Nuclear power plants rely on the Emergency Core Cooling System (ECCS) to cool down the reactor core in case of an accident. Occasionally, air is entrained into the suction piping of ECCS causing voids that decrease pumping efficiency, and consequently damage the pumps. In an attempt to minimize the amount of voids entering the suction side of the pump in ECCS, a Void Recirculation System (VRS) experiment was conducted for a proof of concept purpose. While many studies have been oriented in studying two-component flow behavior in ECCS, none of them propose a solution to minimize air entrainment. As a consequence, there are no simulation models that use computational fluid dynamics to address gas entrainment solutions in ECCS. The objectives of this thesis are to (1) simulate and investigate two-component air-water flow in a VRS that minimizes the amount of air in piping systems, using RELAP5/MOD3 as the computational tool, and (2) to validate the numerical results with respect to experimental results and observations. A one-dimensional model of the VRS was built in RELAP5, in which eight different scenarios (replicating those from the VRS experiment) were simulated for a period of 150 seconds. Four Froude numbers of 0.8, 1.0, 1.3 and 1.6 were evaluated in two different pipe configurations, and the experimental data obtained from the VRS experiment was used to validate the numerical results obtained from these simulations. It was concluded that air recirculation occurs indefinitely throughout the entire 150 seconds of the simulation for Froude numbers up to 1.3; while for a Froude number of 1.6, air recirculation occurs for approximately 100 seconds and ceases after 125 seconds of the simulation. An average air reduction effectiveness of 90% was found for all simulation scenarios. The VRS model was successfully validated and can be used to investigate the effects of air entrainment in suction piping.

Unter K+-limitierenden Wachstumsbedingungen oder, in wesentlich geringerem Ausmaß, unter Salzstress synthetisiert E. coli den KdpFABC-Komplex, ein hoch-affines K+-Transportsystem (Km ~ 2μM). Die Regulation der Expression des kdpFABC-Operons erfolgt durch das Sensorkinase/Antwortregulator-System KdpD/KdpE. Ziel des ersten Teils dieser Arbeit war die Identifizierung des Stimulus, der von der Sensorkinase KdpD wahrgenommen wird. Ausgangspunkt der Untersuchungen war die Beobachtung, dass die K+-Aufnahme über das Kdp-System bei K+-Konzentrationen >5 mM inhibiert wird. Diese wichtige Eigenschaft des Kdp-Systems wurde in der Vergangenheit häufig übersehen, da die Inhibierung des Kdp-Systems bei höheren pH-Werten (pH 7,8) durch eine hohe Rate unspezifischen K+-Transports kompensiert und somit überdeckt wird. Es konnte gezeigt werden, dass einzelne Aspartat-Substitutionen in den periplasmatischen Schleifen der Sensor-Domäne von KdpD ausreichten, um die Inhibierung des Kdp-Systems bei höheren K+-Konzentrationen aufzuheben. Diese KdpD-Derivate zeigten eine, im Vergleich zum KdpD-WT, veränderte Regulation der kdpFABC-Expression bei K+-Konzentrationen >5 mM, die eine adäquate K+-Aufnahme via KdpFABC ermöglichte. Diese Ergebnisse zeigen, dass die Inhibierung der K+-Aufnahme über das Kdp-System bei K+-Konzentrationen >5 mM auf einer Inhibierung der kdpFABC-Expression durch KdpD basiert. Weiterhin konnte gezeigt werde, dass eine Abnahme der extrazellulären K+-Konzentration eine effiziente und sofortige Stimulierung der KdpD/KdpE-Signaltransduktion bewirkt. Aus diesen Ergebnissen wurde geschlussfolgert, dass die extrazelluläre K+-Konzentration als Reiz für die Sensorkinase KdpD dient. Im zweiten Teil dieser Arbeit erfolgte erstmals eine absolute Quantifizierung von KdpD und KdpE, sowie der Untereinheiten des KdpFABC-Komplexes unter induzierenden und nicht-induzierenden Bedingungen mittels hoch-sensitiver und selektiver Massenspektrometrie. Unter nicht-induzierenden Bedingungen liegt die KdpFABC-Synthese in der gleichen Größenordnung wie die KdpD- und KdpE-Synthese vor. Dieser Befund ist eine wichtige Voraussetzung für die postulierte, regulatorische Interaktion zwischen der Sensorkinase KdpD und der K+-Transportuntereinheit KdpB (Kipschull, 2011). Unter induzierenden Bedingungen stieg die KdpFABC-Synthese 100-300-fach, während eine etwa 10-fache Erhöhung der KdpD- und KdpE-Synthese nachgewiesen werden konnte. Diese Beobachtung bestätigt, dass das Zweikomponenten-System KdpD/KdpE unter induzierenden Bedingungen einer Autoregulation unterliegt. Die Autoregulation konnte durch eine räumliche Trennung des kdpFABC- und kdpDE-Operons aufgehoben werden. Die Aufhebung der Autoregulation von KdpD/KdpE hatte jedoch keinen Einfluss auf die Expressionskinetik des kdpFABC-Operons unter induzierenden Bedingungen. Der dritte Teil dieser Arbeit beschreibt die Konstruktion eines E. coli-Stamms, der eine vollständige Deletion des kdpD-Gens trägt. Nach einer zeitlichen Verzögerung konnte in dem daraus resultierenden E. coli-Stamm (LB2240ΔkdpD) unter K+-Limitation eine KdpD-unabhängige Expression des kdpFABC-Operons nachgewiesen werden. Die kdpFABC-Expression befähigte diesen Stamm, in Abwesenheit von KdpD unter K+-Limitation zu wachsen. Es konnte gezeigt werde, dass das K+-limitierte Wachstum von LB2240ΔkdpD eine Phosphorylierung von KdpE voraussetzt, wobei Acetylphosphat nicht als alternativer Phosphodonor diente. Da nur wenige Zellen einer LB2240ΔkdpD-Kultur den beschriebenen Phänotyp zeigten, liegt die Vermutung nahe, dass diese Zellen Träger einer Suppressormutation sind, die eine KdpD-unabhängige Phosphorylierung von KdpE und daraus folgend eine kdpFABC-Expression verursacht.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19476号 / 理博第4136号 / 新制||理||1595(附属図書館) / 32512 / 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 川上 則雄, 教授 石田 憲二, 教授 高橋 義朗 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

No description available.

Pseudomonas aeruginosa is an important opportunistic pathogen. Many P. aeruginosa virulence factors are regulated by the AlgZ/R two component system. AlgZ is the sensor histidine kinase which phosphorylates AlgR, the response regulator. AlgR activates transcription of different gene targets based upon its phosphorylation state. The genes that encode AlgZ and AlgR are transcribed in an operon. While regulation of algR expression has been well studied, regulation of algZ expression has not. Using a pilW mutant in concert with algZTF-lacZ transcriptional fusion, we conducted a transposon mutagenesis to identify algZ regulators. We identified an unknown autoregulatory loop. The type IV pilus minor pilins prevent the phosphorylation of AlgR by AlgZ . This inhibition of the AlgZ/R system subsequently down-regulates both the expression of the fimU operon and the algZ/R operon. Because AlgR regulates virulence, it is possible that virulence can also be reduced by targeting activation of the AlgZ/R system.

Pseudomonas aeruginosa is an important nosocomial infection that has the potential to infect almost every tissue of the human body though it is mainly opportunistic, due to the organism’s intrinsic antibiotic resistance is becoming increasingly difficult to treat. Two-component systems (TCS) rely on a signal sensed from the outside environment by the sensor histidine kinase to initiate phosphotransfer to the response regulator, which may then regulate virulence factors in the organism in response to a changing environment. One important two-component system in P. aeruginosa is the AlgZ/R system. AlgZ, the sensor histidine kinase, has been shown to be co-transcribed with its’ response regulator, AlgR, to affect a myriad of virulence factors, including those related to motility. Pseudomonas species is capable of four types of motility: twitching, swimming, swarming, and gliding. Twitching motility is achieved through the expression of the FimU operon and Type VI pilli, and is most useful when attaching to a solid surface in the initial step of pathogenesis: colonization. Conversely, the swimming phenotype relies on the production of a single polar flagellum upon the activation of the FleQ operon, and allows the organism to move through a fluid environment. A previously unidentified regulator of AlgZ, but not AlgR, has been identified via transposon mutagenesis screening, PA2771, which has a GGDEF domain and predicted diguanylate cyclase activity. The mechanism of PA2771’s action within P. aeruginosa has not been previously studied. The nonpolar deletion mutant was first characterized via various phenotypic assays (including biofilm, rhamnolipid, swimming, and swarming assays) and transcriptional fusions to propose a mechanism in which this predicted diguanylate cyclase (DGC) works with AlgZ to determine the switch in motility from twitching to swimming. When PA2771 is present and active in the cell, cyclic di-GMP levels should be high, leading to the production of Type VI pilli and the upregulation of the FimU operon. In the PA2771 mutant a significant decrease in the expression of the FimU operon, and an increase in the expression of the flagellar genes. Subsequent alterations in swimming and swarming phenotypes were observed, as well as the restoration of these effects via complementation studies. Overexpression of AlgZ in the 2771 mutant showed a restoration of AlgZ expression in the nonmucoid background, and the predicted DGC activity was indirectly verified via a cdrA-lacZ transcriptional fusion.

The morphogenesis of C. albicans is a major aspect of its virulence and is regulated by quorum sensing (QS) molecules they produce, as well as the presence of neighboring microbes.Two mutant transporters, SSU1 and CDR4, were characterized for their ability to form biofilms in the presence of cyclic-di-GMP and 3-oxo-12-homoserine lactone. While homoserine lactone showed a decrease in biofilm density of both mutants compared to the wild-type strain, wild-type and ssu1 biofilm densities increased considerably in the presence of cyclic-di-GMP while testing lower inocula. Additionally, it has been shown that C. albicans mutants lacking the hybrid histidine kinase, Chk1, are refractory to the effects of farnesol, a QS molecule that inhibits morphogenesis.We determined both CDR4 and SSU1 expression is reduced or highly repressed in the chk1, ypd1, and skn7 null strains. Our results suggest these two genes are downstream targets in a pathway regulated by Chk1p.

Signal transduction networks enable organisms to respond to environmental stimuli. Bacteria utilize two-component systems (TCSs) and phosphorelays as their primary means of signal transduction. Histidine kinase (HK) and response regulator (RR) proteins comprise these TCSs and phosphorelays. Previously, signal transduction within TCSs and phosphorelays was thought to only occur through a linear series of phosphotransfers between HKs and RRs. Recently multikinase networks have been shown to be involved in TCS and phosphorelay signal transmission. A multikinase network that includes the HKs RetS and GacS controls the switch between the motile invasive lifestyle and the sessile biofilm lifestyle of the opportunistic human pathogen Pseudomonas aeruginosa. GacS promotes the sessile biofilm lifestyle, while RetS promotes the motile invasive lifestyle via the inhibition of GacS. This inhibition occurs through three distinct mechanisms. Two of the mechanisms are dephosphorylating mechanisms and the third mechanism is a direct interaction between RetS and GacS which results in the inhibition of GacS autophosphorylation. This study examines the direct binding interaction between RetS and GacS using structural biology. We observed a heterodimeric RetS-GacS complex in which the canonical homodimerization interface was replaced with a heterodimeric interface. Heterodimerization between bacterial HKs is currently a novel observation, but it is likely that other HKs heterodimerize. The RetS-GacS direct interaction can serve as a model for HK-HK binding in multikinase networks. / Doctor of Philosophy / The way in which bacteria assess and respond to their environment is of great interest to microbiologists. Bacteria transmit environmental signals via protein interactions. Some of these interactions involve the transfer of phosphate groups, and some involve a direct binding interaction between proteins. We are investigating a direct binding interaction between two proteins, RetS and GacS. These proteins control whether Pseudomonas aeruginosa, an opportunistic pathogen of humans, causes an acute infection, which is characterized by motility and invasiveness, or a chronic infection, which is characterized by a sessile biofilm lifestyle, in a human host. Through the use of structural biology techniques we have visualized the three-dimensional structure of the complex between RetS and GacS. This complex has provided insight into the role of the RetS-GacS interaction in controlling the infection state of P. aeruginosa.

<p> Preparations for an investigation of interfacial momentum transfer in two-phase two-component critical flows have been completed.</p> <p> The experiments involve the measurement of flow rates, axial pressure profiles, axial and transverse void fraction profiles, and axial wall shear stress profiles of steady-state gas-liquid critical flow in a vertical diverging nozzle. A photographic study is to be initiated to record the flow structure. The results of these experiments will be used to develop constitutive relations for interfacial momentum transfer.</p> <p> An experimental loop capable of circulating a gas-liquid mixture in a vertical test section was modified to suit the requirements of this investigation. The void fraction profiles are measured with a traversing gamma densitometer using a 20 mCi Co57 source. The wall shear stress profiles are obtained using the electrochemical method to measure the mass transfer coefficients of electrodes mounted flush with the test section wall. The liquid phase is an electrolyte and the gaseous phase can be air, nitrogen or freon. The latter is used to better approximate the densities of a steam-water flow.</p> <p> This report describes the required theory, measurement techniques, design and operation of the loop, and the experimental procedures.</p> / Thesis / Master of Engineering (MEngr)

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