京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19010号 / 工博第4052号 / 新制||工||1623(附属図書館) / 31961 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 田畑 泰彦, 教授 岩田 博夫, 教授 木村 俊作 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(工学) / 甲第24643号 / 工博第5149号 / 新制||工||1983(附属図書館) / 京都大学大学院工学研究科化学工学専攻 / (主査)教授 河瀬 元明, 教授 大嶋 正裕, 教授 佐野 紀彰 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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<p>The controlled synthesis of functional low molecular weight polyethylene and polyethylene mimics is important in tuning polymer properties and is of great industrial interests. Living polymerization is a method that allows for precise control in polymer structure. Although high molecular weight polymers with controlled structures can be efficiently produced via living polymerization, the production of low molecular weight polymers faces the challenges of the use of large amounts of expensive catalyst and the broadening of polydispersity.</p> <p>The synthesis of well-defined functional low molecular weight polyethylene and polyethylene mimics is studied. Promising polymerization systems, including living ring opening metathesis polymerization (ROMP), living coordination polymerization, coordinative chain transfer polymerization (CCTP), and living C1 polymerization, are identified and are analyzed based on product properties, efficiency, cost, and safety.</p> <p>Within the identified systems, living ROMP is selected for study due to the industrial relevance of ROMP polymers, the availability of raw materials, and the ease of reaction setup. The efficiency of ROMP is challenged by polydispersity broadening resulting from slow initiation and poor reactor volume efficiency due to its implementation as a solution polymerization process. The challenges are addressed by the use of excess phosphine and the realization of ROMP as a bulk polymerization process.</p> <p>Experimental results demonstrate that bulk ROMP with and without phosphines yield product with similar or enhanced molecular weight distribution control as solution ROMP. Kinetic studies confirm living polymerization behaviour of bulk ROMP. A mathematical model is developed for the first time using method of moments to describe the kinetics and development of molecular weight distribution of ROMP. The model is a useful tool in preliminary research and commercialization of ROMP. The success of bulk ROMP and the development of a representative model yield ROMP as a promising method for the production of low molecular weight polymers with controlled architecture.</p> / Master of Applied Science (MASc)

The predicted protein product of open reading frame slr0328 from Synechocystis sp. PCC 6803, SynPTP, possesses significant amino acid sequence similarity with known low molecular weight protein tyrosine phosphatases (PTPs). To determine the gross functional properties of this hypothetical protein, open reading frame slr0328 was cloned, and its predicted protein product was expressed in E. coli. The recombinant protein, SynPTP, was purified by metal ion column chromatography. The catalytic activity of SynPTP was examined toward several exogenous protein substrates that had been phosphorylated on either tyrosine residues or serine residues. SynPTP exhibited phosphatase activity toward tyrosine phosphorylated protein substrates (Vmax toward phosphotyrosyl 32P-casein was 1.5 nmol/min/mg). However, no phosphatase activity was detected toward serine phosphorylated protein substrates. SynPTP displayed phosphohydrolase activity toward several organophosphoesters including para-nitrophenyl phosphate (p-NPP), beta-napthyl phosphate and phosphotyrosine but not toward alpha-napthyl phosphate, phosphoserine, or phosphothreonine. Kinetic analysis indicated that the Km (0.6 mM) and Vmax (3.2 mmole/min/mg) values for SynPTP toward pNPP are similar to those of other known bacterial low molecular weight PTPs. The protein phosphatase activity of SynPTP was inhibited by sodium orthovanadate, a known inhibitor for tyrosine phosphatases, but not by okadaic acid, an inhibitor for many serine/threonine phosphatases. Mutagenic alteration of the predicted catalytic cysteine, Cys7, to serine abolished enzyme activity. Several phosphotyrosine containing proteins were detected from the whole cell extracts of Synechocystis sp. PCC 6803 through immunoreactions using anti-phosphotyrosine antibody. SynPTP was observed to dephosphorylate three of these proteins in vitro. Two of these proteins were identified by peptide-mass fingerprinting analysis, as PsaD (photosystem I subunit II) and CpcD (phycocyanin rod linker protein). In addition, several phosphotyrosine proteins were detected from the soluble and membrane fractions of Synechocystis sp. PCC 6803 cell extracts by in vitro substrate trapping as potential endogenous substrates of SynPTP. Two of these proteins were identified as the alpha and beta subunits of phycocyanin. We therefore speculate that SynPTP might be involved in the regulation of photosynthesis in Synechocystis sp. PCC 6803. / Ph. D.

Soil microorganisms are primarily limited by carbon (C) availability. The majority of C entering belowground food webs comes directly from local flora. Plant derived labile C compounds affect microbial community structure and function, which in turn drive ecosystem function. Research has focused on dissolved organic C (DOC) from litter leachates and root exudates. These compounds are often readily assimilable by soil microorganisms and are precursors for stable soil organic matter formation. Due to diffusion limitation DOC rarely travels far beyond its origin, meaning most soil microorganisms are unable to access these compounds unless they are located near the C source. However, recent studies have illuminated the importance of volatile organic compounds (VOCs) in soil ecosystems. VOCs are produced in abundance and, as vapors, they are able to travel through soil more rapidly than DOC. This dissertation aims to investigate the importance of VOCs commonly produced during the decomposition of leaf litter. We used three separate microcosm experiments to answer the following questions. 1) How do abundant VOCs affect microbial activity in soil? 2) How do VOCs affect nitrogen (N) transformations and the microbes associated with N transformations? 3) How do VOCs affect microbial community composition? 4) Are VOCs from decomposing litter incorporated into soil C pools? In chapter 2, we show that methanol and acetone – common litter derived VOCs – increase microbial activity and labile soil C, while also decreasing available nitrate, and ammonia oxidizing archaea. Interestingly, this decrease in nitrifiers did not affect nitrification rate after VOC addition was ceased. In chapter 3, we demonstrate that soil microbial taxa respond differently to DOC and VOCs at different soil moisture levels. Specifically, DOC primarily affected taxa abundance in wetter soils, while the insoluble VOC α-pinene had the largest impact at lower moisture levels, and methanol affected abundance at all moisture levels. Finally, in chapter 4, we demonstrate that VOCs from decomposing leaf litter altered soil bacterial and fungal communities, and VOC derived C entered all measured soil organic matter pools without direct contact between decomposing litters and the soil. This work demonstrates the importance of VOCs on soil microbial communities and ecosystem function. The VOC induced increase in microbial activity, and the effects of VOCs at low moisture levels suggest that VOCs may function in the bulk soil in a manner similar to DOC in rhizosphere soil. Additionally, the incorporation of VOC-C into soil organic matter pools identifies a hitherto unrecognized mechanism for soil organic matter formation. / Doctor of Philosophy / Soil microorganisms live in an environment where their access to carbon containing compounds limits their growth. In these belowground environments most of the carbon flows from aboveground plant matter through soil microbes into the organisms that consume those microbes. The carbon from plants not only feeds the soil microbes but also changes the type of microbes and how those microbes process important chemicals in the environment – e.g., carbon and nitrogen. Previously, research has focused on carbon compounds that are able to dissolve in water. Often, these compounds originate from liquids that plants release from their roots, or dissolve like tea when leaves are soaked in water. Soil microorganisms can often use these dissolved carbon compounds and directly incorporate them into their biomass. Additionally, these compounds can be stored in soil - sequestering that carbon in the soil, potentially long term. However, dissolved compounds are unable to move very quickly through soil, and the soil microorganisms that live far from the source of these compounds do not have access to them. However, recent studies have found that another form of carbon, volatile organic compounds, are also produced in abundance in the soil environment. These compounds can travel through the air in the soil, as well as in the soil water. When in the air, VOCs travel very quickly and can also travel farther than dissolved compounds. This dissertation aims to investigate the importance of volatile organic compounds that are produced during the decomposition of leaves. We carried out three experiments using small volumes of soil under controlled conditions in the laboratory. We aimed to answer the following questions. 1) How do abundant volatile organic compounds affect microbial activity in soil? 2) How do volatile organic compounds affect microbial processing of nitrogen containing compounds, and the populations of microorganisms that process those compounds? 3) How do volatile organic compounds affect the composition of microorganism in the soil? 4) Are volatile organic compounds from decomposing leaves able to be stabilized in the soil. In chapter 1, we show that methanol and acetone – common volatile compounds produced during the decomposition of leaves– increase microbial activity, and microbial available carbon in soil. Methanol and acetone also decreased available nitrate (an important N containing compound) and a group of organisms that produce nitrate called ammonia oxidizing archaea. Interestingly, once we stopped adding methanol and acetone to the soil the production of nitrate did not differ, meaning that the nitrate producing community was able to recover from the reduction in ammonia oxidizing archaea. In chapter 2, we demonstrated that soil microbial taxa respond differently to dissolved carbon and volatile organic compounds across a gradient of soil moisture. Specifically, dissolved carbon primarily affected taxa abundance in wetter soils, while the insoluble volatile α-pinene had the largest impact at lower moisture levels, and the volatile compound methanol affected abundance of microbial taxa at all moisture levels. Finally, in chapter 3, we demonstrate that volatile organic compounds produced during the decomposition of leaves altered the composition of both bacterial and fungal communities in the soil. Also, and possibly most interestingly, carbon from those volatile organic compounds was stored in all of the pools of carbon that we measured. Together these chapters demonstrate the importance of volatile organic compounds on soil microbial communities and ecosystem function. Since volatile organic compounds induced an increase in microbial activity we are able to infer that soil microorganisms are using these compounds; paired with our observation that volatile organic compounds affected microbial taxa at lower moisture levels than the dissolved compounds did, we can infer that volatile compounds may function as a carbon source in parts of the soil that do not have access to dissolved carbon. Additionally, the incorporation of carbon from volatile organic compounds into soil identified a hitherto unrecognized mechanism for soil carbon sequestration.

The components of soil CO2 efflux are affected by many soil properties including temperature, moisture, microbial abundance and activity, and other soil physical and chemical properties. Changes in these factors can result in high spatial and temporal variability of total soil CO2 efflux. Low molecular weight organic acids (LMWOAs), dissolved organic carbon (DOC) and dissolved organic nitrogen (DON), microbial biomass and activity were measured to evaluate the impact of intercropping switchgrass (Panicum virgatum L.) in a loblolly pine (Pinus taeda L.) plantation. Surface soil samples (0-15 cm) were collected on the bed (PSG-B), interbed (PSG-I) and edge (PSG-E) of pine-switchgrass intercropped treatments, as well as pine only (P-B) and switchgrass only (SG-I) treatments. Differences in most soil properties and processes of intercropped treatments were sporadic and most did not show clear trends. However, significant correlations between DOC, soil temperature, oxalic and acetic acids and soil CO2 efflux were present. In an multiple regression model these factors explained 57% of the variance in total soil CO2 efflux. Therefore we think that LMWOAs, as a labile component of DOC, are influencing total CO2 efflux because they are being consumed by microbial community, increasing heterotrophic respiration and as a result overall total CO2 efflux. The amount and distribution of labile C controls microbial community dynamics, heterotrophic respiration as well as the stabilization of soil C. / Master of Science

Sulfated low molecular weight lignins (LMWLs), CDSO3 and FDSO3, designed recently as macromolecular mimetics of heparin, were found to exhibit potent anticoagulant activity. Small molecules based on the same scaffold, SBD and SBT, showed promising thrombin inhibition. We were able to address the mechanism of the inhibition using Michaelis-Menten kinetics. All the molecules were found to be allosterically impairing thrombin activity using either noncompetitive or uncompetitive mechanism. Absence of competition with hirugen, an exosite 1 ligand, and competition with polymeric heparin points to exosite 2 as the site of interaction for these inhibitors. Yet mixed competition results with other exosite 2 ligands indicated that the molecules utilize different sub-sites within exosite 2 for interaction. Site-directed mutagenesis was used to pin point the key residues important for inhibition. All of all positively charged exosite 2 residues were mutated one at a time to alanine to abolish its charge. The data showed that Arg93 and Arg175 are the major residues involved in CDSO3 binding. FDSO3 showed a progressively greater defect in inhibition with double point mutations, the triple mutant Arg93,97,101Ala displayed a 50 fold drop in inhibition. A single mutant, Arg173Ala, displayed 22-fold reduction in IC50 of SBD, while Arg233Ala was the only mutation that impaired SBT inhibition. This proves the fact that inspite of the structural similarity between the two polymers and the two small molecules, thtey do not share the same binding space in exosite 2. To understand the types of interactions involved in thrombin interaction with the polymers, we resorted to salt-dependence studies. This showed that CDSO3 had fewer ionic contacts with thrombin, with most of its binding energy derived from non-ionic interactions. FDSO3 on the other hand had a balanced contribution of ionic and non-ionic forces. Thermodynamic studies showed that both polymers have a positive ΔCp of binding, which proves the involvement of electrostatic forces and signals the burial of the polar residues on thrombin exosite 2. These molecules offer a rare chance to study thrombin allostery. Little is known about the interplay between exosite 2, active site and sodium binding site. The allosteric nature of inhibition indicated that, for the first time, a link is proven to exist between exosite 2 and the active site that could be used to inhibit the enzyme. The presence of sodium was found to enhance the binding of FDSO3 at exosite 2, which establish the energetic coupling between exosite 2 and sodium binding site. The results identify novel binding sub-sites within exosite 2 that are energetically coupled to thrombin’s catalytic function and linked to the sodium binding site. The design of high affinity small molecules based on LMWLs scaffold presents major opportunities for developing clinically relevant, allosteric modulators of thrombin.

A esquistossomose é uma doença tropical negligenciada que atinge cerca de 200 milhões de pessoas em todo o mundo, abrangindo a América, a África, as Antilhas, o Oriente Médio e Próximo, além do Sudeste Asiático. A espécie encontrada no Brasil é a Schistosoma mansoni, onde se tem como tratamento típico a administração do Praziquantel ou da Oxamniquina. No entanto, sua característica de infecção se associa a saneamento básico precário e baixos padrões sócio-econômicos, de maneira que a reinfecção de doentes apresenta altas taxas de ocorrência, o que motiva a busca por fármacos ou vacinas antihelmíticas que superem esta dificuldade. Neste trabalho são utilizadas técnicas proteômicas para a identificação de proteínas que estejam potencialmente envolvidas na diferenciação entre os sexos, na interação entre parasitas de diferentes sexos ou com o hospedeiro. São estudadas preparações de amostras de sincício e vermes inteiros adultos machos e fêmeas por eletroforese bidimensional e frações de baixo peso molecular de sincício de vermes adultos machos e fêmeas por gel-LC. A expressão diferencial de proteínas de sincício investigada por gel-LC foi avaliada por análise estatítica, sendo detectadas 5 proteínas mais abundantes em machos e 2 em fêmeas, além de 6 proteínas identificadas somente em machos e 21 somente em fêmeas. Estas informações de expressão diferencial possibilitam a investigação dos recursos de sobrevivência e reprodução desenvolvidos evolutivamente por estes parasitas. / Schistosomiasis is a neglected tropical disease that affects approximately 200 million people around the world, occurring in America, Africa, the Antilles, Middle East and Near East, besides Southeast Asia. The species found in Brazil is Schistosoma mansoni, the typical treatment being administration of either Praziquantel or Oxamniquine. Although, the infection characteristics of this disease is associated with poor sanitation and hardened socio-economic conditions, resulting in high reinfection rates, which motivates the search for antihelmintic drugs and vaccines that overcome this situation. In this study proteomics techniques are used in the search of proteins potencially involved in the differentiation of individuals of both sexes, in the interactions between them and between the worms and the host. Samples of worm syncytium and adult whole worms of both male and female are studied by two-dimentional electrophoresis, while low molecular weight syncytium proteins from male and female adult worms were investigated by gel-LC. The differential protein expression in the syncytium investigated by gel-LC was analyzed statistically, being detected 5 proteins most abundant in males, and 2 in females, while 6 were identified solely on males and 21 on females. The information concerning protein differential expression allows the investigation of survival strategies developed evolutionarily by these parasites.