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<div> <div> <div> <p>Prokaryotic Argonautes (pAgos) have been proposed as more flexible tools for gene- editing as they do not require sequence motifs adjacent to their targets for function. One promising pAgo candidate from the halophilic archaeon Natronobacterium gregoryi (NgAgo) has been the subject of intense debate regarding its potential in eukaryotic systems. NgAgo was initially claimed to edit genes in mammalian cells, but the report was retracted due to replication failure. Due to low solubility, subsequent studies refolded NgAgo and suggested that it cuts RNA but not DNA; however, mutation of the conserved active site does not abolish cleavage activity, raising the possibility of nuclease contamination. Another independent study demonstrated gene-editing via NgAgo in bacteria. These inconsistent results underscore the knowledge gap and roadblock for NgAgo-based gene-editing tool development. <br><br> </p><div> <div> <div> <p>In this work, I revisit this enzyme and characterize its function in vitro and in a bacterial system. The halophilic features of NgAgo have been neglected in the literature, leading to inconclusive results. Like other halophilic proteins, NgAgo has modified amino acid composition, leading to failure of domain identification/function prediction via sequence alignment. Indeed, using more sensitive structural alignments, I identified a new single-stranded DNA binding domain, repA, in NgAgo and other halophilic pAgos. Due to its halophilic nature, NgAgo expresses poorly in low-salt environments, with the majority of protein being insoluble and inactive even after refolding. However, soluble NgAgo indeed cuts DNA. NgAgo DNA-cleaving activity can only be abolished via mutation in the canonical PIWI domain and repA deletion, revealing a new catalytic behavior in pAgos. Moreover, NgAgo requires both repA and PIWI domains to create double- stranded DNA breaks, leading to cell death or enhancing homologous recombination, or gene- editing, at a modest level in bacteria. Rational protein engineering of NgAgo was also pursued to increase solubility. Although three out of seven mutants showed significant increases in solubility, they lost the ability to cleave DNA in E.coli. Structural modeling revealed some subtle but important differences in the protein structures, explaining why the mutants lose their function. Besides, a selection system for improving endonuclease activity was optimized for future pAgo optimization. Collectively, this work revealed that NgAgo possesses unique catalytic behavior in the pAgo family and has some gene-editing application potential. More importantly, this work expands knowledge of the pAgo family, providing a foundation for future pAgo-based gene- editing tool development. </p> </div> </div> </div> </div> </div> </div>

Esta investigación, cuya finalidad es diseñar una plataforma virtual en la que las personas puedan tener acceso a un editor de videos amigable por las herramientas que posee y fácil de utilizar; así como también, tener la posibilidad de subir sus propias creaciones a la plataforma para poder compartirlo con sus amigos o contactos, se presenta como un proyecto factible que si puede ser rentable en el tiempo y soluciona los problemas de jóvenes y emprendedores que aún no dominan el manejo de las herramientas en las plataformas de edición de video, es por ello que les proponemos como solución un editor de videos con herramientas básicas, video tutoriales cortos explicando las funciones de las herramientas, plantillas de video para una mejor edición, formatos dependiendo la red social a la que va dirigido el video, entre otras opciones, con el objetivo que el usuario se familiarice con la plataforma. La solución a este problema fue validada mediante entrevistas tanto a posibles usuarios como a expertos en el tema, tomando en cuenta las opiniones y recomendaciones que van a permitir realizar modificaciones y así llegar a desarrollar una página y prototipo de editor de video acorde a lo que nuestro segmento de clientes está buscando. Del mismo modo, se realizaron publicaciones por medio de las redes sociales como Instagram y Facebook para que nuestro proyecto llegue a más usuarios y así tener una comunicación más directa con ellos. Asimismo, se realizaron los cálculos financieros a 3 años de nuestro proyecto tomando en cuenta las simulaciones de órdenes de compra en las últimas semanas y la proyección de nuestras ventas según las inversiones en marketing que se realizarán cada cierto tiempo. De igual manera, los financiamientos a los cuales vamos a acceder por parte del banco, de los accionistas y de un financiamiento no tradicional; finalmente, mencionar nuestros ratios e indicadores financieros que sustentan la viabilidad de TOMA4. / This research, whose purpose is to design a virtual platform in where people can have access to a friendly video editor due to the tools it has and easy to use; as well as having the possibility of uploading your own creations to the platform and to share it with your friends or contacts, it is presented as a feasible project that can be profitable over time and solves the problems of young people and entrepreneurs who have not yet mastered the management of tools in video editing platforms, that is why we propose a video editor with basic tools as a solution, short video tutorials explaining the functions of the tools, video templates for better editing, formats dependent on the network social which the video is directed, among other options, with the aim that the user becomes familiar with the platform. The solution to this problem was validated through interviews with both possible users and experts on the subject, taking into account the opinions and recommendations that will allow modifications, in that way develop a page and video editor prototype according to what our customer segment is looking for. In the same way, publications were made through social networks such as Instagram and Facebook so that our project reaches more users and to have more direct communication with them. Likewise, the financial calculations for 3 years of our project were carried out taking into account the simulations of purchase orders in recent weeks and the projection of our sales according to the marketing investments that will be made from time to time. In the same way, the financing to which we are going to access from the bank, the shareholders and non-traditional financing; finally, mention our financial ratios and indicators that support the viability of TOMA4. / Trabajo de investigación

This thesis focuses on the increasingly popular shareware Blender which has growing application possibilities in various branches. The thesis aims at a general description of basic modelling techniques working with mesh objects as well as curves used for creating models and computer graphics. Part of the thesis is dedicated to a description of animation abilities of the programme, an outline of dierent methods and a more detailed description of correspondent modules. The central point of the thesis is to introduce the reader to the basic and advanced modelling techniques and their use in Blender, in the form of a tutorial.