Dissertations / Theses on the topic 'Proofreading'

The Twin Arginine Translocase (Tat) is one of two protein translocation mechanisms in E. coli to move proteins across the inner bacterial membrane, from the cytosol to the periplasm. A unique feature of the Tat pathway is its ability to translocate fully folded proteins, indeed, in E. coli the Tat pathway preferentially transports correctly folded proteins. This 'proofreading' mechanism, as it has been dubbed, is of interest to the biopharmaceutical industry, however little is known of the mechanism by which Tat proofreads a substrates conformational state. Initial studies (chapter 3) addressed if the Tat proofreading mechanism sensed the surface charge or hydrophobicity of a substrate. To this end, surface residues of an scFv were mutated to create areas of charge and hydrophobicity without altering tertiary structure. Expression of these variants in E. coli revealed that Tat proofreading is tolerant of surface charge and hydrophobicity, but dependent on conformational flexibility. Further studies utilising a maquette in various folding states, confirmed Tat proofreading is sensitive to the structural rigidity of substrates (chapter 4). Investigations then went on to assess the quality of protein entering the periplasm via the Tat pathway by comparing it to the same protein transported by the General Secretory (Sec) pathway (chapter 5). This revealed, at least for a relatively simple biotheraputic, Tat-translocated protein is of the same quality to Sec-translocated protein. Finally, the question of what is responsible for the proofreading ability of Tat began to be addressed through C-terminal truncation studies of the Tat components that attempted to restore export of export-incompatible substrates (chapter 6).

The twin-arginine (Tat) system is a specialised translocation machine found in prokaryotes and chloroplasts which serves to export fully folded proteins across the cytoplasmic membrane. Proteins are specifically targeted to this system by N-terminal signal peptides which bear a conserved SRRxFLK motif. Tat dependent proteins often contain redox factors that must be inserted prior to translocation. The E. coli Tat substrates trimethylamine Noxide reductase (Tor) and periplasmic nitrate reductase (Nap) are two such substrates which require the molybdenum cofactor Mo-bis-MGD in order to function as anaerobic reductases. Pre-export assembly of Tor and Nap are subject to a process called Tat proofreading, where the substrate specific chaperones, TorD and NapD, bind tightly to the signal peptides of these substrates, TorA and NapA, respectively, assisting in the assembly of the proteins. Work described in this thesis demonstrates that TorD simultaneously binds to two independent sites present on the TorA, one present in the mature domain of the substrate and one in the signal peptide. Biophysical and structural analysis shows that the pre-export complexes of TorD and TorA appear identical regardless of whether the TorA signal peptide is present or absent. However, the complex purified in the absence of the TorA signal peptide is able to interact with extrinsically-added signal peptide, confirming the presence of a separate signal peptide binding site. By contrast to TorD, NapD was shown to bind exclusively to the signal peptide of its substrate, NapA. Pre-export complexes of TorAD and NapDA showed similar low resolution structures, with both NapA and TorA exhibiting flexibility in Domain IV. It is hypothesised that this Domain acts as a gate, allowing access of the cofactor, and that closing of this gate is the final step in the folding of the substrate.

The problem of this study was to examine errors located by subjects proofreading hardcopy versus softcopy business documents. Hardcopy refers to a business document typed or printed on paper; softcopy refers to a business document displayed on a computer screen. Data were obtained by 61 Southwest Virginia high school business students completing a background information sheet and proofreading the same three documents—a letter, a report, and a memo—on both hardcopy and softcopy media; for each media, the students proofread each document for 15 to 20 minutes over a 1-hour period. The number and types of errors found, the number of times each document was proofread, and personal characteristics of the subjects were analyzed. The following outcomes are based on the results of the study: (a) subjects located the same number of errors when proofreading from hardcopy and softcopy media; (b) subjects located one to two more errors in the letters and reports than in the memos; (c) the medium was not related to the specific types of errors found; (d) subjects who proofread a document five times located one to two more errors than those who proofread fewer times; (e) subjects with 0 to 2 years of computer experience located one more error than those with more experience. The two main conclusions of the study were: (a) students need not print hardcopies of documents in an attempt to locate more errors than in softcopy documents; and (b) teachers should be aware that students are more likely to locate some types of errors than others.
Master of Science

In the paper we will analyze the Italian localization of the first 5 chapters of the videogame The Last of Us. We will also offer alternative translations and will correct the localization mistakes. The paper consists of five chapters, introduction and conclusion. Each of the chapters represents a category of the revised translation: done badly, omitted translations, done well, could be improved, wrong register or wrong swear words translations. We will observe and analyze the Italian localization of the first five chapters of the videogame and propose the alternatives in the cases of errors and ambiguities, as well as highlight well done translations that reflect the differences between English and Italian grammar, syntax and semantics. The revised parts of the localization primarily include the translation of the subtitles and the artifacts (written documents, not voiced) within the game. The dubbing and the lip sync are not analyzed, unless they influence the translation in one way or another. The orthography and punctuation of the subtitles are kept the same in the thesis as they are in the game. The swear words will be fully spelled for transparency. In the conclusion we will close with the state of the Italian localization of videogames in general and of this one in particular.

This mixed model study informed by a multi-modal approach investigated the relationship between reading aloud and the student revision process. Participants for this study were undergraduate juniors and seniors enrolled in any one of four sections of English 393 (Writing Competency Course) at Ball State University during the summer semester of 2003. These students had previously failed the Writing Competency Exam at Ball State; therefore, they had to complete English 393 successfully to fulfill Ball State's writing competency requirement and, ultimately, to graduate. Specifically, this study examined what types of surface and global features that these English 393 students noticed when reading their initial essay drafts aloud to themselves and what global revisions they made, if any, based upon these initial observations. Methods used were audio recordings, observation logs, multiple copies of student drafts, pre- and postattitudinal surveys, read-aloud surveys, post-revision surveys, introductory and concluding instructor surveys, additional instructor surveys, and reviews of composition/rhetoric textbooks.Results of this study indicated that students enrolled in English 393 courses at Ball State University during the summer of 2003 predominantly noticed surface features in their essays as a result of reading their initial drafts aloud to themselves. Therefore, using this read-aloud method did not prompt the large majority of these junior- and senior-level English 393 students to make global revisions in their drafts. They predominantly made surface-level revisions, indicative of the types of revisions that freshman college writers make. While one student from the population did make global revisions as a result of using the read-aloud method, the researcher attributed this anomaly to the student's probable oral learning style and/or the student's previous experience using the read-aloud method.
Department of English

Thesis (Ph. D.)--Illinois State University, 2006.
Title from title page screen, viewed on June 8, 2007. Dissertation Committee: Janice Neuleib (chair), Ronald Fortune, Bob Broad. Includes bibliographical references (leaves 270-280) and abstract. Also available in print.

Mon travail de thèse s'est focalisé sur la machinerie enzymatique impliquée dans la réplication du génome ARN du Syndrome Respiratoire Aigu Sévère-Coronavirus (SRAS-CoV). J'ai montré in vitro que l'activité ARN polymérase ARN-dépendante (RdRp) portée par nsp12 nécessite le complexe nsp7/nsp8, qui agit comme facteur de processivité. Grâce à ce complexe polymérase hautement actif, j'ai pu en suite étudier le mécanisme de "proofreading" (correction d'épreuve) associé aux coronavirus, pour lequel seulement des preuves indirectes avaient été assemblées. En effet, les coronavirus codent pour une activité exonucléase 3'-5' (nsp14-ExoN) qui lorsqu'elle est absente, entraine 14-fois plus d'erreurs de réplication en contexte cellulaire. In vitro, nous avons pu montrer que nsp14-ExoN est capable d'exciser l'ARN double brin ainsi qu'un nucléotide mésapparié en 3' de l'ARN en cours d'élongation. J'ai pu apporter pour la première fois une preuve directe de l'existence d'un système de réparation des erreurs au cours de la synthèse, mené par le complexe nsp7/nsp8/nsp12/nsp14. En effet, le complexe nsp7/nsp8/nsp12 ralentit jusqu'à 30-fois quand il rajoute une base mésappariée. Par sequençage, nous avons pu montrer la réparation de cette base mésappariée en presence de nsp14. Enfin, grâce à ce système in vitro nous avons une base pour comprendre l'inefficacité de la ribavirine sur des patients atteints du SRAS. En effet, la ribavirine, incorporée par le complexe polymérase, serait également excisée par nsp14, annihilant tout potentiel effet mutagenique. En conclusion, ce système va permettre de guider le développement d'antiviraux de type nucleoside analogues contre les coronavirus
This work focused on the enzymatic machinery involved in Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) RNA replication and transcription. Firstly, I established a robust in vitro polymerase assay with the canonical SARS-CoV RNA-dependent RNA polymerase (RdRp) nsp12. I showed that nsp12, in order to engage processive RNA synthesis, needs two viral proteins, i.e. nsp7 and nsp8. This nsp7/nsp8 complex not only activates nsp12-RdRp, but also acts as a processivity factor. Thus, using this processive polymerase complex, I could investigate SARS-CoV proofreading for which only indirect evidences were reported. Indeed, coronaviruses encode for a 3'-5' exonuclease (nsp14-ExoN), putatively involved in a mechanism that proofreads coronavirus RNA during viral replication. We first showed in vitro that nsp14-ExoN, which is stimulated by nsp10, is able to excise specifically dsRNA as well as all primer/templates bearing a 3' mismatch on the primer. Moreover, we could confirm by sequencing that a RNA 3' mismatch was indeed corrected in vitro by the nsp7/nsp8/nsp12/nsp14 complex. We provide for the first time direct evidence that nsp14-ExoN, in coordination with the polymerase complex, is able to proofread RNA. Interestingly, using this in vitro system we found an element that could possibly explain the inefficacy of ribavirin therapeutic treatment on SARS-patients: ribavirin, which is incorporated by the SARS-CoV polymerase complex, would also be excised by nsp14. In conclusion, this system will drive future development of antivirals, particularly of the nucleoside analogue type, against coronaviruses

Abstract Replicative DNA polymerases (pols) synthesize chromosomal DNA with high accuracy and speed during cell division. In eukaryotes the process involves three family B pols (α, δ, ε), whereas in Archaea, two types of pols, families B and D, are involved. In this study the B-subunits of replicative pols were analysed at the DNA, RNA and protein levels. By cloning the cDNAs for the B-subunits of human and mouse pol ε we were able to show that the encoded proteins are not only homologous to budding yeast pol ε, but also to the second largest subunit of pol α. Later studies have revealed that the B-subunits are conserved from Archaea to human, and also that they belong to the large calcineurin-like phosphoesterase superfamily consisting of a wide variety of hydrolases. At the mRNA level, the expression of the human pol ε B-subunit was strongly dependent on cell proliferation as has been observed for the A-subunit of pol ε and also for other eukaryotic replicative pols. By analysing the promoter of the POLE2 gene encoding the human pol ε B-subunit we show that the gene is regulated by two E2F-pocket protein complexes associated with the Sp1 and NF-1 transcription factors. Comparison of the promoters of the human pol ε and the pol α B-subunit indicates that the genes for the B-subunits may be generally regulated through E2F-complexes whereas adjustment of the basal activity may be achieved by distinct transcription factors. To clarify the function of the B-subunits, we screened through the expression of 13 different recombinant B-subunits. Although they were mainly expressed as insoluble proteins in E. coli, we were able to optimize the expression and purification for the B-subunit (DP1) of Methanococcus jannaschii pol D (MjaDP1). We show that MjaDP1 alone was a manganese dependent 3'-5' exonuclease with a preference for mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreader of archaeal pol D. So far, pol D is the only pol family utilising an enzyme of the calcineurin-like phosphoesterase superfamily as a proofreader.

We have developed a novel, sensitive and less time-consuming method to detect activity of the SecA-dependent protein-conducting channels. Nanogram levels of E. coli inverted membrane vesicles were injected into Xenopus oocytes, and ionic currents were recorded using the two-electrode voltage clamp. Currents were observed only in the presence of E. coli SecA in conjunction with E. coli membranes. The observed currents showed outward rectification in the presence of KCl as permeable ions and were significantly enhanced by coinjection with the precursor protein, proOmpA, or active LamB signal peptide. Channel activity was blockable with sodium azide or adenylyl 5’-(β, γ-methylene)-diphosphonate, a non-hydrolyzable ATP analog, both of which are known to inhibit SecA protein activity. Channel activity was also stimulated by oocyte endogenous precursor proteins, which could be inhibited by puromycin. In the presence of puromycin, exogenous proOmpA or LamB signal peptides, but not defective signal peptides, stimulated the ionic currents. We also measured SecA-dependent currents with membranes depleted of SecYEG. Wild-type LamB signal peptides, or precursor proteins stimulated ionic currents following a co-injection of SecYEG¯ membranes with puromycin. Excess exogenous SecA stimulated ionic currents through SecYEG¯ membranes. Similar activities of added SecA were observed with reconstituted membranes depleted of SecYEG. Currents through such SecYEG-depleted membranes were also stimulated by addition of defective LamB signal peptides and unfolded mature PhoA protein. In contrast, currents produced by the membranes containing wild-type SecYEG were not so stimulated, but ionic currents were stimulated through mutant strains, similar to PrlA (SecY) suppressors, e.g. PrlA4, or PrlA665 membranes, suggesting that the proofreading function of SecY was bypassed in these membranes. We have observed that azide can inhibit ionic currents when E. coli wild-type MC4100 membranes were injected with proOmpA or LamB signal peptides into Xenopus oocytes. However, such inhibition was lost when observed with oocyte-endogenous signal peptides in the absence of bacterial signal peptides. Moreover, azide did not show complete inhibition upon using SecYEG¯ membranes or SecYEG¯ reconstituted membranes plus excess SecA in the presence or absence of LamB signal peptides. Such conformational alterations reflect different sensitivity in response to azide during the opening of protein-conducting channels.

Este trabalho estuda o campo da edição de texto na produção editorial de livros no Brasil. O objetivo é apontar algumas distinções necessárias às pesquisas acadêmicas sobre edição de textos, e importantes para a formação de pesquisadores e profissionais. A proposta centra-se nas principais etapas (edição de texto, preparação de originais e revisão de provas) que compõem o processo por que passa um texto a ser transformado em livro. Parte-se de análise dos manuais de editoração e de pesquisas acadêmicas que tangenciam o tema. E articula-se um diálogo entre alguns trabalhos acadêmicos das áreas de psicolinguística e psicologia cognitiva sobre leitura em suas relações possíveis com os estudos em editoração e a experiência empírica dos editores de texto. Contribui-se, assim, para o aprimoramento dos conhecimentos sobre a edição de texto, entre eles: as etapas, os objetivos principais e secundários, as estratégias, as tarefas e um conjunto de elementos necessários para que um profissional atue como editor de texto consciente de sua intervenção.
It is the purpose of this thesis to study the field of text-editing in the Brazilian book publishing industry. Its main aim is to point out a few distinctions deemed necessary not only to the academic research but also to the formation of researchers and professionals in this particular field of study and work. In order to achieve this goal, the main phases the text goes through on its way to becoming a book -- text-editing, copy-editing and proofreading -- are here taken into account and characterized. Examining the available manuals and academic papers on the book editing field is the starting point of this research. It then goes on to promoting a dialogue between the book editing field and other areas of expertise, such as psycholinguistics and cognitive psychology, that also deal with the process of reading. The possible relationships this dialogue reveals and the actual praxis of text editing are then analyzed. Contributing to a better knowledge of what text-editing actually involves is therefore a primary aim of this thesis. It intends to shed some light on the specific phases, primary and secondary goals and strategies text-editing comprises, as well as on a series of elements needed not only for the praxis of text-editing itself but also for a more conscientious practice of this activity.

The diploma thesis is focused on creation of application for managing translation for content management systems. Application will help translation leader with distribution of requests for translation to translators and then to proofreader. Translation leader will be able to import needed articles from WordPress and export after translation. Editor will be able to write articles directly in application. For managing and granting access will be responsible user with administration rights.

Orientador: Maria Viviane do Amaral Veras
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Estudos da Linguagem
Made available in DSpace on 2018-08-25T09:37:27Z (GMT). No. of bitstreams: 1 Pereira_JulianaCristinaFernandes_M.pdf: 1767935 bytes, checksum: cc5ac8a2f3da9afe0ab9dafb9186bb4b (MD5) Previous issue date: 2014
Resumo: Nos últimos anos, têm sido divulgados inúmeros trabalhos com enfoque na representação do tradutor e do intérprete na literatura e no cinema, mas não tantos focam o revisor. E é justamente na fronteira entre realidade e ficção que se encontra o revisor-detetive Daniel Hernández, personagem central desta pesquisa, uma vez que seu autor Rodolfo Walsh (revisor, tradutor, ensaísta e, posteriormente, militante político) retira-o do mundo real e transporta-o para o ficcional. A presente pesquisa, portanto, tem por objetivo analisar a narrativa A aventura das provas de prelo, do escritor Rodolfo Walsh, como uma metáfora da tarefa do revisor, de seus rastros, de sua escrita, de seu trabalho de tradutor. Junto com o passo a passo do ofício do revisor, apresentamos o trajeto desta pesquisa: uma breve apresentação da vida de Rodolfo Walsh, bem como de sua literatura policial; conceitos relacionados à revisão, diretamente ligados ao dia a dia do revisor; a fundamentação teórica para a análise da tarefa do revisor e de sua criação como personagem de ficção, acompanhado de outros dois personagens: o revisor Raimundo Silva e o tradutor Gallus (abordados segundo uma visão mais conservadora, ou tradicional, de tradução e revisão) e o intérprete, senhor Kapasi (em cujas características reconhecemos uma visão denominada pós-estruturalista); a análise do personagem central criado por Rodolfo Walsh, o revisor-detetive Daniel Hernández. A título de desfecho, o ofício do revisor se apresenta como sempre mais ou menos terminado, sempre sujeito a falhas, confirmando que, ao menos no caso aqui estudado, não há crime perfeito
Abstract: Over the last years, a great number of papers have been written focusing on the part translators and interpreters play in literature and in the movies, but not many have concentrated on the proofreader's role. And precisely on the borderline between reality and fiction, we find proofreader-detective Daniel Hernández, the central character of this research, since his creator, author Rodolfo Walsh (proofreader, translator, essayist, and eventually, political activist), takes him out of the real world and plants him in the fictional. The scope of this paper, therefore, is to analyze the novel A aventura das provas de prelo (The Adventure of the Print Proofs), by author Rodolfo Walsh, as a metaphor of a proofreader¿s job, his tracks, his writings, and his work as a translator. Along with a step-by-step description of an proofreader¿s routine, we present the trajectory of this research: a brief presentation of the life of Rodolfo Walsh, as well as his police literature; concepts related to editing, directly connected to an proofreader¿s daily routine; the theoretical basis for the analysis of an proofreader¿s job and his creation as a fictional character, accompanied by two other characters: proofreader Raimundo Silva and translator Gallus (viewed through a more conservative or traditional lens of translation and editing) and the interpreter, Mr. Kapasi (in whose traits one can perceive a post-structuralist viewpoint); the analysis of the central character created by Rodolfo Walsh, proofreader-detective Daniel Hernández. By way of conclusion, the position of the proofreader always presents itself as more or less finished, always subject to flaws, confirming that, at least in the case studied here, there is no perfect crime
Mestrado
Mestra em Linguística Aplicada

La production documentaire en contexte professionnel entraîne généralement un processus de révision dans lequel les documents doivent être relus avant validation et publication. Cette tâche importante fait face à de nouvelles difficultés avec le numérique. En effet, trois propriétés de l'écriture numérique sont problématiques : les documents évoluent très fréquemment et ne peuvent pas être relus entièrement à chaque version ; les interactions hypertextuelles rendent la tâche laborieuse, voire impossible ; la rééditorialisation documentaire augmente le nombre de formes documentaires à relire. En tant que technologie d'écriture numérique avancée, les chaînes éditoriales XML sont un cadre pertinent pour l'étude de la relecture de documents numériques. Partant du constat que les formes documentaires qu'elles proposent, à savoir les formes génératrices (sources XML modifiables via un éditeur WYSIWYM) et les formes publiées (documents issus de la transformation des sources XML), font défaut à la relecture, nous envisageons la conception de formes documentaires dédiées à cette activité selon deux approches : la linéarisation, qui consiste à restaurer une certaine linéarité matérielle des contenus pour faciliter leur relecture exhaustive ; et la tabulation, qui vise à paralléliser, afin de mieux les comparer, les différents contextes de rééditorialisation d'un document. Une partie des propositions faites dans ce mémoire a mené à la réalisation de prototypes ayant été expérimentés dans des situations d'usage des chaînes éditoriales Scenari en contexte pédagogique. Ces prototypes s'appuient sur des formes linéaires de relecture permettant notamment la comparaison de deux versions du document en se basant sur un algorithme de différentiel
Documentary production in a professional context often involves a revising process in which documents need to be proofread before validation and publication. This important task faces new challenges when dealing with digital documents. Indeed, three features of digital writing are problematic: documents evolve very frequently and cannot be proofread each time as a whole; interactions provided by hypertexts make the task laborious or even impossible; document repurposing increases the views of content to proofread. As an advanced digital writing technology, XML publishing chains are a relevant framework for studying proofreading of digital documents. Observing that the views of content proposed by publishing chains, namely the generative views (XML sources that can be modified through a WYSIWYM editor) and the published views (documents obtained by transformation of the XML sources), are not adapted for proofreading, we consider designing new views of content dedicated for this activity based on two approaches: linearization, which consists in restoring some material linearity among contents; and tabulation, which aims at parallelizing different repurposing contexts so that they can be better compared. Part of the contribution presented here has led to the development of prototypes that have been experimented in the use of Scenari publishing chains in a pedagogical context. These prototypes rely on linear proofreading views allowing in particular the comparison between two versions of the document based on a diff algorithm

Successivement veuve de Berthold Rembolt et de Claude Chevallon, Charlotte Guillard hérite en 1537 du plus ancien atelier typographique français, le Soleil d'Or, dont elle conduit l'activité durant près de vingt années. Sous sa direction, l'atelier parvient à accaparer deux marchés spécifiques : textes de droit savant et ouvrages des Pères de l'Église. La thèse vise à interroger les conditions de réalisation de ce programme éditorial. On y présente les modalités matérielles de production et de commercialisation des ouvrages. On met ainsi en évidence la forte implication de la parentèle de Charlotte Guillard à tous les niveaux de la chaîne éditoriale, et la coexistence de réseaux de collaborateurs qui, en dépit de motivations intellectuelles et idéologiques parfois divergentes, parviennent à faire œuvre commune. À travers une enquête mobilisant à la fois les sources archivistiques, l'analyse matérielle des ouvrages imprimés et la lecture des préfaces et épîtres liminaires, la monographie permet d'écrire une histoire concrète de l'activité intellectuelle qui tienne compte des conditions idéologiques, sociales et économiques de sa mise en œuvre
Widow of Berthold Rembolt first, then of Claude Chevallon, Charlotte Guillard became in 1537 heiress of France's oldest typography workshop. With Charlotte Guillard at its head, the Soleil d'Or managed to monopolise two specific markets, the law texts and the works of the Church Fathers. The purpose of our thesis is to investigate the practical conditions which made these publications possible. It will highlight the material arrangements of the production and selling of those books, and focus at the people who stayed at Charlotte Guillard's side. This will allow us to demonstrate the importance of her relatives at every step of the process, and to show the coexistence of various networks of collaborators who manage to work on a common basis despite, at times, opposite intellectual and ideological motivations. Calling on manuscript archives, physical bibliography, and an analysis of the prefaces and liminary epistles, this monograph allows us to write a holistic history of the intellectual endeavour, taking into account all the ideological, social and economic conditions entering in its construction

Mestrado em Estudos Editoriais
O presente relatório refere-se às atividades concretizadas durante o estágio em edição realizado no Departamento Editorial das Edições Almedina entre outubro de 2015 e março de 2016. Começar-se-á com uma visão geral da história da editora e do grupo editorial. De seguida, são descritas as particularidades da política editorial e da gestão dentro da Almedina. Na última secção são relatadas as várias tarefas concretizadas durante o estágio, divididas em quatro categorias principais.
The present report refers to the activities carried out during the publishing internship that took place at Edições Almedina’s Editorial Department from October 2015 to March 2016. To begin with, an overview of the history of the publishing house and the editorial group is provided. Secondly, the particularities of Almedina’s editorial policies and management are outlined. In the final and main section, an account is given concerning the diverse tasks accomplished during the internship, divided in four main categories.

碩士
國立臺灣大學
醫學檢驗暨生物技術學研究所
104
DNA polymerases play an important role in various cellular processes including DNA replication, DNA repair, genetic recombination, reverse transcription and maintenance of genomic stability. The proofreading function of DNA polymerase is critical in preventing mutations. The fidelity of DNA polymerases depends on its ability to incorporate the appropriate nucleotides into the growing DNA strand and removing mis-incorporated nucleotide at template-primer junction. Traditional methods for the detection of DNA polymerases proofreading ability include gel-based assays and radioisotopic labeling. However, these methods are generally time-consuming and labor-intensive and require necessary safety measures to control radioactive exposure. Luminescent-based methods for the detection of polymerase proofreading activity were also developed by using intrinsically fluorescent nucleotide analogs , such as 2-aminopurine (2-AP) which has been extensively employed to study polymerase proofreading activity in vitro. However, these methods suffer from low specificity due to the ability of 2-AP to form thermodynamically stable base pairs with cytosine and 2-AP formed non-canonical base pair is subject to selective discrimination by some DNA polymerases. The high resolution of MALDI-TOF (matrix-assisted laser desorption ionization mass spectrometry with time-of-flight) for DNA detection has been employed in the PinPoint assay in which the primer extension reactions with four unlabeled ddNPTs followed by an analysis by MALDI-TOF identify the polymorphism at a given locus. We suppose the concept of this approach can be modified to study DNA polymerase proofreading. We employed double strand synthetic oligonucleotides with all 12 possible terminal mismatches at the template-primer junction to mimic mis-incorporated nucleotide as proofreading substrates for E. coli DNA polymerase I (Pol I). From MALDI-TOF analysis, we demonstrate that all 12 mismatches can be actively corrected by Pol I. This method is faster and less laborious than non-mass spectrophotometric methods. The results in mass spectrum also provide additional information for reaction mechanism. The mass spectroscopy analysis of proofreading was also tested for other DNA polymerases such as T4 and T7 DNA polymerases with success.

碩士
國立臺北大學
古典文獻與民俗藝術研究所古典文獻組
100
Yu Yue (1821-1907) was born in Deqing County of Zhejiang Province, with a surname of Yinfu, nickname as Quyuan, and he was one of the most famous Masters on the Han School of classical philology in the end of Qing Dynasty. The discussions made by Yu Yue on the studies of various philosophies were concentrated on three books, including “Zhu Zi Ping Y(Reason and Principles of Various Philosophies)i”, “Quyuan Za Zuan”, and “Yu Lou Za Zuan”, and amongst them, there were proofreading of fifteen books from various philosophies, such as “Guan Zi”, “Yan Zi”, and “Lao Zi” in the “Zhu Zi Ping Yi”, which was his representational work on the studies of various virtuous men in his early stage. Below is the summary of the paper: The paper is divided into seven chapters, and the beginning and ending chapters are respectively as Introduction and Conclusions. In the second chapter, to talk about the flourishing background of the Han School in the end of Qing Dynasty, as well as to talk briefly on Yu Yue and his life in relation to academic field.In the third chapter, to state the motivation and process of how Yu Yue came to write “Zhu Zi Ping Yi( Reason and Principles of Various Philosophies)”. In the fourth chapter, by way of “reasons of error committing”, to make analysis on the situations of proofreading for “Zhu Zi Ping Yi”. In the fifth chapter, firstly to make discussion on three key points of how Yu Yue pursued his studies, afterwards, using “Four Methods of Collation” from Chen Yuan, to make investigations on the practices of emendation. In the sixth chapter, to make conclusions on the values and examinations of “Zhu Zi Ping Yi”.

碩士
國立臺灣大學
醫學檢驗暨生物技術學研究所
107
Insertion/deletion mutation is one of common point mutation during DNA replication . The occurrence of insertion/deletion mutation was thought due to repeated sequence being prone to slippage during DNA replication, so that the replicated product will gain or loss a few nucleotides.i.e. insertion/deletion errors. The insertion/deletion errors in replication can be corrected by the proofreading activity of the polymerase. Previous in vitro DNA synthesis study using template with simple repetitive sequences found that the proofreading efficiency of the polymerase is affected by the loop out position within repeat sequence, being the further the loop out occurs far away from the 3'' end, the less extend to proofread. It was suggested insertion/deletion loops embed upstream of the primer may not be proofread since the matched paring of primer template junction tend to be stabilized and can be successfully extended by DNA polymerase. In order to provide direct evidence for DNA polymerase producing insertion/deletion loop during slipped DNA replication as well as insertion/deletion loop proofreading efficiency of insertion/deletion loop, this study employed proofreading proficient and deficient Klenow polymerase to react with repeat sequences containing oligonucleotides. The resulting products were analyzed by MALDI-TOF MS assay. This study designed DNA substrates with different number (2 to 10) of repeating A．T pairs in primer-template junction. In reactions using proofreading deficient Klenow fragment (3''→5'' exo-) and imbalanced dNTPs pool containing only dATP , this study found that mis-incorporation of A occurred; and the higher number of A．T repeat the greater extend of mis-incorporation in 20 min. Kinetic analysis was performed to confirm above reactions. In comparison, no mis-incorporation were found in the reaction using proofreading proficient klenow as well as reactions using imbalanced dNTPs pool containing dCTP, dTTP, dGTP but not dATP. We then examined proofreading of the Klenow fragment (KF) with DNA containing single nucleotide insertion/deletion loop at different position. Followed by MALDI-TOF MS analysis, the proofreading at the insertion/deletion site is identified by the mass change of the primer. The result indicated that insertion/deletion error within 4 nucleotides upstream from primer terminus could be proofread effectively. We further tested the proofreading efficiency to insertion/deletion loop in slippage strand using DNA substrates containing insertion/deletion loop in nine to fourteen repeating A．T pairs sequence. In reactions using proofreading proficient klenow fragment in the presence of dATP, dCTP and dGTP, we found that the insertion/deletion error substrate with nine to fourteen repeating A．T pairs can be proofread.

碩士
國立臺灣大學
醫學檢驗暨生物技術學研究所
106
Insertion/deletion mismatch is a common point mutation during DNA replication, being associated to human disease such as cancer. Strand slippage known as repetitive sequence may lead to insertion/deletion mutation. DNA polymerase proofreads errors and the proofreading efficiency depends on the contents of repetitive sequence during DNA replication. It’s been known that insertion/deletion loops occurring upstream from the primer terminus may not be proofread by DNA polymerase since the matched paring of primer terminus will be recognized as correct substrate for extension by DNA polymerase. In order to understand the proofreading efficiency of insertion/deletion loop, we designed oligonucleotides containing insertion/deletion to perform proofreading assay. We examined proofreading of the Klenow fragment (KF) with DNA containing single nucleotide insertion/deletion loop at different position. Proofreading products were analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). The result indicated that insertion/deletion error within five nucleotides upstream from primer terminus could be proofread. The insertion/deletion error from six to nine nucleotides upstream of primer terminus escaped proofread. We further tested the proofreading efficiency to insertion/deletion loop in slippage strand using DNA substrates containing insertion/deletion loop in four, ten and fifteen repetitive sequence. Adding KF and ddNTPs, we found that insertion/deletion loop in both the four and ten repetitive sequences could be proofread by KF. However addition of two or three matched nucleotides at primer terminus of ten repetitive sequence, some insertion/deletion loops escaped proofreading. We also examined interference of insertion/deletion mismatch to polymerase activity. We used the KF(3''→5'' exo-), dATP, dGTP and dTTP for the assay. In the reaction lack of dCTP, the primer extension of KF (3''→5'' exo-) would be terminated at the position opposite to a guanine in template. We found KF(3''→5'' exo-) could not extend primer with a terminal mismatch. However, in the ten repetitive sequence containing an insertion/deletion mismatch could be extended by KF(3''→5'' exo-). We conclude that when insertion/deletion loop form at the position over four nucleotides upstream from the primer terminus in repetitive sequence could escape proofreading.

碩士
國立臺灣大學
醫學檢驗暨生物技術學研究所
103
DNA is subjected to deamination at a physiologically significant rate. Deoxyinosine in DNA can arise from deamination of deoxyadenosine, which can be spontaneous or promoted by exposure of DNA to ionizing radiation, UV light, or nitrous acid. Deoxyinosine in DNA can pair with cytosine during replication resulting in A:T to G:C transitions. Specific mechanisms for removal of deaminated bases have been evolved. Several bacterial, archaeal and eukaryotic organisms contain an evolutionary conserved enzyme, endonuclease V (endoV) that recognizes deaminated adenine in DNA. In E. coli, endoV incises DNA at the second phosphodiester bond 3'' to the dI lesion, leaving a 3'' OH and a 5'' P termini. Previously, we developed an in vitro assay to score the dI repair with cell extracts and purified proteins. We found that dI lesions were repaired by endoV mediated repair with high efficiency. In a purified system, endoV, DNA polymerase I (Pol I) and ligase are sufficient for the repair. We also demonstrated that 3'' to 5'' exonuclease (exo) activity of Pol I was very important for processing deoxyinosine lesions. To get better understanding how the dI lesion is processed by Pol I 3'' exo, in this study we designed dI-containing oligo-duplex and utilized proofreading in trans concept supplementing Pol I to proofreading deficient polymerase based reaction mixture in pyro-sequencing dI-containing procedure. We also utilized single nucleotide extension approach to map excision path during dI processing by mass spectrometry. We found Pol I could remove mismatched terminal base and activate sequencing reaction for the reaction otherwise to be inactive when containing proofreading deficient polymerase alone. The correction patch was mapped to the last mismatched base-pair, and no extra paired nucleotide was removed. This study might provide valuable information regarding the excision role of Pol I in endoV DNA repair pathway.

碩士
國立臺灣大學
醫學檢驗暨生物技術學研究所
99
DNA is genetic information for all organisms. It is important for maintaining fidelity during replicating. There are three steps that maintain the high fidelity. The first is structure switch for ensuring the right dNTP on the right place. The second is proofreading activity. DNA polymerase can remove the mismatch at the end of the primer then extension continually. The other is repair systems. Based on the above, the error frequency can decrease to about 10-10. There is no evidence showing that the second last mismatch can activate 3’ to 5’ exonuclease proofreading activity of DNA polymerase I. Besides, we known that after proofreading, Pol I can replicate DNA continually and form correct base pairs. For searching the detail of proofreading, we used the specificity of restriction enzyme to monitor whether the substrate was repaired or not. We designed different mismatch substrates. Besides, there was a nick downstream from the mismatch. In order to search the preliminary reaction of DNA polymerase I, we designed the reaction time within ten minutes and then monitored every two minutes. In addition, we compared the difference of proofreading efficiency and analysis of enzyme kinetic between different DNA substrates. Our results showed that DNA polymerase I could react with DNA substrates including T-T, T-G, and T-C. We also used linear substrate to test DNA polymerase I proofreading activity. It showed that this assay can ignore the extent of nick translation. These results suggested that the proofreading activity of DNA polymerase I can also be activated even when the mismatch located not on the end.

碩士
國立臺灣大學
醫學檢驗暨生物技術學研究所
105
Proofreading activity of DNA polymerase is very important factor in maintaining the high fidelity of genetic information during DNA replication. Traditional methods for detection of DNA polymerase proofreading ability include gel-based assays and radioisotopic labeling. However, these methods are generally time-consuming and labor-intensive and require necessary safety measures to avoid radioactive exposing. Fluorometric techniques have been used in kinetic analyses of proofreading activities. Intrinsically fluorescent nucleotide analogs such as 2-aminopurine (2-AP), which are incorporated into the product by DNA polymerase, have been extensively employed to study polymerase proofreading activity in vitro. However, these methods suffer from low specificity due to the ability of thermodynamically stable base pair formation between 2-AP and cytosine. Moreover, some DNA polymerases are able to selectively discriminate the 2-AP:T base pair from the normal A:T base pair. The high resolution of MALDI-TOF MS (matrix-assisted laser desorption ionization time of flight mass spectrometry) for DNA detection has been employed in the PinPoint assay in which the primer extension reactions with four unlabeled ddNTPs followed by MALDI-TOF analysis identify the polymorphism at a given locus. We propose the concept of this approach can be modified to study DNA polymerase proofreading. Herein, we designed a non-labeled and non-radioisotope simple method to measure proofreading. An oligonucleotide primer is annealed to a template DNA forming a mismatched site and is proofread by a DNA polymerase in the presence of all four dideoxyribonucleotide triphosphates or single deoxyribonucleotide triphosphates. The proofreading excision intermediates and re-synthesis products of single base extension are denatured, desalted and subjected to MALDI-TOF mass spectrometry. The proofreading at the mismatched site is identified by the mass change of the primer. We examined proofreading of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus can be proofread efficiently. Internal single mismatches can also be proofread at different efficiencies, with the best correction for mismatches located 2 to 4 nucleotides from the primer terminus. For mismatches located 5 nucleotides from the primer terminus, there was partial correction and extension. No significant proofreading was observed for mismatches located 6 to 9 nucleotides from the primer terminus. Moreover, this method can apply to deoxyinosine (dI) proofreading assay as well. For deamination product of deoxyinosine at penultimate position of primer 3’ ends, all of A-I, C-I, G-I, and T-I can be processed by Pol I proofreading exonuclease, with efficiency being T-I≥G-I>A-I>C-I. The correction efficiency might be determined by the base-pair stability at primer-template junctions. We also tested the reaction using NH4Cl instead of NaCl in reaction buffer. We found Klenow fragment retains the same activity and a much improved MS spectra with less background noise. A protocol using HCl for reaction termination could avoid tedious phenol extraction process and should be amendable for automation for mass screening. In parallel, the mass spectrometric method also has the capability to study both excision intermediates and proofreading products making this assay highly versatile.

碩士
銘傳大學
應用中國文學系碩士班
97
Duan Yu-cai spent a lot of spirits to focus on annotating “Shuo Wen Jie Zi”, and he accomplished the greatest works which are admired by scholars. After “Shuo Wen Jie Zi” published, it prevailed deep and widely. It turned into the scripture and the scholars had to read it if they researched “Shuo Wen.” The research of “Shuo Wen Duan Zhu” also became the mode; the special works of renewing and correcting were published in succession. The most valuable works which was corrected and renewed “Shuo Wen Duan Zhu” by Feng Gui-fen is “Shuo Wen Jie Zi Duan Zhu Kao Zheng”, it was helpful for “Shuo Wen Dan Zhu.” Duan Yu-cai based Xu Xuan’s works on annotating “Shuo Wne”, and adopted Xu Kai’s works as the first-hand data to correct it. Duan Yu-cai didn’t explain that which parts he annotated it according to Xu Xuan’s works, which parts that he corrected it follow, or he didn’t follow both of them but depended on ancient books. Feng Gui-fen started to collate because he/ she wanted to know what Duan Yu-cai depended on, Xu Xuan’s or Xu Kai’s, when he annotated “Shuo Wen”, and also want to have a grasp of the reference among “Shuo Wen Duan Zhu”, Xu Xuan and Xu Kai. This article went by the title of “The research of “Shuo Wen Jie Zi Duan Zhu Kao Zheng” which was collated by Feng Gui-fen”. There are six chapters: the first chapter is exordium, we explain the motive, the range, the methods of the research, and then, we introduce the glancing situation of the predecessors’ research. We introduce the background of Feng Gui-fen, and relate the content of “Shuo Wen Jie Zi Duan Zhu Kao Zheng” simply in the second chapter. We compare and contrast Xu Xuan’s works, Xu Kai’s works, the works which was annotated by Duan Yu-cai, and the works which was collated by Feng Gui-fen verbally, analyze them, and generalize them; we also inspect the works which was collated by Feng Gui-fen is correct or not in the third and fourth chapters. We discuss the result which was collated by Feng Gui-fen, and the parts which were needed to correct; we illustrate advantages and disadvantages about “Shuo Wen Jie Zi Duan Zhu Kao ZHeng” in the fifth chapter. The sixth chapter is conclusion, we cite the judgment which was propounded by the predecessors, and then we discourse upon the evidence which was based by Duan Yu-cai when he annotated the works. It exhibits the achievement and the effect of “ Shuo Wen Jie Zi Duan Zhu Kao Zheng”, and proves that “Shuo Wen Jie Zi Duan Zhu Kao Zheng” which was authored by Feng Gui-fen is important to the research of “Shuo Wen Duan Zhu.”

The thesis involves computer simulation and theoretical studies of phase transition in soft-condensed matter systems and theoretical understanding of enzyme kinetics along with kinetic proofreading of tRNA-aminoacylation in biological systems. Based on the system and phenomena of interest, the work has be classified into the following four major parts: I. Surface phenomena and surface energy of vapor-liquid interface. II. Condensation of vapor in two and three dimensions. III. Liquid-solid phase transition in polydisperse systems. IV. Enzyme catalysis and kinetic proofreading in biosynthesis. Above mentioned four parts have further been divided into thirteen chapters. In the following we provide a brief chapter-wise outline of the thesis. Part I deals with surface tension and interfacial properties of vapor-liquid interface for Lennard-Jones (LJ) fluid in both two and three dimensions. In Chapter 1, we provide a brief overview of vapor-liquid interface and existing theoretical and computer simulation studies of surface/line tension. In this chapter we also discuss about the existing experimental studies. In Chapter 2, we present computer simulation studies of surface tension in two dimensional Lennard-Jones system. The sensitivity of line tension on range (potential cut-off) of interparticle interaction is discussed in this chapter. We present Density Functional Theory (DFT) of line tension of vapor-liquid interface based on Weeks-Chandler-Anderson (WCA) and Barker-Hendersen (BH) perturbation techniques. We compare the DFT prediction with the computer simulation results. In general, WCA approach has been found to be successful for 3D system in predicting the surface tension. In 2D, however, it does not give good agreement either for phase diagram or for the line tension. In fact, BH also does not give accurate values of the coexistence parameters, however, it predicts better line tension compared to WCA. In Chapter 3 we present both theoretical and computer simulation studies of gas-liquid surface tension for three dimensional Lennard-Jones fluid. We perform non-equilibrium computer simulation study following Transition Matrix Monte Carlo (TMMC) method to obtain surface tension for various ranges of potential and introduce a new scaling relation of surface tension in order to capture both the temperature and interparticle interaction range dependence. The scaling shows excellent agreement with the simulation result and it can also predict the critical temperature with sufficient accuracy. The width of the gas-liquid interface is found to be insensitive to the range of the potential, whereas the density separation of the bulk vapor and liquid phases increases with increasing range of potential. Thus, the major contribution comes from the increasing density separation of the bulk vapor and liquid phases. Part II consists of four chapters, where we focus on the age old problem of nucleation, from the perspective of thermodynamics and kinetics. We account for the rich history of the problem in the introductory Chapter 4. In this chapter we describe various types and examples of the nucleation phenomena, and a brief account of the major theoretical approaches used so far. We begin with the most successful Classical Nucleation Theory (CNT), and then move on to more recent applications of Density Functional Theory (DFT) and other mean-field types of models. We present various experimental techniques used in the literature to obtain rate of nucleation. We conclude with a comparison between the experiments, theories and computational studies. In the next chapter (Chapter 5) we attempt to understand the mechanism of the gas-liquid nucleation in three dimension at large metastability from microscopic point of view. Here we study the nature of sequential growth of all liquid-like clusters (not just the largest cluster) at different degrees of metastability. Therefore, we have ordered the clusters according to their decreasing sizes and identified them in terms of kth largest cluster where, k = 1 denotes the largest cluster in the system, k = 2 represents the second largest and k = 3 is the third largest and so on. We have studied both the free energies and the trajectories of the liquid-like clusters in this extended set of order parameters. We further define Fkl(n) as the free energy of the kth largest cluster with size n. Classical nucleation theory provides an expression of unconditional free energy of a single cluster, F (n) (the free energy of formation of a cluster of size n), which is an intensive property of the system. The study of our conditional free energy surfaces, Fkl(n), reveals a more detailed, microscopic picture of the system’s cluster size distribution that is necessary to understand the kinetics of nucleation and growth at large metastability. The rate of nucleation shows a cross over at kinetic spinodal (the limit of metastability, ∆F1 l = 0). Below kinetic spinodal only one (largest) cluster crosses the critical size through activation whereas above this point more than one cluster grow simultaneously through barrierless diffusion. We present a theoretical analysis of the free energy of kth largest cluster based on order statistics. The theoretical predictions are in excellent agreement with computer simulation results for the range of supersaturation we studied. While the previous chapter focuses on relatively well-studied nucleation mechanism in 3dimensional (3D) LJ system at large metastability, in Chapter 6 we present our studies on the characteristics of the nucleation phenomena in two dimensional Lennard-Jones fluid for different ranges of interparticle interaction. Using various Monte Carlo (MC) methods, we calculate the free energy barrier of nucleation and bulk densities of equilibrium liquid and vapor phases, and also investigate the size and shape of the critical nuclei. We find an interesting interplay between the range of interaction potential and the extent of metastability. The free energy barrier of nucleation strongly depends on the range of interaction potential. The study is carried out at an intermediate level of supersaturation (away from the kinetic spinodal limit). A surprisingly large cutoff (rc � 7.0�, where � is the diameter of LJ particles) in the truncation of the LJ potential is required to obtain converged results. A lower cutoff leads to a substantial deviation in the values of the nucleation barrier, and characteristics of the critical cluster (with respect to full range of interaction). We observe that in 2D system CNT fails to provide a reliable estimate of the free energy barrier. While it is known to slightly overestimate the nucleation barrier in 3D, it underestimates the barrier by � 50% at the saturation ratio S =1.1 (defined as S = P/Pc, where Pc is the coexistence pressure) and at the reduced temperature T � =0.427 (defined as T � = kBT/�, where � is the depth of the potential well). The reason for the marked inadequacy of the CNT in 2D can be attributed to the non-circular nature of the critical clusters. Although the shape becomes increasingly circular and the clusters become more compact with increase in cutoff radius, an appreciable non-circular nature remains even for full potential (without truncation) to make the predictions of CNT inaccurate. In Chapter 7 we report the computer simulation study of nucleation in three dimensional LJ system. At a fixed supersaturation the free energy barrier of nucleation increases with increasing range of interparticle interaction. On increasing range of intermolecular interaction, the kinetic spinodal where the mechanism of nucleation changes from activated barrier crossing to barrierless diffusion, shifts towards the deep metastable region. Both the critical cluster size and pre-critical minimum in the free energy surface of kth largest cluster shift towards the smaller size at their respective kinetic spinodal as we increase the range of potential. We find only a weak non-trivial (other than supersaturation and surface tension) contribution to the free energy barrier of nucleation. Part III consists of two chapters and focuses on the liquid-solid phase transition of polydisperse fluid. In Chapter 8 we introduce polydisperse systems and their classification based on different identities. We describe the importance and abundance of polydisperse system in nature. The theoretical modeling of different polydisperse systems and their extent of applicability have also been presented. We have discussed about the various factors which control the phase diagram and various phenomena related to the structure and phase transition. In Chapter 9 we present computer simulation study on freezing/melting of Lennard-Jones (LJ) fluid at different polydispersities. The freezing/melting of polydisperse LJ fluids presents an interesting case study, because, as the polydispersity increases the energy-entropy balance becomes increasingly unfavorable for the solid to exist as a stable phase. The energy of the solid increases due to build up of strain energy because of increasing mismatch in size of the neighbors, while the entropy of the liquid increases. These two factors lead to the existence of a terminal polydispersity. We find beyond the terminal ploydispersity, δ. 0.11system remains in the disorder state even at very high pressure and low temperature. The terminal polydispersity obtained in the present study is close to the experimental value (δt. ≈ 12%). Interestingly, contrary to hard sphere polydisperse fluid, LJ fluid does not exhibit reentrant melting. The last part (Part IV) of the thesis consists of three chapters that deal with the enzyme catalysis and kinetic proofreading of tRNA-aminoacyl synthetases. In Chapter 10 we describe protein synthesis process in biological system and corresponding two processes: aminoacylation of tRNA and translation of amino acid in ribosome. Our interest is to understand the enzyme catalysis involved in aminoacylation of tRNA in the process of protein synthesis. We present the classification of 20 aminoacyl-tRNA synthetases into two classes based on their structure and mode of binding to ATP and tRNA. We discuss all the steps involved in whole tRNA-aminoacylation process. Then we introduce kinetic proofreading during aminoacylation reaction. In Chapter 11 we theoretically analyze the single turn over and steady state reaction mechanism of two classes of aminoacyl-tRNA synthetases. Class I enzymes not only differ in their structure but they also differ with respect to the pre-steady kinetics compared to class II enzymes. We find that the strong binding of product to class I enzymes causes the product release step to be rate limiting step leading to the burst of product formation in pre-steady reaction. On the other hand class II enzymes do not show any burst kinetics. The present study based on time dependent probability statistics is successful in explaining all the experimental results quantitatively. In Chapter 12 we present an augmented kinetic scheme and then employ methods of time dependent probability statistics to understand the mechanism of kinetic proofreading of isoleucyl-tRNA synthetase (IRS) which belongs to class I. We investigate that the enhanced hydrolysis of wrong substrate (Val) enables IRS to discriminate the correct substrate (Ile) and wrong substrate (Val) efficiently. It has been observed that an extra CP1 editing domain serves as an activating domain towards enhanced hydrolysis of Val. The present study is able to explain most of the existing experimental observations. In the concluding note, Chapter 13 lists a few relevant problems that may prove worthwhile to be addressed in future. In the Appendices, we present two of the techniques used in our present computer simulation and theoretical studies. Appendix A describes Grand Canonical Transition Matrix Monte Carlo (GC-TMMC) method which is employed in computer simulation studies of nucleation and surface tension. In Appendix B we present the probabilistic method of waiting time distribution computation used in enzyme catalysis and kinetic proofreading.

The thesis involves computer simulation and theoretical studies of phase transition in soft-condensed matter systems and theoretical understanding of enzyme kinetics along with kinetic proofreading of tRNA-aminoacylation in biological systems. Based on the system and phenomena of interest, the work has be classified into the following four major parts: I. Surface phenomena and surface energy of vapor-liquid interface. II. Condensation of vapor in two and three dimensions. III. Liquid-solid phase transition in polydisperse systems. IV. Enzyme catalysis and kinetic proofreading in biosynthesis. Above mentioned four parts have further been divided into thirteen chapters. In the following we provide a brief chapter-wise outline of the thesis. Part I deals with surface tension and interfacial properties of vapor-liquid interface for Lennard-Jones (LJ) fluid in both two and three dimensions. In Chapter 1, we provide a brief overview of vapor-liquid interface and existing theoretical and computer simulation studies of surface/line tension. In this chapter we also discuss about the existing experimental studies. In Chapter 2, we present computer simulation studies of surface tension in two dimensional Lennard-Jones system. The sensitivity of line tension on range (potential cut-off) of interparticle interaction is discussed in this chapter. We present Density Functional Theory (DFT) of line tension of vapor-liquid interface based on Weeks-Chandler-Anderson (WCA) and Barker-Hendersen (BH) perturbation techniques. We compare the DFT prediction with the computer simulation results. In general, WCA approach has been found to be successful for 3D system in predicting the surface tension. In 2D, however, it does not give good agreement either for phase diagram or for the line tension. In fact, BH also does not give accurate values of the coexistence parameters, however, it predicts better line tension compared to WCA. In Chapter 3 we present both theoretical and computer simulation studies of gas-liquid surface tension for three dimensional Lennard-Jones fluid. We perform non-equilibrium computer simulation study following Transition Matrix Monte Carlo (TMMC) method to obtain surface tension for various ranges of potential and introduce a new scaling relation of surface tension in order to capture both the temperature and interparticle interaction range dependence. The scaling shows excellent agreement with the simulation result and it can also predict the critical temperature with sufficient accuracy. The width of the gas-liquid interface is found to be insensitive to the range of the potential, whereas the density separation of the bulk vapor and liquid phases increases with increasing range of potential. Thus, the major contribution comes from the increasing density separation of the bulk vapor and liquid phases. Part II consists of four chapters, where we focus on the age old problem of nucleation, from the perspective of thermodynamics and kinetics. We account for the rich history of the problem in the introductory Chapter 4. In this chapter we describe various types and examples of the nucleation phenomena, and a brief account of the major theoretical approaches used so far. We begin with the most successful Classical Nucleation Theory (CNT), and then move on to more recent applications of Density Functional Theory (DFT) and other mean-field types of models. We present various experimental techniques used in the literature to obtain rate of nucleation. We conclude with a comparison between the experiments, theories and computational studies. In the next chapter (Chapter 5) we attempt to understand the mechanism of the gas-liquid nucleation in three dimension at large metastability from microscopic point of view. Here we study the nature of sequential growth of all liquid-like clusters (not just the largest cluster) at different degrees of metastability. Therefore, we have ordered the clusters according to their decreasing sizes and identified them in terms of kth largest cluster where, k = 1 denotes the largest cluster in the system, k = 2 represents the second largest and k = 3 is the third largest and so on. We have studied both the free energies and the trajectories of the liquid-like clusters in this extended set of order parameters. We further define Fkl(n) as the free energy of the kth largest cluster with size n. Classical nucleation theory provides an expression of unconditional free energy of a single cluster, F (n) (the free energy of formation of a cluster of size n), which is an intensive property of the system. The study of our conditional free energy surfaces, Fkl(n), reveals a more detailed, microscopic picture of the system’s cluster size distribution that is necessary to understand the kinetics of nucleation and growth at large metastability. The rate of nucleation shows a cross over at kinetic spinodal (the limit of metastability, ∆F1 l = 0). Below kinetic spinodal only one (largest) cluster crosses the critical size through activation whereas above this point more than one cluster grow simultaneously through barrierless diffusion. We present a theoretical analysis of the free energy of kth largest cluster based on order statistics. The theoretical predictions are in excellent agreement with computer simulation results for the range of supersaturation we studied. While the previous chapter focuses on relatively well-studied nucleation mechanism in 3dimensional (3D) LJ system at large metastability, in Chapter 6 we present our studies on the characteristics of the nucleation phenomena in two dimensional Lennard-Jones fluid for different ranges of interparticle interaction. Using various Monte Carlo (MC) methods, we calculate the free energy barrier of nucleation and bulk densities of equilibrium liquid and vapor phases, and also investigate the size and shape of the critical nuclei. We find an interesting interplay between the range of interaction potential and the extent of metastability. The free energy barrier of nucleation strongly depends on the range of interaction potential. The study is carried out at an intermediate level of supersaturation (away from the kinetic spinodal limit). A surprisingly large cutoff (rc � 7.0�, where � is the diameter of LJ particles) in the truncation of the LJ potential is required to obtain converged results. A lower cutoff leads to a substantial deviation in the values of the nucleation barrier, and characteristics of the critical cluster (with respect to full range of interaction). We observe that in 2D system CNT fails to provide a reliable estimate of the free energy barrier. While it is known to slightly overestimate the nucleation barrier in 3D, it underestimates the barrier by � 50% at the saturation ratio S =1.1 (defined as S = P/Pc, where Pc is the coexistence pressure) and at the reduced temperature T � =0.427 (defined as T � = kBT/�, where � is the depth of the potential well). The reason for the marked inadequacy of the CNT in 2D can be attributed to the non-circular nature of the critical clusters. Although the shape becomes increasingly circular and the clusters become more compact with increase in cutoff radius, an appreciable non-circular nature remains even for full potential (without truncation) to make the predictions of CNT inaccurate. In Chapter 7 we report the computer simulation study of nucleation in three dimensional LJ system. At a fixed supersaturation the free energy barrier of nucleation increases with increasing range of interparticle interaction. On increasing range of intermolecular interaction, the kinetic spinodal where the mechanism of nucleation changes from activated barrier crossing to barrierless diffusion, shifts towards the deep metastable region. Both the critical cluster size and pre-critical minimum in the free energy surface of kth largest cluster shift towards the smaller size at their respective kinetic spinodal as we increase the range of potential. We find only a weak non-trivial (other than supersaturation and surface tension) contribution to the free energy barrier of nucleation. Part III consists of two chapters and focuses on the liquid-solid phase transition of polydisperse fluid. In Chapter 8 we introduce polydisperse systems and their classification based on different identities. We describe the importance and abundance of polydisperse system in nature. The theoretical modeling of different polydisperse systems and their extent of applicability have also been presented. We have discussed about the various factors which control the phase diagram and various phenomena related to the structure and phase transition. In Chapter 9 we present computer simulation study on freezing/melting of Lennard-Jones (LJ) fluid at different polydispersities. The freezing/melting of polydisperse LJ fluids presents an interesting case study, because, as the polydispersity increases the energy-entropy balance becomes increasingly unfavorable for the solid to exist as a stable phase. The energy of the solid increases due to build up of strain energy because of increasing mismatch in size of the neighbors, while the entropy of the liquid increases. These two factors lead to the existence of a terminal polydispersity. We find beyond the terminal ploydispersity, δ. 0.11system remains in the disorder state even at very high pressure and low temperature. The terminal polydispersity obtained in the present study is close to the experimental value (δt. ≈ 12%). Interestingly, contrary to hard sphere polydisperse fluid, LJ fluid does not exhibit reentrant melting. The last part (Part IV) of the thesis consists of three chapters that deal with the enzyme catalysis and kinetic proofreading of tRNA-aminoacyl synthetases. In Chapter 10 we describe protein synthesis process in biological system and corresponding two processes: aminoacylation of tRNA and translation of amino acid in ribosome. Our interest is to understand the enzyme catalysis involved in aminoacylation of tRNA in the process of protein synthesis. We present the classification of 20 aminoacyl-tRNA synthetases into two classes based on their structure and mode of binding to ATP and tRNA. We discuss all the steps involved in whole tRNA-aminoacylation process. Then we introduce kinetic proofreading during aminoacylation reaction. In Chapter 11 we theoretically analyze the single turn over and steady state reaction mechanism of two classes of aminoacyl-tRNA synthetases. Class I enzymes not only differ in their structure but they also differ with respect to the pre-steady kinetics compared to class II enzymes. We find that the strong binding of product to class I enzymes causes the product release step to be rate limiting step leading to the burst of product formation in pre-steady reaction. On the other hand class II enzymes do not show any burst kinetics. The present study based on time dependent probability statistics is successful in explaining all the experimental results quantitatively. In Chapter 12 we present an augmented kinetic scheme and then employ methods of time dependent probability statistics to understand the mechanism of kinetic proofreading of isoleucyl-tRNA synthetase (IRS) which belongs to class I. We investigate that the enhanced hydrolysis of wrong substrate (Val) enables IRS to discriminate the correct substrate (Ile) and wrong substrate (Val) efficiently. It has been observed that an extra CP1 editing domain serves as an activating domain towards enhanced hydrolysis of Val. The present study is able to explain most of the existing experimental observations. In the concluding note, Chapter 13 lists a few relevant problems that may prove worthwhile to be addressed in future. In the Appendices, we present two of the techniques used in our present computer simulation and theoretical studies. Appendix A describes Grand Canonical Transition Matrix Monte Carlo (GC-TMMC) method which is employed in computer simulation studies of nucleation and surface tension. In Appendix B we present the probabilistic method of waiting time distribution computation used in enzyme catalysis and kinetic proofreading.

碩士
國立臺灣大學
醫學檢驗暨生物技術學研究所
102
The highly mutagenic lesion, deoxyinosine (dI) can be produced in DNA spontaneously, and the process is enhanced by exposing DNA to ionizing radiation, UV light or nitrous ion. In Escherichia coli, deoxyinosine is excised through repair pathway that is initiated by Endonuclease V. According to our previous studies, the purified protein system containing Endonuclease V, DNA polymerase I, and E. coli DNA ligase was sufficient to reconstitute the repair of G-dI, T-dI and A-dI substrate in vitro. In order to evaluate the biological significance of endonuclease V and DNA polymerase I in processing nitrous acid-induced deaminated lesions, we employed nitrous mutagenesis assay to E. coli nfi (endonuclease V deficient) and polAexo (DNA Pol I 3’exonuclease deficient) strains to further understand the importance of these gene products in mutation prevention of nitrous enhanced deamination. Cells were treated with NaNO2 and measured the frequency of streptomycin-resistant mutations in the rpsL gene. The nfi mutant demonstrated 14 times higher mutation frequency than its isogenic wild type. The average frequency of mutants was 57-fold higher in the polAexo strain (KA796 D424A) than that of the wild type strain under nitrous stress. The mutants from the nitrite treatment were collected, and the rpsL gene was sequenced. The most common point mutation after treatment was an A:T→ C:G transvertion. This type of mutation accounted for 97% of total mutation in nfi mutant strains and 60% of total mutation in polAexo mutant strains. The results of mutagenesis assay demonstrated that 3’-5’ exonuclease of Pol I is the integral part of Endo V pathway to protect E. coli against nitrous acid-induced mutagenesis.

碩士
國立臺灣大學
醫學檢驗暨生物技術學研究所
101
DNA carries genetic information in all living organisms. During DNA replication, it is important to maintain genomic integrity. Three mechanisms are involved in maintaining the high fidelity of genome. The first is base selection during replication; the second is the proofreading activities of DNA polymerases, which can remove the mis-incorporated nucleotide at the primer-template junction. The third is DNA mismatch repair systems. According to previous studies, it is known that terminal mismatch and consecutive two mismatches at the 3’ end of the primer can be edited by DNA polymerase I (pol I). Our previous study showed that the proofreading activity of pol I could edit deoxyinosine-containing heteroduplex DNA following the process of endonuclease V which create a strand breakage at the second phosphodiester bond 3’ to the deoxyinosine (DNA Repair 9: 1073-9). To figure out how it works, we constructed twelve heteroduplex DNAs containing single mismatch at the penultimate site of the primer and analyzed the proofreading activity. The results showed that all of the twelve heteroduplex DNAs can be edited by proofreading activity of pol I. However, the overall capacity of pol I proofreading exonuclease toward mismatches embedded upstream of the primer is still not fully understood. Therefore, we designed a series of mismatch substrates containing a strand break at 0 to 7 nucleotides 3’ to the mismatch, which mimic mismatches embedded in primer template junctions, to study proofreading activity of pol I. Mismatches were designed to interrupt a restriction endonuclease recognition sequence so that proofreading activity can be scored by the restriction endonuclease assay. We also placed several restriction endonucleases sequences at 3’ side to the mismatches so that in the same sequence content a series of substrates containing different strand breaks can be prepared. The two mismatches, A-A and T-T, were employed for the proofreading assay. The assay condition was in the presence of 0.1 mM each of the four dNTPs to mimic in vivo replication condition. Kinetic reactions of different substrates were assayed in a 6-min reaction span to obtain the initial rates for the comparison of substrate specificity for pol I proofreading. Our results showed that pol I can actively edit mismatches at -1, -2, -3, and -4 positions of the primer terminus. The correction levels were pol I concentration dependent, and also demonstrated certain degree of substrate specificity. Linearized heteroduplex substrate could also be efficiently proofread by pol I which ruled out the possible interference by non-specific nick translation. The results of this study is consistent with previous X-ray crystallography study that at least 4 nucleotide from 3’ end of the primer were required for transfer from polymerization site to exonuclease active site for editing. In addition, we also found mismatches located more than 5 nucleotides from 3’ end were very difficult to remove by proofreading proficient DNA polymerase. The observation could provide a good guidance for designing oligonucleotides for gapped duplex site-directed mutagenesis.

Thesis (M.Sc.)--York University, 2004. Graduate Programme in Biology.
Typescript. Includes bibliographical references (leaves 53-63). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url%5Fver=Z39.88-2004&res%5Fdat=xri:pqdiss &rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR11766

碩士
國立臺灣大學
醫學檢驗暨生物技術學研究所
100
DNA carries genetic information in all organisms. During DNA replication, it is important to maintain genomic fidelity. Three correlating events operate in maintaining the high fidelity of genome：The first is base selection. The second is the proofreading activities of DNA polymerases, which can remove the last mismatched DNA at the primer-template junction. The third is DNA repair systems. To date, there is no evidence showing that the mismatched DNA at penultimate site of the primer can be edited by DNA polymerase I. Our previous study showed that the proofreading activity of DNA polymerase I could edit deoxyinosine-containing heteroduplex DNA after processing by endonuclease V which created a strand breakage at the second phosphodiester bond 3’ to the deoxyinosine. To figure out how it works, we constructed twelve heteroduplex DNA containing single mismatch at penultimate site of the primer and analysed the proofreading activity. The involvement of nick translation activity of DNA polymerase I was eliminated. Our results showed that all the twelve heteroduplex DNA can be edited by proofreading activity of DNA polymerase I and there were no general roles for trend of ionic strength in our proofreading assay. We identified purine．purine, the most frequently misinserted mismatches, could be edited well. According to the structure analysis, two large purine bases cause considerable strand strain that may lead to proofreading efficiency elevated. However, purine．pyrimidine mismatches were poorly edited probably due to these structures were similar to the correct Watson-Crick base pairs with minor distortion but the C-A could be edited well. Furthermore, the mismatch repair system had high efficiency to repair purine．pyrimidine mismatches can compensate to poorly proofreading activity. On the other hand, the large purine bases have increased stacking ability and the common N7 groups may be preferred to bind with the amino acid residue of exonuclease site. We found that the misbase on the primer strand had the more efficiency of proofreading activity but the T-G was not. Besides, we identified that gap-form substrate had better proofreading activity than nick-form. After removing the wrong base, DNA polymerase I will undergo polymerization. As a result of DNA carrying out polymerization without 5’ to 3’exonuclease activity with the gap-form substrate, it has higher proofreading efficiency. Conclusively, we identified the proofreading activity of DNA polymerase I can edit DNA mismatches at the penultimate site of the primer. In addition to our previous study, the DNA polymerase I actually could edit deoxyinosine-containing heteroduplex DNA which containing a strand breakage at the second phosphodiester bond 3’ to the deoxyinosine.

O presente relatório pretende apresentar as tarefas desenvolvidas no estágio realizado no departamento de revisão da Editorial Presença, entre setembro e dezembro de 2017, para obtenção do grau de Mestre em Edição de Texto, na Faculdade de Ciências Sociais e Humanas da Universidade Nova de Lisboa. As tarefas no estágio desenvolvidas incidiram, sobretudo, na área de preparação, revisão e tradução de texto, sendo apresentados exemplos das várias etapas do processo editorial e do estágio em si. Esta apresentação das atividades desenvolvidas estará acompanhada por uma breve reflexão teórica acerca do trabalho realizado e de todo o processo de revisão. Pretende-se ainda apresentar os diferentes âmbitos e desafios da revisão hoje no mercado editorial, assim como descrever algumas dificuldades e problemas desta área em estudo.
This report aim to present the tasks developed during the internship held at the publishing house Editorial Presença, in the Proofreading Department, between September until December 2017, as part of the degree of Master in Editing and Publishing at the Nova School of Social Sciences and Humanities of the Universidade Nova de Lisboa. The main focus of the tasks developed in the internship was in the preparation, proofreading and translating text. In this report it is described some examples of the multiple steps of the editorial process and of the internship itself. This description of the activities developed is followed by a short critical reflection of the work done and of all the editing and proofreading process.

O presente relatório é o produto do estágio curricular realizado na editora Ponto de Fuga, entre setembro de 2018 e fevereiro de 2019. Neste vou apresentar o testemunho da minha experiência, com todas a dificuldades inerentes que fui descobrindo, e os resultados do meu trabalho e contribuição na áre a da revisão de texto, no desafio da tradução e também uma experiência de participação em feira. Apresento também algumas ponderações sobre o trabalho desta pequena editora e da sua afirmação no mercado editorial português.
With this report I outline my experience as an intern at “Ponto de Fuga” publishing house. Giving an acount of the activities I took part in when proofreading several fictional texts, an experience in traduction and my participation in a weekend fair. As this is the testimony of all the work I did as a culmination in my Masters in Edição de Texto , it will inevitably deal with some problems I had to overcome during this period. I also present some thoughts on the particular characteristics of “Ponto de Fuga” as a publishing house and it's growth in Portugal’s publishing market.

O presente relatório tem como objectivo apresentar o trabalho desenvolvido durante o estágio realizado na editora Relógio d’Água, no âmbito do Mestrado de Edição de Texto da Faculdade de Ciências Sociais e Humanas da Universidade Nova de Lisboa. Com duração de 400 horas, o estágio compreendeu o período entre Setembro e Dezembro de 2019, englobando actividades de revisão textual e de tradução, bem como a observância de demais funções editoriais.
The purpose of the following report is to expound the tasks and responsabilitites developed during the internship at the publishing house Relógio d’Água. This internship was integrated in the master’s degree of Edição de Texto, of Faculdade de Ciências Sociais e Humanas of Universidade Nova de Lisboa. With a total of 400 hours, the internship took place between September and December of 2019, and consisted in proofreading and translation, as long as other editorial duties

O presente relatório tem como objetivo descrever o estágio curricular realizado na Relógio d’Água Editores, que se concentrou principalmente na área de revisão de texto. São apresentados exemplos dos diferentes tipos de revisões efetuadas, que problemáticas apresentaram, outras pequenas tarefas realizadas e como se organiza o trabalho nesta pequena editora. São também oferecidas algumas reflexões sobre o trabalho e qualidade do mesmo, bem como os desafios que enfrenta enquanto editora independente no mercado editorial português actual.
This report’s goal is to describe an internship at Relógio d’Água Editores which took place during the summer of 2015, and whose primary focus was to develop proofreading skills. Different types of books that were proofread, and the problems that arose with them, are presented. Other small tasks that were undertaken during this period are also described, as is the method this particular publishing house employs to edit and publish their works. Some notes and thoughts on this publisher’s work and its quality, as well as the challenges it faces as an independent publisher in today’s Portuguese publishing world.

O presente relatório tem o objetivo de descrever as atividades exercidas durante o estágio curricular na Alêtheia Editores, entre setembro de 2016 e fevereiro de 2017, como parte da componente não-letiva do mestrado em Edição de Texto. Na primeira parte do relatório descrevo de forma breve a editora, a sua linha editorial e os elementos constituintes do grupo Alêtheia Editores. Na segunda parte enumero sumariamente as atividades exercidas ao longo dos cinco meses. Na terceira, e última parte descrevo com mais atenção e rigor as duas atividades que exerci durante mais tempo no estágio: a preparação do texto antes da paginação e a revisão de texto.
The main objective of the present work is to describe the activities that I did during the internship at the publishing house Alêtheia Editores, between September 2016 until February 2017, as part of the master degree in Text Editing. In the first chapter of this internship report I try to describe shortly the publishing house, his editorial views, and the main elements of his editorial group. In the second chapter I try to list summarily all the activities over the five months internship. In the third, and last chapter I try to describe with more attention the two activities that I did during the all internship: text prepping before the pagination of the book, and proofreading.

O presente relatório é produto do estágio curricular realizado na editora Ponto de Fuga entre julho de 2019 e janeiro de 2020. Nele é feita uma exposição das circunstâncias e problemáticas associadas ao estágio, bem como das dinâmicas adotadas para as ultrapassar. São também resumidos os resultados da colaboração da estagiária nas áreas de revisão de texto, apoio à tradução e revisão de tradução. Finalmente, são apresentadas algumas ponderações acerca dos resultados obtidos e da influência do curso de mestrado em Edição de Texto na capacitação da estagiária para atingir esses mesmos resultados
This report is the product of the curricular internship carried out at the Ponto de Fuga publishing house between July 2019 and January 2020. In it there’s a presentation of the circumstances and problems associated with the internship and the dynamics adopted to overcome them. There’s also a summary of the intern’s collaboration in proofreading, translation support and translation revision. Lastly, there’s a presentation of some considerations about the results achieved and the influence of the master’s programme for the qualification of the intern in order to obtain said results.

O presente relatório visa descrever o estágio curricular realizado na Alêtheia Editores, de 1 de agosto a 20 de outubro do presente ano letivo, para obtenção do grau de Mestre em Edição de Texto na Faculdade de Ciências Sociais e Humanas, após a conclusão da componente letiva. O estágio incidiu sobretudo na área de preparação e revisão de texto. Como tal, são apresentados exemplos dos diferentes tipos de trabalho editorial desenvolvidos, além de várias problemáticas que foram encontradas. Descrevem-se as tarefas realizadas durante o período do estágio, além da organização da produção, seguindo-se uma análise crítica que reflete sobre a qualidade dos projetos desenvolvidos e sobre a eficácia das metodologias adotadas, quer em relação à instituição de acolhimento, quer em relação ao mercado editorial português
The present report aims to describe the internship held at Alêtheia Editores, from the 1st of August to the 20th of October of this academic year, for the degree of Master of Text Editing at the Faculty of Social and Human Sciences, after the conclusion of the study program’s course units. The internship focused mainly in the area of text’s preparation and proofreading. As such, there are given some examples of different types of editorial work developed, in addition to several challenges that were found. The tasks carried out during the internship are described as well as the organization of production, followed by a critical analysis that reflects on the quality of the projects executed and on the efficacy of the adopted methodologies, whether in the host institution or in the Portuguese publishing market.