One common limitation of enzymatic reactions is the diffusion of a substrate to the enzyme active site and/or the release of the reaction products. These reactions are known as diffusion –controlled. Overcoming this limitation may enable faster catalytic rates, which in the case of catalytic biosensors can potentially lower limits of detection of specific analyte. Here we created an artificial system to enable deoxyribozyme (Dz) 10-23 based biosensor to overcome its diffusion limit. The sensor consists of the two probe strands, which bind to the analyzed nucleic acid by Watson-Crick base pairs and, upon binding re-form the catalytic core of Dz 10-23. The activated Dz 10-23 cleaves the fluorophore and quencher-labeled DNA-RNA substrate which separates the fluorophore from the quencher thus producing high fluorescent signal. This system uses a Dz 10-23 biosensor strand associated to a DNA antenna tile, which captures the fluorogenic substrate and channels it to the reaction center where the Dz 10-23 cleaves the substrate. DNA antenna tile captures fluorogenic substrate and delivers it to the activated Dz 10-23 core. This allows for lower levels of analyte to be detected without compromising the specificity of the biosensor. The results of this experiment demonstrated that using DNA antenna, we can create a synthetic environment around the Dz 10-23 biosensor to increase its efficiency and allow for lower levels of analyte to be detected without using amplification techniques like PCR.

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京都大学 / 新制・課程博士 / 博士(農学) / 甲第23617号 / 農博第2480号 / 新制||農||1088(附属図書館) / 学位論文||R4||N5365(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 德地 直子, 教授 北島 薫, 教授 舘野 隆之輔 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Small birds likely face energetic challenges in temperate zone winters posed by cold weather coupled with food scarcity. These challenges are often assumed to occur, but are rarely experimentally tested. I hypothesized that the naturally occurring, lower abundances of food in temperate zone winters limit a bird's ability to acquire optimal energy and ultimately limit fitness. In this dissertation, I show that supplementation of food decreased mortality and improved traits potentially associated with future reproductive success of wild swamp sparrows (Melospiza georgiana), supporting the hypothesis that winter food abundance limits fitness. These results come from a replicated and controlled food supplementation experiment conducted over three years. First, I demonstrated that following food addition, immigration increased, leading to higher densities, and that all age/sex classes experienced higher survival and maintained larger energy reserves (Chapter I). Survival was positively related to energy reserves, indicating that food availability limits survival through a bird's ability to maintain sufficient fat. In addition to causing mortality in winter, food limitation of energy reserves may carry over to affect future reproductive success by influencing timing of preparations for breeding, including migration. In Chapter II, I show that swamp sparrows decreased fat reserves over each winter, despite unlimited food availability, indicating that they adaptively regulated fat reserves, potentially to balance starvation and depredation risks. Fat reserves of control birds tracked recent temperature and control birds lost muscle throughout the winter, indicating that they were limited by food and were unable to reach optimal fat levels on a daily basis. These results suggest that limitation of energy reserves by food availability can be influenced by temperature and predator abundance. Lastly, I demonstrated that food abundance limits the timing of molt in the wild (Chapter III), an unprecedented finding. Because molt, migration, and breeding typically do not overlap, early molt might lead to earlier migration and breeding. Therefore, we hypothesize that timing of molt is another mechanism by which winter food abundance can limit reproductive success. These results provide strong evidence that food availability can limit wintering temperate migrants in a variety of ways. / Ph. D.

Les rivières reçoivent de l'azote de leurs bassins versants et elles constituent les derniers sites de transformations des nutriments avant leur livraison aux zones côtières. Les transformations de l’azote inorganique dissous en azote gazeux sont très variables et peuvent avoir un impact à la fois sur l’eutrophisation des côtes et les émissions de gaz à effet de serre à l’échelle globale. Avec l’augmentation de la charge en azote d’origine anthropique vers les écosystèmes aquatiques, les modèles d’émissions de gaz à effet de serre prédisent une augmentation des émissions d’oxyde nitreux (N2O) dans les rivières. Les mesures directes de N2O dans le Lac Saint-Pierre (LSP), un élargissement du Fleuve Saint-Laurent (SLR) indiquent que bien qu’étant une source nette de N2O vers l'atmosphère, les flux de N2O dans LSP sont faibles comparés à ceux des autres grandes rivières et fleuves du monde. Les émissions varient saisonnièrement et inter-annuellement à cause des changements hydrologiques. Les ratios d’émissions N2O: N2 sont également influencés par l’hydrologie et de faibles ratios sont observés dans des conditions de débit d'eau plus élevée et de charge en N élevé. Dans une analyse effectuée sur plusieurs grandes rivières, la charge hydraulique des systèmes semble moduler la relation entre les flux de N2O annuels et les concentrations de nitrate dans les rivières. Dans SLR, des tapis de cyanobactéries colonisant les zones à faible concentration de nitrate sont une source nette d’azote grâce à leur capacité de fixer l’azote atmosphérique (N2). Étant donné que la fixation a lieu pendant le jour alors que les concentrations d'oxygène dans la colonne d'eau sont sursaturées, nous supposons que la fixation de l’azote est effectuée dans des micro-zones d’anoxie et/ou possiblement par des diazotrophes hétérotrophes. La fixation de N dans les tapis explique le remplacement de près de 33 % de la perte de N par dénitrification dans tout l'écosystème au cours de la période d'étude. Dans la portion du fleuve Hudson soumis à la marée, la dénitrification et la production de N2 est très variable selon le type de végétation. La dénitrification est associée à la dynamique en oxygène dissous particulière à chaque espèce durant la marée descendante. La production de N2 est extrêmement élevée dans les zones occupées par les plantes envahissantes à feuilles flottantes (Trapa natans) mais elle est négligeable dans la végétation indigène submergée. Une estimation de la production de N2 dans les lits de Trapa durant l’été, suggère que ces lits représentent une zone très active d’élimination de l’azote. En effet, les grands lits de Trapa ne représentent que 2,7% de la superficie totale de la portion de fleuve étudiée, mais ils éliminent entre 70 et 100% de l'azote total retenu dans cette section pendant les mois d'été et contribuent à près de 25% de l’élimination annuelle d’azote. / Rivers receive nitrogen (N) from their watershed and are the final sites of nutrient processing before delivery to coastal waters. Transformations of dissolved inorganic N (DIN) to gaseous N are highly variable and can impact both coastal eutrophication and greenhouse gas emissions. With anthropogenic N loading to aquatic ecosystems on the rise, nitrous oxide (N2O) emission from rivers should increase. Direct measurements of N2O from lake St. Pierre (LSP), an enlargement of the St. Lawrence River (SLR) indicate that although LSP is a net atmospheric source of N2O to the atmosphere fluxes are low compared to others rivers. Emissions are seasonally and inter-annually highly variable due to changes in hydrological conditions. N2O: N2 is also influenced by hydrology and lower ratios are observed in conditions of higher water discharge and elevated N charge into the ecosystem. In a cross system analysis, hydraulic load mitigates the relation between annual N2O flux and nitrate concentrations in rivers. In SLR, cyanobacterial mats colonizing low nitrate areas are a net source of N with high negative di-nitrogen (N2) fluxes. Given that fixation occurred during daylight and that oxygen concentrations in the water column were supersaturated, we hypothesize that N2 fixation is performed by the dominant cyanobacteria in anoxic micro-zone of the mat and/ or possibly by heterotrophic diazotrophs. Our estimates indicate that N fixation in the mats account for the replacement of up to 33% of the N loss via denitrification in the entire ecosystem during the study period. In the tidal Hudson River N2 production is highly variable between vegetated shallows and was associated with species-driven differences in dissolved oxygen (DO) dynamics during the ebb tide. N2 production was extremely high in invasive floating-leaved plants (Trapa natans) but was insignificant in submersed native vegetation. An estimate of summertime N2 production in Trapa beds suggests that these beds are a major seasonal hotspot for N removal. Large Trapa beds represent only 2.7% of the total area of the tidal Hudson but they remove between 70 and 100% of the total N retained in this section of the river during summer months and contribute to as much as 25% of the annual N removal.

Made available in DSpace on 2016-04-26T20:23:34Z (GMT). No. of bitstreams: 1 Hye Jin Kim.pdf: 2120590 bytes, checksum: f1402761bb7f5f4c3856720ad725672d (MD5) Previous issue date: 2015-03-09 / This work aims to investigate the legal nature as well as the legal regime of intervening limitation provided by article 40, paragraph 4, Tax Execution Law, distinguishing it from the limitation of the article 174, National Internal Tax Code. Before studying limitation itself, we delimit the requirements, highlight the importance of language to understand and to create the reality, including the legal reality, harmonize several legal system studies, conceptualize rule of law and explain the correspondent classifications. We found that there is no general theory of limitation and this figure comes from a legal and positive concept, which means, it depends on a statutory provision to verify its nature. Given this conclusion, we separate the primary rule of limitation (substantive rule) from the secondary rule of limitation (procedure rule). We defend that article 40, paragraph 4, Tax Execution Law, is constitutional, extracting from it, choosing from the many interpretations possible, the one that is compatible with the Constitution. In this context, in order to differentiate these figures, we delimited the legal regime of the substantive rule of limitation, provided for article 174, National Internal Tax Code, with the tax constitutional principles, the requirement of declaratory statute to provide about it, as well as its application before, during and after the tax execution. We also outlined the legal regime of the procedure rule of limitation, provided for article 40, paragraph 4, Tax Execution Law, with the procedural constitutional principles and tax execution principles, the requirement of ordinary law, the adoption of the reception theory, the intertemporal issues, and the applicability of this provision by analogy to other situations / O presente trabalho cinge-se à investigação da natureza jurídica, assim como do regime jurídico da prescrição intercorrente prevista no art. 40, §4º, LEF, tendo em vista a sua confusão com a prescrição do art. 174, CTN. Após a delimitação dos pressupostos, o destacamento da importância da linguagem na compreensão e na constituição da realidade, inclusive a jurídica, a compatibilização dos estudos do sistema jurídico, bem como a conceituação da norma jurídica e as classificações pertinentes, aprofundamos o estudo da prescrição. Verificamos que não há uma teoria geral da prescrição, advindo esta figura de um conceito jurídico-positivo, ou seja, depende de uma previsão legal para verificar a sua conformação. Diante desta conclusão, separamos a norma primária de prescrição (de direito material) da norma secundária de prescrição (de direito processual), pois há previsão legal neste sentido: art. 174, CTN, e art. 40, §4º, LEF. Defendemos a constitucionalidade do art. 40, §4º, LEF, tendo em vista o princípio da interpretação em conforme a Constituição. Neste contexto, com o fim de diferenciar estas figuras, delimitamos o regime jurídico da norma primária de prescrição, prevista no art. 174, CTN, com os princípios constitucionais tributários, a exigência de lei complementar para dispor sobre ela, bem como a sua aplicação antes, durante e depois da execução fiscal. Também delineamos o regime jurídico da norma secundária de prescrição, prevista no art. 40, §4º, LEF, com os princípios constitucionais processuais e de execução, a exigência de lei ordinária, a aplicação da teoria da recepção, as questões intertemporais, bem como a possibilidade de aplicação deste dispositivo por analogia