京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18060号 / 理博第3938号 / 新制||理||1567(附属図書館) / 30918 / 京都大学大学院理学研究科物理学・宇宙物理学専攻 / (主査)教授 石田 憲二, 教授 前野 悦輝, 教授 松田 祐司 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第21247号 / 理博第4417号 / 新制||理||1634(附属図書館) / 京都大学大学院理学研究科物理学・宇宙物理学専攻 / (主査)教授 石田 憲二, 教授 前野 悦輝, 教授 松田 祐司 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Explosive precursors and narcotic salts are chemicals contributing to an undesirabledevelopment of the Swedish society, both in terms of criminal activities and harm to the environment. Reducing the illegal use of these chemicals is important in the work towards a safer society. National Forensic Centre (NFC) is the state agency responsible for forensic investigations for the Swedish Police Authority. The Drug Analysis and ChemistryTechnology section at NFC were both in need for an accurate quantification method to determine the content of Cl in narcotic salts and explosive precursors. Nuclear magnetic resonance (NMR) spectroscopy was assessed to be suitable since a recently published article had shown applicability of 35Cl NMR on narcotic salts. The aim of the method was to find the most appropriate parameter settings for the compounds of interest, including operating frequency, 90° pulse length, number of scans, relaxation time, and relaxation delay. To ensure a reliable and accurate method, the following validation parameters were studied; linearity, limit of detection (LOD), limit of quantification (LOQ), intermediate precision, trueness, repeatability, and ruggedness. Dimethyl sulfoxide (d6-DMSO) was chosen as the preferredsolvent for the Drug Analysis section since it is a common solvent for their 1H-NMR analysis. For explosive precursors results showed advantages of using deuterium oxide (D!O) as solvent, considering accuracy, solubility and shorter analysis time.Concluding, the chosen criteria of signal-to-noise (S/N) ratio >6 resulted in an LOQ of around 0.15g/L, though this was dependent upon the number of scans utilized. Successful pulse length experiments determined exact 90° pulse lengths for each sample and solvent combination. The longitudinal relaxation time T1 was also successfully determined, and since it was multiplied with five to ensure complete relaxation to stable state the relaxation delay D1was assumed as an insignificant parameter for the determination of chloride. Quantification was based upon the pulse-length based concentration determination (PULCON) using an external standard. The ruggedness can be studied additionally by another experienced operator (since trueness was strongly dependent upon the preparation of the external standard solution). The method displayed good linearity over the mass range normally utilized in such quantifications. The conclusion drawn in the thesis is that the method shows great promise but additional analyzes are still required before implementation at NFC

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.

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Mutations in epigenetic regulators such as the SET domain-containing methyltransferase NSD2 are of high interest among the research community nowadays. The involvement of this mutations in multiple diseases put them in the spotlight. Interestingly, the change of the glutamic acid residue in the position 1099 of the NSD2 SET domain for a lysine residue has been recurrently found in multiple myeloma patients. This mutation produces a hyperactive enzyme that hypermethylate the natural enzymatic substrate: the lysine in the position 36 of the basic tail of the histone 3 in the nucleosomal context. Apparently, this hyperactivation may be related to the disruption of a critical salt-bridge that stabilize an autoregulatory loop of the NSD2 catalytic site. However, despite the extensive research that have been done around this phenomenon, the molecular mechanism behind this hyperactivation still remains unknow. For this reason, in this study we addressed this matter from a structural point of view by evaluating the structure and dynamics of the protein in solution by high-resolution Nuclear Magnetic Resonance (NMR) spectroscopy and biophysical techniques. We found increased local segmental motions in us to ms timescale that induced protein flexibility that may correlate with gain-of-function of E1099K miss-sense mutation. Further functional studies with the native substrates in vitro and in vivo are needed to understand this observation.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第24399号 / 理博第4898号 / 新制||理||1700(附属図書館) / 京都大学大学院理学研究科物理学・宇宙物理学専攻 / (主査)教授 石田 憲二, 教授 松田 祐司, 教授 柳瀬 陽一 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

No description available.