Adaptive optics (AO) systems have become an essential part of ground-based telescopes and enable diffraction-limited imaging at near-IR and mid-IR wavelengths. For several key science applications the required wavefront quality is higher than what current systems can deliver. For instance obtaining high quality diffraction-limited images at visible wavelengths requires residual wavefront errors to be well below 100 nm RMS. High contrast imaging of exoplanets and disks around nearby stars requires high accuracy control of low-order modes that dominate atmospheric turbulence and scatter light at small angles where exoplanets are likely to be found. Imaging planets using a high contrast corona graphic camera, as is the case for the Spectro-Polarimetric High-contrast Exoplanet Research (SPHERE) on the Very Large Telescope (VLT), and the Gemini Planet Imager (GPI), requires even greater wavefront control accuracy. My dissertation develops a highly sensitive non-linear Curvature Wavefront Sensor (nlCWFS) that can deliver diffraction-limited (λ/D) images, in the visible, by approaching the theoretical sensitivity limit imposed by fundamental physics. The nlCWFS is derived from the successful curvature wavefront sensing concept but uses a non-linear reconstructor in order to maintain sensitivity to low spatial frequencies. The nlCWFS sensitivity makes it optimal for extreme AO and visible AO systems because it utilizes the full spatial coherence of the pupil plane as opposed to conventional sensors such as the Shack-Hartmann Wavefront Sensor (SHWFS) which operate at the atmospheric seeing limit (λ/r₀). The difference is equivalent to a gain of (D/r₀)² in sensitivity, for the lowest order mode, which translates to the nlCWFS requiring that many fewer photons. When background limited the nlCWFS sensitivity scales as D⁴, a combination of D² gain due to the diffraction limit and D² gain due to telescope's collecting power. Whereas conventional wavefront sensors only benefit from the D² gain due to the telescope's collecting power. For a 6.5 m telescope, at 0.5 μm, and seeing of 0.5", the nlCWFS can deliver for low order modes the same wavefront measurement accuracy as the SHWFS with 1000 times fewer photons. This is especially significant for upcoming extremely large telescopes such as the Giant Magellan Telescope (GMT) which has a 25.4 m aperture, the Thirty Meter Telescope (TMT) and the European Extremely Large Telescope (E-ELT) which has a 39 m aperture.

Eukaryotic cells are characterised by membranes with varied and dynamic compositions and shapes. Consequently, membrane-binding proteins are tuned to recognise and modify these membrane states to perform their functions. To study the curvature sensitivity of proteins, I have developed a single-particle assay using NanoSight technology that tracks the Brownian motion of particles to measure their size. I optimised this system to track fluorescently labelled lipid-binding domains bound to liposomes of different sizes moving freely in solution. The comparison of the size distribution of the total liposomes with the fluorescently labelled population allowed me to determine their curvature preferences. To validate the method I tested proteins from the Bin/Amphiphysin/Rvs (BAR) superfamily, which are inherently curved and have known curvature preferences. My method was capable of recapitulating the behaviour of BAR domains with different curvature preferences. I then expanded the range of targets and showed that this assay is also capable of detecting curvature preferences for a variety of other lipid-binding domain families. As such, I identified AKT PH domain as a new curvature-sensing domain. Finally, using the ENTH domain of Epsin1 that causes vesicle budding, I demonstrated that this method can also be used to study membrane remodelling. Trafficking involves generation and sensing of membrane curvature combined with recognition of specific cargo. Endophilin consists of a curvature-sensitive BAR domain followed by an SH3 (Src-homology 3) domain and has recently been identified in a clathrin-independent endocytosis pathway, FEME (fast endophilin-mediated endocytosis), involved in the uptake of cell surface receptors. Endophilin recognises ligands via its SH3 domain, binding G-protein coupled receptors (GPCRs) directly in their intracellular loop 3 and receptor tyrosine kinase (RTKs) via adaptor proteins. However, a specific recognition motif has not been identified yet. Here, using a combination of biophysical approaches and NMR spectroscopy, I characterised the Endophilin binding motif of ALIX (ALG-2-interacting protein X) adaptor protein and of the GPCR $\alpha$2A adrenergic receptor. Comparison of SH3-peptide models resulted in a putative Endophilin recognition site.

No description available.

[EN] Structural Health Monitoring (SHM) is a discipline that quantitatively assesses the integrity and performance of infrastructures, relying on sensors, and support the development of efficient Maintenance and Rehabilitation (M&R) plans. Optical Multicore Fiber (MCF) Shape Sensors offer an innovative alternative to traditional methods and enable the reconstruction of the deformed shape of structures directly and in real-time, with no need of computation models or visual contact and exploiting all the advantages of Optical Fiber Sensors (OFS) technology. Despite the intense research efforts centered on this topic by research groups worldwide, a comprehensive investigation on the parameters that influence the performance of these sensors has not been conducted yet. The first part of the thesis presents a numerical study that examines the effects of strain measurement accuracy and core position errors on the performance of optical multicore fiber shape sensors in sensing three-dimensional curvature, which is at the basis of shape reconstruction. The analysis reproduces the strain measurement process using Monte Carlo Method (MCM) and identifies several parameters which play a key role in the phenomenon, including core spacing (distance between outer cores and sensor axis), number of cores and curvature measured. Finally, a set of predictive models were calibrated, by fitting the results of the simulations, to predict the sensors performance. Afterward, an experimental study is proposed to evaluate the performance of optical multicore fiber in sensing shape, with particular focus on the influence of strain sensors length. Two shape sensors were fabricated, by inscribing long (8.0 mm) and short (1.5 mm) Fiber Bragg Gratings (FBG) into the cores of a multicore seven-core fiber. Thus, the performance of the two sensors was assessed and compared, at all the necessary phases for shape reconstruction: strain sensing, curvature calculation and shape reconstruction. To conclude, an innovative approach, based on the Saint-Venant's Torsion Theory, is presented to determine the twisting of multicore fiber and to compensate the errors due to twisting during shape reconstruction. The efficiency of the theoretical approach was then corroborated performing a series of twisting tests on a shape sensor, fabricated by inscribing FBGs sensors into an optical spun multicore seven-core fiber. The investigation of the mechanical behavior of multicore optical shape sensors has synergically involved diverse disciplines: Solid Mechanics, Photonics, Statistics and Data Analysis. Such multidisciplinary research has arisen from the prolific cooperation between the Institutes of the Institute of Science and Technology of Concrete (ICITECH) and the Institute of Telecommunications and Multimedia Applications (iTEAM) - Photonics Research Labs (PRL) - of Universitat Politècnica de València (UPV), in addition to valuable collaboration with other members of the European ITN-FINESSE project, to which this work belongs. This research work aims to enhance the performance optical multicore fiber shape sensors and support the development of new sensor geometries, with great potential for structural health monitoring applications. / [ES] La Monitorización de la Salud Estructural (MSE) evalúa cuantitativamente la integridad y el comportamiento de las infraestructuras y permite desarrollar planes eficaces de Mantenimiento y Rehabilitación (M&R), utilizando los datos de los sensores. Sensores de forma basados en fibra óptica multinúcleo ofrecen una alternativa a los métodos tradicionales y permiten la reconstrucción de la deformada de estructuras de forma directa y en tiempo real, sin necesidad de modelos de cálculo o contacto visual y con todas las ventajas de la tecnología de los Sensores de Fibra Óptica (SFO). A pesar de los grandes esfuerzos en la investigación centrada en este tema por parte de los grupos de investigación de todo el mundo, todavía no se ha realizado una investigación exhaustiva que estudie los parámetros que influyen en el comportamiento de estos sensores. En la primera parte de la tesis se presenta un estudio numérico en el que se examinan los efectos de la precisión de la medición de la tensión y los errores de posición del núcleo en el comportamiento de los sensores de forma basados en fibra óptica multinúcleo para definir la curvatura tridimensional, que es la base de la reconstrucción de la forma. El análisis reproduce el proceso de medición de la tensión utilizando el método de Monte Carlo (MC) e identifica una serie de parámetros que desempeñan un papel en el proceso, entre ellos la separación del núcleo (distancia entre los núcleos exteriores y el eje del sensor), el número de núcleos y la curvatura medida. Por último, se calibró un conjunto de modelos de predicción ajustando los resultados de las simulaciones para predecir el comportamiento de los sensores. A continuación, se propone un estudio experimental para evaluar el comportamiento de los sensores de forma basado en fibra óptica multinúcleo, con especial atención en la influencia de la longitud de los sensores de deformación. Se fabricaron dos sensores de forma, inscribiendo Fiber Bragg Gratings (FBG) con longitudes de 8,0 mm y 1,5 mm en los núcleos de una fibra multinúcleo de siete núcleos. Así, se evaluó y comparó el comportamiento de los dos sensores en todas las fases necesarias para la reconstrucción de la forma, incluyendo la medición de la tensión, el cálculo de la curvatura y la reconstrucción de la forma. Para concluir, se presenta un enfoque innovador, basado en la Teoría de la Torsión de Saint-Venant, para determinar la torsión de la fibra multinúcleo y compensar los errores debidos a la torsión durante la reconstrucción de la forma. La eficiencia del enfoque teórico fue verificada realizando una serie de pruebas de torsión en un sensor de forma, fabricado inscribiendo los sensores de FBGs en una fibra óptica multinúcleo torcida y siete núcleos. La investigación del comportamiento mecánico de los sensores ópticos de forma multinúcleo ha involucrado sinérgicamente diversas disciplinas: Mecánica del sólido, Fotónica, Estadística y Análisis de datos. Esta investigación multidisciplinaria ha surgido de la prolífica cooperación entre el Instituto de Ciencia y Tecnología del Hormigón (ICITECH) y el Instituto de Telecomunicaciones y Aplicaciones Multimedia (iTEAM) - Laboratorio de Investigación Fotónica (LIF) - de la Universidad Politécnica de Valencia (UPV), además de la valiosa colaboración con otros miembros del proyecto europeo ITN-FINESSE, al que pertenece este trabajo. Este trabajo de investigación puede permitir mejorar el comportamiento de los sensores de forma basados en fibra óptica multinúcleo y apoyar el desarrollo de nuevas geometrías de sensores, con un gran potencial para aplicaciones de control de la salud estructural. / [CA] Structural Health Monitoring (SHM) avalua quantitativament la integritat i el comportament de les infraestructures i permet desenrotllar plans eficaços de Maintenance and Rehabilitation (M&R), utilitzant les dades dels sensors. Optical Multicore Fiber (MCF) Shape Sensors oferixen una alternativa als mètodes tradicionals i permeten la reconstrucció de la forma de la deformació de les estructures de forma directa i en temps real, sense necessitat de models de càlcul o contacte visual i amb tots els avantatges de l'Optical Fiber Sensors (OFS) Technology. A pesar dels grans esforços en la investigació centrada en aquest tema per part dels grups d'investigació de tot el món, encara no s'ha realitzat una investigació exhaustiva que estudie els paràmetres que influïxen en el comportament d'aquestos sensors. En la primera part de la tesi es presenta un estudi numèric en què s'examinen els efectes de la precisió del mesurament de la tensió i els errors de posició del nucli en el comportament dels sensors de forma basats en fibra òptica multinucli per a definir la curvatura tridimensional, que és la base de la reconstrucció de la forma. L'anàlisi reproduïx el procés de mesurament de la tensió utilitzant el mètode de Monte Carlo (MC) i identifica una sèrie de paràmetres que exercixen un paper en el procés, entre ells la separació del nucli (distància entre els nuclis exteriors i l'eix del sensor), el nombre de nuclis i la mesura de la curvatura. Finalment, es va calibrar un conjunt de models de predicció ajustant els resultats de les simulacions per a predir el comportament dels sensors. A continuació, es proposa un estudi experimental per a avaluar el comportament dels sensors de forma basat en fibra òptica multinucli, amb especial atenció en la influència de la longitud dels sensors de deformació. Es van fabricar dos sensors de forma, inscrivint Fiber Bragg Gratings (FBG) amb longituds de 8,0 mm i 1,5 mm en els nuclis d'una fibra multinucli de set nuclis. Així, es va avaluar i es va comparar el comportament dels dos sensors en totes les fases necessàries per a la reconstrucció de la forma, incloent el mesurament de la tensió, el càlcul de la curvatura i la reconstrucció de la forma. Per a concloure, es presenta un enfocament innovador, basat en la Teoria de la Torsió de Saint-Venant, per a determinar la torsió de la fibra multinucli i compensar els errors deguts a la torsió durant la reconstrucció de la forma. L'eficiència de l'enfocament teòric va ser verificada realitzant una sèrie de proves de torsió en un sensor de forma, fabricat inscrivint els sensors de FBGs en una fibra òptica de set nuclis de filat múltiple. La investigació del comportament mecànic dels sensors òptics de forma multinucli ha involucrat sinèrgicament diverses disciplines: Mecànica del sòlid, Fotónica, Estadística i Anàlisi de dades. Aquesta investigació multidisciplinària ha sorgit de la prolífica cooperació entre l'Institut de Ciència i Tecnologia del Formigó (ICITECH) i l'Institut de Telecomunicacions i Aplicacions Multimèdia (iTEAM) - Laboratori de investigación fotònica (LIF) - de la Universitat Politècnica de València (UPV), a més de la valuosa col·laboració amb altres membres del projecte europeu ITN- FINESSE, al qual pertany aquest treball. Aquest treball d'investigació pot permetre millorar el comportament dels sensors de forma basats en fibra òptica multinucli i ajudar al desenrotllament de noves geometries de sensors, amb un gran potencial per a aplicacions de control de la salut estructural. / Floris, I. (2020). Optical Multicore Fiber Shape Sensors. A numerical and experimental performance assessment [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/148715 / TESIS

Cancer metastasis, the spread of cancer from the primary site to distant regions in the body, is the major cause of cancer mortality, accounting for almost 90% of cancer related deaths. During metastasis, cancer cells from the primary tumor initially probe the surrounding fibrous tumor microenvironment (TME) prior to detaching and subsequently migrating towards the blood vessels for further dissemination. It has widely been acknowledged that the biophysical cues provided by the fibrous TME greatly facilitate the metastatic cascade. Consequently, there has been a tremendous wealth of work devoted towards elucidating different modes of cancer cell migration. However, our knowledge of how cancer cells at the primary tumor site initially sense their fibrous surroundings prior to making the decision to detach and migrate remains in infancy. In part, this is due to the lack of a fibrous in vitro platform that allows for precise, repeatable manipulation of fiber characteristics. In this study, we use the non-electrospinning, Spinneret based Tunable Engineered Parameters (STEP) technique to manufacture suspended nanofiber networks with exquisite control on fiber dimensions and network architecture and use these networks to investigate how single cancer cells biophysically sense fibers mimicking in vivo dimensions. Using high spatiotemporal resolution imaging (63x magnification/1-second imaging interval), we report for the first time, that cancer cells sense individual fibers by coiling (i.e. wrapping around the fiber axis) at the tip of a cell protrusion. We find that coiling dynamics are mediated by both the fiber curvature and the metastatic capacity of the cancer cells with less aggressive cancer cells showing diminished coiling. Based on these results, we explore the possibility of using coiling in conjunction with other key biophysical metrics such as cell migration dynamics and forces exerted in the development of a genetic marker independent, biophysical predictive tool for disease progression. Finally, we identify the membrane curvature sensing Insulin Receptor tyrosine kinase Substrate protein of 53 kDa (IRSp53) as a key regulator of protrusive activity with IRSp53 knockout (KO) cells exhibiting significantly slower protrusion dynamics and diminished coil width compared to their wild-type (WT) counterparts. We demonstrate that the hindered protrusive activity ultimately translates to impaired contractility, alteration in the nucleus shape and slower migration dynamics, thus highlighting the unique role of IRSp53 as a signal transducer – linking the protrusive activity at the cell membrane to changes in cytoskeletal contractility. Overall, these findings offer novel perspectives to our understanding of how cancer cells biophysically sense their fibrous surroundings. The results from this study could ultimately pave the way for elucidating the precise fiber configurations that either facilitate or hinder cancer cell invasion, allowing for the development of new therapeutics in the long term that could inhibit the metastatic cascade at a relatively nascent stage and yield a more promising prognosis in the perennial fight against cancer. / Doctor of Philosophy / Cancer is a leading cause of death worldwide. Almost ninety percent of cancer related deaths arise from the spreading of cancer cells from the primary tumor site to secondary sites in the body – a processed termed as metastasis. The environment surrounding a tumor (tumor microenvironment) is highly fibrous in nature and can assist in the metastatic process by providing biophysical cues to the cells at the tumor boundary. These cells sense the presence of the surrounding fibers by extending "arms" termed as protrusions, and then eventually detach from the primary tumor and start migrating through the fibrous microenvironment. While numerous studies have investigated the various modes of cell migration in fibrous environments, there is very little information regarding how cancer cells use protrusions to initially sense the fibers prior to detaching. In this study, we used the Spinneret based Tunable Engineered Parameters (STEP) technique to manufacture suspended nanofiber networks with robust control on fiber diameter and network architecture and use these networks to systematically investigate how single cancer cells biophysically sense fibers that mimic in vivo dimensions. We discovered that cancer cells sense individual fibers by "wrapping-around" the axis of the fiber at the tip of the protrusion – a phenomenon we refer to as coiling. We found both the fiber diameter as well as the invasive capacity of cells can influence the coiling mechanics. Based on these results, we explored the use of coiling in conjunction with other key biophysical metrics such as the cell migration speed and how much force a cell can exert to develop a biophysical predictor for cancer cell aggressiveness. Finally, given that cells sense the fiber curvature by coiling, we explored the role of a key curvature sensing protein Insulin Receptor tyrosine kinase Substrate protein of 53 kDa (IRSp53) in mediating coiling activity and found that knocking out (KO) IRSp53 results in reduced coiling and slower protrusions compared to wild-type (WT) cells. Furthermore, IRSp53 KO cells showed impaired contractility which led to an alteration in the nucleus shape and slower migration dynamics thus highlighting the role of IRSp53 in linking changes at the cell membrane to the underlying cell cytoskeleton. The results from this study could ultimately help us understand what type of fiber conditions around a primary tumor would either help or delay the emergence of the tumor boundary cells and thus allow for the development of therapeutics that could significantly slow down the metastatic process at a relatively early stage.