Doctor of Philosophy, Case Western Reserve University, 2024, Pharmacology

Dynamin superfamily proteins (DSPs) are present in all organisms, mediating critical membrane remodeling events throughout the cell. Despite the decades of structural and functional studies across the superfamily, no structure has been determined of a DSP demonstrating the conformational changes required to transition from a cytosolic, solution state to a helical assembly. This gap in knowledge limits the field's understanding of the mechanisms required for regulation and functional assembly into membrane remodeling complexes. Dynamin-related protein 1 (Drp1) is the master regulator of outer membrane fission for mitochondria. Proper mitochondrial dynamics is essential for cellular health, and an imbalance in this cycle has been implicated in many diseases, from heart failure to prion-related neurodegeneration. Drp1 has been identified as a potential therapeutic target; however, the underlying mechanisms governing its regulation are largely unclear. Drp1 exists predominantly as a mixture of dimers and tetramers in solution, but the specific interactions that stabilize these solution forms and prevent the assembly of larger complexes are not known. Using cryo-EM, we have observed significant conformational rearrangements in native solution structures for both dimer and tetramer states when compared to existing DSP crystal structures. Additionally, we have identified a helical lattice which demonstrates unique GTPase domain assembly between adjacent filaments, providing insight into the mechanisms of constriction. Finally, we studied the impact of terminal tags on the structure and function of Drp1 and identified a newly appreciated intrinsically disordered region of regulation. Together, these observations provide insight into regulatory interactions that stabilize oligomer states and mediate the activation of assembly into a functional fission machinery.

Committee: Jason Mears (Advisor); Edward Yu (Committee Chair); Marcin Golczak (Committee Member); Rajesh Ramachandran (Committee Member); Beata Jastrzebska (Committee Member) Subjects: Biology; Biophysics; Molecular Biology; Pharmacology

Doctor of Philosophy, The Ohio State University, 2023, Biophysics

This dissertation explores the use of applying molecular dynamics (MD) and enhanced sampling methods towards understanding dynamics of the cardiac troponin complex (cTn) and the thermodynamic and functional consequences introduced by cardiomyopathic mutations. Chapter 2 explores an idea from the Davis group at Ohio State that cardiomyopathic mutations in the cTnI inhibitory peptide region (cTnIIP) can cause a reduced effective concentration of the cTnI switch peptide (cTnISP) to the cTnC hydrophobic patch region (cTnCHP). We utilized the Ca2+-unbound cTn structure produced by Yamada and colleagues to simulate both a normal cTn complex (tethered) and a model of the cTn complex with the cTnIIP removed and a free cTnISP (untethered) using molecular dynamics. Our results showed that the tether was essential in producing an effective concentration of cTnISP necessary for physiological function. We also observed that cardiomyopathic mutations did not significantly affect the effective concentration of cTnISP to the cTnCHP but did cause alterations to the dynamics and flexibility of the cTnIIP region. We observed in our simulations from chapter 2 that the cTnCHP never opened for any significant amount of time. Therefore, in the third chapter we sought to produce a trajectory of the transition event between the Ca2+-unbound and Ca2+-bound cTn forms by again using structures produced by Yamada. We successfully performed this using Targeted MD (TMD) and were able to observe a transition from a Ca2+-unbound, cTnCHP closed, cTnISP unbound form of cTn to a Ca2+-bound, cTnCHP open, cTnISP bound cTn. We then selected windows from the trajectory that correlated strongly with the cTnCHP opening and cTnISP binding transition events and performed umbrella sampling (US) simulations. Our results show near perfect replication of NMR studies on the cTnISP binding event and strong correlation with previous computational studies on the cTnCHP opening event. We then introduced mutations to (open full item for complete abstract)

Committee: Steffen Lindert (Advisor); Mark Ziolo (Committee Member); Marcos Sotomayor (Committee Member); Xiaolin Cheng (Committee Member) Subjects: Biophysics

Doctor of Philosophy, Case Western Reserve University, 2023, Chemistry

Positive Strand RNA (PSR) viruses, such as coronaviruses and enteroviruses, cause serious health and economic threats worldwide, as seen with the COVID-19 pandemic. This has drawn attention to the importance of identifying new antivirals and molecular targets in RNA viruses. The multifunctionality of PSR genomes make them desirable targets for therapeutic intervention. Here, we present a class of antivirals that can inhibit SARS-CoV-2 replication in vitro by targeting conserved viral RNA structures at the 5'-end. Specifically, stem loops (SLs) 1, 4, 5a, and 6 of the viral 5'-region have shown a degree of binding with dimethyl amiloride molecules as determined by NMR structural analysis. These results open the door to potentially develop specific small molecules against SARS-CoV-2 and related coronaviruses. Upon investigating SL6, interesting structural dynamics features were observed at the budge region when exposed to different temperatures. From various Nuclear Magnetic Resonance (NMR) and single angle x-ray scattering (SAXS) experiments, experimental restrains were obtained in order to generate a 3D structure of SL6 using molecular dynamics simulations. In SARS-CoV-2, stem-loop 3, which contains the transcriptional regulatory sequence, was proven to bind to the host Unwinding Protein 1 (UP1) using electrophoretic mobility shift assay (EMSA), isothermal titration chromatography (ITC), and NMR, which possibly suggests that UP1 participates in the mechanism of transcription of sub-genomic RNA. In addition, another PSR virus, Enterovirus A71 (EV-A71), which is the etiological agent of the hand, foot, and mouth disease, has caused severe morbidity and high mortality rates in children for decades. Thus, understanding the mechanisms by which EV-A71 replicates within the cellular environment can bring to light efficient drug targets for viral inhibition. The 5'-untranslated region (5'-UTR) of the RNA genome is the control hub of viral replication and transcription in EV- (open full item for complete abstract)

Committee: Blanton S. Tolbert (Advisor); Fu-Sen Liang (Committee Chair); Thomas Gerken (Committee Member); Robert Salomon (Committee Member); Divita Mathur (Committee Member) Subjects: Biochemistry; Biophysics; Chemistry; Virology

Doctor of Philosophy, Case Western Reserve University, 2022, EMC - Mechanical Engineering

Cellular processes strongly regulate the biophysical and biomechanical properties of each blood cell including density, adhesion, and motility, all of which can rapidly change during various healthy and pathological states. To have a better understanding of the cellular processes, an assessment of the biophysical and biomechanical signatures of single cells is needed. Microfluidics has the remarkable ability to mimic the physiological environment of microvasculature, providing a means to better characterize biomechanical and biophysical signatures of blood cells, such as adhesive interactions between different blood cells. As a result, microfluidics has found applications in studies of many different pathophysiologies that stem from altered biomechanical and biophysical properties of blood cells. Despite the advances in microfluidics for the characterization of the biophysical and biomechanical properties of blood cells, the use of microfluidics for mechanistic and mechanobiology studies remains limited due to a lack of standardization and single-cell level imaging. Microfluidics can harness image analysis for standardized characterization of fundamental properties of single cells and reveal subpopulations in heterogeneous cell populations to unfold the mechanobiology and mechanisms of physiology reliably. This dissertation presents the design and engineering of standardized microfluidic systems that utilize image analysis at various technical complexity levels (i.e., manual pixel-to-pixel distance measurements, and automated computer vision with and without machine learning). These microfluidic single-cell level imaging systems demonstrate effective multi-variate measurement of blood cells' biophysical and biomechanical properties and inform the investigation of microcirculatory cellular action mechanisms. The specific aims of this dissertation were: 1) To develop a microfluidic magnetic levitation platform that can perform size and density measurements of single r (open full item for complete abstract)

Committee: Umut Gurkan (Advisor); Bryan Schmidt (Committee Chair); Michael Hinczewski (Committee Member); Ozan Akkus (Committee Member) Subjects: Biomechanics; Biomedical Engineering; Biophysics

PhD, University of Cincinnati, 2022, Medicine: Molecular Genetics, Biochemistry, & Microbiology

Bone Morphogenetic Proteins (BMPs) are the largest subgroup of the Transforming Growth Factor ß (TGFß) superfamily, one of the fundamental protein signaling pathways in biology. BMPs are involved in regulating numerous biological functions, with a particular focus on development, immune modulation, cell homeostasis and wound healing. When dysregulated, aberrant BMP signaling can indue a host of developmental and autoimmune disorders, as well as many different cancers. Given the wide array of biological functions BMPs regulate, precise regulation of signaling is a key component of their biology. Mechanistically, these secreted dimeric signaling proteins function by forming complex with two type 1 and two type 2 serine/threonine kinase receptors on the cell surface to drive signaling by intracellular SMAD proteins. Accordingly, the regulation of these potent signaling molecules in the extracellular space is a vital area of study. The purpose of the work outlined in this thesis is to explore certain understudied mechanisms of extracellular modulation of BMP signaling. We particularly focused on studying these mechanisms not in isolation, but rather as they actually exist in nature, as part of a complex environment with many competing biomolecules. We present studies contrasting related protein antagonists with different function in an attempt to gain insight into the key components needed for BMP inhibition. In addition, we explore a newly discovered interaction between the BMP and Wnt signaling pathways, where BMP ligands may directly antagonize canonical Wnt signaling. Lastly, we describe a procedure for the production of artificial BMP heterodimeric signaling molecules using chains with differential activity with respect to receptor preference, antagonist targeting, and affinity to the extracellular matrix. These asymmetrical signaling molecules were then used to isolate the key components of biological function across multiple different experimental systems. Overal (open full item for complete abstract)

Committee: Thomas Thompson Ph.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member); Aaron Zorn Ph.D. (Committee Member); James Wells Ph.D. (Committee Member); Michael Tranter Ph.D. (Committee Member) Subjects: Biochemistry

Master of Science (MS), Ohio University, 2022, Physics and Astronomy (Arts and Sciences)

Neurofilaments are the most abundant structures in the axonal cytoskeleton. They are synthesized in the cell body then shipped across the axon in an active transport with average velocities that are amongst the slowest in the axon. Neurofilaments are known to be the main determinants of axonal caliber. Their accumulation in the axon serves a space-filling role that leads to its radial expansion. An increase in the number of neurofilaments in the cytoskeleton and a slowing in their transport along the axon are the two experimentally observed phenomena that correlated between axonal radial growth and neurofilaments. However, the exact mechanism by which neurofilaments accumulate remains unknown. Guided by existing morphometric analysis and experimental data correlating axon caliber with cytoskeletal content during axonal growth, a computational model was developed to simulate a growing axon by injecting an increasing influx of neurofilaments into the proximal end of the axon. In parallel, a study was modeled where neurofilaments assembled in the cell body were labeled with a radioactive marker so that their axonal transport along the nerve could be observed and the involvement of their slowing in the axon with its caliber growth was evaluated. Results indicate that both the increasing influx of neurofilaments from the cell body and the slowing of neurofilament transport both contribute to the growth of axonal caliber during development.

Committee: Peter Jung (Advisor); Alexander Neiman (Committee Member); David Tees (Committee Member) Subjects: Biology; Biophysics; Neurobiology; Neurosciences; Physics

Doctor of Philosophy, Case Western Reserve University, 2022, Chemistry

Enterovirus 71 (EV71), represents a persistent threat to global health and economies, as outbreaks are reported in the United States and globally each year. Infections are self-limited; however, prolonged infection in the immunocompromised can lead to severe neurological disorders and, eventually, death. As of the time of writing, there are no FDA-approved treatments against this pathogen. Thus, there is an immediate urgency to determine the mechanisms regulating host-virus interactions. EV71 utilizes a type I IRES element to initiate viral translation in a cap-independent pathway by recruiting multiple host proteins through a poorly understood mechanism. This thesis seeks to define the molecular and specificity determinants regulating the formation of IRES-protein complexes that modulate cap-independent translation. Herein, we studied the interactions of the negative translation modulator AUF1 with the conserved stem loop II (SLII) IRES domain. In chapter 2 we demonstrate that AUF1 and its isolated RRMs bind to the SLII bulge motif via a monophasic thermodynamic transition, where the bulk of the thermodynamic stability is conferred by the first RRM. Building on this knowledge, in chapter 3 we screened a library of RNA-targeting small molecules against the SLII IRES domain and found a potent inhibitor (DMA-135) of EV71 translation and replication. A combination of biophysical and functional studies revealed that DMA-135 functions by inducing a conformational change on SLII which stabilizes the formation of the repressive SLII:DMA-135:AUF1 complex. In chapter 4, we validated the proposed mechanism of action by generating (DMA-135)-EV71 resistant mutants, where the suppressor mutations mapped to SLII. Biophysical studies revealed that the suppressor mutations changed the local RNA structure around the SLII bulge which impaired DMA-135 and AUF1 binding. In chapter 5, we gathered the knowledge obtained in all previous chapters to delineate a pipeline for the identificat (open full item for complete abstract)

Committee: Blanton Tolbert (Advisor); Fu-Sen Liang (Committee Chair); Robert Salomon (Committee Member); Thomas Gerken (Committee Member); Shane Parker (Committee Member) Subjects: Biophysics; Chemistry; Virology

Doctor of Philosophy, Case Western Reserve University, 2022, Physics

In the last two decades, advances in experimental techniques have opened up new vistas for understanding bio-molecules and their complex networks of interactions in the cell. In this thesis, we use theoretical modeling and machine learning to explore two surprising aspects that have been revealed by recent experiments: (i) the discovery that many different types of cellular signaling networks, in both prokaryotes and eukaryotes, can transmit at most 1 to 3 bits of information; (ii) the observation that single bio-molecules can exhibit multiple, stable conformational states with extremely heterogeneous functional properties. The first part of the thesis investigates how the energetic costs of signaling in biological networks constrain the amount of information that can be transferred through them. The focus is specifically on the kinase-phosphatase enzymatic network, one of the basic elements of cellular signaling pathways. We find a remarkably simple analytical relationship for the minimum rate of ATP consumption necessary to achieve a certain signal fidelity across a range of frequencies. This defines a fundamental performance limit for such enzymatic systems, and we find evidence that a component of the yeast osmotic shock pathway may be close to this optimality line. By quantifying the evolutionary pressures that operate on these networks, we argue that this is not a coincidence: natural selection is capable of pushing signaling systems toward optimality, particularly in unicellular organisms. Our theoretical framework is directly verifiable using existing experimental techniques, and predicts that many more examples of such optimality should exist in nature. In the second part of the thesis, we develop two machine learning methods to analyze data from single-molecule AFM pulling experiments: a supervised (deep learning) and an unsupervised (non-parametric Bayesian) algorithm. These experiments involve applying an increasing force on a bio-molecul (open full item for complete abstract)

Committee: Michael Hinczewski (Committee Chair); Peter Thomas (Committee Member); Harsh Mathur (Committee Member); Lydia Kisley (Committee Member) Subjects: Biophysics; Physics

Doctor of Philosophy, The Ohio State University, 2021, Biophysics

A defining characteristic of complex life is the capacity to integrate a variety of stimuli from the environment and, through the interaction of a dizzying array of biomolecules, to properly interpret and respond to such stimuli. Gene duplications and random mutations, when filtered through the sieve of natural selection, provides living organisms with the creative power needed to produce the suite of highly specialized molecules required to perform the vital task of sensing environmental cues. From maintaining balance by harnessing the power of gravity in vertebrates, to the detection of a dangerous shift from diffusive equilibrium in yeast cells, proteins play a vital role in detecting changes in the environment of all living organisms. Central to the function of many of these proteins is the transmission or detection of mechanical forces applied to the proteins themselves, or to surrounding cellular structures and tissues in which they are embedded. Such force sensitive proteins are thus ideal subjects of single-molecule, quantitative biophysical approaches to better understand the molecular basis of force transduction. Examples of such proteins are cadherin-23 (CDH23) and protocadherin-15 (PCDH15), two proteins required for vertebrate hearing; desmoglein (DSG) and desmocollin (DSC), two proteins that are vital for maintaining tissue integrity in the presence of constant mechanical stress; and transient receptor potential yeast 1 (TRPY1), a mechanically-sensitive ion channel in yeast that restores osmotic balance in response to hyperosmotic shock. While these proteins are involved in unrelated biological processes, they all have evolved the ability to detect and respond to force generated by environmental sources, and their proper function is vital to the survival of the host organism. Here, a multidisciplinary approach is used to provide fundamental insights into the structure, mechanical properties, and dynamics of these specific proteins, as well as into pro (open full item for complete abstract)

Committee: Marcos Sotomayor Dr. (Advisor); Steffen Lindert Dr. (Committee Member); Rafael Brüschweiler Dr. (Committee Member); Charles Bell Dr. (Committee Member) Subjects: Biochemistry; Biophysics

Doctor of Philosophy, The Ohio State University, 2021, Biochemistry Program, Ohio State

Development and functionality of multicellular organisms relies on precise and strong adhesion between cells. Members of the cadherin superfamily of proteins are involved in calcium-dependent cell-cell adhesion in animals and have been shown to play vital roles in various relevant biological processes. The cadherin superfamily can be largely classified into three subfamilies: the classical cadherins, the non-clustered protocadherins, and the clustered protocadherins. The typical cadherin protein consists of a single-pass transmembrane domain, a cytoplasmic domain, and multiple non-identical extracellular cadherin (EC) repeats. These ECs are defined by their Greek-key fold and an EC linker region with highly conserved calcium-binding sites. The binding of calcium helps to provide the rigidity necessary for proper protein-protein interaction. Within this work I focus on cadherins responsible for mechanotransduction, both from the classical and non-clustered subfamilies. Adherens junctions are formed by classical members of the cadherin superfamily and provide strong adhesion between cells. Past experiments have determined that interactions between individual cadherins are weak and therefore the strength provided by epithelial cadherin (CDH1), the major cadherin of adherens junctions, must come about through the formation of cadherin complexes. These cadherin complexes are composed of multiple CDH1 molecules binding through trans- (ectodomains originating from adjacent cells) and cis-interactions (ectodomains originating from the same cell) as seen in x-ray crystal structures and cryo-electron tomography images. While most experiments have focused on single homodimers, the mechanical unbinding events of cadherin junctional complexes and their effect on the membrane and associated cytoplasmic proteins remains unexplored. My work on CDH1 junctional complexes and their associated proteins utilizes large-scale all-atom molecular dynamics (MD) simulations to probe the ad (open full item for complete abstract)

Committee: Marcos Sotomayor (Advisor); Mark Foster (Committee Member); Steffen Lindert (Committee Member); Charles Bell (Committee Member) Subjects: Biochemistry; Biomechanics; Biophysics

Doctor of Philosophy, The Ohio State University, 2021, Physics

The ability to apply and measure high forces (≥10pN) on the nanometer scale is critical to the ongoing development of nanomedicine, molecular robotics, and the understanding of biological processes such as chromatin condensation, membrane deformation, and molecular motors [1] [2] [3]. Current force spectroscopy techniques rely on micron-sized handles to apply forces, which can limit applications within nanofluidic devices or cellular environments [4]. To overcome these limitations, I used deoxyribonucleic (DNA) origami to self-assemble a nanocaliper, building on previous designs[5] [6]. I characterize the nanocaliper via a short double-stranded (ds)DNA with each strand attached to opposite arms of the device, via device equilibrium state, output force, and dynamics, to understand the effects of sequence, vertex design, and strut length on the device properties. I also produce nucleosomes, hexasomes, and an alternate dsDNA, which were then measured in the device, yielding mechanistic insight into the free energy landscape of each. I measure forces greater than 20 pN applied by the device with a nanometer dynamic range and 1 to 10 pN/nm stiffness. These high performing characteristics which expand the capabilities of existing force spectroscopy techniques as well as those of DNA origami devices.

Committee: Michael Poirier (Advisor); Ralf Bundschuh (Committee Member); Carlos Castro (Committee Member); Ezekiel Johnston-Halperin (Committee Member) Subjects: Biochemistry; Biophysics; Nanoscience; Physics

Doctor of Philosophy in Engineering, Cleveland State University, 2019, Washkewicz College of Engineering

Development of nervous system has been greatly explored in the framework of genetics, biochemistry and molecular biology. With the growing evidence that mechanobiology plays a crucial role in morphogenesis, current studies are geared towards understanding the role of mechanical cues in nervous system development and progression of neurological disorders. Formation, maturation and differentiation of various development related cells are sensitive to extrinsic and intrinsic perturbations. Based on this hypothesis, the objective of this study was to investigate the effects of environmental toxicants, mutations in molecular clutch proteins, and matrix stiffness cues on the biophysical, biomechanical, and phenotypic changes in brain-derived neural progenitor cells (NPCs) and microglia. In the first aim, we established the utility of biophysical and biomechanical properties of NPCs as indicators of developmental neurotoxicity. Significant compromise (p < 0.001) in NPC mechanical properties was observed with increase in concentration (p < 0.001) and exposure duration (p < 0.001) of four distinct classes of toxic compounds. We propose the utility of mechanical characteristics as a crucial maker of developmental neurotoxicity (mechanotoxicology). In the second aim, we elucidated the critical role of molecular clutch proteins, specifically that of kindlin-3 (K3) in murine brain-derived microglia, on the cell membrane mechanics and physical characteristics. Using genetic knockouts of K3 and AFM analysis, we established the role of K3 in regulating microglia membrane mechanics.Mutation at the K3-β1 integrin binding site revealed that the connection serves as the major contributor of membrane to cortex attachment (MCA). Finally, in aim 3, we identified the molecular mechanisms (non-muscle myosin II) by which NPCs transduce mechanical input from external substrate into fate decisions such as differentiation and phenotype. We established cell mechanics as a label-free marker of d (open full item for complete abstract)

Committee: Chandra Kothapalli (Advisor); Moo-Yeal Lee (Committee Member); Nolan Holland (Committee Member); Xue-Long Sun (Committee Member); Parthasarathy Srinivasan (Committee Member) Subjects: Biomechanics; Biomedical Engineering; Biomedical Research; Biophysics; Engineering; Materials Science; Mechanics; Neurosciences

Doctor of Philosophy, The Ohio State University, 0, Physics

Magnetotactic bacteria (MTB) are a group of motile prokaryotes that synthesize chains of lipid-bound, magnetic nano-particles called magnetosomes, which provide each cell with a magnetic moment. This work exploits their innate magnetism to perform novel experiments in three principal areas i) bacterial hydrodynamics at surfaces, ii) measuring the power output of the bacterial flagellar motor and iii) as a model system for exploring the non-equilibrium, many-body behavior of active matter. In each of these cases, the foundation of the method is based on the interplay of the magnetic and hydrodynamic forces acting on the cell. Through use of external magnetic fields and micro-magnetic patterns, the relative strength of hydrodynamic and magnetic forces is tuned through magnetic control of the bacteria's orientation relative to a surface. The resultant swimming behaviors provide a means of experimentally determining hydrodynamic parameters which are relevant to the physical and behavioral dynamics of a broad range of analogous microswimmers. Furthermore, magnetic manipulation allows a high degree of control over the positions of large numbers of cells, by uncovering dynamic states in which the orientation of the external field leads to systematic differentiation of microscopic swimming paths. Many microorganisms swim by rotating helical filaments, called flagella. Determining the physical properties of this propulsive system is hence crucial to understanding the overall behavior of these organisms. Additionally, the ability to dynamically monitor the activity of the flagellar motor provides a valuable indicator of the cell metabolism. By confining and selectively releasing MTB from micromagnetic traps the flagellar thrust force and swimming speed may be directly measured. The technique permits determination of the ratio of propulsive force to swimming speed (the hydrodynamic resistance) as well as the power output of the flagellar motor for individual cells, over (open full item for complete abstract)

Committee: Ratnasingham Sooryakumar Dr. (Advisor) Subjects: Biomechanics; Biophysics; Condensed Matter Physics; Physics

Bachelor of Science (BS), Ohio University, 2019, Engineering Physics

In this thesis, I probed mechanical properties of MDA-MB-231 human breast cancer cells. To do this, I optimized the fabrication process of microfluidic devices using soft lithography methods. The flowrate inside these devices was found to be linear with respect to change in pressure. The same procedure was then used on the MDA-MB-231 cells to find entry time data. Using entry time data and change in pressure data, power law rheology was used to calculate the cell fluidity of the breast cancer cells.  Cell fluidity was found to be 0.21+/-0.02, which is well inside the realm of reasonable values for fluidity. Using this value and the intercept of the log-log plot of the power law rheology, the elastic modulus of MDA-MB-231 was calculated to be 278+/-6 Pa. This also agrees with most values of the elastic modulus found in the literature. These microfluidic devices in tandem with power law rheology have proven to be an effective way of calculating both cell fluidity and elastic modulus.

Committee: David Tees PhD (Advisor) Subjects: Biophysics; Physics

Bachelor of Science (BS), Ohio University, 2019, Engineering Physics

Neurofilaments (NFs) are a major component of the cytoskeleton of the axons of neurons and they contribute to the establishment of the axon's cross-sectional area. NFs are also a cargo of slow transport along microtubules (MTs) thus relating NF movement with axon caliber. Studies in rats have reported that the NF transport velocity slows significantly and continuously prior to and after sexual maturity (approximately 4 to 5 weeks in rats) while NF slowing continues past 12 weeks of age. Direct experimentation has given insight into the kinetics of NF transport, but the exact mechanism by which NFs transport is regulated leading to the reported slowing remains unknown. Computational modeling aids in the exploration of possible mechanisms of regulation of NF transport and evaluation of possible contributions of NF transport velocity to changes in axon diameter. Our hypothesis is that growth of the axonal cross-sectional area requires a combination of change of NF kinetics, associated with a change of the densities of NFs and MTs, and an increase in their abundance through increased NF assembly rate in the cell body. This research aims to develop models which would provide new insights into the mechanism of axon growth in normal development, which could help researchers understand the mechanisms of abnormal axon swelling in specific neurodegenerative diseases. The primary conclusions of this research were that the velocity obtained from radio-labeled NF pulse experiments may not be representative of the average velocity and therefore the reported NF slowing on a timescale of days and weeks may have been mainly due to an incorrect interpretation of the outcomes of radio-pulse-labeling experiments.

Committee: Peter Jung PhD (Advisor) Subjects: Biology; Biophysics; Neurosciences; Physics

PhD, University of Cincinnati, 2019, Arts and Sciences: Chemistry

Noble metal nanoparticles are excellent candidates for sensing and biophysical characterization of lipid membranes and proteins, as they offer high sensitivity and are label-free. Noble metal nanoparticles facilitate sensing predominantly through two-mechanisms: the localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering (SERS). Both methods are robust well-developed sensing methods that require the analyte to be positioned at or near the metal nanoparticle surface. The interaction of biomolecules such as protein and lipid bilayers with metal surfaces, however, is often perturbative and can alter the structure and function of biomolecules. Thus, for biomolecular sensing with metal nanoparticles it is essential to develop substrates that impart biocompatibility while maintaining sensitivity. This work reports on the development of substrates composed of gold or silver nanoparticles that are passivated with ultrathin silica films to enable the biocompatible measurement of supported lipid bilayers and towards the biocompatible measurement of protein. A silica sol-gel method was developed to prepare thin films over nanodisks for the quantitation of cholera toxin binding to gm1 containing lipid bilayers. A modified sol-gel procedure was used to coat silver film over nanosphere substrates with silica to enable SERS measurement of supported lipid bilayer formation and lipid exchange. In addition to silica coating strategies a liposome-based nanoparticle-on-mirror substrate was developed to measure dye molecules contained within a liposome using SERS, which may be useful for the biocompatible measurement of single protein molecules by SERS. Finally, liposomes coated with metal nanoparticles and metal nanoshells were developed for their potential use as dynamic SERS biosensors.

Committee: Laura Sagle Ph.D. (Committee Chair); Michael Baldwin Ph.D. (Committee Member); Patrick Limbach Ph.D. (Committee Member) Subjects: Chemistry

Doctor of Philosophy, The Ohio State University, 2018, Physics

Individual cells decipher and react to both their chemical and mechanical environment. Clathrin-mediated endocytosis (CME) is a major process by which cells internalize macromolecules. The triskelion-shaped clathrin protein assembles on the membrane as a spherical lattice enveloping the membrane until scission begets internalization. The membrane curvature generated by the invaginations during endocytosis associate CME with the mechanical environment of the cell. Fluorescence microscopy is used to study the dynamics of CME, and in particular to discern the time it takes for CME events to complete (i.e. their lifetime). It is our hypothesis that the lifetime of CME events relates inversely to the cell membrane tension. We will support this hypothesis with live-cell imaging on glass substrates and in living organisms. We suggest a new methodology for studying CME dynamics that enables higher spatial and temporal resolution than lifetime analysis. We will also characterize the tension response of CME by using various cell manipulation techniques. In addition, we will demonstrate the ability of CME dynamics to predict cell movement and relate gradients in clathrin coat growth rates to previously established tension gradients in cultured cells and living organisms. Finally, we will demonstrate that this process is in a state of continual curvature generation.

Committee: Comert Kural (Advisor); Ralph Bundschuh (Committee Member); Michael Poirier (Committee Member); Sooryakumar Ratnasingham (Committee Member) Subjects: Biophysics; Physics

Doctor of Philosophy, The Ohio State University, 2018, Physics

We investigate aspects of RNA secondary structure from the view point of theoretical biophysics and from view point of bioinformatics. From the existence of a novel thermodynamic phase transition to the fundamental mechanisms of life, RNA continues to act as wellspring of new discoveries. RNA forms elaborate secondary structures through intramolecular base pairing. These structures perform critical biological functions within each cell. Due to the availability of a polynomic algorithm to calculate the partition function, they are also a suitable model system for the statistical physics of disordered systems. In this model, below the denaturation temperature random RNA secondary structures can exist in one of two phases: a strongly disordered, low-temperature glass phase and a weakly disordered, high-temperature molten phase. The probability of two bases pairing in these phases has been shown to decay with the distance between the two bases with an exponent 3/2 and 4/3 in the molten and glass phases, respectively. Drawing on previous results from a renormalized field theory of the glass transition, we numerically study this transition and introduce two order parameters that determines the location of the critical point, and explore the driving mechanism behind this transition. Within a cell's genome regulatory elements can often be found within the vicinity of the genes they regulate. In prokaryotes, a common translational regulatory element, the Shine Dalgarno sequence, has been found to be largely absent from entire phyla of bacteria. This sequence element is part of the textbook model of translation initiation. To understand how Shine Dalgarno independent bacteria, such as F. johnsoniae, a member of the phylum Bacteroidetes, initiates translation, we used high-thoughput RNA sequencing and ribosome profiling data to investigate the impact of mRNA secondary structure near a gene's initiation site. We found evidence that strongly implicates the role that unst (open full item for complete abstract)

Committee: Ralf Bundschuh (Advisor) Subjects: Bioinformatics; Biophysics

Doctor of Philosophy, The Ohio State University, 2017, Biophysics

Magnetic tweezers provides a versatile toolkit supporting the mechanistic investigation of DNA substrates and protein association. First, the fundamental concepts of magnetic force are explained in detail. A custom magnetic tweezers system is assembled and optimized using optical components. A DNA substrate capable of interacting with a surface and magnetic particle is constructed from smaller DNA pieces. A flow cell for single molecule experiments is diagramed, including functionalization of the flow cell surface. Finally, tests are performed measuring the lateral and axial resolution limits of the system. Retroviruses must integrate their linear viral cDNA into the host genome for a productive infection. Integration is catalyzed by the retrovirus-encoded integrase (IN), which forms a tetramer complex with the two viral cDNA ends (intasome), removes two 3'-nucleotides and catalyzes end-joining (strand transfer) of the recessed 3'-hydroxyls separated by 4-6 bp of the target DNA. Structures of prototype foamy virus (PFV) integrase tetramers suggest the two strand transfer events may not be coordinated. Here we have used multiple single molecule imaging systems to determine that the Prototype Foamy Virus (PFV) intasome searches for a target site by 1-dimensional (1D) rotational diffusion while in continuous contact with the target DNA. It then catalyzes the two-step strand transfer in 470 ms. The vast majority of PFV intasome search events on a target DNA were non-productive. These observations suggest that target site-selection is rate limiting during retroviral integration, identifying a separate IN function that might be pursued for therapeutic intervention. Eukaryotic telomeres consist of tandem repeats containing 3-4 guanine nucleotides (G-strand) that commonly terminate in a 100-1000 nt single stranded DNA (ssDNA) 3'-tail. Four adjacent G-strand ssDNA repeats characteristically fold into flattened 0.63 nm x 4.1 nm parallel G-quadruplex disc-like st (open full item for complete abstract)

Committee: Richard Fishel (Advisor); Ralf Bundschuh (Committee Member); Marcos Sotomayor (Committee Member); Charles Bell (Committee Member) Subjects: Biophysics

Doctor of Philosophy, The Ohio State University, 2017, Molecular, Cellular and Developmental Biology

Selective cell-to-cell adhesion is essential for normal development of the vertebrate brain, contributing to coordinated cell movements, regional partitioning and synapse formation. Members of the cadherin superfamily mediate calcium-dependent cell adhesion, and selective adhesion by various family members is thought to contribute to the development of neural circuitry. Members of the δ-protocadherin subfamily of cadherins are differentially expressed in the vertebrate nervous system and have been implicated in a range of neurodevelopmental disorders: schizophrenia, mental retardation, and epilepsy. However, little is known about how the δ-protocadherins contribute to the development of the nervous system, nor how this development is disrupted in the disease state. Here I focus on one member of the δ-protocadherin family, protocadherin-19 (pcdh19), since it has the clearest link to a neurodevelopmental disease, being the second most clinically relevant gene in epilepsy. Using pcdh19 transgenic zebrafish, we observed columnar modules of pcdh19-expresing cells in the optic tectum. In the absence of Pcdh19, the columnar organization is disrupted and visually guided behaviors are impaired. Furthermore, similar columns were observed in pcdh1a transgenic zebrafish, located both in the tectum and in other brain regions. This suggests protocadherin defined columns may be a theme of neural development. Our X-ray crystal structure of Pcdh19 reveals the adhesion interface for Pcdh19 and infers the molecular consequences of epilepsy causing mutations. We found several epilepsy causing mutations were located at the interface and disrupted adhesion, which further validated the interface and revealed a possible biochemical cause of Pcdh19 dysfunction. Furthermore, sequence alignments of other δ-protocadherins with Pcdh19 suggest that this interface may be relevant to the entire δ-protocadherin subfamily. We used the information gained about Pcdh19 to design PCDH19-FE mut (open full item for complete abstract)

Committee: James Jontes (Advisor); Marcos Sotomayor (Advisor); Heithem El-Hodiri (Committee Member); Sharon Amacher (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Biophysics; Cellular Biology; Developmental Biology; Molecular Biology; Neurosciences