京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18318号 / 農博第2043号 / 新制||農||1021(附属図書館) / 学位論文||H26||N4825(農学部図書室) / 31176 / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 本田 与一, 教授 星野 敏, 教授 縄田 栄治 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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Detailed analysis of the site-specific phosphorylation of a glucan phosphate from Saccharomyces cerevesiae has been carried out by 2D NMR techniques. Phosphorylation has been shown to be limited to the C-6 and C-2 positions, with the C-6 resonance showing two slightly different environments. Phosphorylation at C-4 is hindered due to proximity effects with the neighboring glucose ring oxygen. Noncovalently bound, nondialyzable phosphate appears to be coordinated to the nonphosphorylated HO-2 group of the helical polymer.

(1,3;1,4)-β- D-glucan (β-glucan) is a plant cell wall hemicellulose and a main component of endosperm cell walls. The Cellulose Synthase F family of genes is involved in the synthesis of β-glucan. In this study full-length genomic sequences of CslF6 were obtained from multiple Avena species. Three unique alleles were found in each A. sativa line. Comparisons of these alleles to diploid Avena species allowed for identification of the genomic origin of each allele. The A and D genome alleles had identical amino acid sequences while the C-genome had 13 different amino acids. Global expression of CslF6 was completed at three developmental time point and three tissue types. RNAseq technology was utilized to determine genome specific expression patterns. Differential expression of genome specific-copies of CslF6 was found at all time points tested. Lower levels of C-genome expression of CslF6 were associated with increased levels of B-glucan.

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Mixed Linkage Glucan (MLG, or (1,3;1,4)-ß-D glucan) is a component of cell walls for major cereal crops and is significant to food and beverage industries. To better understand genetic factors affecting MLG content in oats, this study investigates the presence of glucosidases likely to participate in MLG production. A glucosidase showing co-expression with CslF6—the primary gene responsible for MLG synthesis—could indicate a hand in MLG production by association. Reference genes for expression analysis as well as glucosidase candidates were first selected using in silico methods. In both cases, barley was used as model species because it has abundant public bioinformatic resources for in silico data mining, and it generates large amounts of MLG, like oats. Actin, malate dehydrogenase, and elongation factor 2, were validated in oat and barley as top reference genes. They were then used to compare the expression activity of the top glucosidase candidate gene, ßglux, with CslF6. ßglux was found to have increased activity with CslF6 during caryopsis development. It is a strong candidate for future transgenic experiments regarding its effect on MLG production.

In Southeast Asia, the family Pangasiidae is important for commercial fisheries and aquaculture. Pangasianodon hypophthalmus (striped catfish) is the most economically important species farmed in Vietnam, with a total export value of 1.7 billion USD in 2012. Intensive aquaculture can lead to problems with major outbreaks of disease and Edwardsiella ictaluri and Aeromonas hydrophila represent two important bacterial pathogens in P. hypophthalmus aquaculture. Immunostimulants have proven to be a very useful food additive for the aquaculture industry, since they can be easily fed to fish to enhance their immune response at times of stress and to improve resistance to disease. The immune system of pangasius catfish has not been fully described, despite the recent growth in aquaculture for this species, and little is known about the effects of immunostimulants on disease resistance. Understanding the immune response is very important in order to evaluate the health status of the fish and assist in control of disease (including prevention) so that production levels by the aquaculture industry can be sustained. The aims of this thesis were to develop and standardise methods to elucidate and measure immune responses in P. hypophthalmus and then to use these with relevant disease models (A. hydrophila and E. ictaluri) and immunomodulators (β-glucans from different sources and at different doses) to determine if bacterial diseases can be controlled, and which functional immune responses and immune genes could be correlated with disease resistance. As a variety of different species from family Pangasiidae are economically important for aquaculture, initial work focused on the characterisation of the immunoglobulin IgM molecule in these species, and anti-P. hypophthalmus IgM mAbs were tested to determine if they cross-reacted between different Pangasiidae species (Chapter 2). Although affinity purification of IgM from the different fish species resulted in a purer preparation ammonium sulphate precipitation (14% w/w), the latter proved faster and easier to perform. The heavy (H) and light (L) chains of IgM from P. hypophthalmus were estimated to be 70-72 kDa and 25-26 kDa, respectively, using SDS-PAGE (12.5%). The L chains of IgM in the other Asian fish species examined were similar in molecular weight to P. hypophthalmus, while the H chains varied (P. gigas and P. larnaudii 76kDa, P. sanitwongsei 69kDa, H. filamentus 73kDa, P. borcoti and H. wyckioides 75kDa, C. bactracus 74kDa, C. macrocephalus 73kDa and C. carpio 70kDa), as did the native IgM molecules. Sedimentation velocity ultracentrifugation was used to determine the molecular weight of the whole IgM molecule from P. hypophthalmus as an alternative to the more commonly used native gels that are run under non-denaturing conditions, although this technique proved more complex. Anti–P. hypophthalmus IgM monoclonal antibodies (mAbs) cross reacted with all of the Pangasiidae species and were successfully applied in an enzyme-linked immunosorbent assay (ELISA) using mAb 23 to measure serum antibody response of P. hypoophthalmus following experimental infection with A. hydrophila by interperitoneal (I.P.) injection in Chapter 3 and E. ictaluri by immersion in Chapter 4. As P. hypophthalmus is a relatively new aquaculture species, there are few reports evaluating its immune response to pathogens. Thus, functional assays were standardised to evaluate both innate and adaptive immune responses of this species and then these assays used to compare immune response following stimulation with live and killed A. hydrophila. (Chapter3). Four treatment groups of 40 fish per group (53.2 ± 14.8g.) consisting of an untreated control group, a group injected I.P. with adjuvant (Montanide ISA 760 VG) only, a group injected with heat-killed A. hydrophila (1 x109 cfu ml-1 mixed with adjuvant), and a group injected with a subclinical dose of live A. hydrophila 2.7 x105 cfu ml-1 were used in the study. Samples were collected 0, 1, 3, 7, 14 and 21 days post injection (d.p.i.) to assess the immune response of fish. The results indicated that challenge with live or/and dead bacteria stimulated the immune response in P. hypophthalmus significantly above control groups with respect to specific antibody titre, lysozyme activity, phagocytosis and plasma peroxidase at 7 or/and 14 d.p.i. Moreover, on 21 d.p.i. total IgM, specific antibody titre and lysozyme activity from both live and dead A. hydrophila challenge groups were significantly different to the control groups. Differential immune responses between live and dead bacterial challenges were also observed as only live A. hydrophila significantly stimulated WBC counts and plasma peroxidase at 3 d.p.i. with the greatest increase in WBC counts noted at 21 d.p.i. and in phagocytosis at 14 d.p.i. By 21 d.p.i. only the macrophages from fish challenged with dead A. hydrophila showed significantly stimulated respiratory burst activity. Immunostimulants are food additives used by the aquaculture industry to enhance the immune response, and β-glucan is now commonly used for this purpose in aquaculture. In Chapter 4 the effect of the prebiotic β-glucan on the immune response and disease resistance of P. hypophthalmus was evaluated. The fish (60.3 ± 11.7 g.) were fed with a basal diet (control) or diets supplemented with fungal derived β-glucan at concentrations of 0.05 %, 0.1 %, or 0.2 % g/kg for four weeks. Fish fed 0.1 % commercial yeast derived β-glucan were also included as a positive control group. Samples were collected from fish on Days 0, 1, 3, 7, 14, 21 and 28. The results showed that fish fed with the highest two levels of fungal derived β-glucan had enhanced immune responses compared to the control group, with respiratory burst activity on all days examined and lysozyme activity on 7 days post feeding (d.p.f.) being significantly elevated (P<0.05) in the group fed with 0.2 % fungal derived β-glucan, while plasma anti-protease activity on 21 d.p.f., natural antibody titre on 3 d.p.f. and complement activity 7 d.p.f. and 14 d.p.i. were significantly enhanced (P<0.05) in the group fed 0.1 % fungal derived β-glucan. The lowest dose of fungal derived β-glucan (0.05 %) appeared insufficient to effectively stimulate the fish’s immune response. WBC count, respiratory burst, lysozyme activity and complement were useful as an early indication of immunostimulation (1 to 7 days). Four weeks after feeding with the different diets, the fish were experimentally infected with E. ictaluri by immersion using 8 x104 cfu ml-1 for 1 h and mortalities were monitored for 14 days. There was a great deal of variation in the level of mortalities within the four replicate tanks for each dietary group. Although the in vivo challenge results showed no statistical differences between the groups fed on the different diets, the highest mortalities were observed in group fed with the control diet and the lowest mortalities were observed in the groups fed with commercial yeast derived β-glucan and 0.2 % fungal derived β glucan. Immune gene expression following stimulation with β-glucan and challenge with E. ictaluri was investigated in Chapter 5.

Thirty-five volunteers were randomized and twenty-nine completed the study. Mean plasma and red blood cell (RBC) total DHA concentrations, which were analyzed among all participants as a measure of adherence, increased significantly in the DHA-enriched treatment compared to control oil-control flour. The plasma and RBC n-6: n-3 ratio was reduced after consumption of HOCODHA-control flour compared to control oil- control flour. The present study failed to see differences in body composition with the HOCODHA-barley flour treatment versus control oil-control flour treatment. In conclusion, significant increases in plasma EPA and DHA levels, as well as the omega-3 index, provide evidence supporting the cardioprotective effects of HOCODHA. The present study demonstrated that in the context of current Western macronutrient intakes, altering the dietary fatty acid composition and adding β-glucan had no major effect on body composition during the 28 days controlled dietary intervention. / February 2017