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Leung, Shui-on. "Transcription and translation of the human #theta#-globin gene." Thesis, University of Oxford, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.291083.
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Sinclair, Mark. "Speech segmentation and speaker diarisation for transcription and translation." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/20970.
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This dissertation outlines work related to Speech Segmentation – segmenting an audio recording into regions of speech and non-speech, and Speaker Diarization – further segmenting those regions into those pertaining to homogeneous speakers. Knowing not only what was said but also who said it and when, has many useful applications. As well as providing a richer level of transcription for speech, we will show how such knowledge can improve Automatic Speech Recognition (ASR) system performance and can also benefit downstream Natural Language Processing (NLP) tasks such as machine translation and punctuation restoration. While segmentation and diarization may appear to be relatively simple tasks to describe, in practise we find that they are very challenging and are, in general, ill-defined problems. Therefore, we first provide a formalisation of each of the problems as the sub-division of speech within acoustic space and time. Here, we see that the task can become very difficult when we want to partition this domain into our target classes of speakers, whilst avoiding other classes that reside in the same space, such as phonemes. We present a theoretical framework for describing and discussing the tasks as well as introducing existing state-of-the-art methods and research. Current Speaker Diarization systems are notoriously sensitive to hyper-parameters and lack robustness across datasets. Therefore, we present a method which uses a series of oracle experiments to expose the limitations of current systems and to which system components these limitations can be attributed. We also demonstrate how Diarization Error Rate (DER), the dominant error metric in the literature, is not a comprehensive or reliable indicator of overall performance or of error propagation to subsequent downstream tasks. These results inform our subsequent research. We find that, as a precursor to Speaker Diarization, the task of Speech Segmentation is a crucial first step in the system chain. Current methods typically do not account for the inherent structure of spoken discourse. As such, we explored a novel method which exploits an utterance-duration prior in order to better model the segment distribution of speech. We show how this method improves not only segmentation, but also the performance of subsequent speech recognition, machine translation and speaker diarization systems. Typical ASR transcriptions do not include punctuation and the task of enriching transcriptions with this information is known as ‘punctuation restoration’. The benefit is not only improved readability but also better compatibility with NLP systems that expect sentence-like units such as in conventional machine translation. We show how segmentation and diarization are related tasks that are able to contribute acoustic information that complements existing linguistically-based punctuation approaches. There is a growing demand for speech technology applications in the broadcast media domain. This domain presents many new challenges including diverse noise and recording conditions. We show that the capacity of existing GMM-HMM based speech segmentation systems is limited for such scenarios and present a Deep Neural Network (DNN) based method which offers a more robust speech segmentation method resulting in improved speech recognition performance for a television broadcast dataset. Ultimately, we are able to show that the speech segmentation is an inherently ill-defined problem for which the solution is highly dependent on the downstream task that it is intended for.
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Mudd, E. A. "Transcription and translation from a symbiotic plasmid of Rhizobium leguminosarum." Thesis, University of East Anglia, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.355533.
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Richardson, Nathan Joseph. "Transcription and Translation of the 1658 Jesuit Annual Letter, Vietnam." BYU ScholarsArchive, 2018. https://scholarsarchive.byu.edu/etd/6870.
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Transcription and Translation of the 1658 Jesuit Annual Letter, VietnamNathan Joseph RichardsonDepartment of Spanish and Portuguese, BYUMaster of ArtsThis project provides a translation and two transcriptions (semi-diplomatic and normalized) of the 1658 Jesuit Annua letter sent from the Tonkin kingdom (now Vietnam) to Jesuit authorities back in Portugal. Specifically, the letter, which is housed in the archives of the worldwide Society of Jesus in Rome (folder 89, Japonica Sinica series, fols 286-290v), reports the progress of the Jesuit mission in that kingdom. However, it also contains a fascinating account of contemporary political and other events there. The purpose of this project is to make this letter accessible to a variety of readers. The English translation makes the letter's contents available to an English readership interested in Portugal's expansion in Asia, especially the activities of Jesuit missionaries in Vietnam; the normalized transcription is aimed at those with similar interests who read Portuguese; and the semi-diplomatic transcription, together with a facsimile of the original manuscript, is intended for those who study the history of the Portuguese language and are particularly concerned with the edition of early modern texts.
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Jaafari-Dehaghi, Mahmoud. "Dādestān ī Dēnīg, chapters 1-35 : transcription, translation and commentary." Thesis, SOAS, University of London, 1997. http://eprints.soas.ac.uk/29121/.
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The Dadestan i Denig 'Religious Judgments' consists of ninety-one answers given by ManuScihr i Juwanjaman, the Zoroastrian high priest in the ninth century A.C., to the questions put to him by Mihr Xwarsed i Adurmahan and other members of his community. The first part of the text, which contains forty questions and answers, deals chiefly with the following matters: Why is the righteous man important and what is the purpose of the creation of the perfect man? Why do the good suffer more than the evil in this world? The sin of those who leave the Mazda-worshipping religion for the evil religion; meritorious deeds; the vision of Ohrmazd and Ahreman by the departed soul. How does the soul depart from the body and where do the righteous and wicked souls go? Ceremonies in honour of Sros to be performed during the SedoS (i.e. three days after death). The nature of heaven and hell; individual eschatology; the renovation of the universe; the sacred cord and the sacred shirt. The text is one of the most important surviving books of the ninth century and as a whole is a valuable source for the history of the Zoroastrian community under Islamic influence. Because the subjects discussed in the text cover a wide range of Zoroastrian religious doctrine, mythology and traditions, it shows the extensive knowledge of its author in different areas of study. A characteristic feature of this text is the difficult style of writing. Manuscihr's style is sophisticated but sometimes ambiguous and obscure, so his writing demands to be read carefully. My edition of the first part of the book (chapters 1-35) is based on the text edited by the late B.T. Anklesaria in which all the surviving manuscripts have been carefully examined. I have given a transcription of the text with critical apparatus, following the method of transcription proposed by Prof. D.N. MacKenzie. I have also provided a translation and a very brief commentary.
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Stevenson-Jones, Flint Ruben. "Control of gene expression through coupling of transcription and translation." Thesis, University of Newcastle upon Tyne, 2017. http://hdl.handle.net/10443/3848.
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Transcription and translation form the basis of gene expression in all cells. In prokaryotes they are linked both spatially and temporally as the ribosomes begin translation of the RNA before the RNAP has finished transcribing the entire region, a process known as coupling. Interplay between the two machineries is highly complex and plays an important role in gene expression. To date, most of the studies into transcription-translation coupling have been carried out in vivo, and have focused on the indirect interactions such as attenuation. Due to the many accessory factors for both transcription and translation present within the cell, there is currently no known technique to study direct interactions between the RNAP and the ribosome. Recently, an in vitro transcription-translation system was developed in our lab that is formed from only the pure components required for transcription and translation. This allows the stepwise control of the RNAP and the ribosome. The aim of this study was to determine how close the RNAP and the ribosome can become on the same nascent RNA. The coupled in vitro system was redesigned and optimised to measure the distance between the actively transcribing RNAP and the ribosome translating the same transcript. We show that the ribosome can approach the RNAP as close as 26 nts between the A-site of the ribosome and the active site of the RNAP. This distance is far shorter than was previously thought and reveals a very close contact between the two machineries.
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Friberg, Markus. "Algorithms for analyzing signals in DNA : applications to transcription and translation /." Zürich : ETH, 2007. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=17096.
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Purton, Saul. "Genes for components of the transcription-translation apparatus of pea chloroplasts." Thesis, University of Cambridge, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.257299.
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Silvestre, Cerdà Joan Albert. "Different Contributions to Cost-Effective Transcription and Translation of Video Lectures." Doctoral thesis, Universitat Politècnica de València, 2016. http://hdl.handle.net/10251/62194.
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[EN] In recent years, on-line multimedia repositories have experiencied a strong
growth that have made them consolidated as essential knowledge assets, especially
in the area of education, where large repositories of video lectures have been
built in order to complement or even replace traditional teaching methods.
However, most of these video lectures are neither transcribed nor translated
due to a lack of cost-effective solutions to do so in a way that gives accurate
enough results. Solutions of this kind are clearly necessary in order to make
these lectures accessible to speakers of different languages and to people with
hearing disabilities. They would also facilitate lecture searchability and
analysis functions, such as classification, recommendation or plagiarism
detection, as well as the development of advanced educational functionalities
like content summarisation to assist student note-taking.
For this reason, the main aim of this thesis is to develop a cost-effective
solution capable of transcribing and translating video lectures to a reasonable
degree of accuracy. More specifically, we address the integration of
state-of-the-art techniques in Automatic Speech Recognition and Machine
Translation into large video lecture repositories to generate high-quality
multilingual video subtitles without human intervention and at a reduced
computational cost. Also, we explore the potential benefits of the exploitation
of the information that we know a priori about these repositories, that is,
lecture-specific knowledge such as speaker, topic or slides, to create
specialised, in-domain transcription and translation systems by means of
massive adaptation techniques.
The proposed solutions have been tested in real-life scenarios by carrying out
several objective and subjective evaluations, obtaining very positive results.
The main outcome derived from this thesis, The transLectures-UPV
Platform, has been publicly released as an open-source software, and, at the
time of writing, it is serving automatic transcriptions and translations for
several thousands of video lectures in many Spanish and European
universities and institutions.[ES] Durante estos últimos años, los repositorios multimedia on-line han experimentado un gran crecimiento que les ha hecho establecerse como fuentes fundamentales de conocimiento, especialmente en el área de la educación, donde se han creado grandes repositorios de vídeo charlas educativas para complementar e incluso reemplazar los métodos de enseñanza tradicionales. No obstante, la mayoría de estas charlas no están transcritas ni traducidas debido a la ausencia de soluciones de bajo coste que sean capaces de hacerlo garantizando una calidad mínima aceptable. Soluciones de este tipo son claramente necesarias para hacer que las vídeo charlas sean más accesibles para hablantes de otras lenguas o para personas con discapacidades auditivas. Además, dichas soluciones podrían facilitar la aplicación de funciones de búsqueda y de análisis tales como clasificación, recomendación o detección de plagios, así como el desarrollo de funcionalidades educativas avanzadas, como por ejemplo la generación de resúmenes automáticos de contenidos para ayudar al estudiante a tomar apuntes. Por este motivo, el principal objetivo de esta tesis es desarrollar una solución de bajo coste capaz de transcribir y traducir vídeo charlas con un nivel de calidad razonable. Más específicamente, abordamos la integración de técnicas estado del arte de Reconocimiento del Habla Automático y Traducción Automática en grandes repositorios de vídeo charlas educativas para la generación de subtítulos multilingües de alta calidad sin requerir intervención humana y con un reducido coste computacional. Además, también exploramos los beneficios potenciales que conllevaría la explotación de la información de la que disponemos a priori sobre estos repositorios, es decir, conocimientos específicos sobre las charlas tales como el locutor, la temática o las transparencias, para crear sistemas de transcripción y traducción especializados mediante técnicas de adaptación masiva. Las soluciones propuestas en esta tesis han sido testeadas en escenarios reales llevando a cabo nombrosas evaluaciones objetivas y subjetivas, obteniendo muy buenos resultados. El principal legado de esta tesis, The transLectures-UPV Platform, ha sido liberado públicamente como software de código abierto, y, en el momento de escribir estas líneas, está sirviendo transcripciones y traducciones automáticas para diversos miles de vídeo charlas educativas en nombrosas universidades e instituciones Españolas y Europeas.
[CAT] Durant aquests darrers anys, els repositoris multimèdia on-line han experimentat un gran creixement que els ha fet consolidar-se com a fonts fonamentals de coneixement, especialment a l'àrea de l'educació, on s'han creat grans repositoris de vídeo xarrades educatives per tal de complementar o inclús reemplaçar els mètodes d'ensenyament tradicionals. No obstant això, la majoria d'aquestes xarrades no estan transcrites ni traduïdes degut a l'absència de solucions de baix cost capaces de fer-ho garantint una qualitat mínima acceptable. Solucions d'aquest tipus són clarament necessàries per a fer que les vídeo xarres siguen més accessibles per a parlants d'altres llengües o per a persones amb discapacitats auditives. A més, aquestes solucions podrien facilitar l'aplicació de funcions de cerca i d'anàlisi tals com classificació, recomanació o detecció de plagis, així com el desenvolupament de funcionalitats educatives avançades, com per exemple la generació de resums automàtics de continguts per ajudar a l'estudiant a prendre anotacions. Per aquest motiu, el principal objectiu d'aquesta tesi és desenvolupar una solució de baix cost capaç de transcriure i traduir vídeo xarrades amb un nivell de qualitat raonable. Més específicament, abordem la integració de tècniques estat de l'art de Reconeixement de la Parla Automàtic i Traducció Automàtica en grans repositoris de vídeo xarrades educatives per a la generació de subtítols multilingües d'alta qualitat sense requerir intervenció humana i amb un reduït cost computacional. A més, també explorem els beneficis potencials que comportaria l'explotació de la informació de la que disposem a priori sobre aquests repositoris, és a dir, coneixements específics sobre les xarrades tals com el locutor, la temàtica o les transparències, per a crear sistemes de transcripció i traducció especialitzats mitjançant tècniques d'adaptació massiva. Les solucions proposades en aquesta tesi han estat testejades en escenaris reals duent a terme nombroses avaluacions objectives i subjectives, obtenint molt bons resultats. El principal llegat d'aquesta tesi, The transLectures-UPV Platform, ha sigut alliberat públicament com a programari de codi obert, i, en el moment d'escriure aquestes línies, està servint transcripcions i traduccions automàtiques per a diversos milers de vídeo xarrades educatives en nombroses universitats i institucions Espanyoles i Europees.
Silvestre Cerdà, JA. (2016). Different Contributions to Cost-Effective Transcription and Translation of Video Lectures [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/62194
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Leen, Eoin. "Structural insights into the transcription and translation of murine norovirus RNA." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/11173.
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Noroviruses are positive-sense single-stranded RNA viruses which, in humans, cause rapid onset diarrhoea and vomiting. There is an estimated 21 million cases of noroviral gastroenteritis in the United States per year. The work in this thesis has focused on two noroviral proteins, the viral protein genome-linked (VPg), and the viral RNA dependent RNA polymerase (NS7pol). The VPg protein is covalently linked to the 5' end of the noroviral genome and is a key component of translation and replication initiation in noroviruses. Using Nuclear magnetic resonance spectroscopy (NMR) we have analysed the murine norovirus (MNV) and a human norovirus VPg (Lordsdale virus (LDV)) proteins. The VPg protein of both viruses has a small structured helical core with extensive N and C-terminal flexible regions. We has also determined the structure of the MNV NS7pol as well as two high fidelity mutants (P72S and E75S) using X-ray crystallography. The NS7pol protein has a very similar “right drinking hand” structure to other RNA dependent RNA polymerases. The fidelity mutants were structurally identical to the wild type protein but had subtle changes in local hydrogen bonding networks. This is consistent with similar studies performed with picornaviral polymerases. In addition we used NMR and surface plasmon resonance to characterise the interaction of the MNV VPg protein with the NS7pol protein. The interaction between full length VPg and MNV NS7pol is weak, (KD ~160 μM). In addition the NS7pol interacts with both the full length VPg and the core domain of this protein.
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Maake, Takalani Whitney. "Development of an actinobacteria based in vitro transcription and translation systems." University of the Western Cape, 2015. http://hdl.handle.net/11394/4750.
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>Magister Scientiae - MScHeterologous metagenomic screening strategies have relied largely on the construction of DNA libraries and screening in Escherichia coli to access novel enzymes. There is an increased demand for the identification of novel lignocellulose degrading enzymes with enhanced biochemical properties which are suitable for applications in industrial processes; biofuels being one of them. The use of heterologous gene expression in function based metagenomic studies has resulted in the discovery of enormous novel bioactive compounds. However, there are limitations associated with using E. coli as a heterologous host which does not allow transcription and translation of all genes in the metagenome. E. coli can only express 40% of the environmental DNA because of promoter recognition, codon usage, and host toxicity of gene products. Therefore alternative strategies for expressing or producing novel enzymes are needed, which can also be employed in metagenomic gene discovery. In vitro protein synthesis is an important tool in molecular biology and used to obtain proteins from genes for functional and expression studies. These systems may hold the key to unlock more of the potential in metagenomic DNA. The broader aim of the study is to develop non- E. coli based cell-free protein synthesis systems to further the metagenomics screening. In this study, Rhodococcus erythropolis H8 was evaluated for its suitability in cell-free expression. Crude extracts containing the macromolecular components (70S or 80S ribosomes, tRNAs, initiation, elongation and termination factors) fromR. erythropolis were prepared using existing crude extract based cell-free protein synthesis (CFPS) protocols. Three genes were selected and used as templates for synthesis: cell11, xp12 and acetyl xylan esterase (axe10), all previously isolated from metagenomic libraries screened inE. coli. As judged by zymograms and enzyme assays, all enzymes were successfully expressedfrom their native promoters and in recombinants clones using the PtipA promoter, and wereactive. Furthermore, the amounts of XP12 protein produced using pFos-XP_12 was 1.2mg/mlfrom E. coli and 1.67mg/ml from R. erythropolis CFPS, showing that the R. erythropolismachinery was more efficient in the expression of XP12 than the E. coli machinery. To the best of our knowledge this is the first demonstration of a cell-free expression using an actinomycete.
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O'Dwyer, Rebecca Amy. "Regulation of P-glycoprotein expression at the levels of transcription and translation." Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.428605.
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Sham, Mai Har. "Regulation of transcription and translation of phloem proteins in cucurbitaceae during differentiation." Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.329169.
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Chen, Menglin. "Studies of Translation Elongation and its Relationship to Transcription Elongation in Bacteria." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1586468314499281.
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Davidson, Alexander F. "Elucidating the mechanism of localised mDNA translation during Drosophila oogenesis." Thesis, University of Oxford, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711933.
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Haines, Bryan Peter. "Alternate transcription and translation of the LIF gene produces a novel intracellular protein /." Title page, contents and summary only, 1997. http://web4.library.adelaide.edu.au/theses/09PH/09phh1518.pdf.
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Mellenius, Harriet. "Speed and accuracy in transcription and translation : Modelling of transcript and polypeptide elongation." Doctoral thesis, Uppsala universitet, Struktur- och molekylärbiologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-262698.
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Bernstein, Jeffrey Robert. "Horizontal pathway transfer to Escherichia coli from Rhodopseudomonas palustris transcription, translation, pathway extension /." Diss., Restricted to subscribing institutions, 2007. http://proquest.umi.com/pqdweb?did=1495958891&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Valor, Miró Juan Daniel. "Evaluation of innovative computer-assisted transcription and translation strategies for video lecture repositories." Doctoral thesis, Universitat Politècnica de València, 2017. http://hdl.handle.net/10251/90496.
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Nowadays, the technology enhanced learning area has experienced a strong growth with many new learning approaches like blended learning, flip teaching, massive open online courses, and open educational resources to complement face-to-face lectures. Specifically, video lectures are fast becoming an everyday educational resource in higher education for all of these new learning approaches, and they are being incorporated into existing university curricula around the world.
Transcriptions and translations can improve the utility of these audiovisual assets, but rarely are present due to a lack of cost-effective solutions to do so. Lecture searchability, accessibility to people with impairments, translatability for foreign students, plagiarism detection, content recommendation, note-taking, and discovery of content-related videos are examples of advantages of the presence of transcriptions.
For this reason, the aim of this thesis is to test in real-life case studies ways to obtain multilingual captions for video lectures in a cost-effective way by using state-of-the-art automatic speech recognition and machine translation techniques. Also, we explore interaction protocols to review these automatic transcriptions and translations, because unfortunately automatic subtitles are not error-free. In addition, we take a step further into multilingualism by extending our findings and evaluation to several languages. Finally, the outcomes of this thesis have been applied to thousands of video lectures in European universities and institutions.Hoy en día, el área del aprendizaje mejorado por la tecnología ha experimentado un fuerte crecimiento con muchos nuevos enfoques de aprendizaje como el aprendizaje combinado, la clase inversa, los cursos masivos abiertos en línea, y nuevos recursos educativos abiertos para complementar las clases presenciales. En concreto, los videos docentes se están convirtiendo rápidamente en un recurso educativo cotidiano en la educación superior para todos estos nuevos enfoques de aprendizaje, y se están incorporando a los planes de estudios universitarios existentes en todo el mundo. Las transcripciones y las traducciones pueden mejorar la utilidad de estos recursos audiovisuales, pero rara vez están presentes debido a la falta de soluciones rentables para hacerlo. La búsqueda de y en los videos, la accesibilidad a personas con impedimentos, la traducción para estudiantes extranjeros, la detección de plagios, la recomendación de contenido, la toma de notas y el descubrimiento de videos relacionados son ejemplos de las ventajas de la presencia de transcripciones. Por esta razón, el objetivo de esta tesis es probar en casos de estudio de la vida real las formas de obtener subtítulos multilingües para videos docentes de una manera rentable, mediante el uso de técnicas avanzadas de reconocimiento automático de voz y de traducción automática. Además, exploramos diferentes modelos de interacción para revisar estas transcripciones y traducciones automáticas, pues desafortunadamente los subtítulos automáticos no están libres de errores. Además, damos un paso más en el multilingüismo extendiendo nuestros hallazgos y evaluaciones a muchos idiomas. Por último, destacar que los resultados de esta tesis se han aplicado a miles de vídeos docentes en universidades e instituciones europeas.
Hui en dia, l'àrea d'aprenentatge millorat per la tecnologia ha experimentat un fort creixement, amb molts nous enfocaments d'aprenentatge com l'aprenentatge combinat, la classe inversa, els cursos massius oberts en línia i nous recursos educatius oberts per tal de complementar les classes presencials. En concret, els vídeos docents s'estan convertint ràpidament en un recurs educatiu quotidià en l'educació superior per a tots aquests nous enfocaments d'aprenentatge i estan incorporant-se als plans d'estudi universitari existents arreu del món. Les transcripcions i les traduccions poden millorar la utilitat d'aquests recursos audiovisuals, però rara vegada estan presents a causa de la falta de solucions rendibles per fer-ho. La cerca de i als vídeos, l'accessibilitat a persones amb impediments, la traducció per estudiants estrangers, la detecció de plagi, la recomanació de contingut, la presa de notes i el descobriment de vídeos relacionats són un exemple dels avantatges de la presència de transcripcions. Per aquesta raó, l'objectiu d'aquesta tesi és provar en casos d'estudi de la vida real les formes d'obtenir subtítols multilingües per a vídeos docents d'una manera rendible, mitjançant l'ús de tècniques avançades de reconeixement automàtic de veu i de traducció automàtica. A més a més, s'exploren diferents models d'interacció per a revisar aquestes transcripcions i traduccions automàtiques, puix malauradament els subtítols automàtics no estan lliures d'errades. A més, es fa un pas més en el multilingüisme estenent els nostres descobriments i avaluacions a molts idiomes. Per últim, destacar que els resultats d'aquesta tesi s'han aplicat a milers de vídeos docents en universitats i institucions europees.
Valor Miró, JD. (2017). Evaluation of innovative computer-assisted transcription and translation strategies for video lecture repositories [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90496
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Schaerli, Yolanda. "DNA amplification and in vitro transcription/translation in microfluidic droplets for protein engineering." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608746.
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László, Csaba F. "Translation Regulation of UV-induced Transcription Factor NF-κB and Oncogene COX-2." Ohio University / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1229961185.
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Spevak, Christina C. "Specific requirements for translational regulation by a nascent peptide that stalls ribosomes in response to arginine." Full text open access at:, 2006. http://content.ohsu.edu/u?/etd,46.
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Lysaght, Andrew Christopher. "Characterization of cochlear transcription, translation and energy extraction in aging and noise-induced pathology." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/95864.
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Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2014.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 147-163).
Success in otologic practice is currently limited by the diagnostic tools and treatment options available to address an individual's specific presentation of hearing loss. This limitation results from insufficient characterization of the inner ear's biochemical environment as well as physical hurdles associated with accessing inner ear tissues. The encapsulation of the hearing organ within a bony shell and delicate nature of its tissues make standard tissue biopsy techniques impossible and leave many imaging methods impractical. This thesis sought to approach these clinical limitations in two ways: (1) performing novel transcriptional and translational characterizations of inner ear tissues and (2) development of a novel technique to access and communicate diagnostic information from within the inner ear. The first part of this thesis employs whole transcriptome shotgun sequencing to study murine inner ear transcriptional activity in young, healthy animals as well as changes associated with organ aging and noise-induced auditory neuropathy, an important mechanism of hearing impairment in humans. Knowledge of the inner ear's transcriptional behavior (Part I) is coupled with novel translational insights provided by high-throughput tandem mass-spectrometry (Part III) studies of human inner ear fluids obtained from healthy and pathologic populations. These studies illuminate homeostatic mechanisms employed by the highly specialized inner ear tissues, providing a critical knowledge-base for inner ear scientists and pharmacologists, and identify important expression-level changes which occur during the onset and progression of inner ear pathologies. While these high-throughput studies offer the powerful ability to gain a wealth of knowledge into which genes are active within the inner ear, functional assessment of the specific role these genes play must be assessed in a more focused manner. Phenotypic characterization of mice with specific genetic mutations (Part II) has been performed to provide critical insight into the specific role Fgf23 plays in development and maintenance of the auditory system. The second arm of this thesis seeks to provide clinical practicality to the above work by developing a method to safely access the inner ear environment to gather and communicate diagnostic information (Part IV). A guinea pig model was utilized to develop an approach to insert microelectrodes into the fluid spaces of the inner ear in order to harness and monitor the natural electrochemical gradient of the organ. The useful energy extracted from this "biological battery" was used to power a combined microchip/radio transmitter capable of performing voltage-sensing operations within endolymph and wirelessly relaying this information to an external receiver. This study was the first to utilize a mammalian electrochemical potential to power an electronic device. By performing this task while preserving the integrity of the hearing organ this work provides the first, critical proof-of-concept demonstration toward clinically-applicable sensing and therapeutic devices powered by the inner ear. Further refinement of this technique into a long-term, fully-implantable device will enable previously impossible longitudinal studies of organ behavior in awake, behaving subjects and the incorporation of sensing modalities into current inner ear prostheses to monitor biochemical changes and maximize patient benefits.
by Andrew Christopher Lysaght.
Ph. D.
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Baboo, Sabyasachi. "Nuclear translation." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:5266f049-d576-44fd-ab26-11cf7a27f678.
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In bacteria, protein synthesis can occur tightly coupled to transcription. In eukaryotes, it is believed that translation occurs solely in the cytoplasm; I test whether some occurs in nuclei and find: (1) L-azidohomoalanine (Aha) – a methionine analogue (detected by microscopy after attaching a fluorescent tag using ‘click’ chemistry) – is incorporated within 5 s into nuclei in a process sensitive to the translation inhibitor, anisomycin. (2) Puromycin – another inhibitor that end-labels nascent peptides (detected by immuno-fluorescence) – is similarly incorporated in a manner sensitive to a transcriptional inhibitor. (3) CD2 – a non-nuclear protein – is found in nuclei close to the nascent RNA that encodes it (detected by combining indirect immuno-labelling with RNA fluorescence in situ hybridization using intronic probes); faulty (nascent) RNA is destroyed by a quality-control mechanism sensitive to translational inhibitors. I conclude that substantial translation occurs in the nucleus, with some being closely coupled to transcription and the associated proof-reading. Moreover, most peptides made in both the nucleus and cytoplasm are degraded soon after they are made with half-lives of about one minute. I also collaborated on two additional projects: the purification of mega-complexes (transcription ‘factories’) containing RNA polymerases I, II, or III (I used immuno-fluorescence to confirm that each contained the expected constituents), and the demonstration that some ‘factories’ specialize in transcribing genes responding to tumour necrosis factor α – a cytokine that signals through NFκB (I used RNA fluorescence in situ hybridization coupled with immuno-labelling to show active NFκB is found in factories transcribing responsive genes).
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Deraze, Jérôme. "Epigenetic control of ribosome biogenesis homeostasis." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066342/document.
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La traduction est une activité cellulaire essentielle, réalisée par les ribosomes. Ces particules sont synthétisées dans le nucléole, ce qui nécessite l'expression coordonnée de 4 ARN ribosomaux, 80 protéines ribosomales, et plus de 200 facteurs d'assemblage. Leur biogenèse est complexe et sollicite plus de la moitié de l'énergie des cellules en prolifération. La quantité de ribosomes varie selon les conditions environnementales et métaboliques et de ce fait, leur synthèse est modulée en réponse à de nombreux stimuli. Plusieurs mécanismes coordonnent la biogenèse des ribosomes avec l'homéostasie cellulaire. L'un d'eux est la capacité des protéines ribosomiques à réguler l'expression des gènes à plusieurs niveaux. Ces fonctions effectuées hors du ribosome sont dites extraribosomales. Notre équipe a mis en évidence l'une de ces fonctions de la protéine ribosomale uL11 chez la Drosophile. Quand sa lysine 3 est triméthylée (uL11K3me3), elle interagit avec Corto, un facteur de transcription de la famille des Enhancers de Trithorax et Polycomb. L'étude de leur fixation à la chromatine montre que ces protéines se répartissent différemment à l'échelle du génome, et que uL11K3me3 est présente au niveau de gènes actifs enrichis en composants du ribosome. Nous avons généré les premiers allèles génétiques du gène uL11 chez la Drosophile, et décrivons la stratégie de crible moléculaire employée pour leur isolation. Finalement, nous avons étudié les allèles de uL11 dont la lysine 3 est mutée. Leurs phénotypes ressemblent à ceux des mutants Minute, suggérant que le domaine N-terminal de uL11 possède une fonction essentielle, mais peut-être indépendante d'une interaction avec CortoTranslation is an essential metabolic activity carried by ribosomes. These complexes are synthetized in the nucleolus, and require the coordinated expression of 4 ribosomal RNA, 80 ribosomal proteins, and more than 200 assembly factors. Indeed, their biogenesis is complex and expensive, consuming more than half of the energy in proliferating cells. As the cellular need for ribosomes varies with environmental or metabolic conditions, their synthesis is tightly regulated in response to a number of cues. Many mechanisms ensure that the intensity of ribosome biogenesis is coupled to cell homeostasis. Such is the ability of ribosomal proteins to regulate gene expression at many levels, from translation specificity to activation or repression of transcription. Many such functions are carried off the ribosome, and are thus termed extraribosomal. Our team discovered a new extraribosomal function of ribosomal protein uL11 in Drosophila. Indeed, when trimethylated on lysine 3 (uL11K3me3), it associates with Corto, a transcription factor of the Enhancers of Trithorax and Polycomb family. By studying their genome-wide binding profile on chromatin, we show that these proteins are distributed along different patterns, and that uL11K3me3 specifically binds a subset of active genes enriched in ribosome biogenesis components. Additionally, we generated the first genetic alleles for Drosophila uL11 and describe the molecular screening method that we employed. Last, we studied the uL11 alleles that delete or replace lysine 3. We describe that their Minute-like phenotypes suggest an essential role for the N-terminal domain of uL11, though it may be independent of its association with Corto
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László, Csaba F. "Translation regulation of UV-light-induced transcription factor NF-kappa-B and oncogene COX-2." View abstract, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:3353542.
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Banov, Debra Taylor. "Transcription and Translation of Annuae 1626-1645, from the Jesuit Annual Letters in Tonkin Vietnam." BYU ScholarsArchive, 2015. https://scholarsarchive.byu.edu/etd/5464.
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This project consists of a transcription and translation of the Annuae 1626-1645, written by an unidentified Jesuit missionary in Tonkin (Vietnam). The document appears to have been used as a source text for António Cardim's book Batalhas da Companhia de Jesus, and, as a result, there are many similarities between the two works. Despite these similarities, the Annuae contains new and insightful information on the state of the Tonkin mission as well as an interesting outsider's perspective on Vietnamese politics in the early 17th century.
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Patalano, Solenn. "The translation regulator UNR performs opposite sex-specific functions for dosage compensation in Drosophila." Nice, 2008. http://www.theses.fr/2008NICE4099.
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La compensation de dose est le processus qui permet d’égaliser le niveau d’expression des gènes liés au X entre les mâles et les femelles. Chez la Drosophile, ce processus a lieu par le doublement de la transcription des gènes grâce à la fixation du complexe de compensation de dose sur le chromosome X des mâles. Chez les femelles, la traduction de l’ARN msl-2, codant pour une sous unité du complexe, est réprimée par la protéine SXL empêchant ainsi la formation du complexe. Pour élucider les mécanismes moléculaires de cette répression, des expériences de traduction in vitro ont permis la mise en évidence d’une région requise pour l’inhibition de msl-2 reconnue par UNR, un co-facteur de SXL, préalablement identifié comme impliqué dans le mécanisme de répression de la compensation de dose chez les femelles. L’étude in vivo d’un mutant hypomorphe de UNR a montré qu’une partie de msl-2 était transcrite chez les femelles mutantes, capable ainsi de former le complexe de compensation sur les sites d’affinité des chromosomes X. Curieusement, l’ensemble des membres du complexe était cependant faiblement recruté sur le chromosome X des mutants mâles dont la chromatine était anormalement relaxée. En inactivant la fonction de UNR par RNAi sur des cellules mâles en culture, ces phénotypes ont été reproduits sans que la distribution des composants du complexe ne soit perturbée. Ces résultats ont permis de découvrir une nouvelle fonction pour UNR chez la Drosophile qui, en participant à l’inhibition de la compensation de dose par la répression de msl-2 chez les femelles, agit de manière opposée chez les males en contribuant à l’assemblage du DCC sur le chromosome XDosage compensation is the process by which the expression levels of X-linked genes are equalized between males and females. In Drosophila, binding of the dosage compensation complex to the single X chromosome of males causes a twofold increase of gene expression levels on this chromosome, thereby achieving the same level of expression as in females. In female flies, in contrast, the complex cannot be assembled because of the translational repression of one of its subunits, MSL2, by the binding of the female specific protein, SXL, to its RNA untranslated region. To understand the molecular mechanisms of msl-2 translational repression, in vitro translation experiments have identified a region necessary for msl-2 inhibition where UNR, a SXL co-factor, can indeed bind, highlighting its importance for dosage compensation repression in female flies. Strong evidence in support of such a function for UNR was provided by an in vivo study of a UNR hypomorphic mutant. Despite the low levels of MSL2 in mutant females, they were sufficient for the assembly and binding of the dosage compensation complex on the X “high affinity sites”. Curiously, in mutant males, the complex was weakly recruited to the single X chromosome where X chromatin was highly decondensed. RNAi inactivation of UNR in cultured male cells reproduced this phenotype without affecting the distribution of the complex components suggesting a major role for UNR in dosage compensation in males effected through chromatin regulation. Taken together, these observations uncover dual, sex-specific functions of UNR in X chromosome dosage compensation: repressing msl-2 expression in female flies while promotion DCC recruitment in males
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McCloskey, Nicholas. "Transcriptional Effects of Adaptive Synonymous Mutations in Pseudomonas fluorescens." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/37903.
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Synonymous mutations have traditionally been thought to have no significant effect on fitness. However, a growing body of recent research has shown that this is not always the case. In an experimentally evolved population of Pseudomonas fluorescens grown in minimal glucose media, synonymous mutations arose in a glucose transport gene that resulted in beneficial fitness effects comparable to those of non-synonymous mutations. We found that the increase in fitness was a direct result of increased gene expression; however, the precise mechanism was unclear. Synonymous mutations have been shown to affect gene expression on transcriptional and translational levels through changes in mRNA secondary structure and codon usage.
Our study investigates the underlying mechanisms in which these evolved synonymous mutations lead to increased gene expression. In addition to the evolved mutations, we have a library of 42 strains with single synonymous mutations within the glucose transport gene and found a positive correlation between fitness and gene expression. To determine whether these mutations affect transcript levels, translational efficiency or a combination of both, we systematically incorporated transcriptional and translational fusions of a yellow fluorescent protein within the glucose transport operon. We found that the evolved mutations predominantly act on the level of transcription and have strong polar downstream effects. Additionally, through manipulation of the local genetic sequence, we investigated the specific molecular requirements necessary for the increased expression. We found that for one of our evolved synonymous mutants, mRNA secondary structure does not play an essential role, but we speculate that the mutation may strengthen a weak internal promoter sequence to confer its increased expression. Our study provides evidence of the adaptive mechanisms of beneficial synonymous mutations in an experimentally evolved setting.
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Carpenter, Oliver L. "Ultraviolet Light-Induced Regulation of Transcription and Translation, COX-2 Expression and Noncanonical NF-κB Activation." Ohio University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1382624015.
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Öktem, Alp. "Incorporating prosody into neural speech processing pipelines: applications on automatic speech transcription and spoken language machine translation." Doctoral thesis, Universitat Pompeu Fabra, 2019. http://hdl.handle.net/10803/666222.
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In this dissertation, I study the inclusion of prosody into two applications that involve speech understanding:~automatic speech transcription and spoken language translation. In the former case, I propose a method that uses an attention mechanism over parallel sequences of prosodic and morphosyntactic features. Results indicate an $F_1$ score of 70.3\% in terms of overall punctuation generation accuracy. In the latter problem I deal with enhancing spoken language translation with prosody. A neural machine translation system trained with movie-domain data is adapted with pause features using a prosodically annotated bilingual dataset. Results show that prosodic punctuation generation as a preliminary step to translation increases translation accuracy by 1\% in terms of BLEU scores. Encoding pauses as an extra encoding feature gives an additional 1\% increase to this number. The system is further extended to jointly predict pause features in order to be used as an input to a text-to-speech system.En aquesta tesi estudio la inclusió de la prosòdia en dues aplicacions que involucren la comprensió de la parla:~la transcripció automàtica de la parla i la traducció de la llengua oral. En el primer cas, proposo un mètode que utilitza un mecanisme d’atenció sobre seqüències paral·leles de característiques prosòdiques i morfosintàctiques. Els resultats indiquen una precisió de $F_1$=70.3\% en la generació de la puntuació. En el segon cas m'ocupo de la millora de la traducció de la llengua oral utilitzant la prosòdia. Un sistema neural de traducció automàtica format amb un corpus de text en el domini del cinema s’adapta amb característiques de pauses afegides utilitzant un conjunt de dades bilingües prosòdicament anotada. Els resultats mostren que la generació de puntuació prosòdica com a pas previ a la traducció augmenta la precisió de la traducció en un 1\% en termes de BLEU. La codificació de les pauses com a característica addicional encara incrementa la precisió en un altre 1\%. A més a més, amplio el sistema de traducció per a predir conjuntament les característiques de pausa i poder-les utilitzar com a entrada en un sistema de síntesi de veu.
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Djekic, Uros V. "Coupling selection of the HIV-1 tRNA primer used for reverse transcription with viral translation and encapsidation." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2008r/djekic.pdf.
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Terrazas, Serena Rachelle. "Transcription and Translation of a Letter from the Japonica Sinica 85 of the Archivum Romanum Societatis Iesu." BYU ScholarsArchive, 2016. https://scholarsarchive.byu.edu/etd/6076.
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This project is a transcription and translation of a letter from the Japonica Sinica 85 collection of the Archivum Romanum Societatis Iesu. It was written by an unidentified Jesuit who recounts three years of history (1655-1657) of the Tonkin kingdom (in present-day Vietnam), replacing the annual letters from those years that had been lost at sea. The account includes descriptions of their wars with Cochinchina, the succession of the kingship, and the funeral and burial of the Lê-Triṇh lord, Triṇh Tráng.
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Beus, Annalyn. "Translation and Transcription of a Passage from the Baduem Manuscript: An Eighteenth-Century Portuguese Embassy to China." BYU ScholarsArchive, 2013. https://scholarsarchive.byu.edu/etd/4016.
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This project is a diplomatic transcription and English translation of a passage from an 18-century manuscript that chronicles a remarkable Portuguese embassy to China (Macau). The embassy embarked from Lisbon in February 1752, sailing in a luxuriously outfitted ship (Nossa Senhora da Conceição e Lusitânia Grande), in convoy with a warship (Nossa Senhora das Brotas). The English translation is important because it makes the account accessible to scholars who lack familiarity with Portuguese.This voyage to China is remarkable in light of the long history of maritime loss by the Portuguese. Although the normal projected loss of life on this route was 20%, this journey was made without one death. Some of the most fascinating aspects of the journey include the following: a) how the intrepid crew of the Nossa Senhora (most of whom were novices) and the passengers dealt with bad weather at sea; b) the religious rites conducted during the voyage by Jesuit priests en route to the Far East missions, which the passengers firmly believed mitigated the dangers and were thus responsible for their safe journey; c) the intriguing political maneuvering between the Portuguese and Chinese in Macau; and d) the meticulous descriptions of the different cultures, peoples and places encountered on the journey.
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Kummer, Susann. "A temporal, spatial and quantitative study on the influenza A virus transcription, translation and virus-host interaction." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16372.
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Die Vermehrung des Influenza A Virus umfasst, neben anderen wichtigen Schritten, die Transkrption der viralen mRNA und die ribosomale Translation der viralen Proteine. Mit großem Aufwand wurde bereits an der Entwicklung von Methoden zur Untersuchung des zeitlichen Verlaufs der Synthese viraler mRNA während des Vermehrungszyklusses in der Wirtszelle geforscht. In der vorliegenden Arbeit wurden sequenzspezifische FIT-PNA-Sonden, welche einen einzelnen, als künstliche fluoreszente Nukleobase dienenden Interkalator tragen, auf die quantitative RT-PCR sowie die Lebendzellmikroskopie angewandt. Die FIT-PNA-Sonden bieten dabei eine hohe Sensitivität und eine enorme Zielspezifität unter nichtstringenten Hybridisierungsbedingungen. Im Speziellen wurden FIT-PNA Sonden mit Sequenzspezifität zur mRNA der Neuraminidase und des Matrixproteins 1 entworfen und untersucht. Die somit erhaltenen Ergebnisse besitzen eine hohe biologische Relevanz und weisen diese Sonden als vielversprechende Methodik in der Virologie und der Zellbiologie aus. Ihre Anwendung konnte bereits auf das Vesikular Stomatitis Virus ausgeweitet werden. Die Kombination aus biologischer Expertise mit modernen Proteomstudien und detaillierten statistischen Analysen ermöglichte einen systemumfassenden Blick auf die durch eine Infektion bedingten Auswirkungen auf die Wirtszelle. Die Markierung von Aminosäuren mit stabilen Isotopen in Zellkultur wurde hierfür benutzt. Es wurden Proben zu verschiedene Zeitpunkten im Infektionszyklus in die Untersuchungen einbezogen, um zeitaufgelöste Detailstudien der zellulären Proteinbiosynthese und Degradation durchzuführen.Replication of the influenza A virus involves, amongst other critical steps, the transcription of viral mRNA and ribosomal translation of viral proteins. Significant efforts have been devoted to the development of methods that allow the investigation of viral mRNA progression during the replication cycle inside the host cell. In the present thesis sequence specific FIT-PNA probes which contain a single intercalator serving as artificial fluorescent nucleobase were introduced for quantitative RT-PCR and live cell imaging. FIT-PNAs provide for both high sensitivity and high target specificity at nonstringent hybridisation conditions (where both matched and mismatched probetarget complexes coexist). In particular, FIT-PNAs specific to the neuraminidase and matrix protein 1 were successfully designed and examined. The obtained results are of high biological importance and suggest the FIT-PNA technique as promising tool in the field of virology and cell biology as this approach was readily applied to Vesicular Stomatitis Virus as well. By combining biological expertise with modern high throughput quantitative proteomics and detailed statistical analysis a system wide view of the effects and dynamics of the early H1N1 infection on the cell proteome was generated. Stable isotope labelling of amino acids in cell culture (SILAC) was employed to globally track changes in gene expression at the protein level. Furthermore, samples at various time points post infection enabling a more detailed timeresolved analysis of host cell protein biosynthesis and degradation during the infection cycle were included. As a result the specific expression characteristics of single genes and functional gene subsets in response to viral infection were bioinformatically analysed.
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Laible, Allyson Marie. "Modeling green fluorescent protein transcription, translation and modification as a method to obtain NF-kappaB activation profiles." Thesis, [College Station, Tex. : Texas A&M University, 2007. http://hdl.handle.net/1969.1/ETD-TAMU-1443.
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Knight, Helen Coral. "Alternative non-canonical translation initiation codons are used to synthesise novel isoforms of the transcription factor GATAD1." Thesis, University of Southampton, 2017. https://eprints.soton.ac.uk/413444/.
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Alternative translation initiation from upstream non-AUG codons contributes towards the diversity of the eukaryotic proteome; protein isoforms with varying N-terminal extensions can be generated from one mRNA transcript. Investigations are being carried out in order to elucidate how N-terminal extensions affect subcellular localisation, binding partners and thus the function of a protein as well as how the choice of alternative initiation codon (AIC) is regulated. This thesis is focussed on GATAD1 (GATA Zinc Finger Domain-Containing 1), which is a ubiquitously expressed transcription factor, forming part of a transcriptionally repressive histone demethylase complex. GATAD1 regulates the expression of specific genes by forming an indirect interaction with the activating trimethyl marker of lysine four on histone three (H3K4me3); this interaction is made through a lysine demethylase chromatin ‘reader’, KDM5A (Jarid1A). GATAD1 is also involved in retinal development and heart disease, whereby a single mutation in the gene is the cause of dilated cardiomyopathy (DCM). The GATAD1 mRNA transcript can be translated at alternative translation initiation codons resulting in the synthesis of three protein isoforms. The two isoforms with N-terminal extensions are initiated from a CUG and an unusual AUU codon. CRISPR genome editing has been used to tag genomic GATAD1, confirming endogenous expression of all three isoforms. Translation from the AICs is regulated by various factors, including eukaryotic initiation factors (eIFs) 1, 1A and 5, the context and position of the AICs, as well as secondary structure downstream of the AUU codon. Cell type and stresses such as hypoxia also influence the use of each GATAD1 AIC. Although all three GATAD1 isoforms complex with KDM5A, it has been observed that the extended isoforms have a greater tendency to remain in the cytoplasm, potentially forming part of a cytoplasmic demethylase complex, whilst the annotated protein functions as a nuclear transcription factor.
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Ring, Christine. "Optimization of in vitro transcription/translation conditions for in vitro compartmentalization studies and synthesis of 4-fluorohistidine." VCU Scholars Compass, 2017. http://scholarscompass.vcu.edu/etd/4807.
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Genetic code expansion allows the incorporation of non-canonical amino acids with a variety of new functional groups: fluorescent amino acids,1-3 azides,4-6 alkynes,5-10 and photocrosslinkers.4,11,12 This incorporation requires the evolution of new tRNA/aminoacyl tRNA sythetase pairs. Traditionally screenings of novel tRNA/aminoacyl tRNA synthetase pairs have been done in vivo. While these in vivo screenings have proven robust, they are limited in multiple ways: non-canonical amino acids (ncAAs) must be nontoxic and bioavailable. Furthermore, library size is limited by transformation efficiency. Lastly, in vivo screenings require substantial amounts of the target ncAA, which is often not available in large masses. In vitro screenings bypass these limitations: toxicity and bioavailibilty are no longer concerns. Library size can be expanded by several orders of magnitude as we are no longer limited by transformation efficiency. Lastly, because in vitro transcription/translation reactions are routinely conducted on the μL scale, ncAA usage can be minimized. We set out to use in vitro compartmentalization to further expand the code. In an in vitro compartmentalization screening, the water droplets in a water-in-oil emulsion serve as separate reaction chambers in which individual library members are transcribed and translated. Here we report optimization of S30 transcription/translation reactions. Optimizations include cell lysis method, reaction temperature, template amount, and T7 RNA polymerase amounts. Yields remained low and we transistioned into the use of PURExpress.
Fluorohistidines are isosteric with histidine, but not isoelectronic.13 This change in environment results in a reduction of pKa. We set out to synthesize 4-fluorohistidine to use as a pH probe in several target proteins. A synthesis of 4-fluorohistidine was published in 1973.14,15 We were able to improve upon this synthesis by reducing cost and improving yield of a key step in the reaction. Next, small peptides with polyhistidine tags were translated in vitro using our 4-fluorohistidine. We are calling this polyhistidine tag incorporating 4-fluorohistidine our “hexafluorohistag.” Because of the reduced pKa of the 4-fluorohistidine, the hexafluorohistag showed affinity to Nickel-NTA resin even at reduced pH. This allowed for the purification of hexafluorohistagged peptides in the presence of traditional polyhistidine-tagged peptides.
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Sharma, Mansi. "Regulatory mechanisms of Leishmania Aquaglyceroporin AQP1." FIU Digital Commons, 2015. http://digitalcommons.fiu.edu/etd/2300.
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Pentavalent antimonials [Sb(V)] are the primary drug of choice against all forms of leishmaniasis. Emergence of antimony unresponsiveness is a major issue. There is a dire need of understanding antimony resistance mechanisms in Leishmania. One important mechanism is the down regulation of the trivalent antimony [Sb(III)] (the active form of Sb(V)) uptake system. To date, Leishmania aquaglyceroporin AQP1 is the only reported facilitator of Sb(III). Leishmania do not have promoters. They primarily regulate their genes at post-transcriptional and/or post-translational levels. We reported that mitogen activated protein kinase 2 (MPK2) positively regulated AQP1 stability through the phosphorylation of the threonine 197 (T197) residue of AQP1. The goal of this study was to elucidate the regulatory mechanism(s) of AQP1 in Leishmania in order to advance our understanding about the physiological role(s) of AQP1 in Leishmania biology. When Leishmania promastigotes were treated with the proteasome inhibitor MG132, SbIII accumulation was increased due to upregulation of AQP1. Alteration of lysine 12 of AQP1 to either alanine or arginine improved protein stability. Cells co-expressing a dominant-negative MPK2 mutant exhibited severely reduced AQP1 expression, which was reversed upon addition of MG132. Interestingly, the dominant-negative MPK2 mutant could not destabilize either AQP1K12A /AQP1K12R. Stabilization of AQP1 by MPK2 led to its relocalization from the flagellum to the entire surface of the parasite. Both altered AQP1K12A and AQP1K12R were restricted to the flagellum only. The data demonstrated that lysine12 was targeted for AQP1 proteasomal degradation playing an integral role in subcellular localization of AQP1 as well as its interaction with MPK2.
This study also demonstrated that the stability of AQP1 mRNA in different Leishmania species was regulated by their respective 3’-untranslated regions. Cutaneous leishmaniasis causing species accumulated more antimonite and therefore, exhibited higher sensitivity to antimonials than species responsible for visceral leishmaniasis. This species-specific differential sensitivity to antimonite was found to be directly proportional to the expression levels of AQP1 mRNA. The differential regulation of AQP1 mRNA explained the distinct antimonial sensitivity of each species. This study will help us to identify new drugs for treatment in the future and also lead to a novel understanding of parasite biology aspects such as integral membrane protein trafficking and regulation.
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Hanson, Paul J. "Viral protease disruption of host transcription and translation factors in the pathogenesis of coxsackievirus B3 induced viral myocarditis." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/57694.
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Myocarditis, inflammation of the heart muscle, is a spectrum of conditions causing significant morbidity and mortality, yet scientific and clinical knowledge related to this entity is limited. One of the most common and best studied causes of myocarditis is infection by coxsackievirus B3 (CVB3). An improved understanding of the science behind CVB3 myocarditis is critical to establishing better diagnostic and therapeutic strategies for affected individuals.
CVB3 infection redirects numerous cellular pathways from physiologic processes to viral replication, often mediated by viral proteases. Two viral targets in this process are death associated protein 5 (DAP5) and nuclear pore complex protein 98 (Nup98). DAP5 is a translation initiation factor specific to internal ribosome entry site (IRES) mediated translation. Nup98 is a component of the nuclear pore complex and a transcription factor.
In this thesis, I hypothesize that viral proteases contribute to the pathogenesis of viral myocarditis through interaction with DAP5 and Nup98, redirecting translation and transcription towards viral replication.
Using in vitro (plasmid expressed viral proteases), in situ (CVB3 infection in cell culture), and in vivo (mouse myocarditis model) models, I demonstrate that viral protease 2A is responsible for the cleavage of DAP5 and Nup98 during CVB3 infection. Both cleavage events I show to be integral to the viral lifecycle using over expression of recombinant fragments and siRNA inhibition of that expression.
These results suggest two previously unidentified targets for improved diagnostics and therapeutics for myocarditis, both areas for future research.Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate
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NandyMazumdar, Monali. "RfaH CONTACTS TO DNA, RNA POLYMERASE AND RIBOSOME ACTIVATE GENE EXPRESSION." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1482155208767278.
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Sarkissian, Madathia. "Signaling Events Leading to CPEB-Mediated Translation: a Dissertation." eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/305.
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Fully grown oocytes' of the African clawed frog, Xenopus laevis, are arrested at the diplotene stage of meiotic prophase I, which resembles the G2 phase of the mitotic cell cycle. Re-entry into the meiotic divisions is initiated by hormonal signaling normally provided by progesterone. Progesterone signaling leads to the activation of maturation promoting factor (MPF), a heterodimer consisting of the protein kinase cdk1 and cyclin B1; this complex promotes the oocyte's entry into M phase of meiosis I. A crucial event required for MPF activation is cytoplasmic polyadenylation element (CPE)-mediated translation of specific dormant mRNAs such as c-mos and cyclin B1. The CPE, which resides in mRNA 3' untranslated region (UTR), is bound by the CPE binding protein (CPEB), which in turn is bound by Maskin. Maskin is bound to the 5' cap binding protein eIF4E. This type of closed-loop mRNA structure inhibits the recruitment and assembly of the translation initiation complex at the 5'UTR of CPE containing mRNAs. To alleviate this inhibition, CPEB undergoes phosphorylation on S174 by the serine/threonine kinase Aurora A. Phosphorylated CPEB promotes the recruitment of specific polyadenylation factors leading to the polyadenylation of the dormant mRNA, resulting in the disassociation of Maskin from eIF4E. eIF4E is subsequently bound by translation initiation factors leading to mRNA assembly into polysomes and synthesis of the encoded protein.
Insulin signaling has also been shown to induce oocyte maturation. However, this signaling cascade uniquely requires the activation of two upstream components, PI3 kinase and PKC zeta. In this thesis, I show that insulin induced oocyte maturation requires the same CPE-mediated mRNA translation mechanism as had been described for progesterone signaling. I also show that Aurora A kinase activation and S174 phosphorylation play an essential role in insulin-induced CPE-mediated mRNA translation. Interestingly, inhibition of PI3 kinase and PKC zeta inhibits CPE-mediated polyadenylation only in the insulin-signaling pathway; the progesterone pathway is unaffected. These results clearly indicate that different upstream signaling components control CPE-mediated translation between progesterone and insulin signaling cascades. However, both pathways are antagonized by over expressed GSK-3, leading to inhibition of oocyte maturation. Furthermore, I found that GSK-3 inhibits Aurora A kinase activity by directly phosphorylating Aurora A on serine 290/291, promoting an inhibitory autophosphorylation event on serine 349. The importance of a GSK-3/Aurora A interaction is underscored by the finding that GSK-3, Axin, and Aurora A reside in a complex in immature oocytes. During progesterone or insulin signaling, GSK-3 dissociates from Aurora A allowing Aurora A to become active, leading to CPEB phosphorylation, CPE-mediated mRNA translation and oocyte maturation.
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Nieß, Alexander [Verfasser], and Ralf [Akademischer Betreuer] Takors. "Modeling in vitro and in vivo transcription and translation with different levels of granularity / Alexander Nieß ; Betreuer: Ralf Takors." Stuttgart : Universitätsbibliothek der Universität Stuttgart, 2018. http://d-nb.info/1159570280/34.
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Neal, Allison R. B. "An examination, through drawing, of the text of Gilgamesh, and how translation and transcription can inform contemporary drawing practice." Thesis, University of Gloucestershire, 2017. http://eprints.glos.ac.uk/6081/.
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This PhD began by attempting to locate the Epic of Gilgamesh within a contemporary landscape and using a comparison of Michael Ayrton and Sidney Nolan as a means of creating a body of narrative based drawing. Initial work, however, illustrated rather than illuminated the text. As the research evolved, analysis of the text as a model for thinking and the different approaches to landscape from Ayrton and Nolan, clarified that the metaphorical journey of Gilgamesh required a different drawing practice. The Epic of Gilgamesh was written in cuneiform script on clay tablets some four thousand years ago. The fragmented and incomplete tablets have survived by chance. Failure, evident in Gilgamesh’s quest, suggested exploring contingency and failure as agents of creative practice. The opportunity to draw directly from the clay tablets in the collection of the British Museum generated the insight that the apprehension of physical objects and their recording as image through drawing, also works as a process of visual translation. The original clay tablets became a source for making drawings possessing a physical equivalence beyond the normative approaches to translation and narrative. This was the central aspect of the final research, superseding the narrative drive that had been the original starting point. Models of working allusively with narrative and landscape were also provided by unique access to the archives of Sidney Nolan at The Rodd, in Herefordshire, and by analysing in parallel the work of Michael Ayrton. This aspect of the research developed as a way of asserting that in the liminal space of the studio, equivalence can be found with the complex and contingent aspects of quest narrative as exemplified by Gilgamesh. Working large scale, the final works produced for this PhD explore translation and transcription in drawing through the surface accretions of material, gesture, intuition and fold.
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Mazwi, Ntombomzi R. "Transcription, edition, translation and critical analysis of biographical poems contributed by S E K Mqhayi to early IsiXhosa newspapers." Thesis, Rhodes University, 2017. http://hdl.handle.net/10962/18570.
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During the nineteenth century secular creative literature produced by missionaries and publishers was designed for the educational market and for school children and there was nothing for adults. Works of isiXhosa literature was controlled in content and freely edited by the missionaries to satisfy the demands of educational syllabuses. As a result, students at universities, scholars of literature and academics in higher education are lacking primary documents on this literature and therefore are forced to study the limited and unavailable literature books. This thesis concentrates on the work of a particular isiXhosa writer, namely that of S.E.K. Mqhayi. The earlier writers like S.E.K. Mqhayi, J.J.R. Jolobe, G.B. Sinxo and others made their mark in South African literature and culture. Their works were published in journals and newspapers in isiXhosa by the missionaries. This means isiXhosa literature can be found in abundance in the earlier newspapers. What needs to be addressed is how the South African community and literature scholars mentioned above could have access to that work. Mqhayi is well known as the father of the isiXhosa language because of his substantial literary and linguistic contribution to the development of the language. As already mentioned he made his contribution through written work which was published in various newspapers of his time and unfortunately most people are unable to access this material, hence the focus of this thesis. The vast majority of his journalism remains as yet uncollected. However, scholars like Opland (1983) and Saule (1989) made some effort to bring this information to the public through their extensive research. S.E.K. Mqhayi’s popular poems have been published and analyzed over the last century and more recently (Qangule 1979; Kuse 1979; Opland 1983; Saule 1989 & 1996; Ntuli & Swanepoel 1993 and Opland 2009). However, in terms of quantity and value, these are negligible compared to what Mqhayi has published. There are still numerous of Mqhayi’s poems that would add value to the study and history of isiXhosa literature. The main aim of this research is to carry on from where these scholars left off and to bring to the fore the legacy Mqhayi left to the South African people. Hence, thirty (30) poems on people by S.E.K. Mqhayi have been transcribed from the old newspapers, re- typed, translated into English and analysed. These poems are largely published in newspapers but have never been subsequently republished, and hence they are almost completely unknown. The thirty (30) poems have been selected with the assistance of Professor Jeff Opland, a retired Professor from the University of London’s School of Oriental and African Studies (SOAS). The poems are from his Opland Collection of Xhosa Literature housed in Godalming, United Kingdom. They are presented in the manner in which they appeared originally, that is, in terms of isiXhosa orthography during the times of Mqhayi’s writing (Diplomatic Presentation). The data is analysed and discussed in relation to how Mqhayi’s biographical poems can give insight not only to Mqhayi’s subjects of praise, but into how he uses historical, political and sociocultural contexts in the praises of his subjects, meaning that the discussion revolves around the practice of the Historical-Biographical Criticism. The poems are translated into English to allow for this literature not only to be disseminated among isiXhosa speakers, but also to speakers of other languages who understand English. The translation method chosen is the one believed to produce the originality of the source text and sameness of meaning in the target text which is regarded as equivalence. This thesis therefore is an investigation into 30 poems selected from biographical poems written by S.E.K. Mqhayi in newspapers during the period 1899-1944. In essence this thesis presents an in-depth analysis of Mqhayi’s poems against the backdrop of oral literary theory as expounded by theorists who have grappled with the orality-literacy debate, a debate which directly informs Mqhayi’s poetry as he was the first oral poet to transition between orality and literacy.Uncwadi oluveliswe kwishumi elinesithoba lamakhulu eminyaka ziimishinari nabapapashi lwalwenzelwe izifundiswa kunye nabantwana besikolo kwaye akukho msebenzi mninzi ofumanekayo owawenzelwe abantu basekuhlaleni. Umsebenzi omninzi wokubhaliweyo kuncwadi lwesiXhosa wawulawulwa, uhlelwa kwaye ulungiselelwa uqingqo lwezifundo. Oko ke kuye kwabangela ukuba abafundi abakumaziko emfundo ephakamileyo, iimfundi zoncwadi kunye nabahlohli zingabinawo amaxwebhu okwenene oncwadi lwesiXhosa, kwaye loo nto yenza ukuba kufundwe kwaye kwenziwe uphando ngeencwadi ezimbalwa. Olu phando ke ngoko luza kuqwalasela lugxininise kumsebenzi wombhali wesiXhosa onguS.E.K. Mqhayi. Ababhali bangaphambili abafana noo-S.E.K. Mqhayi, J.J.R. Jolobe, G.B. Sinxo nabanye bashiya ifuthe elikhulu kuncwadi nenkcubeko yoMzantsi Afrika. Imisebenzi yabo yayipapashwe ziimishinari ngesiXhosa kuluhlu lwemibhalo namaphephandaba. Oko ke kuthetha ukuthi le misebenzi yesiXhosa iyafumaneka kumaphepha-ndaba angaphambili. Okufuneka kulungisiwe yindlela abemi boMzantsi Afrika kunye nezi mfundi zoncwadi zikhankanywe ngentla zingathi ziwufumane lo msebenzi waba babhali bangaphambili. UMqhayi waziwa ngokuba yinkcuba- buchopho yolwimi lwesiXhosa kwaye udlale indima enkulu kakhulu ekuphuhliseni ulwimi lwesiXhosa. Njengokuba sele ikhankanyiwe ngentla, umsebenzi wakhe upapashwe kumaphephandaba awohlukeneyo wela xesha wayesaphila kwaye kungelishwa ke ukuba abantu abaninzi abakwazi ukuwufumana loo msebenzi. Eminye yemisebenzi yakhe emininzi ke kodwa ayiqokelelwanga. Iingcali ezifana noo-Opland (1983) noSaule (1989) zaye zenza uphando olukhulu zizama ukuzisa olu lwazi eluntwini, kodwa oko akwanelanga. Kwiminyaka edlulileyo imibongo edumileyo ka-S.E.K. Mqhayi sele yapapashwa (Qangule, 1979; Kuse, 1979; Opland, 1983 & 2009; Saule, 1989 & 1996; Ntuli & Swanepoel, 1993). Nangona kunjalo ke isekhona eminye imibongo kaMqhayi engekaveli nenokuthi ibe nenxaxheba kakhulu ekufundeni nasekufundiseni uncwadi lwesiXhosa. Olu phando ke kukuqhubeka apho aba babhali bakhankanyiweyo bayeke khona ukuzisa phambili umsebenzi nelifa elashiywa nguMqhayi kubemi baseMzantsi Afrika. Kungoko ke imibongo engabantu engama-30 kaMqhayi iza kuthi ikhutshelwe isuka kumaphephandaba akudala, iguqulelwe esiNgesini ze ihlahlelwe. Uninzi lwale mibongo ipapashwe kumaphephandaba akudala kwaye zange iphinde ipapashwe kwenye indawo, kungoko ke ingaziwa kakhulu. Ukukhethwa kwale mibongo ingama-30 kuncediswe nguNjingalwazi uJeff Opland, uNjingalwazi odla umhlala-phantsi weYunivesithi yase-London kwiSikolo seZifundo ngezaseAfrika naseMpumalanga (SOAS). Le mibongo isuka kuluhlu lwakhe athe waluqokelela nolubizwa ngokuba yi-Opland Collection of Xhosa Literature oluse- Godalming, e-United Kingdom. Indlela le mibongo eza kuthi ibhalwe ngayo yileyo uMqhayi wayeyibhale ngayo ngexesha lakhe. Le mibongo ihleliwe kwaye oko kubhaliweyo malunga nayo kuquka indlela apho imibongo kaMqhayi ngobomi babantu kuthi kubonise indlela abonga nabonisa ngayo izinto zoPolitiko, zakudala nezasekuhlaleni ezazisenzeka ngela xesha. Indlela ethi konke oku kuthi kuvele kule mibongo kaMqhayi kwaye kuya kuthi kuvezwe kolu phando. Ukuguqulelwa kwale mibongo esiNgesini kuya kuthi kuncede ukuba nabo bangasithethiyo isiXhosa bakwazi ukufumana le mibongo. Indlela esetyenzisiweyo yoguqulo-lwimi yale mibongo yileyo ivumela ukuba umbhalo uguqulelwe ngokufanayo nombhalo-ntsusa kwaye intsingiselo kumbhalo ekuguqulelwa kuwo ingatshintshi. Lo misebenzi ke ngoko uluphando lwemibongo engabantu ekhethiweyo engama30 ebhalwe nguSEK Mqhayi kumaphephandaba kwimiminyaka u1899 - 1944. Umongo wolu phando kukwenza uhlalutyo - nzulu lwemibongo kaMqhayi eyaleka kuphando - lwazi osele lwenziwe njengoko lucaciswa ziingcali ezithe zazamana nengxoxo yoncwadi lomlomo, ngxoxo leyo ethe yachaphazela ngokumandla umongo wezibongo zikaMqhayi njengembongi yokuqala ukuwela ukusuka kuncwadi lomlomo ukuya kuncwadi olubhaliweyo.
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Dhiman, Sunil Kumar [Verfasser], and Paul [Akademischer Betreuer] Galland. "Magnetoreception in Arabidopsis thaliana : Effects of geomagnetic fields on transcription and translation [[Elektronische Ressource]] / Sunil Kumar Dhiman. Betreuer: Paul Galland." Marburg : Philipps-Universität Marburg, 2015. http://d-nb.info/1074639162/34.
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Heidrich, Corina [Verfasser], and Andreas [Akademischer Betreuer] Burkovski. "Tetracycline regulates transcription and translation of tetO by stabilizing alternative folds of the leader RNA / Corina Heidrich. Gutachter: Andreas Burkovski." Erlangen : Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 2014. http://d-nb.info/106504531X/34.
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Kerins, Michael John, Ajay Amar Vashisht, Benjamin Xi-Tong Liang, Spencer Jordan Duckworth, Brandon John Praslicka, James Akira Wohlschlegel, and Aikseng Ooi. "Fumarate Mediates a Chronic Proliferative Signal in Fumarate Hydratase-Inactivated Cancer Cells by Increasing Transcription and Translation of Ferritin Genes." AMER SOC MICROBIOLOGY, 2017. http://hdl.handle.net/10150/624216.
Full textAbstract:
Germ line mutations of the gene encoding the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) cause a hereditary cancer syndrome known as hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC-associated tumors harbor biallelic FH inactivation that results in the accumulation of the TCA cycle metabolite fumarate. Although it is known that fumarate accumulation can alter cellular signaling, if and how fumarate confers a growth advantage remain unclear. Here we show that fumarate accumulation confers a chronic proliferative signal by disrupting cellular iron signaling. Specifically, fumarate covalently modifies cysteine residues on iron regulatory protein 2 (IRP2), rendering it unable to repress ferritin mRNA translation. Simultaneously, fumarate increases ferritin gene transcription by activating the NRF2 (nuclear factor [erythroid-derived 2]-like 2) transcription factor. In turn, increased ferritin protein levels promote the expression of the promitotic transcription factor FOXM1 (Forkhead box protein M1). Consistently, clinical HLRCC tissues showed increased expression levels of both FOXM1 and its proliferation-associated target genes. This finding demonstrates how FH inactivation can endow cells with a growth advantage.
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Alghoul, Fatima. "The mechanism of inhibition of cap-dependent translation by the Translation Inhibitory Elements (TIE) a3 and a11 in Hox mRNAs." Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ031.
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Chez les eucaryotes, les ARNm cellulaires subissent une traduction dépendante de la coiffe qui nécessite des facteurs appelés eIFs pour produire des protéines, mais les ARNm Hox sont traduits dans un mécanisme non canonique en raison de deux régulateurs d'ARN dans l'élément 5'UTR appelé Internal Ribosome Entry Site (IRES) qui recrute le ribosome sans le besoin de coiffe, et un élément inhibiteur de traduction (TIE) qui empêche la translation dépendant de coiffe. L'objectif de ma thèse est de déchiffrer le mécanisme de deux éléments TIE a3 et a11 dans les ARNm Hox. Pour cela, nous avons utilisé le système de traduction sans cellules RRL. Notre modèle pour TIE a3 suggère qu'il inhibe la traduction en uORF qui se traduit par un ARNm Hox a3 UTR 5'UTR de pleine longueur et produit un peptide de 9 KDa avec l'implication de eIF2D, un facteur d'initiation non canonique indépendant du GTP. Pour TIE a11, il séquestre le ribosome 80S sur une combinaison de codons start-stop à 19 nucléotides en amont d'une structure de boucle de tige riche en GC qui bloque le 80S au codon stopIn eukaryotes, cellular mRNAs undergo cap-dependent translation which requires factors called eIFs to produce proteins.However, Hox mRNAs are translated in a non-canonical mechanism due to two RNA regulons in the 5’UTR called Internal Ribosome Entry Site (IRES) element which recruits the ribosome without the need of a cap, and a Translation Inhibitory Element (TIE) which inhibits cap-dependent translation. The objective of my PhD is to decipher the mechanism of two TIE elements a3 and a11 in Hox mRNAs. For that, we used RRL cell-free translation system. Our model for TIE a3 suggests that it inhibits translation to a uORF which translates through full length 5’UTR Hox a3 mRNA and produces a peptide of 9 KDa with the involvement of eIF2D, a non-canonical GTP-independent initiation factor. For TIE a11, it sequesters 80S ribosome on a start-stop codon combination at 19 nucleotides upstream of a GC-rich stem loop structure which blocks the 80S at the stop codon
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Castro-Roa, Daniel Augusto. "Development of an in vitro, coupled transcription-to-translation system for analysis of the interactions between the ribosome and RNA polymerase." Thesis, University of Newcastle Upon Tyne, 2012. http://hdl.handle.net/10443/1466.
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The various properties of actively transcribing RNA polymerase (RNAP) complexes with nucleic acids during different stages involve various types of regulation and different cross-talk with other cellular entities and with RNAP itself. For instance, transcription and translation are coupled in bacteria, meaning that translation takes place co-transcriptionally. The interactions of transcriptional apparatus with the translational machinery have been focused mainly in terms of gene expression, whereas the study of the physical interaction of the ribosome and the RNA polymerase remains obscure due to the lack of a system which allows such observations. In this study we have developed a pure, transcription-coupled-to-translation system in which the translocation of the ribosome can be performed in a step-wise manner towards RNAP allowing the observation of the outcomes of the interactions between the two machineries at colliding and non-colliding distances; the system also allows the positioning of RNAP in any desired elongation complex such as paused, roadblocked, backtracked, etc. We show the study of the interactions of the ribosome on different aspects of transcription elongation and also the effects on translation caused by RNAP.