UMD Theses and Dissertations

Permanent URI for this collectionhttp://hdl.handle.net/1903/3

New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a given thesis/dissertation in DRUM.

More information is available at Theses and Dissertations at University of Maryland Libraries.

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    The small mechanosensitive channel: Adaptive gating and timing during hypoosmotic shock.
    (2011) Boer, Miriam Sara; Sukharev, Sergei I.; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Mechanosensation is the ability to respond to mechanical stimuli. It is present in many organisms. In Escherichia coli (E. coli), mechanosensation manifests in two membrane channels, the large mechanosensitive channel (MscL) and the small mechanosensitive channel (MscS). Both osmoregulatory channels sense membrane tension. MscS, the subject of these studies, consists of a transmembrane region and a cytoplasmic cage. It opens at tensions below those which can cause immediate damage to membranes, contrasting with MscL which opens at near-lytic tensions. Because it opens at non-threatening tensions, MscS not only opens and closes but also inactivates. Inactivation is non-conductive and tension insensitive, and this adaptive behavior was first observed on patch clamp. With the aid of carefully constructed molecular models, the first part of this dissertation evaluates whether inactivation is merely a patch clamp artifact or if it is indeed a part of in vivo MscS function. Working with wild type (WT) alongside fast inactivating and noninactivating mutants proved inactivation does confer a survival advantage to hypoosmotically shocked bacteria. Additionally, light scattering was used to view the swelling and channel response events in WT and knock out (KO) cells lacking mechanosensitive channels upon instantaneous hypoosmotic shock by way of stopped flow.
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    A Biophysical Study of Clathrin Utilizing Light Scattering, Neutron Scattering and Structure Based Computer Modeling
    (2007-04-27) Ferguson, Matthew Lee; Nossal, Ralph J; Losert, Woflgang; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    A principal component in the protein coats of certain post-golgi and endocytic vesicles is clathrin, which appears as a three-legged heteropolymer (known as a triske- lion) that assembles into polyhedral baskets principally made up of pentagonal and hexagonal faces. In vitro, this assembly depends on the pH, with baskets forming more readily at low pH and less readily at high pH. We have developed procedures, based on static and dynamic light scattering, to determine the radius of gyration, Rg, and hydrodynamic radius, RH, of isolated triskelia under conditions where basket assembly occurs. Calculations based on rigid molecular bead models of a triskelion show that the measured values can be accounted for by bending of the legs and a puckering at the vertex. We also show that the values of Rg and RH measured for clathrin triskelia in solution are qualitatively consistent with the conformation of an individual triskelion that is part of a "D6 barrel" basket assembly measured by cryo-EM tomography. We extended this study by performing small angle neutron scattering (SANS) experiments on isolated triskelia in solution under conditions where baskets do not assemble. SANS experiments were consistent with previous static light scattering ex- periments but showed a shoulder in the scattering function at intermediate q-values just beyond the central diffraction peak (the Guinier regime). Theoretical calcula- tions based on rigid bead models of a triskelion showed well-defined features in this region different from the experiment. A flexible bead-spring model of a triskelion and Brownian dynamics simulations were used to generate a time averaged scattering function. This model adequately described the experimental data for flexibilities close to previous estimates from the analysis of electron micrographs.