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Dinámica de la acetilación de "Escherichia coli" para mejorar sus capacidades biotecnológicas
Full text linkE. coli es uno de los microorganismos más utilizados con fines biotecnológicos y como modelo de investigación, pues ha sido ampliamente estudiado y se conoce su genoma completo, así como su proteoma y transcriptoma. Sin embargo, el uso de E. coli como organismo huésped en bioprocesos (producción de proteínas recombinantes) puede conllevar una serie de inconvenientes, como la carga metabólica, el desbordamiento del acetato y el conocimiento limitado de algunas de sus modificaciones postraduccionales, como la acetilación de lisinas. La Nε-acetilación de las lisinas es una modificación postraduccional que está relacionada con el metabolismo del acetato a través de los intermediarios acetil-CoA y acetil-P, que actúan como dadores de acetilos. Esta modificación se produce en muchas enzimas del metabolismo central y está implicada en la regulación de los procesos metabólicos, los procesos de traducción, la localización celular y las interacciones de las proteínas con otras moléculas.
El principal objetivo de esta Tesis Doctoral fue comprender los factores que gobiernan la dinámica de la acetilación de proteínas y su efecto sobre el control del metabolismo central de E. coli, con el fin de diseñar estrategias que garanticen la optimización celular para la producción de proteínas recombinantes. Así, se estudiaron las implicaciones del uso de distintos medios de cultivo en la producción de proteínas recombinantes, y en el perfil proteico y el nivel de acetilación en E. coli (Capítulos 3 y 6). Además, también se emplearon distintos sistemas de expresión y cepas deficientes en genes involucrados en el metabolismo del acetato y en la acetilación de lisinas, para optimizar la producción de proteínas recombinantes y conocer su nivel de acetilación (Capítulos 3 y 4). Por último, se estudiaron las implicaciones de la acetilación en una importante ruta metabólica, la ruta de biosíntesis de pirimidinas de novo (Capítulo 5). Los resultados obtenidos permitirán diseñar estrategias racionales, tanto por ingeniería de cepas como por la selección de medios de cultivo, para aumentar las capacidades biotecnológicas de la bacteria y conocer las implicaciones de la acetilación en estos procesos.
A partir de los resultados obtenidos en esta Tesis Doctoral, las principales conclusiones extraídas son: 1) La producción de proteínas recombinantes se ve favorecida utilizando un medio complejo y glicerol como fuente de carbono. 2) La utilización de la cepa ΔackA supone una reducción en el desbordamiento del acetato y un aumento de 5 veces en la producción de proteínas recombinantes cuando se emplea el sistema de expresión óptimo. 3) La carga metabólica producida por la transcripción de los vectores de expresión conduce a una disminución de la producción de proteínas recombinantes cuando se emplean sistemas de expresión de alto número de copias con promotores fuertes. 4) La ruta de biosíntesis de pirimidinas de novo es regulada por la acetilación de lisinas en la enzima OPRTasa. La acetilación de la OPRTasa en las lisinas 26 y 103 produce una disminución de la actividad que puede ser revertida por la desacetilasa CobB. 5) La abundancia relativa de proteínas en E. coli depende de las condiciones de cultivo, y la identidad y el número de lisinas acetiladas depende de la abundancia relativa de las proteínas. 6) El nivel de acetilación en E. coli depende principalmente del desbordamiento de acetato, siendo mayor en medio complejo. 7) Se han encontrado lisinas con diferencias en su nivel de acetilación entre las distintas condiciones de cultivo en el ciclo del glioxilato, el ciclo de los ácidos tricarboxílicos y la asimilación del nitrógeno, por lo que estas vías podrían estar reguladas por acetilación de forma diferencial.
En conclusión, los resultados de esta Tesis Doctoral ponen de manifiesto la interconexión de la regulación por acetilación de las rutas del metabolismo central con las condiciones de cultivo en E. coli. Por lo tanto, el empleo de distintas condiciones de cultivo determina los flujos del metabolismo central en general, y en concreto del desbordamiento del acetato, lo que tiene repercusiones en la abundancia relativa de las proteínas, en el nivel de acetilación de lisinas y, en consecuencia, en la producción de proteínas recombinantes.E. coli is one of the most widely used microorganisms for biotechnological purposes and as a research model, as it has been extensively studied and its complete genome, proteome and transcriptome are known. However, the use of E. coli as a host organism in bioprocesses (recombinant protein production) can lead to several drawbacks, such as metabolic burden, acetate overflow and limited knowledge of some of its posttranslational modifications, such as lysine acetylation. Nε-acetylation of lysines is a posttranslational modification that is related to acetate metabolism via acetyl-CoA and acetyl-P intermediates, which act as acetyl donors. This modification occurs in many enzymes of central metabolism and is involved in the regulation of metabolic processes, translation processes, cellular localisation and interactions of proteins with other molecules.
The main objective of this PhD Thesis was to understand the factors that govern the dynamics of protein acetylation and its effect on the control of central metabolism in E. coli, in order to design strategies that guarantee cell optimisation to produce recombinant proteins. Thus, the implications of the use of different culture media on the production of recombinant proteins, and on the protein profile and acetylation level in E. coli were studied (Chapters 3 and 6). In addition, different expression systems and deficient strains in genes involved in acetate metabolism and lysine acetylation were also used to optimise the production of recombinant proteins and to determine their acetylation level (Chapters 3 and 4). Finally, the implications of acetylation in an important metabolic pathway, the de novo pyrimidine biosynthesis pathway, were studied (Chapter 5). The results obtained will allow the design of rational strategies, both by strain engineering and culture media selection, to increase the biotechnological capabilities of the bacteria and to understand the implications of acetylation in these processes.
From the results obtained in this PhD Thesis, the main conclusions drawn are: 1) The production of recombinant proteins is favoured by using a complex medium and glycerol as a carbon source. 2) The use of the ΔackA strain results in a reduction in acetate overflow and a 5-fold increase in recombinant protein production when the optimal expression system is employed. 3) The metabolic burden produced by transcription of expression vectors leads to a decrease in recombinant protein production when using high copy number expression systems with strong promoters. 4) The de novo pyrimidine biosynthesis pathway is regulated by the acetylation of lysines in the OPRTase enzyme. Acetylation of OPRTase at lysines 26 and 103 results in a decrease in activity that can be reversed by CobB deacetylase. 5) The relative abundance of proteins in E. coli depends on culture conditions, and the identity and number of acetylated lysines depends on the relative abundance of proteins. 6) The level of acetylation in E. coli depends mainly on acetate overflow, being higher in complex medium. 7) Lysines with differences in their level of acetylation between culture conditions have been found in the glyoxylate cycle, the tricarboxylic acid cycle and nitrogen assimilation, so these pathways could be differentially regulated by acetylation.
In conclusion, the results of this PhD Thesis show the interconnection of acetylation regulation of central metabolic pathways with culture conditions in E. coli. Thus, the use of different culture conditions determines the fluxes of central metabolism in general, and specifically of acetate overflow, which has an impact on the relative abundance of proteins, the level of lysine acetylation and, consequently, the production of recombinant proteins
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
Author Under Sail The Imagination of Jack London, 1893-1902
No full textIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Intro -- Title Page -- Copyright Page -- Dedication -- Contents -- Acknowledgments -- Introduction -- 1. Spirit Truth -- 2. From Absorption to Theatricality and Back Again -- 3. "I Will Build a New Present" -- 4. Sons as Authors -- 5. Fathers as Publishers -- 6. The Daughter as Author -- 7. Lovers as Authors -- 8. At Sea with the Family -- 9. Yellow News, Yellow Stories -- 10. The Return Home -- Notes -- Bibliography -- Index -- About Jay WilliamsIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Description based on publisher supplied metadata and other sources.Electronic reproduction. Ann Arbor, Michigan : ProQuest Ebook Central, YYYY. Available via World Wide Web. Access may be limited to ProQuest Ebook Central affiliated libraries
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