Novel fluorescence-based tools and applications for characterizing emerging pathways of α-synuclein amyloid aggregation, disaggregation and inhibition

Habitualmente se denomina agregación amiloide a aquel proceso de malplegamiento proteico que comprende la transición de una proteína soluble y funcional a especies oligoméricas intermedias y, en última instancia, fibras insolubles con una estructura característica llamada de lámina β cruzada. Varias enfermedades neurodegenerativas se encuentran asociadas a este proceso, entre las que se encuentra la enfermedad de Párkinson (PD en inglés). Esta se caracteriza por unos depósitos intracelulares, denominados cuerpos o neuritas de Lewy, ricos en α-sinucleína (αS) en forma de agregados amiloides. αS es una proteína intrínsecamente desordenada de 140 aminoácidos que se expresa ampliamente en el cuerpo humano, especialmente en el sistema nervioso central. Su agregación amiloide también está vinculada con otras sinucleinopatías como demencia con cuerpos de Lewy, atrofia sistémica múltiple y enfermedad de Alzheimer (AD en inglés). A pesar de que los factores que provocan el autoensamblado amiloide de αS in vivo son desconocidos, algunos estudios in vitro son capaces de reproducir tal agregación. Habitualmente, lo hacen mediante el uso de interfases de carácter hidrofóbico/hidrofílico que catalizan los primeros contactos entre proteínas en un proceso llamado nucleación primaria. Las estructuras amiloides formadas mediante este mecanismo de nucleación heterogénea poseen una disposición inter-molecular paralela. En este trabajo, hemos logrado inducir y analizar el autoensamblado amiloide de αS en ausencia de interfases bajo condiciones de hidratación limitada. La agregación ocurre en el seno de la disolución mediante una nucleación, por tanto, homogénea. Mediante el empleo de la espectroscopia de fluorescencia de pireno hemos demostrado que, siguiendo este nuevo mecanismo de nucleación, los agregados adoptan una topología antiparalela. Además, hemos observado que este tipo de nucleación podría estar favorecida en el interior de condensados biomoleculares de αS generados a través de separación de fases líquido líquido (LLPS en inglés), donde la hidratación de la proteína se ve reducida. Aplicando una combinación de técnicas biofísicas, hemos estudiado cuantitativamente la capacidad de αS y de la proteína Tau para sufrir LLPS. Entre estas técnicas, hemos empleado microscopía de tiempo de vida fluorescente (FLIM en inglés), al nivel de condensados individuales, para demostrar la maduración en el tiempo de estos, gracias a la exquisita resolución temporal y espacial de FLIM. Hemos descubierto que αS y Tau sí forman condensados biomoleculares mixtos y que, con el tiempo, forman heteroagregados amiloides en el interior de estos coacervados mediante la denominada transición de fases líquido-solido (LSPT en inglés). Cabe destacar que hemos esclarecido que el principal factor que regula esta LSPT es la valencia y ocupación de las interacciones heterotípicas,y no la dinámica de las cadenas polipeptídicas como se ha descrito frecuentemente paraotros sistemas. Nuestros resultados ayudan a establecer un escenario relevante para la co-agregación de ambas proteínas que podría explicar el hecho de que se observen, conjuntamente, tanto en PD como en AD. Además, hemos contribuido al campo de LLPS-LSPT proporcionando una descripción detallada y cuantitativa de sistemas αS/policatión con técnicas avanzadas y complementarias, incluyendo el estudio de coacervados individuales. Esto podría servir como base para el estudio de una amplia variedad de condensados biomoleculares y de especial interés para caracterizar la relación entre estos y la agregación amiloide.Por otra parte, encontrar moléculas con potencial terapéutico o diagnóstico en enfermedades neurodegenerativas es de una importancia extrema. Sin embargo, la complejidad y heterogeneidad en el paisaje conformacional de la agregación amiloide hacen de esta una diana destacablemente complicada para los estudios habituales de cribado de fármacos basados en ensayos de interacción molecular. En esta tesis hemos establecido una estrategia experimental que combina la espectroscopia de correlación cruzada de fluorescencia y la espectroscopia de fluorescencia de partícula individual de dos colores (dcFCCS/dcSPFS en inglés), y la hemos empleado para investigar la unión de pequeñas moléculas a especies amiloides neurotóxicas de αS con resolución de partícula individual y con independencia de las heterogeneidades moleculares del sistema de estudio. Gracias a la observación de los eventos de interacción de uno en uno, hemos resuelto de manera directa la especificidad, afinidad y estequiometría de unión de varios pequeños péptidos inhibidores de la agregación amiloide de αS, entre los cuales se incluye un péptido humano. Hemos descrito en detalle su mecanismo molecular de actuación y desentrañado las propiedades físico-químicas que respaldan la interacción, contribuyendo al diseño racional de otros péptidos candidatos a fármaco.El uso dcFCCS/dcSPFS puede ampliarse a otras situaciones de interacción multiligando/ receptor multimérico y convertirse en una plataforma experimental para el descubrimiento de nuevos fármacos y marcadores diagnósticos específicos de amiloide. Por último, además de inhibir el proceso de autoensamblado, la desagregación de fibras amiloides puede ser una herramienta para combatir la neurodegeneración. Dentro de las células, esta tarea es llevada a cabo sobre fibras de αS por una maquinaria especializada de chaperonas conocida como la desagregasa humana. Sin embargo, el mecanismo preciso por el cual este complejo proteico procesa las fibras, así como el posible vínculo entre la toxicidad y estructura de un agregado y la actividad desagregasa sobre el mismo es un tema todavía bajo intenso debate. Uno de los principales retos es la obtención de datos cinéticos fiables y de calidad de la reacción de desensamblado, debido a artefactos de la técnica más extensamente usada: la fluorescencia de la sonda tioflavina-T. En nuestro trabajo hemos aplicado la fluorescencia de pireno junto con la desextinción de fluorescencia para afrontar este problema. Hemos logrado probar un mecanismo de desagregación de todo o nada, por el cual cada fibra se desensambla y libera monómeros solubles de αS mediante un mecanismo de cremallera. Nuestros datos cinéticos han permitido el modelado cuantitativo del mecanismo de desagregación sobre diferentes estructuras amiloides de αS. Estos resultados han revelado que, probablemente, la desagregasa humana ha evolucionado para actuar específicamente sobre agregados pequeños y citotóxicos.En resumen, hemos implementado nuevas herramientas y aplicaciones de fluorescencia, incluyendo técnicas de fluorescencia resueltas en el tiempo y de partícula única de dos colores, para el estudio detallado de la agregación amiloide, transición de fases, inhibición y desagregación de αS. En conjunto, nuestros resultados contribuyen a responder preguntas clave de la agregación amiloide de αS y de la búsqueda de estrategias terapéuticas contra las sinucleinopatías. Además, los enfoques experimentales presentados en esta tesis se pueden aplicar para comprender y actuar sobre otros sistemas amiloides, siendo por tanto herramientas metodológicas relevantes en el campo de la agregación amiloide y la neurodegeneración.Amyloid aggregation is typically referred to as a protein misfolding process involving the transition from a functional, soluble protein into oligomeric intermediates and, eventually, insoluble fibrils with a hallmark cross-β structure. A number of neurodegenerative diseases are associated to this process, including Parkinson’s disease (PD), which is characterized by intracellular deposits rich in α-synuclein (αS) in the form of amyloid aggregates, which are referred to as Lewy bodies (LB) or neurites. αS is an intrinsically disordered 140-aminoacid protein widely expressed throughout the body, particularly in the central nervous system. Its amyloid aggregation is also associated with other synucleinopathies such as dementia with Lewy bodies (DLB), multiple system atrophy (MSA) or Alzheimer’s disease (AD). While the factors triggering the amyloid self-assembly of αS in vivo are still obscure, in vitro studies are able to reproduce the aggregation, typically using hydrophobic/hydrophilic interfaces to trigger the first protein-protein contacts, a process termed primary nucleation. The amyloid structures resulting from this (heterogeneous) nucleation show a parallel inter-molecular arrangement. In this work, we were able to induce and analyze the amyloid self-assembly of αS in the absence of interfaces in the bulk of the solution (homogeneous nucleation) under limited hydration conditions. By using pyrene fluorescence spectroscopy we proved that, via this new type of nucleation, the aggregates adopt an antiparallel topology. Moreover, we have observed that this type of nucleation could be favorable in the interior of αS condensates generated by liquid-liquid phase separation (LLPS). By using a combination of biophysical techniques, we quantitatively interrogated the ability of αS and the protein Tau to undergo LLPS. Among these techniques, we used fluorescence lifetime imaging microscopy (FLIM) down to the single-coacervate level, to resolve their maturation without ambiguity, owing to the exquisite temporal and spatial resolution of FLIM. We found that, indeed, αS and Tau form mixed biomolecular condensates by complex elecotrostatic coacervation and, over time, they form amyloid heteroaggregates through liquid-to-solid phase transition (LSPT) in the interior of the condensates. Interestingly, we proved that the valence and occupancy of the heterotypic interactions, and not the polypeptide dynamics, are the main factor governing LSPT. Our results help establishing a relevant scenario for the co-aggregation of both proteins which could explain their joint presence in both PD and AD. Besides, we have contributed to the LLPS-LSPT field by providing a thourough, quantitative description of αS/polycation systems with advanced and complementary techniques and by looking at single coacervates. This could serve as a framework to be used in a wide array of biomolecular condensates, and of particular interest for characterizing the link between these and amyloid aggregation. Finding molecules with therapeutic or diagnosic potential in neurodegenerative disorders is of utter importance. However, the complexity and heterogeneity of the amyloid conformational landscape, makes amyloid aggregation a tremendously challenging target for typical drug screens based in molecular interaction assays. Here, we established an experimental strategy which combines dual-color fluorescence correlation spectroscopy and single-particle fluorescence spectroscopy (dcFCCS/dcSPFS) to investigate the binding of small molecules to amyloid species of αS with single-particle resolution and regardless of molecular heterogeneity. By observing binding events individually, we gained direct access to the binding specificity, affinity and stoichiometry of several small amyloid inhibitory peptides, including a human peptidic molecule. We demonstrated its molecular mechanism of action and disentangled the minimum physico-chemical properties behind the binding properties, thus aiding in the rational design of other peptide drug candidates. dcFCCS/dcSPFS could be extended to other multi-ligand/multimeric receptor interaction scenarios and serve as a platform for finding new drugs and amyloid-specific diagnostic probes. Besides inhibiting the self-assembly process, the disaggregation of amyloid fibrils can be a tool for fighting neurodegeneration. In the cell, such task is performed on αS fibrils by an evolutionary refined chaperone machinery termed the human disaggregase. However, the exact mechanism by which this proteic complex processes the fibrils as well as what is the relationship between aggregate toxicity, structure and disaggregase activity remains under debate. A major challenge is to obtain reliable kinetic data of the disassembly reaction due to artifacts related to the most commonly used amyloid probe, thioflavin-T (ThT). In our work, we have applied pyrene fluorescence together with fluorescence dequenching to solving this problem. We demonstrated an all-or-none disassembly mechanism, where a fibril disassembles entirely into soluble monomers by an unzipping mechanism. Our kinetic data enabled to quantitatively model the disaggregation mechanism on different amyloid assemblies of αS. Our results revealed that the chaperone machinery has likely evolved to tackle small cytotoxic aggregates specifically. In summary, we have implemented new fluorescence-based tools and applications, including time-resolved and single-particle dual-color fluorescence techniques, to the detailed investigation of amyloid aggregation, phase separation, inhibition and disaggregation of αS. Collectively, our results help to understand key questions of αS amyloid aggregation and potential therapeutic strategies against synucleinopathies. In addition, the experimental approaches presented in this thesis can be also easily extended to understand and tackle other amyloid systems, representing important methodological tools in the fields of amyloid aggregation and neurodegeneration.<br /

Recent Advances in Signal Processing

The signal processing task is a very critical issue in the majority of new technological inventions and challenges in a variety of applications in both science and engineering fields. Classical signal processing techniques have largely worked with mathematical models that are linear, local, stationary, and Gaussian. They have always favored closed-form tractability over real-world accuracy. These constraints were imposed by the lack of powerful computing tools. During the last few decades, signal processing theories, developments, and applications have matured rapidly and now include tools from many areas of mathematics, computer science, physics, and engineering. This book is targeted primarily toward both students and researchers who want to be exposed to a wide variety of signal processing techniques and algorithms. It includes 27 chapters that can be categorized into five different areas depending on the application at hand. These five categories are ordered to address image processing, speech processing, communication systems, time-series analysis, and educational packages respectively. The book has the advantage of providing a collection of applications that are completely independent and self-contained; thus, the interested reader can choose any chapter and skip to another without losing continuity

Optics in Our Time

Optics, Lasers, Photonics, Optical Devices; Quantum Optics; Popular Science in Physics; History and Philosophical Foundations of Physic

Differential aspects of spatial vision in subjects presenting with either macular degeneration or visual snow

The aim of this study was to investigate differential aspects of spatial vision in subjects presenting with macular degeneration and visual snow. Emphasis was placed on the effects of aging and/or ocular disease on binocular summation and the measurement of differences between the two eyes in a number of visual tasks. Accommodation performance was measured for both pre-presbyopic and presbyopic observers. The results showed no binocular advantage for either group for far or intermediate stimulus vergences. The effects of visual crowding upon visual acuity were tested using a Landolt C optotype with surrounding distractors. Binocular advantage was found to be higher along the line of sight and to decrease in the near periphery. The presence of distractors reduced visual resolution significantly at every eccentricity, with the effect becoming more pronounced in the periphery. The latter was observed for both monocular and binocular viewing conditions leading to the suggestion that the involvement of binocularly driven neurons may not be essential for visual crowding. The effects of healthy aging and ocular disease on spatial and chromatic vision were also investigated. It was found that stimulus presentation time in a gap acuity task affects visual acuity differently in patients with Age-related Macular Degeneration (AMD) when compared to normal subjects. The processing of briefly presented optotypes appears to be severely reduced in AMD patients. Visual performance was also investigated in patients who experience ‘visual snow’. While gap acuity at any stimulus duration and the strength of chromatic afterimages were found to be unaffected, involuntary pupil recovery following brief exposure to chromatic stimuli was found to be delayed in 3 of the 6 visual snow (VS) patients examined. The absence of a normal pupil recovery is consistent with abnormally slow signals that may also play a part in VS. The novel findings reported in this thesis suggest that advanced vision tests can be used to quantify the effects of normal aging and to detect and monitor the earliest changes in diseases of the eye

ESSE 2017. Proceedings of the International Conference on Environmental Science and Sustainable Energy

Environmental science is an interdisciplinary academic field that integrates physical-, biological-, and information sciences to study and solve environmental problems. ESSE - The International Conference on Environmental Science and Sustainable Energy provides a platform for experts, professionals, and researchers to share updated information and stimulate the communication with each other. In 2017 it was held in Suzhou, China June 23-25, 2017

Optoelectronics – Devices and Applications

Optoelectronics - Devices and Applications is the second part of an edited anthology on the multifaced areas of optoelectronics by a selected group of authors including promising novices to experts in the field. Photonics and optoelectronics are making an impact multiple times as the semiconductor revolution made on the quality of our life. In telecommunication, entertainment devices, computational techniques, clean energy harvesting, medical instrumentation, materials and device characterization and scores of other areas of R&D the science of optics and electronics get coupled by fine technology advances to make incredibly large strides. The technology of light has advanced to a stage where disciplines sans boundaries are finding it indispensable. New design concepts are fast emerging and being tested and applications developed in an unimaginable pace and speed. The wide spectrum of topics related to optoelectronics and photonics presented here is sure to make this collection of essays extremely useful to students and other stake holders in the field such as researchers and device designers