The Outer Chloroplast Envelope Protein OEP16-1 for Plastid Import of NADPH:Protochlorophyllide Oxidoreductase A in Arabidopsis thaliana

The outer plastid envelope protein OEP16-1 was previously identified as an amino acid-selective channel protein and translocation pore for NADPH:protochlorophyllide oxidoreductase A (PORA). Reverse genetic approaches used to dissect these mutually not exclusive functions of OEP16-1 in planta have led to descriptions of different phenotypes resulting from the presence of several mutant lines in the SALK_024018 seed stock. In addition to the T-DNA insertion in the AtOEP16-1 gene, lines were purified that contain two additional T-DNA insertions and as yet unidentified point mutations. In a first attempt to resolve the genetic basis of four different lines in the SALK_024018 seed stock, we used genetic transformation with the OEP16-1 cDNA and segregation analyses after crossing out presumed point mutations. We show that AtOEP16-1 is involved in PORA precursor import and by virtue of this activity confers photoprotection onto etiolated seedlings during greening

A Web Implementation of A Generalized NEP

The Networks of Evolutionary Processors (NEPs) are computing mechanisms directly inspired from the behavior of cell populations more specifically the point mutations in DNA strands. These mechanisms are been used for solving NP-complete problems by means of a parallel computation postulation. This paper describes an implementation of the basic model of NEP using Web technologies and includes the possibility of designing some of the most common variants of it by means the use of the web page design which eases the configuration of a given problem. It is a system intended to be used in a multicore processor in order to benefit from the multi thread use.

Implementation of the Heated Pulsed Theory using actively heated fiber optics: measurements of soil volumetric water content and soil volumetric heat capacity

Existe una creciente necesidad de hacer el mejor uso del agua para regadío. Una alternativa eficiente consiste en la monitorización del contenido volumétrico de agua (θ), utilizando sensores de humedad. A pesar de existir una gran diversidad de sensores y tecnologías disponibles, actualmente ninguna de ellas permite obtener medidas distribuidas en perfiles verticales de un metro y en escalas laterales de 0.1-1,000 m. En este sentido, es necesario buscar tecnologías alternativas que sirvan de puente entre las medidas puntuales y las escalas intermedias. Esta tesis doctoral se basa en el uso de Fibra Óptica (FO) con sistema de medida de temperatura distribuida (DTS), una tecnología alternativa de reciente creación que ha levantado gran expectación en las últimas dos décadas. Específicamente utilizamos el método de fibra calentada, en inglés Actively Heated Fiber Optic (AHFO), en la cual los cables de Fibra Óptica se utilizan como sondas de calor mediante la aplicación de corriente eléctrica a través de la camisa de acero inoxidable, o de un conductor eléctrico simétricamente posicionado, envuelto, alrededor del haz de fibra óptica. El uso de fibra calentada se basa en la utilización de la teoría de los pulsos de calor, en inglés Heated Pulsed Theory (HPP), por la cual el conductor se aproxima a una fuente de calor lineal e infinitesimal que introduce calor en el suelo. Mediante el análisis del tiempo de ocurrencia y magnitud de la respuesta térmica ante un pulso de calor, es posible estimar algunas propiedades específicas del suelo, tales como el contenido de humedad, calor específico (C) y conductividad térmica. Estos parámetros pueden ser estimados utilizando un sensor de temperatura adyacente a la sonda de calor [método simple, en inglés single heated pulsed probes (SHPP)], ó a una distancia radial r [método doble, en inglés dual heated pulsed probes (DHPP)]. Esta tesis doctoral pretende probar la idoneidad de los sistemas de fibra óptica calentada para la aplicación de la teoría clásica de sondas calentadas. Para ello, se desarrollarán dos sistemas FO-DTS. El primero se sitúa en un campo agrícola de La Nava de Arévalo (Ávila, España), en el cual se aplica la teoría SHPP para estimar θ. El segundo sistema se desarrolla en laboratorio y emplea la teoría DHPP para medir tanto θ como C. La teoría SHPP puede ser implementada con fibra óptica calentada para obtener medidas distribuidas de θ, mediante la utilización de sistemas FO-DTS y el uso de curvas de calibración específicas para cada suelo. Sin embargo, la mayoría de aplicaciones AHFO se han desarrollado exclusivamente en laboratorio utilizando medios porosos homogéneos. En esta tesis se utiliza el programa Hydrus 2D/3D para definir tales curvas de calibración. El modelo propuesto es validado en un segmento de cable enterrado en una instalación de fibra óptica y es capaz de predecir la respuesta térmica del suelo en puntos concretos de la instalación una vez que las propiedades físicas y térmicas de éste son definidas. La exactitud de la metodología para predecir θ frente a medidas puntuales tomadas con sensores de humedad comerciales fue de 0.001 a 0.022 m3 m-3 La implementación de la teoría DHPP con AHFO para medir C y θ suponen una oportunidad sin precedentes para aplicaciones medioambientales. En esta tesis se emplean diferentes combinaciones de cables y fuentes emisoras de calor, que se colocan en paralelo y utilizan un rango variado de espaciamientos, todo ello en el laboratorio. La amplitud de la señal y el tiempo de llegada se han observado como funciones del calor específico del suelo. Medidas de C, utilizando esta metodología y ante un rango variado de contenidos de humedad, sugirieron la idoneidad del método, aunque también se observaron importantes errores en contenidos bajos de humedad de hasta un 22%. La mejora del método requerirá otros modelos más precisos que tengan en cuenta el diámetro del cable, así como la posible influencia térmica del mismo. ABSTRACT There is an increasing need to make the most efficient use of water for irrigation. A good approach to make irrigation as efficient as possible is to monitor soil water content (θ) using soil moisture sensors. Although, there is a broad range of different sensors and technologies, currently, none of them can practically and accurately provide vertical and lateral moisture profiles spanning 0-1 m depth and 0.1-1,000 m lateral scales. In this regard, further research to fulfill the intermediate scale and to bridge single-point measurement with the broaden scales is still needed. This dissertation is based on the use of Fiber Optics with Distributed Temperature Sensing (FO-DTS), a novel approach which has been receiving growing interest in the last two decades. Specifically, we employ the so called Actively Heated Fiber Optic (AHFO) method, in which FO cables are employed as heat probe conductors by applying electricity to the stainless steel armoring jacket or an added conductor symmetrically positioned (wrapped) about the FO cable. AHFO is based on the classic Heated Pulsed Theory (HPP) which usually employs a heat probe conductor that approximates to an infinite line heat source which injects heat into the soil. Observation of the timing and magnitude of the thermal response to the energy input provide enough information to derive certain specific soil thermal characteristics such as the soil heat capacity, soil thermal conductivity or soil water content. These parameters can be estimated by capturing the soil thermal response (using a thermal sensor) adjacent to the heat source (the heating and the thermal sources are mounted together in the so called single heated pulsed probe (SHPP)), or separated at a certain distance, r (dual heated pulsed method (DHPP) This dissertation aims to test the feasibility of heated fiber optics to implement the HPP theory. Specifically, we focus on measuring soil water content (θ) and soil heat capacity (C) by employing two types of FO-DTS systems. The first one is located in an agricultural field in La Nava de Arévalo (Ávila, Spain) and employ the SHPP theory to estimate θ. The second one is developed in the laboratory using the procedures described in the DHPP theory, and focuses on estimating both C and θ. The SHPP theory can be implemented with actively heated fiber optics (AHFO) to obtain distributed measurements of soil water content (θ) by using reported soil thermal responses in Distributed Temperature Sensing (DTS) and with a soil-specific calibration relationship. However, most reported AHFO applications have been calibrated under laboratory homogeneous soil conditions, while inexpensive efficient calibration procedures useful in heterogeneous soils are lacking. In this PhD thesis, we employ the Hydrus 2D/3D code to define these soil-specific calibration curves. The model is then validated at a selected FO transect of the DTS installation. The model was able to predict the soil thermal response at specific locations of the fiber optic cable once the surrounding soil hydraulic and thermal properties were known. Results using electromagnetic moisture sensors at the same specific locations demonstrate the feasibility of the model to detect θ within an accuracy of 0.001 to 0.022 m3 m-3. Implementation of the Dual Heated Pulsed Probe (DPHP) theory for measurement of volumetric heat capacity (C) and water content (θ) with Distributed Temperature Sensing (DTS) heated fiber optic (FO) systems presents an unprecedented opportunity for environmental monitoring. We test the method using different combinations of FO cables and heat sources at a range of spacings in a laboratory setting. The amplitude and phase-shift in the heat signal with distance was found to be a function of the soil volumetric heat capacity (referred, here, to as Cs). Estimations of Cs at a range of θ suggest feasibility via responsiveness to the changes in θ (we observed a linear relationship in all FO combinations), though observed bias with decreasing soil water contents (up to 22%) was also reported. Optimization will require further models to account for the finite radius and thermal influence of the FO cables, employed here as “needle probes”. Also, consideration of the range of soil conditions and cable spacing and jacket configurations, suggested here to be valuable subjects of further study and development.

Complejidad Esencial: Un argumento por la complementariedad entre ciencia y sensibilidad = Essential Complexity: An argument for complementarity between science and sensibility

Esta investigación recoge un cúmulo de intereses en torno a un modo de generar arquitectura muy específico: La producción de objetos con una forma subyacente no apriorística. Los conocimientos expuestos se apoyan en condiciones del pensamiento reciente que impulsan la ilusión por alimentar la fuente creativa de la arquitectura con otros campos del saber. Los tiempos del conocimiento animista sensible y el conocimiento objetivo de carácter científico son correlativos en la historia pero casi nunca han sido sincrónicos. Representa asimismo un intento por aunar los dos tipos de conocimiento retomando la inercia que ya se presentía a comienzos del siglo XX. Se trata por tanto, de un ensayo sobre la posible anulación de la contraposición entre estos dos mundos para pasar a una complementariedad entre ambos en una sola visión conjunta compartida. Como meta final de esta investigación se presenta el desarrollo de un sistema crítico de análisis para los objetos arquitectónicos que permita una diferenciación entre aquellos que responden a los problemas de manera completa y sincera y aquellos otros que esconden, bajo una superficie consensuada, la falta de un método resolutivo de la complejidad en el presente creativo. La Investigación observa tres grupos de conocimiento diferenciados agrupados en sus capítulos correspondientes: El primer capítulo versa sobre el Impulso Creador. En él se define la necesidad de crear un marco para el individuo creador, aquel que independientemente de las fuerzas sociales del momento presiente que existe algo más allá que está sin resolver. Denominamos aquí “creador rebelde” a un tipo de personaje reconocible a lo largo de la Historia como aquel capaz de reconocer los cambios que ese operan en su presente y que utiliza para descubrir lo nuevo y acercarse algo al origen creativo. En el momento actual ese tipo de personaje es el que intuye o ya ha intuido hace tiempo la existencia de una complejidad creciente no obviable en el pensamiento de este tiempo. El segundo capítulo desarrolla algunas Propiedades de Sistemas de actuación creativa. En él se muestra una investigación que desarrolla un marco de conocimientos científicos muy específicos de nuestro tiempo que la arquitectura, de momento, no ha absorbido ni refleja de manera directa en su manera de crear. Son temas de presencia casi ya mundana en la sociedad pero que se resisten a ser incluidos en los procesos creativos como parte de la conciencia. La mayoría de ellos hablan de precisión, órdenes invisibles, propiedades de la materia o la energía tratados de manera objetiva y apolítica. La meta final supone el acercamiento e incorporación de estos conceptos y propiedades a nuestro mundo sensible unificándolos indisociablemente bajo un solo punto de vista. El último capítulo versa sobre la Complejidad y su capacidad de reducción a lo esencial. Aquí se muestran, a modo de conclusiones, la introducción de varios conceptos para el desarrollo de un sistema crítico hacia la arquitectura de nuestro tiempo. Entre ellos, el de Complejidad Esencial, definido como aquella de carácter inevitable a la hora de responder la arquitectura a los problemas y solicitaciones crecientes a los que se enfrenta en el presente. La Tesis mantiene la importancia de informar sobre la imposibilidad en el estado actual de las cosas de responder de manera sincera con soluciones de carácter simplista y la necesidad, por tanto, de soluciones necesarias de carácter complejo. En este sentido se define asimismo el concepto de Forma Subyacente como herramienta crítica para poder evaluar la respuesta de cada arquitectura y poder tener un sistema y visión crítica sobre lo que es un objeto consistente frente a la situación a la que se enfrenta. Dicha forma subyacente se define como aquella manera de entender conjuntamente y de manera sincrónica aquello que percibimos de manera sensible inseparable de las fuerzas ocultas, creativas, tecnológicas, materiales y energéticas que sustentan la definición y entendimiento de cualquier objeto construido. ABSTRACT This research includes a cluster of interests around a specific way to generate architecture: The production of objects without an a priori underlying form. The knowledge presented is based on current conditions of thought promoting the illusion to feed the creative source of architecture with other fields of knowledge. The sensible animist knowledge and objective scientific knowledge are correlative in history but have rarely been synchronous. This research is also an attempt to combine both types of knowledge to regain the inertia already sensed in the early twentieth century. It is therefore an essay on the annulment of the opposition between these two worlds to move towards complementarities of both in a single shared vision. The ultimate goal of this research is to present the development of a critical analysis system for architectural objects that allows differentiation between those who respond to the problems sincerely and those who hide under an agreed appearance, the lack of a method for solving the complexity of the creative present. The research observes three distinct groups of knowledge contained in their respective chapters: The first chapter deals with the Creative Impulse. In it is defined the need to create a framework for the creative individual who, regardless of the current social forces, forebodes that there is something hidden beyond which is still unresolved. We define the "rebel creator" as a kind of person existing throughout history who is able to recognize the changes operating in its present and use them to discover something new and get closer to the origin of creation. At present, this type of character is the one who intuits the existence of a non obviable increasing complexity in society and thought. The second chapter presents some systems, and their properties, for creative performance. It describes the development of a framework composed of current scientific knowledge that architecture has not yet absorbed or reflected directly in her procedures. These are issues of common presence in society but are still reluctant to be included in the creative processes even if they already belong to the collective consciousness. Most of them talk about accuracy, invisible orders, properties of matter and energy, always treated from an objective and apolitical perspective. The ultimate goal pursues the approach and incorporation of these concepts and properties to the sensible world, inextricably unifying all under a single point of view. The last chapter deals with complexity and the ability to reduce it to the essentials. Here we show, as a conclusion, the introduction of several concepts to develop a critical approach to analyzing the architecture of our time. Among them, the concept of Essential Complexity, defined as one that inevitably arises when architecture responds to the increasing stresses that faces today. The thesis maintains the importance of reporting, in the present state of things, the impossibility to respond openly with simplistic solutions and, therefore, the need for solutions to complex character. In this sense, the concept of Underlying Form is defined as a critical tool to evaluate the response of each architecture and possess a critical system to clarify what is an consistent object facing a certain situation. The underlying form is then defined as a way to synchronously understand what we perceive sensitively inseparable from the hidden forces of creative, technological, material and energetic character that support the definition and understanding of any constructed object.

Comparison of micrometeorological techinques for estimating ammonia emission from covered slurry storage and land spreading of cattle slurry

The micrometeorological mass-balance integrated horizontal flux (IHF) technique has been commonly employed for measuring ammonia (NH3) emissions inon-field experiments. However, the inverse-dispersion modeling technique, such as the backward Lagrangian stochastic (bLS) modeling approach, is currently highlighted as offering flexibility in plot design and requiring a minimum number of samplers (Ro et al., 2013). The objective of this study was to make a comparison between the bLS technique with the IHF technique for estimating NH3 emission from flexible bag storage and following landspreading of dairy cattle slurry. Moreover, considering that NH3 emission in storage could have been non uniform, the effect on bLS estimates of a single point and multiple downwind concentration measurements was tested, as proposed by Sanz et al. (2010).

Modelo de computación conexionista inspirado en las redes de procesadores evolutivos y su aprendizaje

La informática teórica es una disciplina básica ya que la mayoría de los avances en informática se sustentan en un sólido resultado de esa materia. En los últimos a~nos debido tanto al incremento de la potencia de los ordenadores, como a la cercanía del límite físico en la miniaturización de los componentes electrónicos, resurge el interés por modelos formales de computación alternativos a la arquitectura clásica de von Neumann. Muchos de estos modelos se inspiran en la forma en la que la naturaleza resuelve eficientemente problemas muy complejos. La mayoría son computacionalmente completos e intrínsecamente paralelos. Por este motivo se les está llegando a considerar como nuevos paradigmas de computación (computación natural). Se dispone, por tanto, de un abanico de arquitecturas abstractas tan potentes como los computadores convencionales y, a veces, más eficientes: alguna de ellas mejora el rendimiento, al menos temporal, de problemas NPcompletos proporcionando costes no exponenciales. La representación formal de las redes de procesadores evolutivos requiere de construcciones, tanto independientes, como dependientes del contexto, dicho de otro modo, en general una representación formal completa de un NEP implica restricciones, tanto sintácticas, como semánticas, es decir, que muchas representaciones aparentemente (sintácticamente) correctas de casos particulares de estos dispositivos no tendrían sentido porque podrían no cumplir otras restricciones semánticas. La aplicación de evolución gramatical semántica a los NEPs pasa por la elección de un subconjunto de ellos entre los que buscar los que solucionen un problema concreto. En este trabajo se ha realizado un estudio sobre un modelo inspirado en la biología celular denominado redes de procesadores evolutivos [55, 53], esto es, redes cuyos nodos son procesadores muy simples capaces de realizar únicamente un tipo de mutación puntual (inserción, borrado o sustitución de un símbolo). Estos nodos están asociados con un filtro que está definido por alguna condición de contexto aleatorio o de pertenencia. Las redes están formadas a lo sumo de seis nodos y, teniendo los filtros definidos por una pertenencia a lenguajes regulares, son capaces de generar todos los lenguajes enumerables recursivos independientemente del grafo subyacente. Este resultado no es sorprendente ya que semejantes resultados han sido documentados en la literatura. Si se consideran redes con nodos y filtros definidos por contextos aleatorios {que parecen estar más cerca a las implementaciones biológicas{ entonces se pueden generar lenguajes más complejos como los lenguajes no independientes del contexto. Sin embargo, estos mecanismos tan simples son capaces de resolver problemas complejos en tiempo polinomial. Se ha presentado una solución lineal para un problema NP-completo, el problema de los 3-colores. Como primer aporte significativo se ha propuesto una nueva dinámica de las redes de procesadores evolutivos con un comportamiento no determinista y masivamente paralelo [55], y por tanto todo el trabajo de investigación en el área de la redes de procesadores se puede trasladar a las redes masivamente paralelas. Por ejemplo, las redes masivamente paralelas se pueden modificar de acuerdo a determinadas reglas para mover los filtros hacia las conexiones. Cada conexión se ve como un canal bidireccional de manera que los filtros de entrada y salida coinciden. A pesar de esto, estas redes son computacionalmente completas. Se pueden también implementar otro tipo de reglas para extender este modelo computacional. Se reemplazan las mutaciones puntuales asociadas a cada nodo por la operación de splicing. Este nuevo tipo de procesador se denomina procesador splicing. Este modelo computacional de Red de procesadores con splicing ANSP es semejante en cierto modo a los sistemas distribuidos en tubos de ensayo basados en splicing. Además, se ha definido un nuevo modelo [56] {Redes de procesadores evolutivos con filtros en las conexiones{ , en el cual los procesadores tan solo tienen reglas y los filtros se han trasladado a las conexiones. Dicho modelo es equivalente, bajo determinadas circunstancias, a las redes de procesadores evolutivos clásicas. Sin dichas restricciones el modelo propuesto es un superconjunto de los NEPs clásicos. La principal ventaja de mover los filtros a las conexiones radica en la simplicidad de la modelización. Otras aportaciones de este trabajo ha sido el dise~no de un simulador en Java [54, 52] para las redes de procesadores evolutivos propuestas en esta Tesis. Sobre el término "procesador evolutivo" empleado en esta Tesis, el proceso computacional descrito aquí no es exactamente un proceso evolutivo en el sentido Darwiniano. Pero las operaciones de reescritura que se han considerado pueden interpretarse como mutaciones y los procesos de filtrado se podrían ver como procesos de selección. Además, este trabajo no abarca la posible implementación biológica de estas redes, a pesar de ser de gran importancia. A lo largo de esta tesis se ha tomado como definición de la medida de complejidad para los ANSP, una que denotaremos como tama~no (considerando tama~no como el número de nodos del grafo subyacente). Se ha mostrado que cualquier lenguaje enumerable recursivo L puede ser aceptado por un ANSP en el cual el número de procesadores está linealmente acotado por la cardinalidad del alfabeto de la cinta de una máquina de Turing que reconoce dicho lenguaje L. Siguiendo el concepto de ANSP universales introducido por Manea [65], se ha demostrado que un ANSP con una estructura de grafo fija puede aceptar cualquier lenguaje enumerable recursivo. Un ANSP se puede considerar como un ente capaz de resolver problemas, además de tener otra propiedad relevante desde el punto de vista práctico: Se puede definir un ANSP universal como una subred, donde solo una cantidad limitada de parámetros es dependiente del lenguaje. La anterior característica se puede interpretar como un método para resolver cualquier problema NP en tiempo polinomial empleando un ANSP de tama~no constante, concretamente treinta y uno. Esto significa que la solución de cualquier problema NP es uniforme en el sentido de que la red, exceptuando la subred universal, se puede ver como un programa; adaptándolo a la instancia del problema a resolver, se escogerín los filtros y las reglas que no pertenecen a la subred universal. Un problema interesante desde nuestro punto de vista es el que hace referencia a como elegir el tama~no optimo de esta red.---ABSTRACT---This thesis deals with the recent research works in the area of Natural Computing {bio-inspired models{, more precisely Networks of Evolutionary Processors first developed by Victor Mitrana and they are based on P Systems whose father is Georghe Paun. In these models, they are a set of processors connected in an underlying undirected graph, such processors have an object multiset (strings) and a set of rules, named evolution rules, that transform objects inside processors[55, 53],. These objects can be sent/received using graph connections provided they accomplish constraints defined at input and output filters processors have. This symbolic model, non deterministic one (processors are not synchronized) and massive parallel one[55] (all rules can be applied in one computational step) has some important properties regarding solution of NP-problems in lineal time and of course, lineal resources. There are a great number of variants such as hybrid networks, splicing processors, etc. that provide the model a computational power equivalent to Turing machines. The origin of networks of evolutionary processors (NEP for short) is a basic architecture for parallel and distributed symbolic processing, related to the Connection Machine as well as the Logic Flow paradigm, which consists of several processors, each of them being placed in a node of a virtual complete graph, which are able to handle data associated with the respective node. All the nodes send simultaneously their data and the receiving nodes handle also simultaneously all the arriving messages, according to some strategies. In a series of papers one considers that each node may be viewed as a cell having genetic information encoded in DNA sequences which may evolve by local evolutionary events, that is point mutations. Each node is specialized just for one of these evolutionary operations. Furthermore, the data in each node is organized in the form of multisets of words (each word appears in an arbitrarily large number of copies), and all the copies are processed in parallel such that all the possible events that can take place do actually take place. Obviously, the computational process just described is not exactly an evolutionary process in the Darwinian sense. But the rewriting operations we have considered might be interpreted as mutations and the filtering process might be viewed as a selection process. Recombination is missing but it was asserted that evolutionary and functional relationships between genes can be captured by taking only local mutations into consideration. It is clear that filters associated with each node allow a strong control of the computation. Indeed, every node has an input and output filter; two nodes can exchange data if it passes the output filter of the sender and the input filter of the receiver. Moreover, if some data is sent out by some node and not able to enter any node, then it is lost. In this paper we simplify the ANSP model considered in by moving the filters from the nodes to the edges. Each edge is viewed as a two-way channel such that the input and output filters coincide. Clearly, the possibility of controlling the computation in such networks seems to be diminished. For instance, there is no possibility to loose data during the communication steps. In spite of this and of the fact that splicing is not a powerful operation (remember that splicing systems generates only regular languages) we prove here that these devices are computationally complete. As a consequence, we propose characterizations of two complexity classes, namely NP and PSPACE, in terms of accepting networks of restricted splicing processors with filtered connections. We proposed a uniform linear time solution to SAT based on ANSPFCs with linearly bounded resources. This solution should be understood correctly: we do not solve SAT in linear time and space. Since any word and auxiliary word appears in an arbitrarily large number of copies, one can generate in linear time, by parallelism and communication, an exponential number of words each of them having an exponential number of copies. However, this does not seem to be a major drawback since by PCR (Polymerase Chain Reaction) one can generate an exponential number of identical DNA molecules in a linear number of reactions. It is worth mentioning that the ANSPFC constructed above remains unchanged for any instance with the same number of variables. Therefore, the solution is uniform in the sense that the network, excepting the input and output nodes, may be viewed as a program according to the number of variables, we choose the filters, the splicing words and the rules, then we assign all possible values to the variables, and compute the formula.We proved that ANSP are computationally complete. Do the ANSPFC remain still computationally complete? If this is not the case, what other problems can be eficiently solved by these ANSPFCs? Moreover, the complexity class NP is exactly the class of all languages decided by ANSP in polynomial time. Can NP be characterized in a similar way with ANSPFCs?

Unificación de los protocolos de multipunto fiable optimizando la escalabilidad y el retardo

Las aplicaciones distribuidas que precisan de un servicio multipunto fiable son muy numerosas, y entre otras es posible citar las siguientes: bases de datos distribuidas, sistemas operativos distribuidos, sistemas de simulación interactiva distribuida y aplicaciones de distribución de software, publicaciones o noticias. Aunque en sus orígenes el dominio de aplicación de tales sistemas distribuidos estaba reducido a una única subred (por ejemplo una Red de Área Local) posteriormente ha surgido la necesidad de ampliar su aplicabilidad a interredes. La aproximación tradicional al problema del multipunto fiable en interredes se ha basado principalmente en los dos siguientes puntos: (1) proporcionar en un mismo protocolo muchas garantías de servicio (por ejemplo fiabilidad, atomicidad y ordenación) y a su vez algunas de éstas en distintos grados, sin tener en cuenta que muchas aplicaciones multipunto que precisan fiabilidad no necesitan otras garantías; y (2) extender al entorno multipunto las soluciones ya adoptadas en el entorno punto a punto sin considerar las características diferenciadoras; y de aquí, que se haya tratado de resolver el problema de la fiabilidad multipunto con protocolos extremo a extremo (protocolos de transporte) y utilizando esquemas de recuperación de errores, centralizados (las retransmisiones se hacen desde un único punto, normalmente la fuente) y globales (los paquetes solicitados se vuelven a enviar al grupo completo). En general, estos planteamientos han dado como resultado protocolos que son ineficientes en tiempo de ejecución, tienen problemas de escalabilidad, no hacen un uso óptimo de los recursos de red y no son adecuados para aplicaciones sensibles al retardo. En esta Tesis se investiga el problema de la fiabilidad multipunto en interredes operando en modo datagrama y se presenta una forma novedosa de enfocar el problema: es más óptimo resolver el problema de la fiabilidad multipunto a nivel de red y separar la fiabilidad de otras garantías de servicio, que pueden ser proporcionadas por un protocolo de nivel superior o por la propia aplicación. Siguiendo este nuevo enfoque se ha diseñado un protocolo multipunto fiable que opera a nivel de red (denominado RMNP). Las características más representativas del RMNP son las siguientes; (1) sigue una aproximación orientada al emisor, lo cual permite lograr un grado muy alto de fiabilidad; (2) plantea un esquema de recuperación de errores distribuido (las retransmisiones se hacen desde ciertos encaminadores intermedios que siempre estarán más cercanos a los miembros que la propia fuente) y de ámbito restringido (el alcance de las retransmisiones está restringido a un cierto número de miembros). Este esquema hace posible optimizar el retardo medio de distribución y disminuir la sobrecarga introducida por las retransmisiones; (3) incorpora en ciertos encaminadores funciones de agregación y filtrado de paquetes de control, que evitan problemas de implosión y reducen el tráfico que fluye hacia la fuente. Con el fin de evaluar el comportamiento del protocolo diseñado, se han realizado pruebas de simulación obteniéndose como principales conclusiones que, el RMNP escala correctamente con el tamaño del grupo, hace un uso óptimo de los recursos de red y es adecuado para aplicaciones sensibles al retardo.---ABSTRACT---There are many distributed applications that require a reliable multicast service, including: distributed databases, distributed operating systems, distributed interactive simulation systems and distribution applications of software, publications or news. Although the application domain of distributed systems of this type was originally confíned to a single subnetwork (for example, a Local Área Network), it later became necessary extend their applicability to internetworks. The traditional approach to the reliable multicast problem in internetworks is based mainly on the following two points: (1) provide a lot of service guarantees in one and the same protocol (for example, reliability, atomicity and ordering) and different levéis of guarantee in some cases, without taking into account that many multicast applications that require reliability do not need other guarantees, and (2) extend solutions adopted in the unicast environment to the multicast environment without taking into account their distinctive characteristics. So, the attempted solutions to the multicast reliability problem were end-to-end protocols (transport protocols) and centralized error recovery schemata (retransmissions made from a single point, normally the source) and global error retrieval schemata (the requested packets are retransmitted to the whole group). Generally, these approaches have resulted in protocols that are inefficient in execution time, have scaling problems, do not make optimum use of network resources and are not suitable for delay-sensitive applications. Here, the multicast reliability problem is investigated in internetworks operating in datagram mode and a new way of approaching the problem is presented: it is better to solve to the multicast reliability problem at network level and sepárate reliability from other service guarantees that can be supplied by a higher protocol or the application itself. A reliable multicast protocol that operates at network level (called RMNP) has been designed on the basis of this new approach. The most representative characteristics of the RMNP are as follows: (1) it takes a transmitter-oriented approach, which provides for a very high reliability level; (2) it provides for an error retrieval schema that is distributed (the retransmissions are made from given intermedíate routers that will always be closer to the members than the source itself) and of restricted scope (the scope of the retransmissions is confined to a given number of members), and this schema makes it possible to optimize the mean distribution delay and reduce the overload caused by retransmissions; (3) some routers include control packet aggregation and filtering functions that prevent implosión problems and reduce the traffic flowing towards the source. Simulation test have been performed in order to evalúate the behaviour of the protocol designed. The main conclusions are that the RMNP scales correctly with group size, makes optimum use of network resources and is suitable for delay-sensitive applications.

Genomic evolution during a 10,000-generation experiment with bacteria

Molecular methods are used widely to measure genetic diversity within populations and determine relationships among species. However, it is difficult to observe genomic evolution in action because these dynamics are too slow in most organisms. To overcome this limitation, we sampled genomes from populations of Escherichia coli evolving in the laboratory for 10,000 generations. We analyzed the genomes for restriction fragment length polymorphisms (RFLP) using seven insertion sequences (IS) as probes; most polymorphisms detected by this approach reflect rearrangements (including transpositions) rather than point mutations. The evolving genomes became increasingly different from their ancestor over time. Moreover, tremendous diversity accumulated within each population, such that almost every individual had a different genetic fingerprint after 10,000 generations. As has been often suggested, but not previously shown by experiment, the rates of phenotypic and genomic change were discordant, both across replicate populations and over time within a population. Certain pivotal mutations were shared by all descendants in a population, and these are candidates for beneficial mutations, which are rare and difficult to find. More generally, these data show that the genome is highly dynamic even over a time scale that is, from an evolutionary perspective, very brief.

Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis

Multiple lipoxygenase sequence alignments and structural modeling of the enzyme/substrate interaction of the cucumber lipid body lipoxygenase suggested histidine 608 as the primary determinant of positional specificity. Replacement of this amino acid by a less-space-filling valine altered the positional specificity of this linoleate 13-lipoxygenase in favor of 9-lipoxygenation. These alterations may be explained by the fact that H608V mutation may demask the positively charged guanidino group of R758, which, in turn, may force an inverse head-to-tail orientation of the fatty acid substrate. The R758L+H608V double mutant exhibited a strongly reduced reaction rate and a random positional specificity. Trilinolein, which lacks free carboxylic groups, was oxygenated to the corresponding (13S)-hydro(pero)xy derivatives by both the wild-type enzyme and the linoleate 9-lipoxygenating H608V mutant. These data indicate the complete conversion of a linoleate 13-lipoxygenase to a 9-lipoxygenating species by a single point mutation. It is hypothesized that H608V exchange may alter the orientation of the substrate at the active site and/or its steric configuration in such a way that a stereospecific dioxygen insertion at C-9 may exclusively take place.

The Ti plasmid increases the efficiency of Agrobacterium tumefaciens as a recipient in virB-mediated conjugal transfer of an IncQ plasmid

The T-DNA transfer apparatus of Agrobacterium tumefaciens mediates the delivery of the T-DNA into plant cells, the transfer of the IncQ plasmid RSF1010 into plant cells, and the conjugal transfer of RSF1010 between Agrobacteria. We show in this report that the Agrobacterium-to-Agrobacterium conjugal transfer efficiencies of RSF1010 increase dramatically if the recipient strain, as well as the donor strain, carries a wild-type Ti plasmid and is capable of vir gene expression. Investigation of possible mechanisms that could account for this increased efficiency revealed that the VirB proteins encoded by the Ti plasmid were required. Although, with the exception of VirB1, all of the proteins that form the putative T-DNA transfer apparatus (VirB1–11, VirD4) are required for an Agrobacterium strain to serve as an RSF1010 donor, expression of only a subset of these proteins is required for the increase in conjugal transfer mediated by the recipient. Specifically, VirB5, 6, 11, and VirD4 are essential donor components but are dispensable for the increased recipient capacity. Defined point mutations in virB9 affected donor and recipient capacities to the same relative extent, suggesting that similar functions of VirB9 are important in both of these contexts.

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.

The crystal structure of human interferon β at 2.2-Å resolution

Type I interferons (IFNs) are helical cytokines that have diverse biological activities despite the fact that they appear to interact with the same receptor system. To achieve a better understanding of the structural basis for the different activities of α and β IFNs, we have determined the crystal structure of glycosylated human IFN-β at 2.2-Å resolution by molecular replacement. The molecule adopts a fold similar to that of the previously determined structures of murine IFN-β and human IFN-α2b but displays several distinct structural features. Like human IFN-α2b, human IFN-β contains a zinc-binding site at the interface of the two molecules in the asymmetric unit, raising the question of functional relevance for IFN-β dimers. However, unlike the human IFN-α2b dimer, in which homologous surfaces form the interface, human IFN-β dimerizes with contact surfaces from opposite sides of the molecule. The relevance of the structure to the effects of point mutations in IFN-β at specific exposed residues is discussed. A potential role of ligand–ligand interactions in the conformational assembly of IFN receptor components is discussed.

Arabidopsis thaliana mutants altered in homologous recombination

Homologous recombination contributes both to the generation of allelic diversity and to the preservation of genetic information. In plants, a lack of suitable experimental material has prevented studies of the regulatory and enzymatic aspects of recombination in somatic and meiotic cells. We have isolated nine Arabidopsis thaliana mutants hypersensitive to x-ray irradiation (xrs) and examined their recombination properties. For the three xrs loci described here, single recessive mutations were found to confer simultaneous hypersensitivities to the DNA-damaging chemicals mitomycin C (MMCs) and/or methyl methanesulfonate (MMSs) and alterations in homologous recombination. Mutant xrs9 (Xrays, MMSs) is reduced in both somatic and meiotic recombination and resembles yeast mutants of the rad52 epistatic group. xrs11 (Xrays, MMCs) is deficient in the x-ray-mediated stimulation of homologous recombination in somatic cells in a manner suggesting a specific signaling defect. xrs4 (Xrays, MMSs, MMCs) has a significant deficiency in somatic recombination, but this is accompanied by meiotic hyper-recombination. A corresponding phenotype has not been reported in other systems and thus this indicates a novel, plant-specific regulatory circuit linking mitotic and meiotic recombination.

Enhanced stability of maize endosperm ADP-glucose pyrophosphorylase is gained through mutants that alter subunit interactions

Temperature lability of ADP-glucose pyrophosphorylase (AGP; glucose-1-phosphate adenylyltransferase; ADP: α-d-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27), a key starch biosynthetic enzyme, may play a significant role in the heat-induced loss in maize seed weight and yield. Here we report the isolation and characterization of heat-stable variants of maize endosperm AGP. Escherichia coli cells expressing wild type (WT) Shrunken2 (Sh2), and Brittle2 (Bt2) exhibit a reduced capacity to produce glycogen when grown at 42°C. Mutagenesis of Sh2 and coexpression with WT Bt2 led to the isolation of multiple mutants capable of synthesizing copious amounts of glycogen at this temperature. An increase in AGP stability was found in each of four mutants examined. Initial characterization revealed that the BT2 protein was elevated in two of these mutants. Yeast two-hybrid studies were conducted to determine whether the mutant SH2 proteins more efficiently recruit the BT2 subunit into tetramer assembly. These experiments showed that replacement of WT SH2 with the heat-stable SH2HS33 enhanced interaction between the SH2 and BT2 subunits. In agreement, density gradient centrifugation of heated and nonheated extracts from WT and one of the mutants, Sh2hs33, identified a greater propensity for heterotetramer dissociation in WT AGP. Sequencing of Sh2hs33 and several other mutants identified a His-to-Tyr mutation at amino acid position 333. Hence, a single point mutation in Sh2 can increase the stability of maize endosperm AGP through enhanced subunit interactions.

Semaphorin 3A growth cone collapse requires a sequence homologous to tarantula hanatoxin

Axonal guidance is key to the formation of neuronal circuitry. Semaphorin 3A (Sema 3A; previously known as semaphorin III, semaphorin D, and collapsin-1), a secreted subtype of the semaphorin family, is an important axonal guidance molecule in vitro and in vivo. The molecular mechanisms of the repellent activity of semaphorins are, however, poorly understood. We have now found that the secreted semaphorins contain a short sequence of high homology to hanatoxin, a tarantula K+ and Ca2+ ion channel blocker. Point mutations in the hanatoxin-like sequence of Sema 3A reduce its capacity to repel embryonic dorsal root ganglion axons. Sema 3A growth cone collapse activity is inhibited by hanatoxin, general Ca2+ channel blockers, a reduction in extracellular or intracellular Ca2+, and a calmodulin inhibitor, but not by K+ channel blockers. Our data support an important role for Ca2+ in mediating the Sema 3A response and suggest that Sema 3A may produce its effects by causing the opening of Ca2+ channels.