Marine ascidian metabolite perophoramidine has since its isolation in 2002 attracted the interest of several research groups. The complex polycyclic structure with vicinal quaternary carbon stereocenters and two amidine functionalities constitutes a formidable synthetic challenge. In this project a new synthetic approach to dehalogenated perophoramidine has been investigated. The vicinal quaternary carbon stereocenters were introduced via a samarium mediated reductive dialkylation, in which the stereochemistry was determined by the metal chelate that formed, as outlined below. Several of the steps suggested in the synthetic approach have been successfully evaluated. Future work is needed to assess the remaining steps of the suggested route to dehalogenated perophoramidine.

Tetrachloroethylene and its daughter-products represent a group of contaminations which are frequently found at sites with industrial activities, such as metal processing, electrotechnical and pharmaceutical industries as well as dry cleaning of clothing and the production of colours, paints and laquers. Due to their toxicity and persistence under natural conditions "denser-than-water" non aqueous phase liquids are substantial threats to the subsurface environment as well as the surface ecosystems including human beings. During the last two decades a number of technical solutions has been presented to enhance the situation of contaminated areas. One of the more recently established concepts are permeable reactive barriers. Permeable reactive barriers are passive in situ treatment zones containing a reactive material suitable to remove the contamination from the groundwater. They are installed downgradient from the pollution source perpendicular to the groundwater flow direction to immobilise or degrade the dissolved pollutants in the groundwater as it flows through. This project was organised in two main parts. The first part assessed seven different iron powders in batch experiments to determine the most efficient powder in terms of degradation velocity. The second part of the study employed this powder in a column experiment using different mixing ratios with sand to evaluate its performance under simulated subsurface conditions in a permeable reactive barrier. The aim of this experiment was to obtain a more detailed description of the behaviour and performance of the selected material. In the batch experiment the most promissing iron powder produced a half-life of tetrachloroethylene of 2.36 h. The column study demonstrated that cis-dichloroethylene has the longest half-life compared to tetrachlorethylene and trichloroethylene with 1.65 h. Having the longest half-life of all chloroethylenes included in this investigation the cis-dichloroethylene concentration will determine the dimensioning of a permeable barrier for remediation purposes.

Les cytochromes P450 sont des enzymes mononucléaires de fer qui catalysent l’oxydation de liaisons C-H en utilisant le dioxygène de l’air. L’activation de O2 nécessite sa réduction partielle par l'apport de deux protons et deux électrons. Ces derniers sont fournis par le réducteur naturel NADPH par l'intermédiaire d'une flavoprotéine qui permet de convertir l’adduit fer(II)-O2 en un intermédiaire de type fer(III)-peroxo dont les protonations successives permettent la rupture hétérolytique de la liaison O—O et l’obtention d’un intermédiaire de type fer-oxo responsable de l’oxydation du substrat.1 Ce projet s’inscrit dans le cadre de la chimie bio-inspirée, il a pour objectif de développer des catalyseurs de fer non-hémiques afin de réaliser l’oxydation de petites molécules organiques en utilisant le dioxygène de l’air. L’activation du dioxygène est assurée par le biais d’une électrode qui sert de source d’électrons mais également de sonde mécanistique. Pour comprendre le mécanisme de l’activation du dioxygène il est nécessaire (i) de générer les intermédiaires réactionnels oxydants (fer-oxo, fer-(hydro)peroxo) par le biais d’oxydants chimiques comme le PhIO et H2O2 puis (ii) de les caractériser par spectroscopie et électrochimie à basse température. Enfin (iii) l’étude expérimentale par voltampérométrie cyclique de la réaction du complexe de Fe(II) en présence de dioxygène couplée à des simulations de voltampérogrammes de cette même réaction (à l’aide de données thermodynamiques et cinétiques obtenues en (ii)) permettent ainsi de valider le mécanisme de l’activation réductrice du dioxygène de ce système. Ce travail a démontré que les complexes de FeII avec des ligands amines/pyridine peuvent activer le dioxygène à la surface d’une électrode en suivant un mécanisme proche de celui du cytochrome P450. A présent, la difficulté principale est de s’affranchir de la réduction prématurée des intermédiaires lorsqu’ils sont générés à l’électrode. En se basant sur ces résultats, l’essentiel du travail est à présent ciblé sur la compréhension des différents paramètres (première sphère de coordination, conditions expérimentales, …) qui peuvent modifier la stabilité des intermédiaires et donc l’efficacité de la catalyse. / Cytochrome P450 is a mononuclear iron enzyme, which catalyzes the oxidation of robust C-H bonds using O2. Activation of O2 is achieved at the Fe(II) center and requires an electron transfer to convert the iron(II)-dioxygen adduct into an iron(III)peroxo intermediate. After protonation, this latter may directly oxidize substrates or evolves to yield a powerful high valent iron-oxo moiety. In such natural systems, the necessary electrons are provided by a co-substrate NAD(P)H and are conveyed through a reductase.1The aim of this project is to develop non-heme iron(II) complexes as catalysts for the oxidation of small organic molecules by O2. Our objective is to use an electrode to deliver the electrons while providing mechanistic information at the same time thanks to a combined experimental/simulation approach using cyclic voltammetry. This work has shown that simple Fe(II) complexes bearing amine/pyridine ligands can activate O2 at an electrode surface following a mechanism that is reminiscent of the one of P450. However, the main scientific lock is to avoid the fast reduction of the reaction intermediates when they are generated at the electrode. We are currently studying how alterations of the first coordination sphere of the metal center and experimental conditions modulate the formation and the stability of these intermediates and thus, the efficiency of the catalysts.

Nearly all multicellular organisms show changes in redox balance with aging leading to oxidative damage of macromolecules. This study investigated the role of the [NADP+]/[NADPH] redox couple in aging. This redox couple plays an important role in maintaining tissue redox balance and becomes slightly more oxidized in aged tissues. NADPH is a major source of reducing equivalents for enzymes that detoxify hydrogen peroxide. However, catalase detoxifies hydrogen peroxide independently of NADPH. But catalase is absent from mitochondria, a major source of hydrogen peroxide, where instead glutathione plays the major role in hydrogen peroxide detoxification in an NADPH-dependent manner. Three major cytoplasmic and three major mitochondrial NADPH generating enzymes were knocked down by the RNAi feeding technique in C. elegans, but little to no significant effects on lifespan were observed. The C. elegans genome contains two predicted, yet uncharacterized, NAD kinase (NADK) genes nadk-1 and nadk-2 to synthesize NADP+. Data suggest that NADK-1 is cytoplasmic, while NADK-2 is mitochondrial. NADK activity assays strongly suggested that both NADK-1 and NADK-2 possess NADK activity. Knockdown of NADKs did not affect the rate of body bending in young nematodes. However, knockdown of nadk-2 in nadk-1 mutant worms slightly decreased lifespan. Deficiency of NADKs increased the [NADP+]/[NADPH] and decreased the [NAD+]/[NADH], similar to redox changes that occur with aging. Unexpectedly, nadk-1 or nadk-2 knockdown resulted in decreased reactive oxygen species (ROS) levels and increased survival in young adult nematodes in the presence of juglone, a superoxide generator. The antioxidant response generated upon NADK knockdown required the transcription factors DAF-16/FOXO, SKN-1/Nrf2, and HSF-1 in nadk-2 knockdown nematodes, but only required HSF-1 in nadk-1 knockdown nematodes. NADK-1 or NADK-2 deficiency led to increased catalase activity and a strong trend for increased cytoplasmic catalase-1 (ctl-1) gene expression. Peroxisomal catalase-2 (ctl-2) mutant worms showed increased SOD activity when either NADK gene was knocked down, while ctl-1 mutant worms showed increased glutathione peroxidase or glutathione reductase activities following NADK knockdown. In summary, NADK knockdown oxidizes the [NADP+]/[NADPH] to compromise the antioxidant system, but young nematodes are able to mount a compensatory broad antioxidant response leading to decreased ROS levels.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第24432号 / 理博第4931号 / 新制||理||1704(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 依光 英樹, 教授 若宮 淳志, 教授 畠山 琢次 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Amoebozoa is the eukaryotic supergroup sister to Obazoa, the lineage that contains the animals (including us humans) and Fungi. Amoebozoa is extraordinarily diverse, encompassing important model organisms and significant pathogens. Although amoebozoans are integral to global nutrient cycles and present in nearly all environments, they remain vastly understudied. Here we have isolated a naked eukaryotic amoeba with filose subpseudopodia, and a simple life cycle consisting of a trophic amoeba and a cyst stage. Using a wholistic approach including light, electron, fluorescence microscopy and SSU rDNA, we find that this amoeboid organism fails to match any previously described eukaryote genus. Our isolate amoebae are most similar to some variosean amoebae which also possess acutely pointed filose subpseudopodia. Maximum likelihood and Bayesian tree of the SSU-rDNA gene places our isolate in Variosea of Amoebozoa as a novel lineage with high statistical support closely related to the highly diverse protosteloid amoebae Protostelium. This novel variosean is herein named “Hodorica filosa” n. g. n. sp. We present a robust phylogeny of Amoebozoa based on a broad representative set of taxa in a phylogenomic framework (325 genes). By sampling 61 taxa using culture-based and single-cell transcriptomics, our analyses show two major clades of Amoebozoa, Discosea and Tevosa. Overall, the main macroevolutionary patterns in Amoebozoa appear to result from the parallel losses of homologous characters of a multiphase life cycle that included flagella, sex, and sporocarps rather than independent acquisition of convergent features Integrins are transmembrane receptors that activate signal transduction pathways upon extracellular matrix binding. The Integrin Mediated Adhesion Complex (IMAC), mediates various cell physiological processes and are key elements that are associated animal multicellularity. The IMAC was thought to be specific to animals. Over the last decade however, the IMAC complexes were discovered throughout Obazoa. We show the presence of an ancestral complex of integrin adhesion proteins that predate the evolution of the Amoebozoa. Co-option of an ancient protein complex was key to the emergence of animal multicellularity. The role of the IMAC in a unicellular context is unknown but must also play a critical role for at least some unicellular organisms.

With increasing global population comes an increase in the need to safe and clean drinking water. Contaminants can arise in drinking water either naturally, or by the interaction of disinfection chemicals with naturally occurring materials, or simply due to by-products of the disinfection mechanism itself. Due to the oxidative nature of our disinfection treatments, these species are in highly oxidized states, and in some cases require chemical reduction to become less harmful. The present work demonstrates the capabilities of aqueous phase corona plasma in reductive treatment of oxidized contaminants found in drinking water. This study focuses on the treatment of the nitrate ion, bromate ion, chlorate ion and monobromoacetic acid, all of which can be found in typical drinking water systems. The second and third chapters within this thesis establish the optimal water matrix conditions for the treatment of bromate, chlorate and nitrate. These experiments investigate the influence of pH, temperature, presence and types of oxidative scavengers, dissolved gases and by-products that are made by this treatment process with these compounds. The main conclusion of these works is that aqueous phase corona discharge is capable of producing chemical compounds with sufficient energy to chemically reduce the nitrate, bromate and chlorate anions. Acidic conditions, under low dissolved oxygen scenarios facilitated the highest amount of reduction of the target contaminants, as well as having the presence of oxidative species scavengers. It was also observed that the anoxic environment could be obtained by introducing alcohols into the contaminated solution which generated sufficient cavitation and bubbling to strip the oxygen from solution. Through a comparison of various carbonaceous compounds as oxidative species scavengers, it was determined that the volatile alcohols provided a better performance than other soluble carbon sources, due to the decrease in dissolved oxygen. The fourth chapter considers different methods of introducing argon, oxygen and nitrogen into the test solution for the effect they would have on the treatment of solutions containing the bromate anion or monobromoacetic acid. The optimal pH for the treatment of monobromoacetic acid was also established, where again the acidic conditions prevailed. Tests were conducted to consider the effect of having the solution pre-saturated with the test gas, continually sparged, or with the gas passing through a hollow discharge electrode. The tests in which gas was blown through the discharge electrode greatly surpassed all other treatment regimes, where nitrogen provided the best removal for both contaminants under acidic conditions for bromate and under acidic and basic conditions for monobromoacetic acid. The fifth chapter provides conclusions for the overall thesis and recommendations for future work. / Thesis / Doctor of Philosophy (PhD)

Fundamental research is required to support the commercialization of 3rd Generation Advanced High Strength steels (3G AHSS). Mid-manganese 3G AHSS steels can contain up to 11wt% manganese and are expensive if traditional ferroalloying practices are used; reductive alloying is a promising alternative. This study has researched the fundamental science behind possible processing methods. Silicothermic reduction of MnO from slag was studied. The reaction is fast but can be blocked by a stagnant layer of SiO bubbles cutting the rate of reaction by one order of magnitude. A theoretical model for mixed mass transport control was tested against original experimental data. Across nine datasets, the mass transfer coefficient for metal species, kMetal, was 2.3∙10-4m/s and the slag mass transfer coefficient, kSlag, was 6.7∙10-4m/s. In real industrial systems, gas blockage should not have an effect because stirring will dislodge these bubbles. Carbothermic reduction is dramatically different and has been qualitatively documented in this work. The reaction occurs in two stages: the first approximately three times faster than the second. The first stage is characterized by internal CO nucleation and growth and is rate-limited by the formation and growth of these CO bubbles. The second stage occurs along the metal interface and shows that the slag and metal are essentially separated by an intermediary gas phase. This reaction is controlled by decomposition of metal oxides at the gas-slag boundary, decomposition of CO2 at the gas-metal boundary, and transport of CO2 across the gas bubble; this mechanism is nearly identical to the carbothermic reduction of FeO. Reductive alloying can be utilized with the silicothermic reduction process to obtain high levels of manganese in steel but the carbothermic reduction may be too slow to be a viable process. / Thesis / Doctor of Philosophy (PhD) / 3rd Generation Advanced High Strength steels (3G AHSS) are a promising opportunity to produce steels with improved mechanical properties. These steels are alloyed with up to 11wt% manganese; traditional alloy additions are added as ferroalloys which may not be the most economical solution to achieve the required concentrations of manganese. Reductive alloying is a potential method for achieving high concentrations of manganese in the metal. By adding manganese oxide to slag, and reductants like carbon or silicon to the molten metal, manganese can be reduced from slag to metal. This work has determined the kinetics (rate of reaction) during the silicothermic and carbothermic reduction of manganese oxide from slag. The silicothermic reduction of manganese oxide is fast and can achieve high levels of manganese in the metal. The carbothermic reduction is much slower with questionable viability.

Reductive dechlorination is a significant natural attenuation process in chloroethene-contaminated aquifers where organic carbon combined with reducing redox conditions support active dechlorinating microorganisms. At sites where natural organic carbon (NOC) associated with the aquifer matrix provides fermentable organics, the ability to measure the NOC is needed to assess the potential for the long-term sustainability of reductive dechlorination. This study focused on developing a method to measure the potentially bioavailable organic carbon (PBOC) associated with aquifer sediment. To measure NOC and evaluate its biodegradability, liquid extraction techniques on aquifer sediment were investigated. Single extractions with different extracting solutions showed that extractable organic carbon associated with the sediment ranged from 1-38% of the total organic carbon content (TOCs). Bioassay experiments demonstrated that 30-60% of the extractable organic carbon can be utilized by a microbial consortium. Alternating between 0.1% pyrophosphate and base solutions over multiple extractions increased the rate of removal efficiency and targeted two organic carbon pools. The result of the investigation was a laboratory method to quantify organic carbon from the aquifer matrix in terms of the PBOC. In the second part, the extractable PBOC was shown to biodegrade under anaerobic conditions, to produce H2 at levels necessary to maintain reductive dechlorination, and to support reductive dechlorination in enrichment cultures. For the third part of the research, the difference in extractable organic carbon inside and outside of a chloroethene-contaminated plume was examined through the combination of PBOC laboratory data and field parameters. Supported by ground-water constituent data, the PBOC extraction and bioassay studies showed that less extractable organic carbon was present inside than outside of the chloroethene plume. The final part of the research investigated the distribution of PBOC extractions across six contaminated sites. PBOC extractions were directly correlated to the TOCs, soft carbon content, and level of reductive dechlorination activity at the sites. Based on these correlations, a range for organic carbon potentially available to subsurface microorganisms was proposed where the upper bound consisted of the soft carbon and the lower bound consisted of the PBOC. / Ph. D.

Groundwater remediation strategies have advanced toward more effective and economical remedial technologies. Monitored natural attenuation (MNA) has become accepted by federal regulatory agencies as a viable remediation strategy for contaminants under site-specific conditions. At chloroethene contaminated sites where MNA is used as a remediation strategy, microbially-mediated reductive dechlorination is typically the dominant pathway for natural attenuation. The efficacy of reductive dechlorination at sites with no anthropogenic carbon sources is often influenced by the availability of readily-biodegradable natural organic carbon along with favorable geochemical conditions for supporting microbial dehalogenation. Recent research studies have suggested that the pool of labile natural organic carbon, operationally defined as potentially bioavailable organic carbon (PBOC), may be a critical component related to sustaining reductive dechlorination at MNA sites. The objective of this study was to evaluate PBOC as a quantitative measure of the labile organic carbon fraction of aquifer sediments in relation to microbial reductive dechlorination of chlorinated solvents. In the first phase of this study, the variability of PBOC in aquifer sediments was examined among 15 chloroethene contaminated sites. Results showed that PBOC displayed considerable variability among the study sites, ranging over four orders of magnitude. Regression results demonstrated that a positive correlation existed between PBOC, solid phase total organic carbon (TOCs), and reductive dechlorination activity at the sites. Results supported that greater levels of PBOC and TOCs corresponded to higher reductive dechlorination activity at the sites. Composition results showed that 6-86% of PBOC consisted of proteins and amino acids. Results also suggested a positive relationship existed between PBOC, concentrations of potentially bioavailable organic compounds present in the aquifer system, expressed as hydrolyzable amino acids (HAA), and the natural attenuation capacity (NAC) at the sites. Higher PBOC levels were consistently observed at sites with greater NAC and levels of HAA. The results of this study suggested that the variability of PBOC in the aquifer sediments exhibited a reasonable correlation with TOCs, hydrolyzable amino acids, and chloroethene transformation among the selected sites. In the second phase of this study, the relationship between PBOC in aquifer sediments and site specific performance data was evaluated among 12 chloroethene contaminated sites. Results demonstrated that PBOC in aquifer sediments was directly correlated to independent field metrics associated with reductive dechlorination. Levels of PBOC demonstrated direct relationships with hydrogen (H2) and dissolved oxygen (DO) concentrations within the groundwater system at the selected study sites. Results also indicated that PBOC demonstrated positive relationships with reductive dechlorination activity and the natural attenuation capacity of the sites. The findings of this study suggested that the level of PBOC in aquifer sediments may be a key factor in sustaining conditions favorable for microbial reductive dechlorination. In the third phase of this study, the distribution of PBOC was investigated at a chloroethene contaminated site. PBOC was measured in surficial aquifer sediment samples collected at varying depths in the vicinity of a chloroethene plume. Results demonstrated that levels of PBOC were consistently higher in aquifer sediments with minimal chloroethene exposure relative to samples collected in the PCE-contaminated source zone. Regression results demonstrated that a statistically significant inverse correlation existed between PBOC levels and chloroethene concentrations for selected temporary wells in the contaminated source zone at the study site. Consistent with these findings, results also indicated a similar trend of increased PBOC in aquifer sediments outside the chloroethene plume relative to aquifer sediments inside the plume. Results from this study further suggested that differences in extracted carbon levels at the site for surficial aquifer sediment samples in the PCE-contaminated source zone could impact the extent of reductive dechlorination within the hydrographic unit. / Ph. D.