京都大学 / 新制・課程博士 / 博士(農学) / 甲第24247号 / 農博第2526号 / 新制||農||1094(附属図書館) / 学位論文||R4||N5418(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 井上 和生, 教授 谷 史人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Programmed cell death is a widespread phenomenon in the developing nervous system. During early development, neurons are initially produced in excess and up to 70% of them are eliminated in later stages of development, during a period of synapse formation with their targets. However, the mechanisms that initiate the death of neurons are not clear. In the avian ciliary ganglion, neurons go through the period of target-dependent cell loss between E8 and E14; however, almost all neurons in the ganglion are prevented from dying by the chronic in ovo treatment with α7-nAChRs specific antagonists, α- bungarotoxin or MLA. Since α7-nAChRs are implicated in the cell death of ciliary ganglion neurons, I tested whether the activation of these receptors directly on the ciliary ganglion neurons facilitates cell death by inducing large increases in intracellular Ca2+. I found that the ciliary ganglion neurons are heterogeneous with respect to their surface α7-nAChR density and, as a result, activation of these receptors by nicotine leads to large increases in [Ca2+]i in some neurons but not in others. Furthermore, immature E8 neurons exhibit slower rates of Ca2+ decay after nicotine stimulation than E13 neurons, suggesting that E8 neurons do not clear [Ca2+]i efficiently and could be more susceptible to Ca2+ overload. Expressing the αbtx that is tethered to the cell membrane via the glycosylphosphatidylinositol anchor (GPIαbtx) in the ciliary ganglion neurons inhibits the increases in [Ca2+]i induced by nicotine through α7-nAChRs specifically. This cellautonomous inhibition of α7-nAChRs prevents cell death of ciliary and choroid neurons. For this to happen, GPIαbtx must be expressed in neurons; the expression of this construct in the surrounding non-neural tissue does not prevent neuronal loss in the ciliary ganglion. Later in development, α7-nAChRs are prevented from inducing cell death by the chicken PSCA molecule that is significantly upregulated in the ciliary ganglion between E8 and E15. The chicken PSCA is neuronal specific molecule that belongs to the Ly-6/neurotoxin superfamily that includes αbtx and lynx1 and compared to other tissues, it is highly expressed in the ciliary ganglion. The expression of the PSCA mRNA in tissues correlates with the expression of α7-nAChR mRNA, suggesting that PSCA modulates the signaling via these receptors. In fact, overexpressing the PSCA in the ciliary ganglion neurons prevents nicotine-induced increases in [Ca2+]i through α7- nAChRs. Misexpressing the PSCA in E8 ciliary ganglion prevents choroid but not ciliary neurons from dying. Therefore afferent inputs can induce cell death by activation of α7- nAChRs in the developing ciliary ganglion by increasing the [Ca2+]i over the threshold for cell death. Upregulation of endogenous prototoxins, such as PSCA, opposes the large increases in [Ca2+]i via α7-nAChRs and prevents these channels from facilitating cell death after the final numbers of neurons have been established. These results indicate that the control of cell death is more complex than originally proposed by the neurotrophic hypothesis and present the mechanism by which cell death in the developing ciliary ganglion is regulated, thus, further highlighting the importance of non-traditional roles of α7-nAChRs during the development of the nervous system.

>Magister Scientiae - MSc / Determination of trace quantities of boron is required in various studies of materials and in geology. Using 11B(p, α)8Be nuclear reaction with focused proton beam of 670 keV energy is one of the few microanalytical techniques capable of achieving detection limits in the 5-10 ppm range. The set-up for the determination of boron with high sensitivity was developed at iThemba LABS nuclear microprobe (NMP) facility by using a PIN photodiodes detector. The trace elements such as boron and lithium were analyzed using the nuclear reaction analysis (NRA) method. This project aims at performing microanalysis of boron by NRA method and reaching the developmental phase in which routine, non-destructive boron analyses would be possible at detection limits below 5 ppm. A 228Th source was used for energy calibration of the detector. Five set of standards and unknown samples with a wide range of boron concentrations were analyzed in order to demonstrate the effectiveness of the 11B(p, α)8Be reaction by NRA technique. The standards were used to calibrate the boron yield with respect to their concentration in order to determine the boron concentration of the unknown specimen from the calibrated curve. The determination of the detection limit was also addressed. The concentrations of boron from Mts+Tu 950 glass samples were ranging between 0.17-1.05 wt % and the detection limit of 8.6 ppm for the minimum counts of 100 for 1μC accumulated charge was obtained.

Typical Parkinson’s disease (PD) is thought to be caused by a combination of genetic and environmental factors. α-Synuclein (SNCA) is central to PD pathogenesis; however, functions of SNCA outside the brain remain largely unknown. We, and others, have found that wild-type Snca expression confers anti-microbial effects in mice by reducing the severity of viral infections. Our aim is to further characterize a role of SNCA in systemic and brain health of the host during infection. We hypothesize that SNCA plays a role in innate defenses and that SNCA gene dosage will modulate outcomes of infection in the brain following pathogen exposure. Intranasal delivery of reovirus in mouse pups causes systemic illness, leading to encephalitis. In this study, intracranial inoculations of reovirus are used to differentiate the relative contribution of Snca-mediated protection in the brain versus the periphery. Two outcomes are monitored: survival and viral titres in select organs. When comparing wild-type Snca, heterozygous, and knock-out mice, I found that Snca expression did not confer any protection with respect to survival or regarding viral brain titres. These results are paralleled by cellular overexpression models. Unexpectedly, the anti-viral property of Snca, which was previously observed systemically with three distinct dsRNA viruses, did not extend to a paradigm where neural cells were directly exposed to reovirus. These results suggest a complex, anti-viral role for Snca in host defenses that may be mediated, in part, outside the central nervous system. Future studies will address whether this occurs in peripheral neurons or cells of hematopoietic lineages.

Previous studies have suggested that T cell contact‐dependent signaling of macrophages (MΦ) is mediated by membrane tumor necrosis factor‐α (memTNF‐α), based on the observation that anti‐TNF‐α could inhibit T cell‐mediated MΦ activation. The current report confirms that anti‐TNF‐α does inhibit activation of interferon‐γ (IFN‐γ)‐primed MΦ by paraformaldehyde‐fixed activated T cells. However, the involvement of membrane molecules other than memTNF‐α in the contact‐dependent signaling is suggested by two lines of evidence. First, the TH2 clone, AK8, displayed neither secreted TNF‐α/β nor memTNF‐α/β detectable by bioassay or immunofluorescence. Nonetheless, AK8 cells were equally effective, on a per cell basis, in contact‐dependent signaling of MΦ activation as TH2 and TH1 cells which do express memTNF‐α. Second, the expression of memTNF‐α by the TH clone, D10.G4, is maximal 24 h after activation, whereas the ability of this clone to activate MΦ is maximal at 6–8 h of activation and declines thereafter. Since TNF‐α is known to play a critical role in activation of MΦ effector function, it was hypothesized that T cell membrane components other than memTNF‐α might signal MΦ production of TNF‐α, thus allowing autocrine TNF‐α stimulation of MΦ effector function. In support of this, it is demonstrated that paraformaldehyde‐fixed activated TH2 cells can induce de novo production and release of TNF‐α by MΦ. This effect was not an artifactual result of paraformaldehyde fixation since paraformaldehyde‐fixed resting T cells did not induce TNF‐α gene expression. Previous studies have demonstrated a role for autocrine TNF‐α stimulation in LPS induction of effector function in recombinant IFN‐γ‐primed MΦ. The current study confirms that TNF‐α plays a critical role in T cell contact‐dependent signaling of MΦ but indicates that memTNF on the T cells may not be a sine qua non factor for contact‐dependent signaling. The data suggest that other T cell membrane molecules contribute to activation of MΦ effector function by stimulation of MΦ TNF‐α production.

In view of the unexpected behavior of α-(benzenesulfonyl)-acetophenone toward hydrolysis and because of the possible physiological importance of its derivatives it was deemed of interest to make a further study of the hydrolysis of this compound. It was decided to study both the acid and basic hydrolysis of this compound. The problem consisted of finding a satisfactory means of analyzing the hydrolysis products, and carrying out the hydrolysis under the different conditions.

En este artículo usamos la definición de los conjuntos (α,β)-sg-abiertos para definir la (α,β) -sg-compacidad y la (α,β)-sg-conexidad de un espacio topológico (X, T) sobre el cual se tienen operadores α ,β asociados a T. Se estudian y se caracterizan los espacios (α,β)-sg-compactos y los espacios (α,β)-sg-conexos además buscamos condiciones bajo el cual se preserva la imagen de espacios (α,β)-sg-compactos y (α,β)-sg-conexos mediante funciones.

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A lo largo del tiempo, con el incremento de la población mundial y la necesidad de producción de mas energía para satisfacer las necesidades del día a día, ha habido un creciente interés a nivel mundial por encontrar soluciones al problema de la eliminación de óxidos de nitrógeno (NOx), y otros residuos gaseosos, en fuentes móviles y estacionarias ya que estos gases son contaminantes atmosféricos que contribuyen a la formación de la lluvia ácida y de la niebla tóxica fotoquímica. La aplicación de los catalizadores de tres vías ha permitido controlar con éxito la emisión de gases de escape de motores que consumen naftas como combustible. La actual generación de estos catalizadores usa principalmen-te Pd como metal activo para las reacciones de oxidación y reducción, recurriéndose al agregado de promotores para aumentar su eficiencia en la eliminación de NOx en condicio-nes oxidantes y en la operación a baja temperatura. La re-ducción de NOx con CO es una de las principales reacciones que tiene lugar sobre los mencionados catalizadores, recurrién-dose al agregado de óxidos reducibles como Cu, Cr, MoOx, La2O3 y principalmente CeOx para aumentar su efectividad. El notable aumento de la velocidad de reacción con el agregado de CeOx se ha atribuido a un aumento de la capacidad de di-sociación del NO, como así también a la mayor activación del CO, resultante de la interacción Pd-Ce. Por lo tanto, la locali-zación del Ce con relación al Pd, es un tema de considerable importancia. La influencia del Ce y el V en la actividad, selecti-vidad y estabilidad del Pd para la reacción NO + CO a 300 oC ha sido motivo de estudio en esta tesis. Para este trabajo se han preparado catalizadores con cargas de Pd y Ce inferiores al 1 %, agregando el promotor al catalizador Pd/g-Al2O3, mientras que la influencia del V se ha estudiado sobre una muestra donde el Pd fue incorporado a una alúmina dopada previamente con el promotor. En la caracterización de las muestras frescas y usadas hemos aplicado las técnicas de AA, ICP, quimisorción de H2, TEM, XPS, TPR y FTIR de CO y NO adsorbido. En general, los estudios publicados sobre la in-fluencia del Ce en la reacción NO + CO sobre Pd, fueron reali-zados sobre catalizadores con una carga de Ce igual o mayor a la necesaria para formar una monocapa sobre el material soporte, depositada antes del agregado del Pd. También, a diferencia de otros trabajos se han utilizado concentraciones más bajas de NO y CO. Continuando estudios previos de nues-tro laboratorio, se ha investigado el efecto de VOx como óxido reducible, demostrándose que conduce a una disminución de la selectividad a N2 favoreciendo la formación de N2O. Este comportamiento se ha explicado por la existencia de un esta-do de oxidación del Pd en condiciones de reacción, detectado por FTIR y XPS, que no se presenta en ausencia del promo-tor. Si bien la reacción de reducción de NO con CO es aparen-temente simple, desde el punto de vista de las etapas elemen-tales requeridas para la formación de N2, N2O y CO2, a baja temperatura se forman especies adsorbidas estables a partir de las cuales se generan productos secundarios. La identifi-cación de dichas especies y productos, y su influencia en la actividad de los catalizadores se ha logrado mediante la aplicación de espectroscopía FTIR a la caracterización de los catalizadores en condiciones de reacción y el correspondiente análisis de la fase gas. El estudio de las especies adsorbidas en condiciones de reacción se efectuó alimentando a la celda de IR la corriente de salida del reactor a 300 oC, la cual cir-culó en contacto con una pastilla de catalizador. Es bien co-nocido que la reacción NO + CO da lugar a la formación de la especie isocianato, que puede ubicarse sobre el soporte y/o sobre el metal pero su participación en la selectividad de la reacción global es aún motivo de discusión. Como se demues-tra en esta tesis otras especies adsorbidas estables son generadas a partir del isocianato con la participación de los grupos OH del material soporte, como el ácido isociánico (HNCO). La posterior hidrólisis de este ácido da lugar a la formación de NH3, proceso que se intensifica en presencia de H2O. Poniendo especial atención al proceso de formación descomposición de las especies adsorbidas como así también a su reactividad con NO se ha comprobado que dichas especies no participan de la formación de N2O sobre Pd. / During these times, together with the world population increase and the consequently mounting needs of energy production to satisfy the demands of day after day, there has been an upsurge of interest worldwide in finding solutions to the problem of the elimination of gaseous contaminants. Nitrogen oxides (NOx), and other gaseous residues, derived from mobile and stationary sources are atmospheric pollutants that contribute to the formation of the acid rain and the toxic photochemical fog. The application of three way catalysts has allowed us to control successfully the gas emissions of gasoline engines. The current generation of these catalysts uses mainly Pd as active metal for the reactions of oxidation and reduction, with the addition of promoters to increase their efficiency for NOx s elimination under oxidizing conditions in the low temperature range.NOxs inreduction with CO is among the main reactions that take place on the mentioned catalysts, with the addition of reducible oxides as Cu, Cr, MoOx, La2O3 and principally CeOx. The notable increase of reaction rate with CeOx addition has been attributed to a higher NO dissociation activity and to CO activation due to a PdCe interaction. Therefore, Ce location related to Pd is a topic of considerable importance. The influence of Ce and V on the activity, selectivity and stability of Pd for the NO + CO reaction at 300 C has been studied in this thesis. In this work catalysts with Pd and Ce loadings lower than 1 % have been prepared by adding the promoter to the Pd/g-Al2O3 catalyst, whereas V influence has been studied on a sample where Pd was incorporated into an alumina previously doped with the promoter.In the published literature about the influence of Ce on the NO + CO reaction over Pd, the catalysts had a Ce amount higher than the quantity needed to form a monolayer, which was added before Pd. Another difference with other studies is the use of lower concentrations of CO and NO in the reactive mixture. For the characterization of fresh and used samples the techniques of AA, ICP, H2 chemisorption, TEM, XPS, TPR and FTIR of adsorbed CO and NO were used. As a continuation of our previous laboratory studies, the effect of VOx as reducible oxide has been investigated, in order to demonstrate that it leads to a decreasing N2 selectivity, thus favoring the N2O formation. This behavior has been explained by the existence of an oxidized Pd state under reaction conditions, which was detected by FTIR and XPS measurements; this state is not observed if the promoter is absent. Although the reaction of NO reduction with CO is apparently simple from the viewpoint of the elementary steps involved in N2, N2O and CO2s formation, at low temperature adsorbed stable species are formed thus leading to secondary products. The identification of the above mentioned species and products and the determination of its influence in the catalysts activity have been carried out by the application of FTIR spectroscopy for the characterization of the catalysts under reaction conditions together with the corresponding analysis of the gas phase. The study of the species adsorbed under reaction conditions was accomplished by feeding the stream emerging from the reactor at 300o to the IR cell that contained a catalyst disk kept at the same temperature. It is well-known that the NO + CO reaction on supported metals gives place to the formation of isocyanate species, which can be located on the support and/or on the metal although its participation in the selectivity of the global reaction is still under discussion. As demonstrated in this thesis, other adsorbed stable species are generated from the isocyanate by the participation of -OH groups of the material support, like isocyanic (HNCO) acid. The hydrolysis of this acid gives place to NH3 formation, which is a process that is enhanced by the H2O presence. Paying special attention to the process of formationdecomposition of the species adsorbed and also to their reactivity with NO, it was verified that the adsorbed species do not take part in N2O formation on Pd.

This diploma thesis deals with application of newly synthesized α-cyclodextrins derivatives, 2I -O-cinnamyl-α-cyclodextrin and 3I -O-cinnamyl-α-cyclodextrin, in capillary electrophoresis. Their unique feature lies in formation of cyclodextrin aggregates in an aqueous solution by inclusion of phenyl moiety of one molecule into cavity of another one. The influence of addition of 2I -O-cinnamyl-α-CD and 3I -O-cinnamyl-α-CD to background electrolyte (BGE) and its impact on effective mobilities of eighteen selected analytes were tested. Nine analytes were measured in the form of cations (aniline, antipyrine, L-histidine, D,L-tyrosine, D,L- phenylalanine, N-(1-naphtyl)ethylenediamine, 4-nitroaniline, p-aminoaceto-phenon and tyramine) and nine in the form of anions (N-acetyl-D,L-phenylalanine, N-acetyl-D,L-tryptophan, N-benzoyl-D,L-phenylalanine, N-boc-D,L-tryptophan, N-FMOC-D,L-valine, N-FMOC-alanine, N-FMOC-D,L-leucine, D,L-3-phenyllactic acid and (R)-(-)-mandelic acid). Electrophoretic mobilities of cations were tested in BGE at pH 2.2 and anions at pH 8.0. The measurements were conducted at 25 and 50 řC. At the beginning the buffer containing 2.5 mM TRIS was adjusted with phosphoric acid to pH 2.2. However, it was found, that phosphate anions might enter cyclodextrin cavity and disable potential...