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).

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

Background and aims: It is known that smooth muscle possesses substantial mechanical plasticity in that it is able to adapt to large changes in length without compromising its ability to generate force. It is believed that structural malleability of the contractile apparatus underlies this plasticity. There is strong evidence suggesting that myosin thick filaments of the muscle are relatively labile and their length in vivo is determined by the equilibrium between monomeric and filamentous myosin. The equilibrium in turn is governed by the state of phosphorylation of the 20-kD regulatory myosin light chain (MLC20, or RLC). It is known that phosphorylation of the myosin light chain favors formation of the filaments; it is not known how the light chain phosphorylation affects the lability of the filaments. The major aim of this thesis was to measure the mechanical integrity of the filaments formed from purified myosin molecules from bovine airway smooth muscle, and to determine whether the integrity was influenced by phosphorylation of the myosin light chain. Methods: Myosin was purified from bovine trachealis to form filaments, in ATP containing zero-calcium solution during a slow dialysis that gradually reduced the ionic strength. Sufficient myosin light chain kinase and phosphatase, as well as calmodulin, were retained after the myosin purification and this enabled phosphorylation of RLC within 20-40 s after addition of calcium to the filament suspension. The phosphorylated and non-phosphorylated filaments were then partially disassembled by ultrasonification. The extent of filament disintegration was visualized and quantified by atomic force microscopy. Results: RLC phosphorylation reduced the diameter of the filaments and rendered the filaments more resistant to ultrasonic agitation. Electron microscopy revealed a similar reduction in filament diameter in intact smooth muscle when the cells were activated. Conclusion: Our results suggest that RLC phosphorylation is a key regulatory step in modifying the structural properties of myosin filaments in smooth muscle, where formation and dissolution of the filaments are required in the cells’ adaptation to different cell length. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

This thesis describes a novel reconstitution of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) for the purposes of structural characterization by atomic force microscopy (AFM) and functional characterization by electrical recordings of lipid bilayers. AMPARs are glutamate gated ion channels, ubiquitous in the vertebrate central nervous system where they mediate fast excitatory neurotransmission. In a healthy brain, AMPARs are involved in memory formation and learning and their dysfunction has been related to numerous neurological disorders such as Alzheimer's disease, epilepsy, schizophrenia and many others. AMPARs were reconstituted at high and low densities. Densely reconstituted samples contained >100 receptors per μm2, a value comparable to the AMPAR density at synapses. This allowed, for the first time, the imaging of full length tetrameric AMPARs in native-like conditions and with clearly assigned domains: the extracellular domains extended 14 nm above the membrane in agreement with electron microscopy (EM) and X-ray crystallography data. Lipid-protein interactions were studied in samples with low protein density with the receptors showing preference for lipids in the liquid crystalline phase. The activity of the reconstituted receptors was confirmed through single-channel recordings. This is the first case in which an AMPAR has been reconstituted and given (a) single-channel recordings with (b) physiologically plausible conductance levels and (c) pharmacological and no-protein controls and (d) structure. As a result, previously reported biochemistry and EM are now for the first time available in concert with AFM and single-channel recordings for a purified AMPAR of known composition.

Several members of the ABC-6 zeolite family, namely offretite, zeolite T, ZSM-34 and zeolite L, were synthesised with a view to studying the effect of synthetic parameters on the resulting crystal properties. A range of procedures for producing offretite and ZSM-34 were tested, and crystals with the optimal morphology, purity, crystallinity and size identified. A standard procedure for synthesising zeolite T was used, before the effect of systematically altering the cation concentration, Si/Al ratio and water content in the synthesis mixture was studied, and the optimal composition identified. Ex-situ AFM was used to study the morphology of the resulting crystals, as well as to determine information about surface features, such as the shape and height of terraces. This information could be used to infer the growth mechanism for each crystal. The AFM studies of zeolite L were compared with those of previous studies and found to agree. For offretite, zeolite T and ZSM-34, 1.2 nm high terraces elongated along the length of the {100} face of the crystals were observed. Where the crystals exhibited a micro-crystalline morphology without distinct faces HR-SEM was used to study their morphology and terracing. In-situ AFM was used to study the dissolution of the crystals in basic media. In each case the terraces were observed to dissolve primarily length-wise. The dissolution of terraces on zeolite T was considered in particular detail. The terrace height was measured during dissolution, and three distinct measurements were observed, 1.2, 0.8 and 0.2 nm. These heights were related to framework features. Lateral force AFM measurements were used to show the relationship between decreased terrace height and increased lateral deflection, indicating that dissolving areas of crystal exhibited increased tip-sample friction. The rate of dissolution of terraces on zeolite T was also considered. It was found that dissolution rate increased with increasing NaOH concentration, and with increasing tip-sample force in the AFM. These observations allowed order of reaction for the dissolution process of zeolite T to be estimated at 2.54. A magnesium substituted aluminophosphate ABC-6 family zeotype material, MgAPO-CJ60, was also synthesised and analysed. Solid-state NMR was used to determine the distribution of aluminium and phosphorous within the framework, which was found to be non-random.

In this report we detail our work fabricating and measuring graphene quantum dots. We investigate a technique, relatively widely used in several other materials but not yet well investigated in graphene, known as Atomic Force Microscope Lithography (AFML). We then use AFML to fabricate graphene quantum dot systems. Transport measurements are carried out on our graphene quantum dots at low temperatures and high parallel magnetic fields and we try to understand the behaviour of spins in graphene. In our initial investigations into AFML we use graphene samples electrically contacted using standard electron-beam lithography. We were able to cut the graphene lattice by applying a negative voltage to the AFM tip and moving the tip across a grounded graphene surface. We have shown, by measuring the current through the AFM tip during lithography, that cutting of graphene is not current driven. Using a combination of transport measurements and scanning electron microscopy we show that , while indentations accompanied by tip current appear in the graphene lattice for a range of tip voltages, real cuts are characterized by a strong reduction of the tip current above a threshold voltage. The flexibility of the technique was then demonstrated by the fabrication, measurement, modification and re-measurement of graphene nanodevices with resolution down to 15 nm. We subsequently developed a shadow-masking technique to electrically contact graphene samples thus eliminating the use of chemical resists and the associated contamination of the graphene surface. With these pristine samples we were able to oxidise and hydrogenate the graphene using AFML. A graphene quantum dot was then fabricated using AFML oxidation. We also fabricated a graphene quantum dot using e-beam lithography in combination with oxygen plasma etching. We studied electron spin physics in these structures by J:1pplying large parallel magnetic fields at low temperatures and performing electrical transport measurements. We do not find an ordered filling sequence of spin states, which we assign to edge disorder and surface charge impurities.

The diploma thesis deals with characterization of adhesion of plasma polymer films deposited on silicon wafers. The samples included organosilicon thin films based on tetravinylsilane monomer prepared by plasma-enhanced chemical vapour deposition. Scratch test was used to characterize film adhesion employing nanoindentation measurements. Adhesion of plasma polymer films of different mechanical properties and film thickness was analyzed by normal and lateral forces, friction coefficient, and scratch images obtained by scanning probe microscope working in atomic force microscopy mode.

The doctoral thesis deals with the study of morphology and mechanical properties of thin plasma polymer films based on tetravinylsilane monomer and its mixtures with oxygen and argon. Thin films were prepared by plasma-enhanced chemical vapour deposition on silicon and glass substrates. Atomic force microscopy was used for characterization of thin film surface and for depiction of composite interphase with functional interlayer. Mechanical properties of thin films, namely Young’s modulus and hardness, were studied by cyclic nanoindentation technique. Nanoindentation device was also used to carry out scratch test, which was helpful to describe adhesion of films to substrate. In this thesis the influence of deposition conditions on surface and mechanical properties of thin films prepared in continual and pulse wave on planar substrates is discussed. Also, the suitability of few atomic force microscopy techniques for depiction of composite interphase was reviewed.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21106号 / 工博第4470号 / 新制||工||1695(附属図書館) / 京都大学大学院工学研究科材料工学専攻 / (主査)教授 杉村 博之, 教授 邑瀬 邦明, 教授 山田 啓文 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM