylinder may be considered as a simplified model for several geometries found in industrial applications such as aircrafts’ fuselages or submarines. Understanding the complex flow phenomena that surrounds this particular geometry is therefore of major industrial interest. This thesis presents an investigation of the origin and evolution of the complex flow pattern; i.e. separation bubbles, horn vortices and leeward vortices, around the hemisphere-cylinder under separated flow conditions. To this aim, threedimensional Direct Numerical Simulations (DNS) and experimental tests, using Particle Image Velocimetry (PIV) techniques, have been performed for a variety of Reynolds numbers (Re) and angles of attack (AoA). Critical point theory has been applied to the numerical simulations to provide, for the first time for this geometry, a bifurcation diagram that classifies the different flow topology regimes as a function of the Reynolds number and the angle of attack. A complete characterization about the origin and evolution of the complex structural patterns of this geometry has been put in evidence. Surface critical points and surface and volume streamlines were able to describe the main flow structures and their strong dependence with the flow conditions up to reach the structurally stable state. This state was associated with the pattern of the horn vortices, found on ranges from low to high Reynolds numbers and from incompressible to compressible regimes. In addition, different structural analysis techniques have been employed: Proper Orthogonal Decomposition (POD), Dynamic Mode Decomposition (DMD) and Fourier analysis. These techniques have been applied to the experimental and numerical data to extract flow structure information (i.e. modes and frequencies). Experimental and numerical modes are shown to be in good agreement. A dominant frequency associated with an instability of the leeward vortices has been identified in both, experimental and numerical results.

Trade-off aproaches for the vibroacoustic analysis of trains

Passengers’ comfort in terms of acoustic noise levels is a key design driver for train design. The problem is especially relevant for high speed trains, where the aerodynamic induced noise is dominant, but it is also important for medium speed trains where the mechanical sources of noise may have more influence. The numerical interior noise prediction inside the train is a very comp lex problem, involving many different parameters: complex geometries and materials, different noise sources, com- plex interactions among those sources, broad range of frequencies where the phenomenon is important, etc. In this paper, the main findings of this work developed at IDR/UPM (Instituto de Microgravedad “Ignacio Da Riva”, Universidad Politécnica de Madrid) are presented, concentrat ing on the different modelling methodologies used for the different frequency ranges of interest, from FEM-BEM models, hybrid FEM-SEA to pure SEA models. The advantages and disadvantages of the different approaches are summarized. Different modelling techniques have also been evaluated and compared, taking into account the various and specific geometrical configurations typical in this type of structures, and the material properties used in the models. The critical configuration of the train inside a tunnel is studied in order to evaluate the external loads due to noise sources of the train. In this work, a SEA-model composed by periodic characteristic sections of a high spee d train is analysed inside a tunnel.

Nuevas arquitecturas de transpondedores de microondas = New transponder architectures at microwave frequencies = Nouvelles architectures des transpondeurs d’hyperfréquences

En la última década, los sistemas de telecomunicación de alta frecuencia han evolucionado tremendamente. Las bandas de frecuencias, los anchos de banda del usuario, las técnicas de modulación y otras características eléctricas están en constante cambio de acuerdo a la evolución de la tecnología y la aparición de nuevas aplicaciones. Las arquitecturas de los transceptores modernos son diferentes de las tradicionales. Muchas de las funciones convencionalmente realizadas por circuitos analógicos han sido asignadas gradualmente a procesadores digitales de señal, de esta manera, las fronteras entre la banda base y las funcionalidades de RF se difuminan. Además, los transceptores inalámbricos digitales modernos son capaces de soportar protocolos de datos de alta velocidad, por lo que emplean una elevada escala de integración para muchos de los subsistemas que componen las diferentes etapas. Uno de los objetivos de este trabajo de investigación es realizar un estudio de las nuevas configuraciones en el desarrollo de demostradores de radiofrecuencia (un receptor y un transmisor) y transpondedores para fines de comunicaciones y militares, respectivamente. Algunos trabajos se han llevado a cabo en el marco del proyecto TECRAIL, donde se ha implementado un demostrador de la capa física LTE para evaluar la viabilidad del estándar LTE en el entorno ferroviario. En el ámbito militar y asociado al proyecto de calibración de radares (CALRADAR), se ha efectuado una actividad importante en el campo de la calibración de radares balísticos Doppler donde se ha analizado cuidadosamente su precisión y se ha desarrollado la unidad generadora de Doppler de un patrón electrónico para la calibración de estos radares. Dicha unidad Doppler es la responsable de la elevada resolución en frecuencia del generador de “blancos” radar construido. Por otro lado, se ha elaborado un análisis completo de las incertidumbres del sistema para optimizar el proceso de calibración. En una segunda fase se han propuesto soluciones en el desarrollo de dispositivos electro-ópticos para aplicaciones de comunicaciones. Estos dispositivos son considerados, debido a sus ventajas, tecnologías de soporte para futuros dispositivos y subsistemas de RF/microondas. Algunas demandas de radio definida por software podrían cubrirse aplicando nuevos conceptos de circuitos sintonizables mediante parámetros programables de un modo dinámico. También se ha realizado una contribución relacionada con el diseño de filtros paso banda con topología “Hairpin”, los cuales son compactos y se pueden integrar fácilmente en circuitos de microondas en una amplia gama de aplicaciones destinadas a las comunicaciones y a los sistemas militares. Como importante aportación final, se ha presentado una propuesta para ecualizar y mejorar las transmisiones de señales discretas de temporización entre los TRMs y otras unidades de procesamiento, en el satélite de última generación SEOSAR/PAZ. Tras un análisis exhaustivo, se ha obtenido la configuración óptima de los buses de transmisión de datos de alta velocidad basadas en una red de transceptores. ABSTRACT In the last decade, high-frequency telecommunications systems have extremely evolved. Frequency bands, user bandwidths, modulation techniques and other electrical characteristics of these systems are constantly changing following to the evolution of technology and the emergence of new applications. The architectures of modern transceivers are different from the traditional ones. Many of the functions conventionally performed by analog circuitry have gradually been assigned to digital signal processors. In this way, boundaries between baseband and RF functionalities are diffused. The design of modern digital wireless transceivers are capable of supporting high-speed data protocols. Therefore, a high integration scale is required for many of the components in the block chain. One of the goals of this research work is to investigate new configurations in the development of RF demonstrators (a receiver and a transmitter) and transponders for communications and military purposes, respectively. A LTE physical layer demonstrator has been implemented to assess the viability of LTE in railway scenario under the framework of the TECRAIL project. An important activity, related to the CALRADAR project, for the calibration of Doppler radars with extremely high precision has been performed. The contribution is the Doppler unit of the radar target generator developed that reveals a high frequency resolution. In order to assure the accuracy of radar calibration process, a complete analysis of the uncertainty in the above mentioned procedure has been carried out. Another important research topic has been the development of photonic devices that are considered enabling technologies for future RF and microwave devices and subsystems. Some Software Defined Radio demands are addressed by the proposed novel circuit concepts based on photonically tunable elements with dynamically programmable parameters. A small contribution has been made in the field of Hairpin-line bandpass filters. These filters are compact and can also be easily integrated into microwave circuits finding a wide range of applications in communication and military systems. In this research field, the contributions made have been the improvements in the design and the simulations of wideband filters. Finally, an important proposal to balance and enhance transmissions of discrete timing signals between TRMs and other processing units into the state of the art SEOSAR/PAZ Satellite has been carried out obtaining the optimal configuration of the high-speed data transmission buses based on a transceiver network. RÉSUMÉ Les systèmes d'hyperfréquence dédiés aux télécommunications ont beaucoup évolué dans la dernière décennie. Les bandes de fréquences, les bandes passantes par utilisateur, les techniques de modulation et d'autres caractéristiques électriques sont en constant changement en fonction de l'évolution des technologies et l'émergence de nouvelles applications. Les architectures modernes des transcepteurs sont différentes des traditionnelles. Un grand nombre d’opérations normalement effectuées par les circuits analogiques a été progressivement alloué à des processeurs de signaux numériques. Ainsi, les frontières entre la bande de base et la fonctionnalité RF sont floues. Les transcepteurs sans fils numériques modernes sont capables de transférer des données à haute vitesse selon les différents protocoles de communication utilisés. C'est pour cette raison qu’un niveau élevé d'intégration est nécessaire pour un grand nombre de composants qui constitue les différentes étapes des systèmes. L'un des objectifs de cette recherche est d'étudier les nouvelles configurations dans le développement des démonstrateurs RF (récepteur et émetteur) et des transpondeurs à des fins militaire et de communication. Certains travaux ont été réalisés dans le cadre du projet TECRAIL, où un démonstrateur de la couche physique LTE a été mis en place pour évaluer la faisabilité de la norme LTE dans l'environnement ferroviaire. Une contribution importante, liée au projet CALRADAR, est proposée dans le domaine des systèmes d’étalonnage de radar Doppler de haute précision. Cette contribution est le module Doppler de génération d’hyperfréquence intégré dans le système électronique de génération de cibles radar virtuelles que présente une résolution de fréquence très élevée. Une analyse complète de l'incertitude dans l'étalonnage des radars Doppler a été effectuée, afin d'assurer la précision du calibrage. La conception et la mise en oeuvre de quelques dispositifs photoniques sont un autre sujet important du travail de recherche présenté dans cette thèse. De tels dispositifs sont considérés comme étant des technologies habilitantes clés pour les futurs dispositifs et sous-systèmes RF et micro-ondes grâce à leurs avantages. Certaines demandes de radio définies par logiciel pourraient être supportées par nouveaux concepts de circuits basés sur des éléments dynamiquement programmables en utilisant des paramètres ajustables. Une petite contribution a été apportée pour améliorer la conception et les simulations des filtres passe-bande Hairpin à large bande. Ces filtres sont compacts et peuvent également être intégrés dans des circuits à micro-ondes compatibles avec un large éventail d'applications dans les systèmes militaires et de communication. Finalement, une proposition a été effectuée visant à équilibrer et améliorer la transmission des signaux discrets de synchronisation entre les TRMs et d'autres unités de traitement dans le satellite SEOSAR/PAZ de dernière génération et permettant l’obtention de la configuration optimale des bus de transmission de données à grande vitesse basés sur un réseau de transcepteurs.

Influence of a liquid on the natural frequencies of almost circular plates

In this work, the influence of the surrounding fluid on the dynamic characteristics of almost circular plates is investigated. First the natural frequencies and normal modes for the plates in vacuum are calculated by a perturbation procedure. The method is applied for the case of elliptical plates with a low value of eccentricity. The results are compared with other available methods for this type of plates with good agreement. Next, the effect of the fluid is considered. The normal modes of the plate in vacuum are used as a base to express the vibration mode of the coupled plate-fluid system. By applying the Hankel transformation the nondimensional added virtual mass 2 increment (NAVMI) are calculated for elliptical plates. Results of the NAVMI factors and the effect of the fluid on the natural frequencies are given and it is shown that when the eccentricity of the plate is reduced to zero (circular plate) the known results of the natural frequencies for circular plates surrounded by liquid are recovered.

Vibroacoustic Problems in High Speed Trains

Passengers comfort in terms of acoustic noise levels is a key train design parameter, especially relevant in high speed trains, where the aerodynamic noise is dominant. The aim of the work, described in this paper, is to make progress in the understanding of the flow field around high speed trains in an open field, which is a subject of interest for many researchers with direct industrial applications, but also the critical configuration of the train inside a tunnel is studied in order to evaluate the external loads arising from noise sources of the train. The airborne noise coming from the wheels (wheelrail interaction), which is the dominant source at a certain range of frequencies, is also investigated from the numerical and experimental points of view. The numerical prediction of the noise in the interior of the train is a very complex problem, involving many different parameters: complex geometries and materials, different noise sources, complex interactions among those sources, broad range of frequencies where the phenomenon is important, etc. During recent years a research plan is being developed at IDR/UPM (Instituto de Microgravedad Ignacio Da Riva, Universidad Politécnica de Madrid) involving both numerical simulations, wind tunnel and full-scale tests to address this problem. Comparison of numerical simulations with experimental data is a key factor in this process.

A simplified spectral approachfor impedance-based damage identification of frp-strengthened rc beams

Hoy en día, el refuerzo y reparación de estructuras de hormigón armado mediante el pegado de bandas de polímeros reforzados con fibras (FRP) se emplea cada vez con más frecuencia a causa de sus numerosas ventajas. Sin embargo, las vigas reforzadas con esta técnica pueden experimentar un modo de fallo frágil a causa del despegue repentino de la banda de FRP a partir de una fisura intermedia. A pesar de su importancia, el número de trabajos que abordan el estudio de este mecanismo de fallo y su monitorización es muy limitado. Por ello, el desarrollo de metodologías capaces de monitorizar a largo plazo la adherencia de este refuerzo a las estructuras de hormigón e identificar cuándo se inicia el despegue de la banda constituyen un importante desafío a abordar. El principal objetivo de esta tesis es la implementación de una metodología fiable y efectiva, capaz de detectar el despegue de una banda de FRP en una viga de hormigón armado a partir de una fisura intermedia. Para alcanzar este objetivo se ha implementado un procedimiento de calibración numérica a partir de ensayos experimentales. Para ello, en primer lugar, se ha desarrollado un modelo numérico unidimensional simple y no costoso representativo del comportamiento de este tipo vigas de hormigón reforzadas con FRP, basado en un modelo de fisura discreta para el hormigón y el método de elementos espectrales. La formación progresiva de fisuras a flexion y el consiguiente despegue en la interface entre el hormigón y el FRP se formulan mediante la introducción de un nuevo elemento capaz de representar ambos fenómenos simultáneamente sin afectar al procedimiento numérico. Además, con el modelo propuesto, se puede obtener de una forma sencilla la respuesta dinámica en altas frecuencias de este tipo de estructuras, lo cual puede hacer muy útil su uso como herramienta de diagnosis y detección del despegue en su fase inicial mediante una monitorización de la variación de las características dinámicas locales de la estructura. Un método de evaluación no destructivo muy prometedor para la monitorización local de las estructuras es el método de la impedancia usando sensores-actuadores piezoeléctricos (PZT). La impedancia eléctrica de los sensores PZT se puede relacionar con la impedancia mecánica de las estructuras donde se encuentran adheridos Ya que la impedancia mecánica de una estructura se verá afectada por su deterioro, se pueden implementar indicadores de daño mediante una comparación del espectro de admitancia (inversa de la impedancia) a lo largo de distintas etapas durante el periodo de servicio de una estructura. Cualquier cambio en el espectro se podría interpretar como una variación en la integridad de la estructura. La impedancia eléctrica se mide a altas frecuencias con lo cual esta metodología debería ser muy sensible a la detección de estados de daño incipiente local, tal como se desea en la aplicación de este trabajo. Se ha implementado un elemento espectral PZT-FRP como extensión del modelo previamente desarrollado, con el objetivo de poder calcular numéricamente la impedancia eléctrica de sensores PZT adheridos a bandas de FRP sobre una viga de hormigón armado. El modelo, combinado con medidas experimentales captadas mediante sensores PZT, se implementa en el marco de una metodología de calibración de modelos para detectar cuantitativamente el despegue en la interfase entre una banda de FRP y una viga de hormigón. El procedimiento de optimización se resuelve empleando el método del enjambre cooperativo con un algoritmo bagging. Los resultados muestran una gran aproximación en la estimación del daño para el problema propuesto. Adicionalmente, se ha desarrollado también un método adaptativo para el mallado de elementos espectrales con el objetivo de localizar las zonas dañadas a partir de los resultados experimentales, el cual contribuye a aumentar la robustez y efectividad del método propuesto a la hora de identificar daños incipientes en su aparición inicial. Finalmente, se ha llevado a cabo un procedimiento de optimización multi-objetivo para detectar el despegue inicial en una viga de hormigón a escala real reforzada con FRP a partir de las impedancias captadas con una red de sensores PZT instrumentada a lo largo de la longitud de la viga. Cada sensor aporta los datos para definir cada una de las funciones objetivo que definen el procedimiento. Combinando el modelo previo de elementos espectrales con un algoritmo PSO multi-objetivo el procedimiento de detección de daño resultante proporciona resultados satisfactorios considerando la escala de la estructura y todas las incertidumbres características ligadas a este proceso. Los resultados obtenidos prueban la viabilidad y capacidad de los métodos antes mencionados y también su potencial en aplicaciones reales. Abstract Nowadays, the external bonding of fibre reinforced polymer (FRP) plates or sheets is increasingly used for the strengthening and retrofitting of reinforced concrete (RC) structures due to its numerous advantages. However, this kind of strengthening often leads to brittle failure modes being the most dominant failure mode the debonding induced by an intermediate crack (IC). In spite of its importance, the number of studies regarding the IC debonding mechanism and bond health monitoring is very limited. Methodologies able to monitor the long-term efficiency of bonding and successfully identify the initiation of FRP debonding constitute a challenge to be met. The main purpose of this thesisis the implementation of a reliable and effective methodology of damage identification able to detect intermediate crack debonding in FRP-strengthened RC beams. To achieve this goal, a model updating procedure based on numerical simulations and experimental tests has been implemented. For it, firstly, a simple and non-expensive one-dimensional model based on the discrete crack approach for concrete and the spectral element method has been developed. The progressive formation of flexural cracks and subsequent concrete-FRP interfacial debonding is formulated by the introduction of a new element able to represent both phenomena simultaneously without perturbing the numerical procedure. Furthermore, with the proposed model, high frequency dynamic response for these kinds of structures can also be obtained in a very simple and non-expensive way, which makes this procedure very useful as a tool for diagnoses and detection of debonding in its initial stage by monitoring the change in local dynamic characteristics. One very promising active non-destructive evaluation method for local monitoring is impedance-based structural health monitoring(SHM)using piezoelectric ceramic (PZT) sensor-actuators. The electrical impedance of the PZT can be directly related to the mechanical impedance of the host structural component where the PZT transducers are attached. Since the structural mechanical impedance will be affected by the presence of structural damage, comparisons of admittance (inverse of impedance) spectra at various times during the service period of the structure can be used as damage indicator. Any change in the spectra might be an indication of a change in the structural integrity. The electrical impedance is measured at high frequencies with which this methodology appears to be very sensitive to incipient damage in structural systems as desired for our application. Abonded-PZT-FRP spectral beam element approach based on an extension of the previous discrete crack approach is implemented in the calculation of the electrical impedance of the PZT transducer bonded to the FRP plates of a RC beam. This approach in conjunction with the experimental measurements of PZT actuator-sensors mounted on the structure is used to present an updating methodology to quantitatively detect interfacial debonding between a FRP strip and the host RC structure. The updating procedure is solved by using an ensemble particle swarm optimization approach with abagging algorithm, and the results demonstrate a big improvement for the performance and accuracy of the damage detection in the proposed problem. Additionally, an adaptive strategy of spectral element mesh has been also developed to detect damage location with experimental results, which shows the robustness and effectiveness of the proposed method to identify initial and incipient damages at its early stage. Lastly, multi-objective optimization has been carried out to detect debonding damage in a real scale FRP-strengthened RC beam by using impedance signatures. A net of PZT sensors is distributed along the beam to construct impedance-based multiple objectives under gradually induced damage scenario. By combining the spectral element model presented previously and an ensemble multi-objective PSO algorithm, the implemented damage detection process yields satisfactory predictions considering the scale and uncertainties of the structure. The obtained results prove the feasibility and capability of the aforementioned methods and also their potentials in real engineering applications.

Contribution to the analysis and design of reflectarray antennas for reconfigurable beam applications at frequencies above 100 GHz using liquid crystal technology

El trabajo contenido en esta tesis doctoral está encuadrado en el desarrollo de antenas reconfigurables electrónicamente capaces de proporcionar prestaciones competitivas a las aplicaciones cada vez más comunes que operan a frecuencias superiores a 60 GHz. En concreto, esta tesis se centra en el estudio, diseño, e implementación de las antenas reflectarray, a las que se introduce la tecnología de cristal líquido como elemento característico con el que se consigue reconfigurabilidad de haz de forma electrónica. Desde un punto de vista muy general, se puede describir un cristal líquido como un material cuya permitividad eléctrica es variable y controlada por una excitación externa, que generalmente suele corresponderse con un campo eléctrico quasi-estático (AC). Las antenas reflectarray de cristal líquido se han escogido como objeto de estudio por varias razones. La primera de ellas tiene que ver con las ventajas que los reflectarrays, y en especial aquellos realizados en configuración planar, proporcionan con respecto a otras antenas de alta ganancia como los reflectores o los “phased-arrays”. En los reflectarrays, la alimentación a través de una fuente primaria común (característica de reflectores) y el elevado número de grados de libertad de las celdas que los componen (característica de arrays) hacen que estas antenas puedan proporcionar prestaciones eléctricas iguales o mejores que las anteriores, a un coste más reducido y con estructuras de antena más compactas. La segunda razón radica en la flexibilidad que ofrece el cristal líquido a ser confinado y polarizado en recintos de geometría variada, como consecuencia de su fluidez (propiedad de los líquidos). Por ello, la tecnología de cristal líquido permite que el propio elemento reconfigurable en las celdas de reflectarray se adapte a la configuración planar de manera que en sí mismo, el cristal líquido sea una o varias de las capas características de esta configuración. Esto simplifica de forma drástica la estructura y la fabricación de este tipo de antenas, incluso si se comparan con reflectarrays reconfigurables basados en otras tecnologías como diodos, MEMS, etc. Por tanto, su coste y desarrollo es muy reducido, lo que hace que se puedan fabricar reflectarrays reconfigurables eléctricamente grandes, a bajo coste, y en producción elevada. Un ejemplo claro de una estructura similar, y que ha tenido éxito comercial, son las pantallas de cristal líquido. La tercera razón reside en el hecho de que el cristal líquido es, hasta la fecha, de las pocas tecnologías capaces de ofrecer reconfigurabilidad del haz a frecuencias superiores a 60 GHz. De hecho, el cristal líquido permite reconfigurabilidad en un amplio margen de frecuencias, que va desde DC a frecuencias del espectro visible, incluyendo las microondas y los THz. Otras tecnologías, como los materiales ferroeléctricos, el grafeno o la tecnología CMOS “on chip” permiten también conmutar el haz en estas frecuencias. Sin embargo, la tecnología CMOS tiene un elevado coste y actualmente está limitada a frecuencias inferiores a 150 GHz, y aunque los materiales ferroeléctricos o el grafeno puedan conmutar a frecuencias más altas y en un rango más amplio, tienen serias dificultades que los hacen aún inmaduros. En el caso de los materiales ferroeléctricos, los elevados voltajes para conmutar el material los hacen poco atractivos, mientras que en el caso del grafeno, su modelado aún está en discusión, y todavía no se han arrojado resultados experimentales que validen su idoneidad. Estas tres razones hacen que los reflectarrays basados en cristal líquido sean atractivos para multitud de aplicaciones de haz reconfigurable a frecuencias superiores a 60 GHz. Aplicaciones como radar de escaneo de imágenes de alta resolución, espectroscopia molecular, radiómetros para observación atmosférica, o comunicaciones inalámbricas de alta frecuencia (WiGig) son algunas de ellas. La tesis está estructurada en tres partes. En la primera de ellas se describen las características más comunes de los cristales líquidos, centrándonos en detalle en aquellas propiedades ofrecidas por este material en fase nemática. En concreto, se estudiará la anisotropía dieléctrica (Ae) de los cristales líquidos uniaxiales, que son los que se emplean en esta tesis, definida como la diferencia entre la permitividad paralela (£//) y la perpendicular (e±): Ae = e,, - e±. También se estudiará la variación de este parámetro (Ae) con la frecuencia, y el modelado electromagnético macroscópico más general que, extraído a partir de aquella, permite describir el cristal líquido para cada tensión de polarización en celdas de geometría planar. Este modelo es de suma importancia para garantizar precisión en el desfasaje proporcionado por las diferentes celdas reconfigurables para reflectarrays que se describirán en la siguiente parte de la tesis. La segunda parte de la tesis se centra en el diseño de celdas reflectarray resonantes basadas en cristal líquido. La razón por la que se escogen estos tipos de celdas reside en el hecho de que son las únicas capaces de proporcionar rangos de fase elevados ante la reducida anisotropía dieléctrica que ofrecen los cristales líquidos. El objetivo de esta parte trata, por tanto, de obtener estructuras de celdas reflectarray que sean capaces de proporcionar buenas prestaciones eléctricas a nivel de antena, mejorando sustancialmente las prestaciones de las celdas reportadas en el estado del arte, así como de desarrollar una herramienta de diseño general para aquellas. Para ello, se estudian las prestaciones eléctricas de diferentes tipos de elementos resonantes de cristal líquido que van, desde el más sencillo, que ha limitado el estado de la técnica hasta el desarrollo de esta tesis y que está formado por un sólo resonador, a elementos que constan de varios resonadores (multi-resonantes) y que pueden ser monocapa o multicapa. En un primer paso, el procedimiento de diseño de estas estructuras hace uso de un modelo convencional de cristal líquido que ha venido siendo usado en el estado del arte para este tipo de celdas, y que considera el cristal líquido como un material homogéneo e isótropo cuya permitividad varía entre (e/7) y (e±). Sin embargo, en esta parte de la tesis se demuestra que dicho modelado no es suficiente para describir de forma genérica el comportamiento del cristal líquido en las celdas tipo reflectarray. En la tesis se proponen procedimientos más exactos para el análisis y diseño basados en un modelo más general que define el cristal líquido como un material anisótropo e inhomogeneo en tres dimensiones, y se ha implementado una técnica que permite optimizar celdas multi-resonantes de forma eficiente para conseguir elevadas prestaciones en cuanto a ancho de banda, rango de fase, pérdidas, o sensibilidad al ángulo de incidencia. Los errores cometidos en el uso del modelado convencional a nivel de celda (amplitud y fase) se han analizado para varias geometrías, usando medidas de varios prototipos de antena que usan un cristal líquido real a frecuencias superiores a 100 GHz. Las medidas se han realizado en entorno periódico mediante un banco cuasi-óptico, que ha sido diseñado especialmente para este fin. Uno de estos prototipos se ha optimizado a 100 GHz para conseguir un ancho de banda relativamente elevado (10%), pérdidas reducidas, un rango de fase mayor de 360º, baja sensibilidad al ángulo de incidencia, y baja influencia de la inhomogeneidad transversal del cristal líquido en la celda. Estas prestaciones a nivel de celda superan de forma clara aquellas conseguidas por otros elementos que se han reportado en la literatura, de manera que dicho prototipo se ha usado en la última parte de la tesis para realizar diversas antenas de barrido. Finalmente, en esta parte se presenta una estrategia de caracterización de la anisotropía macroscópica a partir de medidas de los elementos de reflectarray diseñados en banco cuasi-óptico, obteniendo resultados tanto en las frecuencias de interés en RF como en AC, y comparándolas con aquellas obtenidas mediante otros métodos. La tercera parte de la tesis consiste en el estudio, diseño, fabricación y medida de antenas reconfigurables basadas en cristal líquido en configuraciones complejas. En reflectarrays pasivos, el procedimiento de diseño de la antena se limita únicamente al ajuste en cada celda de la antena de las dimensiones de las metalizaciones que se emplean para el control de fase, mediante procesos de optimización bien conocidos. Sin embargo, en el caso de reflectarrays reconfigurables basados en cristal líquido, resulta necesario un paso adicional, que consiste en calcular de forma adecuada las tensiones de control en cada celda del reflectarray para configurar la fase requerida en cada una de ellas, así como diseñar la estructura y los circuitos de control que permitan direccionar a cada elemento su tensión correspondiente. La síntesis de tensiones es por tanto igual o más importante que el diseño de la geometría de las celdas, puesto que éstas son las que están directamente relacionadas con la fase. En el estado del arte, existen varias estrategias de síntesis de tensiones que se basan en la caracterización experimental de la curva de fase respecto al voltaje. Sin embargo, esta caracterización sólo puede hacerse a un solo ángulo de incidencia y para unas determinadas dimensiones de celda, lo que produce que las tensiones sintetizadas sean diferentes de las adecuadas, y en definitiva que se alcancen errores de fase mayores de 70º. De esta forma, hasta la fecha, las prestaciones a nivel de antena que se han conseguido son reducidas en cuanto a ancho de banda, rango de escaneo o nivel de lóbulos secundarios. En esta última parte de la tesis, se introduce una nueva estrategia de síntesis de tensiones que es capaz de predecir mediante simulaciones, y con alta precisión, las tensiones que deben introducirse en cada celda teniendo en cuenta su ángulo de incidencia, sus dimensiones, la frecuencia, así como la señal de polarización definida por su frecuencia y forma de onda AC. Esta estrategia se basa en modelar cada uno de los estados de permitividad del cristal líquido como un sustrato anisótropo con inhomogeneidad longitudinal (1D), o en ciertos casos, como un tensor equivalente homogéneo. La precisión de ambos modelos electromagnéticos también se discute. Con el objetivo de obtener una herramienta eficiente de cálculo de tensiones, también se ha escrito e implementado una herramienta de análisis basada en el Método de los Momentos en el Dominio Espectral (SD-MoM) para sustratos estratificados anisótropos, que se usa en cada iteración del procedimiento de síntesis para analizar cada una de las celdas de la antena. La síntesis de tensiones se ha diseñado además para reducir al máximo el efecto del rizado de amplitud en el diagrama de radiación, que es característico en los reflectarrays que están formados por celdas con pérdidas elevadas, lo que en sí, supone un avance adicional para la obtención de mejores prestaciones de antena. Para el cálculo de los diagramas de radiación empleados en el procedimiento de síntesis, se asume un análisis elemento a elemento considerando periodicidad local, y se propone el uso de un método capaz de modelar el campo incidente de forma que se elimine la limitación de la periodicidad local en la excitación. Una vez definida la estrategia adecuada de cálculo de las tensiones a aplicar al cristal líquido en cada celda, la estructura de direccionamiento de las mismas en la antena, y diseñados los circuitos de control, se diseñan, fabrican y miden dos prototipos diferentes de antena de barrido electrónico a 100 GHz usando las celdas anteriormente presentadas. El primero de estos prototipos es un reflectarray en configuración “single offset” con capacidad de escaneo en un plano (elevación o azimut). Aunque previamente se realizan diseños de antenas de barrido en 2D a varias frecuencias en el rango de milimétricas y sub-milimétricas, y se proponen ciertas estrategias de direccionamiento que permiten conseguir este objetivo, se desarrolla el prototipo con direccionamiento en una dimensión con el fin de reducir el número de controles y posibles errores de fabricación, y así también validar la herramienta de diseño. Para un tamaño medio de apertura (con un numero de filas y columnas entre 30 y 50 elementos, lo que significa un reflectarray con un número de elementos superior a 900), la configuración “single offset” proporciona rangos de escaneo elevados, y ganancias que pueden oscilar entre los 20 y 30 dBi. En concreto, el prototipo medido proporciona un haz de barrido en un rango angular de 55º, en el que el nivel de lóbulos secundarios (SLL) permanece mejor de -13 dB en un ancho de banda de un 8%. La ganancia máxima es de 19.4 dBi. Estas prestaciones superan de forma clara aquellas conseguidas por otros autores. El segundo prototipo se corresponde con una antena de doble reflector que usa el reflectarray de cristal líquido como sub-reflector para escanear el haz en un plano (elevación o azimut). El objetivo básico de esta geometría es obtener mayores ganancias que en el reflectarray “single offset” con una estructura más compacta, aunque a expensas de reducir el rango de barrido. En concreto, se obtiene una ganancia máxima de 35 dBi, y un rango de barrido de 12º. Los procedimientos de síntesis de tensiones y de diseño de las estructuras de las celdas forman, en su conjunto, una herramienta completa de diseño precisa y eficiente de antenas reflectarray reconfigurables basados en cristales líquidos. Dicha herramienta se ha validado mediante el diseño, la fabricación y la medida de los prototipos anteriormente citados a 100 GHz, que consiguen algo nunca alcanzado anteriormente en la investigación de este tipo de antenas: unas prestaciones competitivas y una predicción excelente de los resultados. El procedimiento es general, y por tanto se puede usar a cualquier frecuencia en la que el cristal líquido ofrezca anisotropía dieléctrica, incluidos los THz. Los prototipos desarrollados en esta tesis doctoral suponen también unas de las primeras antenas de barrido real a frecuencias superiores a 100 GHz. En concreto, la antena de doble reflector para escaneo de haz es la primera antena reconfigurable electrónicamente a frecuencias superiores a 60 GHz que superan los 25 dBi de ganancia, siendo a su vez la primera antena de doble reflector que contiene un reflectarray reconfigurable como sub-reflector. Finalmente, se proponen ciertas mejoras que aún deben se deben realizar para hacer que estas antenas puedan ser un producto completamente desarrollado y competitivo en el mercado. ABSTRACT The work presented in this thesis is focused on the development of electronically reconfigurable antennas that are able to provide competitive electrical performance to the increasingly common applications operating at frequencies above 60 GHz. Specifically, this thesis presents the study, design, and implementation of reflectarray antennas, which incorporate liquid crystal (LC) materials to scan or reconfigure the beam electronically. From a general point of view, a liquid crystal can be defined as a material whose dielectric permittivity is variable and can be controlled with an external excitation, which usually corresponds with a quasi-static electric field (AC). By changing the dielectric permittivity at each cell that makes up the reflectarray, the phase shift on the aperture is controlled, so that a prescribed radiation pattern can be configured. Liquid Crystal-based reflectarrays have been chosen for several reasons. The first has to do with the advantages provided by the reflectarray antenna with respect to other high gain antennas, such as reflectors or phased arrays. The RF feeding in reflectarrays is achieved by using a common primary source (as in reflectors). This arrangement and the large number of degrees of freedom provided by the cells that make up the reflectarray (as in arrays), allow these antennas to provide a similar or even better electrical performance than other low profile antennas (reflectors and arrays), but assuming a more reduced cost and compactness. The second reason is the flexibility of the liquid crystal to be confined in an arbitrary geometry due to its fluidity (property of liquids). Therefore, the liquid crystal is able to adapt to a planar geometry so that it is one or more of the typical layers of this configuration. This simplifies drastically both the structure and manufacture of this type of antenna, even when compared with reconfigurable reflectarrays based on other technologies, such as diodes MEMS, etc. Therefore, the cost of developing this type of antenna is very small, which means that electrically large reconfigurable reflectarrays could be manufactured assuming low cost and greater productions. A paradigmatic example of a similar structure is the liquid crystal panel, which has already been commercialized successfully. The third reason lies in the fact that, at present, the liquid crystal is one of the few technologies capable of providing switching capabilities at frequencies above 60 GHz. In fact, the liquid crystal allows its permittivity to be switched in a wide range of frequencies, which are from DC to the visible spectrum, including microwaves and THz. Other technologies, such as ferroelectric materials, graphene or CMOS "on chip" technology also allow the beam to be switched at these frequencies. However, CMOS technology is expensive and is currently limited to frequencies below 150 GHz, and although ferroelectric materials or graphene can switch at higher frequencies and in a wider range, they have serious difficulties that make them immature. Ferroelectric materials involve the use of very high voltages to switch the material, making them unattractive, whereas the electromagnetic modelling of the graphene is still under discussion, so that the experimental results of devices based on this latter technology have not been reported yet. These three reasons make LC-based reflectarrays attractive for many applications that involve the use of electronically reconfigurable beams at frequencies beyond 60 GHz. Applications such as high resolution imaging radars, molecular spectroscopy, radiometers for atmospheric observation, or high frequency wireless communications (WiGig) are just some of them. This thesis is divided into three parts. In the first part, the most common properties of the liquid crystal materials are described, especially those exhibited in the nematic phase. The study is focused on the dielectric anisotropy (Ac) of uniaxial liquid crystals, which is defined as the difference between the parallel (e/7) and perpendicular (e±) permittivities: Ae = e,, - e±. This parameter allows the permittivity of a LC confined in an arbitrary volume at a certain biasing voltage to be described by solving a variational problem that involves both the electrostatic and elastic energies. Thus, the frequency dependence of (Ae) is also described and characterised. Note that an appropriate LC modelling is quite important to ensure enough accuracy in the phase shift provided by each cell that makes up the reflectarray, and therefore to achieve a good electrical performance at the antenna level. The second part of the thesis is focused on the design of resonant reflectarray cells based on liquid crystal. The reason why resonant cells have been chosen lies in the fact that they are able to provide enough phase range using the values of the dielectric anisotropy of the liquid crystals, which are typically small. Thus, the aim of this part is to investigate several reflectarray cell architectures capable of providing good electrical performance at the antenna level, which significantly improve the electrical performance of the cells reported in the literature. Similarly, another of the objectives is to develop a general tool to design these cells. To fulfill these objectives, the electrical yields of different types of resonant reflectarray elements are investigated, beginning from the simplest, which is made up of a single resonator and limits the state of the art. To overcome the electrical limitations of the single resonant cell, several elements consisting of multiple resonators are considered, which can be single-layer or multilayer. In a first step, the design procedure of these structures makes use of a conventional electromagnetic model which has been used in the literature, which considers that the liquid crystal behaves as homogeneous and isotropic materials whose permittivity varies between (e/7) y (e±). However, in this part of the thesis it is shown that the conventional modelling is not enough to describe the physical behaviour of the liquid crystal in reflectarray cells accurately. Therefore, a more accurate analysis and design procedure based on a more general model is proposed and developed, which defines the liquid crystal as an anisotropic three-dimensional inhomogeneous material. The design procedure is able to optimize multi-resonant cells efficiently to achieve good electrical performance in terms of bandwidth, phase range, losses, or sensitivity to the angle of incidence. The errors made when the conventional modelling (amplitude and phase) is considered have been also analysed for various cell geometries, by using measured results from several antenna prototypes made up of real liquid crystals at frequencies above 100 GHz. The measurements have been performed in a periodic environment using a quasi-optical bench, which has been designed especially for this purpose. One of these prototypes has been optimized to achieve a relatively large bandwidth (10%) at 100 GHz, low losses, a phase range of more than 360º, a low sensitivity to angle of incidence, and a low influence of the transversal inhomogeneity of the liquid crystal in the cell. The electrical yields of this prototype at the cell level improve those achieved by other elements reported in the literature, so that this prototype has been used in the last part of the thesis to perform several complete antennas for beam scanning applications. Finally, in this second part of the thesis, a novel strategy to characterise the macroscopic anisotropy using reflectarray cells is presented. The results in both RF and AC frequencies are compared with those obtained by other methods. The third part of the thesis consists on the study, design, manufacture and testing of LCbased reflectarray antennas in complex configurations. Note that the design procedure of a passive reflectarray antenna just consists on finding out the dimensions of the metallisations of each cell (which are used for phase control), using well-known optimization processes. However, in the case of reconfigurable reflectarrays based on liquid crystals, an additional step must be taken into account, which consists of accurately calculating the control voltages to be applied to each cell to configure the required phase-shift distribution on the surface of the antenna. Similarly, the structure to address the voltages at each cell and the control circuitry must be also considered. Therefore, the voltage synthesis is even more important than the design of the cell geometries (dimensions), since the voltages are directly related to the phase-shift. Several voltage synthesis procedures have been proposed in the state of the art, which are based on the experimental characterization of the phase/voltage curve. However, this characterization can be only carried out at a single angle of incidence and at certain cell dimensions, so that the synthesized voltages are different from those needed, thus giving rise to phase errors of more than 70°. Thus, the electrical yields of the LCreflectarrays reported in the literature are limited in terms of bandwidth, scanning range or side lobes level. In this last part of the thesis, a new voltage synthesis procedure has been defined and developed, which allows the required voltage to be calculated at each cell using simulations that take into account the particular dimensions of the cells, their angles of incidence, the frequency, and the AC biasing signal (frequency and waveform). The strategy is based on the modelling of each one of the permittivity states of the liquid crystal as an anisotropic substrate with longitudinal inhomogeneity (1D), or in certain cases, as an equivalent homogeneous tensor. The accuracy of both electromagnetic models is also discussed. The phase errors made by using the proposed voltage synthesis are better than 7º. In order to obtain an efficient tool to analyse and design the reflectarray, an electromagnetic analysis tool based on the Method of Moments in the spectral domain (SD-MoM) has also written and developed for anisotropic stratified media, which is used at each iteration of the voltage synthesis procedure. The voltage synthesis is also designed to minimize the effect of amplitude ripple on the radiation pattern, which is typical of reflectarrays made up of cells exhibiting high losses and represents a further advance in achieving a better antenna performance. To calculate the radiation patterns used in the synthesis procedure, an element-by-element analysis is assumed, which considers the local periodicity approach. Under this consideration, the use of a novel method is proposed, which avoids the limitation that the local periodicity imposes on the excitation. Once the appropriate strategy to calculate the voltages to be applied at each cell is developed, and once it is designed and manufactured both the structure to address the voltages to the antenna and the control circuits, two complete LC-based reflectarray antennas that operate at 100 GHz have been designed, manufactured and tested using the previously presented cells. The first prototype consists of a single offset reflectarray with beam scanning capabilities on one plane (elevation and azimuth). Although several LC-reflectarray antennas that provide 2-D scanning capabilities are also designed, and certain strategies to achieve the 2-D addressing of the voltage are proposed, the manufactured prototype addresses the voltages in one dimension in order to reduce the number of controls and manufacturing errors, and thereby validating the design tool. For an average aperture size (with a number of rows and columns of between 30 and 50 elements, which means a reflectarray with more than 900 cells), the single offset configuration provides an antenna gain of between 20 and 30 dBi and a large scanning range. The prototype tested at 100 GHz exhibits an electronically scanned beam in an angular range of 55º and 8% of bandwidth, in which the side lobe level (SLL) remains better than -13 dB. The maximum gain is 19.4 dBi. The electrical performance of the antenna is clearly an improvement on those achieved by other authors in the state of the art. The second prototype corresponds to a dual reflector antenna with a liquid crystal-based reflectarray used as a sub-reflector for beam scanning in one plane (azimuth or elevation). The main objective is to obtain a higher gain than that provided by the single offset configuration, but using a more compact architecture. In this case, a maximum gain of 35 dBi is achieved, although at the expense of reducing the scanning range to 12°, which is inherent in this type of structure. As a general statement, the voltage synthesis and the design procedure of the cells, jointly make up a complete, accurate and efficient design tool of reconfigurable reflectarray antennas based on liquid crystals. The tool has been validated by testing the previously mentioned prototypes at 100 GHz, which achieve something never reached before for this type of antenna: a competitive electrical performance, and an excellent prediction of the results. The design procedure is general and therefore can be used at any frequency for which the liquid crystal exhibits dielectric anisotropy. The two prototypes designed, manufactured and tested in this thesis are also some of the first antennas that currently operate at frequencies above 100 GHz. In fact, the dual reflector antenna is the first electronically scanned dual reflector antenna at frequencies above 60 GHz (the operation frequency is 100 GHz) with a gain greater than 25 dBi, being in turn the first dual-reflector antenna with a real reconfigurable sub-reflectarray. Finally, some improvements that should be still investigated to make these antennas commercially competitive are proposed.

Recessive and dominant mutations in the ethylene biosynthetic gene ACS5 of Arabidopsis confer cytokinin insensitivity and ethylene overproduction, respectively

We identified a set of cytokinin-insensitive mutants by using a screen based on the ethylene-mediated triple response observed after treatment with low levels of cytokinins. One group of these mutants disrupts ACS5, a member of the Arabidopsis gene family that encodes 1-aminocyclopropane-1-carboxylate synthase, the first enzyme in ethylene biosynthesis. The ACS5 isoform is mainly responsible for the sustained rise in ethylene biosynthesis observed in response to low levels of cytokinin and appears to be regulated primarily by a posttranscriptional mechanism. Furthermore, the dominant ethylene-overproducing mutant eto2 was found to be the result of an alteration of the carboxy terminus of ACS5, suggesting that this domain acts as a negative regulator of ACS5 function.

Retardation of skeletal development and cervical abnormalities in transgenic mice expressing a dominant-negative retinoic acid receptor in chondrogenic cells

Skeletal formation is a fundamental element of body patterning and is strictly regulated both temporally and spatially by a variety of molecules. Among these, retinoic acid (RA) has been shown to be involved in normal skeletal development. However, its pleiotropic effects have caused difficulty in identifying its crucial target cells and molecular mechanisms for each effect. Development of cartilage primordia is an important process in defining the skeletal structures. To address the role of RA in skeletal formation, we have generated mice expressing a dominant-negative retinoic acid receptor (RAR) in chondrogenic cells by using the type II collagen α1 promoter, and we have analyzed their phenotypes. These mice exhibited small cartilage primordia during development and retarded skeletal formation in both embryonic and postnatal periods. They also showed selective degeneration in their cervical vertebrae combined with homeotic transformations, but not in their extremities. The cervical phenotypes are reminiscent of phenotypes involving homeobox genes. We found that the expression of Hoxa-4 was indeed reduced in the cartilage primordia of cervical vertebrae of embryonic day 12.5 embryos. These observations demonstrate that endogenous RA acts directly on chondrogenic cells to promote skeletal growth in both embryonic and growing periods, and it regulates the proper formation of cervical vertebrae. Furthermore, RA apparently specifies the identities of the cervical vertebrae through the regulation of homeobox genes in the chondrogenic cells. Great similarities of the phenotypes between our mice and reported RAR knockout mice revealed that chondrogenic cells are a principal RA target during complex cascades of skeletal development.

Variations in frequencies of drug resistance in Plasmodium falciparum

Continual exposure of malarial parasite populations to different drugs may have selected not only for resistance to individual drugs but also for genetic traits that favor initiation of resistance to novel unrelated antimalarials. To test this hypothesis, different Plasmodium falciparum clones having varying numbers of preexisting resistance mechanisms were treated with two new antimalarial agents: 5-fluoroorotate and atovaquone. All parasite populations were equally susceptible in small numbers. However, when large populations of these clones were challenged with either of the two compounds, significant variations in frequencies of resistance became apparent. On one extreme, clone D6 from West Africa, which was sensitive to all traditional antimalarial agents, failed to develop resistance under simple nonmutagenic conditions in vitro. In sharp contrast, the Indochina clone W2, which was known to be resistant to all traditional antimalarial drugs, independently acquired resistance to both new compounds as much as a 1,000 times more frequently than D6. Additional clones that were resistant to some (but not all) traditional antimalarial agents acquired resistance to atovaquone at high frequency, but not to 5-fluoroorotate. These findings were unexpected and surprising based on current views of the evolution of drug resistance in P. falciparum populations. Such new phenotypes, named accelerated resistance to multiple drugs (ARMD), raise important questions about the genetic and biochemical mechanisms related to the initiation of drug resistance in malarial parasites. Some potential mechanisms underlying ARMD phenotypes have public health implications that are ominous.

Dominant transformation by mutated human ras genes in vitro requires more than 100 times higher expression than is observed in cancers

The gene-mutation-cancer hypothesis holds that mutated cellular protooncogenes, such as point-mutated proto-ras, “play a dominant part in cancer,” because they are sufficient to transform transfected mouse cell lines in vitro [Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K. & Watson, J. D. (1994) Molecular Biology of the Cell (Garland, New York)]. However, in cells transformed in vitro mutated human ras genes are expressed more than 100-fold than in the cancers from which they are isolated. In view of the discrepancy between the very low levels of ras transcription in cancers and the very high levels in cells transformed in vitro, we have investigated the minimal level of human ras expression for transformation in vitro. Using point-mutated human ras genes recombined with different promoters from either human metallothionein-IIA or human fibronectin or from retroviruses we found dominant in vitro transformation of the mouse C3H cell line only with ras genes linked to viral promoters. These ras genes were expressed more than 120-fold higher than are native ras genes of C3H cells. The copy number of transfected ras genes ranged from 2–6 in our system. In addition, nondominant transformation was observed in a small percentage (2–7%) of C3H cells transfected with ras genes that are expressed less than 20 times higher than native C3H ras genes. Because over 90% of cells expressing ras at this moderately enhanced level were untransformed, transformation must follow either a nondominant ras mechanism or a non-ras mechanism. We conclude that the mutated, but normally expressed, ras genes found in human and animal cancers are not likely to “play a dominant part in cancer.” The conclusion that mutated ras genes are not sufficient or dominant for cancer is directly supported by recent discoveries of mutated ras in normal animals, and in benign human tissue, “which has little potential to progress” [Jen, J., Powell, S. M., Papadopoulos, N., Smith, K. J., Hamilton, S. R., Vogelstein, B. & Kinzler, K. W. (1994) Cancer Res. 54, 5523–5526]. Even the view that mutated ras is necessary for cancer is hard to reconcile with (i) otherwise indistinguishable cancers with and without ras mutations, (ii) metastases of the same human cancers with and without ras mutations, (iii) retroviral ras genes that are oncogenic without point mutations, and (iv) human tumor cells having spontaneously lost ras mutation but not tumorigencity.

A variant of DNA polymerase β acts as a dominant negative mutant

In eukaryotic cells, DNA polymerase β (polβ) carries out base-excision repair (BER) required for DNA maintenance, replication, recombination, and drug resistance. A specific deletion in one allele in the coding sequence of the polβ gene occurs in colorectal and breast carcinomas. The 87-bp deleted region encodes amino acid residues 208–236 in the catalytic domain of the enzyme. Here, we report evidence for expression of the wild-type (WT) and the truncated polβ proteins in colorectal tumors. To elucidate the potential functional consequences of polβ truncation, stable HeLa cell lines were established from cloned WT and variant polβΔ208–236. Cells expressing the variant protein exhibited substantially decreased BER activity. To test our hypothesis that truncated polβ may disrupt the function of the WT enzyme, we stably transfected mouse embryonic fibroblast 16.3 cells with polβΔ208–236 cDNA. Reverse transcription–PCR and Western blot analyses showed that the new cell line, 16.3ΔP, expresses the WT and the truncated polβ mRNA and proteins. BER and binding activities were undetectable in these cells. Furthermore, in vivo the 16.3ΔP cells were more sensitive to N-methyl-N′-nitro-N-nitrosoguanidine than the 16.3 cells. On adding increasing amounts of 16.3ΔP protein extracts, the BER and DNA binding activities of extracts of the parent 16.3 cell line progressively declined. These results strongly suggest that truncated polβ acts as a dominant negative mutant. The defective polβ may facilitate accumulation of mutations, leading to the expression of a mutator phenotype in tumor cells.

A dominant form of inherited retinal degeneration caused by a non-photoreceptor cell-specific mutation

We have isolated a dominant mutation, night blindness a (nba), that causes a slow retinal degeneration in zebrafish. Heterozygous nba fish have normal vision through 2–3 months of age but subsequently become night blind. By 9.5 months of age, visual sensitivity of affected fish may be decreased more than two log units, or 100-fold, as measured behaviorally. Electroretinographic (ERG) thresholds of mutant fish are also raised significantly, and the ERG b-wave shows a delayed implicit time. These defects are due primarily to a late-onset photoreceptor cell degeneration involving initially the rods but eventually the cones as well. Homozygous nba fish display an early-onset neuronal degeneration throughout the retina and elsewhere in the central nervous system. As a result, animals develop with small eyes and die by 4–5 days postfertilization (pf). These latter data indicate that the mutation affecting nba fish is not in a photoreceptor cell-specific gene.

Abnormalities of pancreatic islets by targeted expression of a dominant-negative KATP channel

ATP-sensitive K+ (KATP) channels are known to play important roles in various cellular functions, but the direct consequences of disruption of KATP channel function are largely unknown. We have generated transgenic mice expressing a dominant-negative form of the KATP channel subunit Kir6.2 (Kir6.2G132S, substitution of glycine with serine at position 132) in pancreatic beta cells. Kir6.2G132S transgenic mice develop hypoglycemia with hyperinsulinemia in neonates and hyperglycemia with hypoinsulinemia and decreased beta cell population in adults. KATP channel function is found to be impaired in the beta cells of transgenic mice with hyperglycemia. In addition, both resting membrane potential and basal calcium concentrations are shown to be significantly elevated in the beta cells of transgenic mice. We also found a high frequency of apoptotic beta cells before the appearance of hyperglycemia in the transgenic mice, suggesting that the KATP channel might play a significant role in beta cell survival in addition to its role in the regulation of insulin secretion.

Isolation and characterization of iron-independent positive dominant mutants of the diphtheria toxin repressor DtxR

It is well known that the functional activity of the diphtheria toxin repressor DtxR is controlled by iron, which serves as an essential cofactor necessary for activation of target DNA binding by this regulatory element. In this communication, we describe the isolation and characterization of a unique series of DtxR mutants that are constitutively active and repress the expression of β-galactosidase from a diphtheria tox promoter/operator–lacZ transcriptional fusion, even in the absence of iron. These self-activating mutants of DtxR (SAD) were isolated through the use of a positive selection system for the cloning of functional dtxR alleles and target DNA operator sites. Of the four independently isolated SAD mutants that were characterized, two (SAD2 and SAD11) were found to carry a single missense mutation (E175K) in their respective C-terminal SH3-like domains. In contrast, the mutant allele encoding SAD3 was found to carry a total of six missense mutations distributed throughout the N- and C-terminal domains of the repressor. Partial diploid analysis of strains carrying both native dtxR and alleles encoding either SAD2 or SAD3 demonstrate that these iron-independent mutants possess a positive dominant phenotype in the regulation of β-galactosidase expression from a diphtheria tox promoter/operator–lacZ transcriptional fusion.