Implantable Low-Noise Fiberless Optoelectrodes for Optogenetic Control of Distinct Neural Populations

The mammalian brain is often compared to an electrical circuit, and its dynamics and function are governed by communication across different types neurons. To treat neurological disorders like Alzheimer’s and Parkinson’s, which are characterized by inhibition or amplification of neural activity in a particular region or lack of communication between different regions of the brain, there is a need to understand troubleshoot neural networks at cellular or local circuit level. In this work, we introduce a novel implantable optoelectrode that can manipulate more than one neuron type at a single site, independently and simultaneously. By delivering multi-color light using a scalable optical waveguide mixer, we demonstrate manipulation of multiple neuron types at precise spatial locations in vivo for the first time. We report design, micro-fabrication and optoelectronic packaging of a fiber-less, multicolor optoelectrode. The compact optoelectrode design consists of a 7 μm x 30 μm dielectric optical waveguide mixer and eight electrical recording sites monolithically integrated on each shank of a 22 μm-thick four-shank silicon neural probe. The waveguide mixers are coupled to eight side-emitting injection laser diodes (ILDs) via gradient-index (GRIN) lenses assembled on the probe backend. GRIN-based optoelectrode enables efficient optical coupling with large alignment tolerance to provide wide optical power range (10 to 3000 mW/mm2 irradiance) at stimulation ports. It also keeps thermal dissipation and electromagnetic interference generated by light sources sufficiently far from the sensitive neural signals, allowing thermal and electrical noise management on a multilayer printed circuit board. We demonstrated device verification and validation in CA1 pyramidal layer of mice hippocampus in both anesthetized and awake animals. The packaged devices were used to manipulate variety of multi-opsin preparations in vivo expressing different combinations of Channelrhodopsin-2, Archaerhodopsin and ChrimsonR in pyramidal and parvalbumin interneuron cells. We show effective stimulation, inhibition and recording of neural spikes at precise spatial locations with less than 100 μV stimulation-locked transients on the recording channels, demonstrating novel use of this technology in the functional dissection of neural circuits.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/137171/1/kkomal_1.pd

Bidirectional Neural Interface Circuits with On-Chip Stimulation Artifact Reduction Schemes

Bidirectional neural interfaces are tools designed to “communicate” with the brain via recording and modulation of neuronal activity. The bidirectional interface systems have been adopted for many applications. Neuroscientists employ them to map neuronal circuits through precise stimulation and recording. Medical doctors deploy them as adaptable medical devices which control therapeutic stimulation parameters based on monitoring real-time neural activity. Brain-machine-interface (BMI) researchers use neural interfaces to bypass the nervous system and directly control neuroprosthetics or brain-computer-interface (BCI) spellers. In bidirectional interfaces, the implantable transducers as well as the corresponding electronic circuits and systems face several challenges. A high channel count, low power consumption, and reduced system size are desirable for potential chronic deployment and wider applicability. Moreover, a neural interface designed for robust closed-loop operation requires the mitigation of stimulation artifacts which corrupt the recorded signals. This dissertation introduces several techniques targeting low power consumption, small size, and reduction of stimulation artifacts. These techniques are implemented for extracellular electrophysiological recording and two stimulation modalities: direct current stimulation for closed-loop control of seizure detection/quench and optical stimulation for optogenetic studies. While the two modalities differ in their mechanisms, hardware implementation, and applications, they share many crucial system-level challenges. The first method aims at solving the critical issue of stimulation artifacts saturating the preamplifier in the recording front-end. To prevent saturation, a novel mixed-signal stimulation artifact cancellation circuit is devised to subtract the artifact before amplification and maintain the standard input range of a power-hungry preamplifier. Additional novel techniques have been also implemented to lower the noise and power consumption. A common average referencing (CAR) front-end circuit eliminates the cross-channel common mode noise by averaging and subtracting it in analog domain. A range-adapting SAR ADC saves additional power by eliminating unnecessary conversion cycles when the input signal is small. Measurements of an integrated circuit (IC) prototype demonstrate the attenuation of stimulation artifacts by up to 42 dB and cross-channel noise suppression by up to 39.8 dB. The power consumption per channel is maintained at 330 nW, while the area per channel is only 0.17 mm2. The second system implements a compact headstage for closed-loop optogenetic stimulation and electrophysiological recording. This design targets a miniaturized form factor, high channel count, and high-precision stimulation control suitable for rodent in-vivo optogenetic studies. Monolithically integrated optoelectrodes (which include 12 µLEDs for optical stimulation and 12 electrical recording sites) are combined with an off-the-shelf recording IC and a custom-designed high-precision LED driver. 32 recording and 12 stimulation channels can be individually accessed and controlled on a small headstage with dimensions of 2.16 x 2.38 x 0.35 cm and mass of 1.9 g. A third system prototype improves the optogenetic headstage prototype by furthering system integration and improving power efficiency facilitating wireless operation. The custom application-specific integrated circuit (ASIC) combines recording and stimulation channels with a power management unit, allowing the system to be powered by an ultra-light Li-ion battery. Additionally, the µLED drivers include a high-resolution arbitrary waveform generation mode for shaping of µLED current pulses to preemptively reduce artifacts. A prototype IC occupies 7.66 mm2, consumes 3.04 mW under typical operating conditions, and the optical pulse shaping scheme can attenuate stimulation artifacts by up to 3x with a Gaussian-rise pulse rise time under 1 ms.PHDElectrical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/147674/1/mendrela_1.pd

Outan: An On-Head System for Driving micro-LED Arrays Implanted in Freely Moving Mice

In the intact brain, neural activity can be recorded using sensing electrodes and manipulated using light stimulation. Silicon probes with integrated electrodes and micro-LEDs enable the detection and control of neural activity using a single implanted device. Miniaturized solutions for recordings from small freely moving animals are commercially available, but stimulation is driven by large, stationary current sources. We designed and fabricated a current source chip and integrated it into a headstage PCB that weighs 1.37 g. The proposed system provides 10-bit resolution current control for 32 channels, driving micro-LEDs with up to 4.6 V and sourcing up to 0.9 mA at a refresh rate of 5 kHz per channel. When calibrated against a micro-LED probe, the system allows linear control of light output power, up to 10 micro-W per micro-LED. To demonstrate the capabilities of the system, synthetic sequences of neural spiking activity were produced by driving multiple micro-LEDs implanted in the hippocampal CA1 area of a freely moving mouse. The high spatial, temporal, and amplitude resolution of the system provides a rich variety of stimulation patterns. Combined with commercially available sampling headstages, the system provides an easy to use back-end, fully utilizing the bi-directional potential of integrated opto-electronic arrays.Comment: 11 pages, 9 figure

Feasibility Assessment of an Optically Powered Digital Retinal Prosthesis Architecture for Retinal Ganglion Cell Stimulation

Clinical trials previously demonstrated the notable capacity to elicit visual percepts in blind patients affected with retinal diseases by electrically stimulating the remaining neurons on the retina. However, these implants restored very limited visual acuity and required transcutaneous cables traversing the eyeball, leading to reduced reliability and complex surgery with high postoperative infection risks. To overcome the limitations imposed by cables, a retinal implant architecture in which near-infrared illumination carries both power and data through the pupil to a digital stimulation controller is presented. A high efficiency multi-junction photovoltaic cell transduces the optical power to a CMOS stimulator capable of delivering flexible interleaved sequential stimulation through a diamond microelectrode array. To demonstrate the capacity to elicit a neural response with this approach while complying with the optical irradiance limit at the pupil, fluorescence imaging with a calcium indicator is used on a degenerate rat retina. The power delivered by the laser at the permissible irradiance of 4 mW/mm2 at 850 nm is shown to be sufficient to both power the stimulator ASIC and elicit a response in retinal ganglion cells (RGCs), with the ability to generate of up to 35 000 pulses per second at the average stimulation threshold. This confirms the feasibility of generating a response in RGCs with an infrared-powered digital architecture capable of delivering complex sequential stimulation patterns at high repetition rates, albeit with some limitations.Comment: 11 pages, 13 figure

Real-time in vivo optogenetic neuromodulation and multielectrode electrophysiologic recording with NeuroRighter

Optogenetic channels have greatly expanded neuroscience’s experimental capabilities, enabling precise genetic targeting and manipulation of neuron subpopulations in awake and behaving animals. However, many barriers to entry remain for this technology – including low-cost and effective hardware for combined optical stimulation and electrophysiologic recording. To address this, we adapted the open-source NeuroRighter multichannel electrophysiology platform for use in awake and behaving rodents in both open and closed-loop stimulation experiments. Here, we present these cost-effective adaptations, including commercially available LED light sources; custom-made optical ferrules; 3D printed ferrule hardware and software to calibrate and standardize output intensity; and modifications to commercially available electrode arrays enabling stimulation proximally and distally to the recording target. We then demonstrate the capabilities and versatility of these adaptations in several open and closed-loop experiments, demonstrate spectrographic methods of analyzing the results, as well as discuss artifacts of stimulation.Emory University. School of Medicine (Emory Neurosciences Initiative seed grant)American Epilepsy SocietyEpilepsy Foundation of America (Predoctoral fellowship)National Science Foundation (U.S.) (NSF GRFP Fellowship 08-593)National Science Foundation (U.S.) (NSF IGERT Fellowship DGE-0333411)National Science Foundation (U.S.) (NSF EFRI #1238097)National Institutes of Health (U.S.) (NIH 1R01NS079757-01)American Society for Engineering Education (SMART Fellowship

Advances in Integrated Circuits and Systems for Wearable Biomedical Electrical Impedance Tomography

Electrical impedance tomography (EIT) is an impedance mapping technique that can be used to image the inner impedance distribution of the subject under test. It is non-invasive, inexpensive and radiation-free, while at the same time it can facilitate long-term and real-time dynamic monitoring. Thus, EIT lends itself particularly well to the development of a bio-signal monitoring/imaging system in the form of wearable technology. This work focuses on EIT system hardware advancement using complementary metal oxide semiconductor (CMOS) technology. It presents the design and testing of application specific integrated circuit (ASIC) and their successful use in two bio-medical applications, namely, neonatal lung function monitoring and human-machine interface (HMI) for prosthetic hand control. Each year fifteen million babies are born prematurely, and up to 30% suffer from lung disease. Although respiratory support, especially mechanical ventilation, can improve their survival, it also can cause injury to their vulnerable lungs resulting in severe and chronic pulmonary morbidity lasting into adulthood, thus an integrated wearable EIT system for neonatal lung function monitoring is urgently needed. In this work, two wearable belt systems are presented. The first belt features a miniaturized active electrode module built around an analog front-end ASIC which is fabricated with 0.35-µm high-voltage process technology with ±9 V power supplies and occupies a total die area of 3.9 mm². The ASIC offers a high power active current driver capable of up to 6 mAp-p output, and wideband active buffer for EIT recording as well as contact impedance monitoring. The belt has a bandwidth of 500 kHz, and an image frame rate of 107 frame/s. To further improve the system, the active electrode module is integrated into one ASIC. It contains a fully differential current driver, a current feedback instrumentation amplifier (IA), a digital controller and multiplexors with a total die area of 9.6 mm². Compared to the conventional active electrode architecture employed in the first EIT belt, the second belt features a new architecture. It allows programmable flexible electrode current drive and voltage sense patterns under simple digital control. It has intimate connections to the electrodes for the current drive and to the IA for direct differential voltage measurement providing superior common-mode rejection ratio (CMRR) up to 74 dB, and with active gain, the noise level can be reduced by a factor of √3 using the adjacent scan. The second belt has a wider operating bandwidth of 1 MHz and multi-frequency operation. The image frame rate is 122 frame/s, the fastest wearable EIT reported to date. It measures impedance with 98% accuracy and has less than 0.5 Ω and 1° variation across all channels. In addition the ASIC facilitates several other functionalities to provide supplementary clinical information at the bedside. With the advancement of technology and the ever-increasing fusion of computer and machine into daily life, a seamless HMI system that can recognize hand gestures and motions and allow the control of robotic machines or prostheses to perform dexterous tasks, is a target of research. Originally developed as an imaging technique, EIT can be used with a machine learning technique to track bones and muscles movement towards understanding the human user’s intentions and ultimately controlling prosthetic hand applications. For this application, an analog front-end ASIC is designed using 0.35-µm standard process technology with ±1.65 V power supplies. It comprises a current driver capable of differential drive and a low noise (9μVrms) IA with a CMRR of 80 dB. The function modules occupy an area of 0.07 mm². Using the ASIC, a complete HMI system based on the EIT principle for hand prosthesis control has been presented, and the user’s forearm inner bio-impedance redistribution is assessed. Using artificial neural networks, bio-impedance redistribution can be learned so as to recognise the user’s intention in real-time for prosthesis operation. In this work, eleven hand motions are designed for prosthesis operation. Experiments with five subjects show that the system can achieve an overall recognition accuracy of 95.8%

Smart Embedded Systems for Biomedical Applications

L'abstract è presente nell'allegato / the abstract is in the attachmen

A Consumer-tier based Visual-Brain Machine Interface for Augmented Reality Glasses Interactions

Objective.Visual-Brain Machine Interface(V-BMI) has provide a novel interaction technique for Augmented Reality (AR) industries. Several state-of-arts work has demonstates its high accuracy and real-time interaction capbilities. However, most of the studies employ EEGs devices that are rigid and difficult to apply in real-life AR glasseses application sceniraros. Here we develop a consumer-tier Visual-Brain Machine Inteface(V-BMI) system specialized for Augmented Reality(AR) glasses interactions. Approach. The developed system consists of a wearable hardware which takes advantages of fast set-up, reliable recording and comfortable wearable experience that specificized for AR glasses applications. Complementing this hardware, we have devised a software framework that facilitates real-time interactions within the system while accommodating a modular configuration to enhance scalability. Main results. The developed hardware is only 110g and 120x85x23 mm, which with 1 Tohm and peak to peak voltage is less than 1.5 uV, and a V-BMI based angry bird game and an Internet of Thing (IoT) AR applications are deisgned, we demonstrated such technology merits of intuitive experience and efficiency interaction. The real-time interaction accuracy is between 85 and 96 percentages in a commercial AR glasses (DTI is 2.24s and ITR 65 bits-min ). Significance. Our study indicates the developed system can provide an essential hardware-software framework for consumer based V-BMI AR glasses. Also, we derive several pivotal design factors for a consumer-grade V-BMI-based AR system: 1) Dynamic adaptation of stimulation patterns-classification methods via computer vision algorithms is necessary for AR glasses applications; and 2) Algorithmic localization to foster system stability and latency reduction.Comment: 15 pages,10 figure

Electronic Devices for the Combination of Electrically Controlled Drug Release, Electrostimulation, and Optogenetic Stimulation for Nerve Tissue Regeneration

[ES] La capacidad de las células madre para proliferar formando distintas células especializadas les otorga la potencialidad de servir de base para terapias efectivas para patologías cuyo tratamiento era inimaginable hasta hace apenas dos décadas. Sin embargo, esta capacidad se encuentra mediada por estímulos fisiológicos, químicos, y eléctricos, específicos y complejos, que dificultan su traslación a la rutina clínica. Por ello, las células madre representan un campo de estudio en el que se invierten amplios esfuerzos por parte de la comunidad científica. En el ámbito de la regeneración nerviosa, para modular su desarrollo y diferenciación el tratamiento farmacológico, la electroestimulación, y la estimulación optogenética son técnicas que están consiguiendo prometedores resultados. Es por ello por lo que en la presente tesis se ha desarrollado un conjunto de sistemas electrónicos para permitir la aplicación combinada de estas técnicas in vitro, con perspectiva a su aplicación in vivo. Hemos diseñado una novedosa tecnología para la liberación eléctricamente controlada de fármacos. Esta tecnología está basada en nanopartículas de sílice mesoporosa y puertas moleculares de bipiridina-heparina. Las puertas moleculares son electroquímicamente reactivas, y encierran los fármacos en el interior de las nanopartículas, liberándolos ante un estímulo eléctrico. Hemos caracterizado esta tecnología, y la hemos validado mediante la liberación controlada de rodamina en cultivos celulares de HeLa. Para la combinación de liberación controlada de fármacos y electroestimulación hemos desarrollado dispositivos que permiten aplicar los estímulos eléctricos de forma configurable desde una interfaz gráfica de usuario. Además, hemos diseñado un módulo de expansión que permite multiplexar las señales eléctricas a diferentes cultivos celulares. Además, hemos diseñado un dispositivo de estimulación optogenética. Este tipo de estimulación consiste en la modificación genética de las células para que sean sensibles a la radiación lumínica de determinada longitud de onda. En el ámbito de la regeneración de tejido mediante células precursoras neurales, es de interés poder inducir ondas de calcio, favoreciendo su diferenciación en neuronas y la formación de circuitos sinápticos. El dispositivo diseñado permite obtener imágenes en tiempo real mediante microscopía confocal de las respuestas transitorias de las células al ser irradiadas. El dispositivo se ha validado irradiando neuronas modificadas con luz pulsada de 100 ms. También hemos diseñado un dispositivo electrónico complementario de medida de irradiancia con el doble fin de permitir la calibración del equipo de irradiancia y medir la irradiancia en tiempo real durante los experimentos in vitro. Los resultados del uso de los bioactuadores en procesos complejos y dinámicos, como la regeneración de tejido nervioso, son limitados en lazo abierto. Uno de los principales aspectos analizados es el desarrollo de biosensores que permitiesen la cuantización de ciertas biomoléculas para ajustar la estimulación suministrada en tiempo real. Por ejemplo, la segregación de serotonina es una respuesta identificada en la elongación de células precursoras neurales, pero hay otras biomoléculas de interés para la implementación de un control en lazo cerrado. Entre las tecnologías en el estado del arte, los biosensores basados en transistores de efecto de campo (FET) funcionalizados con aptámeros son realmente prometedores para esta aplicación. Sin embargo, esta tecnología no permitía la medición simultánea de más de una biomolécula objetivo en un volumen reducido debido a las interferencias entre los distintos FETs, cuyos terminales se encuentran inmersos en la solución. Por ello, hemos desarrollado instrumentación electrónica capaz de medir simultáneamente varios de estos biosensores, y la hemos validado mediante la medición simultánea de pH y la detección preliminar de serotonina y glutamato.[CA] La capacitat de les cèl·lules mare per a proliferar formant diferents cèl·lules especialitzades els atorga la potencialitat de servir de base per a teràpies efectives per a patologies el tractament de les quals era inimaginable fins fa a penes dues dècades. No obstant això, aquesta capacitat es troba mediada per estímuls fisiològics, químics, i elèctrics, específics i complexos, que dificulten la seua translació a la rutina clínica. Per això, les cèl·lules mare representen un camp d'estudi en el qual s'inverteixen amplis esforços per part de la comunitat científica. En l'àmbit de la regeneració nerviosa, per a modular el seu desenvolupament i diferenciació el tractament farmacològic, l'electroestimulació, i l'estimulació optogenética són tècniques que estan aconseguint prometedors resultats. És per això que en la present tesi s'ha desenvolupat un conjunt de sistemes electrònics per a permetre l'aplicació combinada d'aquestes tècniques in vitro, amb perspectiva a la seua aplicació in vivo. Hem dissenyat una nova tecnologia per a l'alliberament elèctricament controlat de fàrmacs. Aquesta tecnologia està basada en nanopartícules de sílice mesoporosa i portes moleculars de bipiridina-heparina. Les portes moleculars són electroquímicament reactives, i tanquen els fàrmacs a l'interior de les nanopartícules, alliberant-los davant un estímul elèctric. Hem caracteritzat aquesta tecnologia, i l'hem validada mitjançant l'alliberament controlat de rodamina en cultius cel·lulars de HeLa. Per a la combinació d'alliberament controlat de fàrmacs i electroestimulació hem desenvolupat dispositius que permeten aplicar els estímuls elèctrics de manera configurable des d'una interfície gràfica d'usuari. A més, hem dissenyat un mòdul d'expansió que permet multiplexar els senyals elèctrics a diferents cultius cel·lulars. A més, hem dissenyat un dispositiu d'estimulació optogenètica. Aquest tipus d'estimulació consisteix en la modificació genètica de les cèl·lules perquè siguen sensibles a la radiació lumínica de determinada longitud d'ona. En l'àmbit de la regeneració de teixit mitjançant cèl·lules precursores neurals, és d'interés poder induir ones de calci, afavorint la seua diferenciació en neurones i la formació de circuits sinàptics. El dispositiu dissenyat permet obtindré imatges en temps real mitjançant microscòpia confocal de les respostes transitòries de les cèl·lules en ser irradiades. El dispositiu s'ha validat irradiant neurones modificades amb llum polsada de 100 ms. També hem dissenyat un dispositiu electrònic complementari de mesura d'irradiància amb el doble fi de permetre el calibratge de l'equip d'irradiància i mesurar la irradiància en temps real durant els experiments in vitro. Els resultats de l'ús dels bioactuadors en processos complexos i dinàmics, com la regeneració de teixit nerviós, són limitats en llaç obert. Un dels principals aspectes analitzats és el desenvolupament de biosensors que permeteren la quantització de certes biomolècules per a ajustar l'estimulació subministrada en temps real. Per exemple, la segregació de serotonina és una resposta identificada amb l'elongació de les cèl·lules precursores neurals, però hi ha altres biomolècules d'interés per a la implementació d'un control en llaç tancat. Entre les tecnologies en l'estat de l'art, els biosensors basats en transistors d'efecte de camp (FET) funcionalitzats amb aptàmers són realment prometedors per a aquesta aplicació. No obstant això, aquesta tecnologia no permetia el mesurament simultani de més d'una biomolècula objectiu en un volum reduït a causa de les interferències entre els diferents FETs, els terminals dels quals es troben immersos en la solució. Per això, hem desenvolupat instrumentació electrònica capaç de mesurar simultàniament diversos d'aquests biosensors i els hem validat amb mesurament simultani del pH i la detecció preliminar de serotonina i glutamat.[EN] The stem cells' ability to proliferate to form different specialized cells gives them the potential to serve as the basis for effective therapies for pathologies whose treatment was unimaginable until just two decades ago. However, this capacity is mediated by specific and complex physiological, chemical, and electrical stimuli that complicate their translation to clinical routine. For this reason, stem cells represent a field of study in which the scientific community is investing a great deal of effort. In the field of nerve regeneration, to modulate their development and differentiation, pharmacological treatment, electrostimulation, and optogenetic stimulation are techniques that are achieving promising results. For this reason, we have developed a set of electronic systems to allow the combined application of these techniques in vitro, with a view to their application in vivo. We have designed a novel technology for the electrically controlled release of drugs. This technology is based on mesoporous silica nanoparticles and bipyridine-heparin molecular gates. The molecular gates are electrochemically reactive and entrap the drugs inside the nanoparticles, releasing them upon electrical stimulus. We have characterized this technology and validated it by controlled release of rhodamine in HeLa cell cultures. For combining electrostimulation and controlled drug release we have developed devices that allow applying the different electrical stimuli in a configurable way from a graphical user interface. In addition, we have designed an expansion module that allows multiplexing electrical signals to different cell cultures. In addition, we have designed an optogenetic stimulation device. This type of stimulation consists of genetically modifying cells to make them sensitive to light radiation of a specific wavelength. In tissue regeneration using neural precursor cells, it is interesting to be able to induce calcium waves, favoring the cell differentiation into neurons and the formation of synaptic circuits. The designed device enable the obtention of real-time images through confocal microscopy of the transient responses of cells upon irradiation. The device has been validated by irradiating modified neurons with 100 ms pulsed light stimulation. We have also designed a complementary electronic irradiance measurement device to allow calibration of the irradiator equipment and measuring irradiance in real time during in vitro experiments. The results of using bioactuators in complex and dynamic processes, such as nerve tissue regeneration, are limited in an open loop. One of the main aspects analyzed is the development of biosensors that would allow quantifying of specific biomolecules to adjust the stimulation provided in real time. For instance, serotonin secretion is an identified response of neural precursor cells elongation, among other biomolecules of interest for the implementation of a closed-loop control. Among the state-of-the-art technologies, biosensors based on field effect transistors (FETs) functionalized with aptamers are promising for this application. However, this technology did not allow the simultaneous measurement of more than one target biomolecule in a small volume due to interferences between the different FETs, whose terminals are immersed in the solution. This is why we have developed electronic instrumentation capable of simultaneously measuring several of these biosensors, and we have validated it with the simultaneous pH measurement and the preliminary detection of serotonin and glutamate.Monreal Trigo, J. (2023). Electronic Devices for the Combination of Electrically Controlled Drug Release, Electrostimulation, and Optogenetic Stimulation for Nerve Tissue Regeneration [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/19384