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Diese Arbeit beschäftigt sich mit der cytoplasmatischen Untereinheit KtrA des K - Transportsystems KtrAB aus V. alginolyticus. KtrA gehört zur Familie der KTN/RCK Proteine. Ligandenbindung an entsprechende Proteine oder Proteindomänen ist direkt mit der Regulation entsprechender Transportsysteme verbunden. Im Verlauf dieser Untersuchung wurde KtrA mittels Affinitätschromatographie aufgereinigt und anstelle der erwarteten NAD(H)-Bindung, da die Aminosäuresequenz von KtrA den konservierten Rossman fold aufweist, eine ATP-Bindung festgestellt. Die Bindung von ATP führt zu einer Konformationsänderung des Proteins. Mittels Flowdialyse konnten Dissoziationskonstanten sowohl für ATP, als auch für andere Nukleotide ermittelt werden. Ebenfalls wurde gezeigt, dass ATP die Assemblierung des KtrAB-Komplexes unterstützt, sowohl unter denaturierenden, als auch unter nativen Bedingungen. Der Bedarf an ATP konnte letztlich durch Energiekopplungsexperimente in vivo bestätigt werden.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20242号 / 医博第4201号 / 新制||医||1020(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 渡邊 直樹, 教授 岩井 一宏, 教授 楠見 明弘 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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It is well known that TCP performs poorly in wireless mesh networks. There has been intensive research in this area, but most work uses simulation as the only evaluation method; however, it is not clear whether the performance gains seen with simulation will translate into benefits on real networks. To explore this issue, we have implemented ATP (Ad-hoc Transport Protocol), a transport protocol designed specifically for wireless ad hoc networks. We have chosen ATP because it uses a radically different design from TCP and because reported results claim significant improvement over TCP. We show how ATP must be modified in order to be implemented in existing open-source wireless drivers, and we perform a comprehensive performance evaluation on mesh testbeds under different operating conditions. Our results show that the performance of ATP is highly sensitive to protocol parameters, especially the epoch timeout value. To improve its performance we design an adaptive version that utilizes a self-adjustable feedback mechanism instead of a fixed parameter. A comprehensive measurement study demonstrates the advantages of our adaptive ATP under various operating conditions. For networks with high bit-rate, low quality links, our adaptive version of ATP demonstrates an average of more than 50% gain in goodput over the default ATP for a single flow case. With respect to fairness, the adaptive ATP generally outperforms the default ATP by an order of magnitude in most results.

The Food Safety Modernization Act (FSMA), specifically the Produce Safety Rule (PSR), requires growers to clean and sanitize food-contact surfaces to protect against produce contamination. The ATP monitoring device is a potential sanitation tool to monitor the efficacy of an on-farm cleaning and sanitation program that could help growers meet regulatory expectations mandated by PSR. The ATP device uses bioluminescence to detect all ATP (found in bacteria and produce matter cells) from a swabbed surface. Little work has been done to test the efficacy of these tools under postharvest conditions. The present study evaluated ATP measurement for postharvest surface cleanliness evaluation. Concentrations of leafy greens (spinach, romaine, red cabbage) (with/without L. innocua) were used as organic matter inocula onto stainless steel, HDPE plastic, and bamboo wood coupons to represent postharvest surfaces. The ATP levels on the coupons were measured using swabs and ATP monitoring device. Results showed that the concentration of L. innocua and leafy greens on a surface had a highly significant effect on the ATP device reading (PL. innocua at 4.5 log CFU/coupon where the ATP device could no longer detect ATP from L. innocua. The type of leafy green on a food-contact surface did not affect the ATP reading (P=0.88). Leafy greens with L. innocua had a higher ATP reading when compared to saline and L. innocua, demonstrating the presence of leafy green matter contributes to ATP reading when combined with L. innocua. The different food-contact surfaces had different ATP readings (P=0.03) and the ATP device did not detect bacterial or leafy green ATP from bamboo wood surfaces (P=0.16). Based upon our results, ATP measurement is an appropriate tool to measure produce or bacterial contamination on stainless steel or HDPE plastic surfaces, however it is not recommended for wood surfaces.

Stora Enso is a well-established provider of renewable solution packaging and board materials.Board products for liquid packaging and folding cartons for food are particularly sensitive tothe microbiological contaminants. In the manufacturing process, microbiological growth occursdue to environmental favourable conditions. Most of the microorganisms eventually die in theboard machine. The microflora in the process consists mainly of bacteria from genera Bacillusand Paenibacillus, and these species have a unique characteristic of forming endospores. Thespores are non-vegetative capsules transformed from the dying bacteria, with its purpose ofprotecting the bacterial genome from the unfriendly environment as it passes through in theboard machine. The levels of spores found in the board products are usually low, but suddenpeaks of spores in the board products are unwanted for food and health safety. Bacterial andspore cultivation is a standard method to investigate microbiological activity. The method isinaccessible due to 48-hour incubation time for obtaining test results and does not provide anyoverview in a short time frame of where in the process that might triggered sporulation. In thelong run, it can cause production losses that affect the prospects of the board production.The purpose of this study is to investigate a new analysis method to facilitate identification andquantification of microbiological activity with ATP bioluminescence (AdenosineTriphosphate) measurement and redox potential, and as well investigate the main process sitethat possibly causes the issue. ATP bioluminescence measures the concentration of ATP inrelation to the number of bacterial cells. The study was limited to the broke system due to theclosed system and accumulation of microorganisms. The broke system manages the reuse ofdowngraded new paper board. For the laboratory work, 15 process positions were selected inthe broke system. Pulp were sampled from each process position for each laboratory workday,and measurements of ATP bioluminescence, redox potential, pH, temperature, TOC (totalorganic carbon), retention time, and bacterial and spore cultivation were completed as well. Thecollected data were studied in a multivariate analysis and correlograms were produced for eachprocess position. In addition, a parallel study of a lab scale broke tower simulation wasperformed to further investigate if the broke tower could be the source of the microbiologicalactivity.The obtained results showed low correlations between the process parameters and the totalbacterial and spore concentration. Similar results were obtained for ATP bioluminescence andredox potential, resulted in a low correlation to the total bacterial and spore concentration aswell. The simulation gave additional insight in the function of ATP bioluminescence and redoxpotential.The conclusion is that ATP bioluminescence is an accessible method due to high repeatability,however the reliability is lacking. The analysis is not reliable because of low correlationbetween the total bacterial and spore concentration. Redox potential is both a reliable andaccessible method to identify and quantify the microbiological growth in the system, becauseit signifies the environmental conditions for the bacterial growth. It has been proven in the brokesimulation. Further research is needed to understand the representation in relation to thebacterial growth behind each analysis in order to fully consider the analysing measurementapplicable. Due to low correlations between the variables in each process position, anyassumptions cannot be considered in any specific process position that might be main cause ofraised spore values in board products. / Stora Enso är en väletablerad leverantör av förnybara lösningar för förpackningar ochkartongmaterial. Kartongprodukter för vätskeförpackningar och vikkartong för livsmedel ärsärskilt känslig för mikrobiologisk aktivitet. I tillverkningsprocessen sker mikrobiologisktillväxt på grund av gynnsamma miljöförhållanden. De flesta av de vegetativa cellerna dör såsmåningom i kartongmaskinen, men en stor del av mikrofloran består av bakterier från släktenaBacillus och Paenibacillus. Dessa arter har en unik egenskap att bilda endosporer. Sporerna äricke-vegetativa kapslar som transformerats från de döende bakterierna, med syftet att skyddabakteriegenomet från den ogynnsamma miljön som passeras i genom maskinen. Nivåerna avsporer som finns i kartongprodukterna är låga, men plötsliga förhöjda värden av sporer ikartongprodukterna är oönskade för livsmedels- och hälsosäkerheten. Bakterie- och sporodlingär en standardmetod att undersöka den mikrobiologiska aktiviteten. Metoden är otillgängligvars resultat visas efter 48 timmar inkubation. Metoden ger ingen översikt var i processen somkan ha framkallats sporulering, och detta bildar en diffus överblick var i processen inom korttidsram som kan ha bidragit till sporuleringen. På lång sikt kan det orsakas produktionsförlustersom påverkar kartongproduktionens framtidsutsikter.Syftet med denna studie är att undersöka nya analysmetoder för att underlätta identifiering ochkvantifiering av mikrobiologisk aktivitet med en ATP bioluminescens (Adenosin Trifosfat)mätning och redoxpotential och även undersöka den huvudsakliga processposition som orsakardet huvudsakliga problemet. ATP bioluminescens mäter koncentrationen av ATP i förhållandetill antalet vegetativa celler. Studien begränsades till utskottssystemet på grund av slutet systemoch ansamling av mikroorganismer. För laborationsarbetet valdes 15 processpunkter ut iutskottssystemet. Utskottsmassan togs från varje processpunkt för varje laborationsdag, ochmätningar av ATP bioluminescens, redoxpotential, pH, temperatur, TOC (totalt organiskt kol),retentionstid och bakterie- och sporodling genomfördes också. De insamlade data studerades ien multivariat analys och korrelogram togs fram för varje processpunkt. En parallellundersökning genomfördes med en labbsaklig simulering av utskottstornet för att ytterligareundersöka om tornet är möjligtvis källan till den mikrobiologiska aktiviteten.De erhållna resultaten visade låg korrelation mellan processparametrarna och den totalabakterie- och sporkoncentrationerna i samtliga processpositioner. ATP bioluminescens ochredox potential erhöll låg korrelation mellan den totala bakterie- och sporkoncentrationen, mensimuleringen gav tydligare förståelse i de båda mätningarnas funktion.Slutsatsen är att ATP är en tillgänglig metod eftersom det är repeterbar. Däremot har det visatsatt den är opålitlig, på grund av den låga korrelationen mellan den totala bakteriella ochsporhalten. Redox potential är pålitlig att använda, vilket har visats i simuleringen. Denförklarar de miljömässiga förhållandena i systemet och på så sätt stärks förståelsen om denbakteriella tillväxten. Vidare forskning behövs för att förstå betydelsen i samband med denbakteriella tillväxten bakom varje mätning för att analysmetoden ska vara applicerbar. På grundav låga korrelationer mellan variablerna i varje processposition kan några antaganden intebeaktas i någon specifik processposition som kan vara huvudorsaken till förhöjda sporvärden ikartongprodukter.

Traumatic brain injury (TBI) induces a state of microglialactivation, which includes upregulation of macrophage functions and release inflammatory mediators such as certain inflammatory cytokines. Current literature suggests that interleukin-1Beta is an important cytokine mediator, which is dramatically increased after brain injury. Previous studies indicate that ATP is released by traumatically injured astrocytes and serves as a cell-to-cell mediator through purinergic receptors after in vitro injury. However, the mechanism of interleukin-1Beta release after traumatic brain injury remains poorly defined and is difficult to study using in vivo models. Using an in vitro model for traumatic brain injury (cell strain or stretch), we investigated the role of the extracellular nucleotides (ATP) in regulation of interleukin-1Beta release in rat cortical brain cells. We now report that activated microglia constitute the major source of interleukin-1Beta release after in vitro trauma. ATP is a powerful stimulus for interleukin-1Beta release from microglial cultures. Glutamate inhibits interleukin-1Beta release. ATP-induced interleukin-1Beta release was blocked completely by the P2X7 receptor antagonist, oxidized ATP, and partially by the P2X7 receptor antagonist suramin, suggesting that ATP stimulates interleukin-1Beta release from microglia via purinergic receptor and the P2X7 receptor is responsible for the interleukin-1Beta release. Blockage of interleukin-1Beta release by the purinergic receptor antagonists oATP and suramin decreased cell damage in uninjured mixed organotypic brain cell culture exposed to activated microglia. Taken together, these results suggest that interleukin-1Beta mediated inflammatory events are regulated in activated microglia by extracellular nucleotides (ATP) via purinergic receptors in central nervous system after in vitro trauma.

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