Physics

Permanent URI for this communityhttp://hdl.handle.net/1903/2269

Browse

Subject: search.filters.subject.Microscopy

Search Results

Now showing 1 - 4 of 4
  • Thumbnail Image
    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.
  • Thumbnail Image
    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.
  • Thumbnail Image
    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.
  • Thumbnail Image
    Item
    Measurements of Doping-Dependent Microwave Nonlinearities in High-Temperature Superconductors
    (2004-04-26) Lee, Sheng-Chiang; Anlage, Steven M.; Physics
    I 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.