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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23813号 / 医博第4859号 / 新制||医||1059(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 中本 裕士, 教授 花川 隆, 教授 大森 孝一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The sugarcane crop is the major agricultural activity in the coastal tableland ecosystems in Alagoas State, exerting a remarkable influence in the economical and social aspects. This activity has been continuously and intensively conducted for several years and the necessity of environmental conservation and preservation of the several natural resources in this agroecosystem makes the adoption of sustainable practices mandatory for this activity. Among these resources, soil assumes a primary role and the evaluation of its quality through the observation and analysis of physical, chemical and biological indicators that are sensitive to management practices and robust to seasonal variations may be used to define this agroecosystem sustainability. Therefore, the objective of this work was to select indicator variables of soil conservation that fulfill the criteria of sensitivity to management and robustness to seasonal variability. For this, ten composite soil samples (0-10 cm) were taken from areas grown with organic sugarcane (OS), unburned sugarcane (US), and burned sugarcane (BS), as well as from an adjacent native forest (NF) used as a reference of the undisturbed condition. Sampling was carried out at two dates: July 2005 (rainy season) and March 2006 (dry season). The following variables were analyzed: microbial biomass-C (MBC), total organic C (TOC), soil enzyme activity by the fluorescein diacetate (FDA) hydrolysis technique, the weighed mean diameter of water stable soil aggregates (WSA), and the percent of macroaggregates (PMA). MBC and TOC were higher in NF than in the cultivated areas. No differences were observed in these C pools between the three sugarcane management treatments. The response of FDA to the crop management was dependent on the sampling time. In the rainy period, the activity followed the order: NF > OS > US > BS; in the dry season, only NF differed from the other treatments. Irrespective to the sampling time, WSA and PMA decreased according with the order NF > OS = US > BS. The variables WSA and PMA were quite sensitive to discriminate between sugarcane managements, and, at the same time, robust to seasonal variations. As an additional study, we analyzed the distribution of enzyme activity (fluorescein diacetate hydrolisys) across different diameter classes of water stable aggregates in soil samples from NF and US in order to verify the relationship between these variables. It was suggested the occurrence of two pools of fluorescein diacetate catalyzing enzymes in soils from NF and US. A non-monotonic relationship was observed between aggregate diameter and FDA. For both soils, intermediate aggregate size classes (0.25 to 1.00 mm) had lower FDA values than the classes bellow and above this diameter range. / No ecossistema dos tabuleiros costeiros do Estado de Alagoas a cultura da cana-deaçúcar destaca-se como a principal atividade agrícola exercendo influência nos aspectos econômico e ambiental da região. Esta atividade tem sido praticada de forma contínua e intensa ao longo do tempo e a necessidade de conservação ambiental e preservação dos diversos recursos presentes neste agroecossistema têm exigido que a sua utilização seja sustentável. Entre os recursos existentes, o solo assume papel importante e a avaliação da sua qualidade através da observação e análise dos indicadores físicos, químicos e biológicos do solo que sejam sensíveis às práticas de manejo e resistentes a variações sazonais podem ser utilizadas para definir a sustentabilidade deste agroecossistema. Assim o objetivo deste trabalho foi selecionar variáveis indicadoras de conservação do solo que atendam aos critérios de sensibilidade ao manejo e robustez as variações sazonais. Para tanto, dez amostras compostas de solo (0-10 cm) foram coletadas de áreas sob cana-orgânica (CO), cana-crua (CC), e cana-queimada (CQ), bem como de uma mata nativa (MN) adjacente usada como referência da condição não-manejada. A amostragem foi realizada em duas datas: julho de 2005 (chuva) e março de 2006 (seca). Avaliaram-se o C da biomassa microbiana (CBM), o C orgânico total (COT), a atividade enzimática do solo pela taxa de hidrólise do diacetato de fluoresceína (FDA), o diâmetro médio ponderado de agregados estáveis em água (DMA) e a percentagem de macroagregados (PMA). O CBM e COT foram mais elevados na MN que nas áreas cultivadas, não diferindo, no entanto entre os três sistemas de manejo da cana-deaçúcar. A resposta de FDA aos manejos foi dependente da época de amostragem. No período chuvoso, os valores desta variável seguiram a ordem: MN > CO > CC > CQ; na época seca, apenas MN diferiu dos demais tratamentos. Independentemente da data de coleta, DMA e PMA decresceram na ordem MN > CO = CC > CQ. As variáveis DMA e PMA apresentaram sensibilidade satisfatória para diferenciar manejos de cana-deaçúcar e robustez à data de coleta. Após estes estudos foi analisada a distribuição da atividade enzimática medida pela hidrólise de FDA nas diferentes classes de agregados estáveis em água em amostras de solos de MN e cultivo de cana crua CC. Buscou-se com esta análise, averiguar o grau de associação entre o estado de agregação do solo e a atividade enzimática. Foi evidenciada a existência de uma compartimentalização diferenciada dos reservatórios de enzimas catalisadoras da hidrólise do FDA entre CC e MN. Observou-se uma relação não-monotônica entre estas duas variáveis. Para ambos os solos, classes de agregados intermediárias (0,25 a 1,00 mm) apresentaram menores valores de FDA que as classes abaixo e acima desta faixa de diâmetros.

The diploma thesis deals with resonance energy transfer in hydrogel matrix enviroment. In the theoretical part, all the important features of these systems and their components are summarized and all the principles for explaining the method used are described. The work also includes a short literary background research. In the experimental part, the resonance energy transfer was measured in micellar solutions and hydrogels in 0.15 M NaCl medium. The hydrogels were formed on the basis of interaction of the surfactant with the oppositely charged polyelectrolyte. As a positively charged surfactant, carbethopendecinium bromide (Septonex) was chosen and hyaluronan as the negatively charged polyelectrolyte. Perylene with fluorescein and perylene were selected as fluorescent pairs for RET. It has been found that increasing concentration of the acceptor molecule increases the intensity of RET, in other word the rate of acceptor binding to the donor. In the study of fluorescent pair of perylene with DiO was observed, that it was not appropriate to use fluorescence probe DiO at higher concentrations. Experiments were carried out to determine whether RET changes in the time from the point of mixing the donor and acceptor fluorescence probes together. This phenomenon has not been confirmed. In hydrogels, the effect of increasing surfactant concentration and molecular weight of hyaluronan was observed. The resulting analysis showed that the molecular weight of hyaluronan has a significant effect on RET efficiency in hydrogels.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20986号 / 医博第4332号 / 新制||医||1027(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 富樫 かおり, 教授 羽賀 博典, 教授 別所 和久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No / We describe a series of experiments designed to investigate the conformational transition that highly-branched polymers with ligands undergo when interacting with bacteria, a process that may provide a new sensing mechanism for bacterial detection. Fluorescent highly-branched poly(N-isopropyl acrylamide)s (HB-PNIPAM) were prepared by sequential self-condensing radical copolymerizations, using anthrylmethyl methacrylate (AMMA) and fluorescein-O-acrylate (FA) as fluorescent comonomers and 4-vinylbenzyl pyrrole carbodithioate as a branch forming monomer. Differences in reactivity necessitated to first copolymerize AMMA then react with FA in a separate sequential monomer feed step. Modifications of the chain ends produced vancomycin-functional derivatives (HB-PNIPAM-Van). The AMMA and FA labels allow probing of the conformational behaviour of the polymers in solution via Forster resonance energy transfer experiments. It was shown that interaction of this polymer's end groups with Staphylococcus aureus induced a macromolecular collapse. The data thus provide conclusive evidence for a conformational transition that is driven by binding to a bacterium.

Fluorescent labeling is a method used for visualization of various types of biomolecules including proteins and protein complexes. However, the effect of protein labeling on protein structure and functions has not been investigated so far. The goal of the diploma thesis was to examine an influence of NHS-fluorescein binding on structure and function of human carbonic anhydrase I (hCA-I). The particular aims of this work were to prepare recombinant 15N-hCA-I which was used for NMR structure analysis of carbonic anhydrase upon fluorescent labeling. Furthermore, enzyme activity was measured in order to find out a correlation between the concentration of NHS- fluorescein and protein function. In addition, the reaction mixtures were systematically analyzed by ESI FT-ICR mass spectrometry. The analysis revealed experimental conditions for fluorescent labeling of human carbonic anhydrase I with minimal effect on protein structure and function. The results of this study show that the calculation of molar excess of NHS-fluorescein cannot rely on a simple procedure provided by manufacturer. However, due to decrease of enzyme activity upon fluorescent labeling, it is better to take into count the influence of NHS-fluorescein concentration on the relative enzymatic activity. Moreover, the calculation of molar...

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Um estudo prospectivo, randomizado, duplo-cego, com grupos em paralelo e controle ativo foi conduzido para comparar os efeitos cardiovasculares da soluÃÃo aquosa de fenilefrina a 2,5% versus a 10% em pacientes diabÃticos do tipo 2, submetidos a angiografia fluoresceÃnica, e tambÃm para comparar a eficÃcia dessas soluÃÃes na dilataÃÃo pupilar. Os voluntÃrios foram atendidos no Hospital Santo InÃcio, em Juazeiro do Norte, CearÃ, e randomicamente alocados em dois grupos. Os pacientes no grupo A receberam uma gota de proximetacaÃna a 0,5%, de tropicamida a 1% e de fenilefrina a 2,5% em ambos os olhos, enquanto os do grupo B receberam proximetacaÃna a 0,5%, tropicamida a 1% e fenilefrina a 10%. A pressÃo arterial e a freqÃÃncia cardÃaca foram mensuradas cinco minutos antes e em vÃrios instantes apÃs a instilaÃÃo de fenilefrina no fÃrnice conjuntival ao longo de um perÃodo de trÃs horas. As alteraÃÃes no ritmo cardÃaco foram gravadas por uma unidade de Holter em todos os pacientes. A Ãrea pupilar foi calculada antes e sessenta minutos apÃs a instilaÃÃo de fenilefrina. Dos quarenta e trÃs pacientes envolvidos, 22 foram alocados no grupo A e 21, no grupo B. NÃo houve diferenÃa estatisticamente significante entre os grupos na mÃdia da pressÃo arterial sistÃlica apÃs a instilaÃÃo de fenilefrina. Essa observaÃÃo tambÃm foi vÃlida para todos os instantes em relaÃÃo à mÃdia da pressÃo diastÃlica, menos em um (150 minutos), onde o grupo da fenilefrina a 10% registrou valor mais alto (P = 0,0460). Um aumento significativo na mÃdia da pressÃo arterial (sistÃlica e diastÃlica - P < 0,001) foi igualmente verificado no momento da injeÃÃo endovenosa de fluoresceÃna nos dois grupos. NÃo se observou diferenÃa estatisticamente significante entre os grupos na mÃdia da freqÃÃncia cardÃaca. As mudanÃas no ritmo cardÃaco nÃo se relacionaram com o uso da fenilefrina em qualquer dos grupos. A magnitude da dilataÃÃo pupilar sessenta minutos apÃs o uso da fenilefrina nÃo foi diferente entre os grupos. Diante destes resultados, pÃde-se concluir que, apÃs uma Ãnica instilaÃÃo, a fenilefrina a 2,5% comparada à fenilefrina a 10% foi igualmente capaz de induzir e de manter adequada midrÃase em pacientes diabÃticos do tipo 2, submetidos a angiografia fluoresceÃnica. O aumento na pressÃo arterial que seguiu a injeÃÃo de fluoresceÃna, principal mudanÃa nos parÃmetros cardiovasculares analisados, provavelmente nÃo se relacionou diretamente com qualquer das concentraÃÃes de fenilefrina usadas / A prospective, randomized, double-blind study with groups in parallel and active control was conducted to compare the cardiovascular effects of aqueous solution of phenylephrine 2.5% versus 10% in diabetic type 2 patients undergoing fluorescein angiography, and also to compare their efficacy on pupillary dilation. The volunteers at the Santo InÃcio Hospital, in Juazeiro do Norte, Cearà were randomized into two groups. Patients in group A received one drop of 0.5% proxymetacaine, 1% tropicamide, and 2.5% phenylephrine in both eyes, whereas those in group B received 0.5% proxymetacaine, 1% tropicamide, and 10% phenylephrine. Blood pressure and heart rate were measured five minutes before and several times after phenylephrine eyedrop instillation for a period of three hours. Alterations in cardiac rhythm were recorded by Holter in all patients. Pupil surface was measured before and sixty minutes after phenylephrine instillation. Forty three patients were allocated into 22 patients (group A) and 21 patients (group B). There was no significant difference in the mean systolic blood pressure after phenylephrine instillation between the two groups. This observation was also valid for all but one instant where the mean diastolic blood pressure (150 minutes) was significantly higher in 10% phenylephrine group (P = 0.0460). A significant raise in mean systolic and diastolic blood pressure was registered at the moment of intravenous fluorescein injection in both groups (P < 0.001). No statistically significant difference was detected in the mean heart rate. Changes in cardiac rhythm could not be attributed to phenylephrine in either group. The amount of pupillary dilation at sixty minutes did not differ between the groups. In conclusion, after a single dose instillation, 2.5% phenylephrine compared to 10% phenylephrine was equally able to induce and keep adequate pupil dilation in diabetic type 2 patients undergoing fluorescein angiography. The raise in blood pressure following fluorescein injection was the main change observed in the cardiovascular parameters analyzed and probably did not relate to the use of any phenylephrine concentration

To effectively design systems of microchannels it is necessary for scientists and engineers to understand thermal transport characteristics of microchannels. To experimentally determine the convective heat transfer coefficient of microchannels it is necessary to measure both the bulk and surface temperature fields. This investigation aims to develop a technique, named Total Internal Reflection Fluorescent Micro-Thermometry (TIR-FMT), to measure the temperature of water within several hundred nanometers of a wall--effectively, the surface temperature of the wall. In TIR-FMT, an evanescent-wave is generated in the water near the wall. The intensity of this evanescent-wave decays exponentially with distance from the wall. A fluorophore if illuminated by the evanescent-wave can absorb a photon. Excited fluorophores subsequently emit red-shifted photons, which are called fluorescence. The probability of a fluorescent emission is temperature-dependent. Therefore, by monitoring the intensity of the fluorescence a correlation can be made to the temperature of the region of illumination. Using the TIR-FMT technique the temperature dependence of the fluorescence intensity from buffered fluorescein (pH=9.2) was determined to be 1.35%/C. TIR-FMT can be used to measure the temperature of a fluorophore solution within 600 nm of a wall across a temperature range of 12.5-55C. The average uncertainties (95% confidence) of the temperature measured was determined to be 2.3C and 1.5C for a single 1.5x1.5 and #956;m pixel and the entire 715x950 and #956;m viewfield. By spatial averaging, average uncertainties of 2.0C and 1.8C were attained with spatial resolutions of 16x16 and 100x100 and #956;m, respectively.

Στην παρούσα Ειδική Ερευνητική Εργασία μελετόνται οι φωτοφυσικές ιδιότητες γνωστών χρωμοφόρων μορίων. Συγκεκριμένα μελετάται η Coumarin 30, η Fluorescein Isothiocyanate και η Dichlorofluorescein, τόσο με φασματοσκοπία σταθερής κατάστασης όσο και με φασματοσκοπία χρονικής ανάλυσης μέσω της τεχνικής Time Correlated Single Photon Counting. Η διερεύνηση γίνεται με τα μόρια αυτά να βρίσκονται σε μορφή διαλυμάτων με στόχο τον προσδιορισμό της συμπεριφοράς τους όταν αυτά βρισκονται σε διαφορετικά περιβάλλοντα (διαλύτες). Επιπλέον ερευνώνται οι αλληλεπιδράσεις των μορίων αυτών μέσω της φασματοσκοπίας σταθερής κατάστασης και χρονικής ανάλυσης όταν βρίσκονται σε μορφή μειγμάτων. / In this Master Thesis, the phophysical properties of chromophrores are investigated. Specifically, Coumarin 30, Fluorescein Isothiocyanate and Dichlorofluorescein are studied with steady – state spectroscopy as well as with time – resolved spectroscopy via the technique of Time Correlated Single Photon Counting. The study of these molecules focuses also on solvent – solute effects. Finally, interactions between them are also studied through steady – state spectroscopy and time resolved spectroscopy.