Physics

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    Understanding Allosteric Communication in Biological Systems using Molecular Dynamics Simulations
    (2024) Samanta, Riya; Matysiak, Silvina; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Allostery is critical to survival in living organisms due to its biological relevance in signal transduction, metabolism, and drug discovery. However, the molecular details of this phenomenon remain unclear. In this thesis, I present my work on two allosteric protein systems, each representative of structure-based (E. coli Biotin Protein Ligase) and dynamics-based (B. taurus S100B) allostery. I examined the structural and dynamic features of the proteins and associated variants subjected to various allosteric triggers (ligand/salt/mutations) to study how external/internal perturbations transmit across large distances using Molecular Dyanmic simulations in conjunction with the experiments carried out by our collaborators. Additionally, I carried out Network analyses on the two systems to characterize communication pathways on the protein/ protein family levels. Together, the structural and dynamic features would help us elucidate the underlying mechanism of allostery. The first chapter introduces the two systems with a brief dive into the history of allostery. In the second chapter, my work on E. coli Biotin Protein Ligase and its variants reveal one possible mechanism by which disorder-to-order transitions at the functional surfaces transmit via local changes around the critical residues in the allosteric network. The third chapter explores how the protein network reconfigures to adopt a new allosteric function by studying the allosteric and non-allosteric Biotin Protein Ligases. The fourth chapter elucidates the structural and dynamical markers in bovine S100B, which help to relay information about an allosteric signal by varying two allosteric triggers - ionic strength and target peptide. The final chapter sums up my conclusions, where I propose additional experiments and computational analyses that could be carried out to further our understanding of allostery.
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    High Resolution Mapping of Intracellular Mechanical Properties during Key Stages of Cancer Progression
    (2022) Nikolic, Milos; Scarcelli, Giuliano; Tanner, Kandice; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The mechanical phenotype of the living cell is critical for survival following deformations due to confinement and fluid flow. Furthermore, in recent years mechanical interaction between cells and the cellular environment has been implicated as one of the key regulators of cancer progression and malignant transformation. Due to the need to better understand the mechanical properties of invasive cells and how the mechanical phenotype plays a role in cancer progression, several microrheology techniques have been applied to study cell mechanics in a range of in vitro environments. However, many of these techniques have been limited either to studying cells in only one type of environment (e.g. 2D), with limited resolution, or with invasive probes. To begin to address this question, in this dissertation we aim to quantify the mechanical state of cells in a broader range of different contexts and geometries. To do this we use Brillouin microscopy, a non-contact, label free, non-invasive technique which enables us to probe the mechanical response of cells in a wide range of complex microenvironments. Here we introduce an improved Brillouin microscope with improved signal and acquisition speed which enables us to perform biological studies at the single cell level. Using the improved Brillouin microscopy, we find that individual cells can be softer as function of the invasive potential, but that cells are able to dynamically change their mechanical properties across many different contexts. We validate our results using complementary microrheology methods such as atomic force microscopy and broadband optical tweezer microrheology. We directly observe changes in cell mechanics in key processes relevant for metastatic migration, as well as a function of external and internal parameters like morphology, ECM properties, intracellular factors, and cell-cell cooperativity during multicellular tissue organization. These results support the paradigm that the mechanical state of a cell is a dynamic parameter that varies as a consequence of the microenvironmental and functional context, in addition to the observable changes in cell’s mechanical properties due to malignant transformation.
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    Detecting heterogeneity in and between breast cancer cell lines
    (Springer Nature, 2020-02-03) Shen, Yang; Schmidt, B. U. Sebastian; Kubitschke, Hans; Morawetz, Erik W.; Wolf, Benjamin; Käs, Josef A.; Losert, Wolfgang
    Cellular heterogeneity in tumor cells is a well-established phenomenon. Genetic and phenotypic cell-to-cell variability have been observed in numerous studies both within the same type of cancer cells and across different types of cancers. Another known fact for metastatic tumor cells is that they tend to be softer than their normal or non-metastatic counterparts. However, the heterogeneity of mechanical properties in tumor cells are not widely studied. Here we analyzed single-cell optical stretcher data with machine learning algorithms on three different breast tumor cell lines and show that similar heterogeneity can also be seen in mechanical properties of cells both within and between breast tumor cell lines. We identified two clusters within MDA-MB-231 cells, with cells in one cluster being softer than in the other. In addition, we show that MDA-MB-231 cells and MDA-MB-436 cells which are both epithelial breast cancer cell lines with a mesenchymal-like phenotype derived from metastatic cancers are mechanically more different from each other than from non-malignant epithelial MCF-10A cells. Since stiffness of tumor cells can be an indicator of metastatic potential, this result suggests that metastatic abilities could vary within the same monoclonal tumor cell line.
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    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.