Therapeutic proteins, a.k.a. biopharmaceuticals, are most commonly produced in expression systems derived from Chinese Hamstery Ovary (CHO) cells, thanks to great capacity of post-translational modifications like secretation, folding and glycosylation. The engineering of cells for regulation of protein expression has many options including knock-in and knock-out of genes, epigenetic studies or improvement of the expression casette of the protein of interest by e.g. promotor variants or modifications of the 5’ and 3’ untranslated region (UTR). The 3’-UTR is therefore a good optimization candidate for attempting to achieve increased expression of therapeutic proteins. The final aim of this study was to identify and design 3’-UTRs for improved expression of therapeutic proteins in HyClone™ CHO cells from GE Healthcare Bio-Sciences AB (GEHC). The impact goal is to increase the efficiency and lower the costs for pharmaceutical companies when producing biopharmaceuticals in the HyClone™ CHO cell line, leading to increased accessibility of monoclonal antibodies (mAbs) on the pharmaceutical market. The study was initiated with bioinformatic analysis of the CHO cell transcriptome from a set of RNA-seq data of HyClone™ CHO to find high expressing, context independent genes. The 3’-UTRs from the best candidate genes were used for construction of plasmids for expression of a Fc-eGFP fusion protein. Nine selected 3’-UTRs were designed, synthesized and cloned into a parent plasmid (pGE0520) creating nine plasmid variants (pGE0523-531). The constructed plasmids were used for evaluation with site directed integration (SDI) into the HyClone™ CHO cell line and expression analysis were performed by flow cytometry and antibody titer measurements from cells with successfully integrated plasmid sorted by fluorescence-activated cell sorting (FACS).   Result show a significant effect on protein expression when using different variants of 3’-UTRs. Two variants, pGE0524 and pGE0526, competing with the parent plasmid in expression levels and integration efficiency from SDI, making them candidates for further investigations against the parent plasmid. Results also show good correlation between flow cytometry data from pre- and post-sorting, which can make research for further 3’-UTRs more efficient by evaluations and prediction of expression levels before cell sorting.

Submitted by PPG Hist?ria (historia-pg@pucrs.br) on 2017-11-03T16:34:10Z No. of bitstreams: 1 TESE - LUCIANA DA COSTA DE OLIVEIRA - VERS?O MENOR.pdf: 14177891 bytes, checksum: 99e2fd93dd19fb5b7b8d954e21be268a (MD5) / Rejected by Caroline Xavier (caroline.xavier@pucrs.br), reason: Devolvido devido a 1) Diferen?a de entre o t?tulo da capa/t?tulo da publica??o cadastrada, e o t?tulo do restante do material (folha de rosto, ficha catalogr?fica, folha de aprova??o da banca). 2) Data de defesa cadastrada na publica??o (31/08/2017), est? diferente da data do arquivo PDF (22/08/2017) on 2017-11-16T17:39:40Z (GMT) / Submitted by PPG Hist?ria (historia-pg@pucrs.br) on 2017-11-20T19:47:40Z No. of bitstreams: 1 final_corrigido.pdf: 13712674 bytes, checksum: 0fae3ff53f2742992410ac18fecb8139 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-11-28T18:59:51Z (GMT) No. of bitstreams: 1 final_corrigido.pdf: 13712674 bytes, checksum: 0fae3ff53f2742992410ac18fecb8139 (MD5) / Made available in DSpace on 2017-11-28T19:05:50Z (GMT). No. of bitstreams: 1 final_corrigido.pdf: 13712674 bytes, checksum: 0fae3ff53f2742992410ac18fecb8139 (MD5) Previous issue date: 2017-08-22 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The main objective of this doctoral thesis is to analyze the way in which the image of the gaucho and the ga?cho was constructed and consecrated in the Argentine, Uruguayan and Brazilian painting of the 20th century, especially in the painting by Ces?reo Bernaldo de Quir?s (1879-1968), Pedro Figari (1861-1938) and Pedro Weing?rtner (1853-1929). In this perspective, starting from theoretical-methodological issues that include not only the dialogues that the images interlock, but, especially, their temporal and mnemic links, it was also aimed to establish a relation between the work that celebrated the gaucho and the ga?cho in the twentieth century with those that preceded it. Thus combining the nascent image of the eighteenth and nineteenth centuries with that which was consecrated in the twentieth century, it was intended to define the diverse and multiple threads that, intertwined, produced a wide and complex network of meanings around the image of the gaucho and the ga?cho. These same threads, woven by times and memories, by yearnings and desires, besides particularizing the image of the man from the pampa, also reveals their uses and their functions. / La presente tesis doctoral, tiene como objetivo principal analizar la forma con lo cual la imagen gaucha y del ga?cho fue construida y consagrada en la pintura argentina, uruguaya y brasile?a del siglo XX, en especial en la pintura de Ces?reo Bernaldo De Quir?s (1879-1968), Pedro Figari (1861-1938) y Pedro Weing?rtner (1853-1929). En ese sentido, y partiendo de las cuestiones te?rico metodol?gicos que perciben no solamente los di?logos que las im?genes se traban entre s?, pero, sobre todo, en sus encadenamientos temporales y mn?micos, se ha objetivado, tambi?n, establecer una relaci?n entre la obra que ha celebrado el gaucho y el ga?cho en el siglo XX junto aquellas que los precedieron. As?, conjugando la imagen de los siglos XVIII y XIX, se pretendi?, pues, definir los diversos y m?ltiples hilos que entrelazados, produjeron una amplia y compleja red de significados acerca de la imagen del gaucho y del ga?cho. Esos mismos hilos, tramados por el tiempo y por memorias, por anhelos y deseos, aparte particularizar la imagen del hombre del pampa, igualmente revela sus usos y funciones. / A presente tese de doutorado tem como objetivo central analisar a forma com a qual a imagem do gaucho e do ga?cho foi constru?da e consagrada na pintura argentina, uruguaia e brasileira do s?culo XX, especialmente na pintura de Ces?reo Bernaldo de Quir?s (1879- 1968), Pedro Figari (1861-1938) e Pedro Weing?rtner (1853-1929). Nesse sentido, partindo de quest?es te?rico-metodol?gicas que percebem n?o apenas os di?logos que as imagens travam entre si, mas, sobretudo, seus encadeamentos temporais e mn?micos, objetivou-se, tamb?m, estabelecer uma rela??o entre a obra que celebrou o gaucho e o ga?cho no s?culo XX junto das que lhe antecederam. Assim conjugando a imagem nascente dos s?culos XVIII e XIX junto daquela que foi consagrada no XX, pretendeu-se, pois, definir os diversos e m?ltiplos fios que, entrela?ados, produziram uma ampla e complexa rede de significa??es no entorno da imagem do gaucho e do ga?cho. Esses mesmos fios, tramados por tempos e mem?rias, por anseios e desejos, afora particularizar a imagem do homem do pampa, igualmente descortina seus usos e suas fun??es.

Made available in DSpace on 2015-04-14T14:41:31Z (GMT). No. of bitstreams: 1 437496.pdf: 12123309 bytes, checksum: 3b8d667c4708e630a619d67c40d65377 (MD5) Previous issue date: 2011-12-19 / The media has changed due to the emergence of new technologies. Traditional media has segmented the content to meet the demand of the population. The popular newspapers were created, reaching readers who did not use to buy printed papers because of the high cost. With a peculiar language, bold colors and eye-catching headlines, such publications caught the attention of part of a less favored population. With the advances in technology, popular journalism has also changed, following the natural order and also gaining ground in the online medium. Di?rio Ga?cho , a popular publication in the state of Rio Grande do Sul, which covers readers from classes "B", "C" and "D" will be analyzed in this academic research in order to show how the popular journalism on the Internet happens. In addition, the readers interactivity relationships of this popular newspaper with the web will be observed. A few years ago, these readers had no access to the network, but due to the increasing purchasing power, they started standing out as one of the classes of higher growth in the Brazilian market. It is clear, then, that class C seeks information in the online medium and contributes to the means by sending content to the popular papers on the web. / Os meios de comunica??o sofreram mudan?as devido ao surgimento de novas tecnologias. As m?dias tradicionais segmentaram o conte?do para atender ? demanda da popula??o. Os jornais populares foram criados e atingiram leitores que, em sua maioria, n?o costumavam comprar impressos devido ao alto custo. Com uma linguagem peculiar, cores arrojadas e manchetes chamativas, esse tipo de publica??o conquistou uma parte da popula??o, at? ent?o, menos favorecida. Com os avan?os tecnol?gicos, o jornalismo popular tamb?m se modificou, mas seguiu a ordem natural e ganhou espa?o, ainda, no meio online. O Di?rio Ga?cho, publica??o popular do Rio Grande do Sul, que abrange um p?blico leitor de classes B, C, e D, ser? analisado, nesta pesquisa acad?mica, com o intuito de destacar como d?-se o jornalismo popular na internet. Ser?o observadas as rela??es de interatividade dos leitores deste jornal popular via web, sendo que, h? poucos anos, estes leitores n?o tinham acesso ? rede, mas, devido ao aumento do poder de consumo, passaram a destacar-se como uma das classes de maior crescimento no mercado brasileiro. Percebe-se, ent?o, que estas pessoas buscam informa??es no meio online e contribuem com os meios atrav?s de envio de conte?dos para os jornais populares na web.

本論文的研究主題在於：透過分析日治時期的「台灣寫真帖」中的視覺圖像，釐析「台灣」如何被觀看？編者如何編排一套「應該觀看台灣的方式」？在此視覺論述中，什麼被選擇？什麼被捨棄？相較先行研究者以後世眼光所分類之「類型」，可能切斷寫真既有的編輯思維及脈絡，本文乃將寫真帖重新置回原初的時代脈絡來觀察。 　　經由本文的分析，有以下幾點發現。首先，攝影傳入台灣及其發展情況，由辭彙轉譯的過程及拍攝、出版者來看，本文論析之「台灣寫真帖」可視為殖民者眼光所看見的「台灣」。而寫真帖的台灣視覺論述，可歸納以下幾項特性：（一）地理空間架構下的台灣，將台灣編制為北而南、由西向東的觀看順序；而東、西台灣的差異，呼應統制者經營台灣的態度及政策。（二）時間遞延下的台灣，歷經明治、大正、昭和不同時期各有不同的時代特性。（三）蕃族、自然資源、台灣風俗等視覺元素，並不特定被擺置於某一地理空間或某段時代，但依舊隱含殖民的凝視。綜論之，台灣寫真帖可以巨視和微觀的角度切入：巨視者與1908年縱貫鐵路通車密切相關，微觀則以1934年《台灣教材寫真教科書》分類框架最具代表性。 　　將寫真帖與其他文類對比參照後發現，其間多有互相複製、挪用的情形，可見殖民者建制台灣視覺論述的用心，並不僅見於寫真帖，亦以各種綜論性的台灣相關論著、教育讀本、美術作品的視覺圖像，相互作用以「召喚」個體（被觀看的「台灣」）進入主體（殖民者的知識體系）。「台灣」在殖民論述下如何被操作及轉化，本文以北白川宮、新高山、香蕉為例討論，援引羅蘭巴特的神話學概念，分析各樣文類構築的「台灣神話」。最後，從「觀看」到「觀光」的實踐，將觀光指南中的「台灣」與寫真對照，以「日月潭杵歌」與「台灣神社」為例，試論神話形構後，傳遞過程中可能的變異性。 / This research aims to interpret how Taiwan was observed by analyzing the visual icons in the “Taiwan shashin cho (Taiwan photo albums/台湾写真帖)” during the Japanese Colonial period. In addition, we will discuss about the way to see Taiwan arranged by the editor, and which factors was chosen or abandoned in the editorial process. Regarding prior researches, the classifications of former researchers possibly break both the thinking and the context of photo-album editors in later versions. We purpose to place the “Taiwan shashin cho” in the original context of the old times. 　　With our analysis, our research findings are: First of all, based on the development of photography in Taiwan, the Taiwanese translation of photography as well as the photographers and publishers, the “Taiwan shashin cho” in this research can represent the “Taiwan” image in the colonial perspective. We generalize three characteristics in the basis of the “Taiwan shashin cho”: A. Geographically, the order to view Taiwan by region was arranged from the north to the south and then the west to the east. The differences between western and eastern Taiwan corresponded to the colonial attitude and policy in this era. B. Chronologically, the “Taiwan shashin cho” implicated individual temporal characteristics in each section of the Japanese Colonial period, respectively. C. Generally, the visual factors such as aborigines, natural resources, and the customs and traditions cannot be categorized into specific regions and period, though they still implied the gaze of the rulers. In summary, there are two kinds of perspectives to analyze “Taiwan shashin cho”: macro and micro. The macro perspective is closely related to the operation of the North-South Railway. On the other hand, the category framework of the “Taiwan kyozai shashin kyokashu (Sources of Photography for Taiwan Textbook/台湾教材写真教科書)” in 1934 is the most typical micro perspective. 　　Secondly, to compare with other archives, numerous visual icons are reproduced and appropriated from the “Taiwan shashin cho”. Thus, we can conclude that the rulers spare no efforts to build up the visual discourse of Taiwan images with the “Taiwan shashin cho”, those treatises, educational textbooks and fine art works. By these archives, the individual (the Taiwan observed) is interpellated to the subject (the knowledge of the rulers). Based on the Mythologies by Roland Barthes, this thesis take the “Kitashirakawa-no-miya Yoshihisa-shinno (Imperial Prince Kitashirakawa Yoshihisa/北白川宮能久親王)”, the “niitakayama(former name of Yushan/新高山)”, and the “bananas” as examples to understand how “Taiwan” was operated and transformed. Furthermore, how the forecited archives constructed the “Taiwan Myths”. 　　Finally, this research compares the “Taiwan shashin cho” with the “Taiwan ryoco annai(Guidebooks of Taiwan Railway Tour/台湾鉄道旅行案内)” in the practice of transforming “seeing” to “sightseeing” for discussing about the divergences in the process of propagating myth with two illustrations, “ Zizugezu tan kineuta(Pestle Dancing in Sun Moon Lake/日月潭杵唄)” as well as “Taiwan Zinzha(Taiwan shrine/台湾神社)”.

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Made available in DSpace on 2014-10-09T12:43:49Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:10:05Z (GMT). No. of bitstreams: 1 06777.pdf: 5273885 bytes, checksum: b88f10c3d25adde0595b62adc866d4ee (MD5) / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

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This thesis is devoted to the study and application of constraint-based metabolic models. The objective was to find simple ways to handle the difficulties that arise in practice due to uncertainty (knowledge is incomplete, there is a lack of measurable variables, and those available are imprecise). With this purpose, tools have been developed to model, analyse, estimate and predict the metabolic behaviour of cells. The document is structured in three parts. First, related literature is revised and summarised. This results in a unified perspective of several methodologies that use constraint-based representations of the cell metabolism. Three outstanding methods are discussed in detail, network-based pathways analysis (NPA), metabolic flux analysis (MFA), and flux balance analysis (FBA). Four types of metabolic pathways are also compared to clarify the subtle differences among them. The second part is devoted to interval methods for constraint-based models. The first contribution is an interval approach to traditional MFA, particularly useful to estimate the metabolic fluxes under data scarcity (FS-MFA). These estimates provide insight on the internal state of cells, which determines the behaviour they exhibit at given conditions. The second contribution is a procedure for monitoring the metabolic fluxes during a cultivation process that uses FS-MFA to handle uncertainty. The third part of the document addresses the use of possibility theory. The main contribution is a possibilistic framework to (a) evaluate model and measurements consistency, and (b) perform flux estimations (Poss-MFA). It combines flexibility on the assumptions and computational efficiency. Poss-MFA is also applied to monitoring fluxes and metabolite concentrations during a cultivation, information of great use for fault-detection and control of industrial processes. Afterwards, the FBA problem is addressed. / Llaneras Estrada, F. (2011). Interval and Possibilistic Methods for Constraint-Based Metabolic Models [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/10528

No / The identification of an agent that is selectively activated by a cytochrome P450 (CYP) has the potential for tissue specific dose intensification as a means of significantly improving its therapeutic value. Towards this goal, we disclose evidence for the pathway of activation of a duocarmycin analogue, ICT2700, which targets CYP1A1 for biological activity.