Thesis (M. S.)--Mechanical Engineering, Georgia Institute of Technology, 2007. / King, William Paul, Committee Chair ; Henderson, Clifford L, Committee Co-Chair ; Gall, Ken, Committee Member.

Thesis (Ph. D.)--Mechanical Engineering, Georgia Institute of Technology, 2006. / Levent Degertekin, Committee Member ; William P. King, Committee Member ; Boris Mizaikoff, Committee Member ; Mark G. Allen, Committee Member ; Peter J. Hesketh, Committee Chair.

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: AFM, Bacterial Adhesion. Includes bibliographical references (p.130-143).

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 221-230).

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 285-298). Also available in electronic version.

Thesis (M.S.)--Texas State University-San Marcos, 2007. / Vita. Appendices: leaves 69-80. Includes bibliographical references (leaves 81-83).

Thesis (Ph. D.)--University of Canterbury, 2006. / Typescript (photocopy). "November 2006." Includes bibliographical references (p. [153]-161). Also available via the World Wide Web.

Thesis (MIng)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

abstract: In eukaryotes, DNA is packed in a highly condensed and hierarchically organized structure called chromatin, in which DNA tightly wraps around the histone octamer consisting of one histone 3-histone 4 (H3-H4) tetramer and two histone 2A- histone 2B (H2A-H2B) dimers with 147 base pairs in an almost two left handed turns. Almost all DNA dependent cellular processes, such as DNA duplication, transcription, DNA repair and recombination, take place in the chromatin form. Based on the critical importance of appropriate chromatin condensation, this thesis focused on the folding behavior of the nucleosome array reconstituted using different templates with various controllable factors such as histone tail modification, linker DNA length, and DNA binding proteins. Firstly, the folding behaviors of wild type (WT) and nucleosome arrays reconstituted with acetylation on the histone H4 at lysine 16 (H4K16 (Ac)) were studied. In contrast to the sedimentation result, atomic force microscopy (AFM) measurements revealed no apparent difference in the compact nucleosome arrays between WT and H4K16 (Ac) and WT. Instead, an optimal loading of nucleosome along the template was found necessary for the Mg2+ induced nucleosome array compaction. This finding leads to the further study on the role of linker DNA in the nucleosome compaction. A method of constructing DNA templates with varied linker DNA lengths was developed, and uniformly and randomly spaced nucleosome arrays with average linker DNA lengths of 30 bp and 60 bp were constructed. After comprehensive analyses of the nucleosome arrays' structure in mica surface, the lengths of the linker DNA were found playing an important role in controlling the structural geometries of nucleosome arrays in both their extended and compact forms. In addition, higher concentration of the DNA binding domain of the telomere repeat factor 2 (TRF2) was found to stimulate the compaction of the telomeric nucleosome array. Finally, AFM was successfully applied to investigate the nucleosome positioning behaviors on the Mouse Mammary Tumor Virus (MMTV) promoter region, and two highly positioned region corresponded to nucleosome A and B were identified by this method. / Dissertation/Thesis / Ph.D. Chemistry 2010

Glutamate is the major excitatory neurotransmitter in the mammalian brain. It binds to three different subclasses of ionotropic glutamate receptors (iGluRs): AMPA, kainate and NMDA receptors, and triggers a cation influx that generates synaptic currents crucial to brain function. Significantly, iGluRs are implicated in various neurological disorders, such as depression, schizophrenia, Alzheimer’s and Parkinson’s diseases, autism, seizure, and stroke. Several crystal structures for intact iGluRs in various functional states (i.e. closed, activated and desensitized) have now been reported. The receptors have also been studied using single-particle cryo-electron microscopy. Together, these studies provide fascinating ‘snap-shots’ of the receptors as they transition between different states. What is lacking, so far, is information about the kinetics underlying these structural transitions, because the techniques used lack time resolution. I have used fast-scan atomic force microscopy (AFM), in some cases in combination with UV photolysis of caged L-glutamate, to study activation-induced structural changes in GluK2 kainate receptors and GluA2 AMPA receptors. AFM provides single-molecule resolution under fluid, permitting the imaging of proteins ‘in action’. Receptors were purified from transfected cells by immunoaffinity chromatography and imaged after integration into supported lipid bilayers. Activation of both receptors caused a rapid ~1-nm vertical compression of the receptor. In both cases, the height reduction did not occur in the presence of receptor antagonists. Further, the D776K mutant of the kainate receptor, which does not desensitize, did not undergo the height change, and cyclothiazide, which blocks desensitization of the AMPA receptor, also blocked the height change. I conclude, therefore, that the vertical compression is associated with receptor desensitization, and suggest that it may reflect a weakening of the interaction between receptor subunits at the LBD dimer interface. When imaged from the ‘top’ by AFM, the receptors appeared as double-blob structures, with each blob representing a pair of ATDs. By measuring the distance between the centres of the blobs in successive AFM images, I was able to monitor the mobility of the ATDs relative to each other before and during receptor stimulation. I found that for both kainate and AMPA receptors, the relative mobility of the ATDs became greater after stimulation. Further, at low glutamate concentrations, the ATDs of the (rapidly desensitizing) flop splice variant of the AMPA receptor were more mobile than those of the (more slowly desensitizing) flip splice variant. I suggest that the greater mobility of the flop splice variant might be connected with its more short-lived functional response to activation. In a final series of experiments, in collaboration with two other groups, I used AFM to measure conformational changes induced by allosterically-bound halide ions. We found that anion substitution (i.e. chloride to bromide, or chloride to iodide) produced vertical compression of AMPA receptors prior to agonist binding, and also (in electrophysiological experiments conducted by collaborators) altered the duration of agonist-evoked channel activity. The anion binding site was identified (in X-ray crystal structures obtained by collaborators) within the ligand binding domain, where flip-flop alternative splicing occurs. Interestingly both anion effects were isoform-dependent.