Theses and Dissertations from UMD

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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 give thesis/dissertation in DRUM

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

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2011 - 20173

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    Development of Nanoparticle-Based Intracellular Dual Sensing and Actuation Modalities
    (2017) Field, Lauren D.; White, Ian M; Medintz, Igor L; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The integration of therapeutics with diagnostic agents, or theranostics, is vital for the development of novel and effective disease treatments. To effectively design new and efficient theranostic materials, a thorough understanding of the carrier ensemble, the interactions within the construct components, and the surrounding environment is required. This dissertation focuses on the development of new strategies to produce an effective ‘toolbox’ of nanoscale theranostics, namely through the use of a central NP scaffold and the visualization technique of Förster Resonance Energy Transfer (FRET). The NP scaffold used throughout this work, the semiconductor quantum dot (QD), is ideal for visualizing sensing modalities due to their high quantum yield (QY), tunable, narrow and symmetric emission profiles with broad, far-UV excitation, and resistance to photobleaching - making them optimal FRET donors. We first examined the intracellular assembly of QDs to proteins by injecting 545 nm emitting QDs, coated with various capping ligands, into cells transfected to express mCherry at two distinct intracellular locations: the cytosol and the plasma membrane. We found that the small, zwitterionic capping ligand CL4 and the cytosolically located mCherry protein assembled into the most efficient FRET complexes. We used this knowledge to design and implement a novel intracellular actuation modality for drug delivery that used a 520 nm emitting QD with the carrier maltose binding protein appended to the surface and carrying drug or dye conjugated to a maltose analog, -cyclodextrin in the binding pocket. Rather than relying on intracellular environmental changes or external stimuli to actuate release, the addition of the innocuous sugar maltose to the medium induced cargo actuation and could be visualized via FRET. Finally, the same methods were implemented to develop a novel pH sensor to report on the extracellular changes that occur in tumor development where the physiological pH is lowered dramatically. Using a 464 nm QD scaffold conjugated to pH-responsive FITC, we successfully monitored changes in extracellular pH and accurately determined unknown pH values. With the work in this thesis, we believe we have contributed greatly to the advancement and development NPs for the design and implementation of sensing and actuation complexes.
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    SINGLE MOLECULE FRET OF LACI-DNA-IPTG LOOP CONFORMATIONS
    (2012) Goodson, Kathy A.; Kahn, Jason D.; English, Douglas S.; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    This work focuses on the Escherichia coli lactose repressor protein (LacI) which represses expression of the lac operon. In order to repress transcription, the tetrameric LacI protein binds a primary promoter-proximal operator, O1, and one of two auxiliary operators, O2 or O3. The binding of these two sites to a single LacI molecule occurs via DNA loop formation. Induction of the lac operon by allolactose reduces the affinity of LacI for DNA, but induction does not completely prevent looping in vivo. The synthetic inducer isopropyl-β-D-thiogalactoside (IPTG) acts similarly to allolactose. Model DNA constructs have been used to demonstrate, through fluorescence resonance energy transfer (FRET) analysis, that LacI may change conformation in order to form more than one loop structure. This work employs single molecule FRET to investigate LacI-induced loop formation in DNA looping constructs, as a function of IPTG concentration, on freely diffusing LacI-DNA complexes. The results include evidence for the persistence of DNA loop formation under at saturating IPTG concentration, and they provide a detailed view of how LacI conformation affects DNA loop formation. In addition, this work explores possible changes in geometry in LacI-induced DNA loops through the use of model DNA constructs that produce alternative loop topologies. We propose that inducer-bound LacI-DNA looped complexes may control the kinetics of induction and re-repression of the operon.
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    USING SINGLE MOLECULE TECHNIQUES TO DETERMINE THE MECHANISM OF DNA TOPOLOGY SIMPLIFICATION BY TYPE IIA TOPOISOMERASES
    (2011) Hardin, Ashley Harris; Thirumalai, Devarajan; Neuman, Keir C; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Type IIA topoisomerases are essential, universally conserved proteins that modify DNA topology by passing one segment of duplex DNA (the transfer, or T-segment) through a transient double strand break in a second segment of DNA (the gate, or G-segment) in an ATP-dependent reaction. Type IIA topoisomerases decatenate, unknot, and relax supercoiling in DNA to levels below equilibrium, resulting in global topology simplification. The mechanism underlying non-equilibrium topology simplification remains speculative, though several plausible models have been proposed. This thesis tests two of these, the bend angle and kinetic proofreading models, using single-molecule techniques. The bend angle model postulates that non-equilibrium topology simplification scales with the bend angle imposed on the G-segment DNA by a type IIA topoisomerase. To test this model, we used atomic force microscopy and single molecule Förster resonance energy transfer to measure the extent of bending imposed on DNA by three type IIA topoisomerases that span the range of topology simplification activity. We found that all proteins bent DNA, but the imposed bends are similar and cannot account for the differences among the enzymes. These data do not support the bend angle model and suggest that DNA bending is not the sole determinant of non-equilibrium topology simplification. Based on the assumption that the rates of collision between DNA segments is higher in knotted, linked, and supercoiled DNA than in topologically free or relaxed DNA, the kinetic proofreading model proposes that two successive binding events between a G-segment bound topoisomerase and a putative T-segment are required to initiate strand passage. As a result of the two step process, the overall rate of strand passage should scale with the square of the collision probability of two DNA segments. To test this model, we used magnetic tweezers to manipulate a paramagnetic bead tethered to the surface by two DNA molecules. By rotating the bead, we varied the proximity, and thus collision rate, of the two molecules to determine the relationship between collision probability and rate of strand passage. Our data indicate that the strand passage rate scales linearly with the collision probability, which is inconsistent with the kinetic proofreading model.