Modal analysis of buildings : quantification of uncertainties and model updating

Una estructura vibra con la suma de sus infinitos modos de vibración, definidos por sus parámetros modales (frecuencias naturales, formas modales y coeficientes de amortiguamiento). Estos parámetros se pueden identificar a través del Análisis Modal Operacional (OMA). Así, un equipo de investigación de la Universidad Politécnica de Madrid ha identificado las propiedades modales de un edificio de hormigón armado en Madrid con el método Identificación de los sub-espacios estocásticos (SSI). Para completar el estudio dinámico de este edificio, se ha desarrollado un modelo de elementos finitos (FE) de este edificio de 19 plantas. Este modelo se ha calibrado a partir de su comportamiento dinámico obtenido experimentalmente a través del OMA. Los objetivos de esta tesis son; (i) identificar la estructura con varios métodos de SSI y el uso de diferentes ventanas de tiempo de tal manera que se cuantifican incertidumbres de los parámetros modales debidos al proceso de estimación, (ii) desarrollar FEM de este edificio y calibrar este modelo a partir de su comportamiento dinámico, y (iii) valorar la bondad del modelo. Los parámetros modales utilizados en esta calibración han sido; espesor de las losas, densidades de los materiales, módulos de elasticidad, dimensiones de las columnas y las condiciones de contorno de la cimentación. Se ha visto que el modelo actualizado representa el comportamiento dinámico de la estructura con una buena precisión. Por lo tanto, este modelo puede utilizarse dentro de un sistema de monitorización estructural (SHM) y para la detección de daños. En el futuro, podrá estudiar la influencia de los agentes medioambientales, tales como la temperatura o el viento, en los parámetros modales. A structure vibrates according to the sum of its vibration modes, defined by their modal parameters (natural frequencies, damping ratios and modal shapes). These parameters can be identified through Operational Modal Analysis (OMA). Thus, a research team of the Technical University of Madrid has identified the modal properties of a reinforced-concrete-frame building in Madrid using the Stochastic Subspace Identification (SSI) method and a time domain technique for the OMA. To complete the dynamic study of this building, a finite element model (FE) of this 19-floor building has been developed throughout this thesis. This model has been updated from its dynamic behavior identified by the OMA. The objectives of this thesis are to; (i) identify the structure with several SSI methods and using different time blocks in such a way that uncertainties due to the modal parameter estimation are quantified, (ii) develop a FEM of this building and tune this model from its dynamic behavior, and (iii) Assess the quality of the model, the modal parameters used in this updating process have been; thickness of slabs, material densities, modulus of elasticity, column dimensions and foundation boundary conditions. It has been shown that the final updated model represents the structure with a very good accuracy. Thus, this model might be used within a structural health monitoring framework (SHM). The study of the influence of changing environmental factors (such as temperature or wind) on the model parameters might be considered as a future work.

Modal liquid crystal microaxicon array

A novel tunable liquid crystal microaxicon array is proposed and experimentally demonstrated. The proposed structure is capable of generating tunable axicons (thousands of elements) of micrometric size, with simple control (four control voltages) and low voltage, and is totally reconfigurable. Depending on the applied voltages, control over the diameter, as well as the effective wedge angle, can be achieved. Controls over the diameter ranging from 107 to 77 μm have been demonstrated. In addition, a control over the phase profile tunability, from 12π to 24π radians, has been demonstrated. This result modifies the effective cone angle. The diameter tunability, as well the effective cone angle, results in a control over the nondiffractive Bessel beam distance. The RMS wavefront deviation from the ideal axicon is only λ∕3. The proposed device has several advantages over the existing microaxicon arrays, including being simple having a low cost. The device could contribute to developing new applications and to reducing the fabrication costs of current devices.

Comparison of implicit and explicit FSI coupling strategies in cardiovascular system = Comparación de las estrategias implícitas y explícitas de acoplamiento FSI en el sistema cardiovascular

Comparación de los esquemas de integración temporal explícito e implícito, en la simulación del flujo sanguíneo y su interacción con la pared arterial. There are two major strategies in FSI coupling techniques: implicit and explicit. The general difference between these methodologies is how many times the data is exchanged between the fluid and solid domains at each FSI time-step. In both coupling strategies, the pressure values coming from fluid domain calculations at each time-step are exported to the solid domain, and consequently, the solid domain is analyzed with these imported forces. In contrast to the explicit coupling, in the implicit approach the fluid and solid domain’s data is exchanged several times until the convergence is achieved. Although this method may boost the numerical stabilization, it increases the computational cost due to the extra data exchanges. In cardiovascular simulations, depending on the analysis objectives, one may choose an explicit or implicit approach. In the current work, the advantage of an explicit coupling strategy is highlighted when simulation of pulsatile blood flow in elastic arteries is desired.

Loose Coupling Based Reference Scheme for Shop Floor-Control-System/Production-Equipment Integration.

Acoplamiento del sistema informático de control de piso de producción (SFS) con el conjunto de equipos de fabricación (SPE) es una tarea compleja. Tal acoplamiento involucra estándares abiertos y propietarios, tecnologías de información y comunicación, entre otras herramientas y técnicas. Debido a la turbulencia de mercados, ya sea soluciones personalizadas o soluciones basadas en estándares eventualmente requieren un esfuerzo considerable de adaptación. El concepto de acoplamiento débil ha sido identificado en la comunidad de diseño organizacional como soporte para la sobrevivencia de la organización. Su presencia reduce la resistencia de la organización a cambios en el ambiente. En este artículo los resultados obtenidos por la comunidad de diseño organizacional son identificados, traducidos y organizados para apoyar en la solución del problema de integración SFS-SPE. Un modelo clásico de acoplamiento débil, desarrollado por la comunidad de estudios de diseño organizacional, es resumido y trasladado al área de interés. Los aspectos claves son identificados para utilizarse como promotores del acoplamiento débil entre SFS-SPE, y presentados en forma de esquema de referencia. Así mismo, este esquema de referencia es presentado como base para el diseño e implementación de una solución genérica de acoplamiento o marco de trabajo (framework) de acoplamiento, a incluir como etapa de acoplamiento débil entre SFS y SPE. Un ejemplo de validación con varios conjuntos de equipos de fabricación, usando diferentes medios físicos de comunicación, comandos de controlador, lenguajes de programación de equipos y protocolos de comunicación es presentado, mostrando un nivel aceptable de autonomía del SFS. = Coupling shop floor software system (SFS) with the set of production equipment (SPE) becomes a complex task. It involves open and proprietary standards, information and communication technologies among other tools and techniques. Due to market turbulence, either custom solutions or standards based solutions eventually require a considerable effort of adaptation. Loose coupling concept has been identified in the organizational design community as a compensator for organization survival. Its presence reduces organization reaction to environment changes. In this paper the results obtained by the organizational de sign community are identified, translated and organized to support the SFS-SPE integration problem solution. A classical loose coupling model developed by organizational studies community is abstracted and translated to the area of interest. Key aspects are identified to be used as promoters of SFS-SPE loose coupling and presented in a form of a reference scheme. Furthermore, this reference scheme is proposed here as a basis for the design and implementation of a generic coupling solution or coupling framework, that is included as a loose coupling stage between SFS and SPE. A validation example with various sets of manufacturing equipment, using different physical communication media, controller commands, programming languages and wire protocols is presented, showing an acceptable level of autonomy gained by the SFS.

ISA S88 / Loose Coupling Based Solution for Production Control Software Reusability

A solution for the problem of reusability of software system for batch production systems is proposed. It is based on ISA S88 standard that prescribes the abstraction of elements in the manufacturing system that is equipment, processes and procedures abstraction, required to make a product batch. An easy to apply data scheme, compatible with the standard, is developed for management of production information. In addition to flexibility provided by the S88 standard, software system reusability requires a solution supporting manufacturing equipment reconfigurability. Toward this end a coupling mechanism is developed. A software tool, including these solutions, was developed and validated at laboratory level, using product manufacturing information of an actual plant.

State space models for multi-setup operational modal analysis

Operational Modal Analysis consists on estimate the modal parameters of a structure (natural frequencies, damping ratios and modal vectors) from output-only vibration measurements. The modal vectors can be only estimated where a sensor is placed, so when the number of available sensors is lower than the number of tested points, it is usual to perform several tests changing the position of the sensors from one test to the following (multiple setups of sensors): some sensors stay at the same position from setup to setup, and the other sensors change the position until all the tested points are covered. The permanent sensors are then used to merge the mode shape estimated at each setup (or partial modal vectors) into global modal vectors. Traditionally, the partial modal vectors are estimated independently setup by setup, and the global modal vectors are obtained in a postprocess phase. In this work we present two state space models that can be used to process all the recorded setups at the same time, and we also present how these models can be estimated using the maximum likelihood method. The result is that the global mode shape of each mode is obtained automatically, and subsequently, a single value for the natural frequency and damping ratio of the mode is computed. Finally, both models are compared using real measured data.

Arquitectura modal o ensamblaje de repertorios, procesos y disposiciones

En muchas ocasiones se ha establecido una crítica sobre la excesiva disposicionalidad estética del arquitecto: el abuso subjetivista, un innatismo desligado del entorno socioambiental o su desafección hacia las necesidades y los requerimientos pertinentes del usuario final, buscando, principalmente, la objetivación cultural como expresión personal e individualista. Criticas que responden a un malestar persistente, que subyace a la práctica arquitectónica. En otras ocasiones se han ideado y propuesto tentativas o aproximaciones para la corrección y regulación de dicho “exceso”. En busca de la “pretendida objetividad”, éstas han recurrido a un pasado prístino, a una tradición inconsciente, colectiva e impersonal, a la ausencia de autoría, a lo artesanal y a lo vernáculo. También hacia un futuro tecnocrático: al mesianismo basado en la máquina, la ciencia, los métodos y el progreso tecnológico. La búsqueda de un lenguaje compartido, de un diseño sistemático y de una adecuación funcional han establecido nuevos excesos –repertoriales-, relegando la creatividad y la concepción estética a un plano casi inexistente, subordinándolo a variables de diseño externas a la autoría y la expresión subjetiva del diseñador. Esta tensión se ha saldado en una pugna de binomios categoriales que reducen la realidad a un dualismo aporético y contrapuesto, difícilmente superable. El propósito articulatorio de dichas posturas, en modelos ontológicos integrales, permite la incorporación de dichas categorías bajo estructuras y ensamblajes que despliegan los modos de relación, mediación y producción arquitectónicas. Estos ensamblajes combinan los distintos agentes –disposicionalidades- y sus repertorios en cada una de las fases del proceso arquitectónico, construyendo modos de acción y relación en base a los acoplamientos diferenciales, que devienen en técnicas y dinámicas de producción arquitectónica. Cada modo integrará diversas variables en base a los modelos propuestos -estratos, períodos y polos- y a la elección intencional –“situada”- de cada práctica: la Autonomía Modal Arquitectónica. El producto final de cada modo –o ensamblaje- será inconmensurable, estableciendo sistemas y métodos diversos para la práctica estética, sin que por ello esté garantizado el valor o éxito del resultado. ABSTRACT On many occasions an aesthetic criticism of the excessive disposition of the architect´s subjectivist abuse, innate posture detached from socioenvironmental issues or disaffection regarding the needs and requirements of who -or what- will finally use it has been brought up mainly in pursuit of a cultural objectification as a personal expression and an individualistic one. It is a criticism which goes hand in hand with a persistent uneasiness which is interwoven in the architectural practice. On other occasions we have seen ideas, attempts and approaches developed and proposed in order to correct, improve or adjust that so-called “excess”. In the search for this " expected objectivity" theorists have resorted to delving into a pristine past, an unconscious tradition both collective and personal, to the absence of authorship, regarding craft and vernacular. The pursuit of a common language, a systematic design and a functional adequacy has established new excesses -repertoires-, leaving behind creativity and aesthetic conception at an almost nonexistent level and subordinating it to variables outside the authoring design and subjective expression of the designer. This tension has resulted in a conflict between categorical binomial, which reduces reality to an aporetic and opposed dualism which is not easily overcome. The purpose of such articulatory positions in comprehensive ontological modals allows for the incorporation of those categories under structures and assemblies which display and unfold different models of relationship, mediation and production of architecture. These assemblies combine different agents - disposition - and their repertoires in each of the phases of the architectural process, building actions and relational modes based on the differential couplings, which become techniques and dynamics in architectural production. Each mode will integrate several variables within the proposed ontological models - strata, periods and poles - based on an intentional choice –“situated knowledge”- of each practice: Architectural Modal Autonomy. The final product of each mode -or assembly- will be immeasurable, establishing different systems and methods for aesthetic practice without the value or success of the outcome ever being guaranteed.

New methods for the synthesis of radiation patterns of coupled antenna arrays = Nuevos métodos de síntesis de diagramas de radiación de agrupaciones de antenas acopladas

El principal objetivo de esta tesis es el desarrollo de métodos de síntesis de diagramas de radiación de agrupaciones de antenas, en donde se realiza una caracterización electromagnética rigurosa de los elementos radiantes y de los acoplos mutuos existentes. Esta caracterización no se realiza habitualmente en la gran mayoría de métodos de síntesis encontrados en la literatura, debido fundamentalmente a dos razones. Por un lado, se considera que el diagrama de radiación de un array de antenas se puede aproximar con el factor de array que únicamente tiene en cuenta la posición de los elementos y las excitaciones aplicadas a los mismos. Sin embargo, como se mostrará en esta tesis, en múltiples ocasiones un riguroso análisis de los elementos radiantes y del acoplo mutuo entre ellos es importante ya que los resultados obtenidos pueden ser notablemente diferentes. Por otro lado, no es sencillo combinar un método de análisis electromagnético con un proceso de síntesis de diagramas de radiación. Los métodos de análisis de agrupaciones de antenas suelen ser costosos computacionalmente, ya que son estructuras grandes en términos de longitudes de onda. Generalmente, un diseño de un problema electromagnético suele comprender varios análisis de la estructura, dependiendo de las variaciones de las características, lo que hace este proceso muy costoso. Dos métodos se utilizan en esta tesis para el análisis de los arrays acoplados. Ambos están basados en el método de los elementos finitos, la descomposición de dominio y el análisis modal para analizar la estructura radiante y han sido desarrollados en el grupo de investigación donde se engloba esta tesis. El primero de ellos es una técnica de análisis de arrays finitos basado en la aproximación de array infinito. Su uso es indicado para arrays planos de grandes dimensiones con elementos equiespaciados. El segundo caracteriza el array y el acoplo mutuo entre elementos a partir de una expansión en modos esféricos del campo radiado por cada uno de los elementos. Este método calcula los acoplos entre los diferentes elementos del array usando las propiedades de traslación y rotación de los modos esféricos. Es capaz de analizar agrupaciones de elementos distribuidos de forma arbitraria. Ambas técnicas utilizan una formulación matricial que caracteriza de forma rigurosa el campo radiado por el array. Esto las hace muy apropiadas para su posterior uso en una herramienta de diseño, como los métodos de síntesis desarrollados en esta tesis. Los resultados obtenidos por estas técnicas de síntesis, que incluyen métodos rigurosos de análisis, son consecuentemente más precisos. La síntesis de arrays consiste en modificar uno o varios parámetros de las agrupaciones de antenas buscando unas determinadas especificaciones de las características de radiación. Los parámetros utilizados como variables de optimización pueden ser varios. Los más utilizados son las excitaciones aplicadas a los elementos, pero también es posible modificar otros parámetros de diseño como son las posiciones de los elementos o las rotaciones de estos. Los objetivos de las síntesis pueden ser dirigir el haz o haces en una determinada dirección o conformar el haz con formas arbitrarias. Además, es posible minimizar el nivel de los lóbulos secundarios o del rizado en las regiones deseadas, imponer nulos que evitan posibles interferencias o reducir el nivel de la componente contrapolar. El método para el análisis de arrays finitos basado en la aproximación de array infinito considera un array finito como un array infinito con un número finito de elementos excitados. Los elementos no excitados están físicamente presentes y pueden presentar tres diferentes terminaciones, corto-circuito, circuito abierto y adaptados. Cada una de estas terminaciones simulará mejor el entorno real en el que el array se encuentre. Este método de análisis se integra en la tesis con dos métodos diferentes de síntesis de diagramas de radiación. En el primero de ellos se presenta un método basado en programación lineal en donde es posible dirigir el haz o haces, en la dirección deseada, además de ejercer un control sobre los lóbulos secundarios o imponer nulos. Este método es muy eficiente y obtiene soluciones óptimas. El mismo método de análisis es también aplicado a un método de conformación de haz, en donde un problema originalmente no convexo (y de difícil solución) es transformado en un problema convexo imponiendo restricciones de simetría, resolviendo de este modo eficientemente un problema complejo. Con este método es posible diseñar diagramas de radiación con haces de forma arbitraria, ejerciendo un control en el rizado del lóbulo principal, así como en el nivel de los lóbulos secundarios. El método de análisis de arrays basado en la expansión en modos esféricos se integra en la tesis con tres técnicas de síntesis de diagramas de radiación. Se propone inicialmente una síntesis de conformación del haz basado en el método de la recuperación de fase resuelta de forma iterativa mediante métodos convexos, en donde relajando las restricciones del problema original se consiguen unas soluciones cercanas a las óptimas de manera eficiente. Dos métodos de síntesis se han propuesto, donde las variables de optimización son las posiciones y las rotaciones de los elementos respectivamente. Se define una función de coste basada en la intensidad de radiación, la cual es minimizada de forma iterativa con el método del gradiente. Ambos métodos reducen el nivel de los lóbulos secundarios minimizando una función de coste. El gradiente de la función de coste es obtenido en términos de la variable de optimización en cada método. Esta función de coste está formada por la expresión rigurosa de la intensidad de radiación y por una función de peso definida por el usuario para imponer prioridades sobre las diferentes regiones de radiación, si así se desea. Por último, se presenta un método en el cual, mediante técnicas de programación entera, se buscan las fases discretas que generan un diagrama de radiación lo más cercano posible al deseado. Con este método se obtienen diseños que minimizan el coste de fabricación. En cada uno de las diferentes técnicas propuestas en la tesis, se presentan resultados con elementos reales que muestran las capacidades y posibilidades que los métodos ofrecen. Se comparan los resultados con otros métodos disponibles en la literatura. Se muestra la importancia de tener en cuenta los diagramas de los elementos reales y los acoplos mutuos en el proceso de síntesis y se comparan los resultados obtenidos con herramientas de software comerciales. ABSTRACT The main objective of this thesis is the development of optimization methods for the radiation pattern synthesis of array antennas in which a rigorous electromagnetic characterization of the radiators and the mutual coupling between them is performed. The electromagnetic characterization is usually overlooked in most of the available synthesis methods in the literature, this is mainly due to two reasons. On the one hand, it is argued that the radiation pattern of an array is mainly influenced by the array factor and that the mutual coupling plays a minor role. As it is shown in this thesis, the mutual coupling and the rigorous characterization of the array antenna influences significantly in the array performance and its computation leads to differences in the results obtained. On the other hand, it is difficult to introduce an analysis procedure into a synthesis technique. The analysis of array antennas is generally expensive computationally as the structure to analyze is large in terms of wavelengths. A synthesis method requires to carry out a large number of analysis, this makes the synthesis problem very expensive computationally or intractable in some cases. Two methods have been used in this thesis for the analysis of coupled antenna arrays, both of them have been developed in the research group in which this thesis is involved. They are based on the finite element method (FEM), the domain decomposition and the modal analysis. The first one obtains a finite array characterization with the results obtained from the infinite array approach. It is specially indicated for the analysis of large arrays with equispaced elements. The second one characterizes the array elements and the mutual coupling between them with a spherical wave expansion of the radiated field by each element. The mutual coupling is computed using the properties of translation and rotation of spherical waves. This method is able to analyze arrays with elements placed on an arbitrary distribution. Both techniques provide a matrix formulation that makes them very suitable for being integrated in synthesis techniques, the results obtained from these synthesis methods will be very accurate. The array synthesis stands for the modification of one or several array parameters looking for some desired specifications of the radiation pattern. The array parameters used as optimization variables are usually the excitation weights applied to the array elements, but some other array characteristics can be used as well, such as the array elements positions or rotations. The desired specifications may be to steer the beam towards any specific direction or to generate shaped beams with arbitrary geometry. Further characteristics can be handled as well, such as minimize the side lobe level in some other radiating regions, to minimize the ripple of the shaped beam, to take control over the cross-polar component or to impose nulls on the radiation pattern to avoid possible interferences from specific directions. The analysis method based on the infinite array approach considers an infinite array with a finite number of excited elements. The infinite non-excited elements are physically present and may have three different terminations, short-circuit, open circuit and match terminated. Each of this terminations is a better simulation for the real environment of the array. This method is used in this thesis for the development of two synthesis methods. In the first one, a multi-objective radiation pattern synthesis is presented, in which it is possible to steer the beam or beams in desired directions, minimizing the side lobe level and with the possibility of imposing nulls in the radiation pattern. This method is very efficient and obtains optimal solutions as it is based on convex programming. The same analysis method is used in a shaped beam technique in which an originally non-convex problem is transformed into a convex one applying symmetry restrictions, thus solving a complex problem in an efficient way. This method allows the synthesis of shaped beam radiation patterns controlling the ripple in the mainlobe and the side lobe level. The analysis method based on the spherical wave expansion is applied for different synthesis techniques of the radiation pattern of coupled arrays. A shaped beam synthesis is presented, in which a convex formulation is proposed based on the phase retrieval method. In this technique, an originally non-convex problem is solved using a relaxation and solving a convex problems iteratively. Two methods are proposed based on the gradient method. A cost function is defined involving the radiation intensity of the coupled array and a weighting function that provides more degrees of freedom to the designer. The gradient of the cost function is computed with respect to the positions in one of them and the rotations of the elements in the second one. The elements are moved or rotated iteratively following the results of the gradient. A highly non-convex problem is solved very efficiently, obtaining very good results that are dependent on the starting point. Finally, an optimization method is presented where discrete digital phases are synthesized providing a radiation pattern as close as possible to the desired one. The problem is solved using linear integer programming procedures obtaining array designs that greatly reduce the fabrication costs. Results are provided for every method showing the capabilities that the above mentioned methods offer. The results obtained are compared with available methods in the literature. The importance of introducing a rigorous analysis into the synthesis method is emphasized and the results obtained are compared with a commercial software, showing good agreement.

Improved modeling of photoluminescent and electroluminescent coupling in multijunction solar cells

The performance of tandem stacks of Group III?V multijunction solar cells continues to improve rapidly, both through improved performance of the individual cells in the stack and throughi ncrease in the number of stacked cells. As the radiative efficiency of these individual cells increases, radiative coupling between the stacked cells becomes an increasingly important factor not only in cell design, but also in accurate efficiency measurement and in determining performance of cells and systems under varying spectral conditions in the field. Past modeling has concentrated on electroluminescent coupling between the cells, although photoluminescent coupling is shown to be important for cells operating near their maximum power point voltage or below or when junction defect recombination is significant. Extension of earlier models i sproposed to allow this non-negligible component of luminescent coupling to be included. Therefined model is validated by measurement of the closely related external emission from both single and double junction cells.

Versatile 5′ phosphoryl coupling of small and large molecules to an RNA

A Ca2+-requiring catalytic RNA is shown to create 5′ phosphate–phosphate linkages with all nucleotides and coenzymes including CoA, nicotinamide adenine dinucleotide phosphate, thiamine phosphate, thiamine pyrophosphate, and flavin mononucleotide. In addition to these small molecules, macromolecules such as RNAs with 5′-diphosphates, and nonnucleotide molecules like Nɛ-phosphate arginine and 6-phosphate gluconic acid also react. That is, the self-capping RNA isolate 6 is an apparently universal 5′ phosphate-linker, reacting with any nucleophile containing an unblocked phosphate. These RNA reactions demonstrate a unique RNA catalytic capability and imply versatile and specific posttranscriptional RNA modification by RNA catalysis.

Translational coupling by modulation of feedback repression in the IF3 operon of Escherichia coli

A pseudoknot formed by a long-range interaction in the mRNA of the initiation factor 3 (IF3) operon is involved in the translational repression of the gene encoding ribosomal protein L35 by another ribosomal protein, L20. The nucleotides forming the 5′ strand of the key stem of the pseudoknot are located within the gene for IF3, whereas those forming the 3′ strand are located 280 nt downstream, immediately upstream of the Shine–Dalgarno sequence of the gene for L35. Here we show that premature termination of IF3 translation at a nonsense codon introduced upstream of the pseudoknot results in a substantial enhancement of L20-mediated repression of L35 expression. Conversely, an increase of IF3 translation decreases repression. These results, in addition to an analysis of the effect of mutations in sequences forming the pseudoknot, indicate that IF3 translation decreases L20-mediated repression of L35 expression. We propose that ribosomes translating IF3 disrupt the pseudoknot and thereby attenuate repression. The result is a novel type of translational coupling, where unfolding of the pseudoknot by ribosomes translating IF3 does not increase expression of L35 directly, but alleviates its repression by L20.

Analysis of exocytosis mutants indicates close coupling between regulated secretion and transcription activation in Tetrahymena

Stimulation of regulated secretory cells promotes protein release via the fusion of cytoplasmic storage vesicles with the plasma membrane. In Tetrahymena thermophila, brief exposure to secretagogue results in synchronous fusion of the entire set of docked dense-core granules with the plasma membrane. We show that stimulation is followed by rapid new dense-core granule synthesis involving gene induction. Two genes encoding granule matrix proteins, GRL1 and GRL4, are shown to undergo induction following stimulation, resulting in ≈10-fold message accumulation within 1 h. The mechanism of induction involves transcriptional regulation, and the upstream region of GRL1 functions in vivo as an inducible promoter in a heterologous reporter construct using the gene encoding green fluorescent protein. Taking advantage of the characterized exocytosis (exo−) mutants available in this system, we asked whether the signals for regranulation were generated directly by the initial stimulation, or whether downstream events were required for transcription activation. Three mutants, with defects at three distinct stages in the regulated secretory pathway, failed to show induction of GRL1 and GRL4 after exposure to secretagogue. These results argue that regranulation depends upon signals generated by the final steps in exocytosis.

Transcriptional coupling between the divergent promoters of a prototypic LysR-type regulatory system, the ilvYC operon of Escherichia coli

The twin-domain model [Liu, L. F. & Wang, J. C. (1987) Proc. Natl. Acad. Sci. USA 84, 7024–7027] suggests that closely spaced, divergent, superhelically sensitive promoters can affect the transcriptional activity of one another by transcriptionally induced negative DNA supercoiling generated in the divergent promoter region. This gene arrangement is observed for many LysR-type-regulated operons in bacteria. We have examined the effects of divergent transcription in the prototypic LysR-type system, the ilvYC operon of Escherichia coli. Double-reporter constructs with the lacZ gene under transcriptional control of the ilvC promoter and the galK gene under control of the divergent ilvY promoter were used to demonstrate that a down-promoter mutation in the ilvY promoter severely decreases in vivo transcription from the ilvC promoter. However, a down-promoter mutation in the ilvC promoter only slightly affects transcription from the ilvY promoter. In vitro transcription assays with DNA topoisomers showed that transcription from the ilvC promoter increases over the entire range of physiological superhelical densities, whereas transcription initiation from the ilvY promoter exhibits a broad optimum at a midphysiological superhelical density. Evidence that this promoter coupling is DNA supercoiling-dependent is provided by the observation that a novobiocin-induced decrease in global negative superhelicity results in an increase in ilvY promoter activity and a decrease in ilvC promoter activity predicted by the in vitro data. We suggest that this transcriptional coupling is important for coordinating basal level expression of the ilvYC operon with the nutritional and environmental conditions of cell growth.

Reducing the flexibility of retinal restores a wild-type-like photocycle in bacteriorhodopsin mutants defective in protein–retinal coupling

The thermal re-isomerization of retinal from the 13-cis to the all-trans state is a key step in the final stages of the photocycle of the light-driven proton pump, bacteriorhodopsin. This step is greatly slowed upon replacement of Leu-93, a residue in van der Waals contact with retinal. The most likely role of this key interaction is that it restricts the flexibility of retinal. To test this hypothesis, we have exchanged native retinal in Leu-93 mutants with bridged retinal analogs that render retinal less flexible by restricting free rotation around either the C10—C11 (9,11-bridged retinal) or C12—C13 (11,13-bridged retinal) single bonds. The effect of the analogs on the photocycle was then determined spectroscopically by taking advantage of the previous finding that the decay of the O intermediate in the Leu-93 mutants provides a convenient marker for retinal re-isomerization. Time-resolved spectroscopic studies showed that both retinal analogs resulted in a dramatic acceleration of the photocycling time by increasing the rate of decay of the O intermediate. In particular, exchange of native retinal in the Leu-93 → Ala mutant with the 9,11-bridged retinal resulted in an acceleration of the decay of the O intermediate to a rate similar to that seen in wild-type bacteriorhodopsin. We conclude that the protein-induced restriction of conformational flexibility in retinal is a key structural requirement for efficient protein–retinal coupling in the bacteriorhodopsin photocycle.

Ab initio calculation of electronic coupling in the photosynthetic reaction center

We have carried out an ab initio electronic structure calculations of electron transfer couplings between chromophores in the bacterial photosynthetic reaction center. The couplings agree remarkably well with parameters obtained from recent quantum dynamical modeling of experimental data assuming an explicit intermediate mechanism. We also have computed couplings on the M-side of the reaction center and have found that the interaction of the primary donor to the M-side intermediate bacteriochlorophyll is quite small because of destructive interference of the two localized coupling matrix elements. This may explain the slow rate of electron transfer down the M-side of the reaction center.