Paisaje de energías: conocimiento estético en el enfoque ambiental del proyecto

Desde hace ya varias décadas la praxis de la ecología ha venido reconociendo la necesidad de estudiar los múltiples sistemas de interacción del ser humano, como especie viva, y su entorno. Entidades espaciales como el paisaje geográfico son empleadas para delimitar sistemas territoriales operados por la sociedad, precisando campos concretos de su acción física, biológica y cultural. La ecología aborda así el conocimiento científico del territorio como asentamiento humano, rastrea sus patrones espaciales y analiza su compleja estructura funcional. En ese contexto, la transferencia de herramientas e instrumentos desde la ecología al ámbito proyectivo posee ya un bagaje de más de cinco décadas. Cada vez con más frecuencia el proyecto emplea parámetros, inventarios, fórmulas, indicadores y tecnologías que tratan de dar una respuesta ambientalmente adecuada a los condicionantes de contorno, por ejemplo aprovechando las condiciones climáticas en la optimización energética o proponiendo programas de usos del suelo que eviten perturbaciones en ecosistemas de interés. Con todo, en el momento presente surgen voces que, ante el dominio indiscutible de los enfoques netamente deterministas, tratan de recordar que los principios del pensamiento ecológico van más allá del mero control cuantitativo de los procesos biofísicos. Recuerdan que la etología demostró a principios del XX que el ser humano, como ser consciente, inviste una relación de intimidad con su entorno que supera tales perspectivas: a través de la correspondencia entre percepción y significación, entre lo físico y lo psíquico, entre interioridad y exterioridad, las personas abrazan la plenitud de aquello que les rodea en un acto de profunda conciliación afectiva. De tal ligadura de intimidad depende, sí o sí, y en toda su profundidad, la aceptación humana del entorno construido. A través de la noción de ambiente [Umwelt] se demuestra que la relación del hombre con su entorno es inseparable, bidireccional y coordinada y, por lo tanto, desde una posición coherente, la experiencia del espacio puede ser examinada a partir de la reciprocidad que constituyen, en continuidad, la persona y el lugar. De esta forma, la tesis dirige su objetivo principal a explorar y considerar, desde el proyecto, el significado y la influencia de la experiencia ambiental del espacio construido en la vida humana. Es más que probable que buena parte de los problemas de desafección del hombre con los paisajes transformados de su contemporaneidad tenga que ver con que tanto las intensidades de la experiencia y percepción humana, como la potestad interpretativa de sus productos culturales, incluyendo la arquitectura, han sido fuertemente reducidas. Ante este problema, la investigación toma como hipótesis la oportunidad que ofrece el pensamiento ecológico de reformular la experiencia estética como un acto de conocimiento, como un evento donde se da el encuentro físico y se construyen significados, donde se sancionan valores sociales y se mira hacia el futuro. Se ha de señalar que la presente tesis doctoral arranca en el Laboratorio de Paisaje del Grupo de Investigación Paisaje Cultural de la Universidad Politécnica de Madrid dirigido por Concha Lapayese y Darío Gazapo, y por tanto hace suyos para el estado del arte los principales conceptos e ideas bajo los que el trabajo teórico y práctico del grupo se viene orientando desde hace años: la consideración del paisaje como acontecimiento; la oscilación de la interpretación entre un paisaje específico y un paisaje genérico en un mundo globalizado; el reconocimiento de la experiencia estética del paisaje como una toma de conciencia social; y en definitiva, la reivindicación de la interioridad en el proyecto contemporáneo. La investigación profundiza en una línea de oportunidad que se abre al promover lo que se ha llamado un conocimiento por lo sentido como estrategia ambiental que permite contrarrestar mitos profundamente arraigados en las estructuras sociales. El primer paso en ese recorrido sería explorar ecológicamente el aporte de la experiencia estética; esto es, su consideración como forma de conocimiento específico. Resultaría pertinente impulsar la idea de la inmersión en el paisaje como fenómeno experiencial, sensual y corporal, y enfrentar, desde ahí, el problema de la aceptación social de lo nuevo y lo trasformado de acuerdo con el momento actual. La exploración sobre la afectividad en el ambiente no es, en cualquier caso, un asunto nuevo. Sin pretensiones de historiografía, dos momentos del siglo XX concentran el interés de la investigación. La primera se corresponde fundamentalmente con la segunda década del siglo, en relación a una serie de influencias que desde los avances científicos determinaron singulares aventuras del arte más experimental. La segunda se posiciona en el entorno de 1970, época en la que es conocido el interés que despertaron las cuestiones ambientales. En ambos casos se han estudiado aportaciones que desvelan conceptos determinantes en la definición de la experiencia estética como un evento de adquisición de conocimiento por lo sentido. Es conveniente adelantar el rol de centralidad que para la investigación tiene el concepto de energía, tal como el propio título subraya. La energía como realidad material y sensible es el sustrato que permite navegar por el principio de unidad epistemológica que subyace al pensamiento ecológico. Sus continuas referencias simbólicas, físicas y metafóricas entre los artistas estudiados no son un mero recurso iconográfico: mantienen inherente el principio de continuidad ambiental en el cual el ser humano y la inmensidad del cosmos navegan indisociables. Un discurso unificado y consistente sobre los aportes de la experiencia estética enfocada como forma de conocimiento por lo sentido hila la lectura histórica, conceptual y práctica de toda la investigación. Con ello se alcanza a hilvanar un diagrama conceptual, modelo de análisis proyectivo, que recoge ideas científicas, filosóficas y proyectivas. De alguna manera, el diagrama trata de dibujar, desde los principios del pensamiento ecológico, la correlación de continuidad que, vacilante, tensa, sutil y frágil se desplaza incesante e irresuelta entre interioridad y exterioridad. ABSTRACT Over the last few decades ecological practice has come to acknowledge a need for studying the multiple systems of interaction between the human being - inasmuch as it is a living species - and its environment. Spatial entities such as the geographic notion of landscape have been used to delimitate the territorial systems operated by society and to describe in detail specific fields of its physical, biological and cultural action. Ecology has thus managed to address the scientific knowledge of the territory as a human settlement, tracking its spatial patterns and analysing its complex functional structure. In this context, the transfer of tools and instruments from the field of ecology to that of design has a tradition already going back more than fifty years. Increasingly more often, design makes use of parameters, inventories, formulas, indicators and technologies to give an environmentally sound response to contour conditions: for instance, taking advantage of the local climate for the optimisation of energy consumption or proposing land uses that avoid disturbing valuable ecosystems. Yet in the present day some voices have arisen that, against the uncontested domination of purely positivistic approaches, are trying to draw attention to the fact that the principles of ecological thought go beyond mere quantitative control of biophysical processes. They point out that, in the early 20th century, ethology proved that the human being, as a conscious entity, invests itself into a relationship of intimacy with its environment that surpasses such perspectives: through the correspondences between perception and signification, between physical and psychological or between inside and outside, people embrace the entirety of their surroundings in an action of deep affective conciliation. It is on this link of intimacy that - fully and unquestionably - human acceptance of the built environment depends. Through the notion of environment [Umwelt] it can be proven that the relationship between the human being and its environment is inseparable, bidirectional and coordinated; and that, therefore, from a coherent position the experience of space can be examined through the reciprocity constituted continuously by person and place. Thus, the main goal in this thesis is to explore and acknowledge, from the standpoint of design, the meaning and influence of the environmental experience in human life. It is extremely likely that many of the issues with mankind’s alienation from the transformed landscapes of the present day arise from the fact that both the intensity of human perception and experience and the interpretive capacity of its cultural products –including architecture - have been greatly reduced. Facing this issue, research has taken as hypothesis the opportunity offered by ecological thought of reformulating aesthetic experience as an act of knowledge – as an event where physical encounter takes place and meanings are constructed; where social values are sanctioned and the path towards the future is drawn. This notwithstanding, the present thesis began in the Landscape Laboratory of the Technical University of Madrid Cultural Landscape Research Group (GIPC-UPM), led by Concha Lapayese and Darío Gazapo; and has therefore appropriated for its state of the art the main concepts and ideas that have been orienting the practical and theoretical work of the latter: the understanding of landscape as an event, the oscillation of interpretation between a specific and a generic landscape within a globalised world; the acknowledgement of the aesthetic experience of landscape as a way of acquiring social awareness; and, all in all, a vindication of interiority in contemporary design. An exploration has been made of the line of opportunity that is opened when promoting what has been termed knowledge through the senses as an environmental strategy allowing to counter myths deeply rooted in social structures. The first step in this path would be an ecological exploration of the contribution of the aesthetic experience; that is, its consideration as a type of specific knowledge. It would be pertinent to further the idea of immersion into the landscape as an experiential, sensual and corporeal phenomenon and, from that point, to face the issue of social acceptance of what is new and transformed according to the values of the present day. The exploration of affectivity in the environment is not, at any rate, a new topic. Without aspiring to make a history of it, we can mark two points in the 20th century that have concentrated the interest of this research. The first coincides with the second decade of the century and relates to a number of influences that, arising from scientific progress, determined the singular adventures of the more experimental tendencies in art. The second is centred around 1970: a period in which the interest drawn by environmental matters is well known. In both cases, contributions have been studied that reveal crucial concepts in defining the aesthetic experience as an event for the acquisition of knowledge through the senses. It is necessary to highlight the role of centrality that the concept of energy has throughout this research, as is evident even in its title. Energy as a material, sensitive reality is the substrate making it possible to navigate through the principle of epistemological unity underlying ecological thought. The continuous symbolic, physical and metaphorical references to it among the artists studied here are not a mere iconographic source: they remind of the inherency of the principle of environmental continuity within which the human being and the immensity of cosmos travel indissociably. A unified, consistent discourse on the contributions of the aesthetic experience addressed as knowledge through the senses weaves together the historic, conceptual and practical reading of the whole research. With it, a conceptual diagram is constructed – a model of design analysis – gathering together scientific, philosophical and design ideas. Somehow, the diagram tries to draw from the principles of ecological thought the correlation of continuity that, vacillating, tense, subtle and fragile, shifts incessantly and unresolved between interiority and exteriority.

Parametric and structural self-adaptation of embedded systems using evolvable hardware

Los sistemas empotrados han sido concebidos tradicionalmente como sistemas de procesamiento específicos que realizan una tarea fija durante toda su vida útil. Para cumplir con requisitos estrictos de coste, tamaño y peso, el equipo de diseño debe optimizar su funcionamiento para condiciones muy específicas. Sin embargo, la demanda de mayor versatilidad, un funcionamiento más inteligente y, en definitiva, una mayor capacidad de procesamiento comenzaron a chocar con estas limitaciones, agravado por la incertidumbre asociada a entornos de operación cada vez más dinámicos donde comenzaban a ser desplegados progresivamente. Esto trajo como resultado una necesidad creciente de que los sistemas pudieran responder por si solos a eventos inesperados en tiempo diseño tales como: cambios en las características de los datos de entrada y el entorno del sistema en general; cambios en la propia plataforma de cómputo, por ejemplo debido a fallos o defectos de fabricación; y cambios en las propias especificaciones funcionales causados por unos objetivos del sistema dinámicos y cambiantes. Como consecuencia, la complejidad del sistema aumenta, pero a cambio se habilita progresivamente una capacidad de adaptación autónoma sin intervención humana a lo largo de la vida útil, permitiendo que tomen sus propias decisiones en tiempo de ejecución. Éstos sistemas se conocen, en general, como sistemas auto-adaptativos y tienen, entre otras características, las de auto-configuración, auto-optimización y auto-reparación. Típicamente, la parte soft de un sistema es mayoritariamente la única utilizada para proporcionar algunas capacidades de adaptación a un sistema. Sin embargo, la proporción rendimiento/potencia en dispositivos software como microprocesadores en muchas ocasiones no es adecuada para sistemas empotrados. En este escenario, el aumento resultante en la complejidad de las aplicaciones está siendo abordado parcialmente mediante un aumento en la complejidad de los dispositivos en forma de multi/many-cores; pero desafortunadamente, esto hace que el consumo de potencia también aumente. Además, la mejora en metodologías de diseño no ha sido acorde como para poder utilizar toda la capacidad de cómputo disponible proporcionada por los núcleos. Por todo ello, no se están satisfaciendo adecuadamente las demandas de cómputo que imponen las nuevas aplicaciones. La solución tradicional para mejorar la proporción rendimiento/potencia ha sido el cambio a unas especificaciones hardware, principalmente usando ASICs. Sin embargo, los costes de un ASIC son altamente prohibitivos excepto en algunos casos de producción en masa y además la naturaleza estática de su estructura complica la solución a las necesidades de adaptación. Los avances en tecnologías de fabricación han hecho que la FPGA, una vez lenta y pequeña, usada como glue logic en sistemas mayores, haya crecido hasta convertirse en un dispositivo de cómputo reconfigurable de gran potencia, con una cantidad enorme de recursos lógicos computacionales y cores hardware empotrados de procesamiento de señal y de propósito general. Sus capacidades de reconfiguración han permitido combinar la flexibilidad propia del software con el rendimiento del procesamiento en hardware, lo que tiene la potencialidad de provocar un cambio de paradigma en arquitectura de computadores, pues el hardware no puede ya ser considerado más como estático. El motivo es que como en el caso de las FPGAs basadas en tecnología SRAM, la reconfiguración parcial dinámica (DPR, Dynamic Partial Reconfiguration) es posible. Esto significa que se puede modificar (reconfigurar) un subconjunto de los recursos computacionales en tiempo de ejecución mientras el resto permanecen activos. Además, este proceso de reconfiguración puede ser ejecutado internamente por el propio dispositivo. El avance tecnológico en dispositivos hardware reconfigurables se encuentra recogido bajo el campo conocido como Computación Reconfigurable (RC, Reconfigurable Computing). Uno de los campos de aplicación más exóticos y menos convencionales que ha posibilitado la computación reconfigurable es el conocido como Hardware Evolutivo (EHW, Evolvable Hardware), en el cual se encuentra enmarcada esta tesis. La idea principal del concepto consiste en convertir hardware que es adaptable a través de reconfiguración en una entidad evolutiva sujeta a las fuerzas de un proceso evolutivo inspirado en el de las especies biológicas naturales, que guía la dirección del cambio. Es una aplicación más del campo de la Computación Evolutiva (EC, Evolutionary Computation), que comprende una serie de algoritmos de optimización global conocidos como Algoritmos Evolutivos (EA, Evolutionary Algorithms), y que son considerados como algoritmos universales de resolución de problemas. En analogía al proceso biológico de la evolución, en el hardware evolutivo el sujeto de la evolución es una población de circuitos que intenta adaptarse a su entorno mediante una adecuación progresiva generación tras generación. Los individuos pasan a ser configuraciones de circuitos en forma de bitstreams caracterizados por descripciones de circuitos reconfigurables. Seleccionando aquellos que se comportan mejor, es decir, que tienen una mejor adecuación (o fitness) después de ser evaluados, y usándolos como padres de la siguiente generación, el algoritmo evolutivo crea una nueva población hija usando operadores genéticos como la mutación y la recombinación. Según se van sucediendo generaciones, se espera que la población en conjunto se aproxime a la solución óptima al problema de encontrar una configuración del circuito adecuada que satisfaga las especificaciones. El estado de la tecnología de reconfiguración después de que la familia de FPGAs XC6200 de Xilinx fuera retirada y reemplazada por las familias Virtex a finales de los 90, supuso un gran obstáculo para el avance en hardware evolutivo; formatos de bitstream cerrados (no conocidos públicamente); dependencia de herramientas del fabricante con soporte limitado de DPR; una velocidad de reconfiguración lenta; y el hecho de que modificaciones aleatorias del bitstream pudieran resultar peligrosas para la integridad del dispositivo, son algunas de estas razones. Sin embargo, una propuesta a principios de los años 2000 permitió mantener la investigación en el campo mientras la tecnología de DPR continuaba madurando, el Circuito Virtual Reconfigurable (VRC, Virtual Reconfigurable Circuit). En esencia, un VRC en una FPGA es una capa virtual que actúa como un circuito reconfigurable de aplicación específica sobre la estructura nativa de la FPGA que reduce la complejidad del proceso reconfiguración y aumenta su velocidad (comparada con la reconfiguración nativa). Es un array de nodos computacionales especificados usando descripciones HDL estándar que define recursos reconfigurables ad-hoc: multiplexores de rutado y un conjunto de elementos de procesamiento configurables, cada uno de los cuales tiene implementadas todas las funciones requeridas, que pueden seleccionarse a través de multiplexores tal y como ocurre en una ALU de un microprocesador. Un registro grande actúa como memoria de configuración, por lo que la reconfiguración del VRC es muy rápida ya que tan sólo implica la escritura de este registro, el cual controla las señales de selección del conjunto de multiplexores. Sin embargo, esta capa virtual provoca: un incremento de área debido a la implementación simultánea de cada función en cada nodo del array más los multiplexores y un aumento del retardo debido a los multiplexores, reduciendo la frecuencia de funcionamiento máxima. La naturaleza del hardware evolutivo, capaz de optimizar su propio comportamiento computacional, le convierten en un buen candidato para avanzar en la investigación sobre sistemas auto-adaptativos. Combinar un sustrato de cómputo auto-reconfigurable capaz de ser modificado dinámicamente en tiempo de ejecución con un algoritmo empotrado que proporcione una dirección de cambio, puede ayudar a satisfacer los requisitos de adaptación autónoma de sistemas empotrados basados en FPGA. La propuesta principal de esta tesis está por tanto dirigida a contribuir a la auto-adaptación del hardware de procesamiento de sistemas empotrados basados en FPGA mediante hardware evolutivo. Esto se ha abordado considerando que el comportamiento computacional de un sistema puede ser modificado cambiando cualquiera de sus dos partes constitutivas: una estructura hard subyacente y un conjunto de parámetros soft. De esta distinción, se derivan dos lineas de trabajo. Por un lado, auto-adaptación paramétrica, y por otro auto-adaptación estructural. El objetivo perseguido en el caso de la auto-adaptación paramétrica es la implementación de técnicas de optimización evolutiva complejas en sistemas empotrados con recursos limitados para la adaptación paramétrica online de circuitos de procesamiento de señal. La aplicación seleccionada como prueba de concepto es la optimización para tipos muy específicos de imágenes de los coeficientes de los filtros de transformadas wavelet discretas (DWT, DiscreteWavelet Transform), orientada a la compresión de imágenes. Por tanto, el objetivo requerido de la evolución es una compresión adaptativa y más eficiente comparada con los procedimientos estándar. El principal reto radica en reducir la necesidad de recursos de supercomputación para el proceso de optimización propuesto en trabajos previos, de modo que se adecúe para la ejecución en sistemas empotrados. En cuanto a la auto-adaptación estructural, el objetivo de la tesis es la implementación de circuitos auto-adaptativos en sistemas evolutivos basados en FPGA mediante un uso eficiente de sus capacidades de reconfiguración nativas. En este caso, la prueba de concepto es la evolución de tareas de procesamiento de imagen tales como el filtrado de tipos desconocidos y cambiantes de ruido y la detección de bordes en la imagen. En general, el objetivo es la evolución en tiempo de ejecución de tareas de procesamiento de imagen desconocidas en tiempo de diseño (dentro de un cierto grado de complejidad). En este caso, el objetivo de la propuesta es la incorporación de DPR en EHW para evolucionar la arquitectura de un array sistólico adaptable mediante reconfiguración cuya capacidad de evolución no había sido estudiada previamente. Para conseguir los dos objetivos mencionados, esta tesis propone originalmente una plataforma evolutiva que integra un motor de adaptación (AE, Adaptation Engine), un motor de reconfiguración (RE, Reconfiguration Engine) y un motor computacional (CE, Computing Engine) adaptable. El el caso de adaptación paramétrica, la plataforma propuesta está caracterizada por: • un CE caracterizado por un núcleo de procesamiento hardware de DWT adaptable mediante registros reconfigurables que contienen los coeficientes de los filtros wavelet • un algoritmo evolutivo como AE que busca filtros wavelet candidatos a través de un proceso de optimización paramétrica desarrollado específicamente para sistemas caracterizados por recursos de procesamiento limitados • un nuevo operador de mutación simplificado para el algoritmo evolutivo utilizado, que junto con un mecanismo de evaluación rápida de filtros wavelet candidatos derivado de la literatura actual, asegura la viabilidad de la búsqueda evolutiva asociada a la adaptación de wavelets. En el caso de adaptación estructural, la plataforma propuesta toma la forma de: • un CE basado en una plantilla de array sistólico reconfigurable de 2 dimensiones compuesto de nodos de procesamiento reconfigurables • un algoritmo evolutivo como AE que busca configuraciones candidatas del array usando un conjunto de funcionalidades de procesamiento para los nodos disponible en una biblioteca accesible en tiempo de ejecución • un RE hardware que explota la capacidad de reconfiguración nativa de las FPGAs haciendo un uso eficiente de los recursos reconfigurables del dispositivo para cambiar el comportamiento del CE en tiempo de ejecución • una biblioteca de elementos de procesamiento reconfigurables caracterizada por bitstreams parciales independientes de la posición, usados como el conjunto de configuraciones disponibles para los nodos de procesamiento del array Las contribuciones principales de esta tesis se pueden resumir en la siguiente lista: • Una plataforma evolutiva basada en FPGA para la auto-adaptación paramétrica y estructural de sistemas empotrados compuesta por un motor computacional (CE), un motor de adaptación (AE) evolutivo y un motor de reconfiguración (RE). Esta plataforma se ha desarrollado y particularizado para los casos de auto-adaptación paramétrica y estructural. • En cuanto a la auto-adaptación paramétrica, las contribuciones principales son: – Un motor computacional adaptable mediante registros que permite la adaptación paramétrica de los coeficientes de una implementación hardware adaptativa de un núcleo de DWT. – Un motor de adaptación basado en un algoritmo evolutivo desarrollado específicamente para optimización numérica, aplicada a los coeficientes de filtros wavelet en sistemas empotrados con recursos limitados. – Un núcleo IP de DWT auto-adaptativo en tiempo de ejecución para sistemas empotrados que permite la optimización online del rendimiento de la transformada para compresión de imágenes en entornos específicos de despliegue, caracterizados por tipos diferentes de señal de entrada. – Un modelo software y una implementación hardware de una herramienta para la construcción evolutiva automática de transformadas wavelet específicas. • Por último, en cuanto a la auto-adaptación estructural, las contribuciones principales son: – Un motor computacional adaptable mediante reconfiguración nativa de FPGAs caracterizado por una plantilla de array sistólico en dos dimensiones de nodos de procesamiento reconfigurables. Es posible mapear diferentes tareas de cómputo en el array usando una biblioteca de elementos sencillos de procesamiento reconfigurables. – Definición de una biblioteca de elementos de procesamiento apropiada para la síntesis autónoma en tiempo de ejecución de diferentes tareas de procesamiento de imagen. – Incorporación eficiente de la reconfiguración parcial dinámica (DPR) en sistemas de hardware evolutivo, superando los principales inconvenientes de propuestas previas como los circuitos reconfigurables virtuales (VRCs). En este trabajo también se comparan originalmente los detalles de implementación de ambas propuestas. – Una plataforma tolerante a fallos, auto-curativa, que permite la recuperación funcional online en entornos peligrosos. La plataforma ha sido caracterizada desde una perspectiva de tolerancia a fallos: se proponen modelos de fallo a nivel de CLB y de elemento de procesamiento, y usando el motor de reconfiguración, se hace un análisis sistemático de fallos para un fallo en cada elemento de procesamiento y para dos fallos acumulados. – Una plataforma con calidad de filtrado dinámica que permite la adaptación online a tipos de ruido diferentes y diferentes comportamientos computacionales teniendo en cuenta los recursos de procesamiento disponibles. Por un lado, se evolucionan filtros con comportamientos no destructivos, que permiten esquemas de filtrado en cascada escalables; y por otro, también se evolucionan filtros escalables teniendo en cuenta requisitos computacionales de filtrado cambiantes dinámicamente. Este documento está organizado en cuatro partes y nueve capítulos. La primera parte contiene el capítulo 1, una introducción y motivación sobre este trabajo de tesis. A continuación, el marco de referencia en el que se enmarca esta tesis se analiza en la segunda parte: el capítulo 2 contiene una introducción a los conceptos de auto-adaptación y computación autonómica (autonomic computing) como un campo de investigación más general que el muy específico de este trabajo; el capítulo 3 introduce la computación evolutiva como la técnica para dirigir la adaptación; el capítulo 4 analiza las plataformas de computación reconfigurables como la tecnología para albergar hardware auto-adaptativo; y finalmente, el capítulo 5 define, clasifica y hace un sondeo del campo del hardware evolutivo. Seguidamente, la tercera parte de este trabajo contiene la propuesta, desarrollo y resultados obtenidos: mientras que el capítulo 6 contiene una declaración de los objetivos de la tesis y la descripción de la propuesta en su conjunto, los capítulos 7 y 8 abordan la auto-adaptación paramétrica y estructural, respectivamente. Finalmente, el capítulo 9 de la parte 4 concluye el trabajo y describe caminos de investigación futuros. ABSTRACT Embedded systems have traditionally been conceived to be specific-purpose computers with one, fixed computational task for their whole lifetime. Stringent requirements in terms of cost, size and weight forced designers to highly optimise their operation for very specific conditions. However, demands for versatility, more intelligent behaviour and, in summary, an increased computing capability began to clash with these limitations, intensified by the uncertainty associated to the more dynamic operating environments where they were progressively being deployed. This brought as a result an increasing need for systems to respond by themselves to unexpected events at design time, such as: changes in input data characteristics and system environment in general; changes in the computing platform itself, e.g., due to faults and fabrication defects; and changes in functional specifications caused by dynamically changing system objectives. As a consequence, systems complexity is increasing, but in turn, autonomous lifetime adaptation without human intervention is being progressively enabled, allowing them to take their own decisions at run-time. This type of systems is known, in general, as selfadaptive, and are able, among others, of self-configuration, self-optimisation and self-repair. Traditionally, the soft part of a system has mostly been so far the only place to provide systems with some degree of adaptation capabilities. However, the performance to power ratios of software driven devices like microprocessors are not adequate for embedded systems in many situations. In this scenario, the resulting rise in applications complexity is being partly addressed by rising devices complexity in the form of multi and many core devices; but sadly, this keeps on increasing power consumption. Besides, design methodologies have not been improved accordingly to completely leverage the available computational power from all these cores. Altogether, these factors make that the computing demands new applications pose are not being wholly satisfied. The traditional solution to improve performance to power ratios has been the switch to hardware driven specifications, mainly using ASICs. However, their costs are highly prohibitive except for some mass production cases and besidesthe static nature of its structure complicates the solution to the adaptation needs. The advancements in fabrication technologies have made that the once slow, small FPGA used as glue logic in bigger systems, had grown to be a very powerful, reconfigurable computing device with a vast amount of computational logic resources and embedded, hardened signal and general purpose processing cores. Its reconfiguration capabilities have enabled software-like flexibility to be combined with hardware-like computing performance, which has the potential to cause a paradigm shift in computer architecture since hardware cannot be considered as static anymore. This is so, since, as is the case with SRAMbased FPGAs, Dynamic Partial Reconfiguration (DPR) is possible. This means that subsets of the FPGA computational resources can now be changed (reconfigured) at run-time while the rest remains active. Besides, this reconfiguration process can be triggered internally by the device itself. This technological boost in reconfigurable hardware devices is actually covered under the field known as Reconfigurable Computing. One of the most exotic fields of application that Reconfigurable Computing has enabled is the known as Evolvable Hardware (EHW), in which this dissertation is framed. The main idea behind the concept is turning hardware that is adaptable through reconfiguration into an evolvable entity subject to the forces of an evolutionary process, inspired by that of natural, biological species, that guides the direction of change. It is yet another application of the field of Evolutionary Computation (EC), which comprises a set of global optimisation algorithms known as Evolutionary Algorithms (EAs), considered as universal problem solvers. In analogy to the biological process of evolution, in EHW the subject of evolution is a population of circuits that tries to get adapted to its surrounding environment by progressively getting better fitted to it generation after generation. Individuals become circuit configurations representing bitstreams that feature reconfigurable circuit descriptions. By selecting those that behave better, i.e., with a higher fitness value after being evaluated, and using them as parents of the following generation, the EA creates a new offspring population by using so called genetic operators like mutation and recombination. As generations succeed one another, the whole population is expected to approach to the optimum solution to the problem of finding an adequate circuit configuration that fulfils system objectives. The state of reconfiguration technology after Xilinx XC6200 FPGA family was discontinued and replaced by Virtex families in the late 90s, was a major obstacle for advancements in EHW; closed (non publicly known) bitstream formats; dependence on manufacturer tools with highly limiting support of DPR; slow speed of reconfiguration; and random bitstream modifications being potentially hazardous for device integrity, are some of these reasons. However, a proposal in the first 2000s allowed to keep investigating in this field while DPR technology kept maturing, the Virtual Reconfigurable Circuit (VRC). In essence, a VRC in an FPGA is a virtual layer acting as an application specific reconfigurable circuit on top of an FPGA fabric that reduces the complexity of the reconfiguration process and increases its speed (compared to native reconfiguration). It is an array of computational nodes specified using standard HDL descriptions that define ad-hoc reconfigurable resources; routing multiplexers and a set of configurable processing elements, each one containing all the required functions, which are selectable through functionality multiplexers as in microprocessor ALUs. A large register acts as configuration memory, so VRC reconfiguration is very fast given it only involves writing this register, which drives the selection signals of the set of multiplexers. However, large overheads are introduced by this virtual layer; an area overhead due to the simultaneous implementation of every function in every node of the array plus the multiplexers, and a delay overhead due to the multiplexers, which also reduces maximum frequency of operation. The very nature of Evolvable Hardware, able to optimise its own computational behaviour, makes it a good candidate to advance research in self-adaptive systems. Combining a selfreconfigurable computing substrate able to be dynamically changed at run-time with an embedded algorithm that provides a direction for change, can help fulfilling requirements for autonomous lifetime adaptation of FPGA-based embedded systems. The main proposal of this thesis is hence directed to contribute to autonomous self-adaptation of the underlying computational hardware of FPGA-based embedded systems by means of Evolvable Hardware. This is tackled by considering that the computational behaviour of a system can be modified by changing any of its two constituent parts: an underlying hard structure and a set of soft parameters. Two main lines of work derive from this distinction. On one side, parametric self-adaptation and, on the other side, structural self-adaptation. The goal pursued in the case of parametric self-adaptation is the implementation of complex evolutionary optimisation techniques in resource constrained embedded systems for online parameter adaptation of signal processing circuits. The application selected as proof of concept is the optimisation of Discrete Wavelet Transforms (DWT) filters coefficients for very specific types of images, oriented to image compression. Hence, adaptive and improved compression efficiency, as compared to standard techniques, is the required goal of evolution. The main quest lies in reducing the supercomputing resources reported in previous works for the optimisation process in order to make it suitable for embedded systems. Regarding structural self-adaptation, the thesis goal is the implementation of self-adaptive circuits in FPGA-based evolvable systems through an efficient use of native reconfiguration capabilities. In this case, evolution of image processing tasks such as filtering of unknown and changing types of noise and edge detection are the selected proofs of concept. In general, evolving unknown image processing behaviours (within a certain complexity range) at design time is the required goal. In this case, the mission of the proposal is the incorporation of DPR in EHW to evolve a systolic array architecture adaptable through reconfiguration whose evolvability had not been previously checked. In order to achieve the two stated goals, this thesis originally proposes an evolvable platform that integrates an Adaptation Engine (AE), a Reconfiguration Engine (RE) and an adaptable Computing Engine (CE). In the case of parametric adaptation, the proposed platform is characterised by: • a CE featuring a DWT hardware processing core adaptable through reconfigurable registers that holds wavelet filters coefficients • an evolutionary algorithm as AE that searches for candidate wavelet filters through a parametric optimisation process specifically developed for systems featured by scarce computing resources • a new, simplified mutation operator for the selected EA, that together with a fast evaluation mechanism of candidate wavelet filters derived from existing literature, assures the feasibility of the evolutionary search involved in wavelets adaptation In the case of structural adaptation, the platform proposal takes the form of: • a CE based on a reconfigurable 2D systolic array template composed of reconfigurable processing nodes • an evolutionary algorithm as AE that searches for candidate configurations of the array using a set of computational functionalities for the nodes available in a run time accessible library • a hardware RE that exploits native DPR capabilities of FPGAs and makes an efficient use of the available reconfigurable resources of the device to change the behaviour of the CE at run time • a library of reconfigurable processing elements featured by position-independent partial bitstreams used as the set of available configurations for the processing nodes of the array Main contributions of this thesis can be summarised in the following list. • An FPGA-based evolvable platform for parametric and structural self-adaptation of embedded systems composed of a Computing Engine, an evolutionary Adaptation Engine and a Reconfiguration Engine. This platform is further developed and tailored for both parametric and structural self-adaptation. • Regarding parametric self-adaptation, main contributions are: – A CE adaptable through reconfigurable registers that enables parametric adaptation of the coefficients of an adaptive hardware implementation of a DWT core. – An AE based on an Evolutionary Algorithm specifically developed for numerical optimisation applied to wavelet filter coefficients in resource constrained embedded systems. – A run-time self-adaptive DWT IP core for embedded systems that allows for online optimisation of transform performance for image compression for specific deployment environments characterised by different types of input signals. – A software model and hardware implementation of a tool for the automatic, evolutionary construction of custom wavelet transforms. • Lastly, regarding structural self-adaptation, main contributions are: – A CE adaptable through native FPGA fabric reconfiguration featured by a two dimensional systolic array template of reconfigurable processing nodes. Different processing behaviours can be automatically mapped in the array by using a library of simple reconfigurable processing elements. – Definition of a library of such processing elements suited for autonomous runtime synthesis of different image processing tasks. – Efficient incorporation of DPR in EHW systems, overcoming main drawbacks from the previous approach of virtual reconfigurable circuits. Implementation details for both approaches are also originally compared in this work. – A fault tolerant, self-healing platform that enables online functional recovery in hazardous environments. The platform has been characterised from a fault tolerance perspective: fault models at FPGA CLB level and processing elements level are proposed, and using the RE, a systematic fault analysis for one fault in every processing element and for two accumulated faults is done. – A dynamic filtering quality platform that permits on-line adaptation to different types of noise and different computing behaviours considering the available computing resources. On one side, non-destructive filters are evolved, enabling scalable cascaded filtering schemes; and on the other, size-scalable filters are also evolved considering dynamically changing computational filtering requirements. This dissertation is organized in four parts and nine chapters. First part contains chapter 1, the introduction to and motivation of this PhD work. Following, the reference framework in which this dissertation is framed is analysed in the second part: chapter 2 features an introduction to the notions of self-adaptation and autonomic computing as a more general research field to the very specific one of this work; chapter 3 introduces evolutionary computation as the technique to drive adaptation; chapter 4 analyses platforms for reconfigurable computing as the technology to hold self-adaptive hardware; and finally chapter 5 defines, classifies and surveys the field of Evolvable Hardware. Third part of the work follows, which contains the proposal, development and results obtained: while chapter 6 contains an statement of the thesis goals and the description of the proposal as a whole, chapters 7 and 8 address parametric and structural self-adaptation, respectively. Finally, chapter 9 in part 4 concludes the work and describes future research paths.

Measurement of plasma electron density generated in an experiment of laser shock processing, utilizing the H­α­-line

In this work we have realized plasma diagnosis produced by Laser (LPP), by means of emission spectroscopy in a Laser Shock Processing (LSP). The LSP has been proposed as an alternative technology, competitive with classical surface treatments. The ionic species present in the plasma together with electron density and its temperature provide significant indicators of the degree of surface effect of the treated material. In order to analyze these indicators, we have realized spectroscopic studies of optical emission in the laser-generated plasmas in different situations. We have worked focusing on an aluminum sample (Al2024) in air and/or in LSP conditions (water flow) a Q-switched laser of Nd:YAG (λ = 1.06 μm, 10 ns of pulse duration, running at 10 Hz repetition rate). The pulse energy was set at 2,5 J per pulse. The electron density has been measured using, in every case, the Stark broadening of H Balmer α line (656.27 nm). In the case of the air, this measure has been contrasted with the value obtained with the line of 281.62 nm of Al II. Special attention has been paid to the self-absorption of the spectral lines used. The measures were realized with different delay times after the pulse of the laser (1–8 μs) and with a time window of 1 μs. In LSP the electron density obtained was between 1017 cm−3 for the shortest delays (4–6 μs), and 1016 cm−3 for the greatest delays (7,8 μs).

Loss of protein kinase C inhibition in the β-T594M variant of the amiloride-sensitive Na+ channel

We previously reported the presence of a novel variant (β-T594M) of the amiloride-sensitive Na+ channel (ASSC) in which the threonine residue at position 594 in the β-subunit has been replaced by a methionine residue. Electrophysiological studies of the ASSC on Epstein–Barr virus (EBV)-transformed lymphocytes carrying this variant showed that the 8-(4-chlorophenylthio) adenosine 3′:5′-cyclic monophosphate (8cpt-cAMP)-induced responses were enhanced when compared to wild-type EBV-transformed lymphocytes. Furthermore, in wild-type EBV-transformed cells, the 8cpt-cAMP-induced response was totally blocked by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). This inhibitory effect of PMA was blocked by a protein kinase C inhibitor, chelerythrine. We now have identified individuals who are homozygous for this variant, and showed that PMA had no effect on the 8cpt-cAMP-induced responses in the EBV-transformed lymphocytes from such individuals. Cells heterozygous for this variant showed mixed responses to PMA, with the majority of cells partially inhibited by PMA. Our results demonstrate that an alteration in a single amino acid residue in the β-subunit of the ASSC can lead to a total loss of inhibition to PMA, and establish the β-subunit as having an important role in conferring a regulatory effect on the ASSC of lymphocytes.

Context-sensitive synaptic plasticity and temporal-to-spatial transformations in hippocampal slices

Hippocampal slices are used to show that, as a temporal input pattern of activity flows through a neuronal layer, a temporal-to-spatial transformation takes place. That is, neurons can respond selectively to the first or second of a pair of input pulses, thus transforming different temporal patterns of activity into the activity of different neurons. This is demonstrated using associative long-term potentiation of polysynaptic CA1 responses as an activity-dependent marker: by depolarizing a postsynaptic CA1 neuron exclusively with the first or second of a pair of pulses from the dentate gyrus, it is possible to “tag” different subpopulations of CA3 neurons. This technique allows sampling of a population of neurons without recording simultaneously from multiple neurons. Furthermore, it reflects a biologically plausible mechanism by which single neurons may develop selective responses to time-varying stimuli and permits the induction of context-sensitive synaptic plasticity. These experimental results support the view that networks of neurons are intrinsically able to process temporal information and that it is not necessary to invoke the existence of internal clocks or delay lines for temporal processing on the time scale of tens to hundreds of milliseconds.

High-frequency, spin-label EPR of nonaxial lipid ordering and motion in cholesterol-containing membranes

The EPR spectra of spin-labeled lipid chains in fully hydrated bilayer membranes of dimyristoyl phosphatidylcholine containing 40 mol % of cholesterol have been studied in the liquid-ordered phase at a microwave radiation frequency of 94 GHz. At such high field strengths, the spectra should be optimally sensitive to lateral chain ordering that is expected in the formation of in-plane domains. The high-field EPR spectra from random dispersions of the cholesterol-containing membranes display very little axial averaging of the nitroxide g-tensor anisotropy for lipids spin labeled toward the carboxyl end of the sn-2 chain (down to the 8-C atom). For these positions of labeling, anisotropic 14N-hyperfine splittings are resolved in the gzz and gyy regions of the nonaxial EPR spectra. For positions of labeling further down the lipid chain, toward the terminal methyl group, the axial averaging of the spectral features systematically increases and is complete at the 14-C atom position. Concomitantly, the time-averaged 〈Azz〉 element of the 14N-hyperfine tensor decreases, indicating that the axial rotation at the terminal methyl end of the chains arises from correlated torsional motions about the bonds of the chain backbone, the dynamics of which also give rise to a differential line broadening of the 14N-hyperfine manifolds in the gzz region of the spectrum. These results provide an indication of the way in which lateral ordering of lipid chains in membranes is induced by cholesterol.

Genetic dissection of dome formation in a mammary cell line: Identification of two genes with opposing action

In this work, we extend the study of the genes controlling the formation of domes in the rat mammary cell line LA7 under the influence of DMSO. The role of the rat8 gene has already been demonstrated. We have now studied two additional genes. The first, called 133, is the rat ortholog of the human epithelial membrane protein 3 (EMP3), a member of the peripheral myelin protein 22 (PMP22)/EMP/lens-specific membrane protein 20 (MP20) gene family that encodes for tetratransmembrane proteins; it is expressed in the LA7 line in the absence of DMSO but not in its presence. The second gene is the β subunit of the amiloride-sensitive Na+ channel. Studies with antisense oligonucleotides show that the formation of domes is under the control of all three genes: the expression of rat8 is required for both their formation and their persistence; the expression of the Na+ channel β subunit is required for their formation; and the expression of gene 133 blocks the expression of the Na+ channel genes, thus preventing formation of the domes. The formation of these structures is also accompanied by the expression of α6β1 integrin, followed by that of E-cadherin and cytokeratin 8. It appears, therefore, that dome formation requires the activity of the Na+ channel and the rat8-encoded protein and is under the negative control of gene 133. DMSO induces dome formation by blocking this control.

A conserved serine-rich stretch in the glutamate transporter family forms a substrate-sensitive reentrant loop

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft. The proteins belong to a large family of secondary transporters, which includes bacterial glutamate transporters. The C-terminal half of the glutamate transporters is well conserved and thought to contain the translocation path and the binding sites for substrate and coupling ions. A serine-rich sequence motif in this part of the proteins is located in a putative intracellular loop. Cysteine-scanning mutagenesis was applied to this loop in the glutamate transporter GltT of Bacillus stearothermophilus. The loop was found to be largely intracellular, but three consecutive positions in the conserved serine-rich motif (S269, S270, and E271) are accessible from both sides of the membrane. Single-cysteine mutants in the serine-rich motif were still capable of glutamate transport, but modification with N-ethylmaleimide blocked the transport activity in six mutants (T267C, A268C, S269C, S270C, E271C, and T272C). Two milimolars l-glutamate effectively protected against the modification of the cysteines at position 269–271 from the periplasmic side of the membrane but was unable to protect cysteine modification from the cytoplasmic side of the membrane. The results indicate that the conserved serine-rich motif in the glutamate transporter forms a reentrant loop, a structure that is found in several ion channels but is unusual for transporter proteins. The reentrant loop is of crucial importance for the function of the glutamate transporter.

Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position

Acyl-acyl carrier protein (ACP) desaturases introduce double bonds at specific positions in fatty acids of defined chain lengths and are one of the major determinants of the monounsaturated fatty acid composition of vegetable oils. Mutagenesis studies were conducted to determine the structural basis for the substrate and double bond positional specificities displayed by acyl-ACP desaturases. By replacement of specific amino acid residues in a Δ6-palmitoyl (16:0)-ACP desaturase with their equivalents from a Δ9-stearoyl (18:0)-ACP desaturase, mutant enzymes were identified that have altered fatty acid chain-length specificities or that can insert double bonds into either the Δ6 or Δ9 positions of 16:0- and 18:0-ACP. Most notably, by replacement of five amino acids (A181T/A200F/S205N/L206T/G207A), the Δ6-16:0-ACP desaturase was converted into an enzyme that functions principally as a Δ9-18:0-ACP desaturase. Many of the determinants of fatty acid chain-length specificity in these mutants are found in residues that line the substrate binding channel as revealed by x-ray crystallography of the Δ9-18:0-ACP desaturase. The crystallographic model of the active site is also consistent with the diverged activities associated with naturally occurring variant acyl-ACP desaturases. In addition, on the basis of the active-site model, a Δ9-18:0-ACP desaturase was converted into an enzyme with substrate preference for 16:0-ACP by replacement of two residues (L118F/P179I). These results demonstrate the ability to rationally modify acyl-ACP desaturase activities through site-directed mutagenesis and represent a first step toward the design of acyl-ACP desaturases for the production of novel monounsaturated fatty acids in transgenic oilseed crops.

Effect of Carbohydrate Position on Lysosomal Transport of Procathepsin L

To study the role of carbohydrate in lysosomal protein transport, we engineered two novel glycosylation signals (Asn-X-Ser/Thr) into the cDNA of human procathepsin L, a lysosomal acid protease. We constructed six mutant cDNAs encoding glycosylation signals at mutant sites Asn-138, Asn-175, or both sites together, in the presence or absence of the wild-type Asn-204 site. We stably transfected wild-type and mutant cDNAs into NIH3T3 mouse fibroblasts and then used species-specific antibodies to determine the glycosylation status, phosphorylation, localization, and transport kinetics of recombinant human procathepsin L containing one, two, or three glycosylation sites. Both novel glycosylation sites were capable of being glycosylated, although Asn-175 was utilized only 30–50% of the time. Like the wild-type glycosylation at Asn-204, carbohydrates at Asn-138 and Asn-175 were completely sensitive to endoglycosidase H, and they were phosphorylated. Mutant proteins containing two carbohydrates were capable of being delivered to lysosomes, but there was not a consistent relationship between the efficiency of lysosomal delivery and carbohydrate content of the protein. Pulse-chase labeling revealed a unique biosynthetic pattern for proteins carrying the Asn-175 glycosylation sequence. Whereas wild-type procathepsin L and mutants bearing carbohydrate at Asn-138 appeared in lysosomes by about 60 min, proteins with carbohydrate at Asn-175 were processed to a lysosome-like polypeptide within 15 min. Temperature shift, brefeldin A, and NH4Cl experiments suggested that the rapid processing did not occur in the endoplasmic reticulum and that Asn-175 mutants could interact with the mannose 6-phosphate receptor. Taken together, our results are consistent with the interpretation that Asn-175 carbohydrate confers rapid transport to lysosomes. We may have identified a recognition domain in procathepsin L that is important for its interactions with the cellular transport machinery.

Purification of the β-cell glucose-sensitive factor that transactivates the insulin gene differentially in normal and transformed islet cells

The β cell-specific glucose-sensitive factor (GSF), which binds the A3 motif of the rat I and human insulin promoters, is modulated by extracellular glucose. A single mutation in the GSF binding site of the human insulin promoter abolishes the stimulation by high glucose only in normal islets, supporting the suggested physiological role of GSF in the glucose-regulated expression of the insulin gene. GSF binding activity was observed in all insulin-producing cells. We have therefore purified this activity from the rat insulinoma RIN and found that a single polypeptide of 45 kDa was responsible for DNA binding. Its amino acid sequence, determined by microsequencing, provided direct evidence that GSF corresponds to insulin promoter factor 1 (IPF-1; also known as PDX-1) and that, in addition to its essential roles in development and differentiation of pancreatic islets and in β cell-specific gene expression, it functions as mediator of the glucose effect on insulin gene transcription in differentiated β cells. The human cDNA coding for GSF/IPF-1 has been cloned, its cell and tissue distribution is described. Its expression in the glucagon-producing cell line αTC1 transactivates the wild-type human insulin promoter more efficiently than the mutated construct. It is demonstrated that high levels of ectopic GSF/IPF-1 inhibit the expression of the human insulin gene in normal islets, but not in transformed βTC1 cells. These results suggest the existence of a control mechanism, such as requirement for a coactivator of GSF/IPF-1, which may be present in limiting amounts in normal as opposed to transformed β cells.

Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines

Induction of wild-type p53 in the ECV-304 bladder carcinoma cell line by infection with a p53 recombinant adenovirus (Ad5CMV-p53) resulted in extensive apoptosis and eventual death of nearly all of the cells. As a strategy to determine the molecular events important to p53-mediated apoptosis in these transformed cells, ECV-304 cells were selected for resistance to p53 by repeated infections with Ad5CMV-p53. We compared the expression of 5,730 genes in p53-resistant (DECV) and p53-sensitive ECV-304 cells by reverse transcription–PCR, Northern blotting, and DNA microarray analysis. The expression of 480 genes differed by 2-fold or more between the two p53-infected cell lines. A number of potential targets for p53 were identified that play roles in cell cycle regulation, DNA repair, redox control, cell adhesion, apoptosis, and differentiation. Proline oxidase, a mitochondrial enzyme involved in the proline/pyrroline-5-carboxylate redox cycle, was up-regulated by p53 in ECV but not in DECV cells. Pyrroline-5-carboxylate (P5C), a proline-derived metabolite generated by proline oxidase, inhibited the proliferation and survival of ECV-304 and DECV cells and induced apoptosis in both cell lines. A recombinant proline oxidase protein tagged with a green fluorescent protein at the amino terminus localized to mitochondria and induced apoptosis in p53-null H1299 non-small cell lung carcinoma cells. The results directly implicate proline oxidase and the proline/P5C pathway in p53-induced growth suppression and apoptosis.

An experimental test for lineage-specific position effects on alcohol dehydrogenase (Adh) genes in Drosophila

Independent transgene insertions differ in expression based on their location in the genome; these position effects are of interest because they reflect the influence of genome organization on gene regulation. Position effects also represent potentially insurmountable obstacles to the rigorous functional comparison of homologous genes from different species because (i) quantitative variation in expression of each gene across genomic positions (generalized position effects, or GPEs) may overwhelm differences between the genes of interest, or (ii) divergent genes may be differentially sensitive to position effects, reflecting unique interactions between each gene and its genomic milieu (lineage-specific position effects, or LSPEs). We have investigated both types of position-effect variation by applying our method of transgene coplacement, which allows comparisons of transgenes in the same position in the genome of Drosophila melanogaster. Here we report an experimental test for LSPE in Drosophila. The alcohol dehydrogenase (Adh) genes of D. melanogaster and Drosophila affinidisjuncta differ in both tissue distribution and amounts of ADH activity. Despite this striking regulatory divergence, we found a very high correlation in overall ADH activity between the genes of the two species when placed in the same genomic position as assayed in otherwise Adh-null adults and larvae. These results argue against the influence of LSPE for these sequences, although the effects of GPE are significant. Our new findings validate the coplacement approach and show that it greatly magnifies the power to detect differences in expression between transgenes. Transgene coplacement thus dramatically extends the range of functional and evolutionary questions that can be addressed by transgenic technology.

Uncoupling proteins 2 and 3 are highly active H+ transporters and highly nucleotide sensitive when activated by coenzyme Q (ubiquinone)

Based on the discovery of coenzyme Q (CoQ) as an obligatory cofactor for H+ transport by uncoupling protein 1 (UCP1) [Echtay, K. S., Winkler, E. & Klingenberg, M. (2000) Nature (London) 408, 609–613] we show here that UCP2 and UCP3 are also highly active H+ transporters and require CoQ and fatty acid for H+ transport, which is inhibited by low concentrations of nucleotides. CoQ is proposed to facilitate injection of H+ from fatty acid into UCP. Human UCP2 and 3 expressed in Escherichia coli inclusion bodies are solubilized, and by exchange of sarcosyl against digitonin, nucleotide binding as measured with 2′-O-[5-(dimethylamino)naphthalene-1-sulfonyl]-GTP can be restored. After reconstitution into vesicles, Cl− but no H+ are transported. The addition of CoQ initiates H+ transport in conjunction with fatty acids. This increase is fully sensitive to nucleotides. The rates are as high as with reconstituted UCP1 from mitochondria. Maximum activity is at a molar ratio of 1:300 of CoQ:phospholipid. In UCP2 as in UCP1, ATP is a stronger inhibitor than ADP, but in UCP3 ADP inhibits more strongly than ATP. Thus UCP2 and UCP3 are regulated differently by nucleotides, in line with their different physiological contexts. These results confirm the regulation of UCP2 and UCP3 by the same factors CoQ, fatty acids, and nucleotides as UCP1. They supersede reports that UCP2 and UCP3 may not be H+ transporters.

A human myeloma cell line suitable for the generation of human monoclonal antibodies

Ever since monoclonal antibodies were produced in 1975 with mouse myeloma cells there has been interest in developing human myeloma cultures for the production of monoclonal antibodies. However, despite multiple attempts, no human myeloma line suitable for hybridoma production has been described. Here we report the derivation of a hypoxanthine–aminopterin–thymidine-sensitive and ouabain-resistant human myeloma cell line (Karpas 707H) that contains unique genetic markers. We show that this line is useful for the generation of stable human hybridomas. It can easily be fused with ouabain-sensitive Epstein–Barr virus-transformed cells as well as with fresh tonsil and blood lymphocytes, giving rise to stable hybrids that continuously secrete very large quantities of human immunoglobulins. The derived hybrids do not lose immunoglobulin secretion over many months of continuous growth. The availability of this cell line should enable the in vitro immortalization of human antibody-producing B cells that are formed in vivo. The monoclonal antibodies produced may have advantages in immunotherapy.