In this thesis, I describe experiments that were designed to provide a better understanding of polymer diffusion during latex film formation. This step leads to the improvement of film mechanical properties. Polymer diffusion in these films was monitored by fluorescence resonance energy transfer. Current paint formulations contain Volatile Organic Compounds (VOCs) as plasticizers to facilitate polymer diffusion. The drawback of this technology is the release of VOCs to the atmosphere. VOCs are deleterious to the environment and contribute to smog and ground level ozone formation. The propensity of water, an indispensible part of any latex dispersion, to promote polymer diffusion was studied. Copolymers of poly (butyl acrylate-co-methyl methacrylate) and poly(ethylhexyl acrylate-co-tertiary butyl methacrylate) with similar glass transition temperatures but different hydrophobicity were compared. Polymer diffusion was monitored for films aged at different relative humidities. Water absorbed by the hydrophobic copolymer film was less efficient in promoting polymer diffusion than in the hydrophilic polymer. Only the fraction of water which is molecularly dissolved in the film participate actively in plasticization. Although water has low solubility in most latex polymers, molecularly dissolved water is more efficient than many traditional plasticizers. The possibility of modifying film formation behavior of acrylic dispersions with oligomers was studied by synthesizing hybrid polymer particles consisting of a high molecular weigh (high-M) polymer and an oligomer with the same composition. Oligomers with lower molecular weight are more efficient as diffusion promoters and have less deleterious effect on high-M polymer viscosity. A different set of hybrid particles were prepared in which the oligomer contained methacrylic acid units. The composition of the oligomer was tuned to be miscible with the high-M polymer when the acid groups were protonated but to phase separate when the acid groups were deprotonated. At basic pH, these particles adopt a core-shell morphology, with a shell rich in neutralized oligomers. After film formation, the oligomer shell retarded polymer diffusion. This retardation is expected to expand the time window during which the paint surface can be altered without leaving brush marks (open time). Short open time is a pressing problem in current technology.

Single-molecule spectroscopy has provided a means to uncover pathways and heterogeneities that were previously hidden beneath the ensemble average. Such heterogeneity, however, is often obscured by the artifacts of experimental noise and the occurrence of undesired processes within the experimental medium. This has subsequently caused in the need for new analytical methodologies. It is particularly important that objectivity be maintained in the development of new analytical methodology so that bias is not introduced and the results improperly characterized. The research presented herein identifies two such sources of experimental uncertainty, and constructs objective approaches to reduce their effects in the experimental results. The first, photoblinking, arises from the occupation of dark electronic states within the probe molecule, resulting in experimental data that is distorted by its contribution. A method based in Bayesian inference is developed, and is found to nearly eliminate photoblinks from the experimental data while minimally affecting the remaining data and maintaining objectivity. The second source of uncertainty is electronic shot-noise, which arises as a result of Poissonian photon collection. A method based in wavelet decomposition is constructed and applied to simulated and experimental data. It is iii found that, while making only one assumption, that photon collection is indeed a Poisson process, up to 75% of the shot-noise contribution may be removed from the experimental signal by the wavelet-based procedure. Lastly, in an effort to connect model-based approaches such as molecular dynamics simulation to model-free approaches that rely solely on the experimental data, a coarse-grained molecular model of a molecular ionic fluorophore diffusing within an electrostatically charged polymer brush is constructed and characterized. It is found that, while the characteristics of the coarse-grained simulation compare well with atomistic simulations, the model is lacking in its representation of the electrostatically-driven behavior of the experimental system.

Eukaryotic genomes are packaged as chromatin, which restricts access to the DNA by critical processes such as DNA replication, repair, and transcription. As a result, eukaryotic cells rely on ATP-dependent chromatin remodeling enzymes (remodelers) to alter the position, structure, and composition of nucleosomes. Understanding the mechanism and regulation of remodeling requires detailed information about transient intermediates of the remodeling process--a challenge ideally suited for single-molecule approaches. In particular, we use single-molecule fluorescence resonance energy transfer (smFRET) to measure nanometer-scale distance changes between strategically placed donor and acceptor dyes to monitor nucleosome translocation in real-time. The mechanism(s) by which remodelers use the free energy of ATP hydrolysis to disrupt histone-DNA contacts and reposition nucleosomes are not well understood. Using smFRET, we show that remodeling by ISWI enzymes begins with a 7 base-pair (bp) step followed by subsequent 3 bp steps toward the exit-side of the nucleosome. These multi-bp steps are actually compound steps composed of 1 bp elementary steps. We discover that DNA movement on the entry side lags behind exit side translocation, which is contrary to previously proposed models. Based on our results, we propose a new integrated mechanism for nucleosome translocation by ISWI enzymes. In the physiological context, remodelers are highly regulated. We study the regulation of human ACF, a prototypical ISWI complex, by critical features of the nucleosomal substrate. First, we dissect how the nucleosome translocation cycle is affected by the linker DNA length and histone H4 tail. Next, we introduce mutations/deletions into conserved enzyme domains to determine the mechanism by which linker length information sensed by the Acfl accessory subunit is allosterically transmitted to the Snf2h catalytic subunit. Interestingly, we find that Acfl modulates the activity of Snf2h indirectly by interacting with the H4 tail in a linker-length dependent fashion. While the majority of our experiments focus on observing changes in nucleosome position, we also develop strategies for site-specific labeling of ISWI enzymes and demonstrate their use in the study of dynamic enzyme-substrate interactions and enzyme conformational changes.

<p>This dissertation hypothesizes,</p><p><italic>"It is possible to design a self-assembled, nanoscale, high-speed, resonance energy transfer device exhibiting non-linear gain with a few molecules."</italic></p><p>The report recognizes DNA self-assembly, a relatively inexpensive and a massively parallel fabrication process, as a strong candidate for self-assembled RET systems. It successfully investigates into the design and simulations of a novel sequential self-assembly process employed to realize the goal of creating large, scalable, fully-addressable DNA nanostructure-substrate for future molecular circuitry. </p><p>As a pre-cursor to the final device modeling various RET wire designs for interconnecting nanocircuits are presented and their modeling and simulation results are discussed. A chromophore RET system using a biomolecular sensor as a proof-of-concept argument that shows it is possible to model and characterize chromophore systems as a first step towards device modeling is also discussed. </p><p>Finally, the thesis report describes in detail the design, modeling, characterization, and fabrication of the Closed-Diffusive Exciton Valve: a self-assembled, nanoscale (area of 17.34 nm<super>2</super>), high-speed (3.5 ps to 6 ps) resonance energy transfer device exhibiting non-linear gain using only 10 molecules, thus confirming the hypothesis. It also recognized improvements that can be made in the future to facilitate better device operation and suggested various applications.</p> / Dissertation

The use of microfluidic tools to develop two novel approaches to surface-based oligonucleotide hybridization assays has been explored. In one of these approaches, immobilized oligonucleotide probes on a glass surface of a microfluidic channel were able to quantitatively hybridize with oligonucleotide targets that were electrokinetically injected into the channel. Quantitative oligonucleotide analysis was achieved in seconds, with nM detection limits and a dynamic range of 3 orders of magnitude. Hybridization was detected by the use of fluorescently labeled target. The fluorescence intensity profile evolved as a gradient that could be related to concentration, and was a function of many factors including hybridization reaction rate, convective delivery speed, target concentration and target diffusion coefficient. It was possible to acquire kinetic information from the static fluorescence intensity profile to distinguish target concentration, and the length and base-pair mismatches of target sequences. Numerical simulations were conducted for the system, and fit well with the experimental data. In a second approach, a solid-phase nucleic acid assay was developed using immobilized Quantum Dot (QD) bioprobes. Hybridization was used to immobilize QDs that had been coated with oligonucleotides having two different sequences. The hybridization of one oligonucleotide sequence conjugated to a QD (a linker sequence) with a complementary sequence that was covalently attached to a glass substrate of a microfluidic channel was shown to be an immobilization strategy that offered flexibility in assay design, with intrinsic potential for quantitative replacement of the sensing chemistry by control of stringency. A second oligonucleotide sequence conjugated to the immobilized QDs provided for the selective detection of target nucleic acids. The microfluidic environment offered the ability to manipulate flow conditions for control of stringency and increasing the speed of analytical signal by introduction of convective delivery of target sequences to the immobilized QDs. This work introduces a stable and adaptable immobilization strategy that facilitates solid-phase QD-bioprobe assays in microfluidic platforms.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第23106号 / 医博第4733号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小柳 義夫, 教授 松田 道行, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The diploma thesis deals with resonance energy transfer in hydrogel matrix enviroment. In the theoretical part, all the important features of these systems and their components are summarized and all the principles for explaining the method used are described. The work also includes a short literary background research. In the experimental part, the resonance energy transfer was measured in micellar solutions and hydrogels in 0.15 M NaCl medium. The hydrogels were formed on the basis of interaction of the surfactant with the oppositely charged polyelectrolyte. As a positively charged surfactant, carbethopendecinium bromide (Septonex) was chosen and hyaluronan as the negatively charged polyelectrolyte. Perylene with fluorescein and perylene were selected as fluorescent pairs for RET. It has been found that increasing concentration of the acceptor molecule increases the intensity of RET, in other word the rate of acceptor binding to the donor. In the study of fluorescent pair of perylene with DiO was observed, that it was not appropriate to use fluorescence probe DiO at higher concentrations. Experiments were carried out to determine whether RET changes in the time from the point of mixing the donor and acceptor fluorescence probes together. This phenomenon has not been confirmed. In hydrogels, the effect of increasing surfactant concentration and molecular weight of hyaluronan was observed. The resulting analysis showed that the molecular weight of hyaluronan has a significant effect on RET efficiency in hydrogels.

Rewritable photochrome-based 3D optical data storage requires photochromic molecules with high two-photon absorption (2PA) cross sections. Currently, the low value of two-photon absorption cross sections of existing photochromes makes them unsuitable for practical application in 3D data storage. Worldwide attempts to increase the cross section of photochromic molecules by altering the chemical structure have yielded poor results. In this work, two ways to increase the two-photon absorption cross sections of photochromes were investigated. In the first method, partial success demonstrated by extending the conjugation of a photochromic molecule, a high two-photon absorption cross section of the closed form isomer and high photoconversion to the closed form were realized. At the same time, a decrease in photoswitching quantum yield and low photoconversion to open form was observed. A discussion is provided to explain the results, suggesting that the proposed method of extending the conjugation may not solve the problem. For this reason a new method for effective two-photon absorption cross section enhancement of photochromes was proposed. As a proof of principle, a new two-photon absorbing dye with a hydrogen bonding moiety was synthesized and used for the formation of supramolecular structures with a photochromic compound. Theoretical reasoning and experimental demonstration of energy transfer from the dye to the photochrome under one and two-photon excitation confirmed the practical value of the method. The effects of a 2PA dye on the photochromic properties of a diarylethene were investigated using a model compound to simplify data analysis. Formation of supramolecular structures was revealed using ¹H NMR spectroscopic methods. The model compound, having the same hydrogen bonding moiety as 2PA dye, has been demonstrated to bind with photochrome molecules at very low concentrations. Photochromic properties of 2,3-bis(2,4,5-trimethyl-3-thienyl)maleimide, including conversions at the photostationary state, extinction coefficients, photoisomerization reaction rates and quantum yields, were shown to be affected by hydrogen bonding with the model compound - 2,6-bis-(acetamido)pyridine. The extent of this change was determined and discussed, demonstrating a balanced supramolecular strategy to modulate photochemical and photophysical properties of this important class of photochromic material.

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