1,347 research outputs found

    Discovering circulating protein biomarkers through in-depth plasma proteomics

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    Plasma, i.e., the liquid component of blood, is one of the most clinically used samples for biomarker measurement. Despite that plasma proteins and metabolites are the most frequently analysed biomarkers in practice, identifying and implementing new circulating protein biomarkers for diagnosis, treatment prediction, prognosis, and disease monitoring has been limited. This PhD thesis compiles the discovery of systemic alterations in the blood plasma proteome and potential biomarkers related to disease status, prognosis, or treatment through plasma proteomics. We analysed plasma and serum samples with global proteomics by high-resolution isoelectric focusing (HiRIEF) and liquid chromatography coupled with mass-spectrometry (LC-MS/MS), and targeted proteomics by antibody-based proximity extension assays (PEA) in three diseases that would benefit from blood biomarkers: stage IV metastatic cutaneous melanoma (mCM), glioblastoma (GBM), and coronavirus disease 2019 (COVID-19). Specifically: a.) New treatment options for mCM substantially prolong overall survival (OS), but multiple patients do not respond to treatment or develop treatment resistance, thus having shorter progression free survival (PFS). Corroborated by the presence of multiple metastases, which makes biomarker sampling difficult, circulating proteins derived from the tumour and in response to treatment could serve as predictive and prognostic biomarkers in mCM. b.) GBM is the most malignant primary brain tumour with limited treatment options and notoriously short OS. Sampling biomarkers for GBM requires an invasive surgical intervention on the skull, which makes GBM a good candidate for circulating protein biomarkers for prognosis and monitoring. c.) COVID-19 is an inflammation-driven infectious disease that affects multiple organs and systems, thus making the plasma proteome a good source to explore systemic biological processes occurring in COVID-19. In papers I and II, using HiRIEF LC-MS/MS and PEA, we explored the treatment-driven plasma proteome alterations in mCM patients treated with anti-PD-1 immune checkpoint inhibitors (ICI) and MAPK-inhibitors (MAPKi), respectively, and identified potential treatment predictive and monitoring biomarkers. mCM patients treated with anti-PD-1 ICI had a strong increase in soluble PD-1 levels during treatment, and upregulation of proteins involved in T-cell response. BRAF[V600]-mutated mCM patients treated with MAPKi had deregulation in proteins involved in immune response and proteolysis. CPB1 had the highest increase in patients treated with BRAF- and MEK-inhibitors and was associated with longer PFS. Higher levels of several proteins involved in inflammation before treatment were associated with shorter PFS regardless of ICI or MAPKi treatment. In paper III, using HiRIEF LC-MS/MS and PEA, we longitudinally analysed the plasma proteome dynamics of GBM patients, collecting plasma samples before surgery and at three timepoints after surgery. Through consensus clustering, based on treatment-naïve plasma protein levels, we identified two patient clusters that differed in median OS. The association between the cluster membership and OS remained consistent after adjustment for age, sex, and treatment. Through machine learning, we identified protein panels that separated the patient clusters and may serve as prognostic biomarkers. The largest alterations in the plasma proteome of GBM patients occurred within two months after surgery, whereas the plasma protein levels at later timepoints had no difference compared to pre- surgery levels. We observed a decrease in glioma-elevated proteins in the blood after surgery, identifying potential monitoring biomarkers. In paper IV, using HiRIEF LC-MS/MS, we analysed serum proteome alterations in hospitalised COVID-19 patients in comparison to healthy controls, and identified a strong upregulation in inflammatory, interferon-induced, and proteasomal proteins. Several protein groups showed association with clinical parameters of COVID-19 severity, including proteasomal proteins. Serum proteome alterations were traceable to proteome alterations induced in a lung adenocarcinoma cell line (Calu-3) by infection with SARS-CoV-2. Finally, we performed the first meta-analysis of global proteomics studies of the soluble blood proteome in COVID-19, providing estimates of standardised mean differences and summary receiver operating characteristics curves. We demonstrate the high accuracy and precision of HiRIEF LC-MS/MS when compared to the meta-analysis estimates and pinpoint proteins that may serve as biomarkers of COVID-19. In summary, this thesis postulates that new circulating protein biomarkers would be clinically useful. By combining mass-spectrometry- and antibody-based-proteomics, we demonstrate the potential of in-depth analyses of the plasma proteome in capturing systemic alterations related to treatment, survival, and disease status, pinpointing potentially novel biomarkers that require validation in larger cohorts

    Combining Tumour-Treating Fields with DNA damage response inhibitors for the improved treatment of glioblastoma

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    Glioblastoma is the most common and deadliest type of primary brain cancer, taking over 2,500 lives each year in the UK. glioblastoma has a median overall survival of 10-16 months, despite treatment consisting of maximal surgical resection followed by chemo- and radio-therapy. glioblastoma survival rates have seen little improvement over the past 40 years and given this devastating prognosis, new treatment options for the management of glioblastoma are urgently needed. Recently, Tumour Treating Fields (TTFields) has emerged as a novel fourth modality for the treatment of high-grade gliomas following its success in clinical trials, where the addition of TTFields to standard care temozolomide was shown to increase median progression-free survival (6.7 versus 4.0 months) and overall survival (20.9 vs 16.0 months) of newly-diagnosed glioblastoma patients compared to temozolomide alone. TTFields are primarily thought to mediate their anti-cancer effects by disrupting tubulin dimer alignment during mitosis, resulting in abnormal chromosomal segregation and mitotic cell death. In addition, recent data suggests that TTFields affect a number of other cellular processes – 1- cell membrane and blood-brain barrier (BBB) permeability, 2- cell migration and invasion, 3- anti�tumour immunity, 4- autophagy, and 5- replication stress and DNA damage repair, the latter of which will be the focus of this project. TTFields has also been shown to downregulate DNA damage response (DDR) proteins and delay the repair of radiotherapy- and chemotherapy-induced DNA lesions, an effect that is thought to be mediated through reduced homologous recombination repair efficiency and induction of a ‘BRCAness’ phenotype. Such vulnerabilities within DNA damage repair pathways provides a rational for the use of TTFields in combinational therapeutic approaches that target the DDR. We therefore aim to assess whether combining TTFields with DDR inhibitors (PARPi, ATMi, ATRi and WEE1i) can enhance the efficacy of TTFields in clinically relevant glioblastoma stem-like cultures (GSCs) using a number of established cell survival assays. Additionally, we aim to investigate the mechanisms by which combination treatments of DDR inhibitors and TTFields affect the DNA damage response. In this thesis we show that combining TTFields with radiation and clinically approved PARP inhibitor therapy leads to significantly increased amounts of DNA damage with concurrent decreased clonogenic survival in GSCs. Furthermore, we have shown similar impressive potency when TTFields treatments are combined with BBB-penetrant ATR inhibitors that are currently being assessed in various global clinical trials for glioblastoma as well as other cancers. Overall, these exciting findings support further assessment of TTFields and DDRi combinations to underpin future clinical trials combining TTFields with clinically approved DDRi to improve outcomes for patients with currently incurable high-grade gliomas

    A Contribution Towards Intelligent Autonomous Sensors Based on Perovskite Solar Cells and Ta2O5/ZnO Thin Film Transistors

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    Many broad applications in the field of robotics, brain-machine interfaces, cognitive computing, image and speech processing and wearables require edge devices with very constrained power and hardware requirements that are challenging to realize. This is because these applications require sub-conscious awareness and require to be always “on”, especially when integrated with a sensor node that detects an event in the environment. Present day edge intelligent devices are typically based on hybrid CMOS-memristor arrays that have been so far designed for fast switching, typically in the range of nanoseconds, low energy consumption (typically in nano-Joules), high density and endurance (exceeding 1015 cycles). On the other hand, sensory-processing systems that have the same time constants and dynamics as their input signals, are best placed to learn or extract information from them. To meet this requirement, many applications are implemented using external “delay” in the memristor, in a process which enables each synapse to be modeled as a combination of a temporal delay and a spatial weight parameter. This thesis demonstrates a synaptic thin film transistor capable of inherent logic functions as well as compute-in-memory on similar time scales as biological events. Even beyond a conventional crossbar array architecture, we have relied on new concepts in reservoir computing to demonstrate a delay system reservoir with the highest learning efficiency of 95% reported to date, in comparison to equivalent two terminal memristors, using a single device for the task of image processing. The crux of our findings relied on enhancing our capability to model the unique physics of the device, in the scope of the current thesis, that is not amenable to conventional TCAD simulations. The model provides new insight into the redox characteristics of the gate current and paves way for assessment of device performance in compute-in-memory applications. The diffusion-based mechanism of the device, effectively enables time constants that have potential in applications such as gesture recognition and detection of cardiac arrythmia. The thesis also reports a new orientation of a solution processed perovskite solar cell with an efficiency of 14.9% that is easily integrable into an intelligent sensor node. We examine the influence of the growth orientation on film morphology and solar cell efficiency. Collectively, our work aids the development of more energy-efficient, powerful edge-computing sensor systems for upcoming applications of the IOT

    Single molecule MATAC-seq reveals key determinants for DNA replication efficiency

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    The stochastic nature of origin activation results in significant variability in the way genome replication is carried out from cell to cell. The reason for the diversity in efficiency and timing of individual origins has remained an unresolved issue for a long time. Cell-to-cell variability has been demonstrated to play a crucial role in cellular plasticity and cancer in mammalian cells. Although population-based methods have provided valuable insight into biological processes, it is necessary to use single molecule techniques to uncover events that are hidden by the population average. Many biological processes, such as DNA replication, transcription, and gene expression, are closely linked to the local chromatin structure. In yeast, although DNA replication origins have conserved DNA sequences, they display remarkable differences in timing and efficiency. Some origins initiate replication earlier during S-phase or more frequently than others, resulting in a high degree of heterogeneity among the cells in a population, with no two cells having the exact same replication profile. Our hypothesis is that the local nucleosomal structure may affect the DNA replication profile of individual origins. To explore this relationship, we have developed Methylation Accessibility of Targeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility maps of specific genomic locations after targeted purification in their native chromatin context. Our analysis of selected early-efficient (EE) and late-inefficient (LI) replication origins in Saccharomyces cerevisiae using MATAC-Seq revealed significant cell-to-cell heterogeneity in their chromatin states. Disrupting the INO80 or ISW2 chromatin remodeling complexes led to changes at individual nucleosomal positions that correspond to changes in replication efficiency. Our results show that a chromatin state with a narrow size of accessible origin DNA in combination with well-positioned surrounding nucleosomes and an open +2 linker region was a strong predictor for efficient origin activation. MATAC-Seq provides a single-molecule assay for chromatin accessibility that reveals the large spectrum of alternative chromatin states that coexist at a given locus, which was previously masked in population-based experiments. This provides a mechanistic basis for origin activation heterogeneity that occurs during DNA replication in eukaryotic cells. As a result, our single-molecule assay for chromatin accessibility will be ideal for defining single-molecule heterogeneity across many biological processes, such as transcription, replication, or DNA repair in vitro and ex vivo.Die stochastische Natur der Aktivierung von Replikationsursprüngen führt zu einer signifikanten Variabilität in der Art und Weise, wie die DNA Replikation von Zelle zu Zelle durchgeführt wird. Der Grund für die Diversität in Effizienz und Zeitpunkt der individuellen Aktivierung von Ursprüngen blieb lange ein ungelöstes Problem. Es wurde gezeigt, dass die Zell-zu-Zell-Variabilität eine entscheidende Rolle bei der zellulären Plastizität und Krebs in Säugetierzellen spielt. Obwohl populationsbasierte Methoden wertvolle Einblicke in biologische Prozesse geliefert haben, ist es notwendig, Einzelmolekültechniken zu verwenden, um Ereignisse aufzudecken, die durch das Durchschnittsverhalten aller Moleküle verborgen sind. Viele biologische Prozesse wie DNA-Replikation, Transkription und Genexpression sind eng mit der lokalen Chromatinstruktur verbunden. Obwohl die DNA-Replikationsursprünge in Hefe konservierte DNA-Sequenzen aufweisen, zeigen sie bemerkenswerte Unterschiede im Zeitpunkt und Effizienz der Replikation. Einige Ursprünge initiieren die Replikation früher während der S-Phase oder häufiger als andere, was zu einem hohen Grad an Heterogenität zwischen den Zellen in einer Population führt, wobei keine zwei Zellen das exakt gleiche Replikationsprofil aufweisen. Unsere Hypothese ist, dass die lokale nukleosomale Struktur das DNA-Replikationsprofil beeinflussen kann. Um diese Beziehung zu untersuchen, haben wir Methylation Accessibility of Targeted Chromatin Domain Sequencing (MATAC-Seq) entwickelt, um Einzelmolekül-Chromatin-Zugänglichkeitskarten spezifischer genomischer Orte nach gezielter Reinigung in ihrem nativen Chromatin-Kontext zu bestimmen. Unsere Analyse ausgewählter früh-effizient (EE) und spät-ineffizient (LI) feuernde Replikationsursprünge in Saccharomyces cerevisiae mit MATAC-Seq ergab eine signifikante Zell-zu-Zell-Heterogenität in ihren Chromatinzuständen. Die genetische Deletion der INO80- oder ISW2-Chromatin-Remodeling Komplexe führte zu Veränderungen an einzelnen nukleosomalen Positionen, die mit Veränderungen der Replikationseffizienz korrespondierten. Unsere Ergebnisse zeigten, dass ein Chromatinzustand mit einem engen Fenster an zugänglicher Replikationsursprungs-DNA in Kombination mit gut positionierten umgebenden Nukleosomen und einer offenen +2-Linkerregion ein starker Prädiktor für eine effiziente Ursprungsaktivierung war. MATAC-Seq bietet einen Einzelmolekül-Assay für die Zugänglichkeit von Chromatin, der das große Spektrum alternativer Chromatinzustände aufzeigt, die an einem bestimmten genomischen Lokus koexistieren, der zuvor in populationsbasierten Experimenten maskiert war. Dies liefert eine mechanistische Grundlage für die Heterogenität der Ursprungsaktivierung, die während der DNA-Replikation in eukaryotischen Zellen auftritt. Infolgedessen ist unser Einzelmolekül-Assay 5 für Chromatin-Zugänglichkeit ideal für die Definition der Einzelmolekül-Heterogenität über viele biologische Prozesse hinweg, wie z. B. Transkription, Replikation oder DNA-Reparatur in vitro und ex vivo

    Planetáris környezetünk veszélyeztetése és megmentése

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    Természeti környezetünkre sokféle emberi tevékenység van hatással. A környezet-károsító folyamatok, az emberi egészségre és általában véve az élővilágra gyakorolt ártalmas visszahatásaik az elmúlt egy évszázadnyi időszakban globális szintűvé váltak. Közös planetáris otthonunkat növekvő mértékben kezdte veszélyeztetni más behatások mellett a toxikus nehézfémek, a veszélyes vegyi anyagok és hulladékok által okozott környezetszennyezés, az ózonkárosító anyagok és az üvegházhatású gázok kibocsátása. Következményeik a jelenlegi és a jövő nemzedékek életét, életminőségét is súlyosan érintik. A kötetben bemutatjuk e környezeti problémák kialakulását, felismerésük történetét, az ezekkel foglalkozó nemzetközi tudományos és politikai együttműködés fejlődését. Áttekintjük a különböző programokat és megállapodásokat, értékeljük azok hatékonyságát és levonjuk a jövőre nézve is legfontosabbnak tartott tanulságokat

    Interplay of DNA replication, repair and chromatin: structure versus function

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    Hierarchical levels of chromatin organization allow different genomic functions to be spatio-temporally regulated within mammalian nuclei. Both DNA replication and DNA repair are global genomic processes. Their chromatin units have remarkable structural similarities and microscopically appear as clusters of nanofocal structures, each in the size range of chromatin loops. The present work aimed at relating genomic functions with the underlying structural organization by the two chromatin architectural proteins CTCF and cohesin, which cooperate to shape the genome into chromatin loops and domains. Here, CTCF was shown to be critical for cellular survival after ionizing irradiation in a CTCF-dose dependent way. The results obtained in different cell lines upon CTCF-depletion were integrated into a biophysical model. The decreased clonogenic potential showed to derive from the increased probability of double strand breaks to cluster in larger chromatin domains lacking CTCF at their borders. Moreover, CTCF proved to be enriched at the sites and at the time of DNA replication. CTCF intensity within replication foci was shown to decrease over a chase time after replication labeling, suggesting the occurrence of CTCF accumulation during ongoing DNA replication. The depletion of CTCF correlated with an impairment in cell cycle progression. CTCF-depleted cells stalled in G1 in a CTCF-dose dependent way, indicating that the chromatin structure provided by CTCF might be needed to properly enter S-phase. Additionally, CTCF resulted to be particularly enriched at the replicating inactive X and Y chromosomes. The depletion of CTCF led to the loss of synchrony in the DNA replication of the Y chromosome. Additionally, Y chromosome architecture showed changes of volume and shape upon CTCF reduction. In the second part of this work, the cohesin subunit RAD21 was shown essential to determine the structure of chromatin loops. RAD21-depleted cells exhibited an increase in the size and shape heterogeneity of chromatin loops. The cohesin component SA1 was investigated for a role in DNA damage signaling. SA1 KO cells showed an impairment of the γH2AX foci at all tested X-ray doses. The repair functional units decreased in number, volume and intensity in the absence of SA1. In conclusion, the results presented here led to propose that the functions of DNA replication and repair are determined by the chromatin architecture, with the structure dictating the function. Future work should further investigate the mechanisms behind the regulation of global genomic functions by these two chromatin architectural proteins and define the precise interplay between cohesin and CTCF within such regulation

    International Academic Symposium of Social Science 2022

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    This conference proceedings gathers work and research presented at the International Academic Symposium of Social Science 2022 (IASSC2022) held on July 3, 2022, in Kota Bharu, Kelantan, Malaysia. The conference was jointly organized by the Faculty of Information Management of Universiti Teknologi MARA Kelantan Branch, Malaysia; University of Malaya, Malaysia; Universitas Pembangunan Nasional Veteran Jakarta, Indonesia; Universitas Ngudi Waluyo, Indonesia; Camarines Sur Polytechnic Colleges, Philippines; and UCSI University, Malaysia. Featuring experienced keynote speakers from Malaysia, Australia, and England, this proceeding provides an opportunity for researchers, postgraduate students, and industry practitioners to gain knowledge and understanding of advanced topics concerning digital transformations in the perspective of the social sciences and information systems, focusing on issues, challenges, impacts, and theoretical foundations. This conference proceedings will assist in shaping the future of the academy and industry by compiling state-of-the-art works and future trends in the digital transformation of the social sciences and the field of information systems. It is also considered an interactive platform that enables academicians, practitioners and students from various institutions and industries to collaborate

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

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    [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
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