O objetivo deste trabalho é discutir a questão da cultura livre a partir dos aspectos relativos ao domínio público. O método utilizado se baseia na observação, na análise e na síntese de dados relacionados aos temas propostos, viabilizando uma discussão baseada no estudo bibliográfico exploratório e explicativo das várias questões abordadas na pesquisa que culminam na problemática desta cultura livre e da noção e dos espaços de domínio público.Por sua vez, recorre-se ao estudo e análise de objetos que compõema pesquisa como, por exemplo, o papel da internet, da \"pirataria\", do direito autoral, da evolução dos meios de comunicação e disseminação do conhecimento, das atividades de cooperação e produção independente pelos usuários da grande rede e das políticas culturais no ambiente digital. Desse modo, são descritas e discutidas experiências de bibliotecas (Digital Public Library of America - DPLA), arquivos históricos (Europeana 1914-1918), música (Jamendo), quadrinhos e games.A partir da análise e estudo do conceito e das práticas de cultura livre estabelecidas principalmente sob o domínio público e da pirataria social, chega-se a conclusão que tais questões são fundamentais no incremento de atividades e relações estabelecidas no ambiente cooperativo da internet, que identificam os usuários como produtores e disseminadores de um ciclo cultural vivo entrelaçado pelas vias da grande rede. Com isso, reforça-se o potencial de políticas culturais a partir do ambiente digital que fortaleçam o ideal de cultura livre a partir de questões tais como a revisão das leis de direito autoral, do incentivo ao domínio público e até mesmo sobre novos entendimentos acerca do universo social-cooperativo da pirataria social em rede. / The mains objective of this paper is to discuss the issue of free culture relating it to the public domain questions. The method used is based on observation, analysis and synthesis of data related to the proposed themes, enabling a discussion based on exploratory and explanatory bibliographical study of the various issues addressed in the research culminating in the issue of this free culture and the notion of public domain.In turn, it is through the study and analysis of objects that make up the research as, for example, the role of the internet, \"piracy\", copyright law, the evolution of media and dissemination of knowledge, cooperative and independent cultural activitiesmade by the users and the large network of cultural policies in the digital environment. Some interesting activities in many cultural fields are discussed and described such as in libraries (Digital Public Library of America - DPLA), historic archives (Europeana 1914-1918), music devices and communities (Jamendo), comics and video gamesFrom the analysis and study of the concept and practice of free culture established primarily in the public domain and social piracy, we conclude that such questions are fundamental in development of activities and relationships established in the collaborative environment of the internet, which identify users as producers and disseminators of a living cultural cycle paths intertwined by the large network.Thus, it reinforces the potential of cultural policies from the digital environment that strengthen the ideal of free culture from issues such as the revision of copyright law, encouraging the public domain and even on new understandings of cooperative and social universe of social piracy.

Células de mamíferos são os hospedeiros mais frequentemente utilizados para a fabricação de proteínas biofarmacêuticas e para a produção de vacinas virais, A qualidade é um elemento-chave para o estabelecimento de um processo de bioconversão eficiente. No presente trabalho utilizamos a linhagem de células BHK- 21C13(Baby Hamster Kidney) adaptadas para cultivo em suspensão. O uso de Soro Fetal Bovino (SFB) é tradicionalmente utilizado, sendo considerado um suplemento universal, pois permite o crescimento em várias linhagens de células de mamíferos; porém, uso de SFB apresenta risco de infecção por prions, variabilidade entre lotes e aumento no custo em etapa de purificação (Downstream processing). O objetivo do presente trabalho foi comparar o cultivo de células BHK-21 C13 entre dois meios suplementados com SFB e sem SFB, através do estudo cinético para cultivo em suspensão estático e agitado com frascoT, frasco spinner e biorreator, respectivamente. Os parâmetros; Xmáx e µmáx, não foram significativamente influenciados pelo meio de cultura em cultivo estático, em cultivo com agitação em frasco spinner e também no cultivo em biorreator. O tempo de duplicação ficou próximo para todas as condições testadas. A produtividade alcançada foi: 0,032x106 cel/mL.h-1 para o meio com SFB e 0,031 X106 cel/mL.h-1 para o meio sem SFB. Ao final do processo foi possível obter uma concentração celular em torno de 4,7x106 cel/mL, tanto para o cultivo com SFB quanto para o cultivo sem SFB. Dessa forma, o uso de meio de cultivo sem SFB não alterou os principais parâmetros cinéticos, não apresentando as desvantagens do uso do SFB. / Mammalian cells are the most frequently used hosts for the production of biopharmaceutical proteins and viral vaccines. Quality is a key element for the establishment of an efficient bioconversion process. In this work, we used the cell line Baby Hamster Kidney C13 (BHK-21 C13) adapted to suspension culture was used. Fetal Bovine Serum (FBS) is traditionally used and it is considered a universal insert due to its power to increase cell growth in this kind of animal cells. However, the utilization of FBS introduces risks of infection from prions, variability between batches and increase in cost associated to purification stages (downstream processing). This work aimed to compare the kinetic behaviors of BHK-21 C13 cells in two media supplemented with FBS and without FBS using both one static and two suspension systems, T-flask, spinner flask and bioreactor respectively. The parameters; Xmax and µmax were not significantly influenced by the culture medium in T- flask culture static, in spinner flask cultivation and were neither significantly influenced by growing in culture media stirred bioreactor. The doubling time was close to all conditions tested. At the end of the growth phase it was possible to obtain a nearby cell concentration of 4.7 x 106 cells / ml, both for cultivation with FBS as for FBS without cultivation. Thus, the use of culture medium without FBS did not affect the main kinetic parameters. Besides, it does not show the disadvantages of culture media using FBS.

A hemofilia A é uma doença ligada ao cromossomo X causada pela deficiência do fator VIII da coagulação sanguínea (FVIII). O tratamento disponível consiste na terapia de reposição da proteína do fator VIII derivada do plasma (FVIIIdp) ou recombinante (FVIIIr). Atualmente, dos 7 produtos recombinantes disponíveis no mercado, 6 são produzidos em linhagens celulares de roedores. A expressão dessa proteína em sistemas celulares não-humanos pode gerar uma molécula com perfil de modificações diferente do endógeno, podendo levar a reações imunogênicas e geração de inibidores anti-FVIIIr. Em função disso, novas estratégias de produção têm sido avaliadas, como a utilização de células hospedeiras mais eficientes no que diz respeito ao potencial de expressão da proteína de interesse. Dentre as linhagens de interesse, a linhagem hepática SK-HEP-1 tem se destacado por apresentar altos níveis de expressão do FVIIIr e potencial para o cultivo em suspensão em meios livres de soro fetal bovino (SFB). Dessa forma, o objetivo deste trabalho foi avaliar a produção de FVIIIr na linhagem celular humana SK-Hep-1 comparando duas estratégias para o estabelecimento de processos de produção em condições livres de soro e em suspensão: Estratégia 1 - adaptação para tais condições da linhagem já modificada geneticamente e Estratégia 2 - modificação gênica para a expressão da proteína já em células previamente adaptadas à tais condições. Para a estratégia 1, foram geradas duas linhagens recombinantes produtoras de FVIIIr, SK-HEP-F8/Neo-E1 e SK-HEP-F8/GFP-E1 aderentes e em cultivos suplementados com SFB. Na caracterização da cinética e produção do FVIIIr as linhagens apresentaram taxas específicas máxima de crescimento (?max) de 0,064 e 0,0031h-1 produzindo 1,0 e 0,78UI/mL de FVIIIr, respectivamente. Diversos protocolos de adaptação foram empregados, entretanto, não foi possível obter sucesso na adaptação das linhagens recombinantes para condições livres de soro e em suspensão. Para a estratégia 2, as células SK-HEP-1 selvagens adaptadas ao meio de cultura livre de SFB SFMII apresentaram um valor de ?max de 0,0186h-1 e Xmax de 1,9x106cels/mL. Para as etapas de modificação gênica da linhagem selvagem foram utilizados os mesmos vetores lentivirais empregados para a geração das células recombinantes aderentes, pLVmpsvFVIII?B-Neo e pLVCMVFVIII?B-GFP. Para o primeiro, não foi possível gerar uma linhagem produtora do FVIIIr. Para o segundo, foi possível obter duas linhagens produtoras do FVIIIr com 0.14 e 0.12IU/mL de atividade pelo ensaio cromogênico. O presente trabalho mostrou que a linhagem humana Sk-Hep-1 é apropriada para a produção de altos níveis de FVIIIr. No entanto, maiores esforços devem ser voltados ao desenvolvimento de meios de cultura livres de soro específicos para a linhagem para possibilitar a produção eficiente do FVIIIr em suspensão em meios livre de soro. / Hemophilia A is a genetic X-linked disorder caused by the coagulation factor VIII (FVIII) deficiency. The current treatment is the replacement therapy with plasma derived FVIII (pdFVIII) or recombinant FVIII (rFVIII) products. Nowadays, of the seven products available in the market, six are produced in rodent expression systems, which can result in a rFVIII molecule with different post-translational modifications and may lead to immune responses to non-human epitopes. Therefore, new production strategies have been evaluated, as the use of more efficient hosts in terms of protein expression potential. Among potential cell lines, the hepatic SK-HEP-1 cell line features high levels of rFVIII production and potential for serum-free suspension culture. In face of the exposed above, the goal of this study was to evaluate rFVIII production in the SK-HEP-1 human cell line comparing two strategies for the establishment of production process in a suspension serum-free condition: strategy 1 - adaptation to these conditions of a genetic modified cell line; strategy 2 - genetic modification of an already adapted cell line to rFVIII protein expression. For strategy 1, two adherent rFVIII producer cell lines were established in serum containing medium, SK-HEP-F8/Neo-E1 e SK-HEP-F8/GFP-E1. Characterization of cell growth and rFVIII production showed a maximum specific growth rate (?max) of 0.064 and 0.00311h-1 with rFVIII production of 1.0 and 0.78UI/mL, respectively. Different adaptation protocols were used; however, it was not possible to adapt the recombinant cell lines to growth in suspension serum-free conditions. For strategy 2, the wildtype SK-HEP-1 cell line adapted growth in SFMII BSF medium, showed a ?max of 0.0186h-1 and a maximum cell concentration (Xmax) of 1.9x106cells/mL. For the genetic modification, it were employed the same lentiviral vectors used for the recombinant adherent cells generation, pLVmpsvFVIII?B-Neo and pLVCMVFVIII?B-GFP. For the first, no attempts were successful. For the second, it was possible to generate two rFVIII producer populations with 0.14 and 0.12IU/mL activity, measured by chromogenic assay. These results demonstrate that the SK-HEP-1 cell line is appropriate for the production of high levels of rFVIII. Nevertheless, efforts should be made in developing specific medium to support efficient rFVIII production in suspension and suspension serum-free conditions.

Células de mamíferos são os hospedeiros mais frequentemente utilizados para a fabricação de proteínas biofarmacêuticas e para a produção de vacinas virais, A qualidade é um elemento-chave para o estabelecimento de um processo de bioconversão eficiente. No presente trabalho utilizamos a linhagem de células BHK- 21C13(Baby Hamster Kidney) adaptadas para cultivo em suspensão. O uso de Soro Fetal Bovino (SFB) é tradicionalmente utilizado, sendo considerado um suplemento universal, pois permite o crescimento em várias linhagens de células de mamíferos; porém, uso de SFB apresenta risco de infecção por prions, variabilidade entre lotes e aumento no custo em etapa de purificação (Downstream processing). O objetivo do presente trabalho foi comparar o cultivo de células BHK-21 C13 entre dois meios suplementados com SFB e sem SFB, através do estudo cinético para cultivo em suspensão estático e agitado com frascoT, frasco spinner e biorreator, respectivamente. Os parâmetros; Xmáx e µmáx, não foram significativamente influenciados pelo meio de cultura em cultivo estático, em cultivo com agitação em frasco spinner e também no cultivo em biorreator. O tempo de duplicação ficou próximo para todas as condições testadas. A produtividade alcançada foi: 0,032x106 cel/mL.h-1 para o meio com SFB e 0,031 X106 cel/mL.h-1 para o meio sem SFB. Ao final do processo foi possível obter uma concentração celular em torno de 4,7x106 cel/mL, tanto para o cultivo com SFB quanto para o cultivo sem SFB. Dessa forma, o uso de meio de cultivo sem SFB não alterou os principais parâmetros cinéticos, não apresentando as desvantagens do uso do SFB. / Mammalian cells are the most frequently used hosts for the production of biopharmaceutical proteins and viral vaccines. Quality is a key element for the establishment of an efficient bioconversion process. In this work, we used the cell line Baby Hamster Kidney C13 (BHK-21 C13) adapted to suspension culture was used. Fetal Bovine Serum (FBS) is traditionally used and it is considered a universal insert due to its power to increase cell growth in this kind of animal cells. However, the utilization of FBS introduces risks of infection from prions, variability between batches and increase in cost associated to purification stages (downstream processing). This work aimed to compare the kinetic behaviors of BHK-21 C13 cells in two media supplemented with FBS and without FBS using both one static and two suspension systems, T-flask, spinner flask and bioreactor respectively. The parameters; Xmax and µmax were not significantly influenced by the culture medium in T- flask culture static, in spinner flask cultivation and were neither significantly influenced by growing in culture media stirred bioreactor. The doubling time was close to all conditions tested. At the end of the growth phase it was possible to obtain a nearby cell concentration of 4.7 x 106 cells / ml, both for cultivation with FBS as for FBS without cultivation. Thus, the use of culture medium without FBS did not affect the main kinetic parameters. Besides, it does not show the disadvantages of culture media using FBS.

Lawrence Lessig在Free Culture一書中探討了法律、新興科技以及媒體產業如何形成許可文化的制度，來控制我們的創作自由和取用公共財的權利，他指向一個核心問題：私益(private interest)與公益(public interest)之爭——在網路科技與智慧財產權交互作用之下的自由文化中，私益與公益如何達到平衡狀態？ 「音樂」具有可被數位化的特性，是傳播自由文化的理想範例，而筆者意欲探求的是：以臺灣的音樂產業為例，是否有可能在自由文化中形成一個讓「獲利」與「分享」並行不悖，甚至相輔相成的運作模式？最重要的是，這個模式要能發揚自由文化帶給大眾的公益，也要保障音樂工作者的私益。而在音樂產業中，「獨立音樂」次領域向來重視創作自主性，而自由文化鼓勵發想創意，兩者或許有媒合的空間，故本研究聚焦於獨立音樂。 本研究視臺灣的獨立音樂產業為資訊內容的生產與消費過程，運用Bourdieu的場域理論、資本理論、再製理論等學說，找出有哪些因素影響自由文化和獨立音樂的發展，並且探討贊成∕質疑自由文化這二種立場之間的辯證過程，以及各種利益如何折衝、妥協，從何達成平衡點。故本研究的目的包括：擬將探討結果回饋至對自由文化的省思，此為研究目的之一；嘗試建立一個自由文化中的音樂商業模式，此為研究目的之二。 本研究以深度訪談法訪談四種不同類型的獨立音樂創唱人（包括完全獨立的個體戶、社運歌曲創作者、大型唱片工業體系以外的獨立廠牌、大型唱片公司釋放出來的音樂人才，共訪談五組個人與團體），以及五家數位授權音樂網站（KKBOX、ezPeer、Omusic、StreetVoice、iNDIEVOX），並輔以參與觀察法和分析次級資料，來說明主要研究發現。 在「場域內行動者對於自由文化的認知」方面，本研究發現，「自由文化」這個名詞還不夠普及，這種現象反映出兩個事實：（一）受訪者對於自由文化的認知是分歧的：Lessig所言之自由文化是「在相當程度上開放他人據以再創造的文化」（Lessig, 2004／劉靜怡譯，2008，頁57）；然而，獨立音樂創唱人的認知則是創作行為上的自由、自由文化要能保障授用雙方的自由、自由文化等同CC授權制度、自由文化是一種行銷廣宣工具、自由文化等同網路賦予大眾使用的自由；授權音樂網站經營者們則認為「自由文化的核心概念是『服務』」。（二）Lessig的自由文化理念與實務有差距：研究結果發現，受訪之獨立音樂創唱人的開放心態和行為，要比Lessig「保守」許多，大多是停留在提供免費聆聽，少數開放下載，而其目的多半是為了廣告與宣傳效益。 在「獨立音樂創唱人的線上／線下活動與資本應用策略」方面，可以看出獨立音樂創唱人經營創唱事業的幾個重點：（一）線上∕線下資本會互相流動和兌換；（二）獨立音樂創唱人專注創作，唱片公司致力發行，二者保持地位對等的平衡關係；（三）獨立音樂創唱人有成為專職的趨勢。 在「想像一個自由文化中的音樂經營模式」方面，本研究根據各家授權音樂網站的經營特色，以及配合獨立音樂創唱人的需求、大眾的公益考量，擘畫了一個自由文化中的獨立音樂商業模式，其規劃重點在於：（一）免費與付費並存；（二）虛擬與實體並行；（三）著作權安定運作秩序；（四）經紀事務拓展人脈。整體而言，此模式試圖建構一個整合網路發表平台、付費授權網站、經紀公司的場域，各行動者之間要維繫的是一種夥伴關係，而非從屬關係。 / In his book Free Culture, Lawrence Lessig investigates how the legal system, modern technology, and media industry shape a permission culture to define our rights of consuming public goods and our freedom of creation. He orientates his core research question towards the dispute over the relative importance of private interests and public interests. In other words, how do we balance private interests against public interests under the dual impacts of the Internet technology and intellectual property rights in a free culture? “Music” can be digitalized, and it is a prime example of free culture propagation. This thesis examines the music industry in Taiwan and thereby explores the possibility of shaping an operational model that makes “profiting” and “sharing” compatible with or even complementary to each other in a free culture. More importantly, such a model should be able to promote the public interests generated from a free culture and, at the same time, secure the private interests of musicians. “Independent music,” as a subfield in the music industry, always emphasizes autonomy in creation, and a free culture encourages creation and innovation. Accordingly, we may couple independent music with free culture, and this thesis primarily deals with their relationship. This thesis treats Taiwanese independent music as a process of information production and consumption. It adopts Bourdieu’s theories about field, capital, and reproduction to identify the effective factors in the development of free culture and independent music. In addition, this thesis investigates the dialectical process between defending and challenging free culture, and how a variety of interests negotiate, compromise, and finally strike a balance among themselves. Therefore, this thesis aims to, on the one hand, use the research findings to reflect on free culture and, on the other hand, establish a music business model in a free culture. This thesis employs in-depth interviews, participant observation, and secondary data analysis to answer my research question. I interviewed four types of independent music composers/singers (a total of five cases of individuals and bands who are wholly-independent individuals, composers of social movement songs, independent brands outside the system of the large-scale record industry, or musical talents released from major record companies) and five licensed digital music websites (KKBOX, ezPeer, Omusic, StreetVoice, and iNDIEVOX). Regarding “the inside-field actors’ understanding about a free culture,” this thesis finds that the term “free culture” is not as popular as it is expected to be. This phenomenon implies two critical points. Firstly, the interviewees perceive the term free culture in different ways. According to Lessig (2004: 30),”Free Cultures are cultures that leave a great deal open for others to build upon.” However, for composers/singers of independent music, a free culture implies the freedom of creation. A free culture should be able to guarantee the freedom and rights of both original creators and users. It is equivalent to the Creative Commons licensing scheme. It is a tool of marketing and advertising. The freedom embedded in a free culture is similar to that offered by the Internet. In contrast, for managers of licensed music websites, “service” is the core concept of a free culture.” Secondly, there exists a gap between Lessig’s idea of free culture and practice. The research findings indicate that the composers/singers of independent music interviewed by the author are more “conservative” than Lessig. With the primary goals of advertising and propagating independent music, most of their works remain free for listening, while some of them are free for downloading. Concerning “the independent music composers/singers’ on-line/off-line activities and their strategies of using capital,” this thesis points out three critical points in their career management. Firstly, the on-line capital and the off-line capital flow to and exchange with each other. Secondly, these composers/singers devote themselves to creation, and record companies concentrate on issuing their works. Composers/singers and record companies maintain a peer status and a balanced relationship. Thirdly, the “composer/singer of independent music” seems to become a potential profession. With regard to “envisioning a music business model in a free culture,” this thesis designs a business model based on the managerial features of licensed music websites, the needs of composers/singers of independent music, and public interests. The model contains the following characteristics. Firstly, non-payment coexists with payment. Secondly, virtuality runs parallel with reality. Thirdly, copyrights stabilize the order of operation. Fourthly, agency transactions help establish connections among independent music composers/singers and people who are able to provide better performance opportunities. In general, this model seeks to open up a field that integrates platforms of Internet publication, paid licensing websites, and agencies. The relationship among the actors is equal rather than hierarchical.

A hemofilia A é uma doença ligada ao cromossomo X causada pela deficiência do fator VIII da coagulação sanguínea (FVIII). O tratamento disponível consiste na terapia de reposição da proteína do fator VIII derivada do plasma (FVIIIdp) ou recombinante (FVIIIr). Atualmente, dos 7 produtos recombinantes disponíveis no mercado, 6 são produzidos em linhagens celulares de roedores. A expressão dessa proteína em sistemas celulares não-humanos pode gerar uma molécula com perfil de modificações diferente do endógeno, podendo levar a reações imunogênicas e geração de inibidores anti-FVIIIr. Em função disso, novas estratégias de produção têm sido avaliadas, como a utilização de células hospedeiras mais eficientes no que diz respeito ao potencial de expressão da proteína de interesse. Dentre as linhagens de interesse, a linhagem hepática SK-HEP-1 tem se destacado por apresentar altos níveis de expressão do FVIIIr e potencial para o cultivo em suspensão em meios livres de soro fetal bovino (SFB). Dessa forma, o objetivo deste trabalho foi avaliar a produção de FVIIIr na linhagem celular humana SK-Hep-1 comparando duas estratégias para o estabelecimento de processos de produção em condições livres de soro e em suspensão: Estratégia 1 - adaptação para tais condições da linhagem já modificada geneticamente e Estratégia 2 - modificação gênica para a expressão da proteína já em células previamente adaptadas à tais condições. Para a estratégia 1, foram geradas duas linhagens recombinantes produtoras de FVIIIr, SK-HEP-F8/Neo-E1 e SK-HEP-F8/GFP-E1 aderentes e em cultivos suplementados com SFB. Na caracterização da cinética e produção do FVIIIr as linhagens apresentaram taxas específicas máxima de crescimento (?max) de 0,064 e 0,0031h-1 produzindo 1,0 e 0,78UI/mL de FVIIIr, respectivamente. Diversos protocolos de adaptação foram empregados, entretanto, não foi possível obter sucesso na adaptação das linhagens recombinantes para condições livres de soro e em suspensão. Para a estratégia 2, as células SK-HEP-1 selvagens adaptadas ao meio de cultura livre de SFB SFMII apresentaram um valor de ?max de 0,0186h-1 e Xmax de 1,9x106cels/mL. Para as etapas de modificação gênica da linhagem selvagem foram utilizados os mesmos vetores lentivirais empregados para a geração das células recombinantes aderentes, pLVmpsvFVIII?B-Neo e pLVCMVFVIII?B-GFP. Para o primeiro, não foi possível gerar uma linhagem produtora do FVIIIr. Para o segundo, foi possível obter duas linhagens produtoras do FVIIIr com 0.14 e 0.12IU/mL de atividade pelo ensaio cromogênico. O presente trabalho mostrou que a linhagem humana Sk-Hep-1 é apropriada para a produção de altos níveis de FVIIIr. No entanto, maiores esforços devem ser voltados ao desenvolvimento de meios de cultura livres de soro específicos para a linhagem para possibilitar a produção eficiente do FVIIIr em suspensão em meios livre de soro. / Hemophilia A is a genetic X-linked disorder caused by the coagulation factor VIII (FVIII) deficiency. The current treatment is the replacement therapy with plasma derived FVIII (pdFVIII) or recombinant FVIII (rFVIII) products. Nowadays, of the seven products available in the market, six are produced in rodent expression systems, which can result in a rFVIII molecule with different post-translational modifications and may lead to immune responses to non-human epitopes. Therefore, new production strategies have been evaluated, as the use of more efficient hosts in terms of protein expression potential. Among potential cell lines, the hepatic SK-HEP-1 cell line features high levels of rFVIII production and potential for serum-free suspension culture. In face of the exposed above, the goal of this study was to evaluate rFVIII production in the SK-HEP-1 human cell line comparing two strategies for the establishment of production process in a suspension serum-free condition: strategy 1 - adaptation to these conditions of a genetic modified cell line; strategy 2 - genetic modification of an already adapted cell line to rFVIII protein expression. For strategy 1, two adherent rFVIII producer cell lines were established in serum containing medium, SK-HEP-F8/Neo-E1 e SK-HEP-F8/GFP-E1. Characterization of cell growth and rFVIII production showed a maximum specific growth rate (?max) of 0.064 and 0.00311h-1 with rFVIII production of 1.0 and 0.78UI/mL, respectively. Different adaptation protocols were used; however, it was not possible to adapt the recombinant cell lines to growth in suspension serum-free conditions. For strategy 2, the wildtype SK-HEP-1 cell line adapted growth in SFMII BSF medium, showed a ?max of 0.0186h-1 and a maximum cell concentration (Xmax) of 1.9x106cells/mL. For the genetic modification, it were employed the same lentiviral vectors used for the recombinant adherent cells generation, pLVmpsvFVIII?B-Neo and pLVCMVFVIII?B-GFP. For the first, no attempts were successful. For the second, it was possible to generate two rFVIII producer populations with 0.14 and 0.12IU/mL activity, measured by chromogenic assay. These results demonstrate that the SK-HEP-1 cell line is appropriate for the production of high levels of rFVIII. Nevertheless, efforts should be made in developing specific medium to support efficient rFVIII production in suspension and suspension serum-free conditions.

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Technology has become closely interwoven with our lives, positioning us as authors of large and diverse databases. These extensive collections of digital assets will be left behind as digital legacies after users eventually die. Addressing the inevitability of death in digital systems, including considerations for pre-configuring, or accessing these digital legacies, calls for thanatosensitivity in design. As a relatively new field, thanatosensitive HCI research on digital legacy has primarily focused on data storage and security as well as social networking systems. However, people might create online content that can be of relevance postmortem beyond the next of kin and private network, such as contributions to digital commons communities. In my research, I explore challenges and opportunities for thanatosensitive design in the context of digital commons communities by examining two design cases as samples of that area: GitHub and the Free Music Archive. Through a process inspired by programmatic design research, I followed a mixed method approach including literature reviews, interviews, workshop sessions, and iterative design synthesis. The outcome is a guidebook consisting of annotated portfolios with design exemplars for each design case, accessible to different stakeholders for further collaboration. Drawing on the annotations and intersections between both cases, I frame the knowledge contributions of this study as insights from the design process, aiming to provide directions for future research on thanatosensitivity in systems for digital commons contributions.

Le cancer du sein est une maladie complexe et difficile à caractériser. Parmi les différents sous-types moléculaires, les tumeurs du sein Triple-Négatives (TN) sont particulièrement agressives et de mauvais pronostic. Elles sont caractérisées par une absence d’expression des récepteurs aux œstrogènes (ER), à la progestérone (PR), l’absence de surexpression du récepteur Human Epidermal growth factor 2 (HER2) et de fréquentes mutations sur les gènes BRCA1/2 (profil « BRCAness »). En absence de thérapies ciblées efficaces, de nombreux traitements ciblés notamment les inhibiteurs de poly-ADP-ribose polymérases (anti-PARPs) sont actuellement en cours de développement, en recherche préclinique et clinique. Basés sur le principe de létalité synthétique, les anti-PARPs ciblent les propriétés BRCAness des tumeurs TN. Dans ce contexte, ces travaux de recherche ont été orientés sur le développement d’outils diagnostics afin d’optimiser l’efficacité des anti-PARPs sur des tumeurs TN. Pour ce faire, dans un premier temps, des cultures cellulaires en 3D via la technique Liquid Overlay ainsi que des tests de cytotoxicités associés ont été développés, à partir des lignées cellulaires MDA-MB-231 et SUM1315 de phénotype TN. Ces deux modèles de sphéroïdes ont ensuite été optimisés/normalisés dans un milieu de culture synthétique intitulé OPTIPASS (BIOPASS). Dans un deuxième temps, l’efficacité d’un co-traitement combinant l’anti-PARP1 Olaparib à faibles et à fortes doses et la radiothérapie fractionnée (5x2 Gy) a été modélisée sur les deux lignées MDA-MB-231 et SUM1315, en conditions 2D et 3D. Ces expériences ont clairement mis en évidence un effet potentialisateur de l’Olaparib sur la radiothérapie (i) en présence de faibles doses de cet anti-PARP (5 µM ou inférieur) (ii) à long terme et (iii) en présence d’un fractionnement maximum (5x2 Gy). De plus, les lignées tumorales TN étudiées présentaient des différences de sensibilité vis-à-vis du co-traitement. Ainsi, une analyse transcriptomique in silico a mis en évidence des profils très différents de ces lignées hautement métastatiques et très agressives. Notamment, la lignée SUM1315 semblait présenter un engagement neuronal, suggérant son origine métastatique cérébrale. Ces résultats encourageants pourraient ouvrir de nouvelles perspectives pour le traitement des métastases cérébrales de tumeurs mammaires TN, très fréquentes chez ce sous-type. Dans un troisième temps, afin de mieux caractériser le mode d’action de l’Olaparib sur ces modèles de sphéroïdes, un dérivé fluorescent de l’Olaparib, l’Ola-FL, a été synthétisé et caractérisé. L’analyse de la pénétration et de la distribution de l’Ola-FL au sein des sphéroïdes MDA-MB-231 et SUM1315 a mis en évidence une distribution rapide et homogène du composé ainsi que sa persistance après 3h d’incubation, dans toute la profondeur des sphéroïdes et notamment dans les zones hypoxiques centrales. Enfin, l’analyse de la co-expression de deux pompes Multidrug Resistance (MDR) majeures, la MRP7 et la P-gp après le traitement des deux lignées TN avec l’Olaparib, a mis en évidence sur les cultures 2D, une expression de type relai de la MRP7 et la P-gp. Sur les sphéroïdes traités avec une faible dose d’Olaparib à long terme, une expression basale de la MRP7 et une surexpression de la P-gp ont été détectées, au sein des cellules résiduelles périphériques des sphéroïdes. Ces résultats mettent clairement en évidence l’implication des pompes d’efflux dans les mécanismes de résistances à l’Olaparib, dans ces tumeurs agressives. L’ensemble des résultats issus de la modélisation de l’action de l’Olaparib sur des sphéroïdes MDA-MB-231 et SUM1315 laissent supposer sa plus grande efficacité à faible dose et à long-terme, notamment dans les zones hypoxiques des sphéroïdes, probablement aussi à l’origine de son effet potentialisateur avec la radiothérapie. / Breast cancer is a very complex and heterogeneous disease. Among the different molecular subtypes, Triple-Negative (TN) breast cancers are particularly aggressive and of poor prognosis. TN tumours are characterized by a lack of estrogen receptors expression (ER), progesterone receptors expression (PR), the absence of Human Epidermal growth factor receptor 2 overexpression (HER2) of the frequent mutations on BRCA1 / 2 genes ("BRCAness" phenotype). In the absence of effective targeted therapies, many targeted therapies including poly-ADP-ribose polymerase inhibitors (anti-PARPs) are currently under development in preclinical and clinical studies. Based on the synthetic lethality concept, the anti-PARPs specifically target the BRCAness properties of TN tumors. In this context, these works were focused on the development of diagnostic tools for the optimization of TN tumours treatment with anti-PARPs. For this, firstly, 3D cell cultures formed with the Liquid Overlay technique as well as associated cytotoxicity tests were developed, from the TN breast cancer cell lines MDA-MB-231 and SUM1315. These two spheroid models were then optimized and standardized in a synthetic culture medium called OPTIPASS (BIOPASS). Secondly, the efficacy of a co-treatment combining anti-PARP1 Olaparib at low and high doses and fractioned radiotherapy (5x2 Gy) was analyzed on the two cell lines MDA-MB-231 and SUM1315 cultured in 2D and 3D conditions. These experiments clearly demonstrated a potentiating effect of Olaparib on radiotherapy (i) in presence of low doses of this anti-PARP (5 μM or inferior) (ii) at long term and (iii) in presence of the maximum fractionation (5x2 Gy). In addition, these two TN cell lines showed a heterogeneous sensitivity to the co-treatment. Thus, an in silico transcriptomic analysis revealed very different profiles of these highly metastatic and highly aggressive cell lines. Notably, the SUM1315 cell line presented a neuronal commitment, suggesting its cerebral metastatic origin. These promising results could open up new perspectives for the treatment of TN tumours brain metastases, which are very common in this subtype. Thirdly, in order to better characterize the mode of action of Olaparib on these spheroid models, a fluorescent derivative of Olaparib, Ola-FL, was synthesized and characterized. The analysis of Ola-FL penetration and distribution in MDA-MB-231 and SUM1315 spheroids showed a rapid and homogeneous distribution of the compound as well as its persistence after 3h of incubation, in all the depth of the spheroids and especially in the central hypoxic zones. Finally, the analysis of the co-expression of two major Multidrug Resistance (MDR) pumps, MRP7 and P-gp after the treatment of the two TN lines with Olaparib, revealed on 2D cultures, a relay type expression of the MRP7 and the P-gp. On spheroids treated with a low dose of Olaparib art long term (10 days), a basal expression of MRP7 and an overexpression of P-gp were detected in the peripheral residual cells of the spheroids. These results clearly highlighted the involvement of these efflux pumps in Olaparib resistance mechanisms, in these aggressive tumors. All the results resulting from the modeling of the action of Olaparib on MDA-MB-231 and SUM1315 spheroids suggest its greater efficacy at low dose and at long-term, especially in the hypoxic zones of the spheroids. This parameter might be probably at the origin of its potentiating effect with radiotherapy.

Copyright is conventionally justified as an incentive to produce and disseminate works of authorship. We can justify and theorise copyright more richly, not least because empirical evidence does not support the incentive narrative. Rather than focussing on quantitative matters such as the number of works incentivised and produced, we should consider copyright's qualitative influence on culture. A threshold objection to such an approach is the risk of cultural paternalism. This objection can be overcome. Rather than specifying paternalistic standards of merit for works, we can target the conditions under which their creation and consumption takes place. I argue, firstly, that we should adopt the following high-level principles: (i) that the conditions of creation and consumption of works should be conducive to democratic deliberation (democracy) and (ii) that they should facilitate the development of human capabilities (autonomy). Secondly, I propose that we pursue three mid-level objectives, which are helpful indicia of democracy and autonomy: - a fair and wide distribution of communicative and cultural power (inclusiveness); - diversity in the content and perspectives available to the public (diversity); and - conditions that permit authors and users of works to engage rigorously with the conventions of the media in which they operate (rigour). It is often said that copyright obstructs important qualitative objectives, like freedom of expression, and that we could better pursue these goals by weakening copyright and relying on non-proprietary alternatives. My approach produces a more optimistic, but also more complicated, view of copyright. While copyright's qualitative influence is not optimal, reductions in the strength and scope of copyright sometimes produces conditions and incentive structures that are worse for inclusiveness, diversity and rigour than stronger copyright. For example, both attention and wealth are highly concentrated in networked information economies driven by free sharing of content, and this is bad for diversity or inclusiveness. Online business models, based on surveillance of users' consumption of free works, are corrosive of autonomy and democracy. Merely removing copyright-based restrictions on the sharing of works is not a panacea for copyright's ills. A qualitative theory such as mine equips us to better understand and calibrate more richly the trade-offs involved in copyright policy decisions, and encourages us to treat copyright as part of a broader, qualitatively-oriented information and cultural policy.