Protein-DNA interactions are central to many biological processes and form the bedrock of gene transcription, DNA replication, and DNA repair processes. Many proteins recognize specific sequences in DNA- a restriction enzyme must only cut at the correct sequence and a transcription factor should bind at its consensus sequence. Some proteins are designed to bind to specific structural or chemical features in DNA, such as DNA repair proteins and some DNA modifying enzymes. Target-specific DNA binding proteins initially bind to non-specific DNA and then search for their target sites through different types of diffusion mechanisms. Atomic force microscopy (AFM) is a single-molecule technique that is specifically well-suited to resolve the distinct states of target-specific as well as nonspecific protein-DNA interactions that are vital for a deeper insight into the target site search mechanisms of these enzymes. In this thesis, protein systems involved in epigenetic regulation, base excision repair (BER), and transcription are investigated by single-molecule AFM analyses complemented by biochemical and biophysical experiments. The first chapter of this thesis narrates the establishment of a novel, user-unbiased MatLab-based tool for automated DNA bend angle measurements on AFM data. This tool has then been employed to study the initial lesion detection step of several DNA glycosylases. These results promoted a model describing the altered plasticities of DNA at the target lesions of DNA glycosylases as the fundamental mechanism for their enhanced efficiency of lesion detection. In the second chapter of this thesis, the novel automated tool has been further extended to provide protein binding positions on the DNA along with corresponding DNA bend angles and applied to the study of DNMT3A DNA methyltransferase. These AFM studies revealed preferential co-methylation at specific, defined distances between two CpG sites by the enzyme and when combined with biochemical analyses and structural modelling supported novel modes of CpG co-methylation by DNMT3A. In the third chapter of this thesis, the role of 8-oxo-guanine glycosylase (hOGG1) in Myc-mediated transcription initiation has been investigated. AFM analyses revealed that in the presence of oxidative damage in DNA, Myc is recruited to its target site (E-box) by hOGG1 through direct protein-protein interactions, specifically under oxidizing conditions. Intriguingly, oxidation of hOGG1 was further observed to result in dimerization of hOGG1, which may also play a role in the mechanism of transcription regulation by hOGG1 under oxidative stress. / Protein-DNA-Wechselwirkungen sind für viele biologische Prozesse von zentraler Bedeutung und bilden die Grundlage der Gentranskription, der DNA-Replikation und der DNA-Reparaturprozesse. Viele Proteine erkennen bestimmte Bassen-Sequenzen in der DNA - ein Restriktionsenzym darf nur an der richtigen Sequenz schneiden, und ein Transkriptionsfaktor sollte an seine Konsenssequenz binden. Einige Proteine sind darauf ausgelegt, an bestimmte strukturelle oder chemische Merkmale der DNA zu binden, wie z. B. DNA-Reparaturproteine und verschiedene DNA-modifizierende Enzyme. Zielspezifische DNA-bindende Proteine binden zunächst an unspezifische DNA und suchen dann durch verschiedene Arten von Diffusionsmechanismen nach ihren Zielstellen in der DNA. AFM ist eine Einzelmolekültechnik, die besonders gut geeignet ist, um die verschiedenen Zustände sowohl der spezifisch gebundenen als auch unspezifischen Protein-DNA-Wechselwirkungen aufzulösen, die für einen tieferen Einblick in die Mechanismen der Zielstellensuche unerlässlich sind. In dieser Arbeit werden Proteinsysteme, die an der epigenetischen Regulation, der Basenexzisionsreparatur (BER) und der Transkription beteiligt sind, durch Einzelmolekül- AFM-Analysen untersucht, und diese Studien werden durch biochemische und biophysikalische Experimente komplementiert. ...

PLE presents an alternative or complementary probe-based tool to DPN, PPL, and NFL. Unlike most scanning probe techniques, where patterning by deposition is usually employed, PLE is unique because it is capable of deposition and removal in one or multiple steps. Therefore, PLE allows rectification of patterning errors, and it can be employed for both additive and subtractive patterning through molecular deposition and chemical and electrochemical etching, respectively. PLE is a technique that exploits the intrinsic porosity of hydrogels like agarose and polyacrylamide. The probes are made by polymerizing a liquid mixture of agarose or acrylamide monomers in a conical or pyramidal master. The polymeric probe is hydrated in deionized water or ink of interest after polymerization. For deposition, PLE has shown promising results in the selective deposition of fluorescent inks on bare or functionalized glass substrates. Erasing via PLE has been done in two ways: the first method involves selectively erasing the fluorescent molecules using a probe loaded with deionized water by bringing the probe in contact with the area of interest. Thus, solvation and transportation of the molecules into the polymeric probes render selective removal of materials (fluorescent inks) from a substrate. On the other hand, erasing or removal of metals deposited on a substrate was demonstrated using redox reactions. Here, the probe is loaded with an etchant, which is selectively delivered onto the substrate by bringing the probe close to or in contact with the surface. Thus, the etchant molecules passively diffuse from the probe to the substrate through a meniscus formed at the probe-substrate interface. Removal of molecules occurs after the redox reaction between the ink, and the substrate is completed. Many in-length microscale complex patterns can be easily made by translocating the probe over the substrate while the probe’s tip is in contact with the surface. Since the probes used in PLE are made of polymers, the probe-substrate contacting area can be easily modulated, and damage to the substrate by the probe is minimum. Moreover, it has been shown that the probes can be used multiple times, a hurdle frequently faced by probes made of hard materials such as silicon-based probes. We explored the capabilities of a polymeric probe made of PAAM to selectively deliver and remove (erase) material deposited on a surface. PLE, pioneered by our group, takes advantage of the hydrophilic and porous nature of polyacrylamide. In addition, the conformability of PAAM hydrogels was exploited to make patterns of various sizes and to the pattern on non-planar surfaces. The main advantage of PLE is removing materials from various substrates. Additionally, selective delivery of material to planar and non-planar substrates was demonstrated. Whereas DPN and sister techniques require multiple steps for patterning through the etching process, PLE can perform etching in one step. Therefore, using PLE, microscale patterning on surfaces can save considerable time, labor, and cost. Further, chemical and supplies waste are minima in PLE. Notably, the deposition and etching at the microscale level can be simultaneously achieved in one single step, providing an extremely high throughput patterning rate (on the order of 1000 mm2/s). The PLE patterning rate is two to three orders larger than DPN-based patterning. However, PLE inherently deposits and removes materials with features much larger (microscale) than that can be achieved with DPN (sub-nanoscale). Therefore, PLE is an alternative to DPN, PPL, and related probe-based deposition and erasing techniques, and in some cases, PLE provides enhanced capabilities than its contemporary techniques. In this dissertation, I intend to demonstrate the potential of PLE for fabricating working devices at a lower cost as an alternative to contemporary fabrication. Chapter 2 involves the fabrication of micro-electrodes on rigid and flexible substrates by selectively removing copper and ITO from a glass and a PET substrate. As proof of concept, substrates coated with the PLE patterned surfaces were used to fabricate a photodetector, and LEDs were assembled on the electrodes made on ITO-PET substrates. Chapter 3 describes a series of experiments involving the evaluation of ink withholding capacity, large area patterning, and the effect of modification of substrate surface energy on PLE patterning. These experiments an increased understanding of processes involved in PLE editing and microscale patterning. A potential pitfall of PLE-based etching was also observed in these experiments, where a thin layer of material was left behind after subtractive editing with a PLE probe. EDS analysis indicated that the material was composed of iron, chlorine, and copper ─ components of the etchant solution and the copper film. The ring structure was attributed to the coffee-ring effect pinning the water meniscus to the substrate. By understanding the potential causes of the formation of the coffee-ring possible solutions to this problem were formulated. Chapter 4 describes the physical and mechanical properties of the hydrogel PAAM probes at the nanoscale. ESEM and AFM were employed to investigate the structural and mechanical properties of the probes after impregnation with metal etchants of various concentrations. The effect of local RH on PLE patterns was also investigated. More importantly, these experiments show critical structural differences of PAAM hydrogels composed of various monomer and crosslinker concentrations. ESEM showed the significant influence exerted by RH on meniscus size and its interaction with the substrate. The behavior of the water meniscus observed in ESEM shows that large RH promotes water spreading on the substrate generating larger patterning features. Chapter 5 describes the capability of PLE to selectively deliver metallic inks on a non-linear curved substrate to fabricate a microscale battery. PLE was used to deposit silver nitrate onto a non-planar flexible substrate which was used to grow a thin electrically conductive copper film via copper electroless deposition. Electrodeposition of zinc on the copper substrate was accomplished. By coupling a zinc electrode to a manganese oxide-graphite composite cathode, we demonstrated a working Zn-MnO2 aqueous microscale battery.

This study uses in situ atomic force microscopy (AFM) to directly observe the atomic scale effects of Sr on the monomolecular layer growth of abiotic calcite. These insights are coupled with quantitative measurements of the kinetics and thermodynamics of growth to determine the direction-specific effects of Sr on the positive and negative surface coordination environments that characterize calcite step edges. Low concentrations of strontium enhance calcite growth rate through changes in kinetics. A new conceptual model is introduced to explain this behavior. Higher concentrations of strontium inhibit and ultimately stop calcite growth by a step blocking mechanism. The critical supersaturation required to initiate growth (sigma*) increases with increasing levels of strontium. At higher supersaturations, strontium causes growth rates to increase to levels greater than those for the pure system. The step blocking model proposed by Cabrera and Vermilyea in 1958 does not predict the experimental data reported in this study because the dependence of sigma* upon strontium concentration is not the same for all supersaturations. Strontium inhibits calcite growth by different mechanisms for positive and negative step directions. Preliminary evidence indicates that strontium is preferentially incorporated into the positive step directions suggesting that impurity concentrations are not homogeneous throughout the crystal structure. Despite geochemical similarities, this study demonstrates that strontium and magnesium have different surface interaction mechanisms. The findings of this study demonstrate the importance of understanding microscopic processes and the significance of interpreting biominerals trace element signatures in the context of direction-specific interactions. / Master of Science

The stochastic dynamics of an array of closely spaced micron scale cantilevers in a viscous fluid is considered. The stochastic cantilever dynamics are due to the constant buffeting of fluid particles by Brownian motion and the dynamics of adjacent cantilevers are correlated due to long range effects of fluid dynamics. The measurement sensitivity of an experimental setup is limited by the magnitude of inherent stochastic motion. However, the magnitude of this noise can be decreased using correlated measurements allowing for improved force resolution. A correlated scheme is proposed using two atomic force microscope cantilevers for the purpose of analyzing the dynamics of single molecules in real time, a regime that is difficult to observe using current technologies. Using a recently proposed thermodynamic approach the hydrodynamic coupling of an array of cantilevers is quantified for precise experimental conditions through deterministic numerical simulations. Results are presented for an array of two readily available micron-scale cantilevers yielding the possible force sensitivity and time resolution of correlated measurements. This measurement scheme is capable of achieving a force resolution that is more than three fold more sensitive than that of a single cantilever when the two cantilevers are separated by 200 nm, with a time scale on the order of tens of microseconds. / Master of Science

Through enhancing aerosol particle measurement accuracy by determining particle shape factors using Atomic Force Microscopy (AFM) combined with machine learning techniques, this study aims to provide a methodology that will improve the precision of aerosol measurements and contribute to the development of more effective filtration technologies. Accurate shape factor measurement is crucial for devices such as the Scanning Mobility Particle Sizer (SMPS), which often assume spherical particles of uniform density. By identifying and analyzing particles in AFM scans using machine learning techniques, this research provides a better understanding of shape factors, improving the quality of aerosol measurements. These advancements contribute to a deeper understanding of aerosol properties and their impact on filtration systems, aiding in the development of more effective filtration technologies and improving our capability to measure and control particulate matter in various environmental and industrial applications.

"The adhesion of bacteria to surfaces has been analyzed in terms of surface charge, surface energy, and the characteristics of polymers on bacteria, to understand the factors that control bacterial adhesion. Pseudomonas aeruginosa has received a great deal of interest because it is responsible for a variety of chronic bacterial infections such as airway infections in cystic fibrosis patients and ulcerative bacterial keratitis in soft contact lens users. Over the past few years, force measurement techniques such as atomic force microscopy (AFM) have made it possible to examine interactions between colloidal particles and surfaces. In the present study, the AFM was used to study the interactions between each of two Pseudomonas aeruginosa strains with proteins. Topographical images and force cycles of bacterial cells and proteins were analyzed. Bovine serum albumin (BSA) and concanavalin A (Con A) were the model proteins chosen to represent protein molecules that might affect bacterial adhesion. In addition, the role of LPS structure in bacterial adhesion was investigated. The magnitude of adhesive forces for two P. aeruginosa stains was not statistically significant when they interact with silicon. Although it is not clear if the pull-off distances are accurate representatives of the absolute length of bacterial surface molecules, the trend indicates that the surface molecules of strain AK1401 are shorter than those of strain PAO1. The semi-rough strain AK1401 was more hydrophobic than the smooth strain PAO1, according to the water contact angle measurements. However, surface free energy components and zeta potential values were not significantly different for both strains. Zeta potential of bacterial cells decreased when they were suspended in HEPES/DTT buffer instead of ultrapure water. The AFM results demonstrate the importance of nano-scale interactions between proteins and bacterial cells. Our results show that the lipid A and core oligosaccharides are the most important molecules influencing the interactions of P. aeruginosa with protein molecules. The interactions of P. aeruginosa with model proteins in our study were weak. Therefore, the role of protein molecules may be inadequate for the purpose of enhancing subsurface delivery for bioremediation. Our results suggest that the semi-rough mutant, AK1401, can adhere to the protein receptors of the epithelial cells or protein coated implants stronger than the smooth strain, PAO1, and therefore can cause serious infections."

Microbial infections of medical implants occur in more than 2 million surgical cases each year in the United States alone. These increase patient morbidity and mortality, as well as patient cost and recovery time. Many treatments are available, but none are guaranteed to remove the infection. The purpose of this work is to examine the initial events in microbial adhesion by simulating the approach and contact between a planktonic cell, immobilized on an Atomic Force Microscope (AFM) cantilever, and a biomaterial or biofilm substrate. Distinct adhesive interactions exist between Candida parapsilosis and both unmodified silicone rubber and Pseudomonas aeruginosa biofilms. Using C. parapsilosis cells immobilized on AFM cantilevers with a silicone substrate, we have measured attractive interactions with magnitude of 2.3 ± 0.5 nN (SD) in the approach portion of the force cycle. On P. aeruginosa biofilms, the magnitude of the attractive force increases to 3.5 ± 0.75 nN (SD), and is preceded by a 2.5 nN repulsion at approximately 175 nm from the cell surface. This repulsion may be attributed to steric and electrostatic interactions between the two microbial polymer brushes. Young's moduli for microbes and biofilms were calculated using Hertzian contact models. These produced values of 0.21 ± 0.003 MPa (SD) for the C. parapsilosis-silicone rubber system, and 0.84 ± 0.015 MPa (SD) for the C. parapsilosis-biofilm system. This technique may be extended to calculate the work per unit contact area involved in the attractions in experimental data. For example, the work of adhesion using a spore probe is an order of magnitude greater for unmodified silicone rubber than for a P. aeruginosa biofilm. This indicates a high affinity for silicone rubber, and suggests that this material is vulnerable to infection by C. parapsilosis in vivo. We have also demonstrated that AFM force curve analysis using established qualitative and quantitative models fails to accurately represent the physical interactions taking place between the probe and sample for the case where a polymer brush exists on the substrate, the probe, or both. As such, an approximate method defining the sample surface as the actual surface plus some vertical dimension associated with the maximum compressible thickness of the polymer brush is discussed. Characterization of cell-biomaterial and cell-cell interactions allows for a quantitative evaluation of the materials used for medical implantation. It also provides a link between the physicochemical and physicomechanical properties of these materials and the nanoscale interactions leading to microbial colonization and infection. The goal of this research is to study this link and determine how best to exploit it to prevent microbial infections of medical implant materials.

"Bacteria during the course of their life undergo a lot of developments on their surface. The changes that occur inside a cell result in the production of a variety of biopolymers on the cell surface. These polysaccharides have been found to play a major role in deciding the adhesive or repulsive nature of a bacterial cell. Based on the application the adhesive nature of a cell sometimes needs to be manipulated such that bacteria are required to have higher adhesions for bioremediation applications and in the case of bioreactors bacteria must not stick to walls to avoid fouling. In order to control adhesions of a cell to a variety of substrates, knowledge of the polysaccharides present on its surface is needed. Therefore the goal of the present study is to detect the sugars present on the surface of Pseudomonas putida KT2442 using Atomic force microscopy and to relate properties of the polysaccharides to bacterial adhesion. Previous experiments suggested that cellulose and other sugars were produced by Pseudomonas putida KT2442. Thus the cells were grown to late exponential phase and treated with cellulase to degrade any cellulose, if present, on the surface of the cells. Control experiments were done on untreated cells and cells that were not treated with cellulase but were centrifuged, since centrifugation is a part of the cellulase treatment and may also affect the bacterial surface. An appropriate (Steric) fitting model for the atomic force microscope (AFM) approach curves was applied to calculate the height and density of the polymer brush layer present on the cell surface. There was a decrease in the density of the polymer brush and increase in the height of the brush upon treatment with cellulase. Centrifugation alone did not affect the approach curves. From looking at the retraction curves it verified the results got from the approach curves and indicated stretching out of the polymer brush to greater distances after the treatment with cellulase. Another batch of cells was treated with dextranase to check for the presence of dextran on the cell surface. Dextranase treated cells behaved identical to the control cells, suggesting that dextran is not one of the polysaccharides present on the bacterial surface. No change was observed in retraction curves data for dextranase treated and untreated cells."

Integrins are heterodimers that mediate cell adhesion in many physiological processes. Binding of integrins to ligands provides anchorage and signals for the cell. However, how force regulates integrin/ligand dissociation is unclear. Atomic force microscopy was used to measure the force dependence of lifetimes of single bonds between a FN fragment and integrin α₅β₁. First, lifetime-force relationships demonstrated that force prolonged bond lifetimes in the 10-30 pN range, a behavior called catch bonds. Changing divalent cations from Ca²⁺/Mg²⁺ to Mg²⁺/EGTA and to Mn²⁺ caused more pronounced catch bonds. A truncated α₅β₁ construct containing the headpiece but not the legs (trα₅β₁-Fc) formed much longer-lived catch bonds in the same force range. Bindings of two activating mAbs, 12G10 and TS2/16, left shift the catch bond and converted catch bonds to slip bonds, respectively. Catch bonds may provide a mechanical mechanism for the cell to regulate adhesion by applying different forces. Second, FNIII₇₋₁₀/α₅β₁-Fc/GG-7 bond was stretched to ~ 30 pN and then relaxed to ~ 7 pN at which the bond's lifetime was measured. The strong bond state induced by the 30 pN stretching stayed stable even after the force was reduced to 7 pN. In other words, lower the force would not weaken FNIII₇₋₁₀/α₅β₁-Fc bond once it had been stretched. Similar behaviors were observed for FNIII₇₋₁₀/trα₅β₁-Fc and FNIII₇₋₁₀/mα₅β₁interactions. In addition, the efficiency of the force to induce such a strong bond state for FNIII₇₋₁₀/α₅β₁-Fc interaction in 2 mM Mg²⁺/EGTA condition was characterized. The probability of force to induce the strong bond state increased as force increased and when the force reached 26 pN, all bonds were transit to the strong state. Moreover, reversible unbending of α₅β₁binding with FNIII₇₋₁₀ under mechanical force were observed, which proved that integrin bending and unbending was dynamic. Importantly, integrin could restore bent conformation even when engaged with its ligand, providing a mechanism for mechanotransduction. Third, structural changes of α₅β₁under force were observed. The structural changes did not change the trend of lifetime-force relationships of FNIII₇₋₁₀/α₅β₁/GG-7 bond. Moreover, the lifetime for the structural changes to occur and molecular length changes caused by them were characterized.

Atomic Force Microscopy (AFM) has become an indispensable metrology tool for nanoscale surface characterization. Today, research and industry demand faster and more accurate metrology and these demands must be met expediently. Traditional AFM cantilevers and associated actuators (i.e. piezoelectric) are limited in regards to actuation speed and resonance frequency presenting the user with an undesired trade-off of speed versus resolution. Based on a pre-existing technology known as the FIRAT (Force Sensing Integrated Readout and Active Tip) AFM probe, this work aims to remedy actuation and response issues by implementing a cantilever-on-cantilever probe as well as a novel seesaw probe. Both cases implement electrostatic actuation, eliminating the need for piezoelectrics while demonstrating large - micron scale - actuation and sensitive displacement detection. These new probe designs can potentially demonstrate a wide bandwidth frequency response (e.g. 100 kHz) ideal for high-speed video-rate imaging. Unlike traditional AFM cantilevers, this is realized by mechanically coupling two physically separate structures to provide a soft resonator sensor atop a stiff actuator structure. Common surface-micromachining techniques are utilized to solve the logistical challenge of fabricating these stacked structures. By manipulating the viscous damping and mechanical mode coupling it becomes feasible to attain the aforementioned desired dynamic characteristics.