abstract: Using an adapted Straussian Grounded Theory approach, and as a participant observer, data from members of a Facebook group made up of parents and caretakers of infants or children with Gastro Esophageal Reflux Disease (GERD) were collected and analyzed. During the first exploratory phase, 31 semi-structured interviews were conducted with 25 theoretically sampled members of the group. During the second phase, 604 postings (original and comments) created by members of the online social media group, for one week, were analyzed. The study explored various dimensions of informal learning in this space. These included what learning strategies members used, what types of knowledge were encouraged and shared, how community within the group was characterized and its role in the learning space, what factors led members to join and share knowledge, and what patterns of participation existed in the group. The findings revealed a core concept of a disconnect between group members and their medical community that drove participation in the online health-related social media group, as well as a substantive theory of learning to survive. A new framework for understanding online informal learning spaces in social media was developed and proposed. It was adapted from Wenger’s Community of Practice and Gee’s Affinity Spaces. Its key components include a disconnect; inherent learning processes; community and space characteristics; and types of knowledge that are encouraged and available. Findings also contributed to a better understanding of online information-seeking behaviors by introducing a new model of information-seeking within online social media groups. This model includes the stages of initiating, lurking, and browsing; requesting information; being guided by a highly knowledgeable member; reconciling; applying; and appraising. The model is a continuous cycle with entry and exit permitted at each stage based on the learner’s needs. In addition, this study’s findings demonstrate that social media spaces are a viable avenue for the transferring of experience-based knowledge. / Dissertation/Thesis / Doctoral Dissertation Educational Technology 2018

abstract: This dissertation shares the results of a study of the community of the mobile augmented reality game Pokémon Go. It also serves to build on and expand the framework of Distributed Teaching and Learning (DTALS), which here is used as a framework through which to explore the game’s community (Gee & Gee, 2016; Holmes, Tran, & Gee, 2017).  DTALS serves to expand on other models which examine learning in out-of-school contexts, and in particular on the connections between classroom and out-of-school learning, which numerous scholars argue is of critical importance (Sefton-Green, 2004; Vadeboncoeur, Kady-Rachid, & Moghtader, 2014). This framework serves to build bridges as well as fill gaps in some key literature on learning in out-of-school contexts, including connected learning (Ito et al., 2009), participatory culture (Jenkins, Purushotma, Weigel, Clinton, & Robison, 2009), learning ecologies (Barron, 2006), and affinity spaces (Gee, 2004; Gee & Hayes, 2012). The model also focuses on teaching in addition to learning in and across informal contexts. While DTALS can be used to examine any number of phenomena, this dissertation focuses on the community around Pokémon Go. The game, with its emphasis on geography and community, presents unique opportunities for research. This research draws on existing video game research which focuses on not only games but their communities, and in particular the learning and literacy activities which occur in these communities (Gee & Hayes, 2012; Hayes & Duncan, 2012; Squire, 2006; Steinkuehler, 2006). The results here are presented as three separate manuscripts. Chapter Two takes a broad view of a local community of players, and discusses different player types and how they teach and learn around the game. Chapter Three focuses on families who play the game together, and in particular three focal parents who share their perceptions of the game's merits, especially its potential to promote family bonding and learning. Chapter Four discusses teaching, in particular guides written about the game and the ways in which they are situated in particular Discourses (Gee, 2014). Finally, Chapter Five offers implications from these three chapters, including implications for designers and researchers as well as calls for future research. / Dissertation/Thesis / Doctoral Dissertation Learning, Literacies and Technologies 2018

abstract: In the real world outside of schools, contemporary students are routinely reading, writing, communicating, acting, and learning internationally, translingually, and multimodally, thanks to the prevalence of digital online communication; this has taken place across students’ racial, ethnic, and linguistic identities and national affiliations. Today, the global online contexts are considered as one of essential literacy environments, and the globally networked online contexts might become a main stage of future literacy practices. In this sense, this study develops new three theories about literacies studies from the perspective of the New Literacy Studies in an increasingly digitalized and globalized contemporary world. To achieve this, first, I introduced the features of a global online affinity space as a new concept. Second, I developed the theoretical claim of “complexified diversity.” Finally, I developed the theoretical concept of “Border-Crossing Discourses” on the basis of Gee’s (1990/2015) seminal idea of capital “D” Discourses. I expanded the concept of capital “D” Discourses, looking across borders at a variety of languages, nations, and broader cultures under the global view. The concept of Border-Crossing Discourses was established on the basis of the new concepts that I put forth previously of global online affinity spaces and complexified diversity. As an example of possible supplementary empirical studies, I conducted a small piece of discourse analysis. I observed and examined literacy practices in two global online affinity spaces. They are sites devoted to K-pop fanfiction sharing (hereafter, Asianfanfics) and to Japanese anime (hereafter, Crunchyroll). In particular, I explored the aspects of multimodal and translingual practices in these spaces. Both theoretical and empirical future research will contribute to the elaboration of these theories. / Dissertation/Thesis / Doctoral Dissertation Learning, Literacies and Technologies 2018

Está estabelecido que o processo de glicosilação é essencial para a conformação estrutural e função efetora dos anticorpos. Entretanto, não está completamente claro como diferenças nos carboidratos ligados aos anticorpos podem interferir na sua atividade biológica. Foi previamente descrito que anticorpos IgG1 murinos podem ser divididos em anafiláticos ou não-anafiláticos, de acordo com a sua capacidade de induzir in vivo reação de anafilaxia. Somado a isso, foi verificado que a cadeia de oligossacarídeos N-ligada à molécula de IgG1 é fundamental para a manutenção da sua função efetora. O objetivo do presente trabalho é estudar diferenças estruturais entre os subtipos de IgG murinos que poderiam determinar a sua atividade biológica. O seqüenciamento dos nucleotídeos que codificam os domínios CH2 e CH3 dos dois subtipos de IgG1 permitiu constatar homologia de 100% dessas regiões nas duas moléculas estudadas. Entretanto, ao analisar o padrão de carboidratos N-ligados aos anticorpos IgG1 foi observado maior conteúdo de ácido siálico e fucose na cadeia N-ligada ao anticorpo anafilático em relação à do não-anafilático. Contudo, a remoção de resíduos de ácido siálico por tratamento enzimático do anticorpo IgG1 anafilático resultou na perda da capacidade desta molécula de induzir desgranulação celular in vitro e reação anafilática in vivo, semelhante ao anticorpo IgG1 deglicosilado. Em contraste, a remoção de fucose não afetou a sua função anafilática. A análise por PCR em tempo real da expressão dos genes das enzimas envolvidas no processo de glicosilação das proteínas revelou menor expressão gênica de algumas glicosidases, principalmente as sialiltransferases, no hibridoma e linfócitos B secretores do subtipo IgG1 não-anafilático em relação ao obtido no hibridoma e linfócitos B que secretam a IgG1 anafilática. Além disto, foi observada menor atividade enzimática das sialiltransferases obtidas do hibridoma produtor da IgG1 não-anafilática em relação à do hibridoma que produz a IgG1 anafilática. Em conjunto, estes resultados comprovam que a capacidade de anticorpos IgG1 murinos de induzir anafilaxia é diretamente dependente do conteúdo de ácido siálico presente na cadeia de oligossacarídeos ligada à região Fc do anticorpo, além disso sugerem fortemente que essa maior sialilação observada no tipo anafilático seja resultante da maior expressão gênica destas enzimas e assim da sua atividade enzimática no momento da síntese dos anticorpos. / It is well established that the glycosylation process is essential for the structural conformation and effector function of the antibodies. However, it is quite clear how differences in the carbohydrates attached to the antibodies may interfere with their biological activities. It was previously reported that murine IgG1 antibodies can be divided into anaphylactic or nonanaphylactic according to their ability to induce anaphylaxis. Furthermore, it was demonstrated that the oligosaccharide chain N-linked to the IgG1 is essential for its conformation and biological activity. The objective of this work is to study structural differences between these subtypes of murine IgG1 that could determine their biological activity. The sequencing of the nucleotides encoding the CH2 and CH3 domains of these two subtypes of IgG1 showed 100% of homology in the Fc regions of these molecules. In contrast, the analysis of the carbohydrates N-linked to the IgG1 antibodies demonstrated higher sialic acid and fucose contents in the chain attached to the anaphylactic antibody than in the nonanaphylactic IgG1. However, the removal of sialic acid residues by enzymatic treatment of anaphylactic IgG1 antibody resulted in the abrogation of its ability to induce mast cells degranulation in vitro and anaphylactic reaction in vivo as observed to deglycosylated IgG1 antibody. On the other hand, the removal of fucose did not change the anaphylactic activity. The analysis by real time PCR of the gene expression of enzymes that are involved in the protein glycosylation showed lower gene expression of some glycosyltransferases, mainly sialyltransferases, in the hybridoma and B lymphocytes that produce the non-anaphylactic IgG1 compared to those verified in the hybridoma and B cells producer of the anaphylactic IgG1. Furthermore, it was verified lower enzymatic activity of sialyltransferases purified from the hybridoma producer of the non-anaphylactic IgG1 in relation to the hybridoma producer of the anaphylactic antibody. Together, these results prove that the ability of murine IgG1 to induce anaphylaxis is directly dependent of the sialic acid content in the carbohydrate core attached to the antibody Fc region. It is also strongly suggested that this higher sialylation observed in the anaphylactic IgG1 may be resultant of the higher gene expression and enzymatic activity of some sialyltransferases during the antibody synthesis.

Orientador: Sonia Maria Alves Bueno / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-08T05:04:16Z (GMT). No. of bitstreams: 1 Ribeiro_MarianaBorsoi_D.pdf: 5252506 bytes, checksum: b9eb50542c7fa4e04b6b4c83526b4ccc (MD5) Previous issue date: 2006 / Resumo: As imunoglobulinas do isotipo G (IgG) são usadas enOlmemente em aplicações terapêuticas e são requeridas, usualmente, com um elevado grau de pureza. V álias técnicas cromatográficas vêm sendo investigadas para a purificação de IgG a partir do plasma humano. Neste trabalho, investigou-se o efeito dos agentes quelantes ácido iminodiácetico (IDA), ácido aspártico carboxi-metilado (CM-Asp) e TIis-2(aminoetil)amina (TREN) na purificação de IgG a partir do plasma humano utilizando a técnica de cromatografia de afinidade com os íons metálicos imobilizados em membranas de fibras ocas. Para tanto, foram realizados experimentos de adsorção em diferentes sistemas tamponantes em membranas de álcool de poli(etileno) vinílico (PEV A) finamente c011adas e em módulos de filtração derivatizados com IDA, TREN e CM-Asp, utilizando-se os íons metálicos níquel e cobre. A seletividade dos adsorventes foi verificada por eletroforese SDS-P AGE e análise nefelométrica. As melhores condições de purificação foram encontradas utilizando-se as membranas PEV A-CM-Asp-Ni(II) em módulo de filtração, em presença do tampão TrisHCI 25 mM pH 7,0 e eluição por acréscimo da concentração de TIis, sendo obtida IgG com aproximadamente 90% de pureza. As membranas de PEV A-CM-Asp-Ni(II) em módulos de filtraçã? apresentaram melhor seletividade, segundo eletroforese SDS-P AGE, e capacidade dinâmica de adsorção maior comparadas às membranas finamente cortadas (capacidade média de adsorção: 53,0 e 7,7 mg de IgG pOT g seca de membrana, respectivamente). O modelo de LangmUir ajustou-se às isotermas de adsorção, à temperatura ambiente, mostrando alta capacidade de adsorção para os sistemas PEV A-IDA-Ni(II) e PEV A-CM Asp-Ni(II) (204,6 e 302,3 mg/g seca de membrana, respectivamente) e constantes de dissociação característica de sistemas de média afinidade (6, I e 10,1 x 10-6 M, respectivamente). A análise dos parâmetros termodinâmicos encontrados para o sistema modelo PEV A-IDA-Ni(II)-IgG, indicaram a complexidade das interações, indicando a existência de interações hidrofóbicas, eletrostáticas e de ligações de coordenação. Neste trabalho, conseguiu-se purificar IgG a partir do plasma humano em! uma única etapa, utilizando-se a técnica da cromatografia em membranas de afinidade, demonstrando a alta potencialidade da utilização do método desenvolvido em processos industriais / Abstract: The use of immunoglobulin G (lgG) in therapeutic applications has growing vastly and it is used in the treatment of a growing number of indications. Several chromatographic techniques have been investigated for IgG purification. In this work, we investigated the effect of the chelators iminodiacetic acid (IDA), carboxymethylaspartate acid (CM-Asp) and Tris-2(aminoethyl)amine (TREN) for IgG purification, from human plasma using Immobilized Metal-ion Affinity Chromatography (IMAC) technique using hollow fibers membranes as support to the chromatographic experiments. For that, adsorption experiments were done, using different buffers systems, on filtration module and on finely cut poly(ethylene) vinyl alcohol (PEV A) membranes containing chelators IDA, TREN and CM-Asp with the metallic ions nickel and copper. The adsorbent selectivity was verified through electrophoresis SDS-PAGE and nefelometric analysis. Best purification conditions were found with PEV A-CM-Asp-Ni(ll) filtration module in the presence of 25 mM Tris HCI pH 7.0 and elution by increasing Tris concentration. In this case, it was possible to obtain IgG with approximately 90% of purity. According to electrophoresis SDS-P AGE and nefelometric analysis, PEV A-CM-Asp-Ni(ll) filtration modules showed better selectivity and superior adsorption dynamic capacity for IgG than those obtained by finely cut membranes (medium adsorption capacity: 53.0 and 7.7 mg of IgG /g of dry membrane, respectively). Lagmüir model adjusted to adsorption isotherms at room temperature (250 C), showed high adsorption capacity for PEV A-IDA-Ni(ll) and PEV A-CM-Asp-Ni(ll) systems (204.6 and 302.3 mg/g dry of membrane, respectively) and showed dissociation constants characteristic of medium affinity systems (6.1 and 10.1 x 10-6 M, respectively). The thermodynamic parameters analysis obtained for PEV A-IDA-Ni(ll)-lgG system indicated the complexity of the interactions, and the existence of hydrophobic and electrostatics interaction besides the coordination bound. In this work, we could purify IgG from human plasma in a single stage, using the affinity chromatographic membranes technique, allowing in this way, the treatment of great volumes per unit of time, showing the high potentiality of this method in industrial processes / Doutorado / Desenvolvimento de Processos Biotecnologicos / Doutor em Engenharia Química

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-11-19T12:25:23Z No. of bitstreams: 2 Dissertação - Gean Carlos Sousa Gonzaga - 2015.pdf: 2479646 bytes, checksum: d85220319554b1544933aa25ea2c672c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-11-19T12:27:30Z (GMT) No. of bitstreams: 2 Dissertação - Gean Carlos Sousa Gonzaga - 2015.pdf: 2479646 bytes, checksum: d85220319554b1544933aa25ea2c672c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-11-19T12:27:30Z (GMT). No. of bitstreams: 2 Dissertação - Gean Carlos Sousa Gonzaga - 2015.pdf: 2479646 bytes, checksum: d85220319554b1544933aa25ea2c672c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-08-06 / This paper addresses the theorem of Napoleon on historical, conceptual perspectives, and focusing on demonstrations and applications properties as well. In the rst chapter, are discussed aspects of the history and Napoleon Bonaparte biography. In the second chapter are addressed notions of Plane Geometry, Linear Algebra, of Rigid Transformation of Complex Numbers and Related Transformations. In the third chapter, statements are presented, generalizations (especially the so-called Barlotti Theorem), properties and applications in exercises. / O presente trabalho aborda o teorema de Napoleão em perspectivas históricas e conceituais, enfocando demonstrações e propriedades. No primeiro capítulo, são abordados aspectos da biogra a de Napoleão Bonaparte. No segundo capítulo são abordadas no- ções de Geometria Plana, de Álgebra Linear, de Transformações Rígidas, de Números Complexos e Transformações A ns. No terceiro capítulo, são apresentadas demonstra ções, generalizações (em especial, o chamado Teorema de Barlotti), propriedades e aplicações em exercícios.

Orientador: Sônia Maria Alves Bueno / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-25T04:00:44Z (GMT). No. of bitstreams: 1 Oliveira_CarlaReis_M.pdf: 2012737 bytes, checksum: 29446d9a4e9ff14301c100dcd3fb82ab (MD5) Previous issue date: 2014 / Resumo: Anticorpos monoclonais (AcM) são imunoglobulinas secretadas por clones de linfócitos B imortalizados obtidos pela tecnologia de hibridomas. Esses clones são cultivados em meios de cultura complexos e, geralmente, liberam baixas concentrações de AcM, dificultando as etapas de purificação. Como a utilização destas proteínas nas áreas terapêutica e analítica requer um alto grau de pureza, diferentes estratégias de purificação vêm sendo desenvolvidas, mas usualmente os anticorpos são purificados por cromatografia de afinidade com proteína A ou G imobilizada, o que representa um elevado custo de produção para processos industriais. No intuito de contribuir para o desenvolvimento de processos de purificação dessas moléculas, estudou-se a purificação do AcM anti-Trypanosoma cruzi isotipo IgG2a em orto-fosfo-serina (OPS) imobilizado em gel de agarose. Contrário ao relatado em literatura, observou-se que o agente quelante apresentou baixa capacidade em imobilizar o íon metalico Ni2+. Além disso, o quelato formado adsorveu a molécula alvo juntamente com impurezas do sobrenadante do meio de cultura. Foi observado também que o OPS se comporta como ligante de troca iônica seletivo para IgG2a em solução tampão Tris-HCl 50 mmol/L pH 7,0, quando imobilizado em gel de agarose ativado com CNBr, porém os ensaios realizados com o ligante imobilizado em gel ativado com bisoxirano demonstraram que a inserção de uma molécula espaçadora reduz a seletividade do adsorvente. O AcM foi recuperado com elevado grau de pureza quando empregado em solução tampão Tris-HCl 50 mmol/L pH 7,0 e dessorção pelo acréscimo de NaCl 1,0 mol/L para promover aumento da força iônica. Os resultados de ensaios dinâmicos revelaram que a eficiência de recuperação do produto foi de 97,3% e a capacidade dinâmica de adsorção foi 0,39 mg de AcM/mL de gel devido. Este trabalho sugere a potencialidade de utilização do ligante OPS para a purificação dos anticorpos anti-Trypanosoma cruzi (IgG2a) / Abstract: Monoclonal antibodies (mAb) are immunoglobulins produced by lynphocyte B clones immortalized by the hibridoma technology. Those clones are cultivated in complex medium and generally produce low levels of mAb, interfering on purificaton steps. Since these antibodies are required for analytical and therapeutical purposes, the need of highly pure antibodies is imperative. Although they are usually purified by chromatografic techniques utilizing immobilized protein A ligands, different strategies for downstream processes have been studied to overcome the high costs of these conventional medias for industrial scale purposes. Aiming to contribute with the development of such processes, the anti-Trypanosoma cruzi mAb (IgG2a isotype) was investigated under chromatographic conditons twards the affinity ligand ortho-phospho-L-serine (OPS) immobilized onto agarose beads. It was observed that despite the fact this ligand has been reported as a chelating agent for the purification of immunoglobulins by IMAC technique, OPS presented low chelating capacity for Ni2+ and the metal complex formed adsorbed the desired protein within culture medium contaminants. It was also observed that under buffering conditions of Tris-HCl 50 mmol/L pH 7.0 OPS behaves as a selective ionic exchanger ligand for IgG2a when immobilized in CNBr activated agarose. On the other hand, when immobilized in bisoxirane activated agarose, OPS has shown that the presence of a spacer arm reduces the selectivity of the adsorbent. The mAb was recuperated in one single step with highly putity degree when operated with adsorption buffer Tris-HCl 50 mmol/L pH 7.0 and elution by increasing the ionic strength with the addition of NaCl 1.0 mol/L. The dynamic tests results showed that the efficiency for product recovery was 97.3% and the dynamic binding capacity was 0.39 mg of mAb/mL swollen agarose. This work suggests the potencial use os OPS ligand affinity for the purification of IgG2a antibodies anti-Trypanosoma cruzi in a single step / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestra em Engenharia Química

Orientador: Sonia Maria Alves Bueno / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-23T20:44:35Z (GMT). No. of bitstreams: 1 Serracchiani_MarcelMafei_M.pdf: 2065791 bytes, checksum: 04a95a39ac0691a669c45efc9e3fc435 (MD5) Previous issue date: 2013 / Resumo: A produção de proteínas recombinantes em plantas transgênicas tem se mostrado uma forma segura, eficiente e barata de obtenção em grande escala de várias proteínas de interesse, tais como vacinas, enzimas industriais, bioativos, biofarmacos e anticorpos e seus fragmentos. Contudo, para que a produção de proteínas recombinantes em plantas se torne viável é necessário o desenvolvimento de um processo de recuperação e separação da molécula alvo que seja eficiente e reprodutivo, pois a produção de biomoléculas em plantas transgênicas, assim como os métodos tradicionais de produção, apresentam componentes indesejáveis que devem ser removidos. Neste trabalho, avaliou-se a purificação de fragmentos Fab de IgG humana adicionados artificialmente (spiking) a extrato protéico de soja não transgênica utilizando-se a técnica de cromatografia de afinidade por íons metálicos imobilizados (IMAC). Estudou-se o efeito dos quelatos IDA-Ni(II) e TREN-Ni(II), dos sistemas tamponantes Tris-HCl, fosfato de sódio e Mes, na presença e na ausência de sal (NaCl) na purificação de fragmentos Fab. Primeiramente foram realizados experimentos cromatográficos com as proteínas nativas do extrato proteico do grão de soja. Dos resultados obtidos, selecionou-se, para o adsorvente agarose-TREN-Ni(II), o sistema tamponante Tris-HCl/Tris-HCl na ausência de NaCl, e para o adsorvente agarose-IDA-Ni(II), selecionou-se o sistema tamponante fosfato de sódio/acetato de sódio contendo NaCl. Estes sistemas tamponantes e adsorventes foram utilizados para purificação dos fragmentos Fab adicionados (spiking) ao extrato proteico de grãos de soja. Em agarose-TREN-Ni(II), os fragmentos Fab foram purificados por cromatografia negativa, obtendo-se 66% dos fragmentos Fab na etapa de flowthrough e lavagem, com um grau de pureza de 91% e fator de purificação de 1,4. Para o adsorvente agarose-IDA-Ni(II), os fragmentos Fab foram purificados por cromatografia tradicional (eluição a pH 5,8), obtendo-se alto grau de pureza do fragmento Fab purificados, com um fator de purificação de 2,5. Este estudo contribuiu para obter conhecimento de base para posterior aplicação da técnica de IMAC na purificação de proteínas recombinantes produzidas em sementes de plantas transgênicas / Abstract: The recombinant proteins production using transgenic plants have been considered a safe, efficient and cheap way of producing on large scale innumerous proteins of interest, such as vaccines, industrial enzymes, bioactives, biopharmaceuticals and antibody and it fragments. However, for the production of recombinant proteins in plants become viable is necessary to develop a process for recovery and separation of the target molecule that is efficient and reproductive, because the production of biomolecules in transgenic plants, as well as traditional production methods, have components reactions which must be removed. In this work, the purification of Fab fragments of human IgG added artificially (spiking) to extract non-transgenic soy protein, using the technique of affinity chromatography on immobilized metal ions (IMAC), was evaluated. The effect of chelating agents IDA-Ni(II) and TREN-Ni(II), and the buffers Tris, sodium phosphate and Mes, in presence and the absent of NaCl, was studied for the Fab purification. First, the chromatographic experiments with the native proteins of soybean extract were performed. From the results obtained, it was selected, for the adsorbent TREN-Ni(II), the buffers system Tris-HCl/Tris-HCl without NaCl, and for the adsorbent IDA-Ni(II), the buffer system sodium phosphate/ sodium acetate with NaCl was selected. These adsorbents and buffer systems were used for purification of Fab fragments added (spiking) to soybean extract proteins. In the agarose adsorbent TREN-Ni (II), Fab fragments were purified by negative chromatography, obtaining 66% of Fab fragments in flowthrough and wash steps with a purity of 91% and purification factor of 1.4. For the adsorbent agarose-IDA-Ni (II), Fab fragments were purified by traditional chromatography (elution at pH 5.8), obtaining a high purity of the purified FAB, with a purification factor of 2.5. This study contributed to obtain knowledge base for application of the technique on the IMAC purification of recombinant proteins produced in transgenic plant seeds / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

Orientador: Sonia Maria Alves Bueno / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-02T17:38:51Z (GMT). No. of bitstreams: 1 Serpa_Gisele_M.pdf: 3619704 bytes, checksum: 1feef42faa8bb785a83f4c9f481a438c (MD5) Previous issue date: 2002 / Resumo: Anticorpos monoc1onais são imunoglobulinas secretadas por uma célula híbrida, chamada hibridoma, que é fonnada pela fusão de um linfócito (produtor de anticorpos) e uma célula de mieloma, o que faz do hibridoma uma célula produtora de anticorpos virtualmente imortal. Os anticorpos monoc1onais têm sido utilizados nas áreas analítica e terapêutica, o que implica na necessidade de obtenção de anticorpos de alta pureza. Muitos estudos têm sido realizados visando a purificação de anticorpos monoc1onais, e destacam-se as técnicas de adsorção seletiva, como as cromatografias de troca iônica, hidrofóbicas e de a:f11Údade. Neste trabalho aplicou-se a cromatografia em membranas de álcool polietileno-vinílico, derivatizadas com ácido iminodiacético (IDA), com íons metálicos imobilizados na purificação de anticorpos monoc1onais IgGl a partir de sobrenadante de cultura celular. Para determinar as melhores condiçôes de adsorção e eluição, foram testados os íons Cu2+, Ni2+, Zn2+ e C02+, na presença de diferentes sistemas tamponantes. A seletividade dos metais, em cada um dos sistemas, foi determinada através de eletroforese SDS-PAGE e testes ELISA das frações dos picos de proteína obtidos. A melhor condição de purificação foi a alimentação de sobrenadante de cultura celular previamente precipitado e dialisado com solução de sulfato de amônio, em coluna contendo PEVA-IDA-Zn2+, em presença de tampão Tris-HCI 50 mM a pH 7,0 e eluição por aumento de concentração de Tris. A partir das isotermas de adsorção, determinou-se a capacidade máxima de adsorção e a constante de dissociação do complexo IDA­Zn2+-IgGl que de acordo com o ajuste dos parâmetros pelo modelo de Langmuir, mostraram uma alta capacidade de adsorção (63,4 mg/g de membrana seca) e uma constante de dissociação (8,lxlO-6 M) característica de sistemas de média afinidade. Foram também determinadas as curvas de ruptura para o processo proposto, através de experimentos de fIltração a diferentes vazões de alimentação, utilizando um módulo contendo as fibras ocas com Zn2+ imobilizado, construído em nosso laboratório / Abstract: Monoc1onal antibodies are immunoglobulins produced by a hybrid cell ca11ed hybridoma. These cells result from the fusion of lymphocytes with malignant myeloma cells. Hybridomas cells express both the lymphocyte's property of specific-antibody production and the immortal character of the myeloma cells. Monoc1onal antibodies have been used in analy1ica1 and therapeutica1 areas. This application needs high1y pure antibodies. Many techniques have been studied focusing monoc1onal antibodies purification. These techniques inc1ude ion exchange, hydrophobic and affinity chromatography. In this study, we applied polyethylenevinyl alcohol (PEV A) membranes in the purification of monoc1onal antibody from cell culture supem.atant. These membranes were derivatized with the quelant agent, iminodiacetic acid (IDA). We evaluated the adsorption and purification of monoc1onal antibodies on the matrix with different immobilized metal ios, Cu2+, Ni2+, Zn2+ and C02+ and with different buffers. According to SDS­PAGE electrophoresis and ELISA analysis, the higher selectivity was obtained in the presence of 50 mM Tris-HCl buffer, pH 7,0 and with elution by increasing Tris concentration, with immobilized Zn2+, wich provided the purification of IgG with traces of albumin. The adsorbent capacity and the dissociation constant of the complex IDA-Zn2+-IgGI were determinated from adsorption isotherms. According to the Langmuir model, the results indicated that the matrix presents high adsorption capacity (63,4 mg/g de chy membrane) and a dissociation constant (8,1 x 10-6 M) characteristic for intermediate affinity systems. We also evaluated the breaktrough curves for PEVA-IDA-Zn2+ membrane chromatography for the antibodies purification using different flow rates. These breaktrough CUIVes are important to sca1e up procedure / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

The power of thermodynamics in the calculation of complex chemical and metallurgical equilibria of importance to industry has, over the last 15 years, been considerably enhanced by the availability of computers. It has resulted in the storage of data in databanks, the use of physical but complex models to represent thermodynamic data, the vast effort spent in the generation of critically assessed data and the development of sophisticated software for their application in equilibrium calculations. This thesis is concerned with the generation and application of metallurgical thermodynamic data in which the computer plays a central and essential role. A very wide range of topics have been covered from the generation of data by experiment and critical assessment through to the application of these data in calculations of importance to industry. Particular emphasis is placed on the need for reliable models and expressions which can represent the molar Gibbs energy as a function of temperature and composition. In addition a new computer program is described and used for the automatic calculation of phase diagrams for binary systems. Measurements of the enthalpies of formation of alloys in the Fe-Ti system are reported. All data for this system have been critically assessed to provide a dataset consistent with the published phase diagram. Critically assessed data for a number of binary alloy systems have been combined in order to perform quantitative calculations in two types of steel system. Firstly data for the Cr-Fe-Ni-Si-Ti system have been used to provide information about the long term stability of alloys used in fast breeder nuclear reactors. Secondly very complex calculations involving nine elements have been made to predict the distribution of carbon and various impurities between competing phases in low alloy steels on the addition of Mischmetall. Finally a new model is developed to represent the thermodynamic data for sulphide liquids and is used in the critical assessment and calculation of data for the Cu-Fe-Ni-S system. The phase diagram and thermodynamic data calculated from the assessed data are in excellent agreement with those observed experimentally. The work reported in this thesis, whilst successful, has also indicated areas which will benefit from further study particularly the development of reliable data and models for pure elements, ordered solid phases and liquid phases for high affinity systems.