京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12822号 / 論農博第2795号 / 新制||農||1025(附属図書館) / 学位論文||H26||N4817(農学部図書室) / 31309 / 京都大学農学研究科食品生物科学専攻 / (主査)教授 喜多 恵子, 教授 三上 文三, 教授 栗原 達夫 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23332号 / 生博第450号 / 新制||生||60(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 片山 高嶺, 教授 永尾 雅哉, 教授 上村 匡 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Research was conducted on thirteen 100% cotton denim samples using an acid wash, established by Murray, to extract oligosaccharides from the cellulosic material. The oligosaccharide ion groups (+, +, and +) for molecules with degrees of polymerization between two and seven (DP2-DP7) were analyzed using liquid chromatography coupled to mass spectrometry with an electrospray ionization interface (LC-ESI-MS). The results were compared using the least-squares means in a Multivariate ANOVA (MANOVA) test followed by Univariate ANOVA and Tukey HSD tests and demonstrated that the method could correctly determine that two samples were statistically different 85.9% of the time when analyzing the amount (ng) of each of the oligosaccharide ion groups separately, and 82.0% when analyzing the total moles of monosaccharide units released. A dye extraction was performed on the denim materials and the extract analyzed using gas chromatography coupled with mass spectrometry (GC-MS). Indigo dye was present in all of the denim samples except one. When these results were combined with the two oligosaccharide statistical analyses, the discriminating power was increased to 88.5% and 85.9%, respectively. Additional cellulosic materials were also investigated including four white 100% cotton t-shirts as well as five raw cotton samples grown in Tajikistan, Uzbekistan, Egypt, Iran, and Benin West Africa. The analytical methodology gave results for the white cotton t-shirts and raw cotton samples that were inconsistent with those obtained from the denim samples.

Glycans and glycoconjugates exert myriad important biological functions, extending and diversifying the functionality of protein molecules. Extensive studies have focused on the protein and gene realms; however, due to the lack of means for external amplification and the inherent heterogeneity of glycans and glycoconjugates, their researches have not adequately informed the understanding of critical biological and pathological processes. Researchers in glycoscience have strived to bridge this gap and redefine our understanding of carbohydrate functions. Glycosaminoglycans (GAGs) represent the most highly charged and poly-disperse animal glycans. GAGs exist on the surfaces of most mammalian cells and in the extracellular matrices. They play critical roles in anticoagulation, angiogenesis, inflammation, metastasis, cell proliferation and differentiation. Heparin and heparan sulfate (HS) are the most highly sulfated and structurally diverse GAGs, regulating a variety of cell functions by interacting directly with many growth factors and their receptors. Examples include fibroblast growth factor, bone morphogenetic protein and Wnt. These interactions rely on the unique structural properties of HS/heparin molecules. Extracellular enzymes (Sulfs and heparanase) also alter the fine structure of HS molecules. In order to investigate to the correlation between structure and function for mature HS/heparin chains, we employed mass spectrometry (MS) coupled with liquid chromatography (LC) as a sensitive and robust platform for composition profiling and detailed structural characterization. We developed a novel HPLC-chip based LC-MS platform to enable HS oligosaccharide profiling. In this thesis work, the chip LC-MS platform was improved for effective and informative tandem MS for HS oligosaccharides. We also advanced electron-based ion dissociation methods for more detailed and reliable sequence determination of HS oligosaccharides. These newly developed methods enable the investigation of the HS/heparin structural changes induced by HS extracellular remodeling enzymes, human Sulfs and heparanase. Application of the methods revealed the recognition preferences of these remodeling enzymes at the oligosaccharide level and led to the discovery of a novel peeling reaction induced by the 3-O-sulfation at the reducing end of HS saccharides.

Introduction: The incidence of food allergy has increased substantially in developed countries, with limited treatment and/or prevention options. Milk oligosaccharides have shown to modulate immune responses by serving as prebiotic substrates for the intestinal microbiota. However, some studies suggest that oligosaccharides may exert direct immunomodulatory effects, suggesting their therapeutic potential in preventing allergic diseases. We hypothesized that specific milk oligosaccharides including 6’sialyllactose, 2’fucosylactose, 3’sialyllactose and lacto-N-neotetraose may directly exert immunomodulatory effects on dendritic cells (DCs) and epithelial cells (ECs) by altering their phenotype and/or function in vitro. Methods: The effects of milk oligosaccharides (MOs) on bone-marrow derived DCs and the T84 and MODE-K epithelial cell lines were studied via direct treatment, in vitro. The expression of immunomodulatory cytokines and maturation markers were assessed to measure the effect of MOs on DC phenotype. Pro- and anti-inflammatory cytokines as well as NFκB p65 activity were measured to assess the effect of MOs on DC and EC function. In addition, in vitro stimulation of CD23 with IgE-Antigen complexes were used to study the effects of MOs on ECs in relation to allergy. Lastly, inhibitory antibodies for Siglec-F and PPARγ were used to elucidate the mechanism used by specific MOs to exert their effects. Results: Of the oligosaccharides studied, 6’siallylactose has direct immunomodulatory effects on DC phenotype and on DC and EC function at high concentrations. 6’sialyllactose increased DC expression of IL-10 and HO-1; it also increased CpG- and LPS- induced IL-10 release and decreased IL-12p70 release. Blocking the PPARγ receptor with GW9662 resulted in attenuation of this latter effect on IL-12p70 release. 6’sialyllactose reduced TNF-α induced IL-8 to a small but statistically significant extent and mKC to a great extent in T84 and MODE-K cells, respectively. In addition, 6’sialyllactose reduced IgE-Antigen stimulated release of IL-8 and CCL20, as well as NFκB p65 activity. Pre-treatment of cells with GW9662 resulted in attenuation of the effect of 6’SL on IL-8 release and p65 activation. In addition, 2’fucosylactose reduced CCL20 release and NFκB activity substantially, but these effects were not exerted via PPARγ. Conclusion: Some oligosaccharides are able to directly modulate the inflammatory response in DCs and ECs, via pathways involving PPARγ activation and/or NFκB inhibition. / Thesis / Master of Science (MSc)

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The impact of yeasts on food production, quality and safety is closely linked with their ecology and biological activities. Recently, as a consequence of the relationship between diet and health, yeasts are becoming relevant as new probiotics or for the production of bioactive compounds. In dairy products, yeasts play a key role in proteolysis, lipolysis and lactose fermentation during cheese ripening, promoting the development of sensory properties, particularly aroma. This thesis focuses on the yeast diversity in artisanal cheeses produced in the Natural Park Serra d¿Espadà (Castelló) from ewes¿ and goats¿ raw milk. Different molecular techniques have been employed in order to characterize yeast isolates. Moreover, the succession of species along the cheese ripening process was studied. The intraspecific variability of the most abundant identified species Debaryomyces hansenii and Kluyveromyces lactis was also assessed. Additionally, the potential of Kluyveromyces marxianus and K. lactis ß-galactosidases to synthetize prebiotic oligosaccharides from lactose and lactulose was tested. Finally, Kluyveromyces and Debaryomyces isolates were investigated for the production of cheese aromatic compounds. / Padilla López, B. (2014). Yeast diversity in artisanal cheeses: biotechnological applications [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/36065

Les objectifs expérimentaux de ce projet étaient en premier lieu de comparer la digestibilité d'une source de cuivre (Cu) et de zinc (Zn) organiques (protéinate de minéral) par rapport à une forme inorganique (sulfate), et en deuxième lieu de vérifier l'impact des mannan-oligosaccharides (MOS) sur la digestibilité des minéraux, le pH intestinal et la production d'acides gras volatiles. Vingt-huit porcs d'environ 25 kg ont reçu aléatoirement quatre traitements enrichis avec 10 ppm de Cu et 100 ppm de Zn. Les traitements impliquaient l'utilisation d'une source inorganique ou organique, et l'absence ou la présence de mannan-oligossacharides. Les résultats montrent que la source organique a amélioré la digestibilité (P<0,01) et l'utilisation nette (P<0,01) du Cu. En revanche, les MOS n'ont eu comme seul effet de montrer une tendance (P<0,10) à augmenter la digestibilité et l'utilisation nette du Cu en présence d'une source inorganique de minéraux. Bref, la source organique a permis une meilleure digestibilité et utilisation du Cu, alors que le MOS n'a engendré aucune modification de l'environnement intestinal qui aurait pu modifier de façon significative l'absorption du Zn et du Cu.