京都大学 / 新制・課程博士 / 博士(農学) / 甲第23943号 / 農博第2492号 / 新制||農||1090(附属図書館) / 学位論文||R4||N5378(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 神﨑 護, 教授 北島 薫, 教授 舘野 隆之輔 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

<p>Biodegradation of petroleum hydrocarbons by microorganisms is one of the most effective methods used to remediate environmental systems. However, much of what is known is based on the ability of (mostly bacterial) species to degrade hydrocarbons under enrichment conditions in a laboratory setting. In order to refine biodegradation as a remediation method, there is a critical need to understand the dynamics and mechanisms of microbial communities under <em>in situ </em>conditions. The goal of this dissertation was to provide insight and knowledge into the function of microbial communities in petroleum-contaminated environments using a combination of DNA, lipid and isotopic analyses. Microbial biomass, community structure, carbon sources were assessed at two study sites: (1) a former industrial facility contaminated by PAHs and (2) coastal salt marshes impacted by the <em>Deepwater Horizon</em> oil spill.</p> <p>Isotopic analyses of soils collected from the PAH-contaminated site revealed that microbial carbon sources were derived from vegetation and/or natural organic matter present in soils matter rather than PAHs. Similarly, microbial community structure remained consistent across samples and there were no observed shifts in phylotype diversity with increasing levels of PAHs. Bioaccessibility assays revealed that a large fraction of soil-borne PAHs at the site are not bioavailable to microorganisms; thus, highlighting the importance of environmental factors to <em>in situ</em> biodegradation.</p> <p>Biodegradation of <em>Deepwater Horizon </em>spilled oil was detected in salt marsh sediments such that petroleum-derived carbon was a primary carbon source for indigenous microbial communities in the months following the spill. Likewise, pyrosequencing of all three microbial domains showed an increase in the relative of abundance of taxonomic groups known to include hydrocarbon-degrading species, such as <em>Sphingomonadales</em>. These results suggest that Gulf of Mexico marsh sediments have considerable biodegradation potential and that natural attenuation may be feasible remediation strategy in this region.</p> / Doctor of Philosophy (PhD)

<p> Deuterium tracer studies, kinetic isotope effect measurements and product composition studies in both ammonia and ammonia-d3 have been used to elucidate the mechanism of the y-elimination reaction of 3-phenylpropyltrimethylammonium iodide with potassium amide in liquid ammonia at -55°. Deuterium tracer studies involving the products from the reaction of 3,3-dideutero-3-phenylpropyltrimethylammonium iodide have excluded the carbene and ylide mechanism. A deuterium exchange test involving the deuterated quaternary salt in ammonia demonstrated that a y-carbanion is formed during the reaction. In addition, a large y-hydrogen isotope effect (kH/kD > 22) and a large nitrogen isotope effect (k^14/K^15 = 1.022) were observed for the reaction. These results are consistent with either an Elcb mechanism in which the rates of ring closure and of return of the carbanion are of comparable magnitudes or a concerted mechanism accompanied by an irrelevant exchange reaction at the y-carbon. The latter has been eliminated on the basis of a deuterium exchange test involving the undeuterated quaternary salt in ammonia-d3 and the relative rates of y- and aelimination of the deuterated and undeuterated quaternary salts in both ammonia and ammonia-d3. </p> / Thesis / Doctor of Philosophy (PhD)

AroA catalyzes the sixth step of the shikimate biosynthetic pathway which produces aromatiG amino acids in plants and bacteria, but is absent in mammals. This makes AroA an attractive antimicrobial target. The transition state (TS) structures of AroA- and acid-catalyzed 5-eno/pyruvyl shikimate-3-phosphate (EPSP) hydrolysis were studied in atomic detail by kinetic isotope effect (KIE) measurement. Enzymes bind their transition states more tightly than any other species, so molecules that closely resemble the transition state would have a high affinity for the enzyme and be good inhibitors. Radiolabelled EPSPs were synthesized and a KIE measurement method was developed. Six KIEs were measured for both the AroA- and acid-catalyzed reactions. KIEs for the AroA reaction indicate a cationic TS structure. The acid-catalyzed reaction may employ a slightly different mechanism with an earlier TS. A computational TS model was found and its KIEs were calculated. It demonstrated good agreement with the experimental values at most positions. The model is being modified to improve the agreement with the experimental KIEs. This TS structure will be a good starting point for inhibitor design. All these efforts, hopefully, can make a positive contribution to the development of antimicrobial drugs. / Thesis / Master of Science (MSc)

<p> AlkA is a monofunctional DNA glycosylase from E.coli. This enzyme catalyzes the hydrolysis of the N-glycosidic bond, initiating the first step in the base excision repair pathway. This activity is crucial to the maintenance of the genetic code, as the persistence of DNA aberrations can have significant cellular consequences including mutation, and inhibition of DNA replication and transcription. This enzyme has a broad substrate specificity catalyzing the excision of various lesions (including alkylation, oxidation and deamination products) from DNA. While biochemical and structural studies have been carried out on AlkA; how this enzyme is able to recognize and excise a variety of structurally diverse lesions from DNA and the mechanism by which this excision occurs remains unknown. In this study we have shown that a stem-loop DNA structure containing a hypoxanthine bulge is an optimal substrate for TS analysis of AlkA-catalyzed N-glycoside hydrolysis. In addition, we have developed methods to synthesize radiolabeled deoxyinosine triphosphate (diTP) and incorporate this radiolabeled nucleotide into the stem-loop DNA structure. We have developed a facile method of purification for his-tagged AlkA and his-tagged AlkA containing a TEV protease recognition site (for removal of the his-tag), and have shown that these proteins display an activity similar to that of wild-type AlkA. The [1'-3H] KIE was measured using liquid scintillation in a proof-of-principle experiment. The observed value of 1.046 is indicative of either a relatively synchronous ANDN (SN2) TS or an early DN*AN (SN1) TS with oxacarbenium ion character in the sugar ring, but significant bond order to the leaving group base still remaining. Future work involves repeat measurements of the [1 '-3H] KIE to validate the accuracy of the measurement observed here, examination of commitment to catalysis and optimization of the hypoxanthine bulge substrate synthesis. Analysis of KIEs at additional sites on the hypoxanthine base and sugar ring will contribute to TS analysis of AlkA-catalyzed N-glycoside hydrolysis and help elucidate the mechanism of hydrolysis. </p> / Thesis / Master of Science (MSc)

In this study ¹³C/¹²C and ¹⁵N/¹⁴N ratios were measured from 𝘔𝘪𝘤𝘳𝘰𝘱𝘵𝘦𝘳𝘶𝘴 𝘥𝘰𝘭𝘰𝘮𝘪𝘦𝘶 tissue, from all stages throughout the fish's lifecycle. The different stages in the life of 𝘔𝘪𝘤𝘳𝘰𝘱𝘵𝘦𝘳𝘶𝘴 𝘥𝘰𝘭𝘰𝘮𝘪𝘦𝘶 are represented also by shifts in the trophic level of the fish. As embryos, the δ¹⁵N and δ¹³C values for 𝘔𝘪𝘤𝘳𝘰𝘱𝘵𝘦𝘳𝘶𝘴 𝘥𝘰𝘭𝘰𝘮𝘪𝘦𝘶 tissues are dependent on the δ¹⁵N and δ¹³C of the mother. Also until the point of metamorphosis, where the yolk sac is completely used up, the isotopes reflected in the tissues represent a combination of the δ¹⁵N and δ¹³C of the yolk and any plankton which the young fish are able to feed on. As the fish grows and its diet changes to crayfish and other smaller fish the isotopes reflected in the 𝘔𝘪𝘤𝘳𝘰𝘱𝘵𝘦𝘳𝘶𝘴 𝘥𝘰𝘭𝘰𝘮𝘪𝘦𝘶 shift as the fish effectively increases its trophic position. Definite trends were seen in the δ¹⁵N values throughout the entire lifecycle of 𝘔𝘪𝘤𝘳𝘰𝘱𝘵𝘦𝘳𝘶𝘴 𝘥𝘰𝘭𝘰𝘮𝘪𝘦𝘶, and while the δ¹³C values did not produce such definite trends it was discovered that there was an overlying spatial trend represented in the δ¹³C values. This means that Micropterus dolomieu of similar age and size display an obvious offset in their δ¹³C values depending on their location in the lake. Also addressed here are the potential problems related to working with preservative chemicals and the effects they have on isotope ratios. Shifts were not only seen in both δ¹⁵N and δ¹³C (as has been addressed in the past) but also between different batches of the same preservative fluid. Different batches of formalin can potentially give different offsets to both δ¹⁵N and δ¹³C, making it necessary to calibrate offsets given by all batches of formalin used throughout the entire procedure. / Thesis / Master of Science (MS)

The thesis is compilation of four manuscripts discussing the stable isotope analysis modem and archaeological faunal material from Namu, British Columbia. These studies concentrate on the application of stable isotopic analysis of biogenic material for paleonvironmental interpretation over the Holocene. The first study addresses the use of phosphate and carbonate associated oxygen isotopes in bioapatites (Sebastes spp. vertebrae) as a proxy for the isotopic composition of water from approximently 6,000 to 2,000 years before present (BP). The second study evaluates sclerochronological sampling strategies as applicable to the study of bivalves with implications for sampling fragmented material such as that found in archaeological deposits. The third study investigates stable isotopes composition of estuarine bivalve carbonate (Saxidomus gigantea) and the controlling environmental and biological factors. Finally, the fourth study uses a 5,000 year record of archaeological S. gigantea to provide a paleoclimatic record at Namu over the mid-late Holocene. / Thesis / Master of Science (MS)

This thesis investigates breastfeeding and weaning patterns in an Iron Age (7th – 4th century BCE) sample of subadults (n=12) and adults (n=9) buried at the sites of Botromagno, Parco San Stefano and Padreterno in southern Italy. Stable isotope analysis of both human tooth dentine and bone collagen for each subadult, and tooth dentine for adults, was undertaken to create early-life feeding histories. The dentine serial sections were used to determine the onset and completion of weaning for each individual, as well as distinguish general trends in early feeding practices at these Iron Age sites. Results indicate that the average onset of weaning in subadults occurred at 8 ± 3.4 months and weaning was completed by 4 years of age at the latest for all individuals; however, the patterns of breastfeeding and weaning were variable in this sample. This study also explores variation in early childhood diet between survivors and non-survivors (i.e., < 4 years of age). Non-survivors were weaned more rapidly than survivors – possibly contributing to their earlier death – and some non-survivors demonstrated elevated δ15N values that may have been a result of physiological stress. It is, however, difficult to distinguish signals of breastfeeding versus stress in young children who were still likely consuming breast milk. Finally, differences in isotope data between dentine serial sampling and bulk-bone sampling of rib and femoral collagen from the same individuals were investigated. The results show that the combined use of dentine and bone data contribute to more nuanced interpretations of weaning. Further, rib samples represent diet closer to the time of death than femoral samples, as faster bone turnover rate in ribs allow for the incorporation of more recent dietary changes. / Thesis / Master of Arts (MA)

Hydroclimate evolution in the northern Caribbean region has been investigated by several studies, but they offer contradicting results and none of them cover a full glacial-interglacial transition to examine orbital climate forcing. Here we present refined U-Th dating and oxygen isotope (δ18O) data from a stalagmite named Katún collected from Rio Secreto cave in the Yucatan Peninsula that discontinuously spans the time interval between 198 thousand years (kyr) before present (BP) to 320 kyr BP. Stalagmite δ18O is interpreted to reflect precipitation amount variability as established by previous studies from this cave. On orbital timescales, we identified marine isotope stages (MIS) 7, 8, and 9 in the Katún stalagmite δ18O record. The average stalagmite δ18O value during MIS 7 and 9 interglacial intervals is ~-4.5‰, suggesting wetter conditions than the glacial MIS 8 with an average δ18O value of ~-3‰. The stalagmite δ18O record exhibits higher amplitude δ18O and thus precipitation variability, and an increase in the frequency and intensity of drought events during the drier glacial interval. The record suggests a strong link between atmospheric greenhouse gases, CO2 andCH4, and precipitation variability in the Caribbean, which affects hydroclimate via changes in tropical Atlantic sea surface and air temperature. We propose that changes in North Atlantic high latitude climate are connected to regional hydroclimate, as evidenced by concurrent Caribbean precipitation and atmospheric methane variability, similar to Greenland D-O events. The broader implication of this study is that human activities may alter the fundamental relationship between Caribbean hydroclimate and greenhouse gases, which have likely sustained tropical wetlands and modulated methane production for millennia.

The parkinsonian inducing drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is bioactivated in a reaction catalyzed by the flavoenzyme monoamine oxidase B (MAO-B) to form the corresponding dihydropyridinium (MPDP+) subsequently pyridinium (MPP+) metabolites. As part of our ongoing studies to characterize the structural features responsible for this unexpected biotransformation, we have synthesized and examined the MAO-B substrate properties of a variety of MPTP analogs bearing various heteroaryl groups at the 4-position of the tetrahydropyridinyl ring. The results of these SAR studies indicate that electronic features, steric features and polar interactions can contribute to the substrate activities. Additionally, isotope effects have been examined to investigate the mechanism and stereoselectivity of the MAO-B catalytic pathway. The synthesis and characterization of regio and stereoselectively deuterated MPTP analogs have been achieved. The results indicate that the catalytic step occurs exclusively at the allylic C-6 position and is rate-determining for both good and poor substrates. The two enantiomers of MPTP bearing a deuterium atom at C-6 have been prepared via chiral aminooxazolinyl derivatives and have been characterized by 2H NMR in a chiral liquid crystal matrix. These enantiomers were used to determine the selectivity of the MAO-B catalyzed a C-H bond cleavage reaction leading to the dihydropyridinium metabolite MPDP+. Some of the cyclopropyl analogs of MPTP have also been synthesized as the potential inhibitors. / Ph. D.