Numerické simulace se staly nedílnou součástí procesu navrhování v nanofotonice, což nevyhnutelně vedlo k vývoji softwaru specializovaného pro tento úkol. Ačkoli je zde celá řada komerčně dostupných produktů, mnohé aplikace vyžadují datovou analýzu, která překračuje standardní výbavu těchto nástrojů. Zpracování výsledků simulací je těžištěm této práce, kdy důraz je kladen zejména na vývoj semianalytických modelů ušitých na míru jednotlivým experimentům. Spolu s lepší shodou mezi teorií a měřeními tyto modely poskytují také cenný vhled do studovaných fyzikálních procesů. Hlavní část této práce je věnována plazmonicky zesílené elektronové paramagnetické rezonanci (PE EPR), nové metodě využívající kovové antény pro zesílení interakce mezi zářením a materiály s magnetickými přechody mezi spinovými stavy. Jsou zde objasněny základní principy řídící tento jev a představen model umožňující rychlou optimalizaci polí antén pro PE EPR spektroskopii tenkých vrstev. Zvláštní pozornost je pak věnována roli indukovaného proudu a možnostem, které nabízí při projekcích do dalekého pole nebo počítání elektromagnetické interakce mezi objekty. Toto je dále demonstrováno na několika aplikacích, jmenovitě fázovém zobrazování metapovrchů pomocí koherencí řízeného holografického mikroskopu, designu optického prvku pro generování pole svazků na bázi metapovrchu a multipólové analýze elektromagnetických vln emitovaných objekty nacházejícími se uvnitř multivrstvy.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23460号 / 理博第4754号 / 新制||理||1681(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 杤尾 豪人, 教授 森 和俊, 教授 望月 敦史 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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This project deals with the optical control of welding glass blank. The blank consists two small parts of made of special glass. This blank is an essential part of the measuring device density of oxygen. Measuring device algorithm is based on paramagnetic properties of oxygen. It is a small and precise piece of the whole mechanism therefore the precision and perfection made strict criteria. For checking compliance with those criteria, developed a computer program written in C# programming language. Its task is to rule on perfection produced parts. It also serves to back up data on individual production units.

In this work two overall subjects were addressed. 1. In recent years high pressure perturbance has become a tool to investigate the folding energy landscape, the volumetric properties and the conformational equilibria of proteins. Conformational states which are not populated at ambient conditions thus become accessible to spectroscopic characterization. In this work a high pressure application was combined with EPR spectroscopy to investigate three spin labeled proteins, BSA from Bos taurus, HisJ from Salmonella enterica serovar Typhimurium and α-synuclein from Homo sapiens. The goal of these studies was to comprehend the influence of pressure on the respective EPR spectra and to identify changes in conformational equilibria and volumetric properties of the investigated proteins. Studies on BSA revealed a negative activation volume for rotational diffusion of the spin labeled site. Moreover, a rotameric equilibrium was derived from the pressure-dependent side chain dynamics and a correlating negative partial molar volume was observed, indicating a shift of the rotameric equilibrium to lesser order. In this regard it was also shown that a chaotropic medium (guanidine hydrochloride) supports the pressure-dependent effect. Spin labeled sites in the substrate binding protein HisJ revealed to be highly influenceable by low pressures between ambient conditions and 200 bar. Pressurization induced oligomerization and precipitation of the protein. Substrate binding revealed differences in pressure-dependence with regard to a decreased precipitation effect but not in relation to oligomerization. The natively unfolded protein α-synuclein plays a key role in Parkinson´s disease and is known for forming β-sheet rich aggregates, so called amyloid fibrils. The experimental data of this work revealed that hydrostatic pressure can induce a non-amyloid aggregation of monomeric α-synuclein which produces an unspecific oligomer. Furthermore, it was shown that α-synuclein amyloid fibrils can be dissolved by hydrostatic pressure. From the pressure dependent conformational equilibrium between the monomer and the fibril form the change of the partial molar volume of the investigated site was determined. 2. The second subject of this work was focused on different import systems, ATP-binding cassette (ABC) transporters and Energy-Coupling-Factor (ECF) transporters, for amino acids, vitamins and metal ions in prokaryotes. Studies on one bacterial ABC and two ECF transporter systems from two different organisms, the histidine ABC-type transporter HisQMP2 from Salmonella enterica serovar Typhimurium, the biotin ECF-type importer BioMNY from Rhodobacter capsulatus and the cobalt-specific ECF-type transporter CbiMNQO from Rhodobacter capsulatus, were performed using DEER and cw EPR spectroscopy. The goal of the studies on HisQMP2 and BioMNY was to shed light on conformations and dynamics connected to their transporter function. Studies on CbiMNQO aimed at the detection of the substrate in the transporter´s substrate binding unit. For HisQMP2 transport cycle dependent conformational changes and interactions with the substrate binding protein HisJ were revealed. Three different distance values between sites H101R1 and H101’R1 in the transporter´s nucleotide binding domains were assigned to the apo-, the ATP-bound and the posthydrolysis state. It was shown that the closed conformation of the nucleotide binding domains is achieved only in the presence of the ligand-bound HisJ which indicates a transmembrane communication of the association of HisJ to the transporter. Furthermore, interspin distances were determined between sites D86R1-A96R1, C197R1-C104R1 and A118R1-G123R1 in the transmembrane domains HisQ and HisM revealing distinguishable conformational states which correlate to the different states of the nucleotide binding sites during the hydrolysis cycle. Measured interspin distances between HisJ and HisM in the HisQMP2 complex showed that interaction only occurred in the closed state of the HisP2 dimer, the nucleotide bound state. Two different, substrate-dependent interactions between site G24R1 in HisJ and site A96R1 in HisQMP2 were observed, revealing that the substrate-free and substrate-bound form of HisJ both associate with HisQMP2. Distance measurements between sites G24R1 and T151R1 in HisJ in the presence and absence of its substrate revealed interspin distance changes that correlate with the proteins open and closed conformation. Investigations on the ECF transporter BioMNY, reconstituted into nanodiscs, revealed a closure and reopening of the nucleotide binding domains between sites H87R1 and H87’R1 using DEER spectroscopy which delivered interspin distance values that correlate with the apo-, the ATP-bound and the posthydrolysis state of the transporter. Further experiments were aimed to shed light on the transporters substrate-translocation mechanism with regard to the so called toppling over mechanism. Unfortunately, the experiments of this work were not able to give a distinct answer with respect to the proposed model because of the transmembrane domains tendency to oligomerize when reconstituted into nanodiscs. In this work we showed that substrate uptake by the substrate binding unit CbiM of the cobalt-specific ECF transporter CbiMNQO depends on the presence of the small transmembrane protein CbiN. Measurements of spin labeled CbiMN in detergent showed oligomerization of CbiM.

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京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18058号 / 理博第3936号 / 新制||理||1567(附属図書館) / 30916 / 京都大学大学院理学研究科物理学・宇宙物理学専攻 / (主査)准教授 池田 隆介, 教授 川上 則雄, 教授 石田 憲二 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development, and nervous system function. Previous studies have suggested that the regulatory domain (RD) of CaN binds Calmodulin (CaM) towards the N-terminal end of CaN. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the regulatory domain, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto inhibitory domain (AID) from the active site of CaN, which activates phosphatase activity. In the absence of calcium-loaded CaM, the RD is at least partially disordered, and binding of CaM induces folding in the RD. Previous studies have shown that an ?-helical structure forms in the N-terminal half of the RD, but organization may occur in the C-terminal region as well. Here, we are presenting a model for the structural transition of the full length RD as it binds to CaM. Using nuclear magnetic resonance (NMR) spectroscopy, we have successfully assigned >85% of the 15N, 13C?, 13C? and HN chemical shifts of the unbound, regulatory domain of CaN. Secondary chemical shifts support a model where the RD is highly disordered. Our study of the CaM and CaN interaction supports the formation of a distal helix in the region between the AID and calmodulin-binding region. Heat capacity changes upon binding predict that 43 residues fold when CaM binds to CaN, consistent with the formation of this distal helix. Paramagnetic relaxation enhancement (PRE) studies of this interaction suggest a potential binding mode where the distal helix binds to CaM near residues I10-A11. Mutagenesis in the distal helix disrupts PREs, further supporting this hypothesis. Together, these data suggest that the interactions between CaM and the distal helix of CaN can be important in regulation of phosphatase activity.