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N-Acylphosphatidylethanoiamine (NAPE) is synthesized in the microsomes of cotton seedlings by a mechanism that is possibly unique to plants, the ATP-, Ca2+-, and CoA-independent acylation ofphosphatidylethanolamine (PE) with unesterified free fatty acids (FFAs), catalyzed by NAPE synthase. A photoreactive free fatty acid analogue, 12-[(4- azidosalicyl)amino]dodecanoic acid (ASD), and its 125I-labeled derivative acted as substrates for the NAPE synthase enzyme.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22304号 / 医博第4545号 / 新制||医||1040(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 坂井 義治, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Finding a source from which high-energy-density biofuels can be derived at an industrial scale has become an urgent challenge for renewable energy production. Some microorganisms can produce free fatty acids (FFA) as precursors towards such high-energy-density biofuels. In particular, photosynthetic cyanobacteria are capable of directly converting carbon dioxide into FFA. However, current engineered strains need several rounds of engineering to reach the level of FFA production for it to be commercially viable. Thus, new chassis strains that require less engineering are needed. Although more than 140 cyanobacterial genomes are sequenced, the natural potential of these strains for FFA production and excretion has not been systematically estimated. In relation to the above-mentioned problems, we developed the first in silico screening method (FFASC) that evaluates the cyanobacterial strains’ potential for FFA production based on the strains’ proteome, which for the first time allows non-experimental selection of the most promising chassis for cyanofactories. The solution is based on the original problem formulation, optimization and ranking. To provide developers and researchers easy means for evaluation and assessment of the cyanobacterial strains potential for production of FFA, we developed the BioPS platform. In addition to being able to compare capacity for FFA production of any novel strain against 140 pre-valuate strains, BioPS can be used to explore characteristics and assessment rules in play for an individual strain. This is the first tool of this type developed. Finally, we developed a novel generic in silico method (PathDES) for ranking and selection of the most suitable pathways / sets of metabolic reactions, which suggests genetic modifications for improved metabolic productivity. The method heavily relies on optimization and integration of disparate information in a novel manner. It has been successfully used in connection with FFASC for design of cyanofactories. In conclusion, this study has contributed novel and unique methods, and tools for the field of bioinformatics, with applications towards the metabolic design of cyanofactories. We believe that these will be of good use to researchers and technology developers in this field.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18327号 / 農博第2052号 / 新制||農||1022(附属図書館) / 学位論文||H26||N4834(農学部図書室) / 31185 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 河田 照雄, 教授 金本 龍平, 教授 入江 一浩 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Student Number : 9404757K - PhD thesis - School of Physiology - Faculty of Science / Natural secretions of oestrogen and progesterone vary according to menstrual phase in eumenorrhoeic women and have an affect on physiological systems that could consequentially influence exercise performance. In a series of menstrual phase comparative investigations (considering mainly the early follicular (EF), late follicular (LF) and mid-luteal (ML) phase), I aimed to elucidate the physiological relation of oestrogen and progesterone to fat metabolism and ventilation during endurance exercise and exercise performance. I measured plasma free fatty acid (FFA) kinetics during prolonged exercise from an intravenous infusion of K+[1-13C]palmitate in eumenorrhoeic women and found the change in palmitate rate of appearance and disappearance between menstrual phases to be related to the oestrogen/progesterone (E/P) ratio. Overall the results from this study suggest that oestrogen promotes increases in FFA availability during exercise, while progesterone mitigates this response. Therefore, a high oestrogen concentration and E/P ratio in the ML phase is required in order to produce a favourable FFA metabolic response for endurance events. In order to estimate plasma FFA oxidation rate using carbon-FFA tracers, the acetate correction factor which accounts for carbon-label retention in secondary metabolic pools, must be applied. When I derived the acetate correction factor by measuring fractional recovery of carbon-13 in expired CO2 from a constant infusion of Na+[1- 13C]acetate during submaximal exercise we found the acetate correction factor to be significantly lower in the ML than EF phase (average change from EF phase -1.8± 0.5%, p<0.05). Failure to account for the lower correction factor in the ML phase will result in a 6% underestimation of plasma FFA oxidation rate. Furthermore, since human serum albumin (HSA) routinely used as a carrier for parenteral delivery of FFA tracers has various disadvantages, I tested the feasibility of using 2-hydroxypropyl-b- cyclodextrin (HP-b-CD) as a possible alternative carrier. A comparison of expired CO2 enrichment following HSA-FFA (7.4±2.0 %o) and HP-b-CD-FFA (8.6±2.1%o) infusion during exercise showed that the HP-b-CD does not compromise natural in vivo behaviour of the FFA tracer (p=0.4). Progesterone-induced hyperventilation is occasionally reported during exercise in the luteal phase. I found that the change in ventilatory parameters (minute ventilation and respiratory rate) during exercise from EF to ML phase is related to both the oestrogen and progesterone concentration in the ML phase. However, the associated increase in respiratory rate throughout prolonged exercise in the ML versus EF phase did not increase metabolic demand and therefore could not be expected to exacerbate fatigue. When exercise performance was evaluated by means of a cycling time trial, I found a trend for best performance in the LF phase versus the EF phase (8 of 11 subjects improved by 5.2±2.9%, p=0.027), while no differences occurred between other menstrual phases. Metabolic and performance benefits of oestrogen may be concealed in the ML phase by the coincident increase in progesterone in this phase and thus a significant effect is often only evident with a high E/P ratio in the ML phase. The transient LF phase, characterised by the pre-ovulatory surge in oestrogen, reveals the maximum benefits of oestrogen on metabolism and performance during submaximal exercise.

Biodiesel is the most successful drop-in biofuel used in transportation. It can reduce GHG emissions in transportation by 50 to 90% depending on the type of feedstock used. Waste cooking oils and fats containing free fatty acids (FFA) are less expensive feedstocks for biodiesel production than refined vegetable oils. The major issue that limits the use of these oils as feedstock is the interference of FFAs with widely used base catalyzed reaction processes. The FFAs consume base catalyst, produce water of neutralization and form soaps that create emulsions downstream in the process reducing process yields. There is an important need to develop technologies that reduce the FFA content in these oils to below 0.5 wt%; the accepted limit for a feedstock to be processed by the base catalysed reaction. Enzymes are an efficient and environmentally friendly catalyst for FFA esterification. However, they are prone to deactivation with methanol and also catalyze the hydrolysis of esters and triglycerides to FFA. Using them to pre-treat oils and fats remains a challenge: in the presence of water, enzymes can readily produce FFAs from lipids. The objective of this work was to investigate two enzymatic processes to pre-treat acidic oil below the FFA requirement of 0.5 wt%. In this study, two different continuous systems, a packed bed reactor (PBR) and membrane reactor (MR) were used in FFA enzymatic esterification to meet the 0.5 wt% requirement, improve the reusability of enzymes and reduce catalyst cost. The esterification in the PBR was carried out using CALB immobilized on a new 2D/3D nanoplatelet support (TAN). The enzyme was covalently bonded to the TAN using a hydrophobic epoxy ligand. Acidic oil containing canola oil and 2.5 wt% FFA was used as the feedstock for the esterification. It was found that the FFA concentration met the quality specification of <0.5 wt% using CALB-TAN, while it did not using the commercial Novozym 435. The surface fluid velocity was found to have an effect on the removal of water from the PBR reactor. When the velocity was too low, water was retained in the reactor and the FFA conversion was low, when it was too high the reaction time for esterification was not sufficient. It was found that feed velocity of 3 to 6 x 10-5 m/s met the 0.5 wt% requirement. In the PBR, the use of CALB-TAN successfully eliminated the hydrolysis of TG and achieved the continuous esterification of FFA for 42 days. In the MR, acidic oil containing canola oil and 10 wt% FFA was used as the feedstock for the esterification. The enzyme adsorbed on the surface of the polar phase containing glycerol and water and was successfully retained in the reactor by a 0.2-micron ceramic membrane. The addition of glycerol increased the polarity of the dispersed phase in the reactor, bounded water, and retained the liquid enzyme in the reactor. However, the added glycerol in the reactor increased the operating pressure of the reactor. The operating pressure was reduced by adding biodiesel to the feedstock prior to treatment. The lowest level of FFA from the 10 wt% FFA feedstock was 0.68 wt%. This would require a second polishing step to reach the required 0.5 wt%. The PBR and MR using CALB are technologies that limit the hydrolysis at low FFA concentrations and are promising for the pre-treatment of acidic feedstocks in base catalysed biodiesel processes.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23088号 / 医博第4715号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 柳田 素子, 教授 岩田 想, 教授 浅野 雅秀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第20311号 / 薬科博第80号 / 新制||薬科||9(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 岡村 均, 教授 竹島 浩, 准教授 平澤 明 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23253号 / 農博第2460号 / 新制||農||1085(附属図書館) / 学位論文||R3||N5343(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 阪井 康能, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM