京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22066号 / 工博第4647号 / 新制||工||1725(附属図書館) / 京都大学大学院工学研究科材料工学専攻 / (主査)教授 辻 伸泰, 教授 安田 秀幸, 教授 宇田 哲也 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

The Actinomycetes, to which the Mycobacterium genus belongs contains many pathogens, such as Mycobacterium tuberculosis, which can cause tuberculosis, so it has become a focus area in modern molecular biology research. Mycobacteria contain many proteins and regulatory factors, including tRNA, ncRNA and sRNA, which can help bacteria better adapt to the environment. Among them, 6C RNA is a stem-loop non-coding RNA, widely found in mycobacteria. According to previous studies, it may be involved in the rapid growth of mycobacteria. We aimed to clone the 6C RNA promoter region into the pIGn plasmid carrying lacZ reporter gene and transform the construct into Mycobacterium marinum, a close relative of M. tuberculosis. Then we analyzed the β-galactosidase activity of the transformed strain under different stress conditions to study the change of 6C RNA expression. At the same time, we recorded the growth curve and analyze expression changes of 6C RNA in the exponential growth phase and stationary phase of the transformed strain. In addition, we tried to clone the 6C RNA overexpression vector and to study the changes of gene expression at different growth stages, which will help us to better understand the role of 6C RNA in M. marinum.

Wnt/β-catenin signaling is activated in the heart after myocardial infarction (MI). This study aims to investigate if β-catenin deletion affects post-MI ion channel gene alterations and ventricular tachycardias (VT). MI was induced by permanent ligation of left anterior descending artery in wild-type (WT) and cardiomyocyte-specific β-catenin knockout (KO) mice. KO mice showed reduced susceptibility to VT (18% vs. 77% in WT) at 8 weeks after MI, associated with reduced scar size and attenuated chamber dilation. qPCR analyses of both myocardial tissues and purified cardiomyocytes demonstrated upregulation of Wnt pathway genes in border and infarct regions after MI, including Wnt ligands (such as Wnt4) and receptors (such as Fzd1 and Fzd2). At 1 week after MI, cardiac sodium channel gene (Scn5a) transcript was reduced in WT but not in KO hearts, consistent with previous studies showing Scn5a inhibition by Wnt/β-catenin signaling. At 8 weeks after MI when Wnt genes have declined, Scn5a returned to near sham levels and K⁺ channel gene downregulations were not different between WT and KO mice. This study demonstrated that VT susceptibility in the chronic phase after MI is reduced in mice with cardiomyocyte-specific β-catenin deletion primarily through attenuated structural remodeling, but not ion channel gene alterations.

Experiment 1 A survey on the β-Carotene status of Holstein cows in different feeding systems In order to make meaningful recommendations with regard to β-Carotene supplementation is it necessary to know whether cows are deficient in β-Carotene. The objective of this study was to generate data on the β-Carotene status of Holstein cows under three different feeding systems in South Africa, namely; pasture-based, silage-based and hay-based feeding systems. A survey was conducted amongst 30 farms with 10 farms utilizing each of the three systems. Twenty multiparous cows were randomly selected from each farm. Blood samples were taken from the tail vein and analysed for plasma β-Carotene using the iCheck™, a hand held spectrophotometer (BioAnalyt, GmbH, Germany). Cows were then classified as deficient (< 1.5 mg/L), marginal (1.5 to 3.5 mg/L) or optimal (>3.5 mg/L). The average plasma β-Carotene levels differed between feeding systems and concentrations were 5.53, 2.98 and 1.71 mg/L for the pasture based, hay-based and silage-based feeding systems respectively. There was a wide variation in average plasma β-Carotene concentrations in cows on farms within the different feeding systems. Average values per farm ranged between 3.84 and 10.81 mg/L for the pasture based farms, 0.91 and 5.00 mg/L for the hay-based farms and between 0.78 and 3.38 mg/L for the silage-based farms. Results suggest cows on a pasture based feeding system have optimal β-Carotene status and do not need supplementation. Cows on hay-based systems are marginal and on farm testing is recommended. Cows on silage-based systems are generally deficient and β-Carotene supplementation is recommended. Experiment 2 Effect of prepartum β-Carotene supplementation on the postpartum β-Carotene status of Holstein cows. It has been recommended that cows be supplemented β-Carotene when blood plasma levels are deficient (< 1.5 mg/L) or marginal (< 3.5 mg/L) especially during the transition period which is characterised by low intakes and significant losses of β-Carotene through colostrum. The objective of this trial was to determine to what extent prepartum β-Carotene supplementation could maintain postpartum plasma β-Carotene concentrations above 3.5 mg/L in cows fed a lucerne hay-based TMR. Twenty multiparous Holstein cows were blocked into two groups of ten cows each and were fed either 8kg/d of a control TMR (DM) or the control diet supplemented with 1200mg of ROVIMIX® β-Carotene 10%. The experimental period was from 60d pre-partum until 56d postpartum; however the period of the β-Carotene supplementation for the one group was only from 60d prepartum until calving. Blood samples were collected from the tail vein once per week and analysed for plasma β-Carotene using the iCheck™, a handheld spectrophotometer (BioAnalyt, GmbH, Germany). Average plasma β-Carotene concentrations prepartum were higher (6.15 mg/L) (P < 0.05) for supplemented cows compared to the control cows (3.10 mg/L). For the first 5 weeks postpartum, plasma β-Carotene was higher (P < 0.05) for supplemented cows compared to control cows (3.00 mg/L vs. 1.39 mg/L), from weeks 6 to 9 there were no differences (P > 0.05). Overall the average postpartum plasma β-Carotene values were 1.50 mg/L for the control cows and 2.43 mg/L for the supplemented cows and did not differ. Supplemented cows maintained sufficient β-Carotene concentrations only for the first 2 weeks postpartum and were either marginal or deficient for the rest of the experimental period. Results suggest a minor carryover effect of β-Carotene after prepartum supplementation. / Dissertation (MSc Agric)--University of Pretoria, 2013. / gm2014 / Animal and Wildlife Sciences / unrestricted

Abstract: Poly (vinylidene fluoride) (PVDF), thanks to its versatile properties, finds many applications ranging from water purification membranes (thermal and chemical stability) to electronic devices (piezoelectric, pyroelectric and ferroelectric properties). Block copolymers of PVDF with other polymers further expand its properties and, consequently, its applications. Toward this line, my thesis investigates the synthesis, molecular characterization and properties of novel PVDF-based copolymers mainly with poly(tert-butyl acrylate) (PtBuA), poly(methyl methacrylate) (PMMA) and polystyrene (PSt). To prepare the block copolymers a living polymerization is needed, which is compatible with the VDF and the comonomer (tBuA, MMA, St). For this purpose, we used iodine transfer polymerization (ITP) with the difunctional chain transfer agent (CTA) C4F8I2. Difunctional macroinitiator (I-PVDF-I) was first obtained by ITP of VDF monomer with C4F8I2, followed by addition of the comonomer tBuA, MMA or St to afford the triblock copolymers poly(tert-butyl acrylate)-block-poly(vinylidene fluoride)-block-poly(tert-butyl acrylate) (PtBuA-b-PVDF-b-PtBuA), poly(methyl methacrylate)-block-poly(vinylidene fluoride)-block-poly(methyl methacrylate) (PMMA-b-PVDF-b-PMMA) and polystyrene-block-poly(vinylidene fluoride)-block-polystyrene (PSt-b-PVDF-b-PSt). The structure of all intermediates and final products were characterized by Nuclear Magnetic Resonance (NMR) and Gel Permeation Chromatography (GPC). The microstructure and polymorphism of all triblock copolymers, characterized by XRD, shown that the PVDF in the first two copolymers exhibits the electroactive β-phase, while in the third copolymer there is the coexistence of α- and γ-phases. Linear PVDF homopolymers, using the free radical and IT polymerizations, were prepared for comparison purposes. All linear polymers possess the α-phase. The thesis is divided into the following five chapters: 1. Introduction, where the scope of this thesis is given with a brief background on PVDF; 2. Literature Review, where a summary of previously published works on PVDF synthesis and polymorphism is presented; 3. Experimental Section, where detailed procedures and characterization methods are given; 4. Results and Discussion, where outcomes of successful experiments are discussed; and 5. Conclusion and Perspective, where the outcomes of this work are summarized and perspective are discussed.

Infectious diseases are a serious concern worldwide especially in immune-compromised patients. Some of these diseases are considered to be contagious and are spread via airborne transmission, while others are not contagious, i.e. non-communicable diseases. The problem is compounded by the emergence of pathogens resistant to currently used antimicrobial drugs. A wide range of microbes including bacteria, fungi, parasites, viruses and protozoans are implicated as causative agents of various diseases. Many patients without ready access to Western medical facilities rely on medicinal plants for the cure of various ailments including infectious diseases. Two plant species used in South African traditional medicine for treating infectious diseases, namely Calodendrum capense Thunb. (Rutaceae) and Lydenburgia cassinoides N. Robson (syn. Catha transvaalensis, Celastraceae) were screened for antimicrobial activities against a range of fungi, bacteria and mycobacteria. The test organisms included Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Mycobacterium smegmatis and M. bovis BCG. The L. cassinoides acetone extract generally had good activity, with MIC values ranging from 0.04 to 0.15 mg/ml while the C. capense extract MIC values ranged from 0.31 to 0.62 mg/ml against the fungi. The hexane extract of L. cassinoides had good activity (MIC = 0.04 mg/ml) against M. smegmatis and the methanol extract had MIC = 0.16 mg/ml against M. bovis BCG. The two plant species had reasonable antibacterial activity against S. aureus and P. aeruginosa, with MIC values ranging from 0.16 to 0.32 mg/ml. Antibacterial activity against Escherichia coli and Enterococcus faecalis resulted in MIC = 0.63 mg/ml for both plants. However in the bioautography assay, the hexane extracts of C. capense and L. cassinoides had good activity against S. aureus, showing active zones of bacterial growth inhibition. The aim of this study was to isolate and characterize the active compounds, with emphasis on antifungal activity, from Calodendrum capense and Lydenburgia cassinoides that may be useful in treating opportunistic infections in immunocompromised patients. Bioassay-guided evaluation of the antimicrobial active components of both hexane fractions using C. neoformans and C. albicans as test organisms led to the isolation and characterization of lupeol from C. capense and ß-amyrin from L. cassinoides. The MIC values of lupeol and ß-amyrin ranged from 1.5 to 6.2 µg/ml against all the tested organisms. Both compounds were also tested against a resistant strain of Candida albicans which resulted in MICs of 3.2 and 6.2 µg/ml respectively. Crude extracts and compounds were also tested for cytotoxicity against human liver (C3A) cells. The crude plant extracts had a low cytotoxicity with average LC50 values of 205.8 ± 8.38 µg/ml for L. cassinoides and 83.07 ± 44.66 µg/ml for C. capense. LC50 values for the isolated compounds were greater than 200 µg/ml, the highest concentration tested. Selectivity index (SI) values were calculated using the formula SI = LC50/MIC. The SI values of the crude extracts of the two plant species ranged between 0.18 and 0.91, showing that these extracts were relatively toxic compared to the antimicrobial activity as the SI values were less than 1. However, the compounds ß-amyrin and lupeol had good activity and low toxicity with SI values greater than 10. In conclusion both plant species showed broad-based antimicrobial activity against the standard ATCC strains of bacterial and laboratory isolates of fungal pathogens. Purified compounds with very good antifungal activity and negligible detectable cytotoxicity, namely lupeol from C. capense and ß-amyrin from L. cassinoides (both pentacyclic triterpenoids) were isolated. This is apparently the first report of these two compounds from these two plant species. In our research group, lupeol has been isolated from various other plant species and it is known to have antimicrobial and anti-inflammatory activities. The second compound, ß-amyrin, reportedly has anti-inflammatory, antitumor, antibacterial, gastroprotective and hepatoprotective effects. The findings from this study show that the two isolated compounds were highly active against fungal and bacterial pathogens, with the lowest MIC value of 0.015 mg/ml. Both compounds showed much better selectivity index values with regard to antifungal activity compared to those of the crude extracts. The compounds also had good activity against the two Mycobacterium strains tested, indicating potential application in antimycobacterial therapy. The results may validate to an extent the use of these two plants as anti-infectious agents in traditional medicine. The compounds have potential for development into therapeutic agents, but various factors will need to be investigated further, including in vivo efficacy and safety, as well as other aspects such as mode of administration. / Dissertation (MSc)--University of Pretoria, 2012. / Paraclinical Sciences / unrestricted

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.

This research explores various immobilized enzyme support materials, including the novel nylon-6 fiber membrane (NFM), observing the increase of surface area and what effect that has on enzyme binding potential. This study also manipulates incubation and reaction conditions and observes what affect that has on activity and stability of β-galactosidase comparing various solid support materials and free enzyme. Nylon-6 fiber membranes were created using the process of electrospinning and were compared with other materials as solid support materials for enzyme binding. The other materials included polyvinylidene fluoride 5 kD nanofiltration dairy membranes, nylon-6 pellets, silica glass beads, and free—dissolved—enzyme. Scanning electron microscopy images exposed the nylon-6 fiber membrane’s large amount of surface area which coordinated with greater enzyme activity as compared to the relatively flatter surfaces of the other solid support materials. Enzyme activity was measured spectrophotometrically with the color-changing substrate ortho-Nitrophenyl-β-galactoside. NFM had greater maximum enzyme binding potential than the other solid supports. Across pH conditions ranging from 3.5 to 6.0., enzyme activity was maintained on the membrane immobilized samples whereas free enzyme did not maintain activity. Altering storage temperature (4, 22, and 50 °C) affected enzyme stability, the ability of the enzyme to maintain activity over time, of free and polyvinylidene fluoride membrane samples. However, nylon-6 fiber membrane samples maintained stability across the varying storage temperatures. Increasing the immobilization solution enzyme concentration above maximum enzyme binding capacity had no significant effect on enzyme stability for membrane immobilized samples. Although, both had lower mean stability than free enzyme by approximately 74% percent. With further development, β-galactosidase immobilized on nylon-6 fiber membranes, or other membranes, could be used in continuous processing in the dairy industry for a combination of filtration and lactose hydrolysis—creating products reduced in lactose and increased in sweetness with no “added sugars” requirement for a nutrition label and no enzyme listed as final product ingredient.

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.