Estudio y realización de un neuroprocesador biológico: métodos de aprendizaje

[SPA] La presente tesis se enmarca dentro de dos campos diferentes pero intrínsecamente unidos entre sí en este caso: neurociencia y computación. El objetivo global de esta tesis es la realización de un neuroprocesador biológico empleando como plataforma redes neuronales biológicas cultivadas sobre matrices de microelectrodos. Este objetivo global resulta en una serie de sub-objetivos: (1) Definición y construcción de una plataforma para el soporte en tiempo real de los sistemas de adquisición de registros neuronales, y estimulación eléctrica de los mismos, que se comunique remotamente con un dispositivo robótico. (2) Estudio y propuesta de un método de guiado robótico basado en una plataforma de lazo cerrado que integre la información de los sensores del robot en el neuroprocesador y, en función de la respuesta de éste, direccione el sistema robótico. (3) Normalización y calibración estadística de los registros del neuroprocesador para su adecuación a los distintos algoritmos de guiado robótico y aprendizaje en los cultivos neuronales. (4) Estudio y definición de técnicas de aprendizaje en cultivos neuronales para la realización de conectividad funcional dirigida con objeto de proporcionar nuevos paradigmas de programación en neuroprocesadores biológicos. Con respecto al sub-objetivo (1), se ha propuesto un sistema de experimentación con cultivos neuronales en lazo cerrado y tiempo real que proporciona las herramientas de filtrado, visualización, procesamiento y estimulación de la respuesta electrofisiológica de poblaciones neuronales y su comunicación con un sistema robótico remoto. Para alcanzar el objetivo (2), se ha adaptado el algoritmo de centro de área para guiado robótico a las respuestas funcionales de las poblaciones de neuronas, identificando aquellos electrodos de la matriz cuyas neuronas incrementan en mayor medida sus disparos, como objetivo para el direccionamiento del robot. El cumplimiento del sub-objetivo (3) se ha conseguido al proporcionar técnicas de calibración y normalización estadística de los registros de poblaciones de neuronas que conforman el neuroprocesador, con objeto de suprimir la variabilidad intrínseca de las mismas y a las distintas características de no-homogeneidad tanto en la densidad del cultivo como en las propiedades eléctricas de los distintos electrodos. Finalmente, atendiendo al sub-objetivo (4), se ha propuesto un paradigma de aprendizaje natural, como es el aprendizaje hebbiano, para la conformación de conexiones funcionales entre electrodos que no se encontraban enlazados previamente y conseguir de esta forma el modelado del cultivo para la implementación en su estructura de las funciones a implementar, en este caso las estructuras de Braitenberg.[ENG] This thesis deals with two different fields, inherently related to each other in this case: neuroscience and computation. The overall objective of this thesis is the development of a biological neuroprocessor with cultured biological neural networks using microelectrode arrays as platform. This objective results in a set of specific subobjectives: (1) Define and build a platform for real time support of acquisition systems and electrical stimulation systems of neural registers, which remotely communicates with a robotic device. (2) Study and propose a robotic guidance method based on a close-loop platform which includes the sensory robot information in the neuroprocessor and, according to its response, guides the robotic system. (3) Normalization and statistic calibration of the registers of the neuroprocessor in order to adapt them to different algorithms of robotic guidance and learning in cultured neural networks. (4) Study and define learning techniques in neural cultures for the development of functional connectivity which allows new programming paradigms in biological neuroprocessors. Regarding objective (1), a real-time close-loop experimentation system with neural cultures has been proposed, which provides a complete solution for filtering, visualization, processing and stimulation of electrophysiological response from neural population and communication with a robotic system. In order to reach objective (2), centre of area algorithm for robotic guidance has been adapted to the functional response of neural populations, identifying those electrodes from the array whose neurons increase the most its firing rate, as target for robotic guidance. Objective (3) has been met giving statistic calibration and normalization techniques of neural population registers that conform the neuroprocessor having in mind the goal of supressing the intrinsic variability of those populations and the different nonhomogeneity characteristics, both in culture density and electrical properties of the electrodes. Finally, regarding objective (4), a natural learning paradigm has been proposed, Hebbian learning, to conform functional connections between previously not connected electrodes. In this way, the cultures can be modelled for implementing the desired behaviour in the biological structure, in this case Braitenberg behaviour.Universidad Politécnica de Cartagen

A new bioavailable fenretinide formulation with antiproliferative, antimetabolic, and cytotoxic effects on solid tumors.

Fenretinide is a synthetic retinoid characterized by anticancer activity in preclinical models and favorable toxicological profile, but also by a low bioavailability that hindered its clinical efficacy in former clinical trials. We developed a new formulation of fenretinide complexed with 2-hydroxypropyl-beta-cyclodextrin (nanofenretinide) characterized by an increased bioavailability and therapeutic efficacy. Nanofenretinide was active in cell lines derived from multiple solid tumors, in primary spheroid cultures and in xenografts of lung and colorectal cancer, where it inhibited tumor growth independently from the mutational status of tumor cells. A global profiling of pathways activated by nanofenretinide was performed by reverse-phase proteomic arrays and lipid analysis, revealing widespread repression of the mTOR pathway, activation of apoptotic, autophagic and DNA damage signals and massive production of dihydroceramide, a bioactive lipid with pleiotropic effects on several biological processes. In cells that survived nanofenretinide treatment there was a decrease of factors involved in cell cycle progression and an increase in the levels of p16 and phosphorylated p38 MAPK with consequent block in G0 and early G1. The capacity of nanofenretinide to induce cancer cell death and quiescence, together with its elevated bioavailability and broad antitumor activity indicate its potential use in cancer treatment and chemoprevention

Contactless acoustic micro/nano manipulation:a paradigm for next generation applications in life sciences

Acoustic actuation techniques offer a promising tool for contactless manipulation of both synthetic and biological micro/nano agents that encompass different length scales. The traditional usage of sound waves has steadily progressed from mid-air manipulation of salt grains to sophisticated techniques that employ nanoparticle flow in microfluidic networks. State-of-the-art in microfabrication and instrumentation have further expanded the outreach of these actuation techniques to autonomous propulsion of micro-agents. In this review article, we provide a universal perspective of the known acoustic micromanipulation technologies in terms of their applications and governing physics. Hereby, we survey these technologies and classify them with regards to passive and active manipulation of agents. These manipulation methods account for both intelligent devices adept at dexterous non-contact handling of micro-agents, and acoustically induced mechanisms for self-propulsion of micro-robots. Moreover, owing to the clinical compliance of ultrasound, we provide future considerations of acoustic manipulation techniques to be fruitfully employed in biological applications that range from label-free drug testing to minimally invasive clinical interventions

Tauopathy-Associated Tau Fragment Ending at Amino Acid 224 Is Generated by Calpain-2 Cleavage

BACKGROUND: Tau aggregation in neurons and glial cells characterizes tauopathies as Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). Tau proteolysis has been proposed as a trigger for tau aggregation and tau fragments have been observed in brain and cerebrospinal fluid (CSF). Our group identified a major tau cleavage at amino acid (aa) 224 in CSF; N-terminal tau fragments ending at aa 224 (N-224) were significantly increased in AD and lacked correlation to total tau (t-tau) and phosphorylated tau (p-tau) in PSP and CBD. OBJECTIVE: Previous studies have shown cleavage from calpain proteases at sites adjacent to aa 224. Our aim was to investigate if calpain-1 or -2 could be responsible for cleavage at aa 224. METHODS: Proteolytic activity of calpain-1, calpain-2, and brain protein extract was assessed on a custom tau peptide (aa 220-228), engineered with fluorescence resonance energy transfer (FRET) technology. Findings were confirmed with in-gel trypsination and mass spectrometry (MS) analysis of brain-derived bands with proteolytic activity on the FRET substrate. Finally, knock-down of the calpain-2 catalytic subunit gene (CAPN2) was performed in a neuroblastoma cell line (SH-SY5Y). RESULTS: Calpain-2 and brain protein extract, but not calpain-1, showed proteolytic activity on the FRET substrate. MS analysis of active gel bands revealed presence of calpain-2 subunits, but not calpain-1. Calpain-2 depletion and chemical inhibition suppressed proteolysis of the FRET substrate. CAPN2 knock-down caused a 76.4% reduction of N-224 tau in the cell-conditioned media. CONCLUSIONS: Further investigation of the calpain-2 pathway in the pathogenesis of tauopathies is encouraged

Bipolar Electroactive Conducting Polymers for Wireless Cell Stimulation

Electrochemical stimulation (ES) promotes wound healing and tissue regeneration in biomedical applications and clinical studies and is central to the emerging field of electroceuticals. Traditional ES such as deep brain stimulation for Parkinson’s disease, utilises metal electrodes that are hard wired to a power supply to deliver the stimulation. Bipolar electrochemistry (BPE) introduces an innovative approach to cell stimulation that is wireless. Developing conducting polymers (CPs)-based stimulation platforms wireless powdered by BPE bipolar will provide an exciting new dimension to medical bionics. In this project, Chapter 2 deals with development of a bipolar electrochemical activity testing system and bipolar electrochemical stimulation (BPES) system. Then, bipolar electroactive and biocompatible CPs grown on FTO substrate are successfully synthesised, modified, and characterised in Chapter 3 and Chapter 4 using the above systems prior to using for wireless cell stimulation. Furthermore, free standing and soft CP templates are developed (Chapter 5). More importantly, all these bipolar electroactive CPs have been applied to wireless cell stimulation using BPE (all research Chapters). Significant increase in both cell number and neurite growth has been demonstated, suggesting that the BPES system is highly efficient for stimulation of animal PC 12 cell and human SH-SY5Y cell. More specific information is presented in each chapter as below. In Chapter 3, a CP-based bipolar electrochemical stimulation (BPES) system for cell stimulation was present. Polypyrrole (PPy) films with different dopants have demonstrated reversible and recoverable bipolar electrochemical activity under a low driving DC voltage