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The purpose of this study was to observe penetration profiles of lysozyme on a variety of contact lens materials by confocal microscopy, to analyze influential factors that are involved in these penetration curves and to suggest possible mechanisms related to the in-eye clinical performance of these materials. An FITC-lysozyme conjugate was synthesized in-house by amine reaction. Contact lenses were incubated in a lysozyme solution with a final concentration of 1.9 mg/mL for various periods before undergoing microscopic analysis. Optimal parameters for confocal scanning were successfully obtained to acquire desired fluorescence signals on various contact lenses. Measurement units were converted into absolute amounts of lysozyme using lysozyme data from ^(125)I gamma counting studies. A rhodamine labeled dextran solution was applied to distingush the surface of the contact lenses under examination. The data from these studies were then used to calculate the theoretical numbers of layers of adsorbed lysozyme on the lens surface. The results show that there were distinct differences in lysozyme penetration in the twelve hydrogel materials examined. A pure pHEMA lens, with a water content of 38%, deposited lysozyme primarily on the lens surface after 24 hours, with full penetration occurring after 4-weeks of incubation. Three types of non-ionic contact lens materials with water contents > 50% exibited rapid penetration within the lens bulk after 24-hours incubation, with increased deposition within the matrix after 4 weeks. Two ionic, high water content polymers (Acuvue 2 and Focus Monthly) exhibited markedly different penetration profiles, particularly after 24 hours, with very rapid and total penetration in Acuvue 2, as compared with partial penetration in Focus Monthly. Modern silicone hydrogel contact lenses can be nominally divided into first generation, plasma-modified materials and second generation materials which incorporate an internal wetting agent such as polyvinyl pyrrolidone (PVP). These materials exhibited different lysozyme deposition profiles. Lysozyme fully penetrated PureVision after 24 hours, whereas no lysozyme penetration occurred on lenses manufactured from Focus Night & Day or O_2Optix, even after 4 weeks. Lenses manufactured from Acuvue Advance and Acuvue OASYS, two second generation silicone hydrogel lenses, also displayed their own characteristic deposition profile. Acuvue Advance always exhibited a partial penetration of lysozyme within the matrix, even after 4 weeks of doping. Interestingly, Acuvue OASYS showed a similar profile to Focus Night & Day and O_2Optix, with predominantly surface deposition occurring. To confirm possible surface adsorption of lysozyme on surface-coated Focus Night & Day and O_2Optix, a rigid polymethylmethacrylate (PMMA) contact lens was used as a model of surface adsorption. A mounting medium containing rhodamine labeled dextran was scanned to distinguish the lens surface, as it was assumed that no surface penetration of the very high molecular weight dextran would occur. Using this model, it was confirmed that surface adsorption of lysozyme occurred on these plasmacoated lens materials, which is similar to that seen with PMMA. In a further experiment, it was seen that lysozyme sorption on Acuvue OASYS exhibits a penetration profile which is different to that seen in Focus Night & Day and O_2Optix, with lysozyme just penetrating the lens surface. The results from the studies described above demonstrated that in 24 hours lysozyme sorption did not achieve a complete monolayer. However, after 4 weeks multi-layer adsorption occurred, with the more hydrophilic materials depositing the most lysozyme. The quantitative measurement of lysozyme penetration on and into contact lens materials by confocal microscopy combined with ^(125)I labelling offers a valuable tool to discover the potential mechanisms of interactions between protein and polymer materials. This study reveals some important information that may be beneficial to contact lens development and will prove to be valuable in other more broad areas of biomedical research in which polymers and biological fluids come into contact. / Thesis / Master of Applied Science (MASc)

The heart, as a vital organ, must pump continuously to deliver oxygenated blood to the tissues of the body. The physical stress of pumping is supported by the extracellular matrix (ECM), a dynamic protein scaffold inside and around the heart. While a regulated ECM is required to maintain heart function, aberrant or excessive ECM remodelling, called fibrosis, is associated with disease states and is a hallmark of cardiovascular disease. One major trigger of cardiovascular disease is obesity, and fibrotic remodelling is known to occur in this context. In order to study the impact of increased body size on heart function and the molecular and biophysical characteristics of the ECM, a larval overgrowth model for obesity in the genetic model Drosophila melanogaster has been developed and characterised. This model produces giant larvae twice as heavy as their wildtype counterparts, and allows a unique opportunity to study changes in the cardiac ECM in a simple genetic model. Results demonstrate a remarkable ability of the ECM to accommodate this increase in size. The muscles of the heart are particularly robust, and there are no obvious observable defects to the matrix. Preliminary results suggest Collagen fibres are thicker and more disperse. When observing heart functionality, the cross-sectional area of the heart lumen is increased significantly in giant larvae, both at diastole and systole. However, giant larvae display defects in contraction of the heart tube, characterised by an inability to contract fully at systole. This results in a less than proportional increase in stroke volume, and an increase in heart rate. Heart function of giant larvae is clearly affected by the increase in body size. To quantify the impact to the biophysical structure of the ECM, an atomic force microscopy protocol is being developed. / Thesis / Master of Science (MSc) / A known side effect of cardiovascular disease is fibrosis of the heart, a form of pathological extracellular matrix (ECM) remodelling. Fibrosis causes the stiffening of heart muscle, leading to impaired cardiac function. One of the main risk factors for the development of cardiovascular disease is obesity, and fibrosis is known to occur in this context. I have characterised changes in the morphology and physiology of the heart in a Drosophila model for obesity. The resulting cardiac hypertrophy reveals significant plasticity in the heart ECM, while heart contraction and output is compromised.

Migratory cells in vivo move within a predominantly fibrous microenvironment through the action of forces. These dynamic interactions facilitate mechanosensing, critical to fundamental biological processes in pathophysiology. Naturally, the field of mechanobiology has evolved over the past several decades to decipher the role of forces in mechanotransduction using a variety of force-measurement platforms. A central challenge that has yet to be overcome in the field is connecting forces with the interplay between cell shape and ever-changing environment. Here, through design of specific fibrous architectures, a mechanobiological understanding of force feed-forward loop accounting for shape shifting of the environment and cells is developed. Using the non-electrospinning Spinneret Tunable Engineered Parameters (STEP) technique, two complementary force measurement platforms of varying physical attributes are developed to investigate how the force feed-forward loop impacts cell fate. Nanonet Force Microscopy (NFM) comprised of aligned nanonets is designed to study anisotropic cell shapes, while Crosshatch Force Microscopy (CM) comprised of orthogonal arrangement of fibers is designed to study cell bodies of broad shapes. The combination of shapes achieved on these networks recapitulate mesenchymal shapes observed in vivo, which are used to describe cell behaviors not reported before. The new findings include (i) discovery of a new biological structure, termed 3D-perpendicular lateral protrusions (3D-PLPs) which is proposed to be the missing biophysical link in the remodeling of the ECM and perpetuation of desmoplasia. Using NFM, seven discreet steps in formation of force-exerting PLPs anywhere along the cell body is documented, which allow cells to spread laterally and increase in contractility. Using a variety of fiber networks, it is shown that aligned fibers are necessary for PLP formation and suitable environments for myofibroblast activation, and (ii) a force dipole that links matrix deformability with cell contractility. Aided by machine learning, CFM automates the process of fiber feature recognition to measure forces as cells change shapes during migration and differentiate to osteogenic and adipogenic lineages. The force platforms are applied to investigate (i) the bioenergetic contributors fueling cellular migration and a surprisingly overwhelming impact of glycolytic energetic pathway over the traditionally thought mitochondrial energy production is found. However, neither pathway has substantial impact over the cellular force production, and (ii) quantitate the migratory and contractile response of enucleated cytoplasmic fragments naturally shed by cells. A peculiar contractility driven oscillatory migratory phenotype is found, capable of lasting over tens of hours, and absent in intact cells. Overall, new high spatiotemporal capabilities are developed in mechanobiology to quantitate the force-feed forward loops between cell shape and ECM in pathophysiology. / Doctor of Philosophy / Pathophysiology is the study of abnormal changes in the regular body functions of an organism that are causes or consequences of disease onset. Research in this area is mainly focused on identifying the different factors that cause and propagate the disease states such as cancer. Central to many of these processes are events such as cell migration and remodeling of their surrounding environment. The native microenvironment surrounding cells is highly complex and is composed of many classes of macromolecules, with fibrous components being one of the most important. How cells interact with these environments through application of forces and how this further regulates cellular behavior is vital to advancing our understanding of many of these pathophysiological processes. Currently, there is a lack in our understanding of how this dynamic process referred to as the "force feed-forward loop", is perpetuated. This limitation in our understanding can be attributed to the lack of an in vivo mimicking platform that captures this dynamic interaction and is capable of measuring the forces. To this end, the development of two novel single cell force measurement platforms: Nanonet Force Microscopy (NFM) and Crosshatch Force Microscopy (CFM) is presented. These platforms are fiber based systems, generated with the utilization of previously established non-electrospinning technique of Spinneret based Tunable Engineered Parameters (STEP) technique. Using NFM and CFM, forces were computed in wide range of cell shapes from anisotropic to all other spread morphologies. These platforms were applied to identify a new biological structure called perpendicular lateral protrusions and shown to have potential role in the spreading of tumor microenvironment. Furthermore, the force dynamics in physiological processes such as stem cell differentiation into fat cells or bone cells is also identified. How cellular processes such as migration and force production is fueled is also investigated and found to be not heavily reliant on the commonly understood mitochondrial activity. Finally, sub-cellular components known as cell fragments, which are devoid of nucleus, are also observed to be contractile and migratory in nature, independent of parent cell body. These platforms and findings can be further utilized to advance our current knowledge of the progression of these physiological and pathological processes and serve as diagnostic tools for the early identification of disease onset. Furthermore, based on these findings, strategies can be developed for early intervention to inhibit disease progression or devise bioengineered scaffolds for applications in tissue engineering.

Ferroelectric (piezoelectric) and ferromagnetic materials have extensively permeated in modern industry. (Na1/2Bi1/2)TiO3-BaTiO3 (NBT-x%BT) single crystals and K1/2Na1/2NbO3 (KNN) textured ceramics are top environment-friendly candidates which have potential to replace the commercial lead zirconate titanate or PZT. High magnetostrictive strain (up to 400 ppm) of Fe-xat.%Ga makes this alloys promising alternatives to existing magnetostrictive materials, which commonly either contain costly rare-earth elements or have undesirable mechanical properties for device applications. These systems have common characteristics: compositional/thermal/ electrical dependent structural heterogeneity and chemical disorder on sub-micron or nano scale, resulting in diverse local structures and different physical properties. In this work, I have investigated domain and local structures of NBT-x%BT crystals, KNN ceramics and Fe-xat.%Ga alloys under various conditions, mainly by scanning probe and electron transmission techniques. In NBT-x%BT single crystals, polarized light, piezo-response force (PFM) and transmission electron (TEM) microscopies were used to study domain structures and oxygen octahedral tiltings. Hierarchical domain structures were found in NBT: a high-temperature tetragonal ferroelastic domain structure is elastically inherited into a lower temperature rhombohedral ferroelectric phase. Nanoscale domain engineering mechanism was found to still work in NBT-x%BT system and a modified phase diagram was proposed based on domain observations. An increased intensity of octahedral in-phase tilted reflections and a decrease in the anti-phase ones was observed, with increasing x as the morphotropic phase boundary (MPB) is approached. It was also found that Mn substituents favor the formation of long range ordered micro-sized ferroelectric domains and octahedral in-phase tilted regions near the MPB. Nano-size heterogeneous regions were observed within submicron domain structure, indicating that the nanoscale polarization dynamics are not confined by domain boundaries, and the high piezoelectricity of NBT-x%BT is due to a polarization dynamics with high sensitivity to electric field and a broadened relaxation time distribution. In KNN textured ceramics, an aging effect was found to exist in the orthorhombic single phase field, not only in the orthorhombic and tetragonal two-phase field as previously reported. No variation of phase structure was revealed between before and after aging states. However, pronounced changes in domain morphology were observed by both PFM and TEM: more uniform and finer domain structures were then found with aging. These changes were even more pronounced after poling the aged state. A large number of sub-micron lamellar domains within micron-domains were observed: suggesting a domain origin for improved piezoelectric properties. In Fe-xat.%Ga alloys, an underlying inhomogeneity from Ga atoms embedded into the α-Fe matrix was believed to be the origin of giant magneostrictive properties. I have systematically investigated the phase structure and nano-size heterogeneity of Fe-xat.%Ga alloys subjected to different thermal treatments using standard TEM and high resolution TEM for 10<x<30. Nano-precipitates were observed in all specimens studied: A2, D03 and B2 phases were found depending on x. Nano-precipitates of D03 were observed to be dominant for compositions near the magnetostriction peaks in the phase diagram. Quenching was found to increase the volume fraction of nanoprecipitates for x=19, near the first magnetostriction peak. With increasing x to 22.5, nanoprecipitates were observed to undergo a D03 – B2 transformation. A high density of D03 precipitates of nanoscale size was found to be the critical factor for the first maximum in the magnetostriction. / Ph. D.

In the first part of this study, frictional and normal forces in aqueous solutions were measured between a glass particle and oligopeptide films grafted from a glass plate. Homopeptide molecules consisting of 11 monomers of different amino acids were each "grafted from" an oxidized silicon wafer using microwave-assisted solid phase peptide synthesis. Oligopeptides increased the magnitude of friction compared to a bare hydrophilic silicon wafer. Friction was a strong function of the nature of the monomer unit and was lower for hydrophilic films. There was a strong adhesion and therefore friction between surfaces of opposite charges. Changes in adhesion and friction depended on the hydrophobicity and electrostatic forces: hydrophobic films and oppositely charged films produced high friction, whereas hydrophilic and like-charges produced low friction. Friction was lower in phosphate buffered saline than in pure water due to the screening of the double layer attraction for oppositely charged surfaces and additional lubrication by hydrated salt ions. We also investigated antimicrobial action of poly (allyl amine) (PA) when covalently bonded to glass. Glass surfaces were prepared by a two-step procedure where the glass was first functionalized with epoxide groups using 3-glycidoxypropyltrimethoxy silane (GOPTS) and then exposed to PA to bind via reaction of a fraction of its amine groups. Antibacterial properties of these coatings were evaluated by spraying aqueous suspensions of bacteria on the functionalized glass slides, incubating them under agar, and counting the number of surviving cell colonies. The PA film displayed strong anti-microbial activity against both Gram-positive and Gram-negative bacteria. Films that were prepared by allowing the PA to self assemble onto the solid via electrostatic interactions were ineffective antimicrobials. Such films had an insufficient positive charge and did not extend far from the solid. Thus we found that antimicrobial activity was correlated with a combination of the ability of the polymer chain to extend into solution and a positive surface potential. / Ph. D.

Yes / Confocal microscopy has been increasingly employed in the field of traceology to acquire metrological data of surface changes on a micro-scale. However, its advantages for a traditional visual inspection of use-wear are rarely highlighted. As traditional optical microscopy (OM) has proven unable to entirely fulfil the prerequisites for an ideal observation of highly reflective and irregular materials, alternative ways for providing better observation conditions must be sought. In this contribution, we explore the combination of laser scanning confocal (LSCM) and scanning electron microscopy (SEM) micro-graphs for the visual characterisation of wear on quartzite and evaluate the potential of both techniques. / AHRC Fragmented Heritage project (AH/L00688X/1) at the University of Bradford, and of the MICINN-FEDER (PGC2018-093925-B-C32), the AGAUR (SGR 2017-1040) and the URV (2018PFR-URV-B2-91) projects at IPHES-URV. One of the authors (A.P.) was beneficiary of a Catalan pre-doctoral grant (2014FI B 00539), at the Rovira i Virgili University (URV), the IPHES and the Muséum national d’Histoire naturelle of Paris.

Advancements in neural interfacing technologies, such as microelectrode arrays, have significantly contributed to understanding brain function and treating neurological disorders. Decoding the intricacies and functioning of neural circuits is key to further unlocking its potential. Two key approaches, electrical neural recording and optical imaging, have been the basis of stimulating and monitoring neural circuits. Despite the remarkable progress, several key issues such as reliable stimulation of neurons, closed-loop stimulation and monitoring, and undesired background fluorescence during widefield optical imaging remain challenging. After giving a brief background on electrode and microLED arrays, the dissertation delves into the design, microfabrication, and characterization of microelectrode arrays for neural electrical stimulation, recordings, and microLED arrays as a light source for improving optical microscopy. We first discuss a dense conformal electrode array for high spatial resolution stimulation in electrosensory systems. The performance metrics of the integrated system are thoroughly examined through meticulous characterization and optimization processes. Special emphasis is placed on evaluating biocompatibility, electrical properties, and spatial resolution to ensure robust and reliable neural stimulation capability. Next, we discuss a microelectrode device that combines simultaneous electrical recording and 2-photon imaging. We use an Indium Tin Oxide (ITO) material to fabricate a transparent electrode array with a design capable of single neuron recordings. The design, microfabrication, and electrooptical characterization are presented to demonstrate the device’s capability. A system integrating the array with a GRIN lens is also presented to record and image deeper into the brain tissue. Combining both the electrical and optical recordings of neuron ensembles and finding correlations can shed further light on the functioning of neural circuits. To address the problem of unwanted background fluorescence during neural cell imaging, two microLED arrays as light sources are presented. With a microstripe array, we implement optical sectioning structured illumination microscopy (OS-SIM), and with the 2D microLED array, we implemented targeted illumination to reject background fluorescence and improve contrast. We examine the capability of the microLED as a light source with luminance-current-voltage, directivity, and transient measurements. Both implementations highlight the novel non-display application of microLED to address challenges in neural imaging. This research represents a significant contribution to the burgeoning field of neural engineering, offering novel methodologies and technologies that promise to revolutionize our approach to understanding brain functions.

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