In a time when the amount of biological data present in the public domain is becoming increasingly vast, the need for good classification systems has never been greater. In the field of glycoscience the necessity of a good classification for the enzymes involved in the biosynthesis, modification and degradation of polysaccharides is even more pronounced than in other fields. This is due to the complexity of the substrates, the polysaccharides, as the theoretical number of possible hexa-oligosaccharides from only hexoses exceeds 1012 isomers!  An initiative to classify enzymes acting on carbohydrates began around 1990 by the French scientist Bernard Henrissat. The resulting database, the Carbohydrate Active enzymes database (CAZy), classifies enzymes by sequence similarity into families allowing the inference of structure and catalytic mechanism. What CAZy does not provide however, are means to understand how members of a family are related, and in what way they differ from each other. The top-down approach used in this thesis, combining phylogenetic analysis of whole CAZy families, or sub-families, with structural determinations and detailed kinetic analysis allows for exactly that.   Finding determinants for transglycosylation versus hydrolysis within the xth gene product family of GH16 as well as restricting the hydrolytic enzymes to a well defined clade are integral parts of paper I. In paper II a new bacterial sub-clade within CE8 was discovered. The structural determination of theEscherichia coli outer membrane lipoprotein YbhC from from the new sub-clade explained the difference in specificity. The information provided in the two papers of this thesis gives a better understanding of the development of different specificities of diverse CAZY families as well as it aids in future gene product annotations. Furthermore this work has begun to fill the white spots uncovered in the phylogenetic trees.

In this study, oligo-porphyrans, obtained by acid hydrolysis of porphyran, were investigated for their H2O2-inducing abilities in the defense responses of P. yezoensis. Oligo-porphyrans with average molecular weights (MWs) lower than 1.43 kDa had H2O2-inducing abilities. In contrast, oligo-porphyrans with average MWs of 6.12 kDa triggered no response. The active oligo-porphyrans were fractioned by anion-exchange chromatography. We found that two distinct mechanisms might be involved in the oligo-porphyran-induced H2O2 release in P. yezoensis. Mixtures of mono-sulfated oligo-galactans with degrees of polymerization (DPs) ranging from 1 to 3 might induce the response through the oxidation of cellular oligosaccharides, which enable P. yezoensis to resist rotting caused by dense incubation. Mixtures of oligo-porphyrans, consisting of 4 ~ 7 monosaccharide residues and 2 ~ 3 sulfate groups, might induce the generation of H2O2 by activation of NADPH oxidase, leading to an oxidative burst in P. yezoensis. The elicitor activity of oligo-porphyrans thus depends on their molecular size.

Members of the genus Malassezia are commensal fungi found on the skin of both human and domestic animals and are associated with skin diseases including dandruff/seborrheic dermatitis, pityriasis versicolor, and atopic eczema (AE) in humans. In this study we have characterized the cell-wall carbohydrates of Malassezia sympodialis, one of the species most frequently isolated from both AE patients and healthy individuals. Cells were grown in liquid Dixon media at 32 °C, harvested, and processed using a standard Fehling's precipitation methodology for the isolation of mannan and a standard base/acid extraction for (1→3)-β-d-glucans. Using these classic extraction methods we were unable to isolate precipitable mannan or insoluble (1→3)-β-d-glucan. However, acidification and addition of methanol to the remaining Fehling's-treated sample resulted in a very clean precipitate. This material was characterized by GPC-MALLS, 1D and 2D NMR, and GC-MS for monomer-type and linkage-type composition. We determined that trace amounts of both mannan and branched (1→3, 1→6)-β-d-glucan were present in the recovered precipitate, but not linear (1→3)-β-d-glucan. Surprisingly, NMR analysis indicated that (1→6)-β-d-glucan was the major carbohydrate component isolated from M. sympodialis cell wall. GC-MS linkage analysis confirmed the (1→6)-β-d-glucan structure. Based on these studies we have determined that the M. sympodialis cell wall contains (1→6)-β-d-glucan as the major carbohydrate component along with trace amounts of mannan and (1→3, 1→6)-β-d-glucan. In addition, these data indicate that modification of the classic mannan isolation methodology may be useful in the simultaneous isolation of both mannan and (1→6)-β-d-glucan from other fungi.

Vitamin A and the T helper 2 cytokines IL-4 and IL-13 play important roles in the induction of mucin gene expression and mucus hypersecretion. However, the effects of these agents on enzymes responsible for mucin glycosylation have received little attention. Here, we report the upregulation of core 2 β1,6 N-acetylglucosaminyltransferase (C2GnT) activity both by all-trans retinoic acid (RA) and by IL-4 and IL-13 in the H292 airway epithelial cell line. Northern blotting analysis showed that the M isoform of C2GnT, which is expressed in mucus-secreting tissues and can form all mucin glycan β1,6-branched structures, including core 2, core 4, and blood group I antigen, was upregulated by both RA and IL-4/13. The L isoform, which forms only the core 2 structure, was moderately upregulated by IL-4/13 but not by RA. Enhancement of the M isoform of C2GnT by RA was abolished by an inhibitor, of RA receptor α, implicating RA receptor α in the effect of RA. Likewise, an inhibitor of the Janus kinase 3 pathway blocked the enhancing effects of IL-4/13 on the L and M isoforms of C2GnT, suggesting a role of this pathway in the upregulation of these two C2GnTs by these cytokines. Taken together, the results suggest that IL-4/13 T helper 2 cytokines and RA can alter the activity of enzymes that synthesize branching mucin carbohydrate structure in airway epithelial cells, potentially leading to altered mucin carbohydrate structure and properties.

Cyclodextrin mediation offers a unique mechanism with the potential for interesting control of reaction parameters. Cyclodextrin mediation of hydrophobic monomers may offer desirable kinetics over conventional free radical polymerizations, and it has been shown in this work that cyclodextrin mediation facilitates polymerization of hydrophobic monomers in aqueous solution and in ethylene glycol. It also may be a facile method for controlling relative reactivity of comonomer mixtures. In addition, complexation of cyclodextrin with guest molecules has been utilized in selective synthesis where the host cyclodextrin has been utilized to sterically hinder the attack of certain reactive sites contained within the host cavity. This aspect of inclusion complexation could also be utilized in free radical polymerizations of monomers with multiple reactive double bonds to preferentially reduce the reactivity of the hindered reactive sites. This thesis involves the use of methylated(1.8)-beta-cyclodextrin (MeCD) as a mediator for polymerizations in solvents that would not facilitate polymerization of the pure monomer in the absence of cyclodextrin. This study focuses on the carbohydrate mediation of a series of methacrylic monomers. t-Butyl methacrylate, n-butyl methacrylate, cyclohexyl methacrylate, and 2-ethylhexyl methacrylate were complexed with methylated(1.8)-beta-cyclodextrin and subsequently dissolved in either water or ethylene glycol. The complexes were studied by 1H and 13C NMR spectroscopy, thin layer chromatography, CPK modeling, and thermogravimetric analysis, and were found to have molar ratios of cyclodextrin to monomer as high as 1.0 to 0.72. These complexes were then free radically polymerized in either water or ethylene glycol and resulted in high molecular weight polymers that precipitated out of solution, allowing for facile polymer isolation through filtration. Isolated yields were found to be as high as 86 %. The majority of the cyclodextrin remained in solution after polymerization. It was also recovered and found to be recyclable. Heterogeneous polymerizations were also performed with 2-ethylhexyl methacrylate in which linear dextrin and methylated (1.8)-beta-cyclodextrin were used in emulsifier quantities. It was found that linear dextrin, at concentrations of 3.0 wt% produced a stable latex product with high molecular weight and an isolated yield of >90%. MeCD on the other hand failed to produce a stable emulsion at concentrations between 0.9-3.0 wt%, but remarkably MeCD at 3.0 wt% gave high molecular weight coagulated polymer with a yield of >90%. It is proposed that a heterogeneous mechanism inconsistent with the four major types discussed by Arshady is taking place. Unlike typical suspension or emulsion polymerizations, the cyclodextrin mediated polymerizations are completely homogeneous at the onset, making them more like a dispersion or precipitation polymerization. However, in dispersion and precipitation polymerizations the pure monomer is soluble in the reaction media. In the absence of cyclodextrin, the monomers utilized in this study possessed no appreciable solubility in the reaction media. Therefore, it is proposed that cyclodextrin acts as a phase transfer agent, effectively solublizing the hydrophobic monomer and allowing for the aqueous dispersion or precipitation type polymerization to occur, depending on the relative solubility of the components. Bulk polymerizations of t-butyl methacrylate, cyclohexyl methacrylate, and 2-ethylhexyl methacrylate and their subsequent use in the preparation of carbohydrate/poly(alkyl methacrylate) blends was also performed in this project. Bulk polymers were utilized as references for physical properties for the polymers produced through polymerization of the MeCD/monomer complexes in either aqueous solution or in ethylene glycol. 1H NMR analysis of the polymers from both the cyclodextrin mediation and bulk polymerizations indicated that the tacticity of the polymers produced in both cases were identical. The bulk polymers were also used in the preparation of carbohydrate/methacrylic blends with potential applications in the areas of selective barriers, biodegradable films. Inclusion of drug molecules or antioxidants into these cyclodextrin containing films also may have potential in drug delivery, or food packaging applications. In addition, the side chain liquid crystalline monomer, 6-(4-hexyloxy-biphenyl-4-yloxy)hexyl methacrylate was synthesized in high purity via a three-step procedure and confirmed by a combination of mass spectrometry, thin layer chromatography, and 1H and 13C NMR. This hydrophobic liquid crystalline monomer was subsequently complexed with 1.0-3.0 equivalents of methylated(1.8)-beta-cyclodextrin in an attempt to alter the water solubility of the monomer. Complexes of this side-chain liquid crystalline monomer have not been studied previously and it is proposed that complexation with cyclodextrin will lead not only to novel polymerizations routes for this monomer, but also to novel smectic phases for this thermotropic liquid crystalline polymer. / Master of Science

No description available.

Coral reefs are the proverbial rainforests of the ocean, but these spectacular structures are under threat from globally rising sea surface temperatures and ocean acidification. The Red Sea and the Persian/Arabian Gulf (PAG) display unusually high sea surface temperatures, and therefore, provide a model for studying environmental change. Corals are so-called holobionts consisting of the coral host, photosynthetic algae (Symbiodiniaceae), along with other microorganisms, such as bacteria, archaea, fungi, and viruses. While the importance of bacteria to coral holobiont functioning is acknowledged, little is known about changes in the microbial communities under natural environmental stressors in the Red Sea and the PAG. Accordingly, I investigated microbial community and mucus differences in bleached, healthy, and diseased corals. Analysis of the composition of mucus-associated microbial communities of bleached and healthy Porites lobata colonies from the Red Sea and the PAG were stable, although some regional differences were present. In a distinct study investigating coral disease, a broad range of corals in the Red Sea were shown to be infected with black band disease (BBD). Investigating the microbial community associated with BBD revealed the presence of the three main indicators for BBD (cyanobacteria, sulfate-reducing bacteria (SRB), and sulfide-oxidizing bacteria (SOB). Last, I investigated the chemical composition (carbohydrates) of the surface mucus layer of a range of Red Sea corals. Given that coral mucus represents a first line of defense, I was interested to examine whether mucus carbohydrate composition would point to a role of adaptation to the extreme environment of the Red Sea. This analysis showed that mucus consists of conserved sugars that are globally conserved. In summary, this thesis characterizes the microbial communities associated with a range of coral species in different health states (bleached, healthy, and diseased). The microbial community patterns I characterized support the notion that bacteria contribute to coral holobiont health and possibly adaptation to extreme environmental conditions in the Red Sea and PAG.

In a completely randomized design with a factorial arrangement, three energy supplements and two species (cows and ewes) were evaluated in two identical experimental periods: gestation and lactation. The three supplement treatments were a control, barley, and sugar beet pulp (SBP). The basal diet was tall wheatgrass straw (5.52% CP). Forage and total dry matter intake (FDMI and TDMI, respectively) interacted (P=.04) with species, supplements, and physiological stages. An interaction of physiological stage and sampling time occurred for pH. Supplement and sampling time interacted for butyric acid proportion and total VFA concentration. Diet digestibility displayed an interaction between species and physiological stage. Digestibility was lower during lactation than gestation for cows, but similar for ewes. Retention time interacted for species, supplement, and physiological stage. Fill of the gastrointestinal tract responded to physiological stage. Fill of the gastrointestinal tract responded to physiological stage, with values of .80 and 1.36% BW for gestation and lactation, respectively. Methane output displayed an interaction between species and physiological stage when data where analyzed as g CH4 d-1kg-1 BW. The highest and more variable values in CH4 losses were from ewes. From gestation to lactation, both species increased CH4 production. Energy lost in feces responded to physiological stage (P=.0008). Across species and supplements, 48 and 60% of the GEI were lost as feces during gestation and lactation, respectively. The use of energetic supplements in ewes depresses FDMI in late gestation, but stimulates it during lactation when a diet based on low-quality forage is fed. For cows, the use of energetic supplements does not affect FDMI during late gestation or lactation. The source of energy does not have an effect, and thus the decision will depend on the cost of supplements. Intake is more strongly affected in ewes than cows when low-quality forage is used. However, for both species, dry matter intake (DMI) is strongly depressed in late gestation.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18324号 / 農博第2049号 / 新制||農||1021(附属図書館) / 学位論文||H26||N4831(農学部図書室) / 31182 / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 髙野 俊幸, 教授 西尾 嘉之, 教授 梅澤 俊明 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19194号 / 農博第2133号 / 新制||農||1034(附属図書館) / 学位論文||H27||N4940(農学部図書室) / 32186 / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 髙野 俊幸, 教授 西尾 嘉之, 教授 梅澤 俊明 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM