Sensitivity and Fragmentation Calibration of the ROSINA Reflectron-type Time-Of-Flight Mass Spectrometer

The goal of this work was to increase the performance and to calibrate one of the ROSINA sensors, the Reflectron-type Time-Of-Flight mass spectrometer, currently flying aboard the ESA Rosetta spacecraft. Different optimization techniques were applied to both the lab and space models, and a static calibration was performed using different gas species expected to be detected in the vicinity of comet 67P/Churyumov-Gerasimenko. The database thus created was successfully applied to space data, giving consistent results with the other ROSINA sensors.

On-Chip Thermal Monitoring: Design, Placement and Interconnection of Temperature Sensors

La temperatura es una preocupación que juega un papel protagonista en el diseño de circuitos integrados modernos. El importante aumento de las densidades de potencia que conllevan las últimas generaciones tecnológicas ha producido la aparición de gradientes térmicos y puntos calientes durante el funcionamiento normal de los chips. La temperatura tiene un impacto negativo en varios parámetros del circuito integrado como el retardo de las puertas, los gastos de disipación de calor, la fiabilidad, el consumo de energía, etc. Con el fin de luchar contra estos efectos nocivos, la técnicas de gestión dinámica de la temperatura (DTM) adaptan el comportamiento del chip en función en la información que proporciona un sistema de monitorización que mide en tiempo de ejecución la información térmica de la superficie del dado. El campo de la monitorización de la temperatura en el chip ha llamado la atención de la comunidad científica en los últimos años y es el objeto de estudio de esta tesis. Esta tesis aborda la temática de control de la temperatura en el chip desde diferentes perspectivas y niveles, ofreciendo soluciones a algunos de los temas más importantes. Los niveles físico y circuital se cubren con el diseño y la caracterización de dos nuevos sensores de temperatura especialmente diseñados para los propósitos de las técnicas DTM. El primer sensor está basado en un mecanismo que obtiene un pulso de anchura variable dependiente de la relación de las corrientes de fuga con la temperatura. De manera resumida, se carga un nodo del circuito y posteriormente se deja flotando de tal manera que se descarga a través de las corrientes de fugas de un transistor; el tiempo de descarga del nodo es la anchura del pulso. Dado que la anchura del pulso muestra una dependencia exponencial con la temperatura, la conversión a una palabra digital se realiza por medio de un contador logarítmico que realiza tanto la conversión tiempo a digital como la linealización de la salida. La estructura resultante de esta combinación de elementos se implementa en una tecnología de 0,35 _m. El sensor ocupa un área muy reducida, 10.250 nm2, y consume muy poca energía, 1.05-65.5nW a 5 muestras/s, estas cifras superaron todos los trabajos previos en el momento en que se publicó por primera vez y en el momento de la publicación de esta tesis, superan a todas las implementaciones anteriores fabricadas en el mismo nodo tecnológico. En cuanto a la precisión, el sensor ofrece una buena linealidad, incluso sin calibrar; se obtiene un error 3_ de 1,97oC, adecuado para tratar con las aplicaciones de DTM. Como se ha explicado, el sensor es completamente compatible con los procesos de fabricación CMOS, este hecho, junto con sus valores reducidos de área y consumo, lo hacen especialmente adecuado para la integración en un sistema de monitorización de DTM con un conjunto de monitores empotrados distribuidos a través del chip. Las crecientes incertidumbres de proceso asociadas a los últimos nodos tecnológicos comprometen las características de linealidad de nuestra primera propuesta de sensor. Con el objetivo de superar estos problemas, proponemos una nueva técnica para obtener la temperatura. La nueva técnica también está basada en las dependencias térmicas de las corrientes de fuga que se utilizan para descargar un nodo flotante. La novedad es que ahora la medida viene dada por el cociente de dos medidas diferentes, en una de las cuales se altera una característica del transistor de descarga |la tensión de puerta. Este cociente resulta ser muy robusto frente a variaciones de proceso y, además, la linealidad obtenida cumple ampliamente los requisitos impuestos por las políticas DTM |error 3_ de 1,17oC considerando variaciones del proceso y calibrando en dos puntos. La implementación de la parte sensora de esta nueva técnica implica varias consideraciones de diseño, tales como la generación de una referencia de tensión independiente de variaciones de proceso, que se analizan en profundidad en la tesis. Para la conversión tiempo-a-digital, se emplea la misma estructura de digitalización que en el primer sensor. Para la implementación física de la parte de digitalización, se ha construido una biblioteca de células estándar completamente nueva orientada a la reducción de área y consumo. El sensor resultante de la unión de todos los bloques se caracteriza por una energía por muestra ultra baja (48-640 pJ) y un área diminuta de 0,0016 mm2, esta cifra mejora todos los trabajos previos. Para probar esta afirmación, se realiza una comparación exhaustiva con más de 40 propuestas de sensores en la literatura científica. Subiendo el nivel de abstracción al sistema, la tercera contribución se centra en el modelado de un sistema de monitorización que consiste de un conjunto de sensores distribuidos por la superficie del chip. Todos los trabajos anteriores de la literatura tienen como objetivo maximizar la precisión del sistema con el mínimo número de monitores. Como novedad, en nuestra propuesta se introducen nuevos parámetros de calidad aparte del número de sensores, también se considera el consumo de energía, la frecuencia de muestreo, los costes de interconexión y la posibilidad de elegir diferentes tipos de monitores. El modelo se introduce en un algoritmo de recocido simulado que recibe la información térmica de un sistema, sus propiedades físicas, limitaciones de área, potencia e interconexión y una colección de tipos de monitor; el algoritmo proporciona el tipo seleccionado de monitor, el número de monitores, su posición y la velocidad de muestreo _optima. Para probar la validez del algoritmo, se presentan varios casos de estudio para el procesador Alpha 21364 considerando distintas restricciones. En comparación con otros trabajos previos en la literatura, el modelo que aquí se presenta es el más completo. Finalmente, la última contribución se dirige al nivel de red, partiendo de un conjunto de monitores de temperatura de posiciones conocidas, nos concentramos en resolver el problema de la conexión de los sensores de una forma eficiente en área y consumo. Nuestra primera propuesta en este campo es la introducción de un nuevo nivel en la jerarquía de interconexión, el nivel de trillado (o threshing en inglés), entre los monitores y los buses tradicionales de periféricos. En este nuevo nivel se aplica selectividad de datos para reducir la cantidad de información que se envía al controlador central. La idea detrás de este nuevo nivel es que en este tipo de redes la mayoría de los datos es inútil, porque desde el punto de vista del controlador sólo una pequeña cantidad de datos |normalmente sólo los valores extremos| es de interés. Para cubrir el nuevo nivel, proponemos una red de monitorización mono-conexión que se basa en un esquema de señalización en el dominio de tiempo. Este esquema reduce significativamente tanto la actividad de conmutación sobre la conexión como el consumo de energía de la red. Otra ventaja de este esquema es que los datos de los monitores llegan directamente ordenados al controlador. Si este tipo de señalización se aplica a sensores que realizan conversión tiempo-a-digital, se puede obtener compartición de recursos de digitalización tanto en tiempo como en espacio, lo que supone un importante ahorro de área y consumo. Finalmente, se presentan dos prototipos de sistemas de monitorización completos que de manera significativa superan la características de trabajos anteriores en términos de área y, especialmente, consumo de energía. Abstract Temperature is a first class design concern in modern integrated circuits. The important increase in power densities associated to recent technology evolutions has lead to the apparition of thermal gradients and hot spots during run time operation. Temperature impacts several circuit parameters such as speed, cooling budgets, reliability, power consumption, etc. In order to fight against these negative effects, dynamic thermal management (DTM) techniques adapt the behavior of the chip relying on the information of a monitoring system that provides run-time thermal information of the die surface. The field of on-chip temperature monitoring has drawn the attention of the scientific community in the recent years and is the object of study of this thesis. This thesis approaches the matter of on-chip temperature monitoring from different perspectives and levels, providing solutions to some of the most important issues. The physical and circuital levels are covered with the design and characterization of two novel temperature sensors specially tailored for DTM purposes. The first sensor is based upon a mechanism that obtains a pulse with a varying width based on the variations of the leakage currents on the temperature. In a nutshell, a circuit node is charged and subsequently left floating so that it discharges away through the subthreshold currents of a transistor; the time the node takes to discharge is the width of the pulse. Since the width of the pulse displays an exponential dependence on the temperature, the conversion into a digital word is realized by means of a logarithmic counter that performs both the timeto- digital conversion and the linearization of the output. The structure resulting from this combination of elements is implemented in a 0.35_m technology and is characterized by very reduced area, 10250 nm2, and power consumption, 1.05-65.5 nW at 5 samples/s, these figures outperformed all previous works by the time it was first published and still, by the time of the publication of this thesis, they outnumber all previous implementations in the same technology node. Concerning the accuracy, the sensor exhibits good linearity, even without calibration it displays a 3_ error of 1.97oC, appropriate to deal with DTM applications. As explained, the sensor is completely compatible with standard CMOS processes, this fact, along with its tiny area and power overhead, makes it specially suitable for the integration in a DTM monitoring system with a collection of on-chip monitors distributed across the chip. The exacerbated process fluctuations carried along with recent technology nodes jeop-ardize the linearity characteristics of the first sensor. In order to overcome these problems, a new temperature inferring technique is proposed. In this case, we also rely on the thermal dependencies of leakage currents that are used to discharge a floating node, but now, the result comes from the ratio of two different measures, in one of which we alter a characteristic of the discharging transistor |the gate voltage. This ratio proves to be very robust against process variations and displays a more than suficient linearity on the temperature |1.17oC 3_ error considering process variations and performing two-point calibration. The implementation of the sensing part based on this new technique implies several issues, such as the generation of process variations independent voltage reference, that are analyzed in depth in the thesis. In order to perform the time-to-digital conversion, we employ the same digitization structure the former sensor used. A completely new standard cell library targeting low area and power overhead is built from scratch to implement the digitization part. Putting all the pieces together, we achieve a complete sensor system that is characterized by ultra low energy per conversion of 48-640pJ and area of 0.0016mm2, this figure outperforms all previous works. To prove this statement, we perform a thorough comparison with over 40 works from the scientific literature. Moving up to the system level, the third contribution is centered on the modeling of a monitoring system consisting of set of thermal sensors distributed across the chip. All previous works from the literature target maximizing the accuracy of the system with the minimum number of monitors. In contrast, we introduce new metrics of quality apart form just the number of sensors; we consider the power consumption, the sampling frequency, the possibility to consider different types of monitors and the interconnection costs. The model is introduced in a simulated annealing algorithm that receives the thermal information of a system, its physical properties, area, power and interconnection constraints and a collection of monitor types; the algorithm yields the selected type of monitor, the number of monitors, their position and the optimum sampling rate. We test the algorithm with the Alpha 21364 processor under several constraint configurations to prove its validity. When compared to other previous works in the literature, the modeling presented here is the most complete. Finally, the last contribution targets the networking level, given an allocated set of temperature monitors, we focused on solving the problem of connecting them in an efficient way from the area and power perspectives. Our first proposal in this area is the introduction of a new interconnection hierarchy level, the threshing level, in between the monitors and the traditional peripheral buses that applies data selectivity to reduce the amount of information that is sent to the central controller. The idea behind this new level is that in this kind of networks most data are useless because from the controller viewpoint just a small amount of data |normally extreme values| is of interest. To cover the new interconnection level, we propose a single-wire monitoring network based on a time-domain signaling scheme that significantly reduces both the switching activity over the wire and the power consumption of the network. This scheme codes the information in the time domain and allows a straightforward obtention of an ordered list of values from the maximum to the minimum. If the scheme is applied to monitors that employ TDC, digitization resource sharing is achieved, producing an important saving in area and power consumption. Two prototypes of complete monitoring systems are presented, they significantly overcome previous works in terms of area and, specially, power consumption.

Gesture recognition using mobile phone's inertial sensors

The availability of inertial sensors embedded in mobile devices has enabled a new type of interaction based on the movements or “gestures” made by the users when holding the device. In this paper we propose a gesture recognition system for mobile devices based on accelerometer and gyroscope measurements. The system is capable of recognizing a set of predefined gestures in a user-independent way, without the need of a training phase. Furthermore, it was designed to be executed in real-time in resource-constrained devices, and therefore has a low computational complexity. The performance of the system is evaluated offline using a dataset of gestures, and also online, through some user tests with the system running in a smart phone.

Uncertainty in optical bio-sensors due to the spectral displacement of the interference modes of the transduction signal. Incertidumbre en bio-sensores ópticos asociada al desplazamiento espectral de los modos de interferencia de la señal de transducción

The analysis of the interference modes has an increasing application, especially in the field of optical biosensors. In this type of sensors, the displacement Δν of the interference modes of the transduction signal is observed when a particular biological agent is placed over the biosensor. In order to measure this displacement, the position of a maximum (or a minimum) of the signal must be detected before and after placing the agent over the sensor. A parameter of great importance for this kind of sensors is the period Pν of the signal, which is inversely proportional to the optical thickness h0 of the sensor in the absence of the biological agent. The increase of this period improves the sensitivity of the sensor but it worsens the detection of the maximum. In this paper, authors analyze the propagation of uncertainties in these sensors when using least squares techniques for the detection of the maxima (or minima) of the signal. Techniques described in supplement 2 of the ISO-GUM Guide are used. The result of the analysis allows a metrological educated answer to the question of which is the optimal period Pν of the signal. El análisis del comportamiento de los modos de interferencia tiene una aplicación cada vez más amplia, especialmente en el campo de los biosensores ópticos. En este tipo de sensores se observa el desplazamiento Δν de los modos de interferencia de la señal de transducción al reconocer un de-terminado agente biológico. Para medir ese desplazamiento se debe detectar la posición de un máximo o mínimo de la señal antes y después de dicho desplazamiento. En este tipo de biosensores un parámetro de gran importancia es el periodo Pν de la señal el cual es inversamente proporcional al espesor óptico h0 del sensor en ausencia de agente biológico. El aumento de dicho periodo mejora la sensibilidad del sensor pero parece dificultar la detección del mínimo o máximo. Por tanto, su efecto sobre la incertidumbre del resultado de la medida presenta dos efectos contrapuestos: la mejora de la sensibilidad frente a la dificultad creciente en la detección del mínimo ó máximo. En este trabajo, los autores analizan la propagación de incertidumbres en estos sensores utilizando herramientas de ajuste por MM.CC. para la detección de los mínimos o máximos de la señal y técnicas de propagación de incertidumbres descritas en el suplemento 2 de la Guía ISO-GUM. El resultado del análisis permite dar una respuesta, justificada desde el punto de vista metrológico, de en que condiciones es conveniente o no aumentar el periodo Pν de la señal.

Application of PZT and FBG impedance sensors for structural health monitoring of reinforced concrete beams strengthened with FRP composite materials

Esta Tesis tiene como objetivo principal el desarrollo de métodos de identificación del daño que sean robustos y fiables, enfocados a sistemas estructurales experimentales, fundamentalmente a las estructuras de hormigón armado reforzadas externamente con bandas fibras de polímeros reforzados (FRP). El modo de fallo de este tipo de sistema estructural es crítico, pues generalmente es debido a un despegue repentino y frágil de la banda del refuerzo FRP originado en grietas intermedias causadas por la flexión. La detección de este despegue en su fase inicial es fundamental para prevenir fallos futuros, que pueden ser catastróficos. Inicialmente, se lleva a cabo una revisión del método de la Impedancia Electro-Mecánica (EMI), de cara a exponer sus capacidades para la detección de daño. Una vez la tecnología apropiada es seleccionada, lo que incluye un analizador de impedancias así como novedosos sensores PZT para monitorización inteligente, se ha diseñado un procedimiento automático basado en los registros de impedancias de distintas estructuras de laboratorio. Basándonos en el hecho de que las mediciones de impedancias son posibles gracias a una colocación adecuada de una red de sensores PZT, la estimación de la presencia de daño se realiza analizando los resultados de distintos indicadores de daño obtenidos de la literatura. Para que este proceso sea automático y que no sean necesarios conocimientos previos sobre el método EMI para realizar un experimento, se ha diseñado e implementado un Interfaz Gráfico de Usuario, transformando la medición de impedancias en un proceso fácil e intuitivo. Se evalúa entonces el daño a través de los correspondientes índices de daño, intentando estimar no sólo su severidad, sino también su localización aproximada. El desarrollo de estos experimentos en cualquier estructura genera grandes cantidades de datos que han de ser procesados, y algunas veces los índices de daño no son suficientes para una evaluación completa de la integridad de una estructura. En la mayoría de los casos se pueden encontrar patrones de daño en los datos, pero no se tiene información a priori del estado de la estructura. En este punto, se ha hecho una importante investigación en técnicas de reconocimiento de patrones particularmente en aprendizaje no supervisado, encontrando aplicaciones interesantes en el campo de la medicina. De ahí surge una idea creativa e innovadora: detectar y seguir la evolución del daño en distintas estructuras como si se tratase de un cáncer propagándose por el cuerpo humano. En ese sentido, las lecturas de impedancias se emplean como información intrínseca de la salud de la propia estructura, de forma que se pueden aplicar las mismas técnicas que las empleadas en la investigación del cáncer. En este caso, se ha aplicado un algoritmo de clasificación jerárquica dado que ilustra además la clasificación de los datos de forma gráfica, incluyendo información cualitativa y cuantitativa sobre el daño. Se ha investigado la efectividad de este procedimiento a través de tres estructuras de laboratorio, como son una viga de aluminio, una unión atornillada de aluminio y un bloque de hormigón reforzado con FRP. La primera ayuda a mostrar la efectividad del método en sencillos escenarios de daño simple y múltiple, de forma que las conclusiones extraídas se aplican sobre los otros dos, diseñados para simular condiciones de despegue en distintas estructuras. Demostrada la efectividad del método de clasificación jerárquica de lecturas de impedancias, se aplica el procedimiento sobre las estructuras de hormigón armado reforzadas con bandas de FRP objeto de esta tesis, detectando y clasificando cada estado de daño. Finalmente, y como alternativa al anterior procedimiento, se propone un método para la monitorización continua de la interfase FRP-Hormigón, a través de una red de sensores FBG permanentemente instalados en dicha interfase. De esta forma, se obtienen medidas de deformación de la interfase en condiciones de carga continua, para ser implementadas en un modelo de optimización multiobjetivo, cuya solución se haya por medio de una expansión multiobjetivo del método Particle Swarm Optimization (PSO). La fiabilidad de este último método de detección se investiga a través de sendos ejemplos tanto numéricos como experimentales. ABSTRACT This thesis aims to develop robust and reliable damage identification methods focused on experimental structural systems, in particular Reinforced Concrete (RC) structures externally strengthened with Fiber Reinforced Polymers (FRP) strips. The failure mode of this type of structural system is critical, since it is usually due to sudden and brittle debonding of the FRP reinforcement originating from intermediate flexural cracks. Detection of the debonding in its initial stage is essential thus to prevent future failure, which might be catastrophic. Initially, a revision of the Electro-Mechanical Impedance (EMI) method is carried out, in order to expose its capabilities for local damage detection. Once the appropriate technology is selected, which includes impedance analyzer as well as novel PZT sensors for smart monitoring, an automated procedure has been design based on the impedance signatures of several lab-scale structures. On the basis that capturing impedance measurements is possible thanks to an adequately deployed PZT sensor network, the estimation of damage presence is done by analyzing the results of different damage indices obtained from the literature. In order to make this process automatic so that it is not necessary a priori knowledge of the EMI method to carry out an experimental test, a Graphical User Interface has been designed, turning the impedance measurements into an easy and intuitive procedure. Damage is then assessed through the analysis of the corresponding damage indices, trying to estimate not only the damage severity, but also its approximate location. The development of these tests on any kind of structure generates large amounts of data to be processed, and sometimes the information provided by damage indices is not enough to achieve a complete analysis of the structural health condition. In most of the cases, some damage patterns can be found in the data, but none a priori knowledge of the health condition is given for any structure. At this point, an important research on pattern recognition techniques has been carried out, particularly on unsupervised learning techniques, finding interesting applications in the medicine field. From this investigation, a creative and innovative idea arose: to detect and track the evolution of damage in different structures, as if it were a cancer propagating through a human body. In that sense, the impedance signatures are used to give intrinsic information of the health condition of the structure, so that the same clustering algorithms applied in the cancer research can be applied to the problem addressed in this dissertation. Hierarchical clustering is then applied since it also provides a graphical display of the clustered data, including quantitative and qualitative information about damage. The performance of this approach is firstly investigated using three lab-scale structures, such as a simple aluminium beam, a bolt-jointed aluminium beam and an FRP-strengthened concrete specimen. The first one shows the performance of the method on simple single and multiple damage scenarios, so that the first conclusions can be extracted and applied to the other two experimental tests, which are designed to simulate a debonding condition on different structures. Once the performance of the impedance-based hierarchical clustering method is proven to be successful, it is then applied to the structural system studied in this dissertation, the RC structures externally strengthened with FRP strips, where the debonding failure in the interface between the FRP and the concrete is successfully detected and classified, proving thus the feasibility of this method. Finally, as an alternative to the previous approach, a continuous monitoring procedure of the FRP-Concrete interface is proposed, based on an FBGsensors Network permanently deployed within that interface. In this way, strain measurements can be obtained under controlled loading conditions, and then they are used in order to implement a multi-objective model updating method solved by a multi-objective expansion of the Particle Swarm Optimization (PSO) method. The feasibility of this last proposal is investigated and successfully proven on both numerical and experimental RC beams strengthened with FRP.

“Switch I” mutant forms of the bacterial enhancer-binding protein NtrC that perturb the response to DNA

NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding protein of 469 residues that activates transcription by σ54-holoenzyme. A region of its transcriptional activation (central) domain that is highly conserved among homologous activators of σ54-holoenzyme—residues 206–220—is essential for interaction with this RNA polymerase: it is required for contact with the polymerase and/or for coupling the energy from ATP hydrolysis to a change in the conformation of the polymerase that allows it to form transcriptionally productive open complexes. Several mutant NtrC proteins with amino acid substitutions in this region, including NtrCA216V and NtrCG219K, have normal ATPase activity but fail in transcriptional activation. We now report that other mutant forms carrying amino acid substitutions at these same positions, NtrCA216C and NtrCG219C, are capable of activating transcription when they are not bound to a DNA template (non-DNA-binding derivatives with an altered helix–turn–helix DNA-binding motif at the C terminus of the protein) but are unable to do so when they are bound to a DNA template, whether or not it carries a specific enhancer. Enhancer DNA remains a positive allosteric effector of ATP hydrolysis, as it is for wild-type NtrC but, surprisingly, appears to have become a negative allosteric effector for some aspect of interaction with σ54-holoenzyme. The conserved region in which these amino acid substitutions occur (206–220) is equivalent to the Switch I region of a large group of purine nucleotide-binding proteins. Interesting analogies can be drawn between the Switch I region of NtrC and that of p21ras.

The Putative “Switch 2” Domain of the Ras-related GTPase, Rab1B, Plays an Essential Role in the Interaction with Rab Escort Protein

Posttranslational modification of Rab proteins by geranylgeranyltransferase type II requires that they first bind to Rab escort protein (REP). Following prenylation, REP is postulated to accompany the modified GTPase to its specific target membrane. REP binds preferentially to Rab proteins that are in the GDP state, but the specific structural domains involved in this interaction have not been defined. In p21 Ras, the α2 helix of the Switch 2 domain undergoes a major conformational change upon GTP hydrolysis. Therefore, we hypothesized that the corresponding region in Rab1B might play a key role in the interaction with REP. Introduction of amino acid substitutions (I73N, Y78D, and A81D) into the putative α2 helix of Myc-tagged Rab1B prevented prenylation of the recombinant protein in cell-free assays, whereas mutations in the α3 and α4 helices did not. Additionally, upon transient expression in transfected HEK-293 cells, the Myc-Rab1B α2 helix mutants were not efficiently prenylated as determined by incorporation of [3H]mevalonate. Metabolic labeling studies using [32P]orthophosphate indicated that the poor prenylation of the Rab1B α2 helix mutants was not directly correlated with major disruptions in guanine nucleotide binding or intrinsic GTPase activity. Finally, gel filtration analysis of cytosolic fractions from 293 cells that were coexpressing T7 epitope-tagged REP with various Myc-Rab1B constructs revealed that mutations in the α2 helix of Rab1B prevented the association of nascent (i.e., nonprenylated) Rab1B with REP. These data indicate that the Switch 2 domain of Rab1B is a key structural determinant for REP interaction and that nucleotide-dependent conformational changes in this region are largely responsible for the selective interaction of REP with the GDP-bound form of the Rab substrate.

Calmodulin kinase is a molecular switch for cardiac excitation –contraction coupling

Signaling between cell membrane-bound L-type Ca2+ channels (LTCC) and ryanodine receptor Ca2+ release channels (RyR) on sarcoplasmic reticulum (SR) stores grades excitation–contraction coupling (ECC) in striated muscle. A physical connection regulates LTCC and RyR in skeletal muscle, but the molecular mechanism for coordinating LTCC and RyR in cardiomyocytes, where this physical link is absent, is unknown. Calmodulin kinase (CaMK) has characteristics suitable for an ECC coordinating molecule: it is activated by Ca2+/calmodulin, it regulates LTCC and RyR, and it is enriched in the vicinity of LTCC and RyR. Intact cardiomyocytes were studied under conditions where CaMK activity could be controlled independently of intracellular Ca2+ by using an engineered Ca2+-independent form of CaMK and a highly specific CaMK inhibitory peptide. CaMK reciprocally enhanced L-type Ca2+ current and reduced release of Ca2+ from the SR while increasing SR Ca2+ content. These findings support the hypothesis that CaMK is required to functionally couple LTCC and RyR during cardiac ECC.

Blue Light and Abscisic Acid Independently Induce Heterophyllous Switch in Marsilea quadrifolia1

In natural habitats Marsilea quadrifolia L. produces different types of leaves above and below the water level. In aseptic cultures growth conditions can be manipulated so that leaves of the submerged type are produced continuously. Under such conditions the application of either blue light or an optimal concentration of abscisic acid (ABA) induced the development of aerial-type leaves. When fluridone, an inhibitor of ABA biosynthesis, was added to the culture medium it did not prevent blue light induction of aerial leaf development. During blue light treatment the endogenous ABA level in M. quadrifolia leaves remained unchanged. However, after the plants were transferred to an enriched medium, the ABA level gradually increased, corresponding to a transition in development from the submerged type of leaves to aerial leaves. These results indicate that the blue light signal is not mediated by ABA. Therefore, in the regulation of heterophyllous determination, discrete pathways exist in response to environmental signals.

Dynamic properties of the Ras switch I region and its importance for binding to effectors

We have investigated the dynamic properties of the switch I region of the GTP-binding protein Ras by using mutants of Thr-35, an invariant residue necessary for the switch function. Here we show that these mutants, previously used as partial loss-of-function mutations in cell-based assays, have a reduced affinity to Ras effector proteins without Thr-35 being involved in any interaction. The structure of Ras(T35S)⋅GppNHp was determined by x-ray crystallography. Whereas the overall structure is very similar to wildtype, residues from switch I are completely invisible, indicating that the effector loop region is highly mobile. 31P-NMR data had indicated an equilibrium between two rapidly interconverting conformations, one of which (state 2) corresponds to the structure found in the complex with the effectors. 31P-NMR spectra of Ras mutants (T35S) and (T35A) in the GppNHp form show that the equilibrium is shifted such that they occur predominantly in the nonbinding conformation (state 1). On addition of Ras effectors, Ras(T35S) but not Ras(T35A) shift to positions corresponding to the binding conformation. The structural data were correlated with kinetic experiments that show two-step binding reaction of wild-type and (T35S)Ras with effectors requires the existence of a rate-limiting isomerization step, which is not observed with T35A. The results indicate that minor changes in the switch region, such as removing the side chain methyl group of Thr-35, drastically affect dynamic behavior and, in turn, interaction with effectors. The dynamics of the switch I region appear to be responsible for the conservation of this threonine residue in GTP-binding proteins.

Azotobacter vinelandii NIFL is a flavoprotein that modulates transcriptional activation of nitrogen-fixation genes via a redox-sensitive switch.

The NIFL regulatory protein controls transcriptional activation of nitrogen fixation (nif) genes in Azotobacter vinelandii by direct interaction with the enhancer binding protein NIFA. Modulation of NIFA activity by NIFL, in vivo occurs in response to external oxygen concentration or the level of fixed nitrogen. Spectral features of purified NIFL and chromatographic analysis indicate that it is a flavoprotein with FAD as the prosthetic group, which undergoes reduction in the presence of sodium dithionite. Under anaerobic conditions, the oxidized form of NIFL inhibits transcriptional activation by NIFA in vitro, and this inhibition is reversed when NIFL is in the reduced form. Hence NIFL is a redox-sensitive regulatory protein and may represent a type of flavoprotein in which electron transfer is not coupled to an obvious catalytic activity. In addition to its ability to act as a redox sensor, the activity of NIFL is also responsive to adenosine nucleotides, particularly ADP. This response overrides the influence of redox status on NIFL and is also observed with refolded NIFL apoprotein, which lacks the flavin moiety. These observations suggest that both energy and redox status are important determinants of nif gene regulation in vivo.

SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants

Metabolic adjustment to changing environmental conditions, particularly balancing of growth and defense responses, is crucial for all organisms to survive. The evolutionary conserved AMPK/Snf1/SnRK1 kinases are well-known metabolic master regulators in the low-energy response in animals, yeast and plants. They act at two different levels: by modulating the activity of key metabolic enzymes, and by massive transcriptional reprogramming. While the first part is well established, the latter function is only partially understood in animals and not at all in plants. Here we identified the Arabidopsis transcription factor bZIP63 as key regulator of the starvation response and direct target of the SnRK1 kinase. Phosphorylation of bZIP63 by SnRK1 changed its dimerization preference, thereby affecting target gene expression and ultimately primary metabolism. A bzip63 knock-out mutant exhibited starvation-related phenotypes, which could be functionally complemented by wild type bZIP63, but not by a version harboring point mutations in the identified SnRK1 target sites.

Oxygen sensors and energy sensors act synergistically to achieve a graded alteration in gene expression: Consequences for assessing the level of neuroprotection in response to stressors

Changes in gene expression are associated with switching to an autoprotected phenotype in response to environmental and physiological stress. Ubiquitous molecular chaperones from the heat shock protein (HSP) superfamily confer neuronal protection that can be blocked by antibodies. Recent research has focused on the interactions between the molecular sensors that affect the increased expression of neuroprotective HSPs above constitutive levels. An examination of the conditions under which the expression of heat shock protein 70 (Hsp70) was up regulated in a hypoxia and anoxia tolerant tropical species, the epaulette shark (Hemiscyllium ocellatum), revealed that up-regulation was dependent on exceeding a stimulus threshold for an oxidative stressor. While hypoxic-preconditioning confers neuroprotective changes, there was no increase in the level of Hsp70 indicating that its increased expression was not associated with achieving a neuroprotected state in response to hypoxia in the epaulette shark. Conversely, there was a significant increase in Hsp70 in response to anoxic-preconditioning, highlighting the presence of a stimulus threshold barrier and raising the possibility that, in this species, Hsp70 contributes to the neuroprotective response to extreme crises, such as oxidative stress. Interestingly, there was a synergistic effect of coincident stressors on Hsp70 expression, which was revealed when metabolic stress was superimposed upon oxidative stress. Brain energy charge was significantly lower when adenosine receptor blockade, provided by treatment with aminophylline, was present prior to the final anoxic episode, under these circumstances, the level of Hsp70 induced was significantly higher than in the pair-matched saline treated controls. An understanding of the molecular and metabolic basis for neuroprotective switches, which result in an up-regulation of neuroprotective Hsp70 expression in the brain, is needed so that intervention strategies can be devised to manage CNS pathologies and minimise damage caused by ischemia and trauma. In addition, the current findings indicate that measurements of HSP expression per se may provide a useful correlate of the level of neuroprotection achieved in the switch to an autoprotected phenotype.

Optical fibre sensors and their interrogation

This thesis describes novel developments in the fabrication and understanding of type IA fibre Bragg gratings, the uses of said gratings as optical sensors and the interrogation of optical sensors using tilted fibre Bragg gratings. This thesis presents the most detailed study of type IA gratings performed to date and provides the basis of a dual grating optical sensor capable of independently measuring strain and temperature. Until this work it was not known how to reliably fabricate type IA gratings or how they would react to high ambient temperatures, nor was it known what effect external parameters such as fibre type, dopant levels, inscription laser intensity, or hydrogenation levels would have on the physical properties of the grating. This comprehensive study has yielded answers to all of these unknowns and produced several unexpected uses for type IA gratings, such as the use of the previously unreported strong loss band at 1400nm to locally heat fibres by optical absorption and thereby fabricate optically tuneable gratings which do not affect directly adjacent standard gratings. Blazed fibre Bragg gratings have been studied in detail and used to produce several high quality prototype sensor interrogation systems yielding stability an accuracy values unsurpassed by similar devices reported in literature. An accurate distribution map of light radiated by blazed gratings is shown for the first time and has been studied in respect of polarisation state showing that for certain easily achievable conditions a blazed grating spectrometer may be deemed to be polarisation insensitive. In a novel implementation of the system, it is shown that the dynamic wavelength range of a blazed grating spectrometer may be at least doubled by superimposing blazed gratings.

Effectively simultaneous temperature and strain measurement utilising a dual-grating sensor formed by type IA and type IIA FBGs

In this paper, we report a systematic investigation of the dependence of both temperature and strain sensitivities on the jiber Bragg grating (FBG) type, including the wellknown Type I, Type IIA, and a new type which we have designated Type 1.4, using both hydrogen-Ji-ee and hydrogenated B/Ge codoped jibers. We have identijed distinct sensitivity characteristics for each grating type, and we have utilised them to implement a novel dual-grating, duul-parameter sensor device. Three dual-grating sensing schemes with different combinations of gruting types have been constructed and compared. The Type IA-Type IIA combination exhibits the best pe$ormance and is superior to that of previously reported gruting-based structures. The characteristics of the measurement errors in such dualgrating sensor systems is also presented in detail.