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1
Ericsson, Olle. « Biomolecular Analysis by Dual-Tag Microarrays and Single Molecule Amplification ». Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distributör], 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8475.
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Hundewale, Nisar. « CAD Tools for DNA Micro-Array Design, Manufacture and Application ». Digital Archive @ GSU, 2006. http://digitalarchive.gsu.edu/cs_diss/13.
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Motivation: As the human genome project progresses and some microbial and eukaryotic genomes are recognized, numerous biotechnological processes have attracted increasing number of biologists, bioengineers and computer scientists recently. Biotechnological processes profoundly involve production and analysis of highthroughput experimental data. Numerous sequence libraries of DNA and protein structures of a large number of micro-organisms and a variety of other databases related to biology and chemistry are available. For example, microarray technology, a novel biotechnology, promises to monitor the whole genome at once, so that researchers can study the whole genome on the global level and have a better picture of the expressions among millions of genes simultaneously. Today, it is widely used in many fields- disease diagnosis, gene classification, gene regulatory network, and drug discovery. For example, designing organism specific microarray and analysis of experimental data require combining heterogeneous computational tools that usually differ in the data format; such as, GeneMark for ORF extraction, Promide for DNA probe selection, Chip for probe placement on microarray chip, BLAST to compare sequences, MEGA for phylogenetic analysis, and ClustalX for multiple alignments. Solution: Surprisingly enough, despite huge research efforts invested in DNA array applications, very few works are devoted to computer-aided optimization of DNA array design and manufacturing. Current design practices are dominated by ad-hoc heuristics incorporated in proprietary tools with unknown suboptimality. This will soon become a bottleneck for the new generation of high-density arrays, such as the ones currently being designed at Perlegen [109]. The goal of the already accomplished research was to develop highly scalable tools, with predictable runtime and quality, for cost-effective, computer-aided design and manufacturing of DNA probe arrays. We illustrate the utility of our approach by taking a concrete example of combining the design tools of microarray technology for Harpes B virus DNA data.
3
李晉鏗 et Chun-hang Lee. « The prose of Tang Shunzhi (1507-1560) ». Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1992. http://hub.hku.hk/bib/B31210417.
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Lindberg, Max. « Fluorescent fusion proteins as probes to characterize tau fibril polymorphism ». Thesis, Linköpings universitet, Kemi, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-158263.
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Alzheimer's disease (AD) is a large and growing problem and while we today lack a full understanding of this disease, we know that the protein tau and the amyloid fibrils it forms play a central role in its development. We also know that these fibrils can have different morphologies in different diseases and that fibrils produced in vitro not necessarily adopt any of the morphologies found in patients. This means there is a need for more pathologically relevant fibrils in vitro to be able to understand this disease better. One approach to satisfy this need is to use fibrils found in patients as seeds and thus transfer their morphology to recombinantly purified protein. To facilitate this process this study has attempted to develop a way to differentiate between different fibril morphologies using a FRET based system. This involves fluorescent fusion proteins (tau-EXFPs) and fluorescent amyloid probes as well as seeding experiments with pseudo wild type tau (PWT) and tau with the P301L mutation. Greater differences in terms of fibrillation rates and ThT fluorescence between PWT and P301L was shown than previously reported between WT and P301L. They were also shown to differ in fibril morphology in TEM. The ThT fluorescence intensity was to a certain degree transferable from PWT to P301L by seeding. Furthermore, this study confirms that the tau-EXFP fusion protein can be incorporated into amyloid fibrils and strongly suggests that a FRET effect between EXFP and BTD14 (as well as X34 and ThT) can be achieved. It also demonstrates differences in FRET efficiency between PWT and P301L fibrils using FLIM. These results indicate that a FRET based approach could be a useful method to discern different fibril morphologies from each other, but further measurements and optimization are needed before this method could be reliably applied. The fusion proteins could also be used to investigate tau spreading in vivo, e.g. in D. melanogaster. To find suitable FRET partners to the fusion proteins, a ligand screen was conducted. This could be used as an alternative to the FRET method. With the right selection of fluorescent amyloid probes, a unique fingerprint for each fibril morphology could maybe be generated and fulfill the same intended function as the FRET method.
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Modise, Kagiso Eagile. « Media Coverage and the Cross Section of Stock Returns A Probe into the JSE ». Master's thesis, Faculty of Commerce, 2019. http://hdl.handle.net/11427/31081.
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Through reaching a wide-ranging population of investors, both institutional and individual, mass media coverage of stocks markets can alleviate financial information frictions and consequently affect the valuation of securities even when it does not present genuine news. The empirical objective of this research is to investigate this hypothesis by studying media reporting and changes in average stock returns. By constructing two portfolios of stocks divided into “stocks without media coverage” and “stocks with media coverage” an investigation can be carried to find out which portfolio outperforms the other and sometimes even after accounting for risk factors. Previous literature news media and the stock market has failed to address African financial markets including the Johannesburg stock exchange (JSE) market. The Johannesburg stock exchange is Africa’s oldest and largest stock market. An opportunity exists to replicate empirical work on news media reporting and changes in average returns in South Africa and Johannesburg stock exchange. The methodology employed in this study is adopted from the widespread research previously conducted in other more developed markets. Media coverage has been derived from the number of headline articles about a stock in a certain month in 23 influential South African print newspapers. Only headline articles are used to proxy for a stocks overall media attention. A systematic search of the LexisNexis database is carried out to find articles published in 23 major, influential newspapers in South Africa. The examination period is from 1 January 2013 to 31 December 2017 (a total of 7620 firm-month observations). The results indicate no statistically significant (at the 95% confidence level) outperformance of stocks without any news media reporting over stocks with news media reporting as found in more developed markets. Further analysis of data indicates that media reporting of the JSE stocks is surprisingly low and 99% of observations having only 6 headlines or less in the media. Therefore, about 1% of the observations are reported at least 7 times in the South African newspaper media.
6
Acton, Sydney. « Design, Synthesis, and Evaluation of Fluorogenic, BODIPY-based Probes for Specific Protein Labelling in Live Cells ». Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39033.
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Visualizing proteins in living cells without perturbing biological function remains a key challenge in chemical biology. A chemical approach to this problem is the synthesis of small molecule fluorophores that react specifically with a protein of interest (POI). We have developed a site-specific labelling method based on a Fluorogenic Addition Reaction (FlARe). The FlARe probe’s fluorescence is quenched until it undergoes thiol addition with a small, genetically encoded dicysteine peptide tag fused to the POI. Recent blue coumarin probes were shown to be highly selective for target proteins over other cellular thiols; however, fluorogens that can label in the red and green channels of the fluorescence microscope are more desirable for cellular imaging, as red light is lower in energy and therefore less photo-toxic. In the work presented herein, we use DFT calculations to guide the design of red-shifted, PeT-quenched BODIPY based dimaleimide fluorogens. Driven by the preliminary results of a FlARe probe (YC29) that emitted in the red channel, we attempted to prepare the hit compound through a new synthetic approach to further evaluate kinetics and in cellulo labelling. Given the time available, this compound was unable to be synthesized through an SNAr or Pd-catalyzed approach. Alternatives probes lacking the red-shifting substituent were synthesized and evaluated in vitro and in cellulo. The fluorescent enhancement and reaction kinetics of these probes were evaluated in detail, in order to determine the suitability of their application to cellular labelling. A green-BODIPY fluorogen was synthesized that exhibits suitable kinetics for labelling and a dramatic fluorescent enhancement of ~800-fold upon tagging. This probe was successfully applied to the specific, fluorescent labelling of a nuclear histone protein in cellulo.
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Lunven, Laurent. « Synthèse et évaluation d'aurones sur des modèles de fibres de tau ». Thesis, Université Grenoble Alpes (ComUE), 2016. http://www.theses.fr/2016GREAV007/document.
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La fibrillation anormale de la protéine tau est un phénomène présent dans de nombreuses maladies neurodégénératives, dont la maladie d’Alzheimer, et conduit à la formation de fibres amyloïdes appelées dégénérescences neurofibrillaires. L’utilisation de molécules organiques capables de marquer ces fibres ou d’inhiber leur formation revêt un intérêt à la fois diagnostic et thérapeutique dans la maladie d’Alzheimer. Une série d’aurones a été synthétisée et leur capacité à interagir et interférer avec le processus de fibrillation a été évaluée in vitro sur des modèles de fibres de tau développés ou optimisés lors de ce projet. Ce travail a permis de montrer que des aurones polyhydroxylées sont capables d’agir comme sonde et/ou comme inhibiteur du processus de fibrillation. Afin d’élargir les applications possibles des aurones, la ligation d’aurones avec des biomolécules ou encore leur radiomarquage a également été évaluéeThe fibrillation of the tau protein occurs in many neurodegenerative diseases, including Alzheimer’s disease, and leads to the formation of amyloid fibres called neurofibrillary tangles. Using organic molecules able to mark these fibres or inhibit their formation provides an interest both in diagnosis and therapy in Alzheimer’s disease. A series of aurones was synthesized and their ability to interfere with the fibrillation process was evaluated in vitro on models of tau fibres developed in this project. This work shows that polyhydroxylated aurones are able to act both as probes and as inhibitors of the fibrillation process. The ligation of these aurones with biomolecules or their radiolabelling has also been investigated
8
陸秀娟 et Sau-kuen Luk. « Li Hua ( ? - c. 766) and his prose ». Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1993. http://hub.hku.hk/bib/B31211720.
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Riley, John D. « Evaluation of Travel Time Estimates Derived From Automatic Vehicle Identification Tags in San Antonio, TX ». Thesis, Virginia Tech, 1999. http://hdl.handle.net/10919/33746.
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The purpose of this research is to examine several aspects of the San Antonio automated vehicle identification (AVI) system, including the reliability and accuracy of the AVI system, travel tag level of market penetration (LMP) trends, and a comparison of aggregated travel time values with probe vehicle travel time values. This thesis serves as a first step toward the modeling of AVI systems in which the effects of travel tag LMP, AVI reader density and AVI reader location are analyzed.
GPS units were first tested as a suitable benchmark for validating AVI reliability and accuracy. A two-part system reliability study was then performed, consisting of overall system reliability and a controlled evaluation of selected AVI reader sites. The accuracy of AVI travel times was also assessed. A LMP analysis was then performed to serve as a reference parameter for the aggregate travel time study. Lastly, the level of aggregation analysis attempted to quantify differences between the individual test vehicle travel times and aggregated travel times of all observed, tag-equipped vehicles.
Overall system reliability was found to be greater than 90%. The controlled reliability study showed that freeway AVI readers slightly outperformed arterial readers for correct tag capture, while total tag capture exceeded the system design parameter of 80%. Tag capture rates were found to be independent of test vehicle speed. The LMP of travel tags at a selected reader site was found to be approximately 0.5% from the morning through the evening peak. Lastly, 5-minute travel time aggregations provided a better estimate of individual test vehicle travel times than 2-minute or 15-minute aggregations.
Master of Science
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Marcoux-Chabot, Gabriel. « Tas-d’roches suivi de Tas-d’roches : entre prose et poésie : la théorie barfieldienne des modes de conscience appliquée à l’analyse d’un roman complexe ». Thesis, Université Laval, 2014. http://www.theses.ulaval.ca/2014/30491/30491.pdf.
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Par le biais de trois voix narratives distinctes, le roman raconte l’histoire de Tas-d’roches, l’homme fort du village de Saint-Nérée. Entre le pastiche rabelaisien, la poésie innue et le roman de chevalerie, multipliant les jeux de mise en page et de typographie, le récit traite des relations du héros avec sa famille, ses amis, le lieu qu’il habite et la femme de sa vie. Partant du constat que Tas-d’roches est un roman difficile à décrire, l’essai qui l’accompagne montre qu’une théorie méconnue et datée - mais étayée par des publications récentes dans les domaines de la neurologie (McGilclirist, 2009), de la phénoménologie (Bortoft, 2012) et de la linguistique (Guillaume, 2007) - selon laquelle tout texte exprime un degré de tension entre modes poétique et prosaïque de conscience (Barfield, 1928) permet de mettre en relation les aspects diégétiques et formels de Tas-d’roches et d’en offrir une description globalement satisfaisante.
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Morrone, Castrese. « Biochemical and structural characterization of proteins involved in prokaryotic DNA metabolism as source of novel drug targets and biotechnological solutions ». Doctoral thesis, Università del Piemonte Orientale, 2021. http://hdl.handle.net/11579/127798.
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The aim of the presented PhD project was the biochemical and structural characterization of proteins and enzymes involved in prokaryotic DNA metabolism as potential target for antitubercular drug discovery as well as for the design of biotechnological tools for application in molecular biology. A variant of the 06-DNA-alkyl-guanine-DNA-alkyl-transferases (OGT) from Saccharolobus solfataricus (HS) has been proposed as a thermostable self-labelling protein tag in order to expand this technology to harsh experimental conditions and extreme environments. We present here the crystal structure of H5 protein in complex with the fluorescent probe functional to a structure based protein engineering aiming at further evolution of the proposed technology based on a chemo-enzymatic approach for the labelling reaction. The second part of the presented thesis focused on the characterization of protein complexes from Mycobacterium tuberculosis (MTB)
involved in the Nucleotide Excision Repair (NER) pathway and the initiation phase of DNA replication. To this aim we obtained a low-resolution model of the UvrA-UvrB complex acting in the damage recognition step along NER pathway. The structural model allow us to map three residues involved in protein-protein interaction whose key role was further confirmed by site directed mutagenesis experiments. On the other hands, we obtained preliminary results on the biochemical characterization of a novel helicase loader acting during DNA replication in MTB, namely DciA. In order to shed light on mechanistic aspects of DciA-dependent helicase in MTB, we investigated the secondary structure element of DciA domains protein using limited proteolysis coupled with native mass spectrometry. Moreover, we determined the kinetic constants of the interaction between DciA loader and the target helicase by means of Surface Plasmon Resonance.
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Nguyen, Tan Hung [Verfasser], Hans Joachim [Akademischer Betreuer] Linke, Ngoc Hieu [Akademischer Betreuer] Nguyen, Boris [Akademischer Betreuer] Lehmann, Carl-Alexander [Akademischer Betreuer] Graubner et Martin [Akademischer Betreuer] Wagner. « Spatial Planning in Flood-prone Areas / Tan Hung Nguyen ; Hans Joachim Linke, Ngoc Hieu Nguyen, Boris Lehmann, Carl-Alexander Graubner, Martin Wagner ». Darmstadt : Universitäts- und Landesbibliothek Darmstadt, 2019. http://d-nb.info/120108654X/34.
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Nguyen, Tan Hung [Verfasser], Hans-Joachim [Akademischer Betreuer] Linke, Ngoc Hieu Akademischer Betreuer] Nguyen, Boris [Akademischer Betreuer] [Lehmann, Carl-Alexander [Akademischer Betreuer] Graubner et Martin [Akademischer Betreuer] Wagner. « Spatial Planning in Flood-prone Areas / Tan Hung Nguyen ; Hans Joachim Linke, Ngoc Hieu Nguyen, Boris Lehmann, Carl-Alexander Graubner, Martin Wagner ». Darmstadt : Universitäts- und Landesbibliothek Darmstadt, 2019. http://nbn-resolving.de/urn:nbn:de:tuda-tuprints-92661.
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Nguyen, Tan Hung [Verfasser], Hans Joachim Akademischer Betreuer] Linke, Ngoc Hieu [Akademischer Betreuer] Nguyen, Boris [Akademischer Betreuer] Lehmann, Carl-Alexander [Akademischer Betreuer] Graubner et Martin [Akademischer Betreuer] [Wagner. « Spatial Planning in Flood-prone Areas / Tan Hung Nguyen ; Hans Joachim Linke, Ngoc Hieu Nguyen, Boris Lehmann, Carl-Alexander Graubner, Martin Wagner ». Darmstadt : Universitäts- und Landesbibliothek Darmstadt, 2019. http://d-nb.info/120108654X/34.
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Sartori, Paola Mondardo. « A extrafiscalidade aplicada ?s energias renov?veis : uma an?lise sob a ?tica da gest?o e prote??o ambiental ». Pontif?cia Universidade Cat?lica do Rio Grande do Sul, 2018. http://tede2.pucrs.br/tede2/handle/tede/8133.
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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES
The environment and its constant degradation are not recent concerns. The possible forms of environmental preservation are subject of constant debate in media, especially because some of the environmental consequences of our inconsequential actions are already palpable. It is undeniable that this debate also orbits the energy sector, given that the sources currently chosen by the Brazilian government for electric power generation aggressively affect the environment. Brazil, despite having hydroelectric plants as its main source of energy, a renewable source although not completely clean, does not have a significant generation through other clean sources, despite having an exorbitant potential, especially with regard to wind and solar energy. Due to the high potential of this type of electric power generation, which is acquired through an inexhaustible and non-polluting source, and the accelerated population growth, which has a significant increase in energy demand, it is undeniable that this type of power generation should be encouraged by the government. Considering this scenario, this research intended, through a dialectical method, to make a legal-dogmatic parallel between the right to an ecologically balanced environment and the current legislation, in order to verify the existing national programs and incentives, as well as to make a juxtaposition between the national and international scenarios, and, finally, discuss the different possibilities of application of the institute of extrafiscality to renewable sources, with the objective of stimulating more effectively this type of power generation in the national territory. In the course of the research, we verified that the range of available alternatives is vast and the application of the extrafiscality to the energy sector has shown to be an excellent tool in the pursuit of constitutional purposes such as the environmental protection. It remains evident, finally, that in order for us to do justice to the constitutional provision that guarantees to everyone the right to an ecologically balanced environment and grant the State duties of environmental protection and promotion, it is necessary that the government actually assume its role of a Socio-environmental State and, urgently, create new and efficient policies, stimulating the national growth of the renewable energy sector, so that we can reach our true potential and guarantee a decent and possible future for the next generations.
N?o ? de hoje a preocupa??o com o meio ambiente e sua constante degrada??o. As poss?veis formas de preserva??o ambiental s?o tema de debate constante nos diversos meios midi?ticos, muito em raz?o de j? serem palp?veis algumas das consequ?ncias ambientais acarretadas pelo nosso agir inconsequente. ? ineg?vel que referido debate tamb?m orbita o setor energ?tico, dado que os meios escolhidos atualmente pelo pa?s para a gera??o de energia afetam agressivamente o ambiente. O Brasil, no que pese ter como principal fonte de gera??o de energia as hidroel?tricas, fonte de energia renov?vel, ainda que n?o completamente limpa, n?o possui gera??o significativa no que se refere as demais energias n?o poluentes, apesar de dispor de um potencial exorbitante, principalmente no que tange ?s fontes e?lica e solar. Em raz?o do alto potencial deste tipo de gera??o de energia, que ? adquirido atrav?s de uma fonte inesgot?vel e n?o poluente, somando-se ainda ao acelerado crescimento da popula??o, que possui como consequ?ncia um aumento significativo da demanda energ?tica, ? incontest?vel que este tipo de gera??o deve ser incentivado. Tendo em vista o mencionado cen?rio, o presente trabalho se prop?s, mediante um m?todo dial?tico, a realizar um cotejo jur?dico-dogm?tico entre o direito fundamental ao meio ambiente ecologicamente equilibrado e a legisla??o vigente, com intuito de verificar quais os incentivos existentes no pa?s at? ent?o, assim como realizar uma compara??o entre o cen?rio nacional e internacional, para, por fim, discorrer sobre as distintas possibilidades de aplica??o, pelo Poder P?blico, do instituto da extrafiscalidade ?s energias renov?veis, para que o mesmo consiga incentivar de modo mais eficaz este tipo de gera??o no territ?rio nacional. Verificamos, no decorrer da pesquisa, que ? imensa a gama de alternativas que est?o dispon?veis ao Estado brasileiro e que a aplica??o da extrafiscalidade ambiental ao setor energ?tico se demonstrou, no direito comparado, um excelente instrumento na persecu??o de fins constitucionais como a prote??o ambiental. Restou evidente, enfim, que, para que possamos fazer jus ? previs?o constitucional que garante a todos o direito a um meio ambiente ecologicamente equilibrado e outorga ao Estado deveres de prote??o e promo??o ambiental, ? preciso que o mesmo vista de fato a roupagem de Estado Socioambiental e, impreterivelmente, crie novas e eficientes pol?ticas p?blicas, induzindo o crescimento real do setor das energias renov?veis de maneira que enfim o nosso verdadeiro potencial seja alcan?ado e, assim, um futuro digno e poss?vel seja garantido ?s pr?ximas gera??es.
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Laos, Roberto. « Protein directed evolution ». Revista de Química, 2012. http://repositorio.pucp.edu.pe/index/handle/123456789/99875.
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Evolución dirigida de proteínas: La evolución dirigida es una técnica que nos permite explorar funciones enzimáticas que no son requeridas en el ambiente natural. Esta técnica, simula procesos genéticos naturales y de selección. Esta estrategia se utiliza cuando un diseño racional es muy complicado. Consiste en una repetición de ciclos de diversificación y selección que llevan a la acumulación de mutaciones benéficas. Aquí se presenta dos ejemplos de evolución dirigida con los cuales se ha trabajado directamente: la ADN polimerasa del organismo Thermus aquaticus usada comúnmente en PCR, y la proteína LacI que regula la expresión de genes usados para el metabolismo de lactosa en E. Coli.Directed evolution allows us to explore protein functionalities not required in the natural environment. It mimics natural genetic processes and selective pressures. This approach is used when the molecular basis is not completely understood and rational design is a difficult task. This approach consists of serial cycles of consecutive diversification and selection which eventually lead to the accumulation of beneficial mutations. Here are presented two cases where directed evolution is used to modify two different proteins: Taq polymerase, enzyme used for DNA extension in PCR, and the LacI repressor protein which regulates gene expression on E.coli.
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Rong, Hengying. « Étude sur l'écriture comme acte politique chez Ouyang Xiu (1007-1072) : pouvoir lettré sous les Song du Nord (960-1127) ». Thesis, Paris, EHESS, 2020. http://www.theses.fr/2020EHES0062.
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Ce travail apporte un éclairage ciblé sur le rapport entre l’action et les écrits de Ouyang Xiu (1007-1072), lettré majeur des Song du Nord (960-1127) et fonctionnaire de première importance par le rôle qu’il occupa dans la consolidation des institutions de cette dynastie. Figure incontournable de l’histoire intellectuelle chinoise, Ouyang Xiu est abordé ici à travers une étude cas de la session 1057 du concours de « doctorat », dont il fut superviseur en chef au niveau métropolitain. À travers la contextualisation minutieuse de cette session, et plus largement la mise en perspective de ce que signifiait, pour les hommes des Song, la sélection des fonctionnaires impériaux par le système des examens, il s’agit d’exposer sous tous ses aspects l’implication réciproque des pratiques lettrées et des enjeux de pouvoir dans le XIe siècle chinois. En démythifiant la légende de 1057 et en redonnant à cette session sa part d’imprévisibilité, l’enquête permet d’en dégager la signification profonde : celle d’un événement tissé d’une pluralité de facteurs, mais dont la dimension exemplaire et la densité d’enjeux ont pu justifier a posteriori une narration rétrospective tendancieuse, indexant la pertinence des choix de Ouyang Xiu sur les accomplissements ultérieurs de ses lauréats. On prend ici le parti d’inverser cette perspective traditionnelle. Plutôt que de partir des conséquences de cet examen singulier, on en revient au moment de ses préparatifs et de son déroulement, afin de comprendre les raisons pour lesquels de tels choix se sont imposés à l’un de ses acteurs-clésThis work sheds light on the relation between action and the writings of Ouyang Xiu (1007-1072), one of the major literati of the Northern Song dynasty (960-1127), and a civil servant of great importance for the role he occupied in the consolidation of institutions of the dynasty. As a key figure in Chinese intellectual history, Ouyang Xiu is approached in the present study through a case study of jinshi examination supervised at the department level by Ouyang Xiu in 1057. Through the detailed contextualization of this session, and more broadly putting into perspective what the selection of officials by the system of examinations meant for the Song literati, we try to expose in all its aspects the reciprocal implication of literate practices and power in the XIth century in China.By demystifying the legend of 1057 and giving back to this session its share of unpredictability, this investigation allows us to identify its deep significance. The event was composed of a plurality of factors, but whose exemplary dimension and density of issues are usually considered to justify a posteriori a tendentious retrospective narrative, indexing the pertinence of Ouyang Xiu's choices on the subsequent achievements of the laureates. However, here we reverse this traditional perspective. Rather than starting from the consequences of this singular examination, we return to the time of its preparations and its development, in order to understand the reasons why such choices were made by one of the key decision makers, Ouyang Xiu
歐陽修作為北宋的重要文人和政治人物，對北宋的一系列以科舉為代表的政治制度的鞏固起到了不可低估的作用。本文試圖針對歐陽修的“從政”與“寫作”之間的關係進行再研究，從嘉佑二年進士科歐陽修任省試知貢舉出發，展開個案研究。通過對歷史背景的深入細緻的考證，尤其是對宋人如何理解科舉取士意義的梳理，來呈現中國十一世紀士人寫作與權力之間的互動。本文旨在整理嘉祐二年貢舉中一系列歷史的不可預見性，去除該事件的神話光環，從而重新解讀其深層的意義。以往對該事件的研究往往從該屆進士的成就出發，解讀嘉祐二年歐陽修選擇的正確性，本文旨在回到該事件的準備階段以及整個進行過程，從而理解作為重要的決策者之一歐陽修,他所作出的重大選擇背後的一系列原因。
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Fung, Cheryl. « The fiscal impacts of use-value taxation in Prince William County, Virginia ». Thesis, Virginia Tech, 1995. http://hdl.handle.net/10919/42232.
Texte intégralRésumé :
Concern that high property taxation of agricultural land encourages its conversion to nonagricultural uses has led to the adoption of use-value taxation practices. Use-value taxation has had mixed results as a deterrent to the conversion of agricultural and open space land. It has been argued that use-value taxation does not succeed in retaining open space along the rura1-urban fringe (Stocker 1975; Ferguson), and further that such programs may actually lower the community's property tax base significantly (Tiebout; Anderson 1993). Additionally, when land is taxed by its usevalue rather than market-value, the local tax base declines curtailing local public services and consequently reducing the attractiveness of the community for residential, commercial and industrial land uses (Abeyratne and Johnson, Bickerdike, Netzer, Oates).
This study seeks to determine the fiscal impacts of use-value taxation and incurred and immediate revenues generated by a particular land use project. By comparing the net impact on the property tax rate of different land uses, the effectiveness of land use taxation policies for communities can be determined. The fiscal impact of alternative land uses are measured using The Virginia Impact Projection (VIP) model. The empirical models employed are based on a static cross-sectional econometric analysis of Virginia counties initially developed by Johnson and Keeling and updated for the current analysis using more recent data. The empirical equations are used to construct a fiscal impact assessment (simulation) model. The simulation model allows the comparison of impact and baseline scenarios developed using alternative land uses.
It was found that the impact offarmland enrollment in use-value assessment programs is not as large when net impacts are considered rather than sole consideration of the direct property tax revenue changes.
Master of Science
19
PANTINI, SARA. « Analysis and modelling of leachate and gas generation at landfill sites focused on mechanically-biologically treated waste ». Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2013. http://hdl.handle.net/2108/203393.
Texte intégralRésumé :
Despite significant efforts have been directed toward reducing waste generation and encouraging alternative waste management strategies, landfills still remain the main option for Municipal Solid Waste (MSW) disposal in many countries. Hence, landfills and related impacts on the surroundings are still current issues throughout the world. Actually, the major concerns are related to the potential emissions of leachate and landfill gas into the environment, that pose a threat to public health, surface and groundwater pollution, soil contamination and global warming effects. To ensure environmental protection and enhance landfill sustainability, modern sanitary landfills are equipped with several engineered systems with different functions. For instance, the installation of containment systems, such as bottom liner and multi-layers capping systems, is aimed at reducing leachate seepage and water infiltration into the landfill body as well as gas migration, while eventually mitigating methane emissions through the placement of active oxidation layers (biocovers). Leachate collection and removal systems are designed to minimize water head forming on the bottom section of the landfill and consequent seepages through the liner system. Finally, gas extraction and utilization systems, allow to recover energy from landfill gas while reducing explosion and fire risks associated with methane accumulation, even though much depends on gas collection efficiency achieved in the field (range: 60-90% Spokas et al., 2006; Huitric and Kong, 2006). Hence, impacts on the surrounding environment caused by the polluting substances released from the deposited waste through liquid and gas emissions can be potentially mitigated by a proper design of technical barriers and collection/extraction systems at the landfill site. Nevertheless, the long-term performance of containment systems to limit the landfill emissions is highly uncertain and is strongly dependent on site-specific conditions such as climate, vegetative covers, containment systems, leachate quality and applied stress. Furthermore, the design and operation of leachate collection and treatment systems, of landfill gas extraction and utilization projects, as well as the assessment of appropriate methane reduction
strategies (biocovers), require reliable emission forecasts for the assessment of system feasibility and to ensure environmental compliance. To this end, landfill simulation models can represent an useful supporting tool for a better design of leachate/gas collection and treatment systems and can provide valuable information for the evaluation of best options for containment systems depending on their performances under the site-specific conditions. The capability in predicting future emissions levels at a landfill site can also be improved by combining simulation models with field observations at full-scale landfills and/or with experimental studies resembling landfill conditions. Indeed, this kind of data may allow to identify the main parameters and processes governing leachate and gas generation and can provide useful information for model refinement.
In view of such need, the present research study was initially addressed to develop a new landfill screening model that, based on simplified mathematical and empirical equations, provides quantitative estimation of leachate and gas production over time, taking into account for site-specific conditions, waste properties and main landfill characteristics and processes. In order to evaluate the applicability of the developed model and the accuracy of emissions forecast, several simulations on four full-scale landfills, currently in operative management stage, were carried out. The results of these case studies showed a good correspondence of leachate estimations with monthly trend observed in the field and revealed that the reliability of model predictions is strongly influenced by the quality of input data. In particular, the initial waste moisture content and the waste compression index, which are usually data not available from a standard characterisation, were identified as the key unknown parameters affecting leachate production. Furthermore, the applicability of the model to closed landfills was evaluated by simulating different alternative capping systems and by comparing the results with those returned by the Hydrological Evaluation of Landfill Performance (HELP), which is the most worldwide used model for comparative analysis of composite liner systems. Despite the simplified approach of the developed model, simulated values of infiltration and leakage rates through the analysed cover systems were in line with those of HELP.
However, it should be highlighted that the developed model provides an assessment of leachate and biogas production only from a quantitative point of view. The leachate and biogas composition was indeed not included in the forecast model, as strongly linked to the type of waste that makes the prediction in a screening phase poorly representative of what could be expected in the field. Hence, for a qualitative analysis of leachate and gas emissions over time, a laboratory methodology including different type of lab-scale tests was applied to a particular waste material. Specifically, the research was focused on mechanically biologically treated (MBT) wastes which, after the introduction of the European Landfill Directive 1999/31/EC (European Commission, 1999) that imposes member states to dispose of in landfills only wastes that have been preliminary subjected to treatment, are becoming the main flow waste landfilled in new Italian facilities. However, due to the relatively recent introduction of the MBT plants within the waste management system, very few data on leachate and gas emissions from MBT waste in landfills are available and, hence, the current knowledge mainly results from laboratory studies. Nevertheless, the assessment of the leaching characteristics of MBT materials and the evaluation of how the environmental conditions may affect the heavy metals mobility are still poorly investigated in literature. To gain deeper insight on the fundamental mechanisms governing the constituents release from MBT wastes, several leaching experiments were performed on MBT samples collected from an Italian MBT plant and the experimental results were modelled to obtain information on the long-term leachate emissions. Namely, a combination of experimental leaching tests were performed on fully-characterized MBT waste samples and the effect of different parameters, mainly pH and liquid to solid ratio (L/S,) on the compounds release was investigated by combining pH static-batch test, pH dependent tests and dynamic up-flow column percolation experiments. The obtained results showed that, even though MBT wastes were characterized by relatively high heavy metals content, only a limited amount was actually soluble and thus bioavailable. Furthermore, the information provided by the different tests highlighted the existence of a strong linear correlation between the release pattern of dissolved organic carbon (DOC) and several metals (Co, Cr, Cu, Ni, V, Zn), suggesting that complexation to DOC is the leaching controlling mechanism of these elements. Thus, combining the results of batch and up-flow column percolation tests, partition coefficients between DOC and metals concentration were derived. These data, coupled with a simplified screening model for DOC release, allowed to get a very good prediction of metal release during the experiments and may
provide useful indications for the evaluation of long-term emissions from this type of waste in a landfill disposal scenario.
In order to complete the study on the MBT waste environmental behaviour, gas emissions from MBT waste were examined by performing different anaerobic tests. The main purpose of this study was to evaluate the potential gas generation capacity of wastes and to assess possible implications on gas generation resulting from the different environmental conditions expected in the field. To this end, anaerobic batch tests were performed at a wide range of water contents (26-43 %w/w up to 75 %w/w on wet weight) and temperatures (from 20-25 °C up to 55 °C) in order to simulate different landfill management options (dry tomb or bioreactor landfills). In nearly all test conditions, a quite long lag-phase was observed (several months) due to the inhibition effects resulting from high concentrations of volatile fatty acids (VFAs) and ammonia that highlighted a poor stability degree of the analysed material. Furthermore, experimental results showed that the initial waste water content is the key factor limiting the anaerobic biological process. Indeed, when the waste moisture was lower than 32 %w/w the methanogenic microbial activity was completely inhibited. Overall, the obtained results indicated that the operative conditions drastically affect the gas generation from MBT waste, in terms of both gas yield and generation rate. This suggests that particular caution should be paid when using the results of lab-scale tests for the evaluation of long-term behaviour expected in the field, where the boundary conditions change continuously and vary significantly depending on the climate, the landfill operative management strategies in place (e.g. leachate recirculation, waste disposal methods), the hydraulic characteristics of buried waste, the presence and type of temporary and final cover systems.
20
Chiu, Troy-Chi, et 邱垂錡. « Design of Vertical Probe for RFID Tag Antenna & ; IC Probe Card ». Thesis, 2006. http://ndltd.ncl.edu.tw/handle/87077555445372614413.
Texte intégralRésumé :
碩士中華大學
機械與航太工程研究所
94
Design of Vertical Probe for RFID Tag Antenna & IC Probe Card ABSTRACT Key Words: Radio Frequency Identification （RFID） Tag, Antenna Dynamic Impedance Measurement, Burn-in Test, Vertical Probe The purpose of this research is for the integrated design and fabrication of the transponder antenna module of RFID on the plastic material, as well as the vertical type probe card for testing. This kind of research was not proposed before, and is very important for practical consideration. The operation range and the output impedance of the RFID tag and antenna are 1 meter, and 50 ohms, respectively. In this research we used both Intellisuite MEMS as well as Ansoft HFSS software packages for the vertical probe card design on either the silicon wafer, or the traditional printed circuit board as the substrate. The probe was a vertical through-hole type, which is linked to the other side with a nickel-plated bump in order to connect to the instrument such as vector network analyzer for impedance measurement. The pitch of the proto-type probe was 50 μm, and the bandwidth was 2 GHz. The conductor of antenna is evaporated by the E-gun in our lab, which has 4 crucibles so that one can evaporate metal such as copper, or other metal such as Cr and Ti to form buried alloy to avoid the moisture corrosion effect. In addition, we have contact-type as well as non-contract laser Doppler interferometer to measure the thickness of the thin film deposited by the E-Gun. The latter will not destroy the surface of the thin film. Both accuracies are about 1 nm. The Intellisuite , Ansoft HFSS , IE3D , MEMS software package can be used for the antenna module design to find the inductance, capacitance as well as resistor to meet the specification of operation range, and output impedance. The Vector Network Analyzer is applied for the output impedance testing. Note also that the wire pull tester in our lab can be used to obtain the wire bond strength of IC pads between the terminals of antenna module. Therefore to sum up the aforementioned analysis, the feasibility of this research is quite well.
21
FROSALI, SIMONE. « Measurement of sigma(Z/gamma* + >= n jets) in the electronic channel with the CMS detector ». Doctoral thesis, 2012. http://hdl.handle.net/2158/630095.
Texte intégral
22
Arnaez, Olivier. « Préparation de la mesure de la section efficace inclusive du Z->e+e- dans ATLAS. Etude des premières données avec le calorimètre électromagnétique ». Phd thesis, 2010. http://tel.archives-ouvertes.fr/tel-00525847.
Texte intégralRésumé :
Les collisions proton-proton à une énergie dans le centre de masse de 7 TeV au LHC produiront des bosons Z en grande quantité dont la mesure de la section efficace de production constitue un test du Modèle Standard. Après avoir décrit le contexte théorique et procédé à une description du détecteur ATLAS et des outils de simulation et d'analyse, une partie de cette thèse est consacrée à l'exploitation des événements cosmiques enregistrés dans la phase de préparation aux collisions afin de valider la simulation des formes de gerbes électromagnétiques dans un contexte proche de celui des collisions. Une deuxième partie de la thèse s'attache à la description des éléments clefs intervenant dans la mesure de la section efficace. Des critères de sélection utilisant des variables d'isolation, alternatifs à la sélection standard, ont été étudiés et ont mené à l'observation des premiers candidats Z->e+e- dans ATLAS. Enfin, les principales sources d'erreurs systématiques intervenant dans la mesure de section efficace ont été étudiées. Il s'agit des incertitudes liées d'une part à l'efficacité de sélection des électrons et d'autre part à l'efficacité géométrique du détecteur.
23
GONZI, SANDRO. « Study of gamma + jets data with the CMS detector in pp collisions at sqrt(s) = 8 TeV ». Doctoral thesis, 2014. http://hdl.handle.net/2158/839694.
Texte intégralRésumé :
The characterization of the production process of prompt photons γ in association with hadronic jets (γ + jets process) at the Large Hadron Collider (LHC), is one of the goals in physics analyses at the Compact Muon Solenoid (CMS) experiment.
This channel provides a way of directly probing the hard scattering process and it is directly sensitive to the gluonic content of the proton. This process is also the starting point for the study of many Standard Model signatures, and constitutes a background for important searches, such as H->γγ, or SUSY and extra-dimensions with energetic photons in the final state.
This thesis presents the measurement of the γ + jets differential cross section production as a function of the photon transverse momentum pT in the high pT kinematic region, in proton-proton collisions at a center-of-mass energy of sqrt(s) = 8 TeV collected by the CMS detector with an integrated luminosity equal to L = (19.7 ± 0.5) fb(-1).
A series of selection cuts have been applied in order to identify the γ + jets process with respect to the background.
The selection efficiencies have been extracted with a data-driven method (Tag & Probe) and have been used to correct the efficiencies extracted from the Monte Carlo simulations.
To perform a precise set of measurements in the γ + jets final state, a background extraction with a data-driven approach has been applied by using a data-driven modeling for signal and background.
The calculation of the differential cross section has then been performed after a bin-by-bin correction of the detector effects on the selected photons.
A detailed treatment of the systematic uncertainties affecting the process has also been performed.
24
Chuang, Pei Hsuan, et 莊沛軒. « Development of Benzylguanine Chemical Probes for SNAP-tag Protein Binding ». Thesis, 2016. http://ndltd.ncl.edu.tw/handle/txx4w9.
Texte intégralRésumé :
碩士國立清華大學
化學系
105
Protein functions and dynamics in living cells can provide important information in biological research and medical diagnosis. The uses of fluorescent probes to study proteins are advantageous, as they allow for fast, high sensitivity, high specificity and ease for observation. In this thesis, I have designed two novel fluorescent probes for SNAP-tag which is an important protein in chemical biology for specific and covalent reaction with probes consisting O6-benzylguanine moiety. The first probe is based on the caged-benzylguanine concept by chemical modification of benzylguanine N-9 position to prevent the reaction of benzylguanine and SNAP-tag. Upon uncaging with the target molecule, the fluorescent dye can then be transferred to the SNAP-tag or MGMT (benzylguanine wild type protein) to emit stronger fluorescence. In the second fluorescent probe, we have successfully developed two near-infrared fluorescent probes based on Nile red and Nile blue, respectively, for fluorescent turn-on labeling of SNAP-tag protein. We have successfully applied these two probes for no-was and highly organelle specific labeling of SNAP-tag proteins in living cells.
25
(8726223), Matthew T. Lerdahl. « Engineering Reporter Tags in Flaviviruses to Probe Viral Structure and Morphogenesis ». Thesis, 2020.
Trouver le texte intégralRésumé :
The family Flaviviridae includes important genera such as flavivirus and hepacivirus which comprise significant human pathogens that affect hundreds of millions annually. The understanding of these viruses, the viral life cycle, and pathogenicity is vital when it comes to developing therapeutics. Flavivirus virions undergo major conformational rearrangements during the life cycle, including the assembly and maturation steps. In order to create a reagent to investigate these processes, luminescent reporter viruses have been constructed. Luminescent reporter tags have yet to be incorporated into the structural proteins of dengue virus (DENV) without significantly affecting replication or infectivity and successful tagging would allow for targeted studies examining access to specific structural epitopes. Engineering tags in DENV structural proteins is particularly difficult because most reporter tags involve large insertions which may create steric hindrance and inhibit proper protein folding. However, the reporter system described here, developed by Promega, is much smaller than a full-size luciferase protein. It involves an eleven amino acid subunit (HiBiT) tagged to a viral protein that creates measurable luminescence when incubated with the larger subunit (LgBiT). Using the structure of the virion as a guide, the HiBiT reporter tag was incorporated into the structural region of the DENV genome including sites in capsid (C) as well as the glycoproteins membrane (M) and envelope (E). Resulting recombinant viruses were characterized and tag sites within the C protein membrane anchor as well as the transmembrane domain of M protein were found to tolerate HiBiT insertion and produce infectious particles. The recombinant virus possessing HiBiT in C protein was found to be stable over three rounds of serial passaging while virus containing the M protein tag site was found to be unstable. HiBiT activity of the capsid tagged virus was also found to directly correlate with purified infectious particles, suggesting the capsid membrane anchor may remain associated with the virus even after polyprotein processing. Additionally, insert composition was found to be a key determinant for the production of infectious virus. The lessons learned from engineering HiBiT in the DENV system were then applied to hepatitis C virus (HCV).
The highly lipophilic and pleiomorphic nature of HCV has made structural studies particularly difficult. However, by constructing multi-tagged reporter viruses containing both HiBiT and various purification tags, researchers will save time and resources in preparation for structural studies which are vital for vaccine development. In this study, HiBiT was incorporated into sites within HCV previously shown to tolerate tags of various sizes. Different insert compositions were engineered within the genome and the construct containing both FLAG and HiBiT tags within the N-terminus of E2 yielded highly infectious and quantifiable, luminescent virus. The recombinant HCV containing FLAG and HiBiT displayed similar peak titer as compared to WT while also demonstrating HiBiT activity. Furthermore, the FLAG peptide was found to be partially surface exposed and capable of being used for virus purification purposes. The multi-tagged reporter virus characterized in this study provides a robust platform for quantification and purification of HCV, two facets of research that are critical for the determination of viral structure via cryo-EM and other imaging techniques. The findings from both the DENV and HCV studies provide a robust foundation for future tagging of viruses within the family Flaviviridae and offer insight on the structural proteins that compose the virion.
26
Kao, Ruo Ching, et 高若晴. « Nonspecific Signal Suppression and Fluorescence Amplification of Fluorescence Probes by SNAP-tag and Avidin ». Thesis, 2016. http://ndltd.ncl.edu.tw/handle/86577102954692305131.
Texte intégralRésumé :
碩士國立清華大學
化學系
104
Despite the promising improvements made recently in fluorescence probes for enzymes and reactive small molecules detection, two fundamental problems remain: weaker fluorescence of many dyes in aqueous buffers and strong nonspecific signals in samples containing high protein levels. In this paper, we introduce a novel fluorescent probe encapsulated in protein cavity (FPEPC) concept as demonstrated by SNAP-tag and avidin protein. The probes were constructed by following the current probe design for enzymes and reactive small molecules, but with an additional benzylguanine moiety for selective SNAP-tag conjugation or a short biotin linker for the conjugation to avidin protein. The SNAP-tag conjugated probes or avidin conjugated probes achieved quantitative nitroreductase and hydrogen sulfide detection in blood plasma and serum, whereas analyte concentrations were overestimated when bare fluorescent probes were employed for detection. Furthermore, detection sensitivity was increased dramatically as our probes displayed fluorescence enhancement upon SNAP-tag conjugation. In contrast to conventional approaches where fluorescent probes are encapsulated into polymers and nanoparticles, our simple and general approach successfully overcome many key issues such as dye leakage, long preparation steps and limited application to detect only small metabolites.
27
Maa, Bao Lien, et 馬寶蓮. « The Study on the Regulated Prose Poetry of the Tang Dynasty ». Thesis, 1993. http://ndltd.ncl.edu.tw/handle/92226599857696566897.
Texte intégral
28
Wang, Wei-ling, et 王維鈴. « The study of characters in Tao Yuan-Ming’s poetry and prose ». Thesis, 2010. http://ndltd.ncl.edu.tw/handle/95495728053274871376.
Texte intégralRésumé :
碩士國立臺南大學
國語文學系碩士班
98
Tao Yuan-Ming got a reputation as hermit poet in the history of Chinese Literature. His insipid and natural words always bring deep emotion and profound contents. Although he lived in political chaos between Wei and Jin dynasties, his simple tranquil life and persistent of law led a good model of sharp sense integrity for later sages, especially the merging between personality and poetry, which admire posterity a lot. Tao Yuan-Ming liked to associate with the ancients, talked about them and their stories. He even cited them as his friend, especially the history characters of his poetry, which is throughout in every dynasty from the far away. Three Sovereigns and Five Emperors till close to the Cao-Wei kingdom in three kingdoms period. He used typical way to connect many sages, which agglomerate them into the conception of alternative group. This journal studies the view of the poet (Tao Yuan-Ming) and explores the history characters in his poetry, which is emphasized at hermit, the poor, confidants, the chastity, emperors, sages and the immortals. By this way, Tao Yuan-Ming’s consolation and spiritual sustenance is found out and is compared to the related fact. The study of characters probes into the deep emotion from heart and symbolizations, which display the unique personality and poetry of Tao Yuan-Ming.
29
Chen, Bo Zheng, et 陳伯政. « Studies of the Writing of Xie-Yin in Tang Dynasty Prose ». Thesis, 2010. http://ndltd.ncl.edu.tw/handle/71886187187761810857.
Texte intégralRésumé :
碩士明道大學
國學研究所
98
The new change and which gradually transferring of old traditions, is the eternal theme of literature and history, Xie-Yin type of prose literature in the completion of Tang-Dynasty, is built on these two points . The research contents of this paper: Against the Tang Dynasty ago , respectively to illustrate how to build a prototype、enhanced content、conversion value , and by Lasswell formula to analyze the patterns of spread. Caused by changes in the different recipients , and ironic nature of the lower , aesthetic ideas into secular , etc . Thus, Xie-Yin literature to enhance the status of history , the more attention than before , is because it is a new model for literary . This study includes six chapters. The first chapter is the introduction including research motivation and goals, research methods, literature review, and outlines, as well as the writing rules of this study. The second chapter explains the formation of Xie-Yin . Contains witty language, ambiguous language, and “Wenxin Diaolong “, from the stylistic point of view of enhanced content . The third chapter discusses formation and development of the Xie-Yin literature , time environment will be the focus of the discussion, which will include the historical facts , the political, and literary theory. The fourth chapter focuses on writing strategy , aesthetic and other functions, Argument the Xie-Yin literature with other literary tradition of the convergence, bind together, transformation , become Tang Dynasty to open up new prose style preparation. The fifth chapter emphasizes the Xie-Yin and narrative literature which shows their “Fusion”, including the application of the type style and biography , As well as the development of the late Tang Dynasty . The seventh chapter is the conclusion that concludes all arguments, and gives explanations to some unmentioned key points in this study.
30
Hua, Shan, et 華珊. « The Plight and Struggle in Tao Yuan-Ming’s Poetry and Prose ». Thesis, 2017. http://ndltd.ncl.edu.tw/handle/dya3u5.
Texte intégralRésumé :
碩士臺北市立大學
中國語文學系
105
The focus of Tao Yuan-Ming's research, most of which focuses on his leisurely pastoral life and noble seclusion. However, Tao Yuan-Ming actually had the great and strong ideal. Tao Yuan-Ming had the great ideal, but ultimately chose a plain living. So, after he chose the seclusion, Tao Yuan-Ming had to faced difficulties in his life, for instance, the economic problem. In addition to the economic problem, the most important thing is Tao Yuan-Ming’s feeling that after he couldn’t realize his political ideal, like anxiety and loneliness. This article hopes to discuss Tao Yuan-Ming's work and the dilemma in his life, so it can reflect how Tao Yuan-Ming got the image of the hermit and the elegant mood through his inner struggle and anxiety in Tao’s poetry. Anxiety and loneliness had prompted Tao Yuan-Ming make his poetry to achieve more profound content and realm.
31
Zeng, Yan Syun, et 曾彥勛. « Labeling Environment-Sensitive Fluorescence Probes to SNAP-tag Protein : Fluorescence Amplification and Nonspecific Signal Suppression Detection in Blood Plasma ». Thesis, 2016. http://ndltd.ncl.edu.tw/handle/rq329p.
Texte intégralRésumé :
碩士國立清華大學
化學系
104
Fluorescent probes are important tools in medical diagnosis, because they allow for rapid and sensitive monitoring target analytes. Although most fluorescent probes show excellent performance in clean buffers, they are easily foiled by nonspecific signals in complex real samples such as blood plasma. The problem is mainly caused by the nonspecific binding of fluorescent probes with other macromolecules. Furthermore, environment-sensitive dyes display weaker emission in aqueous buffers. Therefore, how to solve these two fundamental problems is an important issue. Previously, we’ve developed a BGSBD probe which exhibited dramatic fluorescence turn-on upon covalently labeled to SNAP-tag protein. Based on BGSBD, we design a novel protein-shield concept as demonstrated by SNAP-tag protein for fluorescence amplification and nonspecific signal suppression of fluorescence probes for detection of enzymes and reactive small molecules in blood plasma. Herein, we designed three different probes, BGSBD-NO2, BGSBD-N3 and BGSBD-(OH)2, to detect Nitroreductase (NTRase), Hydrogen Sulfide (H2S) and Hydrogen Peroxide (H2O2). We have successfully amplified the fluorescence signal and suppressed unspecific signal of the probes by our protein-shield strategy in human blood plasma.
32
Afitska, Kseniia. « Výzkum a inhibice agregace alfa-synukleinu ». Doctoral thesis, 2019. http://www.nusl.cz/ntk/nusl-411972.
Texte intégralRésumé :
α-Synuclein (AS) is a small intrinsically disordered protein expressed in neurons and abundantly present in synapses where it is involved in regulation of synaptic vesicle-mediated protein trafficking. Misfolding of AS into amyloid fibrils is a key process in progression of Parkinson's disease (PD), the second most common neurodegenerative disorder which has no cure to date. Inhibition of AS aggregation and blocking of cell-to-cell spreading of AS fibrils is a promising strategy for PD treatment. However, rational design of inhibitors of this type remains complicated due to the lack of thorough knowledge about the mechanisms of aggregation. Therefore, the aim of this thesis was to gain deeper knowledge about AS aggregation and to apply it for developing inhibitors of AS fibrillization. In my work on the mechanisms of AS aggregation, I first determined that the concentration of AS that enables the fibril growth is an order of magnitude lower than the concentration of AS required for initial fibril formation from monomers. I explored fibril disaggregation at AS concentrations below its Kd value, and characterized AS aggregation at low micromolar concentrations. I then investigated how different modifications of AS C-terminus (namely, extensions of various sizes and charges) affect fibril growth and...
33
Yang, Ching-hui, et 楊淨惠. « Ancient Prose Movement and Development of Mencius Doctrine in the Tang Dynasty ». Thesis, 2014. http://ndltd.ncl.edu.tw/handle/53078493097817323469.
Texte intégralRésumé :
碩士國立中山大學
中國文學系研究所
102
This study aims to probe into the development of Mencius Doctrine in the Tang Dynasty, with the Classical Prose Movement (Gu Wen Yun Dong) in the Tang Dynasty as the compass, and to analyze the meanings and the characteristics of Mencius Doctrine in the Classical Prose Movement in the Tang Dynasty, so as to elucidate the thriving Mencius Doctrine in the Song Dynasty. Through the discussion on Mencius Doctrine’s development via the angles of politics, Confucianism, religion, as well as literature, this essay acknowledges the efforts of the pioneers of Classical Prose Movement in Ming Tao (to illuminate the Tao) and Zong Jing (to revere the classics of Confucianism). These pioneers forcefully defended the traditional orthodoxy and were keen to promote those with potential. They therefore revived Confucianism and recalled the scholars to the values of traditional Confucianism. Thus, we will review the growth of Mencius Doctrine in Classical Prose Movement via three distinctive philosophies: the theory of “Width” Han Yu inherited, the theory of “Depth” Li Ao developed, and the theory of “Reason” Liu, Zong-yuan innovated. In addition, we will depict the development of Mencius Doctrine’s annotations in the Tang Dynasty as research methods changed from exegesis on a textual basis to elucidation of arguments within. Last but not least, through the reverence for the scholars leading Classical Prose Movement by the scholars in the late Tang Dynasty and in the Song Dynasty, we will sum up the influences that the Classical Prose Movement in the Tang Dynasty had on Confucian Orthodoxy, Theory of Mind, and the change of research methods, and finally conclude the certainty, suitability, and practicality of Mencius Doctrine’s development in the Tang Dynasty.
34
郭偉廷. « A study on the historical characters in Tao Yuan-ming’s poetry and prose ». Thesis, 1993. http://ndltd.ncl.edu.tw/handle/by3y9a.
Texte intégral
35
Chen, Jing-Ming, et 陳敬明. « Research for Route Tai-15''s Estimation of Accident-prone Location and Improvement Measures ». Thesis, 2000. http://ndltd.ncl.edu.tw/handle/98348934577883702085.
Texte intégralRésumé :
碩士國立交通大學
交通運輸研究所
88
The Western Coastal Penetrated-island Expressway''s improvement plan is one of the government''s fourteen major projects , among of them that Route Tai-15（Kuantu Bridge-Hsiangshan）has been completely finished in July,1998. It has observable positive benefits on behalf of upgrading its traffic efficiency, nevertheless, the sign of accidental rate has risen and has become more serious after operation. Therefore, this study is not only through getting accidental data to analyze, but also to build up the module of estimation for accident-prone location identity, the cause of accident analysis, and that come up with improvement measures to reduce the possibility of accident and its severity. This study is the Western Coastal Expressway in Hsinchu County and in Hsinchu City, and that the accident has taken place within one and one half of year. Initially, according to its performance value of accident-prone jeopardy estimating its location, secondly, based on its accident types and accident causes, there countermeasures are provided by means of method and step after reviewed and then main four-type issues are induced by analyzing its accident causes as well as accident types and road conditions. On the other hand, that questions are too high density of road cross, poor geometrical road condition, linking up on both road type changing and speed shifting, traffic safety is induced distinction between the operation characteristic of Western Coastal Expressway and highway are also offered its an example for improvement, respectively. Besides, owing to current accident-prone location is frequently dissolved by people''s representative''s proposal, people''s appeal or road-inspector, that are odds and ends and out of our plan. Therefore, we need to create a standard operation procedure for accident-prone location improvement, and we pledge that unify the operation proceedings, various forms necessarily. So that, we can reach the goal of standardization as well as process and that will provide as a reference to road administration for improvement.
36
LIN, YU-HSIN, et 林郁馨. « The Study of the Wisdom of Life in Tao Yuan-Ming’s Poetry and Prose ». Thesis, 2019. http://ndltd.ncl.edu.tw/handle/kpxczx.
Texte intégralRésumé :
碩士國立東華大學
中國語文學系
107
This dissertation explores the thoughts and values presented in Tao Yuan-Ming’s(A.D.365-427) poetry and prose. To analyze Tao’s work , his life story is introduced in the first part of this study, including family background, language context of the time,and government service and reclusion. Besides, the display of the writer’s four writing phases through his life time, which are ”pursuing official job,” ”resigning official job,” “living in retirement,” and “rediscovering his true self,” lead us to know Tao in each phase. What’s more, the study discusses Tao’s major decision made in his middle age:he resigned and went home as well as the spirit and meaning of this choice. This has much resemblance to the Western concept of “LOHAS.” Therefore, in the latter part of this thesis, Tao’s poetry is explored through the perspective of LOHAS, especially his Fields and Gardens poetry which leads to the conclusion that these two idea are similar in: “Living in accordance with nature,” “Having passion for life,” and “Going back to innate essence.” Furthermore, the influences of Tao on contemporary times are explored from four aspects: Storytelling expert, Display of Everyday Aesthetics,Home is the root of life, and Discovering true self from the inside out. This study concludes that whether to proceed or to retreat, the poet still stays true to himself,keeps to his primitive simplicity, finds him contented and realized the wisdom of life for better or for worse through the course of the seasons.
37
Huang, Yi-Shan, et 黃羿珊. « 1. Chemoenzymatic synthesis of oligosaccharides with ionic liquids tag-assisted purification2. Synthesis of azido-deoxy glycans as bioactive-probes in protein labeling ». Thesis, 2018. http://ndltd.ncl.edu.tw/handle/nrpvrs.
Texte intégralRésumé :
碩士國立中正大學
化學暨生物化學研究所
106
This thesis is composed of two independent research project. The first project – “chemoenzymatic synthesis of oligosaccharides with ionic liquids tag-assisted purification” – is to facilitate product purification of the enzymatic glycosylation reactions. In this study, we aimed at employing a hydrophobic ionic liquids tag on GlcNAc. Since the ionic liquids tag sugar have affinity with ionic liquids, which is as a solvent for extraction. The target glycan could be extracted from the enzymatic reaction mixture. It provides a highly effective trategy for facile synthesis and purification of glycans. The second project is “Synthesis of azido-deoxy glycans as bioactive-probes in protein labeling.” The potential functions of human milk oligosaccharides (HMOs) are still unknown. In our approach, we aimed to synthesis tetrasaccharides, the core structure of human milk oligosaccharides, with azido group on Galactose at reducing end. The azido group could selectively covalent with fluorescence probes by click reaction for further detection.
38
Tseng, Tzung-yu, et 曾宗宇. « Probe to study and research female image of Du Mu’s collection of poems .（afterlife of Tang dynasty in China） ». Thesis, 2003. http://ndltd.ncl.edu.tw/handle/87946615434076586543.
Texte intégral
39
Zheng, En-Ci, et 鄭恩賜. « A Study on the Commentary and Punctuation of Classical Prose of the Tang-Song School ». Thesis, 2010. http://ndltd.ncl.edu.tw/handle/35428037264028307506.
Texte intégralRésumé :
碩士國立暨南國際大學
中國語文學系
98
This thesis analyzes the commentary and punctuation (ping-dian) of classical prose of Tang-Song School from three perspectives: the construction of canon and the development of ping-dian, the grammar, and the reading activity and the imagination of the world. These three perspectives focus on the inheritance and inauguration of the ping-dian of classical prose of from Tang-Song School from late Song dynasty to late Ming dynasty; moreover, they demonstrate the specialty of the literary theories of Tang-Song School by grasping the differences of the literary theories between early-Ming dynasty and late-Ming dynasty. Finally, this thesis also aims to discuss how the world is presented in ping-dian of classical prose of Tang-Song School. Ping-dian of classical prose of Tang-Song School can be praised as a representative canonic movement in Ming dynasty’s literary field. Intrinsically, the critics of Tang-Song School build the canonic genealogy which can be traced to Qin-Han dynasty by selecting the literary works and by raising something more concrete and more operative. By playing the roles as judges, the ones who do the ping-dian, based on the consciousness of literary competition, not only make the construction of canon practical, but also promote the position of reader. Extrinsically, ping-dian of classical prose highly combines with the printing which develops again during Zheng-de（正德） and Jia-jing（嘉靖） years. This cuases frequent dissemination through giving, selling and making private copies, and ping-dian becomes popular under the systems of symbolic capital, economic capital and education system. Through five codes raised by Roland Barthes in his S/Z, the thesis discovers that ping-dian of classical prose of Tang-Song School concerns language functions in texts and the effects they caused separately, and hermeneutic code and proairetic code are far beyond the others. It shows the critics of Tang-Song School pay much attention on the logic and structure of language in classic literature, and this is the biggest difference between Tang-Song School and Qin-Han School; also, this result follows the development of south-Song dynasty. In addition, other three codes (connotative code, symbolic code and cultural code) are also concerned in ping-dian of classical prose of Tang-Song School, and becomes the forerunner of ping-dian of novels and the Chinese operas. The appropriation from cultural terms into literary criticism in ping-dian of classical prose of Tang-Song School shows the interdisciplinary perspective of people in Ming dynasty. After analyzing two interdisciplinary terms, mai and chi, this thesis discovers that the use of “mai”（脈） , on the one hand, concerns partially texts’ forms and structures; and on the other hand, it also demonstrates those critics of Tang-Song School’s desires for control of the cultural genealogy. Besides, the early understanding to “Mai” has been changed. From early Ming dynasty, “mai” had been regarded as knowledge of origin and as a concept of returning to the ancients, but “mai” has changed to a unique thought of affirming the partial and valuing every stage. The use of “chi”（勢） explains more properly Ping-dian’s expectation for manipulating language, and presents the appreciation of beauty brought by the reading process. Both “mai” and “chi” illustrate the prospect of world from the critical terms of ping-dian. This way, the texts are connected to the world—texts are like the world is watched and the world can be manipulated by texts.
40
« Sequence-specific reactivity of short peptides : Peptide photooxidative fluorogenesis and peptide tags for small molecule fluorescent probes ». UNIVERSITY OF CALIFORNIA, IRVINE, 2009. http://pqdtopen.proquest.com/#viewpdf?dispub=3355899.
Texte intégral
41
Yang, Wan-tzu, et 楊婉慈. « Analyses on the Painting Appreciation and Inscription Interpretation in Lung-Po Prose Collection of Tai Jhing-nung ». Thesis, 2013. http://ndltd.ncl.edu.tw/handle/27705761094467995682.
Texte intégralRésumé :
碩士世新大學
中國文學研究所
101
This paper is an in-depth analysis on the “painting appreciation” and “inscription interpretation” in Lung-Po Prose Collection of Tai Jhing-nung. The main purpose of this study is to present how Tai comprehensively interprets the information in different knowledge fields based on his abundant knowledge and flexible and scholarly ability and further creates inspirational cultural prose filled with knowledge. This paper is organized into 5 chapters: Chapter 1 “Introduction” explains the motivation, scope, current status and framework of this study. Chapters 2 and 3 discuss two examples of “painting appreciation” - “Night Revels of Han Xizai” and “Geng’s Ink Painting of Snow in the Southern Tang Dynasty by Painter in the Song Dynasty,” respectively. Chapter 4 discusses the “inscription interpretation” of “Trip to Liaodong.” These three chapters are the focus of this study, which aims to perform in-depth analyses on painting appreciation and inscription interpretation of Tai, as well as his rich knowledge in various fields. Moreover, this study also discusses the doubts about the prose by Tai. In addition to summarizing the analysis results of the former chapters, Chapter 5 “Conclusion” also indicates that the integrative prose of “painting appreciation” and “inscription interpretation” by Tai reflects his enthusiasm for heritage and promotion of traditional culture. His integrative knowledge in various academic fields of traditional culture is reflected by his superb writings. His writings are undoubtedly the shining examples for the future literati and worthy of appreciation.
42
Chih-Wei, Chung, et 鍾志偉. « A study of prose anthologies of Tang-Sung Pa Ta Chia in the Ming and Ching Dynasties ». Thesis, 2007. http://ndltd.ncl.edu.tw/handle/31630496383845300087.
Texte intégral
43
Hsu, Wan-Shuan, et 許琬瑄. « Climate Adaptation Mechanisms of Urban Communities –A Case Study of Tal-Lao-Keng Flood-prone Area of Hsin-Chuang District ». Thesis, 2016. http://ndltd.ncl.edu.tw/handle/ct8q6z.
Texte intégralRésumé :
碩士中國文化大學
建築及都市設計學系
104
The regional phenomenon of heavy rainfalls under global climate change is extremely likely to induce flash floods in flood-prone urban areas and endangers the lives and safety of residents. Due to the low-lying land and overdevelopment of the urban areas, Hsin-Chuang District in New Taipei City situated in Ta-Liao-Keng river basin faces routine flooding in some of its streets after heavy rainfall. The area is highly exposed to flood risk in a changing climate. Faced with the challenge, this study provides insights into adaptation mechanisms of urban communities of the district in adapting to climate change. On research design, Driver-Force-Pressure-State-Impact-Response （DPSIR） causal model is used as a framework to assess social vulnerability. Our methods include literature review, map and data analysis, and semi-structured interviews and sample analysis. We focus on local residents’ adaptive awareness and actions in response to the uncertain impacts of climate change. It is argued that government flood control measures, such as dikes, gates and pumping stations, have limited utility. Some residents also point out that dikes exacerbate the floods. When there are floods, most residents are accustomed to observing and measuring water levels in the drainage ditches, setting up water control gates in front of their own houses, and elevating living spaces on the ground floors. Early warnings are also broadcast in the communities. But with respect to community engagement in integrated flood risk management, there exists indifference as well as gaps in understanding. The adaptation mechanisms proposed by this study will help increase the levels of knowledge and capabilities of community members as they manage floods as a group and can serve as a reference for the development of resilient communities in Taiwan. The study underlines the construction of social networks and the integration of structural and nonstructural measures to respond comprehensively to the impact of floods under climate change.
44
Yeh, Ya-ping, et 葉雅萍. « Study on the influence of Tao Yuanming to the thought about reclusion of the Sanqu (prose music) of Yuan dynasty ». Thesis, 2012. http://ndltd.ncl.edu.tw/handle/33421181493267635001.
Texte intégralRésumé :
碩士國立臺南大學
國語文學系碩士班
100
Though there was no big feat being achieved during the life of Tao Yuanming, however, his resign from the office to return to the country life and accepted the fate to live in reclusion for the rest of his life have made his name remembered in history. He was the most famous recluse poet in Chinese history and was known as “Pastoral Poet” or “Recluse Poet” to the later generations. His personal character and poem character have drawn highly concern of the later scholars. His turning from a politician to a recluse at certain point of his life and stayed being poor the rest of his life without changing his mind, and the skill of poet art strengthened up by nature have allowed him to present the incomparable sentiment of life and personality condition. The acceptance of Tao Yuanming by the people of Yuan dynasty was mainly due to the similarity on the sentiment of life of the underappreciated of the scholars under the ruling of the other country, besides, they have being impressive on the personal character of Tao Yuanming. Therefore, there were many taking Tao Yuanming as the theme or the praising Tao Yuanming for the whole piece of work on the Sanqu of the Yuan dynasty, the contents of which mostly were description of his life and story, that lauded his character and morality, appreciated his leisure and reclusion on everyday life. These all pointed out how Tao Yuanming took a place in their heart, which Tao can be called their bosom friend at different dynasty or their spiritual destination.
45
Yan, Sergey. « Photoaktivierungsfähige Rhodamine als Bio-Calcium-Sensoren und Markierungen für Tetracystein-Tags in Proteinen ». Doctoral thesis, 2011. http://hdl.handle.net/11858/00-1735-0000-0006-B08B-1.
Texte intégral
46
LIU, CHUNG-HSING, et 劉中興. « To Probe and study the Votes Influence from Interactive Pattern of Local Political Ecology －Case by the Relationships between 2001 and 2005 Elections of Tao-Yuan Master ». Thesis, 2006. http://ndltd.ncl.edu.tw/handle/23837738605317619284.
Texte intégralRésumé :
碩士開南管理學院
公共事務管理學系碩士班
94
The major-population structure is combined of the Taiwan Hans （Fuchienese and Kwangtungese ）and other provinces in Tao-Yuan county. Taiwan Hans have to cooperate with Clan organization to attend political party and basic local faction in order to be local political ecology. The early years, KMT had created local economical source, that planning to balance benefits of basic local faction and difference nationality, because the county master has more administration source, so the object of Fuchienese and Kwangtungese is fight for the Vote, therefore the reason of Taiwan Hans could retuned to be the master. There are planning to raise local faction and balance Clan power, and that county master has the huge administration sources , and Taiwan Hans would like to gain and to pursue the object，It had made the situation that giving power for Taiwan Hans each other 。in basic the policy power will be balanced of Taiwan Hans, that county master and county council speaker who must be different background nationality , and important return should be area from north or south , whatever the condition will ever lost。2001 year county master election that chu, ly-ren who had wined the county source power, and successful to get the county mater position again , which may be exchange the traditional county power belonging to Fuchienese and Kwangtungese each other. The developing history of Tao-Yuan is special condition, mafia group and basic local faction together and to maintain there votes and position ,and attempt to local polity and economical , in periods especially gang criminals, confirm that within the campaigning, they help to do vote-buying, mobilize voters and consolidate voters supports for some certain candidates. some of these candidates are mafia background politicians and made from mafia group or money paying for polity. To observe the election movement for government of center or local，Tao-Yuan county polity ecology appearance or disappearance, because of polity party contort the enterprises. mafia group. basic local faction. Taiwan Hans and Clan group cooperation , the clan group is most strong and will be stead of Taiwan Hans power position and be the most important reason of Tao-Yuan polity development . this kinds background variable and development of local polity ecology, that will know the Tao-Yuan county polity development trending，the other Taiwan polity trend is 50s which had to be married as well , the development information and widely education and national peaceful , one by one and enforce the polity party must to choose the elite in order to get the mid-choicer favor , and the appearance of the situation of polity separation, as the county master election model will be divide the difference satiation. This study is observed the polity ecology for polity party, basic local faction , Taiwan Hans. Clan organization . .mafia group. Enterprise, to find the influences of elections, and case by 2001s and 2005s Vote of Tao-Yuan County Master , in Tao-Yuan polity ecology either appearance or disappearance or not, however the depth interview and export QA to prove the Tao-Yuan election model. as well as the analysis and comparison of their differences. And something to made the conclude and suggestion for the discovery. Key words local political ecology, basic local faction, Taiwan Han and other provinces, major Clan organization, mafia group, political money giving.
47
Lo, Yi-Hsuan, et 羅逸軒. « Synthesis and biological evaluation of nicotinamide/picolinamide derivatives as novel melanoma diagnostic probes and Tau protein imaging as a potential predictive marker in cancers treated with paclitaxel chemotherapy ». Thesis, 2017. http://ndltd.ncl.edu.tw/handle/n37xmg.
Texte intégral
48
YANG, YI-SUNG, et 楊益松. « A probe into the low income elderly people living alone on their demands of home care – a case study in the areas of Feng Yuan city, Taichung county, Da Du Township and Tan Zi Township ». Thesis, 2010. http://ndltd.ncl.edu.tw/handle/24057369227240528030.
Texte intégralRésumé :
碩士玄奘大學
社會福利學系碩士班
98
As the socio-demographic structure is aging, different social demands and problems happen. Taiwan has entered an aging society. Under the changes of social and family structures, more and more elderly people cannot live together with their children, and become the elderly people living alone. The elderly people are always unable to be independent in some of their daily lives, and need other people’s assistance. Those whose healthy conditions are bad have to take medical treatment often. Because of living alone and keeping away from outside world, they have very limited information, and are easy to feel lonesome. Thus, the elderly people living alone become a relative underprivileged group. They need concerns from government and private groups desperately. For taking care of the elderly people living alone, our government has carried out a “Home care” program for almost 30 years. Home care agrees with the principle of “displacement on the spot”, “aging in place” and “welfare community-oriented”, and possesses “accessibility”, “approachability” and “convenience”. This kind of service is more suitable for the elderly people on their living needs. How do these elderly people living alone feel toward the home care? What have they experienced about such home care? Does the current home care need any improvements? All of these questions are the key points that this research wants to probe. This research adopted the qualitative research. In-depth interviews were made by means of semi-structured interviews. The contents of the interviews were written down one word after another. In this way the information was collected. The low income elderly people living alone were the interview subjects in this research. For the research participants, two came from Feng Yuan City, Taichung County, two came from Da Du Township, and one came from Tan Zi Township. In-depth interview method was made to understand the participants on their actual daily lives and the home care status. This research draws a conclusion of 7 points according to the findings of the interviews. 1. There is a high demand on accompanying the elderly people to see a doctor. 2. There is also a high demand on writing service. 3. They need more moral support. 4. They need less leisure service and healthy care. Domestic service is not important for them. 5. The overall services are just satisfactory, but spontaneous service is not enough. 6. They expect innovative services and other services 7. Other demands In accordance with above conclusion, following suggestions were brought up in this research according to the discovery of the problems. 1. Reinforce the ability of home care personnel. 2. Need community-oriented service 3. Spontaneous service and good service manners are needed. 4. A bottom-up adjustment on the roles of government staffs is needed. 5. Develop a long term care policy for the elderly people living alone. 6. For the elderly people, set up three-in-one system - “welfare service”, “health insurance” and “income protection”. 7. Set up a body and mental care system for the dying people. Keywords: low income, elderly people living alone, home care services
49
Cho, Eun-Sang, et 趙殷尚. « A Study on the Vanguards and Their Prose works of the Fu Ku Movement in Tang Dynasty : Focused on Xiao Ying-Shi, Li Hua, Jia Zhi and Yuan Jie ». Thesis, 2003. http://ndltd.ncl.edu.tw/handle/18147270311834834466.
Texte intégral
50
Welegedara, Adarshi Priyanga. « Site-directed labelling of proteins for NMR and EPR studies ». Phd thesis, 2018. http://hdl.handle.net/1885/164198.
Texte intégralRésumé :
Site-specific protein labelling presents an important tool for
protein structural biology by
spectroscopic techniques. This thesis focuses on the development
of new spectroscopic
labels and labelling strategies to improve the sensitivity,
accuracy and scope of NMR and
EPR spectroscopy experiments of proteins.
Double electron–electron resonance (DEER) spectroscopy measures
the distance
between two paramagnetic metal centres introduced by
site-specific attachment of
suitable tag molecules. Good DEER tags possess rigid tethers to
position their
paramagnetic centres at a well-defined location relative to the
protein and deliver narrow
DEER distance distributions with high sensitivity, which can
provide accurate
information about protein flexibility. This thesis introduces
new, cyclen-based Gd 3+ tags
and small, Gd 3+ chelating tags designed to deliver narrow DEER
distance distributions.
Chapter 2 describes the development of two cyclen-based
double-arm Gd 3+ tags
designed for binding to the target protein at two and three
points to obtain the narrowest
Gd 3+ –Gd 3+ DEER distance distributions ever recorded with
proteins. It also describes
DEER distance measurements with the iminodiacetic acid tag
attached to cysteine (Cys),
where tags attached to two neighbouring Cys residues combine to
chelate a single Gd 3+
ion. These results have been published in a journal article
(Welegedara et al., Chem. Eur.
J. 2017, 23, 11694−11702).
Chapter 3 discusses two single-armed Gd 3+ tags, a cyclen-based
Gd 3+ tag and a
PyMTA tag that forms a heptadentate Gd 3+ binding motif. Both
tags deliver the shortest
possible tethers to cysteine residues and are shown to produce
narrow DEER distance
distributions in proteins.
For applications in NMR spectroscopy, proteins can be labelled
site-specifically
with NMR probes such as trimethylsilyl (TMS) probes, which
deliver readily detectable
1D 1 H-NMR signals. Introduction of paramagnetic tags and NMR
probes by attachment
to Cys residues requires mutations of native Cys residues to
achieve site-selectivity,
which is not possible with structurally and functionally
important Cys residues. Chapter
4 demonstrates a solution to this limitation by introducing a
selenocysteine (Sec) residue,
which can be selectively reacted with probe molecules at slightly
acidic pH without
iiiaffecting naturally occuring Cys residues. To achieve this, a
Sec residue was introduced
as a photocaged unnatural amino acid (UAA), PSc, to bypass the
otherwise unavoidable
challenges associated with the natural Sec incorporation
mechanism. UV illumination of
PSc yielded Sec with no evidence for the formation of undesired
dehydroalanine
byproducts. Selective tagging of Sec residues with TMS tags was
shown to deliver a
useful tool for studies of ligand binding to proteins. These
results have also been
published in a journal article (Welegedara et al., Bioconjugate
Chem. 2018, 19,
2257−2264).
Site-selective incorporation of isotope-labelled PSc, photolysis
and anaerobic
deselenization opens an indirect route to labelling a single
specific alanine residue in a
protein with stable isotopes. Such samples would have important
applications in
heteronuclear NMR, as they allow the selective detection of the
labelled alanine residue
with maximal spectral resolution. As shown in Chapter 5,
deselenization of
selenoproteins into alanine is possible but requires extremely
anaerobic conditions to
eliminate serine as the main unwanted byproduct.
A range of UAAs has been developed to serve as spectroscopic
probes or facilitate
the introduction of spectroscopic probes via biorthogonal
reactions. The increasing
demand for proteins with different UAAs and the significant cost
of some of these UAAs
has led to increasing popularity of cell-free protein synthesis
(CFPS) systems, which use
amino acids more sparingly than in vivo expression systems.
Mutants of pyrrolysyl-tRNA
synthetase (PylRS)/tRNA CUA pairs have been developed into a
particularly versatile tool
for the incorporation of many structurally different UAAs, but
most produce
disappointedly poor protein yields in vivo. Chapter 6 describes
attempts to develop an in-
house CFPS system with a PylRS/tRNA CUA pair. In addition, the
polyspecific G2
synthetase has been reported to facilitate the incorporation of
sterically demanding UAAs
and Chapter 7 of this thesis describes attempts to develop a CFPS
system with the G2
synthetase.