An Adeno-Associated Virus-Based Intracellular Sensor of Pathological Nuclear Factor-κB Activation for Disease-Inducible Gene Transfer.

Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS). This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV)-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF) cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF) was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA)-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.

Electrochemical As(III) whole-cell based biochip sensor.

The development of a whole-cell based sensor for arsenite detection coupling biological engineering and electrochemical techniques is presented. This strategy takes advantage of the natural Escherichia coli resistance mechanism against toxic arsenic species, such as arsenite, which consists of the selective intracellular recognition of arsenite and its pumping out from the cell. A whole-cell based biosensor can be produced by coupling the intracellular recognition of arsenite to the generation of an electrochemical signal. Hereto, E. coli was equipped with a genetic circuit in which synthesis of beta-galactosidase is under control of the arsenite-derepressable arsR-promoter. The E. coli reporter strain was filled in a microchip containing 16 independent electrochemical cells (i.e. two-electrode cell), which was then employed for analysis of tap and groundwater samples. The developed arsenic-sensitive electrochemical biochip is easy to use and outperforms state-of-the-art bacterial bioreporters assays specifically in its simplicity and response time, while keeping a very good limit of detection in tap water, i.e. 0.8ppb. Additionally, a very good linear response in the ranges of concentration tested (0.94ppb to 3.75ppb, R(2)=0.9975 and 3.75 ppb to 30ppb, R(2)=0.9991) was obtained, complying perfectly with the acceptable arsenic concentration limits defined by the World Health Organization for drinking water samples (i.e. 10ppb). Therefore, the proposed assay provides a very good alternative for the portable quantification of As (III) in water as corroborated by the analysis of natural groundwater samples from Swiss mountains, which showed a very good agreement with the results obtained by atomic absorption spectroscopy.

An adeno-associated virus-based intracellular sensor of pathological nuclear factor-κB activation for disease-inducible gene transfer.

Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS). This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV)-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF) cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF) was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA)-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.

Position sensorless electrically excited synchronous motor drive for industrial use based on direct flux linkage and torque control

Synchronous machines with an AC converter are used mainly in large drives, for example in ship propulsion drives as well as in rolling mill drives in steel industry. These motors are used because of their high efficiency, high overload capacity and good performance in the field weakening area. Present day drives for electrically excited synchronous motors are equipped with position sensors. Most drives for electrically excited synchronous motors will be equipped with position sensors also in future. This kind of drives with good dynamics are mainly used in metal industry. Drives without a position sensor can be used e.g. in ship propulsion and in large pump and blower drives. Nowadays, these drives are equipped with a position sensor, too. The tendency is to avoid a position sensor if possible, since a sensor reduces the reliability of the drive and increases costs (latter is not very significant for large drives). A new control technique for a synchronous motor drive is a combination of the Direct Flux Linkage Control (DFLC) based on a voltage model and a supervising method (e.g. current model). This combination is called Direct Torque Control method (DTC). In the case of the position sensorless drive, the DTC can be implemented by using other supervising methods that keep the stator flux linkage origin centered. In this thesis, a method for the observation of the drift of the real stator flux linkage in the DTC drive is introduced. It is also shown how this method can be used as a supervising method that keeps the stator flux linkage origin centered in the case of the DTC. In the position sensorless case, a synchronous motor can be started up with the DTC control, when a method for the determination of the initial rotor position presented in this thesis is used. The load characteristics of such a drive are not very good at low rotational speeds. Furthermore, continuous operation at a zero speed and at a low rotational speed is not possible, which is partly due to the problems related to the flux linkage estimate. For operation in a low speed area, a stator current control method based on the DFLC modulator (DMCQ is presented. With the DMCC, it is possible to start up and operate a synchronous motor at a zero speed and at low rotational speeds in general. The DMCC is necessary in situations where high torque (e.g. nominal torque) is required at the starting moment, or if the motor runs several seconds at a zero speed or at a low speed range (up to 2 Hz). The behaviour of the described methods is shown with test results. The test results are presented for the direct flux linkage and torque controlled test drive system with a 14.5 kVA, four pole salient pole synchronous motor with a damper winding and electric excitation. The static accuracy of the drive is verified by measuring the torque in a static load operation, and the dynamics of the drive is proven in load transient tests. The performance of the drive concept presented in this work is sufficient e.g. for ship propulsion and for large pump drives. Furthermore, the developed methods are almost independent of the machine parameters.

Alternative technique for biodiesel quality control using an optical fiber long-period grating sensor

We report the use of an optical fiber sensor to measure the soybean oil concentration in samples obtained from the mixture of pure biodiesel and commercial soybean oil. The operation of the device is based on the long-period grating sensitivity to the surrounding medium refractive index, which leads to measurable modifications in the grating transmission spectrum. The proposed analysis method results in errors in the oil concentration of 0.4% and 2.6% for pure biodiesel and commercial soybean oil, respectively. Techniques of total glycerol, dynamic viscosity, density, and hydrogen nuclear magnetic resonance spectroscopy were also employed to validate the proposed method.

Comparison between dSPACE and NI systems based on real-time intelligent control of a teleoperated hydraulic servo system

The Laboratory of Intelligent Machine researches and develops energy-efficient power transmissions and automation for mobile construction machines and industrial processes. The laboratory's particular areas of expertise include mechatronic machine design using virtual technologies and simulators and demanding industrial robotics. The laboratory has collaborated extensively with industrial actors and it has participated in significant international research projects, particularly in the field of robotics. For years, dSPACE tools were the lonely hardware which was used in the lab to develop different control algorithms in real-time. dSPACE's hardware systems are in widespread use in the automotive industry and are also employed in drives, aerospace, and industrial automation. But new competitors are developing new sophisticated systems and their features convinced the laboratory to test new products. One of these competitors is National Instrument (NI). In order to get to know the specifications and capabilities of NI tools, an agreement was made to test a NI evolutionary system. This system is used to control a 1-D hydraulic slider. The objective of this research project is to develop a control scheme for the teleoperation of a hydraulically driven manipulator, and to implement a control algorithm between human and machine interaction, and machine and task environment interaction both on NI and dSPACE systems simultaneously and to compare the results.

Double-Tagging Polymerase Chain Reaction with a Thiolated Primer and Electrochemical Genosensing based on Gold Nanocomposite Sensor for Food Safety

A novel material for electrochemical biosensing based on rigid conducting gold nanocomposite (nano-AuGEC) is presented. Islands of chemisorbing material (gold nanoparticles) surrounded by nonreactive, rigid, and conducting graphite epoxy composite are thus achieved to avoid the stringent control of surface coverage parameters required during immobilization of thiolated oligos in continuous gold surfaces. The spatial resolution of the immobilized thiolated DNA was easily controlled by merely varying the percentage of gold nanoparticles in the composition of the composite. As low as 9 fmol (60 pM) of synthetic DNA were detected in hybridization experiments when using a thiolated probe. Moreover, for the first time a double tagging PCR strategy was performed with a thiolated primer for the detection of Salmonella sp., one of the most important foodborne pathogens affecting food safety. Ibis assay was performed by double-labeling the amplicon during the PCR with a -DIG and -SH set of labeled primers. The thiolated end allows the immobilization of the amplicon on the nano-AuGEC electrode, while digoxigenin allows the electrochemical detection with the antiDIG-HRP reporter in the femtomole range. Rigid conducting gold nanocomposite represents a good material for the improved and oriented immobilization of biomolecules with excellent transducing properties for the construction of a wide range of electrochemical biosensors such as immunosensors, genosensors, and enzymosensors.

Electrochemical sensor for ranitidine determination based on carbon paste electrode modified with oxovanadium (IV) salen complex

The preparation and electrochemical characterization of a carbon paste electrode modified with the N,N-ethylene-bis(salicyllideneiminato)oxovanadium (IV) complex ([VO(salen)]) as well as its application for ranitidine determination are described. The electrochemical behavior of the modified electrode for the electroreduction of ranitidine was investigated using cyclic voltammetry, and analytical curves were obtained for ranitidine using linear sweep voltammetry (LSV) under optimized conditions. The best voltammetric response was obtained for an electrode composition of 20% (m/m) [VO(salen)] in the paste, 0.10 mol L- 1 of KCl solution (pH 5.5 adjusted with HCl) as supporting electrolyte and scan rate of 25 mV s- 1. A sensitive linear voltammetric response for ranitidine was obtained in the concentration range from 9.9 × 10- 5 to 1.0 × 10- 3 mol L- 1, with a detection limit of 6.6 × 10- 5 mol L- 1 using linear sweep voltammetry. These results demonstrated the viability of this modified electrode as a sensor for determination, quality control and routine analysis of ranitidine in pharmaceutical formulations. © 2013 Published by Elsevier B.V.

MODEL-BASED COMPENSATION FOR BURST MESSAGE LOSS IN WIRELESS NETWORKED CONTROL SYSTEMS: EXPERIMENTAL RESULTS

A recent trend in networked control systems (NCSs) is the use of wireless networks enabling interoperability between existing wired and wireless systems. One of the major challenges in these wireless NCSs (WNCSs) is to overcome the impact of the message loss that degrades the performance and stability of these systems. Moreover, this impact is greater when dealing with burst or successive message losses. This paper discusses and presents the experimental results of a compensation strategy to deal with this burst message loss problem in which a NCS mathematical model runs in parallel with the physical process, providing sensor virtual data in case of packet losses. Running in real-time inside the controller, the mathematical model is updated online with real control signals sent to the actuator, which provides better reliability for the estimated sensor feedback (virtual data) transmitted to the controller each time a message loss occurs. In order to verify the advantages of applying this model-based compensation strategy for burst message losses in WNCSs, the control performance of a motor control system using CAN and ZigBee networks is analyzed. Experimental results led to the conclusion that the developed compensation strategy provided robustness and could maintain the control performance of the WNCS against different message loss scenarios.

The benefits of soft sensor and multi-rate control for the implementation of wireless networked control systems

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Integrating wireless sensor networks and internet of things: A coap-based approach

L'obiettivo su cui è stata basata questa Tesi di Laurea è stato quello di integrare la tecnologia delle Wireless Sensor Networks (WSN) al contesto dell'Internet delle cose (IoT). Per poter raggiungere questo obiettivo, il primo passo è stato quello di approfondire il concetto dell'Internet delle cose, in modo tale da comprendere se effettivamente fosse stato possibile applicarlo anche alle WSNs. Quindi è stata analizzata l'architettura delle WSNs e successivamente è stata fatta una ricerca per capire quali fossero stati i vari tipi di sistemi operativi e protocolli di comunicazione supportati da queste reti. Infine sono state studiate alcune IoT software platforms. Il secondo passo è stato quindi di implementare uno stack software che abilitasse la comunicazione tra WSNs e una IoT platform. Come protocollo applicativo da utilizzare per la comunicazione con le WSNs è stato usato CoAP. Lo sviluppo di questo stack ha consentito di estendere la piattaforma SensibleThings e il linguaggio di programmazione utilizzato è stato Java. Come terzo passo è stata effettuata una ricerca per comprendere a quale scenario di applicazione reale, lo stack software progettato potesse essere applicato. Successivamente, al fine di testare il corretto funzionamento dello stack CoAP, è stata sviluppata una proof of concept application che simulasse un sistema per la rilevazione di incendi. Questo scenario era caratterizzato da due WSNs che inviavano la temperatura rilevata da sensori termici ad un terzo nodo che fungeva da control center, il cui compito era quello di capire se i valori ricevuti erano al di sopra di una certa soglia e quindi attivare un allarme. Infine, l'ultimo passo di questo lavoro di tesi è stato quello di valutare le performance del sistema sviluppato. I parametri usati per effettuare queste valutazioni sono stati: tempi di durata delle richieste CoAP, overhead introdotto dallo stack CoAP alla piattaforma Sensible Things e la scalabilità di un particolare componente dello stack. I risultati di questi test hanno mostrato che la soluzione sviluppata in questa tesi ha introdotto un overheadmolto limitato alla piattaforma preesistente e inoltre che non tutte le richieste hanno la stessa durata, in quanto essa dipende dal tipo della richiesta inviata verso una WSN. Tuttavia, le performance del sistema potrebbero essere ulteriormente migliorate, ad esempio sviluppando un algoritmo che consenta la gestione concorrente di richieste CoAP multiple inviate da uno stesso nodo. Inoltre, poichè in questo lavoro di tesi non è stato considerato il problema della sicurezza, una possibile estensione al lavoro svolto potrebbe essere quello di implementare delle politiche per una comunicazione sicura tra Sensible Things e le WSNs.

Development of a model-based transient calibration process for diesel engine electronic control module tables – Part 1: data requirements, processing, and analysis

This is the first part of a study investigating a model-based transient calibration process for diesel engines. The motivation is to populate hundreds of parameters (which can be calibrated) in a methodical and optimum manner by using model-based optimization in conjunction with the manual process so that, relative to the manual process used by itself, a significant improvement in transient emissions and fuel consumption and a sizable reduction in calibration time and test cell requirements is achieved. Empirical transient modelling and optimization has been addressed in the second part of this work, while the required data for model training and generalization are the focus of the current work. Transient and steady-state data from a turbocharged multicylinder diesel engine have been examined from a model training perspective. A single-cylinder engine with external air-handling has been used to expand the steady-state data to encompass transient parameter space. Based on comparative model performance and differences in the non-parametric space, primarily driven by a high engine difference between exhaust and intake manifold pressures (ΔP) during transients, it has been recommended that transient emission models should be trained with transient training data. It has been shown that electronic control module (ECM) estimates of transient charge flow and the exhaust gas recirculation (EGR) fraction cannot be accurate at the high engine ΔP frequently encountered during transient operation, and that such estimates do not account for cylinder-to-cylinder variation. The effects of high engine ΔP must therefore be incorporated empirically by using transient data generated from a spectrum of transient calibrations. Specific recommendations on how to choose such calibrations, how many data to acquire, and how to specify transient segments for data acquisition have been made. Methods to process transient data to account for transport delays and sensor lags have been developed. The processed data have then been visualized using statistical means to understand transient emission formation. Two modes of transient opacity formation have been observed and described. The first mode is driven by high engine ΔP and low fresh air flowrates, while the second mode is driven by high engine ΔP and high EGR flowrates. The EGR fraction is inaccurately estimated at both modes, while EGR distribution has been shown to be present but unaccounted for by the ECM. The two modes and associated phenomena are essential to understanding why transient emission models are calibration dependent and furthermore how to choose training data that will result in good model generalization.

Hop-to-Hop Reliability in IP-Based Wireless Sensor Networks - A Cross-Layer Approach

To interconnect a wireless sensor network (WSN) to the Internet, we propose to use TCP/IP as the standard protocol for all network entities. We present a cross layer designed communication architecture, which contains a MAC protocol, IP, a new protocol called Hop-to-Hop Reliability (H2HR) protocol, and the TCP Support for Sensor Nodes (TSS) protocol. The MAC protocol implements the MAC layer of beacon-less personal area networks (PANs) as defined in IEEE 802.15.4. H2HR implements hop-to-hop reliability mechanisms. Two acknowledgment mechanisms, explicit and implicit ACK are supported. TSS optimizes using TCP in WSNs by implementing local retransmission of TCP data packets, local TCP ACK regeneration, aggressive TCP ACK recovery, congestion and flow control algorithms. We show that H2HR increases the performance of UDP, TCP, and RMST in WSNs significantly. The throughput is increased and the packet loss ratio is decreased. As a result, WSNs can be operated and managed using TCP/IP.

Soft-computing based visual control for unmanned vehicles

El objetivo principal de esta Tesis es extender la utilización del “Soft- Computing” para el control de vehículos sin piloto utilizando visión. Este trabajo va más allá de los típicos sistemas de control utilizados en entornos altamente controlados, demonstrando la fuerza y versatilidad de la lógica difusa (Fuzzy Logic) para controlar vehículos aéreos y terrestres en un abanico de applicaciones diferentes. Para esta Tesis se ha realizado un gran número de pruebas reales en las cuales los controladores difusos han manejado una plataforma visual “pan-and-tilt”, un helicoptero, un coche comercial y hasta dos tipos de quadrirotores. El uso del método de optimización “Cross-Entropy” ha sido utilizado para mejorar el comportamiento de algunos de los controladores borrosos. Todos los controladores difusos presentados en ésta Tesis han sido implementados utilizando un código desarrollado por el candidato para tal efecto, llamado MOFS (Miguel Olivares’ Fuzzy Software). Diferentes algoritmos visuales han sido utilizados para adquirir la informaci´on visual del entorno, “Cmashift”, descomposición de la homografía y detección de marcas de realidad aumentada, entre otros. Dicha información visual ha sido utilizada como entrada de los controladores difusos para comandar los vehículos en las diferentes applicaciones autonomas. El volante de un vehículo comercial ha sido controlado para realizar pruebas de conducción autónoma en condiciones de tráfico similares a las de una ciudad. El sistema ha llegado a completar con éxito pruebas de más de 6 km sin ninguna interacción humana, mediante el seguimiento de una línea pintada en el suelo. El limitado campo visual del sistema no ha sido impedimento para alcanzar velocidades de hasta 48 km/h y ser guiado autonomamente en curvas de radio reducido. Objetos estáticos y móviles han sido seguidos desde un helicoptero no tripulado, mediante el control de una plataforma visual “pan-and-tilt”. ´Éste mismo helicoptero ha sido controlado completamente para su aterrizaje autonomo, mediante el control del movimiento lateral (roll), horizontal (pitch) y de altitud. El seguimiento de objetos volantes ha sido resulto mediante el control horizontal (pitch) y de orientación (heading) de un quadrirotor. Para tareas de evitación de obstáculos se ha implementado un controlador difuso para el manejo de la orientación (heading) de un quadrirotor. En el campo de la optimización de controladores se ha aportado al estado del arte una extensión del uso del método “Cross-Entropy”. Está Tesis presenta una novedosa implementación de dicho método para la optimización de las ganancias, la posición y medida de los conjuntos de las funciones de pertenecia y el peso de las reglas para mejorar el comportamiento de un controlador difuso. Dichos procesos de optimización se han realizado utilizando “ROS” y “Matlab Simulink” para obtener mejores resultados para la evitación de colisiones con vehículos aéreos no tripulados. Ésta Tesis demuestra que los controladores implementados con lógica difusa son altamente capaces de controlador sistemas sin tener en cuenta el modelo del vehículo a controlador en entornos altamente perturbables con un sensor de bajo coste como es una cámara. El ruido presentes causado por los cambios de iluminación en la adquisición de imágenes y la alta incertidumbre en la detección visual han sido manejados satisfactoriamente por ésta técnica de de “Soft-Computing” para distintas aplicaciones tanto con vehículos aéreos como terrestres.

Wake-up architecture for Wireless sensor nodes based on ultra low power FPGA

In this work a novel wake-up architecture for wireless sensor nodes based on ultra low power FPGA is presented. A simple wake up messaging mechanism for data gathering applications is proposed. The main goal of this work is to evaluate the utilization of low power configurable devices to take advantage of their speed, flexibility and low power consumption compared with traditional approaches, based on ASICs or microcontrollers, for frame decoding and data control. A test bed based on infrared communications has been built to validate the messaging mechanism and the processing architecture.