Aspergillus fumigatusis a ubiquitous environmental soil fungus. With recent development and advancement in medical treatments leading to immunosuppression, there has been an increase in incidence in aspergillosis. With the emergence of antifungal resistance isolates and the continued high mortality rate for invasive aspergillosis, the hunt for new antifungal drug targets is critical. Research on A. fumigatus is still in its infancy, partly due to the relatively recent rise of A. fumigatus as a clinically significant pathogen. The cell wall has been demonstrated to be critical for survival of this fungal organism, with interference of cell wall construction leading to cell death or reduced growth. This, coupled with the lack of shared mechanisms in humans, makes targeting cell wall synthesis for antifungal therapy a reasonable possibility. The cell wall of A. fumigatus shares a few similarities to S. cerevisiae. However, major differences exist, including the presence of β-1,3;1,4-glucan in the cell wall of A. fumigatus. In fact, the presence of β-1,3;1,4-glucan was never previously described in fungi before Latge's group reported it a number of years ago. It comprises about 10% of the glucans in the cell wall of A. fumigatus, but its role in the cell wall is unknown. In 2006 and 2009, two papers were published that demonstrated the role of CslF and CslH(Cellulose like synthases) in the production of β-1,3;1,4-glucan of the cell wall in rice and barley, respectively. Taking both protein sequences for these genes, we blasted it against the A. fumigatus database for any possible orthologues. A single orthologue, albeit with weak homology, was identified that named TFT1. We hypothesize that TFT1a plays a direct role in A. fumigatus β-1,3;1,4-glucan synthesis. Through Agrobacterium tumefaciens mediated transformation, an A. fumigatus strain lacking this enzyme (tft1Δ) was generated. From tft1Δ a revertant strain (revtft1) was created where the gene was reintroduced. Immunofluorescence staining with antibodies against β-1,3;1,4-glucan and biochemical quantification both demonstrated complete loss of β-1,3;1,4-glucan within the cell wall of the tft1Δ strain, with recovery detected in revtft1. This strongly suggests that this enzyme does indeed play a role in β-1,3;1,4-glucan synthesis in A. fumigatus. Growth experiments, spore size determination and an in vitro model of virulence also indicated that the loss of TFT1 leads to additional phenotypes. While the precise mechanism for β-1,3;1,4-glucan synthesis is unknown, the results shown herein indicate a pivotal role forTFT1 in its biosynthesis, and resulting phenotypes upon loss of mixed linkage glucan adds some clues to its role in the cell wall of A. fumigatus.

Barley grain is an important source of energy and protein for ruminant animals. However, feeding must be carefully managed to avoid maladies caused by the rapid breakdown of barley starch in the rumen. The development of slower degrading barley for ruminants may alleviate health problems associated with barley grain consumption. Selection for hard endosperm may result in slower starch degradation and improved feed quality. The objectives of this study were to: examine the effect of grain hardness, variety and environment on dry matter disappearance rate (DMDR); identify accurate and efficient hardness selection tools; and study environmental effects, inheritance and heritability of hardness.<p>To study grain hardness and genetic and environmental effects on DMDR, two genotypes grown at multiple locations in 2004 were analyzed for Single Kernel Characterization System (SKCS) hardness, by scanning electron microscopy (SEM), and for in situ DMDR. Genotype by environment interaction influenced DMDR, while neither SKCS hardness nor SEM analysis accurately differentiated DMDR between genotypes. <p>Eight genotypes were grown at multiple locations during 2003 and 2004 to study grain hardness measurement methodology, and genetic and environmental effects on hardness. Genotypes were analyzed for SKCS hardness, milling energy, endosperm light reflectance, feed particle size, protein and beta-glucan. Hardness measurements ranked genotypes similarly across environments. Feed particle size was correlated with milling energy but not other hardness measurements. Hardness measurements appeared to be influenced by protein and beta-glucan.<p>To examine the inheritance and heritability of barley grain hardness, 245 double haploid (DH) genotypes and parents, grown in 2003 and 2004, were analyzed for SKCS hardness, milling energy, protein, beta-glucan, with 100 evaluated for light reflectance. The population exhibited normal distributions for SKCS hardness, milling energy, protein and beta-glucan, suggesting quantitative inheritance for these traits with no apparent epistatic gene interaction. Narrow-sense heritability was 0.75 for SKCS hardness and 0.41 for protein. Light reflectance was not normally distributed, suggesting complementary gene interaction. Broad-sense heritability was 0.53.<p>Barley grain hardness is highly heritable and an efficient tool in making selections in a breeding program. However, breeding for high beta-glucan and protein may be better selection criteria for indirect selection of DMDR.

Barley grain is an important source of energy and protein for ruminant animals. However, feeding must be carefully managed to avoid maladies caused by the rapid breakdown of barley starch in the rumen. The development of slower degrading barley for ruminants may alleviate health problems associated with barley grain consumption. Selection for hard endosperm may result in slower starch degradation and improved feed quality. The objectives of this study were to: examine the effect of grain hardness, variety and environment on dry matter disappearance rate (DMDR); identify accurate and efficient hardness selection tools; and study environmental effects, inheritance and heritability of hardness.<p>To study grain hardness and genetic and environmental effects on DMDR, two genotypes grown at multiple locations in 2004 were analyzed for Single Kernel Characterization System (SKCS) hardness, by scanning electron microscopy (SEM), and for in situ DMDR. Genotype by environment interaction influenced DMDR, while neither SKCS hardness nor SEM analysis accurately differentiated DMDR between genotypes. <p>Eight genotypes were grown at multiple locations during 2003 and 2004 to study grain hardness measurement methodology, and genetic and environmental effects on hardness. Genotypes were analyzed for SKCS hardness, milling energy, endosperm light reflectance, feed particle size, protein and beta-glucan. Hardness measurements ranked genotypes similarly across environments. Feed particle size was correlated with milling energy but not other hardness measurements. Hardness measurements appeared to be influenced by protein and beta-glucan.<p>To examine the inheritance and heritability of barley grain hardness, 245 double haploid (DH) genotypes and parents, grown in 2003 and 2004, were analyzed for SKCS hardness, milling energy, protein, beta-glucan, with 100 evaluated for light reflectance. The population exhibited normal distributions for SKCS hardness, milling energy, protein and beta-glucan, suggesting quantitative inheritance for these traits with no apparent epistatic gene interaction. Narrow-sense heritability was 0.75 for SKCS hardness and 0.41 for protein. Light reflectance was not normally distributed, suggesting complementary gene interaction. Broad-sense heritability was 0.53.<p>Barley grain hardness is highly heritable and an efficient tool in making selections in a breeding program. However, breeding for high beta-glucan and protein may be better selection criteria for indirect selection of DMDR.

The effects of b-glucan on corn tortilla texture were evaluated. Barley flour (9.7% b-glucan) was substituted at 2.5, 5 and 10% for dry masa flour in corn tortillas. Texture was evaluated after 4 hr and up to 7 d storage at 4°C. Substitution of 2.5-10% barley flour significantly improved tortilla texture. Combined effects of barley flour (0-2.5%), maltogenic amylase (0-1650MAU) and carboxymethylcellulose (0-0.5%) were evaluated using surface response methodology. Barley flour increased rollability, pliability, energy dissipated and reduced rupture force and final stiffness. Overall, maltogenic amylase decreased rupture force and Young's modulus but decreased rupture distance, rollability and pliability at levels above 825 MAU. CMC improved rollability, pliability, and rupture distance. The best response was found using barley flour and CMC with 825 MAU, where rollability, pliability, rupture distance and energy dissipated increased while rupture force, Young's modulus and final stiffness decreased. A 70% barley b-glucan concentrate combined with amylase (550 MAU) or CMC (0-0.5%) was evaluated in corn tortillas. Amylase combined with b-glucan did not improve texture. Tortillas with b-glucan and CMC had significantly improved pliability, rollability, final stiffness and energy dissipated. Texture measurements analysis showed that depending on the stage of storage, objective and subjective methods correlate differently. Subjective and objective measurements of texture were not correlated at 4 hr storage. At the end of storage, pliability had significant correlations with stress relaxation measurements, but rollability had higher correlation coefficients with extensibility measurements. Pliability had higher R2 and lower coefficients of variation compared to rollability. Sensory evaluation was conducted using reheated 14-day-old tortillas of control, 825 MAU with 0.25% CMC, 0.12% b-glucans, 0.18% b-glucan with 0.375% CMC, and 0.24% b-glucan with 0.25% CMC. All tortillas had similar appearance, flexibility, gumminess, flavor and overall quality. Softness and chewiness of treatments with 0.12% b-glucan or 0.24% b-glucan with 0.25% CMC were similar to control. Other tortillas were significantly tougher and chewier. b-glucan may be the active ingredient in barley flour that modifies firming of corn tortillas during storage. Barley flour is inexpensive and effectively improves texture of corn tortillas.

Limited information exists regarding the effects of light pearling on the properties of physical grain characteristics, composition, and technological and sensory properties of selected varieties of Western Canadian barley especially hulless barley genotypes with modified starch characteristics. Nine barley genotypes with different hull (hulled and hulless) and starch characteristics (normal, waxy, and high amylose (HA)) were pearled to three differing levels. Scanning electron micrographs showed that the pericarp, testa, aleurone, and subaleurone layers were completely removed in heavily pearled barley whereas only a few outer layers were removed in minimally pearled barley. Waxy starch genotype Fibar and HA starch genotypes, SH99250 & SB94893 contained high levels of soluble β-glucan (9-11%). Waxy starch genotypes exhibited higher β-glucan solubility when cooked compared to normal and HA starch genotypes. However, HA starch genotypes had lower in vitro starch digestibility which may provide a lower glycemic response in humans.

Limited information exists regarding the effects of light pearling on the properties of physical grain characteristics, composition, and technological and sensory properties of selected varieties of Western Canadian barley especially hulless barley genotypes with modified starch characteristics. Nine barley genotypes with different hull (hulled and hulless) and starch characteristics (normal, waxy, and high amylose (HA)) were pearled to three differing levels. Scanning electron micrographs showed that the pericarp, testa, aleurone, and subaleurone layers were completely removed in heavily pearled barley whereas only a few outer layers were removed in minimally pearled barley. Waxy starch genotype Fibar and HA starch genotypes, SH99250 & SB94893 contained high levels of soluble β-glucan (9-11%). Waxy starch genotypes exhibited higher β-glucan solubility when cooked compared to normal and HA starch genotypes. However, HA starch genotypes had lower in vitro starch digestibility which may provide a lower glycemic response in humans.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22660号 / 工博第4744号 / 新制||工||1741(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 秋吉 一成, 教授 大内 誠, 准教授 佐々木 善浩 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

To investigate the effects of (1→3)-β-D-glucan after inhalation, animals were exposed to different forms of glucan and the number of lung lavage cells was determined 24 h after exposure. None of the different forms assayed caused any increase in cell numbers. In animals exposed to endotoxin, all types of cells were increased after 24 h. A simultaneous exposure to curdlan reduced this increase in a dose-related fashion. The results suggest that (1→3)-β-D-glucan-related acute injury to the lung is induced by mechanisms other than those induced by inflammagenic agents such as endotoxin.

Microglia, the innate immune cells of the central nervous system, feature adaptive immune memory with implications for brain homeostasis and pathologies. However, factors involved in the emergence and regulation of these opposing responses in microglia have not been fully addressed. Recently, we showed that microglia from the newborn brain display features of trained immunity and immune tolerance after repeated contact with pathogens in a dose-dependent manner. Here, we evaluate the impact of developmental stage on adaptive immune responses of brain microglia after repeated challenge with ultra-low (1 fg/ml) and high (100 ng/ml) doses of the endotoxin LPS in vitro. We find that priming of naïve microglia derived from newborn but not mature and aged murine brain with ultra-low LPS significantly increased levels of pro-inflammatory mediators TNF-α, IL-6, IL-1β, MMP-9, and iNOS as well as neurotrophic factors indicating induction of trained immunity (p < 0.05). In contrast, stimulation with high doses of LPS led to a robust downregulation of pro-inflammatory cytokines and iNOS independent of the developmental state, indicating induced immune tolerance. Furthermore, high-dose priming with LPS upregulated anti-inflammatory mediators IL-10, Arg-1, TGF- β, MSR1, and IL-4 in newborn microglia (p < 0.05). Our data indicate pronounced plasticity of the immune response of neonate microglia compared with microglia derived from mature and aged mouse brain. Induced trained immunity after priming with ultra-low LPS doses may be responsible for enhanced neuro-inflammatory susceptibility of immature brain. In contrast, the immunosuppressed phenotype following high-dose LPS priming might be prone to attenuate excessive damage after recurrent systemic inflammation.

Trained immunity and immune tolerance have been identified as long-term response patterns of the innate immune system. The causes of these opposing reactions remain elusive. Here, we report about differential inflammatory responses of microglial cells derived from neonatal mouse brain to increasing doses of the endotoxin LPS. Prolonged priming with ultra-low LPS doses provokes trained immunity, i.e., increased production of pro-inflammatory mediators in comparison to the unprimed control. In contrast, priming with high doses of LPS induces immune tolerance, implying decreased production of inflammatory mediators and pronounced release of anti-inflammatory cytokines. Investigation of the signaling processes and cell functions involved in these memory-like immune responses reveals the essential role of phosphoinositide 3-kinase γ (PI3Kγ), one of the phosphoinositide 3-kinase species highly expressed in innate immune cells. Together, our data suggest profound influence of preceding contacts with pathogens on the immune response of microglia. The impact of these interactions—trained immunity or immune tolerance—appears to be shaped by pathogen dose.