Les hélicases sont des protéines qui utilisent l'énergie fournie par l'hydrolyse de l'ATP ou du GTP pour catalyser la disjonction des doubles hélices d'ADN ou d'ARN. Cette activité de déroulement de double brin leur confère un rôle essentiel dans le métabolisme des acides nucléiques, le maintien de la stabilité du génome et les processus de signalisation cellulaire. En conséquence, ils sont impliqués dans des processus aussi divers que le vieillissement, l'apparition de cancers, l'immunité innée. Cette thèse est axée sur la compréhension de la fonction et des mécanismes moléculaires de deux hélicases différentes et le manuscrit est donc divisé en deux parties. Le premier est dédié à l'hélicase RIG-I, une hélicase à ARN, exprimée lorsque les cellules leucémiques cessent de proliférer et sont induites à se différencier en granulocytes, indispensables à la reconnaissance de l'ARN double brin des virus, initiant la protection des cellules contre la réplication des génomes viraux. Le mécanisme d'action de RIG-I est bien décrit dans le contexte d'une infection virale. Mais dans le cas de la différenciation des cellules myéloïdes, l'intervention de RIG-I et son rôle dans la balance la prolifération / différenciation restent incomplets. En effet, les interactions RIG-I en particulier avec les ligands cellulaires ne sont pas totalement comprises. La première partie de mon travail consistait à tenter d'isoler et de caractériser les partenaires de RIG-I lors de la différenciation des cellules leucémiques NB4. La seconde est consacrée à l'étude des mécanismes sous-jacents aux G-quadruplexes résolus par les hélicases. Plusieurs questions subsistent quant aux interactions entre la structure particulière des G-quadruplexes et ces enzymes. Une hélicase de Bacteroides sp 3_1_23, BsPif1, a été choisie pour comparer et caractériser l'interaction entre les G-quadruplexes et l'ADN canonique de Watson-Crick. Dans les deux parties du travail, les interactions ont été étudiées par des techniques biochimiques utilisant soit une lignée cellulaire ou une protéine purifiée et des acides nucléiques synthétiques. / Helicases are proteins that utilize the energy provided by the hydrolysis of ATP or GTP to catalyse the disjunction of double DNA or RNA helices. This double strand unwinding activity gives them an essential role in the metabolism of nucleic acids, the maintenance of the genome stability and cell signalling processes. As a result, they are involved in processes as diverse as aging, the appearance of cancers, innate immunity. This thesis is focused on the understanding of the function and the molecular mechanisms of two different helicases and the manuscript is therefore divided in two parts. The first one is dedicated to the RIG-I helicase, an RNA helicase, expressed when leukemic cells stop proliferate and are induced to differentiate in granulocytes, which are essential in the recognition of double-stranded RNA of viruses, initiating the protection of the cells against the replication of the viral genomes. The mechanism of action of RIG-I is well described in the context of viral infection. But in the case of the differentiation of myeloid cells, the intervention of RIG-I and its influence on the equilibrium proliferation / differentiation remains incomplete. Indeed, RIG-I interactions in particular with cellular ligands are not fully understood. The first part of my work consisted in an attempt to isolate and characterize RIG-I partners during differentiation of NB4 leukemic cells. The second one is devoted to the study of mechanisms underlying G-quadruplexes resolving by helicases. Several questions remain about the interactions between the particular structure of G-quadruplexes and these enzymes. A Bacteroides sp 3_1_23 helicase, BsPif1, was chosen to compare and characterize the interaction between G-quadruplexes and canonical Watson-Crick DNA. In the two parts of the work, the interactions were investigated by biochemical techniques using either a cell line or purified protein and synthetic nucleic acids.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24984号 / 生博第513号 / 新制||生||68(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 野田 岳志, 教授 朝長 啓造, 教授 今吉 格 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

The EPA Lead and Copper Rule set action limits for lead and copper concentrations in drinking water, but accelerated corrosion of lead in distribution systems due to a galvanic connection to copper. Prior research has demonstrated that the effects of galvanic corrosion can be controlled by water chemistry. This study not only investigated the main effects of alkalinity, natural organic matter (NOM), nitrate, disinfectant and inhibitor to galvanic corrosion, but also the interplay between these factors. A 2-level factorial (2v5-1) design was adopted which resulted in 16 testing conditions. Results of bench-scale experiments using static pipes with lead and copper segments demonstrated that alkalinity, disinfectant, inhibitor and alkalinity-inhibitor interaction had a significant impact on galvanic current. The significant factors affecting total lead release were alkalinity, NOM, disinfectant, alkalinity-inhibitor interaction, NOM-nitrate interaction, NOM-disinfectant interaction, NOM-inhibitor interaction, nitrate-disinfectant interaction and disinfectant-inhibitor interaction.

The EPA Lead and Copper Rule set action limits for lead and copper concentrations in drinking water, but accelerated corrosion of lead in distribution systems due to a galvanic connection to copper. Prior research has demonstrated that the effects of galvanic corrosion can be controlled by water chemistry. This study not only investigated the main effects of alkalinity, natural organic matter (NOM), nitrate, disinfectant and inhibitor to galvanic corrosion, but also the interplay between these factors. A 2-level factorial (2v5-1) design was adopted which resulted in 16 testing conditions. Results of bench-scale experiments using static pipes with lead and copper segments demonstrated that alkalinity, disinfectant, inhibitor and alkalinity-inhibitor interaction had a significant impact on galvanic current. The significant factors affecting total lead release were alkalinity, NOM, disinfectant, alkalinity-inhibitor interaction, NOM-nitrate interaction, NOM-disinfectant interaction, NOM-inhibitor interaction, nitrate-disinfectant interaction and disinfectant-inhibitor interaction.

No description available.

Após o acidente de Macondo (EUA, 2010), cujo prejuízo estimado é da ordem de US$ 42 bilhões, a análise de risco do BOP (Preventor de BlowOut) ganhou grande importância nos projetos de exploração e desenvolvimento de petróleo. O presente trabalho se aprofundou na análise de risco de falha no acionamento do preventor anular de BOP (uma das funções do BOP), mapeando a sensibilidade da manutenção, redundâncias do projeto e da vida útil dos componentes no risco de falha inerente ao seu funcionamento. Este estudo seguiu os fundamentos da inspeção baseada no risco e da manutenção centrada na confiabilidade, a partir das diretrizes das normas ISO 31.000 (gestão do risco) e API 581 (inspeção baseada no risco). Um modelo de confiabilidade foi construído com base na árvore de falhas do equipamento, a qual, por sua vez, foi construída com base no diagrama hidráulico do sistema e do respectivo FMEA (Failure Mode and Effect Analysis). Neste modelo, pressupõe-se uma taxa de falhas constante dos componentes ao longo do tempo para calcular o tempo médio entre falhas do sistema. O trabalho atendeu seus objetivos ao comprovar que o modelo numérico de confiabilidade é aderente às falhas de campo e da literatura. A análise de sensibilidade do modelo por meio da simulação numérica, mostrou que o aumento da qualidade das manutenções preventiva é a melhor estratégia para reduzir o risco de falha do BOP anular. Verificou-se ainda que o aumento da vida útil de alguns componentes é tão relevante quanto o incremento de redundâncias para a confiabilidade do BOP anular. / After Macondo accident (USA, April 2010), with an estimated loss of US $ 42 billion, the failure risk analysis of the BOP (BlowOut Preventor) gained great importance in oil exploration and development projects. This work has deepened in BOP annular preventer (one of BOP functions) failure risk analysis, mapping sensitivity of maintenance of its components in the probability of equipment failure, according to the guidelines of RBI techniques (Risk Based Inspection) and RCM (Reliability Centered Maintenance). Therefore, the methodology used is proposed by API 580 (2009) and API 581 (2008) standards of risk based inspection applied on a reliability model of annular BOP. This reliability model, in turn, was based on fault tree analysis (FTA) and FMEA (Failure Mode and Effect Analysis) over the equipment hydraulic diagram. As hypothesis, it is assumed a constant failure rate of components over time, in order to calculate the mean time between failures. It was proved that reliability calculated by numerical model is coherent to field failures observations and literature data. The sensitivity analysis by numerical simulation shows that improving inspections and preventive maintenance quality in some of BOP components can markedly reduce the risk of failure of annular BOP.

Após o acidente de Macondo (EUA, 2010), cujo prejuízo estimado é da ordem de US$ 42 bilhões, a análise de risco do BOP (Preventor de BlowOut) ganhou grande importância nos projetos de exploração e desenvolvimento de petróleo. O presente trabalho se aprofundou na análise de risco de falha no acionamento do preventor anular de BOP (uma das funções do BOP), mapeando a sensibilidade da manutenção, redundâncias do projeto e da vida útil dos componentes no risco de falha inerente ao seu funcionamento. Este estudo seguiu os fundamentos da inspeção baseada no risco e da manutenção centrada na confiabilidade, a partir das diretrizes das normas ISO 31.000 (gestão do risco) e API 581 (inspeção baseada no risco). Um modelo de confiabilidade foi construído com base na árvore de falhas do equipamento, a qual, por sua vez, foi construída com base no diagrama hidráulico do sistema e do respectivo FMEA (Failure Mode and Effect Analysis). Neste modelo, pressupõe-se uma taxa de falhas constante dos componentes ao longo do tempo para calcular o tempo médio entre falhas do sistema. O trabalho atendeu seus objetivos ao comprovar que o modelo numérico de confiabilidade é aderente às falhas de campo e da literatura. A análise de sensibilidade do modelo por meio da simulação numérica, mostrou que o aumento da qualidade das manutenções preventiva é a melhor estratégia para reduzir o risco de falha do BOP anular. Verificou-se ainda que o aumento da vida útil de alguns componentes é tão relevante quanto o incremento de redundâncias para a confiabilidade do BOP anular. / After Macondo accident (USA, April 2010), with an estimated loss of US $ 42 billion, the failure risk analysis of the BOP (BlowOut Preventor) gained great importance in oil exploration and development projects. This work has deepened in BOP annular preventer (one of BOP functions) failure risk analysis, mapping sensitivity of maintenance of its components in the probability of equipment failure, according to the guidelines of RBI techniques (Risk Based Inspection) and RCM (Reliability Centered Maintenance). Therefore, the methodology used is proposed by API 580 (2009) and API 581 (2008) standards of risk based inspection applied on a reliability model of annular BOP. This reliability model, in turn, was based on fault tree analysis (FTA) and FMEA (Failure Mode and Effect Analysis) over the equipment hydraulic diagram. As hypothesis, it is assumed a constant failure rate of components over time, in order to calculate the mean time between failures. It was proved that reliability calculated by numerical model is coherent to field failures observations and literature data. The sensitivity analysis by numerical simulation shows that improving inspections and preventive maintenance quality in some of BOP components can markedly reduce the risk of failure of annular BOP.

<p>In today’s industrial world, there are high demands on the environmental aspects.</p><p>Siemens Industrial Turbomachinery AB (SIT AB) is a company that is keen about the environment, and therefore spends a lot of effort in developing combustion processes in order to reduce NOx (nitrogen oxides) emissions on their engine products. They are also researching in optional fuels, which are more environment-friendly.</p><p>In order to provide lower emissions the SIT designed a water rig to study the flow dynamics in a DLE (Dry Low Emission) burner.</p><p>An analyze program (GUI horizontal) was developed with new functions and the existing functions were improved. The program’s function was to evaluate different experimental tests of the flow dynamics in the 3rd generation DLE burners, of the SGT-800 gas turbine engine.</p><p>The aim was to ensure repeatability to enhance reliability, of the experimental test results for further comparison, for upcoming projects concerning future DLE burners.</p><p>When repeatability was achieved, implementations of different geometrical modifications were performed in the 3rd generation DLE burner.</p><p>The reason of the geometrical alterations was to look over if better fuel air mixture could be obtained and accordingly (thus) to reduce hotspots in the burner and in that case reduce NOx emissions.</p>

Aichi virus (AiV) is a small, nonenveloped RNA virus categorized to Picornaviridae. AiV infection causes mild gastroenteritis, but in neonates, AiV usually causes the risk of certain enterovirus-related clinical syndromes, such as fever, nausea, vomiting and diarrhea. The first case of AiV infection in Taiwan was diagnosed from a young patient with diarrhea in Kaohsiung Veterans General Hospital, and the AiV was successfully isolated. Antiviral innate immune system of our body plays the major role to defense virus invasion. Because AiV is an emerging picornavirus, the knowledge about its pathogenesis and the interaction with host innate immunity were totally absent. This study aims to investigate the mechanism of AiV regulating innate immune response. We first demonstrated that AiV is a type I IFN sensitive virus. IFN-£\2 treatment potently inhibited AiV replication. Real-time quantitative PCR data indicated that AiV induced only small amout of type I IFN gene expression, and the similar result was observed using IFN-£] luciferase reporter assay. In addition, the AiV triggered IFN-£] luciferase activity was progressively decreased in the late phase of infection. Immunoblotting assay showed that AiV evidently activated IRF-3 and IRF-7, the transcription factors of type I IFN induction. However, the retinoic acid inducible gene I (RIG-I) protein was cleavaged and its activity was downregulated by AiV. This data suggested that AiV triggered low level of type I IFN response may due to the negative feedback regulation of RIG-I activity. This immune evasion might be important for AiV replication in cells. Our study first reveals the status of innate immune response of AiV infection, and provides the basic virological theory for the development of anti-AiV drugs and vaccines in the future.

Bacterial and viral infections affect billions of people each year. While bacterial infections are treated by the use of antibiotics, viral infections are eradicated by the immune system. This thesis comprises two parts. Part I presents the reconstitution of enzymes required for the synthesis of the minimal pharmacophore of moenomycin A (MmA), a molecule with antibacterial activity. Part II details single-particle electron microscopy studies of MDA5 alone and in complex with double-stranded RNA (dsRNA). MmA is a natural product antibiotic from Streptomyces ghanaensis that possesses remarkable potency against clinically relevant Gram-positive bacteria. MmA exerts its antibacterial activity by binding directly to peptidoglycan glycosyltransferases, enzymes involved in the synthesis of the glycan strands of peptidoglycan. The genes responsible for MmA biosynthesis have been identified, and a complete biosynthetic pathway has been proposed. Part I of this thesis describes the reconstitution of enzymes required for the synthesis of two trisaccharide scaffolds of MmA that retain antibacterial activity. It also describes the synthesis of unnatural phosphoglycerate lipid acceptors and UDP-amino sugars that can be used to probe the substrate tolerances of key glycosyltransferases in MmA biosynthesis. This work lays the foundation for the synthesis of unnatural MmA analogs that may possess better pharmacokinetic properties than the parent molecule. MDA5 is a helicase that detects viral dsRNA in the cytoplasm of vertebrate cells. Upon dsRNA recognition, MDA5 initiates a series of signal transduction events that activate the innate immune response. Part II of this thesis presents the structures of apo MDA5 protein and the MDA5-dsRNA complex obtained by using single-particle electron microscopy. Two-dimensional averages of apo MDA5 revealed that the protein is very flexible and can adopt multiple conformations. This finding suggests that MDA5 does not adopt an autorepressed conformation in the absence of viral dsRNA. When MDA5 is incubated with dsRNA, the protein assembles onto the dsRNA to form a linear oligomer. Two-dimensional averages and a three-dimensional reconstruction reveal the complex to consist of seven to eight stacked discs per strand of 112 base pair dsRNA. This work lays the foundation for further structural studies aimed at elucidating the mechanism by which MDA5 is activated. / Chemistry and Chemical Biology