Theses and Dissertations from UMD
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Item REGULATING GENE EXPRESSION: THE ROLE OF TRANSCRIPTION FACTOR DYNAMICS(2023) Wagh, Kaustubh; Upadhyaya, Arpita; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The genetic information encoded within our DNA is converted into RNA in a process called transcription. This is a tightly regulated process where multiple proteins act in concert to activate appropriate gene expression programs. Transcription factors (TFs) are key players in this process, with TF binding being the first step in the assembly of the transcriptional machinery. TFs are sequence-specific DNA binding proteins that bind specific motifs within chromatin. How TFs navigate the complex nuclear microenvironment to rapidly find their target sites remains poorly understood. Technological advances over the past 20 years have enabled us to follow single TF molecules within live cells as they interact with chromatin. Most TFs have been shown to exhibit power law distributed residence times, which arise from the broad distribution of binding affinities within the nucleus. This blurs the line between specific and non-specific binding and renders it impossible to distinguish between different binding modes based on residence times alone. In this dissertation, I combine single molecule tracking (SMT) with statistical algorithms to identify two distinct low-mobility states for chromatin (histone H2B) and bound transcriptional regulators within the nucleus. On our timescales, the TF mobility states represent the mobility of the piece of chromatin that they are bound to. Ligand activation results in a dramatic increase in the proportion of steroid receptors in the lowest mobility state. Mutational analysis revealed that only chromatin interactions in the lowest mobility state require an intact DNA-binding domain as well as oligomerization domains. Importantly, these states are not spatially separated as previously believed but in fact, individual H2B and chromatin-bound TF molecules can dynamically switch between them. Single molecules presenting different mobilities exhibit different residence time distributions, suggesting that the mobility of a TF is intimately coupled with their temporal dynamics. This provides a way to identify different binding modes that cannot be detected by measuring residence times alone. Together, these results identify two unique and distinct low-mobility states of chromatin that appear to represent common pathways for transcription activation in mammalian cells. Next, I demonstrate how SMT can complement genome wide assays to paint a complete picture of gene regulation by TFs using two case studies: corticosteroid signaling and endocrine therapy resistance in breast cancer. Finally, I conclude with a roadmap for future work on examining the role of mechanical cues within the cellular microenvironment (such as stiffness and topography) in regulating TF dynamics and gene expression.Item Three-Dimensional Characterization and Modulation of Corneal Biomechanics via Brillouin Microscopy(2021) Webb, Joshua Norman; Scarcelli, Giuiano; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Corneal mechanical properties are needed for diagnosing and monitoring the progression of ocular disorders such as keratoconus, screening for refractive surgeries, and evaluating treatment procedures including corneal cross-linking. Alterations of these mechanical properties are often localized to a specific area within the cornea. However, there exists a clinical gap of measuring local mechanical properties as current methods are contact-based and provide global measurements. The goal of this dissertation is to close this gap by establishing a three-dimensional, noninvasive characterization method of corneal biomechanics. Previously, our laboratory developed Brillouin microscopy as an imaging modality which can noninvasively extract mechanical measurements of a material. Here, using Brillouin microscopy, we characterized the stiffening effects of accelerated and localized cross-linking procedures with three-dimensional resolution. However, existing procedures to extract elastic modulus information from Brillouin measurements rely on empirical calibrations because a fundamental understanding between the two had not yet been established. In practice, this limits Brillouin measurements to relative softening / stiffening information, which, while useful to compare protocol efficacies, are not optimal for modeling long-term shape behavior of the cornea in clinical settings. Here, we address this shortcoming of Brillouin microscopy. First, we identified that both Brillouin-derived mechanical modulus and traditional elastic modulus are dependent on two major biophysical factors: hydration and the mechanical properties of the solid matrix. We derived and experimentally verified a quantitative relationship to describe the distinct moduli dependencies of such factors. Based on these relationships, we derived a procedure to extract the elastic modulus of the cornea from experimental measurements of Brillouin frequency shift and hydration, two clinically available parameters. Thus, the work presented here establishes a spatially resolved, noninvasive method for measuring corneal elastic modulus.Item DISSECTING THE GENE REGULATORY FUNCTION OF THE MYC ONCOGENE WITH SINGLE-MOLECULE IMAGING(2020) Patange, Simona; Larson, Daniel R; Girvan, Michelle; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The MYC oncogene contributes to an estimated 100,000 cancer-related deaths annually in the United States and is associated with aggressive tumor progression and poor clinical outcome. MYC is a nuclear transcription factor that regulates a myriad of cellular activities and has direct interactions with hundreds of proteins, which has made a unified understanding of its function historically difficult. In recent years, several groups have put forth a new hypothesis that questions the prevailing view of MYC as a gene-specific transcription factor and instead envision it as a global amplifier of gene expression. Instead of being an on/off switch for transcription, MYC is proposed to act as a `volume knob' to amplify and sustain the active gene expression program in a cell. The scope of the amplifier model remains controversial in part because studies of MYC largely consist of cell population-based measurements obtained at fixed timepoints, which makes distinguishing direct from indirect consequences on gene expression difficult. A high-temporal, high-spatial precision viewpoint of how MYC acts in single living cells does not exist. To evaluate the competing hypotheses of MYC function, we developed a single-cell assay for precisely controlling MYC and interrogating the effects on transcription in living cells. We engineered `Pi-MYC,' an optogenetic variant of MYC that is biologically active, can be visualized under the microscope, and can be controlled with light. We combined Pi-MYC with single-molecule imaging methods to obtain the first real-time observations of how MYC affects RNA production and transcription factor mobility in single cells. We show that MYC increases the duration of active periods of genes population-wide, and globally affects the binding dynamics of core transcription factors involved in RNA Polymerase II transcription complex assembly and productive elongation. These findings provide living, single-cell evidence of MYC as a global amplifier of gene expression, and suggests the mechanism is by stabilizing the active period of a gene through interactions with core transcription machinery.Item Microscopy of elongated superfluids(2020) Salces Carcoba, Francisco; Spielman, Ian; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This thesis covers three experiments with cold and ultracold (Bose-Einstein condensate based) alkali Rb-87 gases for quantum simulation. In the first experiment, we quantum simulate Abelian and non-Abelian gauge fields in the parameter space of a four-level quantum system. Then, we describe the experimental framework to perform optimal in-situ microscopy of elongated quantum gases. We then study the thermodynamics of individual one-dimensional Bose gases using in-situ resonant absorption imaging. Finally, we combine holographic microscopy and impulse correlations to digitally enhance our absorption images.Item REAL-TIME INVESTIGATION OF INDIVIDUAL SILICON NANOSTRUCTURED ELECTRODES FOR LITHIUM-ION BATTERIES(2013) Karki, Khim Bahadur; Cumings, John; Material Science and Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Silicon-based anode materials are an attractive candidate to replace today's widely-utilized graphitic electrodes for lithium-ion batteries because of their high gravimetric energy density (3572 mAh/g vs. 372 mAh/g for carbon) and relatively low working potential (~ 0.5V vs. Li/Li+). However, their commercial realization is still far away because of the structural instabilities associated with huge volume changes of ~300% during charge-discharge cycles. Recently, it has been proposed that silicon nanowires and other related one-dimensional nanostructures could be used as lithium storage materials with greatly enhanced storage capacities over that for graphite in the next generation of lithium-ion batteries. However, the studies to date have shown that the nanomaterials, while better, are still not good enough to withstand a large number of lithiation cycles, and moreover, there is little fundamental insight into the science of the improvements or the steps remaining before widespread adoption. This dissertation seeks to understand the basic structural properties and reaction kinetics of one dimensional silicon nanomaterials, including Si-C heterostructures during electrochemical lithiation/delithiation using in-situ transmission electron microscopy (TEM). I present my work in three parts. In part I, I lay out the importance of lithium-ion batteries and silicon-based anodes, followed by experimental techniques using in-situ TEM. In part II, I present results studied on three different nanostructures: Si nanowires (SiNWs), Si-C heterostructures and Si nanotubes (SiNTs). In SiNWs, we report an unexpected two-phase transformation and anisotropic volume expansion during lithiation. We also report an electrochemically-induced weld of ~200 MPa at the Si-Si interface. Next, studies on CNT@α-Si heterostructures with uniform and beaded-string structures with chemically tailored carbon-silicon interfaces are presented. In-situ TEM studies reveal that beaded-string CNT@ α-Si structures can accommodate massive volume changes during lithiation and delithiation without appreciable mechanical failure. Finally, results on lithiation-induced volume clamping effect of SiNTs with and without functional Ni coatings are discussed. In Part III, a conclusion and a brief outlook of the future work are outlined. The findings presented in this dissertation can thus provide important new insights in the design of high performance Si electrodes, laying a foundation for next-generation lithium ion batteries.Item A SCANNING SQUID MICROSCOPE FOR IMAGING HIGH-FREQUENCY MAGNETIC FIELDS(2009) Vlahacos, Constantine Peter; Wellstood, Frederick C.; Electrical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This thesis examines the design and operation of a large-bandwidth scanning SQUID microscope for spatially imaging high frequency magnetic fields. Towards this end, I present results on a cryo-cooled 4.2 K scanning SQUID microscope with a bandwidth of dc to 2 GHz and a sensitivity of about 52.4 nT per sample. By using a thin-film hysteretic Nb dc-SQUID and a pulsed sampling technique, rather than a non-hysteretic SQUID and a flux-locked loop, the bandwidth limitation of existing scanning SQUID microscopes is overcome. The microscope allows for non-contact images of time-varying magnetic field to be taken of room-temperature samples with time steps down to 50 ps and spatial resolution ultimately limited by the size of the SQUID to about 10 micrometers. The new readout scheme involves repeatedly pulsing the bias current to the dc SQUID while the voltage across the SQUID is monitored. Using a fixed pulse amplitude and applying a fixed dc magnetic flux allows the SQUID to measure the applied magnetic flux with a sampling time set by the pulse length of about 400 ps. To demonstrate the capabilities of the microscope, I imaged magnetic fields from 0 Hz (static fields) up to 4 GHz. Samples included a magnetic loop, microstrip transmission lines, and microstrip lines with a break in order to identify and isolate electrical opens in circuits. Finally, I discuss the operation and modeling of the SQUID and how to further increase the bandwidth of the microscope to allow bandwidth of upwards of 10 GHz.Item Measurements of Doping-Dependent Microwave Nonlinearities in High-Temperature Superconductors(2004-04-26) Lee, Sheng-Chiang; Anlage, Steven M.; PhysicsI first present the design and use of a near-field permeability imaging microwave microscope to measure local permeability and ferromagnetic resonant fields. This microscope is then modified as a near-field nonlinear microwave microscope to quantitatively measure the local nonlinearities in high-Tc superconductor thin films of YBa2Cu3O7-d (YBCO). The system consists of a coaxial loop probe magnetically coupling to the sample, a microwave source, some low- and high-pass filters for selecting signals at desired frequencies, two microwave amplifiers for amplification of desired signals, and a spectrum analyzer for detection of the signals. When microwave signals are locally applied to the superconducting thin film through the loop probe, nonlinear electromagnetic response appearing as higher harmonic generation is created due to the presence of nonlinear mechanisms in the sample. It is expected that the time-reversal symmetric (TRS) nonlinearities contribute only to even order harmonics, while the time-reversal symmetry breaking (TRSB) nonlinearities contribute to all harmonics. The response is sensed by the loop probe, and measured by the spectrum analyzer. No resonant technique is used in this system so that we can measure the second and third harmonic generation simultaneously. The spatial resolution of the microscope is limited by the size of the loop probe, which is about 500 mm diameter. The probe size can be reduced to ~ 15 mm diameter, to improve the spatial resolution. To quantitatively address the nonlinearities, I introduce scaling current densities JNL(T) and JNL'(T), which measure the suppression of the super-fluid density as , where J is the applied current density. I extract JNL(T) and JNL'(T) from my measurements of harmonic generation on YBCO bi-crystal grain boundaries, and a set of variously under-doped YBCO thin films. The former is a well-known nonlinear source which is expected to produce both second and third harmonics. Work on this sample demonstrates the ability of the microscope to measure local nonlinearities. The latter is proposed to present doping dependent TRS and TRSB nonlinearities, and I use my nonlinear microwave microscope to measure the doping dependence of these nonlinearities.