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Voltage-gated ion channels play fundamental roles in neural excitability and thus dysfunctional channels can cause disease. Understanding how the voltage-sensor of these channels activate and inactivate could potentially be useful in future drug design of compounds targeting neuronal excitability. The opening and closing of the pore in voltage-gated ion channels is caused by the arginine-rich S4 helix of the voltage sensor domain (VSD) moving in response to an external potential. Exactly how this movement is accomplished is not yet fully known and an area of hot debate. In this thesis I study how the opening and closing in voltage-gated potassium (Kv) channels occurs. Recently, both experimental and computational results have pointed to the possibility of a secondary structure transition from α- to 3(10)-helix in S4 being an important part of the gating. First, I show that the 3(10)-helix structure in the S4 helix of a Kv1.2-2.1 chimera protein is significantly more favorable compared to the α-helix in terms of a lower free energy barrier during the gating motion. Additional I suggest a new gating model for S4, moving as sliding 310-helix. Interestingly, the single most conserved residue in voltage- gated ion channels is a phenylalanine located in the hydrophobic core and directly facing S4 causing a barrier for the gating charges. In a second study, I address the problem of the energy barrier and show that mutations of the phenylalanine directly alter the free energy barrier of the open to closed transition for S4. Mutations can either facilitate the relaxation of the voltage-sensor or increase the free energy barrier, depending on the size of the mutant. These results are confirmed by new experimental data that supports that a rigid, cyclic ring at the phenylalanine position is the determining rate-limiting factor for the voltage sensor gating process. / QC 20110616

Die Ausbreitung antimikrobieller Resistenzen stellt ein zunehmendes gesundheitliches und gesellschaftliches Risiko dar. Alternative antimikrobielle Verfahren mit einem geringen Resistenzpotenzial, breiten Wirkspektrum und geringen Umweltrisiko gewinnen an Bedeutung. Ein solches Verfahren stellt die Photodynamische Inaktivierung (PDI) dar. Ihr Wirkmechanismus beruht auf der photosensibilisierten Anregung von Singulettsauerstoff (1O2), der durch oxidativen Stress zum Zelltod führt. Der für die katalytische Aktivierung des Sauerstoffs verantwortliche Photosensibilisator (PS), muss nicht in die Mikroorganismen eindringen und wird durch sichtbares Licht angeregt. Die Übertragung einer stationär vermittelten PDI auf Oberflächen erscheint daher besonders sinnvoll. In der vorliegenden Arbeit werden erstmalig zwei Ansätze untersucht, die sowohl kommerzielle Substrate als auch industrielle Standardverfahren zur Oberflächenveredelung verwenden: Eine Elektronenstrahl-Funktionalisierung von Mikrofiltrationsmembranen und eine textiltechnologische Funktionalisierung von Polyestergeweben, insbesondere Reinraumtextilien. Für die Charakterisierung der Polymeroberflächen werden neben Versuchen zur Zellviabilität, optisch-spektroskopische Methoden und erstmalig orts- sowie zeit-aufgelöste Messungen der 1O2-Lumineszenz herangezogen. Im Resultat erweisen sich beide Funktionalisierungsansätze als geeignet für eine stationär vermittelte PDI. Dabei sind die textiltechnologisch funktionalisierten Polyestergewebe besonders Wirkungsvoll und erzielen bereits nach kurzer Weißlichtbestrahlung von unter 30 Minuten eine antimikrobielle Wirkung. Die Messungen der 1O2-Lumineszenzkinetik erweisen sich als eine vielversprechende Methode eine mögliche PDI-Aktivität vorab zu bewerten und bei der Entwicklung wichtige Impulse für die Oberflächenfunktionalisierung zu setzen. Schließlich ist eine systematisierte Methodologie zur Bewertung PDI-aktiver Oberflächen ein wesentliches Resultat dieser Arbeit. / The spread of antimicrobial resistance is an increasing health and social risk. Alternative antimicrobial methods with a low resistance potential, broad spectrum of activity and low environmental risk are gaining importance. Photodynamic inactivation (PDI) is one such method. Its mechanism of action is based on the photosensitised excitation of singlet oxygen (1O2), which leads to cell death through oxidative stress. The photosensitizer (PS), which is responsible for the catalytic activation of the oxygen, does not have to penetrate the microorganisms and is excited by visible light. The transfer of a stationary-mediated PDI to surfaces therefore seems particularly useful. In the present work, two approaches are investigated for the first time that use both commercial substrates and standard industrial processes for surface modification: An electron beam functionalisation of microfiltration membranes and a textile-technological functionalisation of polyester fabrics, especially cleanroom textiles. In addition to experiments on cell viability, optical spectroscopic methods and, for the first time, spatially and temporally resolved measurements of 1O2 luminescence are used to characterise the polymer surfaces. As a result, both functionalisation approaches prove to be suitable for a stationary-mediated PDI. The textile-technologically functionalised polyester fabrics are particularly effective and achieve an antimicrobial effect after only a short white light irradiation of less than 30 minutes. Measurements of 1O2 luminescence kinetics are proving to be a promising method of evaluating possible PDI activity in advance and providing important impetus for surface functionalisation during development. Finally, a systematised methodology for the evaluation of PDI-active surfaces is an essential result of this work.

Unsafe sanitation systems poses a risk for pathogen transmission, wherefore it is important to both inactivate pathogens present in human excreta and conduct safe sanitation systems from use to end-use. The Peepoo toilet, using ammonia sanitisation, have been suggested as a low-cost sanitation solution and is implemented in schools in Kibera, an urban slum in Kenya. This master thesis aim to study the inactivation efficiency of ammonia sanitisation when treating human excreta with urea, and to quantify the risks of exposure to microbial hazards from the Peepoo sanitation system using faecal indicator bacteria. Excreta was collected from four schools in Kibera. After adding urea to mimic the inactivation of the Peepoo in the laboratory, the inactivation rate was correlated to temperature and free ammonia concentration for Campylobacter spp., Escherichia coli and Enterococcus spp.. Campylobacter spp. and E. coli both had a high inactivation rate even at low temperature and low addition of urea. Inactivation rate of Enterococcus spp. was lower and close to zero when 1.87 % urea was added for 15 °C. For Enterococcus spp. a lag-phase was observed, which was not affected by temperature but by concentration of free ammonia. For investigated bacteria, inactivation rate increased with increased temperature and free ammonia concentration. Along the Peepoo management chain, several hazardous events were identified such ascontamination during usage, handling and transportation. Sampling showed a higher contamination of Enterococcus spp. than of E. coli. Enterococcus spp. was used as a faecal indicator for Ascaris and E. coli was used as an indicator of E. coli O157:H7 in a quantitative microbial risk assessment (QMRA). Through the QMRA, the risk of infection of Ascaris and E. coli O157:H7 for one exposure event was simulated based on a Exponential and a Beta-Poisson dose-response model respectively. The risk of infection of Ascaris was around 12 % regardless of where exposure occurs, if Ascaris eggs were present. For risk for infection with E. coli O157, the simulated risks were below 10 % at almost all exposure points, with most of the high risk exposure points located in the schools. There are risks of pathogen transmission in the Peepoo management chain that should be further investigated. Ammonia sanitisation permits a high degree of microbial inactivation but to secure a safe end-product it is recommended to be kept in room temperature (24.05±0.62 °C) or higher.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20633号 / 工博第4371号 / 新制||工||1679(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 浜地 格, 教授 跡見 晴幸 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Die Onychomykose ist eine sehr häufige Erkrankung, deren Auftreten weltweit zunimmt und mit einer Pilzinfektion der Nägel einhergeht. Die Ineffizienz der verabreichte Antimykotika motiviert Bemühungen, nach alternativen Behandlungsmethoden zu suchen. Diese Dissertation untersucht die Auswirkungen der photodynamischen Inaktivierung (PDI) auf Dermatophyten und Schimmelpilze. Als Modellorganismen werden drei der Onychomykose auslösenden Pathogene untersucht: Trichophyton rubrum, Trichophyton interdigitale und der Schimmelpilz Scopulariopsis brevicaulis. Um das Potenzial der PDI gegen Onychomykose verursachende Pathogene, abzuschätzen, wurden Phototoxizitätstests mit drei Photosensibilisatoren (PS) durchgeführt: 5,10,15,20-Tetrakis(1-methylpyridinium-4-yl) porphyrintetra(p-toluenesulfonate) (TMPyP) und and 5,10,15-tris-(1-methylpyridinium-2-yl) corrolato-(trans-dihydroxo) phosphorus(V) (PCor+) sowie 4',5',7'-tetrabromo-3',6'-dihydroxyspiro[2-benzofuran-3,9'-xanthene] -1-one (Eosin G). Neben den Phototoxizitätstests wurden zeitaufgelöste Singulettsauerstoff-Lumineszenz Scans aufgenommen, die zur Verifizierung der PDI Effizienz genutzt wurden. Alle drei PS zeigen in vitro eine hohe phototoxische Wirkung. Diese konnte mit Singulettsauerstoff-Lumineszenzmessungen korreliert werden, bei denen ein hohes Singulettsauerstoff-Lumineszenzsignal erfasst wurde. An infizierten menschlichen Nägeln konnten die PS keinen phototoxischen Effekt induzieren. Singulettsauerstoff-Scans, die für einen Einblick in die zugrundeliegenden Ursachen durchgeführt wurden, zeigten fast kein Singulettsauerstoff-Lumineszenzsignal an menschlichen Nägeln. Könnten die verschiedenen bekannten Herausforderungen im Zusammenhang mit PDI an infizierten menschlichen Nägeln bewältigt werden, hätte die PDI das Potenzial, eine schnellwirkende Behandlung dieser Pilzinfektion im Zehennagel zu werden. Diese Studie zeigt erstmals den Zusammenhang zwischen der PDI-Behandlung von Onychomykose und Singulettsauerstoff. / Onychomycosis is a very common illness that befalls an increasing number of individuals worldwide and involves a fungal infection of the nails. The inefficiency of current treatments justifies the efforts to look for alternative treatment modalities. This dissertation investigates the impact of photodynamic inactivation (PDI) against dermatophytes and molds. Three of the causing pathogens of onychomycosis are under investigation: Trichophyton rubrum (T. rubrum), Trichophyton interdigitale (T. interdigitale) and the mold Scopulariopsis brevicaulis (S. brevicaulis). To assess the potential of PDI against onychomycosis causing pathogens, phototoxicity tests were performed using three photosensitizers (PSs): the cationic 5,10,15,20-Tetrakis(1-methylpyridinium-4-yl) porphyrintetra(p-toluenesulfonate) (TMPyP) and 5,10,15-tris-(1-methylpyridinium-2-yl) corrolato-(trans-dihydroxo) phosphorus(V) (PCor+) as well as the anionic 4',5',7'-tetrabromo-3',6'-dihydroxyspiro[2-benzofuran-3,9'-xanthene]-1-one (Eosin Y). Alongside the phototoxicity tests, time resolved singlet oxygen luminescence scans were conducted to serve as a control method of PDI. All three PSs proved to have a high phototoxic effect against the three fungi species in vitro. Those could be correlated with singlet oxygen measurements, where a high singlet oxygen luminescence signal was acquired. Contrary to the expectations from the in vitro experiments were the results obtained ex vivo: On infected human nails, the PSs were not able to induce a phototoxic effect. Singlet oxygen scans conducted to get insight into the reasons behind these results showed nearly no singlet oxygen luminescence signal on human nails. Addressing the various known challenges associated with PDI on infected human nails PDI would have a great impact within short time on treating the toenail fungal infection. This study, for the first time, shows the correlation between PDI treatment of onychomycosis and singlet oxygen.

Biofilme aeroterrestrischer Mikroorganismen stellen im nicht-medizinischen Bereich ein ständig größer werdendes Problem dar. Sie tragen ebenfalls wesentlich zur Verwitterung von Fassaden bei und verursachen durch die daraus resultierende Notwendigkeit der Entfernung des Biofilms und Instandsetzung der Fassaden hohe Kosten. Auch das von Biofilmen auf Innenwänden von Gebäuden ausgehende Gesundheitsrisiko für den Menschen ist schwerwiegend. Solarzellen sind aufgrund ihrer rauen Glasoberfläche ebenfalls ein bevorzugter Siedlungsort für Biofilme. Auf diesen können sie durch Verschattung die Effizienz der Solarzellen deutlich herabsetzen. Die Entfernung von Biofilmen erfordert vor allem den Einsatz von Bioziden, welche ihrerseits eine Belastung für die Umwelt darstellen und zudem oft nur begrenzte Wirksamkeit zeigen. Ziel dieser Arbeit ist daher, das Potenzial der Photodynamischen Inaktivierung (PDI) als alternative Methode zur Beseitigung oder zur Prävention der Bildung von Biofilmen zu bestimmen. Dazu wurden unterschiedliche Photosensibilisatoren (PS) bezüglich ihrer Phototoxizität auf im Rahmen dieser Arbeit etablierte phototrophe Modellkulturen in Suspension, subaquatischen und subaerialen Biofilmen untersucht. Neben der Entwicklung der Biomasse während der PDI wurde mittels spektroskopischer Methoden zudem die Aktivität der Kulturen sowie die EPS-Sekretion und die Biofilmbildung gemessen. Dabei konnte gezeigt werden, dass die kationischen PS PCor+ und TMPyP zur PDI phototropher Kulturen erfolgreich genutzt und die Biofilmbildung verhindert werden kann. Es wurde gezeigt, dass dieses Resultat ohne Aufnahme der PS in die Zellen erzielt wird. Dies ist bezüglich der Vermeidung einer möglichen Resistenzentwicklung der Mikroorganismen von entscheidender Bedeutung. Somit wurden mit dieser Arbeit die Voraussetzungen für die Entwicklung photodynamisch aktiver, antimikrobieller Oberflächenbeschichtungen zur Prävention des Wachstums aeroterrestrischer Biofilme geschaffen. / Biofilms of aeroterrestrial microorganisms are an ever-increasing problem in non-medical applications. They also contribute significantly to the weathering of facades and cause high costs due to the resulting necessity of removing the biofilm and repairing the facades. Furthermore, the health risk for humans caused by biofilms on interior walls of buildings is serious. In addition, solar cells are a preferred location for biofilms due to condensation on their rough glass surface. Growing there, they can significantly reduce the efficiency of the solar cells by shading, a problem which gains even more importance with increasing operating times of solar modules. In the removal of biofilms, biocides are prevalent. They are in turn a burden on the environment and often show only limited effectiveness. The aim of this work is therefore to determine the potential of Photodynamic Inactivation (PDI) as an alternative method to eliminate or prevent the formation of biofilms. Different Photosensitizers (PSs) were investigated with respect to their phototoxicity on phototrophic model cultures established in suspension, subaquatic and subaerial biofilms. In addition to biomass development during PDI, spectroscopic methods were used to measure culture activity, EPS secretion and biofilm formation. It was shown that the cationic PSs PCor+ and TMPyP can be successfully used for PDI of phototrophic cultures and that biofilm formation can be prevented. It has been shown that this result is achieved without uptake of PS into the cells. This is of decisive importance with regard to the avoidance of a possible resistance development in the microorganisms. Thus, this work created the prerequisites for the development of photodynamically active, antimicrobial surface coatings for the prevention of the growth of aeroterrestrial biofilms.

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Low water activity foods (LWAF), specifically nuts and dried fruits, have been generally considered safe because they do not support the growth of foodborne pathogens. However, many pathogens have been noted to survive in LWAF for considerable periods of time, and a number of recent outbreaks and recalls have implicated various types of nuts and dried fruits. The Food Safety Modernization Act requires food processors to develop preventive control plans that make ready-to-eat LWAF safer for consumers. The presented research was designed to investigate several aspects of LWAF safety by evaluating a steam process as a strategy to remove pathogen contamination from LWAF, modeling the inactivation of such treatments, and studying the thermal resistances of two E. coli strains in low-water activity solutions. Low-temperature, vacuum-assisted steam (vacuum-steam) was evaluated as a potential intervention and preventive control to remove pathogens from the surface of LWAF without using high-heat treatments that could damage product quality. The presented work examined the efficacy of vacuum-steam (<85°C) as a means to decontaminate the surface of whole macadamia nuts, dried apricot halves, and raisins from Salmonella spp., Listeria monocytogenes, and Shiga toxin-producing Escherichia coli (STEC) contamination. The low-temperature steam treatments successfully reduced all pathogens by >4 log CFU/g from the surfaces of the foods. Additionally, Pediococcus acidilactici, proved to be a surrogate organism for these pathogens and could be used to challenge and validate similar treatments within processing plants. The data were fit to models, which showed that food type significantly impacted the fit, with the Weibull model best describing bacterial inactivation kinetics on raisins and macadamia nuts, and the Gompertz model best describing reductions on the apricot halves. The models were challenged for validation of their abilities to predict times required for 3-log reductions using internal and external datasets, determining the usefulness to industry members who wish to design similar thermal treatments for LWAF. Comparing predicted values from internally constructed models to observed values generated from external data, models were shown to be limited in scope and application and could only be applied to pathogen inactivation on different LWAF or thermal processes in certain circumstances. First-order and Weibull model predictions of bacterial reductions on dried apricots had varied success in predicting times for 3-log reductions on other thermally treated LWAF. However, the models of bacterial reductions on thermally treated macadamia nuts frequently overestimated the times required for 3-log bacterial reductions for other LWAF. In an effort to understand the effect that reduced water activity has specifically on STEC, two strains were investigated for induced thermal resistance due to osmotic stress. Thermal resistance of STEC strains (O121:H19 and O157:H7) were evaluated on the basis of strain variation, culture preparation, and water activity (D- and z-values). At the lowest treatment temperature (56°C), O121 displayed greater heat resistance than O157, and the broth-grown samples exhibited greater heat resistance than the lawn-grown cells, but significant differences were not observed at higher temperatures. Samples in reduced-water activity solutions displayed reduced thermal resistance at 56°C, but the z-values were 29-43% higher than those of high-water activity samples. While water activity has been shown to impact thermal resistance of pathogens, comparisons of STEC thermal resistance according to the D- and z-values revealed that other factors also play roles in pathogen thermal resistance on LWAF. Results from the collection of experiments conclude that efficacy of thermal treatments is impacted by the physiological state of the cells, stress experienced in the food matrix, and characteristics of the food, including water activity and composition. / Doctor of Philosophy / Consumers expect foods they purchase to be safe to consume by themselves and family members, particularly those that are ready-to-eat with no additional cooking requirements. Many of these foods are low-water activity foods (LWAF), like nuts and dried fruits, with very little water content that could be used by bacteria. These foods may be preferred snack foods due to their affordability, long shelf lives, and health benefits over other types of snack foods. Until recently, LWAF were generally considered safe because they do not support the growth of foodborne pathogens due to the lack of moisture or water within the food. However, a number of recent outbreaks related to various types of nuts and dried fruits have proven that many pathogens can survive in dried foods, even if not actively growing, for considerable amounts of time. Designed to address these types of food safety issues, the Food Safety Modernization Act recognizes risks associated with foods and responded with regulations requiring food processors to take steps to make ready-to-eat LWAF, like nuts and dried fruits, safer for consumers. A popular strategy is to treat foods with heat to destroy pathogens, however the quality attributes of some nuts and dried fruits could be damaged by high-heat treatments like roasting. An alternative process uses a vacuum to form steam at lower temperatures, allowing for efficient heat transfer through water droplets to the surface of the foods, thus causing less damage to the foods without introducing too much moisture. This research evaluated how this process could be used by food processors to remove harmful bacteria from the surfaces of whole macadamia nuts, dried apricot halves, and raisins. Results indicated that the low-temperature steam treatments successfully reduced Salmonella, Listeria monocytogenes, and Shiga toxin-producing Escherichia coli (STEC) by >4 log CFU/g (>99.99%) from the surfaces of the foods. Additionally, a nonpathogenic lactic acid bacterium, Pediococcus acidilactici, exhibited similar or greater heat tolerance, which would allow food processors to use it as a substitute, or surrogate, for in-plant studies without introducing harmful bacteria into the food processing environment. Mathematical models were used to describe the trends of bacterial death due to the steam treatments, and the results indicated that the type of food significantly impacted the reduction of bacteria. The models were tested using additional data collected within our own laboratory, as well as others. Results indicated that some of the models could be used as predictors of bacterial death for similar LWAF but can only be applied with caution and consideration for the type of food and process. Additionally, two different E. coli strains associated with outbreaks (O121:H19 and O157:H7) were investigated to understand impacts of strain variation, growth method, and water activity on thermal resistance. Some differences in heat resistance were observed between the strains and between the growth methods. Additionally, the reduced water activity seemed to decrease the bacteria's ability to withstand some heat treatments. Overall, thermal resistance studies indicated that several factors, in addition to water activity, impact pathogens' development of resistance to heat treatments. The experiments' results show that there are complex relationships between bacteria and the food they inhabit. Food processors must consider these relationships in order to design the best thermal processes to make LWAF safe for consumers.