During nervous system development, growing axons navigate towards their targets using signals from their environment. These signals may be biochemical or mechanical in nature; however, the role of mechanical cues in axon pathfinding in vivo, and the spatiotemporal dynamics of embryonic brain mechanics, are still largely uncharacterised. Here, I have identified a role for tissue mechanics in embryonic axon guidance in vivo, using retinal ganglion cell (RGC) axon outgrowth in the developing Xenopus laevis optic tract (OT) as a model system. Using atomic force microscopy (AFM) to map brain stiffness in vivo, I found that embryonic Xenopus brain tissue was mechanically heterogeneous at both early and later stages of OT outgrowth, i.e. just before RGC axons make a stereotypical turn in the mid-diencephalon, and when they reach their target, respectively. The final path of RGC axon turning followed a clear mechanical gradient: by the later stage, tissue rostral to the OT had become stiffer than tissue caudal to it. This mid-diencephalic stiffness gradient was an intrinsic property of the underlying brain tissue, and correlated with local cell body densities (with higher density rostral to the OT and lower density caudal to it). Crucially, inhibiting cell proliferation in vivo during OT outgrowth abolished the stiffness gradient and reduced OT turning at the later stage. Next, I developed a time-lapse AFM technique to track tissue stiffness and RGC axon behaviour simultaneously in vivo. Using this approach, I followed the evolution of the mid-diencephalic stiffness gradient during intermediate developmental stages, around the time when the OT’s caudal turn is initiated. The stiffness gradient was shallow pre-turn, but increased in magnitude during axon turning (mostly due to an increase in tissue stiffness rostral to the OT). This increase in stiffness gradient preceded the rise in OT turning angle, suggesting that the stiffness gradient is not caused by the invading axons. The observed rise in stiffness gradient correlated with stage-specific increases in local cell density, and was attenuated by blocking mitosis in vivo during time-lapse AFM measurements (which also reduced OT turning). As final confirmation that brain stiffness contributes to RGC axon pathfinding, I disrupted mechanical gradients by artificially stiffening brain tissue in vivo. Importantly, global stiffening via application of transglutaminase eliminated the mid-diencephalic stiffness gradient by increasing tissue stiffness caudal of the OT, and reduced the OT turning angle. Sustained mechanical compression of small areas using an AFM probe stiffened brain locally and repelled RGC axons, consistent with the way they turned away from rapidly stiffening tissue regions during time-lapse AFM experiments. Taken together, these results are consistent with a function for tissue mechanics in axon pathfinding in vivo.

Due to the escalating challenge of antibiotic resistance in bacteria over the past several decades, interest in the identification and development of antibiotic alternatives has intensified. Antimicrobial peptides (AMPs), which serve as part of the innate immune systems of most eukaryotic organisms, are being researched extensively as potential alternatives. However, the mechanism behind their bactericidal capabilities is not well understood. Previous studies have suggested that AMPs may first attach to the cell membranes, leading to pore formation caused by peptide insertion, lipid removal in the form of peptide-lipid aggregates, or a combination of both mechanisms. In addition to the lack of mechanistic knowledge, a significant hurdle in AMP-based drug development is their potential cytotoxicity to mammalian cells. Understanding AMP interactions with eukaryotic model membranes would allow therapeutics to be tailored for preferential action toward specific classes of bacterial membranes. In this study, we developed novel methods of quartz crystal microbalance with dissipation monitoring (QCM-D) data analysis to determine the fundamental mechanism of action between eukaryotic and bacterial membrane mimics and select membrane-active AMPs. A new technique for creating supported membranes composed entirely of anionic lipids was developed to model Gram-positive bacterial membranes. Atomic force microscopy (AFM) imaging was also used to capture the progression of AMP-induced changes in supported lipid membranes over time and to validate our method of QCM-D analysis. QCM-D and AFM were used to investigate the molecular-scale interactions of four peptides, alamethicin, chrysophsin-3, sheep myeloid antimicrobial peptide (SMAP-29) and indolicidin, with a supported zwitterionic membrane, which served as a model for eukaryotic cell membranes. Since established methods of QCM-D analysis were not sufficient to provide information about these interaction mechanisms, we developed a novel method of using QCM-D overtones to probe molecular events occurring within supported lipid membranes. Also, most previous studies that have used AFM imaging to investigate AMP-membrane interactions have been inconclusive due to AFM limitations and poor image quality. We were able to capture high-resolution AFM images that clearly show the progression of AMP-induced defects in the membrane. Each AMP produced a unique QCM-D signature that clearly distinguished their mechanism of action and provided information on peptide addition to and lipid removal from the membrane. Alamethicin, an alpha-helical peptide, predominantly demonstrated a pore formation mechanism. Chrysophsin-3 and SMAP-29, which are also alpha-helical peptides of varied lengths, inserted into the membrane and adsorbed to the membrane surface. Indolicidin, a shorter peptide that forms a folded, boat-shaped structure, was shown to adsorb and partially insert into the membrane. An investigation of rates at which the peptide actions were initiated revealed that the highest initial interaction rate was demonstrated by SMAP-29, the most cationic peptide in this study. The mechanistic variations in peptide action were related to their fundamental structural properties including length, net charge, hydrophobicity, hydrophobic moment, accessible surface area and the probability of alpha-helical secondary structures. Due to the charges associated with anionic lipids, previous studies have not been successful in forming consistent anionic supported lipid membranes, which were required to mimic Gram-positive bacterial membranes. We developed a new protocol for forming anionic supported lipid membranes and supported vesicle films using a vesicle fusion process. Chrysophsin-3 was shown to favor insertion into the anionic lipid bilayer and did not adsorb to the surface as it did with zwitterionic membranes. When introduced to supported anionic vesicle films, chrysophsin-3 caused some vesicles to rupture, likely through lipid membrane disruption. This study demonstrated that molecular-level interactions between antimicrobial peptides and model cell membranes are largely determined by peptide structure, peptide concentration, and membrane lipid composition. Novel techniques for analyzing QCM-D overtone data were also developed, which could enable the extraction of more molecular orientation and interaction dynamics information from other QCM-D studies. A new method of forming supported anionic membranes was also designed, which may be used to further investigate the behavior of bacterial membranes in future studies. Insight into AMP-membrane interactions and development of AMP structure-activity relationships will facilitate the selection and design of more efficient AMPs for use in therapeutics that could impact the lives of millions of people per year who are threatened by antibiotic-resistant organisms.

Pseudomonas aeruginosa is extremely harmful to immunocompromised individuals. An atomic force microscope was used to measure the surface forces of this bacteria’s exopolymers. These forces were characterized with the AdG force model, which is a function of brush length, probe radius, temperature, separation distance and an indefinite density variable, s. This last parameter could represent the root spacing or mesh spacing of the exopolymers. This study aims to clarify s by obtaining force values as a function of temperature. The data suggest that s represents the mesh spacing. If s is the root spacing it should remain constant regardless of the changing polymer lengths, on the other hand if it is the mesh spacing it will vary with changing temperature, as shown by the data presented in this research. This knowledge will aid in understanding and characterizing how bacteria cause infections.

This study presents a systematic investigation of two types of soft biomaterials: phospholipid-based microbubbles (MBs) and agarose hydrogels, using atomic force microscopy (AFM) force-distance curves. Microbubbles are used widely in several applications, especially in medical applications, where they are used as ultrasound contrast agents (UCAs) and as vehicles for transporting the drugs and genes to their targets, which is commonly known as drug/gene delivery. Although plenty of attention has been paid to these materials by medical researchers there is a shortage of engineering research on the properties of these materials. The present study tries to address this gap by studying these materials from the engineering perspective; therefore, the aim of this study is to investigate the mechanical properties of MBs and hydrogels. In this research, phospholipid-based microbubbles (MBs), commercially called SonoVue® microbubbles and used as UCAs, were investigated to measure their mechanical properties using an AFM mode of operation called force-distance curves (or force spectroscopy mode); this mode allows for direct mechanical tests to acquire the force-deformation (F-Δ) behaviour of the MBs. The compression tool was a flat (tipless) cantilever moved at constant speed, whereas the variable was MB size. The MBs behaviour was assessed by calculating several mechanical properties, which were the stiffness, Young's modulus (three different models were applied), hysteresis, plasticity, adhesion forces, nonlinearity and instability. The stiffness and the Young's modulus values were measured to be in the same range as found in similar studies. A phenomenon was observed that the local stiffness of the MB increases after each unstable step provided that the MB stays within the linear elastic region. The Young's modulus was calculated applying three models, two for estimating the elastic modulus of the shell and the third for modulus of elasticity of the whole MB. The stretching component of the membrane theory was found to provide the best prediction of the Young's modulus value. To investigate the effect of the tip geometry on the mechanical properties of the MBs, the MBs were studied with different cantilever/tips, including a conical-tipped cantilever. The study concluded that there is no impact of the contact geometry on the mechanical properties of the MBs if the applied forces and the spring constant of the cantilever are the same. The same phenomenon, increasing the local stiffness of the MB after each unstable step, was found however with a higher rate. Hydrogels were also studied in this research using AFM and adopting a nanoindentation technique. The indenter was a conical tip moving toward the sample surface with constant speed and applying similar forces on all samples, where the variable was the gel concentration. In addition to the previous mechanical properties, other properties were investigated, such as hardness, universal hardness and pressure. An effect of the gel concentration on the mechanical properties of the gels was observed. There is a difference in the results compared to those reported in the literature review, where some of the results are in the same range as those found here, while others were either higher or lower, due to the influence of factors such as the indenter geometry, the applied force and the load rate; moreover, it was found that the viscoelastic behaviour of the gels played a significant role.

Nanoscience is an interdisciplinary science that could be defined as the search and study of new properties (physical, chemical, etc..) that emerge as the size of materials is reduced down to nanometric scale (or nanoscale). A valuable tool for achieving this end is the Scanning Tunneling Microscope (STM). Since its invention by Binnig and Rohrer, the STM has become an essential tool for the characterization and manipulation at the nano-scale. Many other microscopes have been developed rooted in the STM, thus giving birth to the family of Scanning Probe Microscopes (SPM). One of the achievements of this work has been the design and calibration of a SPM which allows a simultaneous measurement of electron transport and mechanical properties (adhesion force and energy dissipation) of nanostructures, in varying conditions of temperature from 1.5K to room temperature. This microscope has a conducting tip fixed at one of the arms of a microfabricated quartz tuning fork used in the resonant detection of forces. This detection system was also implemented and tested on a commercial microscope operating in ultra high vacuum and low temperatures conditions. With the microscopes above electrical and mechanical properties of different nanostructures, including atomic size contacts, surface molecules, nano-capacitors and graphite, have been studied. It is also worth mentioning the experimental development of a new technique of local grapheme electro-exfoliation on graphite and its explanation by means of a theoretical model. / La nanociencia es una ciencia interdisciplinaria que podríamos definir como la búsqueda y estudio de nuevas propiedades (físicas, químicas, etc.) que emergen al reducir el tamaño de los materiales a la escala nanométrica. Un instrumento de gran valor para alcanzar este fin es el Microscopio de Efecto Túnel (STM). Desde su invención por Binnig y Rohrer, el STM se ha convertido en una herramienta esencial para la caracterización y la manipulación en la nano-escala. A partir del STM se han desarrollado otros microscopios generando así la familia de Microscopios de Sonda Local (SPM). Uno de los logros conseguidos en esta tesis de doctorado es el diseño y calibración de un SPM que permite hacer medidas simultáneas de transporte electrónico y propiedades mecánicas (fuerza de adhesión y disipación de energía) de nanoestructuras, en condiciones variables de temperatura desde 1.5K hasta temperatura ambiente. Este microscopio utiliza una punta conductora fijada en uno de los brazos de un diapasón de cuarzo microfabricado, para la detección resonante de fuerzas. Este sistema de detección fue también implementado y probado en un microscopio comercial que opera en condiciones de ultra alto vacío y bajas temperaturas. Con dichos microscopios se han estudiado las propiedades eléctricas y mecánicas diferentes nanoestructuras incluyendo contactos de tamaño atómico, moléculas en superficie, nano-capacitores y grafito. Es de destacar también el desarrollo experimental de una nueva técnica de electroexfoliación local de grafeno sobre grafito y su explicación por medio de un modelo teórico. / This work has been supported by the Departamento de Física of the Universidad Nacional de Costa Rica and the grants MAT2007-65487, 31099-E and CONSOLIDER CSD2007-0010, and partly by the European Union through MolSpinQIP.

Studying physical properties of nanoscale materials has gained a significant attention owing to their applications in the fields such as electronics, medicine, pharmaceutical industry, and materials science. However, owing to size constraints, number of techniques that measures physical properties of materials at nanoscale with a high accuracy and sensitivity is limited. In this context, development of atomic force microscopy (AFM) based techniques to measure physical properties of nanomaterials has led to significant advancements across the disciplines including chemistry, engineering, biology, material science and physics. AFM has recently been utilized in the quantification of physical-chemical properties such as electrical, mechanical, magnetic, electrochemical, binding interaction and morphology, which are enormously important in establishing structure-property relationship. The overarching objective of the investigations discussed here is to gain quantitative insights into the factors that control electrical and mechanical properties of nano-dimensional organic materials and thereby, potentially, establishing reliable structure-property relationships particularly for organic molecular solids which has not been explored enough. Such understanding is important in developing novel materials with controllable properties for molecular level device fabrication, material science applications and pharmaceutical materials with desirable mechanical stability. First, we have studied electrical properties of novel silver based organic complex in which, the directionality of coordination bonding in the context of crystal engineering has been used to achieve materials with structurally and electrically favorable arrangement of molecules for an enhanced electrical conductivity. This system have exhibited an exceptionally high conductivity compared to other silver based organic complexes available in literature. Further, an enhancement in conductivity was also observed herein, upon photodimerization and the development of such materials are important in nanoelecrtonics. Next, mechanical properties of a wide variety of nanocrystals is discussed here. In particular, an inverse correlation between the Young’s modulus and atomic/molecular polarizability has been demonstrated for members of a series of macro- and nano-dimensional organic cocrystals composed of either resorcinol (res) or 4,6-di-X-res (X = Cl, Br, I) (as the template) and trans-1,2-bis(4-pyridyl)ethylene (4,4’-bpe) where cocrystals with highly-polarizable atoms result in softer solids. Moreover, similar correlation has been observed with a series of salicylic acid based cocrystals wherein, the cocrystal former was systematically modified. In order to understand the effect of preparation method towards the mechanical properties of nanocrystalline materials, herein we have studied mechanical properties of single component and two component nanocrystals. Similar mechanical properties have been observed with crystals despite their preparation methods. Furthermore, size dependent mechanical properties of active pharmaceutical ingredient, aspirin, has also been studied here. According to results reduction in size (from millimetre to nanometer) results in crystals that are approximately four fold softer. Overall, work discussed here highlights the versatility of AFM as a reliable technique in the electrical, mechanical, and dimensional characterization of nanoscale materials with a high precision and thereby, gaining further understanding on factors that controls these processes at nanoscale.

Zinc oxide is a very versatile material that can be used in many microsystems and MEMS applications. ZnO thin film has been utilized in a wide variety of MEMS devices because of its unique piezoelectric, optical, and electrical properties. In particular, piezoelectric property of ZnO can be used in numerous applications from resonators and filters to mass sensors and micro-actuators (e.g., micro-valve and micro-pump). Because of its versatility, this research was focused on analyzing some key properties of ZnO thin film achieved by two different deposition techniques, Pulsed Laser Deposition (PLD) and Sputtering. Multiple experiments were conducted in order to identify the best conditions for the growth of ZnO thin film. Under the optimum conditions, the ZnO thin films will provide the best piezoelectric performance in devices such as microcantilevers. In order to find the best deposition conditions in both PLD and Sputtering multiple depositions have been done and then analyzed using the XRD, AFM, FTIR, nanoindenter, and ellipsometer. For the PLD the best conditions were found to be at 200°C with a partial pressure of O2 of 100 millitorr. For the sputtering system the best film formed when the substrate temperature was kept at 400°C along with RF power of 250 Watts, and a flow rate of 25% O2 and 75% Ar. Both experiments were similar in the fact that both a certain amount of O2 in the chamber and an elevated temperature are needed to facilitate the formation of ZnO crystal structure.

In considering the concept of surface-immobilised liposomes as a drug release system, two factors need to addressed, the interfacial surface density of the liposomes for maximum drug loading and the stability of these liposomes to allow for controlled drug release. This thesis investigates a multilayer system for the affinity immobilisation of liposomes and their stability to various applied stresses. In the work presented here an allylamine monomer was used to create plasma coatings that were stable, thin and amine-rich. The aging studies using AFM showed these films to rapidly oxidise on exposure to water. The freshly deposited films were used for further surface modifications, by the covalent grafting of PEG layers of different interfacial densities under the conditions of varying polymer solvation. The AFM was used to measure the interaction forces between the grafted PEG layers and modified silica interfaces. It was found that the polydispersity of the PEG species resulted in bridging interactions of ???brush???-like PEG layers with the silica surface. These interactions were screened minimised by increasing the ionic strength of the solution. Although the densely grafted PEG layers were found to be highly protein-resistant by the XPS and QCM-D some minor protein-polymer adhesions were observed by the AFM. The densely anchored biotinylated PEG chains served as an optimum affinity platform for affinity-docking of NeutrAvidinTM molecules, which assembled in a rigid, 2-D layer as confirmed by the QCM-D. The submonolayer surface density of NeutrAvidin, as determined by Europium-labelling, was attributed to steric hindrance of the immobilised molecules. The final protein layer enabled specific binding of biotin-PEG-liposomes as a highly dissipative, dense and stable layer verified by tapping mode AFM and QCM-D. We found that these liposomes were also stable under a range of stresses induced by the shearing effects of water, silica probe and HSA layer at increased loads and velocities. The frictional response of the liposome layer also demonstrated the viscoelasticity and stability of these surface immobilised liposomes. Finally, the minimal adhesive interaction forces, as measured by the AFM, demonstrated the repellency of these liposomes to commonly found proteins, such as HSA.

Mon projet porte sur l'elaboration d'un microscope optique en champ proche (SNOM) sans ouverture fonctionnant dans le domaine de l'infrarouge et utilisant le rayonnement synchrotron de l'ESRF comme source de lumiere infrarouge. Ce rayonnement a deux particularites bien adaptees aux etudes spectroscopiques: c'est une source de lumiere blanche couvrant la bande du proche infrarouge de 5 a 15 microns alors que les sources lasers accordables sont encore en developpement. Il est tres brillant et stable, a la fois dans le temps et dans l'espace. Une fois elaboree, le microscope sera appliquee a la spectroscopie infrarouge - essentiellement vibrationelle - et le diagnostic des materiaux et des nanostructures qui sont d'inter^et actuel pour l'industrie micro et nanoelectronique [RCB+02]. Comme mon projet est tres instrumental, le debut a ete consacre a la conception de tout un systeme de microscopie, a partir du zero et avec tout le materiel necessaire disponible a la n de la premiere annee. La seconde annee a ete consacree a l'integration et la mise en oeuvre du dispositif experimental, a la comprehension de ses fonctionnalites et a des essais de validation du nouveau outil. Apres nous nous sommes pleinement engages dans la recherche et la comprehension de cet outil unique. Nous avons commence par quelques resultats preliminaires, puis cela a ete essentiellement une question de temps experimental alloue pour obtenir les resultats que nous avions vises. Notre conguration est unique et donc les travaux que nous avons pour reference sont ceux de groupes utilisant les m^emes techniques d'exploitation dans des conditions tres dierentes. Notre principale diculte est de detecter un signal faible. Je montrerai plus loin quelques calculs qui nous y ont fait croire. Notre groupe de reference a reussi a le faire dans des conditions plus simples que les notres, mais il est utile de rappeler que cela leur a pris 3 ans pour adapter leur sensibilite a l'installation, ce qui conrme que ce sont des techniques tres dur. Notre idee de depart etait d'utiliser le rayonnement synchrotron comme source de lumiere dans l'infrarouge en raison de ses caracteristiques. Il s'agit d'une source de lumiere blanche, avec toutes les longueurs d'onde presentes en meme temps, nous permettant d'eectuer une spectroscopie, ce qui signie que nous obtiendrons une information chimique sur l'echantillon [Hil04; MGCS04]. Telle est la grande nouveaute en comparison avec les autres travaux. Il s'est avere que la lumiere est assez faible ce qui rend la recherche du signal dicile. Notre installation doit alors etre testee avec un laser, qui est de plusieurs ordres de grandeur plus puissant que le rayonnement synchrotron, et qui pourrait etre un bon outil de debogage. Cela semble une bonne alternative pour mieux comprendre les points essentiels qui doivent etre ameliores dans notre systeme. Les lasers a utiliser pourraient etre visible, infrarouge (CO2), ou accordable. Pour la spectroscopie, les lasers accordables sont non seulement moins stables mais ils sont aussi plus limites dans la gamme spectrale sur la partie infrarouge proche du spectre, que le rayonnement synchrotron. Le synchrotron de l'ESRF est mon laboratoire d'accueil, et j'ai travaille en collaboration avec le CEA-LETI pour le developpement de cet outil.

Protein aggregation, leading to the formation and depositions of amyloids, is a cause for a number of diseases such as Alzheimer’s and Creutzfeld-Jacob’s disease, systemic amyloidoses, type II diabetes and others . More than 20 proteins are associated with protein misfolding diseases and even a larger number of proteins can self-assemble into amyloid in vitro. Relating structural and functional properties of amyloid is of particular interest, as this will lead to the identification of the main factors and mechanisms involved in the process of protein misfolding and aggregation; consequently, this will provide a basis for developing new strategies to treat protein misfolding diseases. The aim of the thesis is to investigate structural aspects of amyloid formation and relate that to the functional properties of amyloid. The first paper describes the amyloid formation of equine lysozyme (EL). We have demonstrated that EL enters an amyloid forming pathways under conditions where the molten globule state is populated. We have found that the morphology of the amyloids depend on the calcium-binding to lysozyme, specifically the holo-protein assembles into short, linear protofilaments, while the apo-EL forms ring-shaped structures. The morphology of EL amyloid significantly differs from the amyloid fibrils of human and hen lysozymes. We have suggested that the stable alpha-helical core of EL, which remains structured in the molten globule intermediate, may obstruct the formation of fibrilar interface and therefore leads to assembly of short, curly fibrils and rings.In the second paper, we describe the cytotoxicity of EL amyloids. We have analysed the amyloid intermediates on the pathway towards amyloid fibrils. The sizes of amyloid oligomers were determined by atomic force microscopy (AFM) and the formation of cross-beta sheet was shown by thioflavin T (ThT) binding. The toxicity studies show that the oligomers formed during amyloid growth phase are toxic to a range of cell lines and cultures and the toxicity is size-dependant.The last manuscript describes a novel method for manufacturing of silver nanowires by the biotemplating using amyloid fibrils. The amyloid assembled from an abundant and cheap hen egg white lysozyme was used as a scaffold for casting ultrathin silver nanowires. We have manufactured nanowires with a diameter of 1.0-2.5 nm and up to 2 micrometers in length. Up to date, it is the thinnest silver nanowires produced by using biotemplating and at least one order of magnitude thinner than nanowires manufactured by chemical synthesis.