Non-Classical Protein Secretion and Transcriptome Studies during Stationary Phase of Bacillus Subtilis

A cloned esterase and several cytoplasmic proteins which lack a classical cleavable signal-peptide were secreted during late stationary phase in B. subtilis. Several lines of evidence indicate that secretion of enolase, SodA, and Est55 is not due to cell lysis. The extent of possible release of these proteins mediated by membrane vesicles into the medium was also found to be minimal. We have identified a hydrophobic α–helical domain within enolase that contributes to the secretion specificity. Thus, upon the genetic deletion or replacement of a potential membrane-embedding domain, the secretion of plasmid-encoded mutant enolases is totally blocked, while that of the wild-type chromosomal enolase is not affected in the same cultures. However, mutations on the conserved basic residues flanking the hydrophobic core region show no effect. GFP fusion experiments demonstrate that minimal length of N-terminus 140 amino acids and its tertiary structure are required to serve as a functional signal for the export of enolase. Transcriptome analysis revealed several interesting patterns in gene expression when the cell growth switches from exponential phase into stationary phase. As expected, once cell growth enters the stationary phase, expressions of most SigA-dependent house-keeping genes (for syntheses of ATP, amino acids, nucleotides, ribosomes), and surprisingly secY and yidC homolog in the Sec-dependent general protein secretion system were significantly decreased; however, secA and sipT were found progressively induced in the stationary phase. The sigB gene and the SigB regulon exhibited a distinct pattern of transient induction with a peak in transition phase. A total of 62 genes were induced by three fold after cessation of SigB-dependent surge, which includes sigW and many of SigW-depedent genes specifically for antitoxin resistant genes, and some unknown function genes. In addition, oxidative stress response and damage repair genes also dominantly induced in stationary phase implied a high level of oxidant or thio-depleting agents in stationary phase. Besides, induction of fruRAB at T40 and gap operon at T100 suggested a sequential switch of carbon utilization from glucose to fructose. These results indicate a complex adaptation physiology as Bacillus cells change from the fast growing exponential phase toward the stationary phase

クサリヘビの１種における餌生物や競争者の存在に基づいた季節や生息地ごとの待ち伏せ場所調整

京都大学新制・課程博士博士(理学)甲第23563号理博第4757号新制||理||1682(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)准教授 森 哲, 教授 中川 尚史, 教授 中務 真人学位規則第4条第1項該当Doctor of ScienceKyoto UniversityDGA

On the Origin of the Checkerboard Pattern in Scanning Tunneling Microscopy Maps of Underdoped Cuprate Superconductors

The checkerboard pattern in the differential conductance maps on underdoped cuprates appears when the STM is placed above the O-sites in the outermost CuO$_{\text{2}}$-plane. In this position the interference between tunneling paths through the apical ions above the neighboring Cu-sites leads to an asymmetric weighting of final states in the two antinodal regions of ${\boldsymbol{k}}$-space. The form of the asymmetry in the differential conductance spectra in the checkerboard pattern favors asymmetry in the localization length rather than a nematic displacement as the underlying origin.Comment: 8 pages, 5 figures, final versio

PET studies on the mechanisms of action of antidepressant and antipsychotic drugs

Positron emission tomography (PET) is a non-invasive molecular imaging technique suitable for examination of neurochemical biomarkers in the living brain. Among these applications, PET studies are used to facilitate the development of novel psychotropic drugs. The general aim of this thesis work was to develop and implement novel PET imaging paradigms suitable for research and drug development in psychiatry. The work was carried out in nonhuman primates (NHP) with the intention to prepare for future human applications. The first part of the thesis work was to develop improved PET imaging paradigms sensitive to changes in serotonin (5-HT) concentration. In Study I, the binding of [11C]Cimbi-36, a novel 5-HT2A receptor agonist radioligand, was found to be sensitive to fenfluramine-induced 5-HT release. The 5-HT sensitivity of [11C]Cimbi-36 was comparable to that of [11C]AZ10419369, a 5-HT1B receptor radioligand with established 5-HT sensitivity. In Study II, [11C]AZ10419369 binding was found to be sensitive to pretreatment with 5-HT concentration enhancers (amphetamine, MDMA or 5-HTP) at clinically relevant doses that may be safely applied in human studies. After validation, these PET methodologies were used in Study III to assess the mechanisms of action of vortioxetine, a novel antidepressant. At doses with comparable and clinically relevant occupancy of the 5-HT transporter, vortioxetine induced larger reductions in [11C]AZ10419369 binding than citalopram, and had no significant effect on [11C]Cimbi-36 binding. The results suggest that vortioxetine binds to the 5-HT1B receptor when administered at clinically relevant doses. The second part of the thesis work extended the application of PET imaging from neuroreceptors to intracellularly located enzymes, such as phosphodiesterase 10A (PDE10A). In Study IV, we characterized the binding of [11C]Lu AE92686 to PDE10A in the NHP brain and validated the quantification methods. In Study V, we examined the effect of changes in the concentration of 3',5'-cyclic adenosine monophosphate (cAMP) on [11C]Lu AE92686 binding. Decreasing cAMP concentration by administration of SCH 23390 alone or in combination with R-apomorphine significantly decreased striatal [11C]Lu AE92686 binding. The combination of SCH 23390 and R-apomorphine also significantly increased binding in substantia nigra. The decrease in striatal [11C]Lu AE92686 binding may reflect a decrease in PDE10A affinity induced by cAMP depletion. The effect of changes in cAMP concentration on PDE10A binding should thus be considered when [11C]Lu AE92686 is used in human studies. In conclusion, the current thesis work has advanced the PET imaging paradigms for examining changes in 5-HT concentration. The validation of [11C]Lu AE92686 as a PDE10A PET radioligand and understanding the modulatory role of cAMP on [11C]Lu AE92686 binding will facilitate the application of this PDE10A radioligand in future studies. The developed PET methodologies were applied to evaluate the mechanisms of action of psychotropic drugs and can be translated into future human studies

Performance Analysis of a Four-Switch Three-Phase Grid-Side Converter with Modulation Simplification in a Doubly-Fed Induction Generator-Based Wind Turbine (DFIG-WT) with Different External Disturbances

This paper investigates the performance of a fault-tolerant four-switch three-phase (FSTP) grid-side converter (GSC) in a doubly-fed induction generator-based wind turbine (DFIG-WT). The space vector pulse width modulation (SVPWM) technique is simplified and unified duty ratios are used for controlling the FSTP GSC. Steady DC-bus voltage, sinusoidal three-phase grid currents and unity power factor are obtained. In addition, the balance of capacitor voltages is accomplished based on the analysis of current flows at the midpoint of DC bus in different operational modes. Besides, external disturbances such as fluctuating wind speed and grid voltage sag are considered to test its fault-tolerant ability. Furthermore, the effects of fluctuating wind speed on the performance of DFIG-WT system are explained according to an approximate expression of the turbine torque. The performance of the proposed FSTP GSC is simulated in Matlab/Simulink 2016a based on a detailed 1.5 MW DFIG-WT Simulink model. Experiments are carried out on a 2 kW platform by using a discrete signal processor (DSP) TMS320F28335 controller to validate the reliability of DFIG-WT for the cases with step change of the stator active power and grid voltage sag, respectively

Andreev and Single Particle Tunneling Spectroscopies in Underdoped Cuprates

We study tunneling spectroscopy between a normal metal and underdoped cuprate superconductor modeled by a phenomenological theory in which the pseudogap is a precursor to the undoped Mott insulator. In the transparent tunneling limit, the spectra show a small energy gap associated with Andreev reflection. In the Giaever limit, the spectra show a large energy gap associated with single particle tunneling. Our theory semi-quantitatively describes the two gap behavior observed in tunneling experiments.Comment: 5 pages, 4 figures, submitted to Phys. Rev. Lett. minor changes of reference