Contribución al diseño de lazos de realimentación electrónica para microsistemas electromecánicos (MEMS) resonantes: ruido de fase generado en lazos osciladores por sus realimentaciones

En 1966, D. B. Leeson publicó el artículo titulado “A simple model of feedback oscillator noise spectrum” en el que, mediante una ecuación obtenida de forma heurística y basada en parámetros conocidos de los osciladores, proponía un modelo para estimar el espectro de potencia que cuantifica el Ruido de Fase de estos osciladores. Este Ruido de Fase pone de manifiesto las fluctuaciones aleatorias que se producen en la fase de la señal de salida de cualquier oscilador de frecuencia f_0. Desde entonces, los adelantos tecnológicos han permitido grandes progresos en cuanto a la medida del Ruido de Fase, llegando a encontrar una estrecha “zona plana”, alrededor de f_0, conocida con el nombre de Ensanchamiento de Línea (EL) que Leeson no llegó a observar y que su modelo empírico no recogía. Paralelamente han ido surgiendo teorías que han tratado de explicar el Ruido de Fase con mayor o menor éxito. En esta Tesis se propone una nueva teoría para explicar el espectro de potencia del Ruido de Fase de un oscilador realimentado y basado en resonador L-C (Inductancia-Capacidad). Al igual que otras teorías, la nuestra también relaciona el Ruido de Fase del oscilador con el ruido térmico del circuito que lo implementa pero, a diferencia de aquellas, nuestra teoría se basa en un Modelo Complejo de ruido eléctrico que considera tanto las Fluctuaciones de energía eléctrica asociadas a la susceptancia capacitiva del resonador como las Disipaciones de energía eléctrica asociadas a su inevitable conductancia G=1⁄R, que dan cuenta del contacto térmico entre el resonador y el entorno térmico que le rodea. En concreto, la nueva teoría que proponemos explica tanto la parte del espectro del Ruido de Fase centrada alrededor de la frecuencia portadora f_0 que hemos llamado EL y su posterior caída proporcional a 〖∆f〗^(-2) al alejarnos de f_0, como la zona plana o pedestal que aparece en el espectro de Ruido de Fase lejos de esa f_0. Además, al saber cuantificar el EL y su origen, podemos explicar con facilidad la aparición de zonas del espectro de Ruido de Fase con caída 〖∆f〗^(-3) cercanas a la portadora y que provienen del denominado “exceso de ruido 1⁄f” de dispositivos de Estado Sólido y del ruido “flicker” de espectro 1⁄f^β (0,8≤β≤1,2) que aparece en dispositivos de vacío como las válvulas termoiónicas. Habiendo mostrado que una parte del Ruido de Fase de osciladores L-C realimentados que hemos denominado Ruido de Fase Térmico, se debe al ruido eléctrico de origen térmico de la electrónica que forma ese oscilador, proponemos en esta Tesis una nueva fuente de Ruido de Fase que hemos llamado Ruido de Fase Técnico, que se añadirá al Térmico y que aparecerá cuando el desfase del lazo a la frecuencia de resonancia f_0 del resonador no sea 0° o múltiplo entero de 360° (Condición Barkhausen de Fase, CBF). En estos casos, la modulación aleatoria de ganancia de lazo que realiza el Control Automático de Amplitud en su lucha contra ruidos que traten de variar la amplitud de la señal oscilante del lazo, producirá a su vez una modulación aleatoria de la frecuencia de tal señal que se observará como más Ruido de Fase añadido al Térmico. Para dar una prueba empírica sobre la existencia de esta nueva fuente de Ruido de Fase, se diseñó y construyó un oscilador en torno a un resonador mecánico “grande” para tener un Ruido de Fase Térmico despreciable a efectos prácticos. En este oscilador se midió su Ruido de Fase Técnico tanto en función del valor del desfase añadido al lazo de realimentación para apartarlo de su CBF, como en función de la perturbación de amplitud inyectada para mostrar sin ambigüedad la aparición de este Ruido de Fase Técnico cuando el lazo tiene este fallo técnico: que no cumple la Condición Barkhausen de Fase a la frecuencia de resonancia f_0 del resonador, por lo que oscila a otra frecuencia. ABSTRACT In 1966, D. B. Leeson published the article titled “A simple model of feedback oscillator noise spectrum” in which, by means of an equation obtained heuristically and based on known parameters of the oscillators, a model was proposed to estimate the power spectrum that quantifies the Phase Noise of these oscillators. This Phase Noise reveals the random fluctuations that are produced in the phase of the output signal from any oscillator of frequencyf_0. Since then, technological advances have allowed significant progress regarding the measurement of Phase Noise. This way, the narrow flat region that has been found around f_(0 ), is known as Line Widening (LW). This region that Leeson could not detect at that time does not appear in his empirical model. After Leeson’s work, different theories have appeared trying to explain the Phase Noise of oscillators. This Thesis proposes a new theory that explains the Phase Noise power spectrum of a feedback oscillator around a resonator L-C (Inductance-Capacity). Like other theories, ours also relates the oscillator Phase Noise to the thermal noise of the feedback circuitry, but departing from them, our theory uses a new, Complex Model for electrical noise that considers both Fluctuations of electrical energy associated with the capacitive susceptance of the resonator and Dissipations of electrical energy associated with its unavoidable conductance G=1/R, which accounts for the thermal contact between the resonator and its surrounding environment (thermal bath). More specifically, the new theory we propose explains both the Phase Noise region of the spectrum centered at the carrier frequency f_0 that we have called LW and shows a region falling as 〖∆f〗^(-2) as we depart from f_0, and the flat zone or pedestal that appears in the Phase Noise spectrum far from f_0. Being able to quantify the LW and its origin, we can easily explain the appearance of Phase Noise spectrum zones with 〖∆f〗^(-3) slope near the carrier that come from the so called “1/f excess noise” in Solid-State devices and “flicker noise” with 1⁄f^β (0,8≤β≤1,2) spectrum that appears in vacuum devices such as thermoionic valves. Having shown that the part of the Phase Noise of L-C oscillators that we have called Thermal Phase Noise is due to the electrical noise of the electronics used in the oscillator, this Thesis can propose a new source of Phase Noise that we have called Technical Phase Noise, which will appear when the loop phase shift to the resonance frequency f_0 is not 0° or an integer multiple of 360° (Barkhausen Phase Condition, BPC). This Phase Noise that will add to the Thermal one, comes from the random modulation of the loop gain carried out by the Amplitude Automatic Control fighting against noises trying to change the amplitude of the oscillating signal in the loop. In this case, the BPC failure gives rise to a random modulation of the frequency of the output signal that will be observed as more Phase Noise added to the Thermal one. To give an empirical proof on the existence of this new source of Phase Noise, an oscillator was designed and constructed around a “big” mechanical resonator whose Thermal Phase Noise is negligible for practical effects. The Technical Phase Noise of this oscillator has been measured with regard to the phase lag added to the feedback loop to separate it from its BPC, and with regard to the amplitude disturbance injected to show without ambiguity the appearance of this Technical Phase Noise that appears when the loop has this technical failure: that it does not fulfill the Barkhausen Phase Condition at f_0, the resonance frequency of the resonator and therefore it is oscillating at a frequency other than f_0.

Utilización de modelos hidráulicos bidimensionales en la determinación del tiempo de concentración

El tiempo de concentración de una cuenca sigue siendo relativamente desconocido para los ingenieros. El procedimiento habitual en un estudio hidrológico es calcularlo según varias fórmulas escogidas entre las existentes para después emplear el valor medio obtenido. De esta media se derivan los demás resultados hidrológicos, resultados que influirán en el futuro dimensionamiento de las infraestructuras. Este trabajo de investigación comenzó con el deseo de conseguir un método más fiable y objetivo que permitiera obtener el tiempo de concentración. Dada la imposibilidad de poner en práctica ensayos hidrológicos en una cuenca física real, ya que no resulta viable monitorizar perfectamente la precipitación ni los caudales de salida, se planteó llevar a cabo los ensayos de forma simulada, con el empleo de modelos hidráulicos bidimensionales de lluvia directa sobre malla 2D de volúmenes finitos. De entre todos los disponibles, se escogió InfoWorks ICM, por su rapidez y facilidad de uso. En una primera fase se efectuó la validación del modelo hidráulico elegido, contrastando los resultados de varias simulaciones con la formulación analítica existente. Posteriormente, se comprobaron los valores de los tiempos de concentración obtenidos con las expresiones referenciadas en la bibliografía, consiguiéndose resultados muy satisfactorios. Una vez verificado, se ejecutaron 690 simulaciones de cuencas tanto naturales como sintéticas, incorporando variaciones de área, pendiente, rugosidad, intensidad y duración de las precipitaciones, a fin de obtener sus tiempos de concentración y retardo. Esta labor se realizó con ayuda de la aceleración del cálculo vectorial que ofrece la tecnología CUDA (Arquitectura Unificada de Dispositivos de Cálculo). Basándose en el análisis dimensional, se agruparon los resultados del tiempo de concentración en monomios adimensionales. Utilizando regresión lineal múltiple, se obtuvo una nueva formulación para el tiempo de concentración. La nueva expresión se contrastó con las formulaciones clásicas, habiéndose obtenido resultados equivalentes. Con la exposición de esta nueva metodología se pretende ayudar al ingeniero en la realización de estudios hidrológicos. Primero porque proporciona datos de manera sencilla y objetiva que se pueden emplear en modelos globales como HEC-HMS. Y segundo porque en sí misma se ha comprobado como una alternativa realmente válida a la metodología hidrológica habitual. Time of concentration remains still fairly imprecise to engineers. A normal hydrological study goes through several formulae, obtaining concentration time as the median value. Most of the remaining hydrologic results will be derived from this value. Those results will determine how future infrastructures will be designed. This research began with the aim to acquire a more reliable and objective method to estimate concentration times. Given the impossibility of carrying out hydrological tests in a real watershed, due to the difficulties related to accurate monitoring of rainfall and derived outflows, a model-based approach was proposed using bidimensional hydraulic simulations of direct rainfall over a 2D finite-volume mesh. Amongst all of the available software packages, InfoWorks ICM was chosen for its speed and ease of use. As a preliminary phase, the selected hydraulic model was validated, checking the outcomes of several simulations over existing analytical formulae. Next, concentration time values were compared to those resulting from expressions referenced in the technical literature. They proved highly satisfactory. Once the model was properly verified, 690 simulations of both natural and synthetic basins were performed, incorporating variations of area, slope, roughness, intensity and duration of rainfall, in order to obtain their concentration and lag times. This job was carried out in a reasonable time lapse with the aid of the parallel computing platform technology CUDA (Compute Unified Device Architecture). Performing dimensional analysis, concentration time results were isolated in dimensionless monomials. Afterwards, a new formulation for the time of concentration was obtained using multiple linear regression. This new expression was checked against classical formulations, obtaining equivalent results. The publication of this new methodology intends to further assist the engineer while carrying out hydrological studies. It is effective to provide global parameters that will feed global models as HEC-HMS on a simple and objective way. It has also been proven as a solid alternative to usual hydrology methodology.

Conflicts for control of Mapuche-Pehuenche land and natural resources in the Biobío highlands

Análisis de las políticas aplicadas por el Estado chileno y sus efectos en el uso del territorio por parte de las comunidades indígenas de la zona del Alto Bíobio. Se observa que históricamente las comunidades han ido perdiendo el control de sus tierras

Hidrodinamica de planeadores submarinos y su impacto en la funcionalidad de sensores CTD

Los recientes desarrollos tecnológicos permiten la transición de la oceanografía observacional desde un concepto basado en buques a uno basado en sistemas autónomos en red. Este último, propone que la forma más eficiente y efectiva de observar el océano es con una red de plataformas autónomas distribuidas espacialmente y complementadas con sistemas de medición remota. Debido a su maniobrabilidad y autonomía, los planeadores submarinos están jugando un papel relevante en este concepto de observaciones en red. Los planeadores submarinos fueron específicamente diseñados para muestrear vastas zonas del océano. Estos son robots con forma de torpedo que hacen uso de su forma hidrodinámica, alas y cambios de flotabilidad para generar movimientos horizontales y verticales en la columna de agua. Un sensor que mide conductividad, temperatura y profundidad (CTD) constituye un equipamiento estándar en la plataforma. Esto se debe a que ciertas variables dinámicas del Océano se pueden derivar de la temperatura, profundidad y salinidad. Esta última se puede estimar a partir de las medidas de temperatura y conductividad. La integración de sensores CTD en planeadores submarinos no esta exenta de desafíos. Uno de ellos está relacionado con la precisión de los valores de salinidad derivados de las muestras de temperatura y conductividad. Específicamente, las estimaciones de salinidad están significativamente degradadas por el retardo térmico existente, entre la temperatura medida y la temperatura real dentro de la celda de conductividad del sensor. Esta deficiencia depende de las particularidades del flujo de entrada al sensor, su geometría y, también se ha postulado, del calor acumulado en las capas de aislamiento externo del sensor. Los efectos del retardo térmico se suelen mitigar mediante el control del flujo de entrada al sensor. Esto se obtiene generalmente mediante el bombeo de agua a través del sensor o manteniendo constante y conocida su velocidad. Aunque recientemente se han incorporado sistemas de bombeo en los CTDs a bordo de los planeadores submarinos, todavía existen plataformas equipadas con CTDs sin dichos sistemas. En estos casos, la estimación de la salinidad supone condiciones de flujo de entrada al sensor, razonablemente controladas e imperturbadas. Esta Tesis investiga el impacto, si existe, que la hidrodinámica de los planeadores submarinos pudiera tener en la eficiencia de los sensores CTD. Específicamente, se investiga primero la localización del sensor CTD (externo al fuselaje) relativa a la capa límite desarrollada a lo largo del cuerpo del planeador. Esto se lleva a cabo mediante la utilización de un modelo acoplado de fluido no viscoso con un modelo de capa límite implementado por el autor, así como mediante un programa comercial de dinámica de fluidos computacional (CFD). Los resultados indican, en ambos casos, que el sensor CTD se encuentra fuera de la capa límite, siendo las condiciones del flujo de entrada las mismas que las del flujo sin perturbar. Todavía, la velocidad del flujo de entrada al sensor CTD es la velocidad de la plataforma, la cual depende de su hidrodinámica. Por tal motivo, la investigación se ha extendido para averiguar el efecto que la velocidad de la plataforma tiene en la eficiencia del sensor CTD. Con este propósito, se ha desarrollado un modelo en elementos finitos del comportamiento hidrodinámico y térmico del flujo dentro del CTD. Los resultados numéricos indican que el retardo térmico, atribuidos originalmente a la acumulación de calor en la estructura del sensor, se debe fundamentalmente a la interacción del flujo que atraviesa la celda de conductividad con la geometría interna de la misma. Esta interacción es distinta a distintas velocidades del planeador submarino. Específicamente, a velocidades bajas del planeador (0.2 m/s), la mezcla del flujo entrante con las masas de agua remanentes en el interior de la celda, se ralentiza debido a la generación de remolinos. Se obtienen entonces desviaciones significantes entre la salinidad real y aquella estimada. En cambio, a velocidades más altas del planeador (0.4 m/s) los procesos de mezcla se incrementan debido a la turbulencia e inestabilidades. En consecuencia, la respuesta del sensor CTD es mas rápida y las estimaciones de la salinidad mas precisas que en el caso anterior. Para completar el trabajo, los resultados numéricos se han validado con pruebas experimentales. Específicamente, se ha construido un modelo a escala del sensor CTD para obtener la confirmación experimental de los modelos numéricos. Haciendo uso del principio de similaridad de la dinámica que gobierna los fluidos incompresibles, los experimentos se han realizado con flujos de aire. Esto simplifica significativamente la puesta experimental y facilita su realización en condiciones con medios limitados. Las pruebas experimentales han confirmado cualitativamente los resultados numéricos. Más aun, se sugiere en esta Tesis que la respuesta del sensor CTD mejoraría significativamente añadiendo un generador de turbulencia en localizaciones adecuadas al interno de la celda de conductividad. ABSTRACT Recent technological developments allow the transition of observational oceanography from a ship-based to a networking concept. The latter suggests that the most efficient and effective way to observe the Ocean is through a fleet of spatially distributed autonomous platforms complemented by remote sensing. Due to their maneuverability, autonomy and endurance at sea, underwater gliders are already playing a significant role in this networking observational approach. Underwater gliders were specifically designed to sample vast areas of the Ocean. These are robots with a torpedo shape that make use of their hydrodynamic shape, wings and buoyancy changes to induce horizontal and vertical motions through the water column. A sensor to measure the conductivity, temperature and depth (CTD) is a standard payload of this platform. This is because certain ocean dynamic variables can be derived from temperature, depth and salinity. The latter can be inferred from measurements of temperature and conductivity. Integrating CTD sensors in glider platforms is not exempted of challenges. One of them, concerns to the accuracy of the salinity values derived from the sampled conductivity and temperature. Specifically, salinity estimates are significantly degraded by the thermal lag response existing between the measured temperature and the real temperature inside the conductivity cell of the sensor. This deficiency depends on the particularities of the inflow to the sensor, its geometry and, it has also been hypothesized, on the heat accumulated by the sensor coating layers. The effects of thermal lag are usually mitigated by controlling the inflow conditions through the sensor. Controlling inflow conditions is usually achieved by pumping the water through the sensor or by keeping constant and known its diving speed. Although pumping systems have been recently implemented in CTD sensors on board gliders, there are still platforms with unpumped CTDs. In the latter case, salinity estimates rely on assuming reasonable controlled and unperturbed flow conditions at the CTD sensor. This Thesis investigates the impact, if any, that glider hydrodynamics may have on the performance of onboard CTDs. Specifically, the location of the CTD sensor (external to the hull) relative to the boundary layer developed along the glider fuselage, is first investigated. This is done, initially, by applying a coupled inviscid-boundary layer model developed by the author, and later by using a commercial software for computational fluid dynamics (CFD). Results indicate, in both cases, that the CTD sensor is out of the boundary layer, being its inflow conditions those of the free stream. Still, the inflow speed to the CTD sensor is the speed of the platform, which largely depends on its hydrodynamic setup. For this reason, the research has been further extended to investigate the effect of the platform speed on the performance of the CTD sensor. A finite element model of the hydrodynamic and thermal behavior of the flow inside the CTD sensor, is developed for this purpose. Numerical results suggest that the thermal lag effect is mostly due to the interaction of the flow through the conductivity cell and its geometry. This interaction is different at different speeds of the glider. Specifically, at low glider speeds (0.2 m/s), the mixing of recent and old waters inside the conductivity cell is slowed down by the generation of coherent eddy structures. Significant departures between real and estimated values of the salinity are found. Instead, mixing is enhanced by turbulence and instabilities for high glider speeds (0.4 m/s). As a result, the thermal response of the CTD sensor is faster and the salinity estimates more accurate than for the low speed case. For completeness, numerical results have been validated against model tests. Specifically, a scaled model of the CTD sensor was built to obtain experimental confirmation of the numerical results. Making use of the similarity principle of the dynamics governing incompressible fluids, experiments are carried out with air flows. This significantly simplifies the experimental setup and facilitates its realization in a limited resource condition. Model tests qualitatively confirm the numerical findings. Moreover, it is suggested in this Thesis that the response of the CTD sensor would be significantly improved by adding small turbulators at adequate locations inside the conductivity cell.

Propuesta de un método de entrenamiento de la RSA en baloncesto

La capacidad de repetir sprints (RSA) es una cualidad determinante del rendimiento de los deportistas en los deportes de equipo y de raqueta debido a la sucesión de esfuerzos máximos que tienen lugar durante la competición con un tiempo de descanso generalmente incompleto entre las acciones. Debido a esos descansos incompletos se produce una disminución del rendimiento que se manifiesta en la reducción de la velocidad de desplazamiento. Los factores que provocan este hecho están condicionados por la potencia muscular, la condición física aeróbica y la capacidad tampón del músculo. Con toda la información obtenida se realizan dos propuestas de entrenamiento (una para cada objetivo) de la RSA de forma integrada en el baloncesto, planteando diferentes ejercicios para su desarrollo.

Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex

The Arp2/3 complex, a stable assembly of two actin-related proteins (Arp2 and Arp3) with five other subunits, caps the pointed end of actin filaments and nucleates actin polymerization with low efficiency. WASp and Scar are two similar proteins that bind the p21 subunit of the Arp2/3 complex, but their effect on the nucleation activity of the complex was not known. We report that full-length, recombinant human Scar protein, as well as N-terminally truncated Scar proteins, enhance nucleation by the Arp2/3 complex. By themselves, these proteins either have no effect or inhibit actin polymerization. The actin monomer-binding W domain and the p21-binding A domain from the C terminus of Scar are both required to activate Arp2/3 complex. A proline-rich domain in the middle of Scar enhances the activity of the W and A domains. Preincubating Scar and Arp2/3 complex with actin filaments overcomes the initial lag in polymerization, suggesting that efficient nucleation by the Arp2/3 complex requires assembly on the side of a preexisting filament—a dendritic nucleation mechanism. The Arp2/3 complex with full-length Scar, Scar containing P, W, and A domains, or Scar containing W and A domains overcomes inhibition of nucleation by the actin monomer-binding protein profilin, giving active nucleation over a low background of spontaneous nucleation. These results show that Scar and, likely, related proteins, such as the Cdc42 targets WASp and N-WASp, are endogenous activators of actin polymerization by the Arp2/3 complex.

Terpenoid-based defenses in conifers: cDNA cloning, characterization, and functional expression of wound-inducible (E)-α-bisabolene synthase from grand fir (Abies grandis)

(E)-α-Bisabolene synthase is one of two wound-inducible sesquiterpene synthases of grand fir (Abies grandis), and the olefin product of this cyclization reaction is considered to be the precursor in Abies species of todomatuic acid, juvabione, and related insect juvenile hormone mimics. A cDNA encoding (E)-α-bisabolene synthase was isolated from a wound-induced grand fir stem library by a PCR-based strategy and was functionally expressed in Escherichia coli and shown to produce (E)-α-bisabolene as the sole product from farnesyl diphosphate. The expressed synthase has a deduced size of 93.8 kDa and a pI of 5.03, exhibits other properties typical of sesquiterpene synthases, and resembles in sequence other terpenoid synthases with the exception of a large amino-terminal insertion corresponding to Pro81–Val296. Biosynthetically prepared (E)-α-[3H]bisabolene was converted to todomatuic acid in induced grand fir cells, and the time course of appearance of bisabolene synthase mRNA was shown by Northern hybridization to lag behind that of mRNAs responsible for production of induced oleoresin monoterpenes. These results suggest that induced (E)-α-bisabolene biosynthesis constitutes part of a defense response targeted to insect herbivores, and possibly fungal pathogens, that is distinct from induced oleoresin monoterpene production.

Inorganic polyphosphate kinase is required to stimulate protein degradation and for adaptation to amino acid starvation in Escherichia coli

Inorganic polyphosphate (polyP) kinase was studied for its roles in physiological responses to nutritional deprivation in Escherichia coli. A mutant lacking polyP kinase exhibited an extended lag phase of growth, when shifted from a rich to a minimal medium (nutritional downshift). Supplementation of amino acids to the minimal medium abolished the extended growth lag of the mutant. Levels of the stringent response factor, guanosine 5′-diphosphate 3′-diphosphate, increased in response to the nutritional downshift, but, unlike in the wild type, the levels were sustained in the mutant. These results suggested that the mutant was impaired in the induction of amino acid biosynthetic enzymes. The expression of an amino acid biosynthetic gene, hisG, was examined by using a transcriptional lacZ fusion. Although the mutant did not express the fusion in response to the nutritional downshift, Northern blot analysis revealed a significant increase of hisG-lacZ mRNA. Amino acids generated by intracellular protein degradation are very important for the synthesis of enzymes at the onset of starvation. In the wild type, the rate of protein degradation increased in response to the nutritional downshift whereas it did not in the mutant. Supplementation of amino acids at low concentrations to the minimal medium enabled the mutant to express the hisG-lacZ fusion. Thus, the impaired regulation of protein degradation results in the adaptation defect, suggesting that polyP kinase is required to stimulate protein degradation.

Working memory constrains human cooperation in the Prisoner’s Dilemma

Many problems in human society reflect the inability of selfish parties to cooperate. The “Iterated Prisoner’s Dilemma” has been used widely as a model for the evolution of cooperation in societies. Axelrod’s computer tournaments and the extensive simulations of evolution by Nowak and Sigmund and others have shown that natural selection can favor cooperative strategies in the Prisoner’s Dilemma. Rigorous empirical tests, however, lag behind the progress made by theorists. Clear predictions differ depending on the players’ capacity to remember previous rounds of the game. To test whether humans use the kind of cooperative strategies predicted, we asked students to play the iterated Prisoner’s Dilemma game either continuously or interrupted after each round by a secondary memory task (i.e., playing the game “Memory”) that constrained the students’ working-memory capacity. When playing without interruption, most students used “Pavlovian” strategies, as predicted, for greater memory capacity, and the rest used “generous tit-for-tat” strategies. The proportion of generous tit-for-tat strategies increased when games of Memory interfered with the subjects’ working memory, as predicted. Students who continued to use complex Pavlovian strategies were less successful in the Memory game, but more successful in the Prisoner’s Dilemma, which indicates a trade-off in memory capacity for the two tasks. Our results suggest that the set of strategies predicted by game theorists approximates human reality.

Modulatory Roles for Integrin Activation and the Synergy Site of Fibronectin during Matrix Assembly

Initiation of fibronectin (FN) matrix assembly is dependent on specific interactions between FN and cell surface integrin receptors. Here, we show that de novo FN matrix assembly exhibits a slow phase during initiation of fibrillogenesis followed by a more rapid growth phase. Mn2+, which acts by enhancing integrin function, increased the rate of FN fibril growth, but only after the initial lag phase. The RGD cell-binding sequence in type III repeat 10 is an absolute requirement for initiation by α5β1 integrin. To investigate the role of the cell-binding synergy site in the adjacent repeat III9, a full-length recombinant FN containing a synergy mutation, FN(syn−), was tested for its ability to form fibrils. Mutation of this site drastically reduced FN assembly by CHOα5 cells. Only sparse short fibrils were formed even after prolonged incubation, indicating that FN(syn−) is defective in progression of the assembly process. These results show that the synergy site is essential for α5β1-mediated accumulation of a FN matrix. However, the incorporation of FN(syn−) into fibrils and the deoxycholate-insoluble matrix could be stimulated by Mn2+. Therefore, exogenous activation of integrin receptors can overcome the requirement for FN’s synergy site as well as modulate the rate of FN matrix formation.

Accumulation of Major Histocompatibility Complex Class II Molecules in Mast Cell Secretory Granules and Their Release upon Degranulation

To investigate the relationship between major histocompatibility complex (MHC) class II compartments, secretory granules, and secretory lysosomes, we analyzed the localization and fate of MHC class II molecules in mast cells. In bone marrow-derived mast cells, the bulk of MHC class II molecules is contained in two distinct compartments, with features of both lysosomal compartments and secretory granules defined by their protein content and their accessibility to endocytic tracers. Type I granules display internal membrane vesicles and are accessed by exogenous molecules after a time lag of 20 min; type II granules are reached by the endocytic tracer later and possess a serotonin-rich electron-dense core surrounded by a multivesicular domain. In these type I and type II granules, MHC class II molecules, mannose-6-phosphate receptors and lysosomal membrane proteins (lamp1 and lamp2) localize to small intralumenal vesicles. These 60–80-nm vesicles are released along with inflammatory mediators during mast cell degranulation triggered by IgE-antigen complexes. These observations emphasize the intimate connection between the endocytic and secretory pathways in cells of the hematopoietic lineage which allows regulated secretion of the contents of secretory lysosomes, including membrane proteins associated with small vesicles.

Characterization of a replication-incompetent adenovirus type 5 mutant deleted for the preterminal protein gene

An adenovirus type 5 mutant deleted for the preterminal protein (pTP) gene was constructed using cell lines that express pTP. The pTP deletion mutant virus is incapable of replicating in the absence of complementation and does not express detectable levels of viral mRNAs that are expressed only after the onset of replication. Accumulation of early-region mRNAs, including that for E1A, exhibits a lag relative to that observed from the wild-type virus. However, E1A mRNA accumulation attains a steady-state level similar to the level of expression during the early phase of infection with the wild-type virus. In 293-pTP cells (human embryonic kidney cells that express pTP in addition to high levels of adenovirus E1A and E1B proteins), the pTP deletion mutant virus replicates efficiently and yields infectious titers within 5-fold of that of the wild-type virus. The deletion of 1.2 kb of pTP-encoding sequence increases the size of foreign DNA that can be introduced into the virus and, with an absolute block to replication, makes this virus an important tool for gene therapy.

Cyclin E, a redundant cyclin in breast cancer

Cyclin E is an important regulator of cell cycle progression that together with cyclin-dependent kinase (cdk) 2 is crucial for the G1/S transition during the mammalian cell cycle. Previously, we showed that severe overexpression of cyclin E protein in tumor cells and tissues results in the appearance of lower molecular weight isoforms of cyclin E, which together with cdk2 can form a kinase complex active throughout the cell cycle. In this study, we report that one of the substrates of this constitutively active cyclin E/cdk2 complex is retinoblastoma susceptibility gene product (pRb) in populations of breast cancer cells and tissues that also overexpress p16. In these tumor cells and tissues, we show that the expression of p16 and pRb is not mutually exclusive. Overexpression of p16 in these cells results in sequestering of cdk4 and cdk6, rendering cyclin D1/cdk complexes inactive. However, pRb appears to be phosphorylated throughout the cell cycle following an initial lag, revealing a time course similar to phosphorylation of glutathione S-transferase retinoblastoma by cyclin E immunoprecipitates prepared from these synchronized cells. Hence, cyclin E kinase complexes can function redundantly and replace the loss of cyclin D-dependent kinase complexes that functionally inactivate pRb. In addition, the constitutively overexpressed cyclin E is also the predominant cyclin found in p107/E2F complexes throughout the tumor, but not the normal, cell cycle. These observations suggest that overexpression of cyclin E in tumor cells, which also overexpress p16, can bypass the cyclin D/cdk4-cdk6/p16/pRb feedback loop, providing yet another mechanism by which tumors can gain a growth advantage.

Zic2 regulates the kinetics of neurulation

Mutation in human ZIC2, a zinc finger protein homologous to Drosophila odd-paired, causes holoprosencephaly (HPE), which is a common, severe malformation of the brain in humans. However, the pathogenesis is largely unknown. Here we show that reduced expression (knockdown) of mouse Zic2 causes neurulation delay, resulting in HPE and spina bifida. Differentiation of the most dorsal neural plate, which gives rise to both roof plate and neural crest cells, also was delayed as indicated by the expression lag of a roof plate marker, Wnt3a. In addition the development of neural crest derivatives such as dorsal root ganglion was impaired. These results suggest that the Zic2 expression level is crucial for the timing of neurulation. Because the Zic2 knockdown mouse is the first mutant with HPE and spina bifida to survive to the perinatal period, the mouse will promote analyses of not only the neurulation but also the pathogenesis of human HPE.

Dissection of nodulation signaling using pea mutants defective for calcium spiking induced by Nod factors and chitin oligomers

Changes in intracellular calcium in pea root hairs responding to Rhizobium leguminosarum bv. viciae nodulation (Nod) factors were analyzed by using a microinjected calcium-sensitive fluorescent dye (dextran-linked Oregon Green). Within 1–2 min after Nod-factor addition, there was usually an increase in fluorescence, followed about 10 min later by spikes in fluorescence occurring at a rate of about one spike per minute. These spikes, corresponding to an increase in calcium of ≈200 nM, were localized around the nuclear region, and they were similar in terms of lag and period to those induced by Nod factors in alfalfa. Calcium responses were analyzed in nonnodulating pea mutants, representing seven loci that affect early stages of the symbiosis. Mutations affecting three loci (sym8, sym10, and sym19) abolished Nod-factor-induced calcium spiking, whereas a normal response was seen in peas carrying alleles of sym2A, sym7, sym9, and sym30. Chitin oligomers of four or five N-acetylglucosamine residues could also induce calcium spiking, although the response was qualitatively different from that induced by Nod factors; a rapid increase in intracellular calcium was not observed, the period between spikes was lower, and the response was not as sustained. The chitin-oligomer-induced calcium spiking did not occur in nodulation mutants (sym8, sym10, and sym19) that were defective for Nod-factor-induced spiking, suggesting that this response is related to nodulation signaling. From our data and previous observations on the lack of mycorrhizal infection in some of the sym mutants, we propose a model for the potential order of pea nodulation genes in nodulation and mycorrhizal signaling.