Developing Instrumentation for Multi-parametric Investigation of Mechanisms of Mechanosensitivity in Ion Channels

Mechanosensitive (MS) channels are implicated in pathologies of the renal and pulmonary systems. Abnormal activity in MS channel reduces cell viability causing a variety of pathologies. MS channels are also responsible for sensation of pain and hearing. Despite the vital importance of MS channels, very little is known about the gating mechanisms of these channels. Attempts to study the mechanisms are severely limited by the lack of suitable instrumentation. A better understanding of the structure-function interaction of MS channels is necessary to find pharmacological leads for the pathologies. Activation data based on indirect activation of MS channels using hypo- or hyper-osmotic solutions or viscous drag is confounded by factors like membrane stretch and cytoskeletal stress. Traditional patch clamp does not allow direct access to the cell by other probes. While a planar patch clamp chip may allow for such access, most of the existing planar patch clamp chips are focused on high throughput screening for pharmaceutical targets and have designs that limit multi-parametric studies. We present here instrumentation that combines atomic force microscopy with cellular electrophysiology based on planar patch clamp approach. The instrumentation allows multi-parametric studies on single cells and provides unique insights into mechanisms of activation of not just MS channels, but ion channels in general by combining cellular electrophysiology, optical microscopy and atomic force microscopy. Using HaCaT cells as our model system we have obtained functional maps of distribution MS channels across cell surface. The maps reveal that the distribution of MS channels on HaCaT cells is highly non-uniform and that the channels are present in small clusters instead of dispersed as single entities. Our results using direct mechanical stimulation of single cells reveal that threshold stress level is required in order to activate MS channels and that the stress has a limited spatial range. Investigation of kinetics of the electrical response to direct mechanical stimulation reveals that the MS channels respond to the mechanical signal after a small time lag, which we attribute to the conformational changes necessary while the channel is being gated. We hope that the insights gained from studying the mechanosensitive channels of HaCaT cells will also advance the understanding of MS channels in general. Apart from opening new avenues in MS channel research, the instrumentation can also be useful in studying the dynamics and gating of ligand gated channels by appropriately tagging the AFM cantilever. With further improvements in the speed of AFM imaging, it will also be possible to observe the gating of channels in real time at molecular scale by imaging the channel on the cell while the channel is being gated

Development of the in situ forming of a liquid infused preform (ISFLIP) process : a new manufacturing technique for high performance fibre reinforced polymer (FRP) components

A problem is not a problem anymore if no solution exists; therefore, in the present dissertation, a novel manufacturing technique, the In Situ Forming of a Liquid Infused Preform (ISFLIP), is proposed as a solution to some typical problems that manufacturing of Fibre Reinforced Polymer (FRP) parts through Vacuum Infusion (VI) involves, such as not taking advantage of the full potential of FRPs, long processing times and lack of reproducibility. ISFLIP is a hybrid process between VI and diaphragm forming in which a flat preform of a stack of reinforcement fabrics is firstly impregnated with a low viscosity matrix and, then, formed over a mould while the matrix is still in the low viscosity state. Being focused on high performance FRPs and shell components, from simple to complex double curvature shapes, a number of trade-offs between VI and diaphragm forming were overcome to lay the foundations from which ISFLIP ability to manufacture FRP components has been proven. In order to adopt a VI manufacturing methodology that fitted ISFLIP targets, important contributions to more general VI have also been made in terms of part quality optimization, addressing the major concern that void content is in VI, with competitive manufacturing times. An effective vacuum degassing procedure in which bubble formation is enhanced through high speed stirring, and a non-conventional filling and post-filling strategy are proposed for this purpose. Eventually, void content was virtually eliminated and post-filling time minimized without affecting fibre content. In ISFLIP, textile preforms are formed together with a series of auxiliary materials (plastic films and sheets, textile fabrics and knitted meshes), most of them showing different in-plane deformation mechanisms. Forming performance of preforms, as well as final part quality, are severely affected by interactions between all these materials different in nature. Uncertainties on this respect and an initial evaluation of attainable shapes were also addressed to define a more focused research plan to the final goal, still distant, of implementing ISFLIP in a real production environment. Results obtained throughout the research project give cause for reasonable optimism in ISFLIP potential and future prospects.Un problema deja de ser un problema si no existe solución; por lo tanto, en esta disertación, una novedosa técnica de fabricación, el Conformado In Situ de una Preforma Infusionada con resina Líquida (ISFLIP, por sus siglas en inglés), se propone como solución a algunos problemas típicos relacionados con la fabricación de piezas de Polímero Reforzado con Fibra (FRP) a través de la Infusión por Vacío (VI), problemas tales como el desaprovechamiento de todo el potencial de los FRPs, largos tiempos de procesado y falta de reproducibilidad. ISFLIP es un proceso híbrido entre la VI y el conformado por membrana elástica en el que una preforma plana formada a partir de un apilado de tejidos de refuerzo es en primera instancia impregnada con una resina de baja viscosidad y, entonces, conformada sobre un molde mientras que la matriz permanece todavía en el estado de baja viscosidad. Estando centrado en los FRPs de altas prestaciones y en componentes con formas tipo concha, desde curvaturas simples hasta formas con doble curvatura complejas, un número importante de compensaciones entre la VI y el conformado por membrana se han ido superando para asentar las bases a partir de las cuales se ha probado la capacidad de ISFLIP para fabricas componentes de FRP. Con la vista puesta en implementar una metodología de fabricación por VI que cumpliese los objetivos definidos para ISFLIP, también se han realizado importantes contribuciones de carácter más general relacionadas con la VI en términos de optimización de parámetros de calidad de las piezas, abordando la gran preocupación que la porosidad final supone en la VI, y consiguiendo unos tiempos de fabricación competitivos. Con este propósito se han propuesto un proceso de desgasificación por vacío muy efectivo en el que se favorece la nucleación de burbujas mediante la agitación a alta velocidad, y una prometedora y no convencional estrategia de llenado y post-llenado de la preforma. Finalmente, se consiguió virtualmente eliminar la porosidad atrapada en las piezas, minimizando el tiempo de post-llenado sin afectar la fracción de fibra contenida. En ISFLIP las preformas textiles se conforman junto con una serie de materiales auxiliares (films y hojas plásticas, mallas y tejidos textiles), que muestran diferentes mecanismos de deformación en plano. El conformado de las preformas y el acabado final de las piezas se ve severamente afectado por todas las interacciones entre todos estos materiales diferentes en naturaleza. También se han abordado las incertidumbres que surgen al respecto y una evaluación inicial de las geometrías abarcables para definir un plan de investigación más concreto con el que poder afrontar la meta final, todavía distante, de implementar ISFLIP en un entorno productivo real. Los resultados obtenidos a lo largo de este proyecto de investigación permiten ser razonablemente optimistas en cuanto al potencial de ISFLIP y sus expectativas

Modelling, Simulation and Data Analysis in Acoustical Problems

Modelling and simulation in acoustics is currently gaining importance. In fact, with the development and improvement of innovative computational techniques and with the growing need for predictive models, an impressive boost has been observed in several research and application areas, such as noise control, indoor acoustics, and industrial applications. This led us to the proposal of a special issue about “Modelling, Simulation and Data Analysis in Acoustical Problems”, as we believe in the importance of these topics in modern acoustics’ studies. In total, 81 papers were submitted and 33 of them were published, with an acceptance rate of 37.5%. According to the number of papers submitted, it can be affirmed that this is a trending topic in the scientific and academic community and this special issue will try to provide a future reference for the research that will be developed in coming years

Mesh-Free and Finite Element-Based Methods for Structural Mechanics Applications

The problem of solving complex engineering problems has always been a major topic in all industrial fields, such as aerospace, civil and mechanical engineering. The use of numerical methods has increased exponentially in the last few years, due to modern computers in the field of structural mechanics. Moreover, a wide range of numerical methods have been presented in the literature for solving such problems. Structural mechanics problems are dealt with using partial differential systems of equations that might be solved by following the two main classes of methods: Domain-decomposition methods or the so-called finite element methods and mesh-free methods where no decomposition is carried out. Both methodologies discretize a partial differential system into a set of algebraic equations that can be easily solved by computer implementation. The aim of the present Special Issue is to present a collection of recent works on these themes and a comparison of the novel advancements of both worlds in structural mechanics applications

High-Throughput Automated Patch Clamp Investigations on Ion Channels in Erythrocytes

Trotz ihrer morphologischen Einfachheit ist die Membran der roten Blutkörperchen (Erythrozyten) mit einer Reihe von Transportern und Ionenkanälen ausgestattet, die bisher nicht vollständig charakterisiert sind und deren biologische Rolle noch wenig verstanden ist. Die meisten Techniken zur Untersuchung von Ionenkanälen messen summierte Effekte großer Zellpopulationen und verbergen so jede mutmaßliche Variabilität von Zelle zu Zelle. Die Patch-Clamp-Technik hat sich als effektives Werkzeug zur Entdeckung und Charakterisierung von Ionenkanälen auf Einzelzellenebene erwiesen. Dies besonders wichtig für Erythrozyten von Säugetieren, die eine hohe Heterogenität der Leitfähigkeit zwischen verschiedenen Spendern, und auch zwischen Zellen desselben Spenders aufweisen (Kaestner et al., 2004; Minetti et al., 2013). Die Entwicklung des automatisierten Patch-Clamps ermöglichte es, eine hohe Anzahl von Zellen gleichzeitig unter identischen experimentellen Bedingungen zu untersuchen, wodurch Zellheterogenität erstmals umfassend bestimmt wurde. In dieser Arbeit wurden Gárdos- und Piezo1-Kanäle als Hauptuntersuchungsziele ausgewählt, da sie eine prominente Rolle in erythrozytären Erkrankungen, im Einzelnen Gárdos-Kanalopathie (Fermo et al., 2017) und hereditäre Xerozytose (Zarychanski et al., 2012; Bae et al., 2013), spielen. Ziel dieser Arbeit war es, automatisierte Patch-Clamp-Assays zur Charakterisierung dieser Kanäle in Erythrozyten zu entwickeln. Es gibt bisher nur vereinzelte Publikationen zu whole-cell Patch-Clamp-Messungen von Gárdos-Kanälen in Erythrozyten (Grygorczyk et al., 1984; Wolff et al., 1988), wahrscheinlich aufgrund der geringen Expression des Proteins in zirkulierenden Erythrozyten. Der hochparallelisierte Ansatz der automatisierten Patch-Clamp-Technologie ermöglicht zuverlässig die Identifizierung von Gárdos-Strömen in Zelltypen mit einer oft geringen Anzahl von Kanälen und einer großen Heterogenität der Expression, wie bei Erythrozyten. Bisherige Piezo1-Kanaluntersuchungen zeigen, dass die Substanz Yoda1 Piezo1-Ströme bewirken kann, die empfindlich auf GdCl3 (unspezifischer Inhibitor dehnungsaktivierter Kanäle), nicht jedoch auf TRAM-34 (spezifischer Gárdos-Kanalinhibitor) reagieren. Die Anwendung dieses Assays auf Erythrozyten von Patienten mit einer neuartigen PIEZO1 R2110W-Mutation zeigte eine erhöhte Anzahl der Yoda1-empfindlichen Zellen und eine stärkere Antwort auf Yoda1 bei Patienten im Vergleich zu Kontroll-Erythrozyten. In Kombination mit der Untersuchung der Proteinstruktur, die den R2110W-Rests in einem gating-sensitiven Bereich des Kanals lokalisiert, deuten die Patch-Clamp-Ergebnisse darauf hin, dass die neue Piezo1-Mutation eine gain-of-function-Mutation ist (Rotordam et al., 2019). Zusammenfassend zeigt diese Arbeit, dass die automatisierte Patch-Clamp-Methode robuste Assays zur Untersuchung von Ionenkanälen (Gárdos und Piezo1) in Primärzellen liefert. Die Hochdurchsatztechnologie ermöglichte die Entwicklung eines zuverlässigen Assays für gering exprimierte Ionenkanäle bei hoher Heterogenität der Zellen. So war es möglich, eine neuartige Kanalmutation auf funktioneller Ebene direkt in Patientenzellen zu charakterisieren, ohne die Mutation in einem heterologen Expressionssystem exprimieren zu müssen. Dieser Ansatz kann zum Nachweis und zur Charakterisierung weiterer Kanalopathien verwendet werden, die nicht auf Erythrozyten beschränkt sind, und kann generell als zur Gensequenzierung komplementärer Routine-Screening-Assay für Krankheiten dienen, die mit Ionenkanalstörungen zusammenhängen.Despite the morphological simplicity, the Red Blood Cell (RBC) membrane is endowed with a number of transporters and ion channels, yet not fully characterized and whose biological role is still poorly understood. Most of the techniques used to investigate ion channels are addressed to large populations of cells, thus concealing any putative cell-to-cell variability. The patch clamp technique has proven to be a valid tool for the discovery and characterization of ion channels at a single-cell level. This is of particular relevance for mammalian RBCs, which present a high heterogeneity of conductance not only between different donors but also among cells of the same donor (Kaestner et al., 2004; Minetti et al., 2013). The advent of automated patch clamp allowed to probe an increased number of cells at the same time under identical experimental conditions, thus tackling cell heterogeneity issues. In this thesis, Gárdos and Piezo1 channels were selected as main targets of investigation due to their relevance in RBC-related diseases, i.e. Gárdos channelopathy (Fermo et al., 2017) and hereditary xerocytosis (Zarychanski et al., 2012; Bae et al., 2013). The aim of this work was to develop automated patch clamp assays for characterizing those channels in RBCs. As for Gárdos channels, whole cell recordings reported so far are fragmentary probably due to the low expression of the protein in circulating RBCs (Grygorczyk et al., 1984; Wolff et al., 1988). By increasing the number of cells recorded at the same time, the automated patch clamp technology allowed to identify Gárdos-mediated currents in primary cells with a low-copy number of channels and a large heterogeneity of conductance as RBCs. Piezo1 channels investigations confirmed that application of Yoda1 alone is able to elicit currents sensitive to GdCl3 (non-specific stretch-activated channels inhibitor) but not TRAM-34 (specific Gárdos channel blocker). When transferred to patients carrying a novel PIEZO1 R2110W mutation, the assay revealed that the number of responders and the magnitude of the response to Yoda1 increased in patient compared to control RBCs. This result, combined with structural studies identifying the R2110W residue in a gating sensitive area of the channel, suggested that the novel Piezo1 mutation is gain-of-function (Rotordam et al., 2019). Altogether, this work demonstrates that automated patch clamping provides robust assays to investigate ion channels (Gárdos and Piezo1) in primary cells. The high-throughput technology allowed to tackle issues as response heterogeneity and low expression of the channels, and to characterize a novel channel mutation at a functional level directly from patient cells, without having to express the mutation in a heterologous expression system. This approach may be used to detect other channelopathies not limited to RBCs and may serve as routine screening assay for diseases related to ion channel dysfunctions in general, complementary to gene sequencing

Low-frequency Acoustic Noise Mitigation Characteristics of Metamaterials-inspired Vibro-impact Structures

Acoustic absorbers like foams, fiberglass or liners have been used commonly in structures for infrastructural, industrial, automotive and aerospace applications to mitigate noise. However, these conventional materials have limited effectiveness to mitigate low-frequency (LF) acoustic waves with frequency less than $\sim$400 Hz owing to the need for impractically large mass or volume. LF acoustic waves contribute significantly towards environmental noise pollution as well as unwanted structural responses. Therefore, there is a need to develop lightweight, compact, structurally-integrated solutions to mitigate LF noise in several applications. Inspired by metamaterials, which are manmade structural materials that derive their unique dynamic behavior not just from material constituents but more so from engineered configurations, tuned mass-loaded membranes as vibro-impact attachments on a baseline structure are investigated to determine their performance as a LF acoustic barrier. The hypothesis is that the LF incident waves are up-converted via impact to higher modes in the baseline structure which are far more evanescent and may then be effectively mitigated using conventional means. Such Metamaterials-Inspired Vibro-Impact Structures (MIVIS) could be tuned to match the dominant frequency content of LF acoustic sources in specific applications. Prototype MIVIS unit cells were designed and tested to study the energy transfer mechanism via impact-induced frequency up-conversion, and the consequent sound transmission loss. Structural acoustic simulations were done to predict responses using models based on normal incidence transmission loss tests. Experimental proof-of-concept was achieved and further correlations to simulations were utilized to optimize the energy up-conversion mechanism using parametric studies. Up to 36 dB of sound transmission loss increase is obtained at the anti-resonance frequency (326 Hz) within a tunable LF bandwidth of about 200 Hz while impact-induced up-conversion could enable further broadband transmission loss via subsequent dissipation in conventional absorbers. Moreover, this approach while minimizing parasitic mass addition retains or could conceivably augment primary functionalities of the baseline structure. Successful transition to applications could enable new mission capabilities for aerospace and military vehicles and help create quieter built environments.Mechanical & Aerospace Engineerin

Acoustic Properties of Absorbing Materials

Thanks to the progress made in materials research and to the introduction of innovative manufacturing technologies, a wide range of sound-absorbing elements are currently available to adjust the acoustic features of an environment. Nowadays, performance is only one of the required specifications, together with environmental compatibility, longevity, and affordable cost. This book collects the most recent advances in the broad-spectrum characterization of sound-absorbing materials used in civil, industrial, and tertiary applications, by means of experimental, numerical, or theoretical studies

NEW ULTRA-LIGHTWEIGHT STIFF PANELS FOR SPACE APERTURES

Stiff, ultra-lightweight thermal-formed polyimide panels considered in this dissertation are examples of next generation gossamer structures that resolve some of the technology barriers of previous, membrane-dominated gossamer designs while maintaining their low mass and low stowage volume characteristics. The research involved statically and dynamically characterizing and modeling several of these panels to develop validated computer models which can be used to determine the effects of changing manufacturing parameters and scalability. Static characterization showed substantial local nonlinear behavior that was replicated by new physics-based finite element models, and global linear bending behavior that was modeled using classical shell finite elements incorporating effective properties in place of bulk material properties to represent the unique stiffening structure of these panels. Dynamic characterization was performed on individual panels using standard impact hammer and accelerometer testing, enabling successful extraction of several structural natural frequencies and mode shapes. Additionally, the three dimensional time history of the surface of the panels was rendered from video data, and temporal filters were applied to the data to examine the frequency content. These data were also correlated to the shell element numerical models. Overall, the research contributes to the total knowledge base of gossamer technologies, advances stiff panel-based structures toward space qualification, and demonstrates their potential for use in apertures and other spacecraft

Case Studies in the Absorption of Low Frequency Sound in Music Rooms

Room design for music is a special and highly valued skill. The spectrum, timbre and dynamics of music, coupled with our keen sense for musical sounds make these rooms a particular challenge for the acoustician. The goal is to create an acoustic environment whereby the sound field within the space supports the instruments and enhances their tone, with unfavourable colourations and effects minimised. Room dimensions, construction materials and specialised treatments must be chosen carefully to achieve the perfect musical balance for the performance. Architectural Acoustics is the field which brings together art and science to address the challenge of creating an environment suitable for music. The most challenging aspect is to achieve the required spectral balance, particularly at low frequency. This thesis presents a literature review of current knowledge of architectural acoustics for music and traditional acoustics treatments. This is followed by three case studies which each investigate a novel solution to improve the acoustic environment for musicians. The first case study investigated room boundaries as potential absorbers. The study arose at a music college who wanted an evaluation of a beautiful new recital hall. Professional musicians had expressed concern about the unusual acoustic in the hall. A room acoustic assessment is described, and results analysed and compared to standards and guidance. Multiple studies including modal analysis, physical experiments and wave-based computer simulation were undertaken to investigate the mechanism for the low frequency dip in the room response. It was determined that the false walls in the hall were acting as quarter wavelength resonators but for multiple low frequencies thus creating an effective absorber. This absorber design could be repurposed to reduce the boom commonly found in modern music venues. The second case study explored pneumatic absorbers. This investigation arose when a London orchestral rehearsal space wanted an inexpensive, quick to deploy, flexible acoustic which would reduce the low frequency boom in the space thereby balancing the room response. A variety of airbeds were selected for the study and tested in the laboratory with different levels of inflation. Finally, the position and number of the airbeds was investigated for optimisation purposes. The airbeds were installed in Henry Wood Hall and the room response measured. It was found the room response was flattened thus improving the acoustic quality. Conductors from world leading orchestras responded positively to the new condition of the hall. The third case study focused on the Targeted Energy Transfer method as a means of creating a novel low frequency absorber. This approach transferred knowledge from the field of vibration control to architectural acoustics. A test rig was built to measure the vibrational damping in two types of hyperelastic latex materials using a laser vibrometer. A small low frequency non-linear response was found but there was not enough evidence to pursue the research further