Nonlinear seismic behaviour of concrete arch bridges

Arch bridge structural solution has been known for centuries, in fact the simple nature of arch that require low tension and shear strength was an advantage as the simple materials like stone and brick were the only option back in ancient centuries. By the pass of time especially after industrial revolution, the new materials were adopted in construction of arch bridges to reach longer spans. Nowadays one long span arch bridge is made of steel, concrete or combination of these two as "CFST", as the result of using these high strength materials, very long spans can be achieved. The current record for longest arch belongs to Chaotianmen bridge over Yangtze river in China with 552 meters span made of steel and the longest reinforced concrete type is Wanxian bridge which also cross the Yangtze river through a 420 meters span. Today the designer is no longer limited by span length as long as arch bridge is the most applicable solution among other approaches, i.e. cable stayed and suspended bridges are more reasonable if very long span is desired. Like any super structure, the economical and architectural aspects in construction of a bridge is extremely important, in other words, as a narrower bridge has better appearance, it also require smaller volume of material which make the design more economical. Design of such bridge, beside the high strength materials, requires precise structural analysis approaches capable of integrating the combination of material behaviour and complex geometry of structure and various types of loads which may be applied to bridge during its service life. Depend on the design strategy, analysis may only evaluates the linear elastic behaviour of structure or consider the nonlinear properties as well. Although most of structures in the past were designed to act in their elastic range, the rapid increase in computational capacity allow us to consider different sources of nonlinearities in order to achieve a more realistic evaluations where the dynamic behaviour of bridge is important especially in seismic zones where large movements may occur or structure experience P - _ effect during the earthquake. The above mentioned type of analysis is computationally expensive and very time consuming. In recent years, several methods were proposed in order to resolve this problem. Discussion of recent developments on these methods and their application on long span concrete arch bridges is the main goal of this research. Accordingly available long span concrete arch bridges have been studied to gather the critical information about their geometrical aspects and properties of their materials. Based on concluded information, several concrete arch bridges were designed for further studies. The main span of these bridges range from 100 to 400 meters. The Structural analysis methods implemented in in this study are as following: Elastic Analysis: Direct Response History Analysis (DRHA): This method solves the direct equation of motion over time history of applied acceleration or imposed load in linear elastic range. Modal Response History Analysis (MRHA): Similar to DRHA, this method is also based on time history, but the equation of motion is simplified to single degree of freedom system and calculates the response of each mode independently. Performing this analysis require less time than DRHA. Modal Response Spectrum Analysis (MRSA): As it is obvious from its name, this method calculates the peak response of structure for each mode and combine them using modal combination rules based on the introduced spectra of ground motion. This method is expected to be fastest among Elastic analysis. Inelastic Analysis: Nonlinear Response History Analysis (NL-RHA): The most accurate strategy to address significant nonlinearities in structural dynamics is undoubtedly the nonlinear response history analysis which is similar to DRHA but extended to inelastic range by updating the stiffness matrix for every iteration. This onerous task, clearly increase the computational cost especially for unsymmetrical buildings that requires to be analyzed in a full 3D model for taking the torsional effects in to consideration. Modal Pushover Analysis (MPA): The Modal Pushover Analysis is basically the MRHA but extended to inelastic stage. After all, the MRHA cannot solve the system of dynamics because the resisting force fs(u; u_ ) is unknown for inelastic stage. The solution of MPA for this obstacle is using the previously recorded fs to evaluate system of dynamics. Extended Modal Pushover Analysis (EMPA): Expanded Modal pushover is a one of very recent proposed methods which evaluates response of structure under multi-directional excitation using the modal pushover analysis strategy. In one specific mode,the original pushover neglect the contribution of the directions different than characteristic one, this is reasonable in regular symmetric building but a structure with complex shape like long span arch bridges may go through strong modal coupling. This method intend to consider modal coupling while it take same time of computation as MPA. Coupled Nonlinear Static Pushover Analysis (CNSP): The EMPA includes the contribution of non-characteristic direction to the formal MPA procedure. However the static pushovers in EMPA are performed individually for every mode, accordingly the resulted values from different modes can be combined but this is only valid in elastic phase; as soon as any element in structure starts yielding the neutral axis of that section is no longer fixed for both response during the earthquake, meaning the longitudinal deflection unavoidably affect the transverse one or vice versa. To overcome this drawback, the CNSP suggests executing pushover analysis for governing modes of each direction at the same time. This strategy is estimated to be more accurate than MPA and EMPA, moreover the calculation time is reduced because only one pushover analysis is required. Regardless of the strategy, the accuracy of structural analysis is highly dependent on modelling and numerical integration approaches used in evaluation of each method. Therefore the widely used Finite Element Method is implemented in process of all analysis performed in this research. In order to address the study, chapter 2, starts with gathered information about constructed long span arch bridges, this chapter continuous with geometrical and material definition of new models. Chapter 3 provides the detailed information about structural analysis strategies; furthermore the step by step description of procedure of all methods is available in Appendix A. The document ends with the description of results and conclusion of chapter 4.

Dinámicas de ocupación urbana del Anillo Verde metropolitano, desde sus orígenes en el Plan General del Área Metropolitana de Madrid de 1963 hasta el Plan General de Ordenación Urbana de 1997

El Anillo Verde metropolitano, definido por el Plan General de Ordenación Urbana del Área Metropolitana de Madrid en 1963 siguiendo los modelos planteados por la cultura urbanística internacional, como armadura de la estructura urbana del AMM, espacio protagonista dentro del sistema de espacios libres, lugar de uso público destinado al recreo y contacto con la naturaleza de la población madrileña, se convierte en realidad en una reserva de suelo que va entrando en juego motivado por las alianzas entre el poder institucional y la clase social dominante actuando al margen del planeamiento, poniendo en evidencia la escasez de recursos legales y culturales disponibles para la salvaguarda de los intereses comunes y, donde los condicionantes geográficos y naturales del territorio madrileño han influido decisivamente en la especialización funcional y espacial del Área Metropolitana de Madrid. Así pues considerando esta idea como HIPÓTESIS, el objetivo de la TESIS sería demostrarla, para lo cual se hace necesario primero, acotar espacial y temporalmente el objeto de estudio, es decir, del Anillo Verde metropolitano1, segundo, contextualizar histórica y disciplinarmente los presupuestos teóricos que conformaban la idea del Anillo Verde, tercero, reconocer, localizar y documentar las piezas que han ido materializando la ocupación urbana del Anillo Verde, clasificándolas según parámetros temporales, funcionales, urbanísticos y, formales, lo que permite analizar la geografía, uso, instrumentación y forma de su transformación a escala general metropolitana y, cuarto, profundizar a modo de comprobación a escala municipal y urbana en dos escenarios representativos del conjunto metropolitano: el municipio de Pozuelo de Alarcón y el distrito de Hortaleza-Barajas. El contenido del documento se divide en tres bloques, el bloque I, se centra en las bases teóricas, el bloque II sitúa el hilo argumental de la tesis a escala metropolitana y el bloque III comprueba el fenómeno a escala municipal y urbana. De esta forma, se comienza por la comprensión del significado del concepto del Anillo Verde, que va más allá de la dimensión instrumental asignada de límite y contención urbana frente al crecimiento de la ciudad industrial de principios del siglo XX, basada en la descentralización de la ciudad tradicional, para adquirir un significado más complejo, como gran espacio de reserva y salvaguarda de valores naturales y culturales que se expresaban en su territorio y que permitirían alcanzar el equilibrio entre la ciudad y sus habitantes, es decir, entre el hombre y el espacio que habita. Se hace un recorrido por las principales corrientes urbanísticas que se van nutriendo de distintas disciplinas (economía, sociología, geografía, biología, ecología) para plantear teorías que permitieran materializar un nuevo orden urbano según principios de equidad social, económica y ambiental, en una secuencia donde Europa y Estados Unidos realizaban un constante intercambio -el movimiento de la Ciudad Jardín o el Regionalismo, que dieron paso a propuestas como el Greater London o el Gran Berlín, donde la figura del Anillo Verde tenía un papel protagonista, y del que también participaría nuestro país y la ciudad de Madrid, con modelos regionales como el Plan Besteiro y urbanos como el Plan Bidagor, antecedentes directos del Plan General de Ordenación Urbana del Área Metropolitana de Madrid de 1963 que pone en marcha la ordenación del crecimiento metropolitano de Madrid. El hilo argumental de la tesis se organiza en una doble aproximación: un acercamiento a escala metropolitana a partir del reconocimiento del modelo de ciudad definido en los distintos planes generales que acompañaron el desarrollo metropolitano (municipio de Madrid y de los siete términos municipales que rodeaban a este y que tenían suelo destinado a Anillo Verde), haciendo referencia además a las relaciones con el planeamiento regional, concretando en una escala de aproximación municipal que avanza hasta la interpretación urbana detallada. El primer acercamiento tiene lugar en el bloque II y se organiza en tres capítulos. El capítulo 4 se dedica al punto obligado de partida de la geografía local, describiendo las características biofísicas de los terrenos que formaban parte del Anillo Verde, que han marcado históricamente la forma de aprovechamiento del territorio, desde las extensiones de bosques mediterráneos al norte y al oeste continuación del Monte del Pardo, a los distintos tipos de cultivo que se adaptaban al sustrato geológico y la forma del terreno (de las suaves ondulaciones de sedimentos arcósicos al norte a las extensas plataformas arenosas y yesíferas del sur), además de las zonas de huertos aprovechando las depresiones y los cursos de agua (arroyo del Monte Carmelo, arroyo de Valdebebas, arroyo del Quinto, arroyo del Santo, arroyo Butarque, arroyo Meaques y arroyo Pozuelo). Una vez reconocida la realidad física, el capítulo 5, avanza en la descripción de los distintos modelos de ciudad propuestos desde el planeamiento urbanístico, en sus distintas escalas, la regional y la municipal, como respuesta a la coyuntura social, económica y política que ha caracterizado el proceso de ocupación del Anillo Verde al compás de la construcción del AMM. Se han reunido las propuestas de planeamiento municipal de los distintos municipios que disponían de terreno calificado como Anillo Verde: Madrid, Coslada, Getafe, Leganés, Alcorcón, Boadilla del Monte y Pozuelo de Alarcón. Además se han incorporado las distintas propuestas de ordenación territorial que han servido de referencia al planeamiento municipal, en todas sus versiones, desde las sectoriales, de mayor éxito y apoyo institucional, a los distintos intentos de ordenación integral, de mayor complejidad pero de menor calado, precisamente por la dificultad de consenso entre la ordenación física y el desarrollo económico, entre los intereses privados y el beneficio público. El primer horizonte, comienza con la formulación del Plan General de Ordenación Urbana del Área Metropolitana de Madrid de 1963, su desarrollo y la puesta en marcha de los primeros planes municipales en la década de los años setenta, donde se comprueba la necesidad de un marco regional que “ordene” el territorio de forma integral y sirva de referencia a las actuaciones sectoriales que habían marcado el primer desarrollo metropolitano. El segundo, se sitúa dos décadas más tarde con la aprobación del Plan General de Ordenación Urbana de Madrid de 1985 y el conjunto de planes municipales de los términos limítrofes, que siguen su filosofía de austeridad en cuanto a crecimiento territorial. El tercero se inicia en 1997 con la siguiente generación de planes de corte neoliberal que imponen un modelo territorial basado en las grandes operaciones metropolitanas de centralidad, infraestructuras y equipamiento, que consumen de forma indiscriminada la totalidad del territorio madrileño. Será en el último capítulo del segundo bloque (capítulo 6) donde se represente gráficamente a escala metropolitana y se analicen las 229 piezas que han ido colmatando el espacio destinado a Anillo Verde, según los parámetros de estudio, en base a las cuales se plantean las primeras conclusiones generales de la tesis, poniendo de manifiesto que las alianzas entre los agentes soberanos en la construcción de la ciudad y su entorno han trasgredido sucesivamente las determinaciones del Planeamiento en su definición de modelo de ciudad y territorio, acusando la carencia de recursos instrumentales y jurídicos que alentaron el proceso de su desmantelamiento, y revelando la influencia de los condicionantes geográficos y naturales en la especialización funcional y segregación social en el conjunto del Área Metropolitana de Madrid. Se remata el discurso metropolitano con una batería de conclusiones que interpretan el fenómeno de ocupación del anillo de verdor metropolitano confirmando las hipótesis iniciales, reconociendo los valores medioambientales y culturales trasgredidos, sus diversos actores, las numerosas operaciones urbanísticas desarrolladas con distintos usos y envergadura, así como los instrumentos de planeamiento utilizados, en base a las cuales se materializa la construcción del AMM según un modelo extendido (spread), dibujando una mancha de aceite (o grase-spots según Geddes) que precisamente había querido evitarse desde el planeamiento urbanístico con la definición de un Anillo Verde, espacio inmune a la edificación, que se aleja de su papel estructurante (equilibrador entre la ciudad y sus habitantes) para convertirse en armadura de la estructura comunicativa, que una vez consolidada se convierte en la mejor aliada de la máquina inmobiliaria. El último paso, se desarrolla en el bloque III que se divide en los capítulo 7,8 y 9 y supone la comprobación de lo descrito en el conjunto de escala metropolitana, en dos aspectos fundamentales, la falta de consideración por los valores culturales y medioambientales que han modelado el territorio, imprimiéndole un carácter singular y específico y, la estructura del dominio del suelo, donde se reconoce de forma precisa el grupo social y los agentes encargados en cada momento de comercializar los suelos del anillo, que bajo el paraguas de la urgencia social y el engañoso beneficio popular, obtienen importantes beneficios económicos. Con esa intención, se da un salto hacia la escala municipal y urbana, seleccionando dos escenarios de estudio, el municipio de Pozuelo de Alarcón, que representa la materialización del crecimiento suburbano de la élite madrileña ocupando las zonas de mayor valor ecológico del anillo, y el distrito de Hortaleza-Barajas que ofrece su territorio a las grandes operaciones metropolitanas, apoyándose en el eje de actividad marcado por la conexión Madrid-Barcelona y el sistema aeroportuario de escala global, ambos situados al norte de la línea de borde entre la Sierra y la Mancha, ocupando por tanto los lugares más valiosos de la geografía madrileña (estructura funcional anticipada por Bidagor en 1946 en su modelo de ciudad adaptada al territorio madrileño) Una vez descrito este proceso trasgresor de límites, de normas, de conductas, y desde una perspectiva del fenómeno suficientemente documentada, en el capítulo 10, se realiza una reflexión sobre la incidencia real de la propuesta urbanística del Anillo Verde en la construcción del AMM, de la misma forma que se sugieren nuevos roles al planeamiento en un formato intencionado de largo recorrido en oposición a lo inmediato y circunstancial, que permita hacer una nueva lectura de los presupuestos teóricos que conformaban la idea del Anillo Verde, espacio articulador (medioambiental, social y cultural) del territorio madrileño. ABSTRACT The Metropolitan Greenbelt was defined by the 1963 Master Plan for the Madrid Metropolitan Area (MMA), following established international models of urban development, as the structural framework of the MMA, the principal open space within its network of open spaces and a public area of recreation and contact with nature for the residents of Madrid. In reality, however, it ha become a reserve of land in which various alliances between the institutional authorities and the dominant social class have been operating on the margin of the original plan, exposing a scarcity of legal and cultural resources for the safeguarding of common interests, and in which the geographical and natural characteristics of the territory itself have come to play an influential role in the functional specialization and spatial segregation of the MMA. With that idea as its HYPOTHESIS, the aim of this THESIS is to demonstrate its reality. The first step in this is to delineate, temporally and spatially, the object of study; i.e. the Metropolitan Greenbelt2. The second is to contextualize historically and disciplinarily those theoretical ideas which conform to the greenbelt concept. The third is to acknowledge, locate and document the elements which have characterized the urban occupation of the Greenbelt and classify these according to the parameters of time, function, urban development and form, which in turn would enable the geography, use, instrumentation and form of its transformation to be analysed on a general metropolitan scale. The fourth step, as a method of verification, is an in-depth analysis of two representative settings within the metropolitan network: the municipality of Pozuelo de Alarcón and the Hortaleza-Barajas district. The content of the document is divided into three parts. Part I focuses on the study’s theoretical foundations, Part II establishes a line of argument at the metropolitan level and Part III examines the phenomenon from a municipal and urban perspective. The thesis, then, begins with a study of the greenbelt concept itself and its meaning, which is far more complex than the accepted instrumental dimension of limiting and containing urbanization in response to the growth of the industrial city of the early 20th century, and which is based on a decentralization of the traditional city. This wider purpose is the setting aside of a large reserved space to safeguard the natural and cultural values of the region and thereby achieve a balance between the city and its residents; that is to say, between man and the space he inhabits. The principal currents of thought in urban planning will then be examined. These have drawn upon a variety of disciplines (economics, sociology, geography, biology, ecology) to develop theories for establishing a new urban order according to the principles of social, economic and environmental equity, and have involved a constant interchange between Europe and the United States. Thus, the City Garden and Regionalist movements would clear the way for proposals such as Greater London and Great Berlin, Chicago and Washington, in which the greenbelt would play a fundamental role. The participation of our own country and the city of Madrid is also discussed, through regional models such as the Besteiro Plan and urban ones like the Bidagor Plan, direct forerunners of 1963’s General Organizational Plan for the Madrid Metropolitan Area, which would set into motion the organization of Madrid’s metropolitan growth. The line of argument followed in this thesis is two-fold: first, an examination of metropolitan development in keeping with the city model as defined in the various General Plans for the development of both the municipality of Madrid and the seven surrounding municipalities which have land designated for its Greenbelt; and second, an examination of this growth in relation to Regional Planning measures, is detailed on a smaller scale (municipal and district), where the conditioning factors affecting the land property structure and the network of biophysical units may be analysed in depth. The first of these is dealt with in Part II and organized into three chapters (4, 5 and 6). Chapter 4 is dedicated to the obligatory starting point of the geographical setting itself. The biophysical characteristics of the territories set aside for the Greenbelt, and which historically have played a role in the area’s exploitation, are described here. These range from expanses of Mediterranean woodland to the north and west of Monte del Pardo to the various types of farmland that have been adapted to the geological substratum and the contours of the terrain (gentle undulations of arkosic sediment in the north, and wide sandy and gypsiferous tableland in the south), as well as orchards planted in low valleys and along watercourses (the creeks of Monte Carmelo, Valdebebas, Quinto, Santo, Butarque, Meaques and Pozuelo). Once this physical reality ha been detailed, in Chapter 5 will examine the various city models proposed by urban planners, both regionally and municipally, in response to the confluence of social, economic and political interests that have characterized the process of occupation in the Greenbelt area during the construction of the MMA. Municipal planning proposals will be collected and examined for the various municipalities which have land designated for the Greenbelt: Madrid, Coslada, Getafe, Leganés, Alcorcón, Boadilla del Monte and Pozuelo de Alarcón. Furthermore, the various territorial organization proposals which have served as references for municipal planning will also be addressed here, in all of their versions –from the sectorial, which have met with more success and institutional approval, to the many attempts at integration, which have been more complex but less influential, precisely for the difficulty of reconciling physical organization with economic development, and private interest with public benefit. The first period in this process was the development of the General Plan of 1963, followed by the first municipal development plans of the 1970s, in which the need for a regional framework that “organized” the territory in an integral fashion was defined. This would serve as a reference for the sectorial actions that marked the metropolitan area’s initial development. The second came two decades later with the approval of the General Plan of 1985, and the network of municipal plans for the surrounding communities, which followed the same philosophy of austerity with regard to territorial growth. The third would begin to take form in 1997, as a new generation of neo-liberal development plans imposed a territorial model based on centralized large-scale metropolitan operations of infrastructure and equipment, which would indiscriminately consume the totality of Madrid’s land. At the end of the Part II, in Chapter 6, the metropolitan area will be represented graphically and the 229 pieces that have been gradually encroaching upon land designated for the Greenbelt will be analysed. This analysis will be carried out according to the parameters defined for the study, and the first general conclusions of the thesis will be based on its findings. It will show how alliances between the various governing authorities in the construction of the city and its environment have successively violated established plans with regard to the definitions of city and territory, how shortages of instrumental and judicial resources have accentuated the dismantling process, and how natural and geographical factors have influenced functional specialization and social segregation in the Madrid Metropolitan Area. The final step, detailed in Part III, will address two fundamental aspects of what has just been described: the lack of consideration for the cultural and environmental values which have shaped this territory and imprinted upon it a specific and unique character; and the structure of land domination, with a precise identification of the social group and agents responsible at each stage of the Greenbelt’s commercialization, who, under an umbrella of social urgency and deceptive public benefit, have used it to obtain substantial financial rewards. For this purpose, a closer look is taken at two specific areas: the municipality of Pozuelo de Alarcón, representative of the suburban growth of an elite population which has occupied the Greenbelt areas of the greatest ecological value; and the Hortaleza-Barajas district, which has offered its territory to large metropolitan business interests, based on activities centred on the connection between Madrid and Barcelona and the system of international air travel. Both of these settings are located to the north of the line which divides the Sierra from La Mancha, and thus occupy the most valuable land in the Madrid region (a functional structure anticipated by Bidagor in 1946, with his city model adapted to the territory of Madrid). Finally, an attempt will be made to interpret the phenomenon of metropolitan Greenbelt occupation, confirming initial hypotheses, specifying the environmental and cultural values that have been violated, and identifying the various players involved, as well as numerous urbanization operations of varying sizes and interests, and the instruments of planning they have used. It will be seen from this that the construction of the MMA has in fact followed a “spread” model, a “grease spot” (as Geddes calls it) which, from the outset of the planning process and according to the definition of a greenbelt as a construction-free zone, was precisely to be avoided. This structural role (to provide a balance between a city and its residents) has thus been abandoned and the Greenbelt converted instead into a communicative framework which, once consolidated, has become the greatest ally of the real estate machine. After this process of violating limits, norms and established behaviour has been described and solidly documented, a reflection will be made on the real influence of the Greenbelt proposal in the construction of the MMA. At the same time, new roles will be suggested for future planning, roles which are deliberate and long term, in opposition to the immediate and circumstantial. This will enable a new interpretation of the theoretical principles behind the greenbelt concept, a space designed to connect the territory of Madrid environmentally, socially and culturally.

Design of a Semi-analytical Method to Describe Plasma-Wall Interactions

Electric probes are objects immersed in the plasma with sharp boundaries which collect of emit charged particles. Consequently, the nearby plasma evolves under abrupt imposed and/or naturally emerging conditions. There could be localized currents, different time scales for plasma species evolution, charge separation and absorbing-emitting walls. The traditional numerical schemes based on differences often transform these disparate boundary conditions into computational singularities. This is the case of models using advection-diffusion differential equations with source-sink terms (also called Fokker-Planck equations). These equations are used in both, fluid and kinetic descriptions, to obtain the distribution functions or the density for each plasma species close to the boundaries. We present a resolution method grounded on an integral advancing scheme by using approximate Green's functions, also called short-time propagators. All the integrals, as a path integration process, are numerically calculated, what states a robust grid-free computational integral method, which is unconditionally stable for any time step. Hence, the sharp boundary conditions, as the current emission from a wall, can be treated during the short-time regime providing solutions that works as if they were known for each time step analytically. The form of the propagator (typically a multivariate Gaussian) is not unique and it can be adjusted during the advancing scheme to preserve the conserved quantities of the problem. The effects of the electric or magnetic fields can be incorporated into the iterative algorithm. The method allows smooth transitions of the evolving solutions even when abrupt discontinuities are present. In this work it is proposed a procedure to incorporate, for the very first time, the boundary conditions in the numerical integral scheme. This numerical scheme is applied to model the plasma bulk interaction with a charge-emitting electrode, dealing with fluid diffusion equations combined with Poisson equation self-consistently. It has been checked the stability of this computational method under any number of iterations, even for advancing in time electrons and ions having different time scales. This work establishes the basis to deal in future work with problems related to plasma thrusters or emissive probes in electromagnetic fields.

A time-domain finite element method for seakeeping and wave resistance problems

El objetivo de la tesis es la investigación de algoritmos numéricos para el desarrollo de herramientas numéricas para la simulación de problemas tanto de comportamiento en la mar como de resistencia al avance de buques y estructuras flotantes. La primera herramienta desarrollada resuelve el problema de difracción y radiación de olas. Se basan en el método de los elementos finitos (MEF) para la resolución de la ecuación de Laplace, así como en esquemas basados en MEF, integración a lo largo de líneas de corriente, y en diferencias finitas desarrollados para la condición de superficie libre. Se han desarrollado herramientas numéricas para la resolución de la dinámica de sólido rígido en sistemas multicuerpos con ligaduras. Estas herramientas han sido integradas junto con la herramienta de resolución de olas difractadas y radiadas para la resolución de problemas de interacción de cuerpos con olas. También se han diseñado algoritmos de acoplamientos con otras herramientas numéricas para la resolución de problemas multifísica. En particular, se han realizado acoplamientos con una herramienta numérica basada de cálculo de estructuras con MEF para problemas de interacción fluido-estructura, otra de cálculo de líneas de fondeo, y con una herramienta numérica de cálculo de flujos en tanques internos para problemas acoplados de comportamiento en la mar con “sloshing”. Se han realizado simulaciones numéricas para la validación y verificación de los algoritmos desarrollados, así como para el análisis de diferentes casos de estudio con aplicaciones diversas en los campos de la ingeniería naval, oceánica, y energías renovables marinas. ABSTRACT The objective of this thesis is the research on numerical algorithms to develop numerical tools to simulate seakeeping problems as well as wave resistance problems of ships and floating structures. The first tool developed is a wave diffraction-radiation solver. It is based on the finite element method (FEM) in order to solve the Laplace equation, as well as numerical schemes based on FEM, streamline integration, and finite difference method tailored for solving the free surface boundary condition. It has been developed numerical tools to solve solid body dynamics of multibody systems with body links across them. This tool has been integrated with the wave diffraction-radiation solver to solve wave-body interaction problems. Also it has been tailored coupling algorithms with other numerical tools in order to solve multi-physics problems. In particular, it has been performed coupling with a MEF structural solver to solve fluid-structure interaction problems, with a mooring solver, and with a solver capable of simulating internal flows in tanks to solve couple seakeeping-sloshing problems. Numerical simulations have been carried out to validate and verify the developed algorithms, as well as to analyze case studies in the areas of marine engineering, offshore engineering, and offshore renewable energy.

Método experimental para la caracterización de las diferentes tecnologías de integración arquitectónica de la energía fotovoltaica en condiciones de funcionamiento real = Experimental method for characterization of several building integrated photovoltaic technologies under real working conditions

Hoy en día, el proceso de un proyecto sostenible persigue realizar edificios de elevadas prestaciones que son, energéticamente eficientes, saludables y económicamente viables utilizando sabiamente recursos renovables para minimizar el impacto sobre el medio ambiente reduciendo, en lo posible, la demanda de energía, lo que se ha convertido, en la última década, en una prioridad. La Directiva 2002/91/CE "Eficiencia Energética de los Edificios" (y actualizaciones posteriores) ha establecido el marco regulatorio general para el cálculo de los requerimientos energéticos mínimos. Desde esa fecha, el objetivo de cumplir con las nuevas directivas y protocolos ha conducido las políticas energéticas de los distintos países en la misma dirección, centrándose en la necesidad de aumentar la eficiencia energética en los edificios, la adopción de medidas para reducir el consumo, y el fomento de la generación de energía a través de fuentes renovables. Los edificios de energía nula o casi nula (ZEB, Zero Energy Buildings ó NZEB, Net Zero Energy Buildings) deberán convertirse en un estándar de la construcción en Europa y con el fin de equilibrar el consumo de energía, además de reducirlo al mínimo, los edificios necesariamente deberán ser autoproductores de energía. Por esta razón, la envolvente del edifico y en particular las fachadas son importantes para el logro de estos objetivos y la tecnología fotovoltaica puede tener un papel preponderante en este reto. Para promover el uso de la tecnología fotovoltaica, diferentes programas de investigación internacionales fomentan y apoyan soluciones para favorecer la integración completa de éstos sistemas como elementos arquitectónicos y constructivos, los sistemas BIPV (Building Integrated Photovoltaic), sobre todo considerando el próximo futuro hacia edificios NZEB. Se ha constatado en este estudio que todavía hay una falta de información útil disponible sobre los sistemas BIPV, a pesar de que el mercado ofrece una interesante gama de soluciones, en algunos aspectos comparables a los sistemas tradicionales de construcción. Pero por el momento, la falta estandarización y de una regulación armonizada, además de la falta de información en las hojas de datos técnicos (todavía no comparables con las mismas que están disponibles para los materiales de construcción), hacen difícil evaluar adecuadamente la conveniencia y factibilidad de utilizar los componentes BIPV como parte integrante de la envolvente del edificio. Organizaciones internacionales están trabajando para establecer las normas adecuadas y procedimientos de prueba y ensayo para comprobar la seguridad, viabilidad y fiabilidad estos sistemas. Sin embargo, hoy en día, no hay reglas específicas para la evaluación y caracterización completa de un componente fotovoltaico de integración arquitectónica de acuerdo con el Reglamento Europeo de Productos de la Construcción, CPR 305/2011. Los productos BIPV, como elementos de construcción, deben cumplir con diferentes aspectos prácticos como resistencia mecánica y la estabilidad; integridad estructural; seguridad de utilización; protección contra el clima (lluvia, nieve, viento, granizo), el fuego y el ruido, aspectos que se han convertido en requisitos esenciales, en la perspectiva de obtener productos ambientalmente sostenibles, saludables, eficientes energéticamente y económicamente asequibles. Por lo tanto, el módulo / sistema BIPV se convierte en una parte multifuncional del edificio no sólo para ser física y técnicamente "integrado", además de ser una oportunidad innovadora del diseño. Las normas IEC, de uso común en Europa para certificar módulos fotovoltaicos -IEC 61215 e IEC 61646 cualificación de diseño y homologación del tipo para módulos fotovoltaicos de uso terrestre, respectivamente para módulos fotovoltaicos de silicio cristalino y de lámina delgada- atestan únicamente la potencia del módulo fotovoltaico y dan fe de su fiabilidad por un período de tiempo definido, certificando una disminución de potencia dentro de unos límites. Existe también un estándar, en parte en desarrollo, el IEC 61853 (“Ensayos de rendimiento de módulos fotovoltaicos y evaluación energética") cuyo objetivo es la búsqueda de procedimientos y metodologías de prueba apropiados para calcular el rendimiento energético de los módulos fotovoltaicos en diferentes condiciones climáticas. Sin embargo, no existen ensayos normalizados en las condiciones específicas de la instalación (p. ej. sistemas BIPV de fachada). Eso significa que es imposible conocer las efectivas prestaciones de estos sistemas y las condiciones ambientales que se generan en el interior del edificio. La potencia nominal de pico Wp, de un módulo fotovoltaico identifica la máxima potencia eléctrica que éste puede generar bajo condiciones estándares de medida (STC: irradición 1000 W/m2, 25 °C de temperatura del módulo y distribución espectral, AM 1,5) caracterizando eléctricamente el módulo PV en condiciones específicas con el fin de poder comparar los diferentes módulos y tecnologías. El vatio pico (Wp por su abreviatura en inglés) es la medida de la potencia nominal del módulo PV y no es suficiente para evaluar el comportamiento y producción del panel en términos de vatios hora en las diferentes condiciones de operación, y tampoco permite predecir con convicción la eficiencia y el comportamiento energético de un determinado módulo en condiciones ambientales y de instalación reales. Un adecuado elemento de integración arquitectónica de fachada, por ejemplo, debería tener en cuenta propiedades térmicas y de aislamiento, factores como la transparencia para permitir ganancias solares o un buen control solar si es necesario, aspectos vinculados y dependientes en gran medida de las condiciones climáticas y del nivel de confort requerido en el edificio, lo que implica una necesidad de adaptación a cada contexto específico para obtener el mejor resultado. Sin embargo, la influencia en condiciones reales de operación de las diferentes soluciones fotovoltaicas de integración, en el consumo de energía del edificio no es fácil de evaluar. Los aspectos térmicos del interior del ambiente o de iluminación, al utilizar módulos BIPV semitransparentes por ejemplo, son aún desconocidos. Como se dijo antes, la utilización de componentes de integración arquitectónica fotovoltaicos y el uso de energía renovable ya es un hecho para producir energía limpia, pero también sería importante conocer su posible contribución para mejorar el confort y la salud de los ocupantes del edificio. Aspectos como el confort, la protección o transmisión de luz natural, el aislamiento térmico, el consumo energético o la generación de energía son aspectos que suelen considerarse independientemente, mientras que todos juntos contribuyen, sin embargo, al balance energético global del edificio. Además, la necesidad de dar prioridad a una orientación determinada del edificio, para alcanzar el mayor beneficio de la producción de energía eléctrica o térmica, en el caso de sistemas activos y pasivos, respectivamente, podría hacer estos últimos incompatibles, pero no necesariamente. Se necesita un enfoque holístico que permita arquitectos e ingenieros implementar sistemas tecnológicos que trabajen en sinergia. Se ha planteado por ello un nuevo concepto: "C-BIPV, elemento fotovoltaico consciente integrado", esto significa necesariamente conocer los efectos positivos o negativos (en términos de confort y de energía) en condiciones reales de funcionamiento e instalación. Propósito de la tesis, método y resultados Los sistemas fotovoltaicos integrados en fachada son a menudo soluciones de vidrio fácilmente integrables, ya que por lo general están hechos a medida. Estos componentes BIPV semitransparentes, integrados en el cerramiento proporcionan iluminación natural y también sombra, lo que evita el sobrecalentamiento en los momentos de excesivo calor, aunque como componente estático, asimismo evitan las posibles contribuciones pasivas de ganancias solares en los meses fríos. Además, la temperatura del módulo varía considerablemente en ciertas circunstancias influenciada por la tecnología fotovoltaica instalada, la radiación solar, el sistema de montaje, la tipología de instalación, falta de ventilación, etc. Este factor, puede suponer un aumento adicional de la carga térmica en el edificio, altamente variable y difícil de cuantificar. Se necesitan, en relación con esto, más conocimientos sobre el confort ambiental interior en los edificios que utilizan tecnologías fotovoltaicas integradas, para abrir de ese modo, una nueva perspectiva de la investigación. Con este fin, se ha diseñado, proyectado y construido una instalación de pruebas al aire libre, el BIPV Env-lab "BIPV Test Laboratory", para la caracterización integral de los diferentes módulos semitransparentes BIPV. Se han definido también el método y el protocolo de ensayos de caracterización en el contexto de un edificio y en condiciones climáticas y de funcionamiento reales. Esto ha sido posible una vez evaluado el estado de la técnica y la investigación, los aspectos que influyen en la integración arquitectónica y los diferentes tipos de integración, después de haber examinado los métodos de ensayo para los componentes de construcción y fotovoltaicos, en condiciones de operación utilizadas hasta ahora. El laboratorio de pruebas experimentales, que consiste en dos habitaciones idénticas a escala real, 1:1, ha sido equipado con sensores y todos los sistemas de monitorización gracias a los cuales es posible obtener datos fiables para evaluar las prestaciones térmicas, de iluminación y el rendimiento eléctrico de los módulos fotovoltaicos. Este laboratorio permite el estudio de tres diferentes aspectos que influencian el confort y consumo de energía del edificio: el confort térmico, lumínico, y el rendimiento energético global (demanda/producción de energía) de los módulos BIPV. Conociendo el balance de energía para cada tecnología solar fotovoltaica experimentada, es posible determinar cuál funciona mejor en cada caso específico. Se ha propuesto una metodología teórica para la evaluación de estos parámetros, definidos en esta tesis como índices o indicadores que consideran cuestiones relacionados con el bienestar, la energía y el rendimiento energético global de los componentes BIPV. Esta metodología considera y tiene en cuenta las normas reglamentarias y estándares existentes para cada aspecto, relacionándolos entre sí. Diferentes módulos BIPV de doble vidrio aislante, semitransparentes, representativos de diferentes tecnologías fotovoltaicas (tecnología de silicio monocristalino, m-Si; de capa fina en silicio amorfo unión simple, a-Si y de capa fina en diseleniuro de cobre e indio, CIS) fueron seleccionados para llevar a cabo una serie de pruebas experimentales al objeto de demostrar la validez del método de caracterización propuesto. Como resultado final, se ha desarrollado y generado el Diagrama Caracterización Integral DCI, un sistema gráfico y visual para representar los resultados y gestionar la información, una herramienta operativa útil para la toma de decisiones con respecto a las instalaciones fotovoltaicas. Este diagrama muestra todos los conceptos y parámetros estudiados en relación con los demás y ofrece visualmente toda la información cualitativa y cuantitativa sobre la eficiencia energética de los componentes BIPV, por caracterizarlos de manera integral. ABSTRACT A sustainable design process today is intended to produce high-performance buildings that are energy-efficient, healthy and economically feasible, by wisely using renewable resources to minimize the impact on the environment and to reduce, as much as possible, the energy demand. In the last decade, the reduction of energy needs in buildings has become a top priority. The Directive 2002/91/EC “Energy Performance of Buildings” (and its subsequent updates) established a general regulatory framework’s methodology for calculation of minimum energy requirements. Since then, the aim of fulfilling new directives and protocols has led the energy policies in several countries in a similar direction that is, focusing on the need of increasing energy efficiency in buildings, taking measures to reduce energy consumption, and fostering the use of renewable sources. Zero Energy Buildings or Net Zero Energy Buildings will become a standard in the European building industry and in order to balance energy consumption, buildings, in addition to reduce the end-use consumption should necessarily become selfenergy producers. For this reason, the façade system plays an important role for achieving these energy and environmental goals and Photovoltaic can play a leading role in this challenge. To promote the use of photovoltaic technology in buildings, international research programs encourage and support solutions, which favors the complete integration of photovoltaic devices as an architectural element, the so-called BIPV (Building Integrated Photovoltaic), furthermore facing to next future towards net-zero energy buildings. Therefore, the BIPV module/system becomes a multifunctional building layer, not only physically and functionally “integrated” in the building, but also used as an innovative chance for the building envelope design. It has been found in this study that there is still a lack of useful information about BIPV for architects and designers even though the market is providing more and more interesting solutions, sometimes comparable to the existing traditional building systems. However at the moment, the lack of an harmonized regulation and standardization besides to the non-accuracy in the technical BIPV datasheets (not yet comparable with the same ones available for building materials), makes difficult for a designer to properly evaluate the fesibility of this BIPV components when used as a technological system of the building skin. International organizations are working to establish the most suitable standards and test procedures to check the safety, feasibility and reliability of BIPV systems. Anyway, nowadays, there are no specific rules for a complete characterization and evaluation of a BIPV component according to the European Construction Product Regulation, CPR 305/2011. BIPV products, as building components, must comply with different practical aspects such as mechanical resistance and stability; structural integrity; safety in use; protection against weather (rain, snow, wind, hail); fire and noise: aspects that have become essential requirements in the perspective of more and more environmentally sustainable, healthy, energy efficient and economically affordable products. IEC standards, commonly used in Europe to certify PV modules (IEC 61215 and IEC 61646 respectively crystalline and thin-film ‘Terrestrial PV Modules-Design Qualification and Type Approval’), attest the feasibility and reliability of PV modules for a defined period of time with a limited power decrease. There is also a standard (IEC 61853, ‘Performance Testing and Energy Rating of Terrestrial PV Modules’) still under preparation, whose aim is finding appropriate test procedures and methodologies to calculate the energy yield of PV modules under different climate conditions. Furthermore, the lack of tests in specific conditions of installation (e.g. façade BIPV devices) means that it is difficult knowing the exact effective performance of these systems and the environmental conditions in which the building will operate. The nominal PV power at Standard Test Conditions, STC (1.000 W/m2, 25 °C temperature and AM 1.5) is usually measured in indoor laboratories, and it characterizes the PV module at specific conditions in order to be able to compare different modules and technologies on a first step. The “Watt-peak” is not enough to evaluate the panel performance in terms of Watt-hours of various modules under different operating conditions, and it gives no assurance of being able to predict the energy performance of a certain module at given environmental conditions. A proper BIPV element for façade should take into account thermal and insulation properties, factors as transparency to allow solar gains if possible or a good solar control if necessary, aspects that are linked and high dependent on climate conditions and on the level of comfort to be reached. However, the influence of different façade integrated photovoltaic solutions on the building energy consumption is not easy to assess under real operating conditions. Thermal aspects, indoor temperatures or luminance level that can be expected using building integrated PV (BIPV) modules are not well known. As said before, integrated photovoltaic BIPV components and the use of renewable energy is already a standard for green energy production, but would also be important to know the possible contribution to improve the comfort and health of building occupants. Comfort, light transmission or protection, thermal insulation or thermal/electricity power production are aspects that are usually considered alone, while all together contribute to the building global energy balance. Besides, the need to prioritize a particular building envelope orientation to harvest the most benefit from the electrical or thermal energy production, in the case of active and passive systems respectively might be not compatible, but also not necessary. A holistic approach is needed to enable architects and engineers implementing technological systems working in synergy. A new concept have been suggested: “C-BIPV, conscious integrated BIPV”. BIPV systems have to be “consciously integrated” which means that it is essential to know the positive and negative effects in terms of comfort and energy under real operating conditions. Purpose of the work, method and results The façade-integrated photovoltaic systems are often glass solutions easily integrable, as they usually are custommade. These BIPV semi-transparent components integrated as a window element provides natural lighting and shade that prevents overheating at times of excessive heat, but as static component, likewise avoid the possible solar gains contributions in the cold months. In addition, the temperature of the module varies considerably in certain circumstances influenced by the PV technology installed, solar radiation, mounting system, lack of ventilation, etc. This factor may result in additional heat input in the building highly variable and difficult to quantify. In addition, further insights into the indoor environmental comfort in buildings using integrated photovoltaic technologies are needed to open up thereby, a new research perspective. This research aims to study their behaviour through a series of experiments in order to define the real influence on comfort aspects and on global energy building consumption, as well as, electrical and thermal characteristics of these devices. The final objective was to analyze a whole set of issues that influence the global energy consumption/production in a building using BIPV modules by quantifying the global energy balance and the BIPV system real performances. Other qualitative issues to be studied were comfort aspect (thermal and lighting aspects) and the electrical behaviour of different BIPV technologies for vertical integration, aspects that influence both energy consumption and electricity production. Thus, it will be possible to obtain a comprehensive global characterization of BIPV systems. A specific design of an outdoor test facility, the BIPV Env-lab “BIPV Test Laboratory”, for the integral characterization of different BIPV semi-transparent modules was developed and built. The method and test protocol for the BIPV characterization was also defined in a real building context and weather conditions. This has been possible once assessed the state of the art and research, the aspects that influence the architectural integration and the different possibilities and types of integration for PV and after having examined the test methods for building and photovoltaic components, under operation conditions heretofore used. The test laboratory that consists in two equivalent test rooms (1:1) has a monitoring system in which reliable data of thermal, daylighting and electrical performances can be obtained for the evaluation of PV modules. The experimental set-up facility (testing room) allows studying three different aspects that affect building energy consumption and comfort issues: the thermal indoor comfort, the lighting comfort and the energy performance of BIPV modules tested under real environmental conditions. Knowing the energy balance for each experimented solar technology, it is possible to determine which one performs best. A theoretical methodology has been proposed for evaluating these parameters, as defined in this thesis as indices or indicators, which regard comfort issues, energy and the overall performance of BIPV components. This methodology considers the existing regulatory standards for each aspect, relating them to one another. A set of insulated glass BIPV modules see-through and light-through, representative of different PV technologies (mono-crystalline silicon technology, mc-Si, amorphous silicon thin film single junction, a-Si and copper indium selenide thin film technology CIS) were selected for a series of experimental tests in order to demonstrate the validity of the proposed characterization method. As result, it has been developed and generated the ICD Integral Characterization Diagram, a graphic and visual system to represent the results and manage information, a useful operational tool for decision-making regarding to photovoltaic installations. This diagram shows all concepts and parameters studied in relation to each other and visually provides access to all the results obtained during the experimental phase to make available all the qualitative and quantitative information on the energy performance of the BIPV components by characterizing them in a comprehensive way.

Optimization of Dengue immunoassay by label-free interferometric optical detection method

In this communication we report a direct immunoassay for detecting dengue virus by means of a label-free interferometric optical detection method. We also demonstrate how we can optimize this sensing response by adding a blocking step able to significantly enhance the optical sensing response. The blocking reagent used for this optimization is a dry milk diluted in phosphate buffered saline. The recognition curve of dengue virus over the proposed surface sensor demonstrates the capacity of this method to be applied in Point of Care technology.

A genetic method for dissecting the mechanism of transcriptional activator synergy by identical activators

Pairs of transcriptional activators in prokaryotes have been shown to activate transcription synergistically from promoters with two activator binding sites. In some cases, such synergistic effects result from cooperative binding, but in other cases each DNA-bound activator plays a direct role in the activation process by interacting simultaneously with separate surfaces of RNA polymerase. In such cases, each DNA-bound activator must possess a functional activating region, the surface that mediates the interaction with RNA polymerase. When transcriptional activation depends on two or more identical activators, it is not straightforward to test the requirement of each activator for a functional activating region. Here we describe a method for directing a mutationally altered activator to either one or the other binding site, and we demonstrate the use of this method to examine the mechanism of transcriptional activator synergy by the Escherichia coli cyclic AMP receptor protein (CRP) working at an artificial promoter bearing two CRP-binding sites.

Pattern changes of pituitary peptides in rat after salt-loading as detected by means of direct, semiquantitative mass spectrometric profiling

We have established a differential peptide display method, based on a mass spectrometric technique, to detect peptides that show semiquantitative changes in the neurointermediate lobe (NIL) of individual rats subjected to salt-loading. We employed matrix-assisted laser desorption/ionization mass spectrometry, using a single-reference peptide in combination with careful scanning of the whole crystal rim of the matrix-analyte preparation, to detect in a semiquantitative manner the molecular ions present in the unfractionated NIL homogenate. Comparison of the mass spectra generated from NIL homogenates of salt-loaded and control rats revealed a selective and significant decrease in the intensities of several molecular ion species of the NIL homogenates from salt-loaded rats. These ion species, which have masses that correspond to the masses of oxytocin, vasopressin, neurophysins, and an unidentified putative peptide, were subsequently chemically characterized. We confirmed that the decreased molecular ion species are peptides derived exclusively from propressophysin and prooxyphysin (i.e., oxytocin, vasopressin, and various neurophysins). The putative peptide is carboxyl-terminal glycopeptide. The carbohydrate moiety of the latter peptide was determined by electrospray tandem MS as bisected biantennary Hex3HexNAc5Fuc. This posttranslational modification accounts for the mass difference between the predicted mass of the peptide based on cDNA studies and the measured mass of the mature peptide.

Direct isolation of human transcribed sequences from yeast artificial chromosomes through the application of RNA fingerprinting

The identification of cDNA clones from genomic regions known to contain human genes is usually the rate-limiting factor in positional cloning strategies. We demonstrate here that human genes present on yeast artificial chromosomes (YACs) are transcribed in yeast host cells. We have used the arbitrarily primed RNA (RAP) fingerprinting method to identify human-specific, transcribed sequences from YACs located in the 13q12 chromosome region. By comparing the RAP fingerprints generated using defined, arbitrary primers from various fragmented YACs, megaYACs, and host yeast, we were able to identify and map 20 products transcribed from the human YAC inserts. This method, therefore, permits the simultaneous isolation and mapping of novel expressed sequences directly from whole YACs.

Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms

A loxP-transposon retrofitting strategy for generating large nested deletions from one end of the insert DNA in bacterial artificial chromosomes and P1 artificial chromosomes was described recently [Chatterjee, P. K. & Coren, J. S. (1997) Nucleic Acids Res. 25, 2205–2212]. In this report, we combine this procedure with direct sequencing of nested-deletion templates by using primers located in the transposon end to illustrate its value for position-specific single-nucleotide polymorphism (SNP) discovery from chosen regions of large insert clones. A simple ampicillin sensitivity screen was developed to facilitate identification and recovery of deletion clones free of transduced transposon plasmid. This directed approach requires minimal DNA sequencing, and no in vitro subclone library generation; positionally oriented SNPs are a consequence of the method. The procedure is used to discover new SNPs as well as physically map those identified from random subcloned libraries or sequence databases. The deletion templates, positioned SNPs, and markers are also used to orient large insert clones into a contig. The deletion clone can serve as a ready resource for future functional genomic studies because each carries a mammalian cell-specific antibiotic resistance gene from the transposon. Furthermore, the technique should be especially applicable to the analysis of genomes for which a full genome sequence or radiation hybrid cell lines are unavailable.

Direct Visualization of a Protein Nuclear Architecture

Whether the cell nucleus is organized by an underlying architecture analagous to the cytoskeleton has been a highly contentious issue since the original isolation of a nuclease and salt-resistant nuclear matrix. Despite electron microscopy studies that show that a nuclear architecture can be visualized after fractionation, the necessity to elute chromatin to visualize this structure has hindered general acceptance of a karyoskeleton. Using an analytical electron microscopy method capable of quantitative elemental analysis, electron spectroscopic imaging, we show that the majority of the fine structure within interchromatin regions of the cell nucleus in fixed whole cells is not nucleoprotein. Rather, this fine structure is compositionally similar to known protein-based cellular structures of the cytoplasm. This study is the first demonstration of a protein network in unfractionated and uninfected cells and provides a method for the ultrastructural characterization of the interaction of this protein architecture with chromatin and ribonucleoprotein elements of the cell nucleus.

Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry

An approach to analyzing single-nucleotide polymorphisms (SNPs) found in the human genome has been developed that couples a recently developed invasive cleavage assay for nucleic acids with detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The invasive cleavage assay is a signal amplification method that enables the analysis of SNPs by MALDI-TOF MS directly from human genomic DNA without the need for initial target amplification by PCR. The results presented here show the successful genotyping by this approach of twelve SNPs located randomly throughout the human genome. Conventional Sanger sequencing of these SNP positions confirmed the accuracy of the MALDI-TOF MS analysis results. The ability to unambiguously detect both homozygous and heterozygous genotypes is clearly demonstrated. The elimination of the need for target amplification by PCR, combined with the inherently rapid and accurate nature of detection by MALDI-TOF MS, gives this approach unique and significant advantages in the high-throughput genotyping of large numbers of SNPs, useful for locating, identifying, and characterizing the function of specific genes.

“Coarse” stability and bifurcation analysis using time-steppers: A reaction-diffusion example

Evolutionary, pattern forming partial differential equations (PDEs) are often derived as limiting descriptions of microscopic, kinetic theory-based models of molecular processes (e.g., reaction and diffusion). The PDE dynamic behavior can be probed through direct simulation (time integration) or, more systematically, through stability/bifurcation calculations; time-stepper-based approaches, like the Recursive Projection Method [Shroff, G. M. & Keller, H. B. (1993) SIAM J. Numer. Anal. 30, 1099–1120] provide an attractive framework for the latter. We demonstrate an adaptation of this approach that allows for a direct, effective (“coarse”) bifurcation analysis of microscopic, kinetic-based models; this is illustrated through a comparative study of the FitzHugh-Nagumo PDE and of a corresponding Lattice–Boltzmann model.

Fluorescence-based directed termination PCR: direct mutation characterization without sequencing

We describe a fluorescence-based directed termination PCR (fluorescent DT–PCR) that allows accurate determination of actual sequence changes without dideoxy DNA sequencing. This is achieved using near infrared dye-labeled primers and performing two PCR reactions under low and unbalanced dNTP concentrations. Visualization of resulting termination fragments is accomplished with a dual dye Li-cor DNA sequencer. As each DT–PCR reaction generates two sets of terminating fragments, a pair of complementary reactions with limiting dATP and dCTP collectively provide information on the entire sequence of a target DNA, allowing an accurate determination of any base change. Blind analysis of 78 mutants of the supF reporter gene using fluorescent DT–PCR not only correctly determined the nature and position of all types of substitution mutations in the supF gene, but also allowed rapid scanning of the signature sequences among identical mutations. The method provides simplicity in the generation of terminating fragments and 100% accuracy in mutation characterization. Fluorescent DT–PCR was successfully used to generate a UV-induced spectrum of mutations in the supF gene following replication on a single plate of human DNA repair-deficient cells. We anticipate that the automated DT–PCR method will serve as a cost-effective alternative to dideoxy sequencing in studies involving large-scale analysis for nucleotide sequence changes.

Direct Measurement of Calcium Transport across Chloroplast Inner-Envelope Vesicles1

The initial rate of Ca2+ movement across the inner-envelope membrane of pea (Pisum sativum L.) chloroplasts was directly measured by stopped-flow spectrofluorometry using membrane vesicles loaded with the Ca2+-sensitive fluorophore fura-2. Calibration of fura-2 fluorescence was achieved by combining a ratiometric method with Ca2+-selective minielectrodes to determine pCa values. The initial rate of Ca2+ influx in predominantly right-side-out inner-envelope membrane vesicles was greater than that in largely inside-out vesicles. Ca2+ movement was stimulated by an inwardly directed electrochemical proton gradient across the membrane vesicles, an effect that was diminished by the addition of valinomycin in the presence of K+. In addition, Ca2+ was shown to move across the membrane vesicles in the presence of a K+ diffusion potential gradient. The potential-stimulated rate of Ca2+ transport was slightly inhibited by diltiazem and greatly inhibited by ruthenium red. Other pharmacological agents such as LaCl3, verapamil, and nifedipine had little or no effect. These results indicate that Ca2+ transport across the chloroplast inner envelope can occur by a potential-stimulated uniport mechanism.