付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23116号 / 医科博第127号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 藤田 恭之, 教授 渡邊 直樹, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

>Magister Scientiae - MSc / Consolidated bioprocessing (CBP), in which a single organism in a single reactor is responsible for the conversion of pretreated lignocellulosic biomass to bioethanol, remains an attractive option for production of commodity products if an organism fit for this process can be engineered. The yeast Saccharomyces cerevisiae requires engineered cellulolytic activity to enable its use in CBP production of second generation bioethanol. Current recombinant yeast strains engineered for this purpose must overcome the drawback of generally low secretion titres. A promising strategy for directly converting lignocellulose to ethanol is by displaying heterologous cellulolytic enzymes on the cell surface by means of the glycosylphosphatidylinositol (GPI) or similar anchoring systems. Recently, a strain producing cell-adhered enzymes in a ratio-optimized manner was created that showed significant crystalline cellulose hydrolysis.

The master thesis discusses the principles, design and realization of the original device for measuring of the electromagnetic radiation scattering in the structure of solar cells. It follows the results of a previous project "Analýza optických vlastností solárných článku" (ev.n.FT-TA3/142) and as well as knowledge gained from Ing. Vladimir Grundling’s master thesis, which has been done under this project. The subject of this thesis was to make a device for measuring of the electromagnetic radiation scattering in the visible spectrum. The aim of this work is an innovation of the previous device, so that the electromagnetic radiation scattering in the near infrared spectrum can be studied. This makes the possibility to qualify the influence of the rear surface of an active part of solar cell on electromagnetic radiation scattering, i.e., on the conversion efficiency of solar energy into electric. For this reasons it was necessary to modify the device, so that we can change the radiation source and detector because of the transparency of silicon wafers for the near infrared area. The work is supported by the project „Barevné solární články s vysokou účinností pro architektonické aplikace“ (FRTI1/168) in cooperation with Solartec s.r.o.

Signal transducer and activator of transcription (STAT) is a family of intracellular proteins that are responsible for carrying the signal from the cell surface to the nucleus in response to specific ligands. Once in the nucleus, STATs activate the transcription of specific genes. To date, seven human STATs have been identified. Among these STATs, STAT3 is considered as oncogenic. It activates genes that block apoptosis and inhibits antitumor immune responses (1). STAT3 is also essential in early embryogenesis and plays a role in cell growth and survival, differentiation and apoptosis depending on the target tissue. Analysing STAT3 signalling provides insights into pathology and can be used as a tool for diagnosis, prognosis and therapy development. Traditionally, western blot has been used to analyse cell signalling but it is impractical in analysing rare cell populations or providing information at the single cell level. Moreover, it is a demanding and time consuming technique that offers qualitative and less sensitive analysis. The rapid evolution in the multi-parametric flow cytometry and the availability of both epitope specific antibodies and sophisticated software facilitate the wide application of this technology in cell signalling studies. Flow cytometry has the ability to resolve different subcellular sets in a heterogeneous population, collects data at a single cell level and correlates multiple markers simultaneously. However, it requires highly standardized protocols for maximal sensitivity. The aim of this study was to assess the dose and the time response of both total STAT3 and pSTAT3 to in vitro stimulation with either IL-6 or IL-10 in peripheral blood mononuclear cells (PBMC). This assessment was done using both the flow cytometry and the western blot techniques. The results of this study showed that lower doses of IL-6 (1 & 10 ng/ml) were not sufficient to induce phosphorylation of STAT3. However, following stimulation with 100 ng/ml of IL-6, no significant change in the level of total STAT3 could be detected in either lymphocytes or monocytes from 3 different donors using either the FC500 or the Accuri cytometer. Using the FC500 cytometer, a small but insignificant increase in the pSTAT3 was seen in the lymphocytes and monocytes. A significant increase in STAT3 phosphorylation was only observed for monocytes after 15 minutes stimulation with 100 ng/ml of IL-6 using the Accuri flow cytometer. xii When the fluorescent labelled antibodies used in the flow cytometric assays were used for western blot probing, western blot analysis of stimulated cell lysates with 100 ng/ml IL-6 detects proteins of a low molecular weight than STAT3 or pSTAT3 which may explain the flow cytometric results of IL-6 stimulation. In IL-10 stimulation experiments, lower doses (1 and 10 ng/ml) tested by flow cytometric and western blot techniques demonstrated insignificant STAT3 phosphorylation induction. Following stimulation with either 50 or 100 ng/ml IL-10, no significant change in the total STAT3 was seen in either lymphocytes or monocytes when using the Accuri flow cytometer. However, stimulation with 100 ng/ml IL-10 induces STAT3 phosphorylation from 10 minutes through 30 minutes in both lymphocytes and monocytes. Longer times were required and high inter-individual variability was noticed for the activation of STAT3 after stimulation with 50 ng/ml IL-10. By using different antibodies from those used in the flow cytometric assay; the western blot results were comparable with the flow cytometric findings following stimulation with 100 ng/ml IL-10. The addition of phosphatase inhibitors during the flow cytometric protocol didn’t show any increase in the STAT3 phosphorylation. However, using paraformaldehyde for fixation and methanol for permeabilisation significantly decreased the mean fluorescence intensity of the PE conjugated antibodies comparing to the BD commercial fixation and permeabilisation buffers. The onset and the signal intensity of “in house” chemiluminescence mixture for western blot detection of STAT3 were comparable to the commercial ECL reagent used. However, the background of the “in house” mixture increased with time and was higher than with the commercial product. Upon longer exposure, the background increased enough to cause signal loss. In spite of the number of advantages of the flow cytometric assay compared to the western blot assay, these results are highly dependent on the specificity and the selectivity of the used antibodies. Furthermore, flow cytometry requires a highly standardized protocol to be able to assess the normal level of signalling proteins which could be later applied to detect abnormalities. It is suggested that the antibodies used in the flow cytometric assay be tested by western blot to confirm their selective detection of the target protein before their use in the flow cytometric analysis. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Pharmacology / unrestricted

Human polymorphonuclear leukocytes (PMN) are a first line of defense against fungal infections. PMN express numerous pattern recognition receptors (PRR) that facilitate identification of invading microorganisms and ultimately promote resolution of disease. Dectin-1 (β-glucan receptor) is a PRR expressed on several cell types and has been studied on monocytes and macrophages. However, the role played by dectin-1 in the recognition and killing of fungi by PMN is unknown. We investigated the ability of dectin-1 to mediate human PMN phagocytosis and fungicidal activity. Dectin-1 was expressed on the surface of PMN from all subjects tested (n=29) and in an intracellular compartment that co-sedimented with azurophilic granules in Percoll density gradients. Soluble β-glucan and mAb GE2 (anti-dectin-1) inhibited binding and phagocytosis of zymosan by human PMN (e.g., ingestionwas inhibited 40.1% by 3O min, p<0.001), and blocked reactive oxygen species production. Notably, soluble β-glucan and GE2 inhibited phagocytosis and killing of Candida albicans by PMN (inhibition of killing was 54.8% for β-glucan and 36.2% for GE2, p<0.01). Our results reveal a mechanism whereby PMN dectin-1 plays a key role in the recognition and killing of fungal pathogens by the innate immune system.

We identified the C-type-lectin-like receptor, Dectin-1, as the major receptor for fungal β-glucans on murine macrophages and have demonstrated that it plays a significant role in the cellular response to these carbohydrates. Using two novel, isoform-specific mAb, we show here that human Dectin-1, the β-glucan receptor (βGR), is widely expressed and present on all monocyte populations as well as macrophages, DC, neutrophils and eosinophils. This receptor is also expressed on B cells and a subpopulation of T cells, demonstrating that human Dectin-1 is not myeloid restricted. Both major functional βGR isoforms - βGR-A and βGR-B - were expressed by these cell populations in peripheral blood; however, only βGR-B was significantly expressed on mature monocyte-derived macrophages and immature DC, suggesting cell-specific control of isoform expression. Inflammatory cells, recruited in vivo using a new skin-window technique, demonstrated that Dectin-1 expression was not significantly modulated on macrophages during inflammation, but is decreased on recruited granulocytes. Despite previous reports detailing the involvement of other β-glucan receptors on mature human macrophages, we have demonstrated that Dectin-1 acted as the major β-glucan receptor on these cells and contributed to the inflammatory response to these carbohydrates.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18289号 / 工博第3881号 / 新制||工||1595(附属図書館) / 31147 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 岩田 博夫, 教授 木村 俊作, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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Biomarkers are biological characteristics that can be observed or measured during disease conditions, and compared to the healthy state. Biomarkers have been used in medical history to study disease progression, to develop drugs, or to predict drug efficacy. However, in complex diseases such as in cancer, biomarkers vary tremendously among patients and disease stages. Cell surface receptors, proteins that are located at the cell surface and deliver external signals into the cell, are a significant group of easily-detectable biomarkers. Along with the detection of particular biomarkers related to a disease, extensive characterization of expression patterns is necessary to optimize their application. Therefore, we designed a technique to imprint or capture the expression pattern of these receptors on silver nanoparticles. We incorporated branched molecules that can simultaneously bind to the target receptors and the nanoparticle surface. To develop the technique, we used melanocortin receptor 1 (MC1R), a receptor present at high levels on the surface of melanoma cells, as a test system. We determined optimum binding of this molecule in an established melanoma cell line, WM-266-4. We also synthesized a labeled molecule that was used to estimate the number of MC1R proteins on these cells. These studies indicate that this might be a promising approach for developing sensitive and cost-effective tools to characterize cell surface receptors in studying complex diseases and cell mechanisms. / MS / Biomarkers are biological characteristics that can be observed or measured during disease conditions, and compared to the healthy state (e.g. grades of fever during infection). Biomarkers have been used in medical history to study disease progression, to develop drugs, or to predict drug efficacy. However, in complex diseases such as in cancer, biomarkers vary tremendously among patients and disease stages. Cell surface receptors, proteins that are located at the cell surface and deliver external signals into the cell, are a significant group of easily-detectable biomarkers. Along with the detection of particular biomarkers related to a disease, extensive characterization of expression patterns is necessary to optimize their application. Therefore, we designed a technique to imprint or capture the expression pattern of these receptors on silver nanoparticles. We incorporated branched molecules that can simultaneously bind to the target receptors and the nanoparticle surface. To develop the technique, we used melanocortin receptor 1 (MC1R), a receptor present at high levels on the surface of melanoma cells, as a test system. We determined optimum binding of this molecule in an established melanoma cell line, WM-266-4. We also synthesized a labeled molecule that was used to estimate the number of MC1R proteins on these cells. These studies indicate that this might be a promising approach for developing sensitive and cost-effective tools to characterize cell surface receptors in studying complex diseases and cell mechanisms.