Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-12-05T17:32:40Z No. of bitstreams: 1 Manoel Leoncio da Penha Filho Anticorpos monoclonais... 2003.pdf: 40157874 bytes, checksum: a8a843890d6d1f967434db64a38014bb (MD5) / Made available in DSpace on 2012-12-05T17:32:40Z (GMT). No. of bitstreams: 1 Manoel Leoncio da Penha Filho Anticorpos monoclonais... 2003.pdf: 40157874 bytes, checksum: a8a843890d6d1f967434db64a38014bb (MD5) Previous issue date: 2003 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / O diagnóstico de certeza da leishmaniose visceral (LV) só é conseguido através da demonstração do parasito diretamente ou em cultura o que implica em procedimentos demoradc» envolvendo certo risco e/ou dor. Este estudo tem como objetivos a produção de AcMc diVí^ã-Leishmania chagasi, padronização de um ensaio de imunohistoquímica para visualização de amastigotas de Leíshmania utilizando AcMc e verificação do potencial de um ELISA de inibição utilizando AcMc em quantificar amastigotas de Leishmania. [MATERIAIS E MÉTODOS] Camundongos BALB/c foram imunizados com antígeno de amastigotas de L chagasi ou com a proteína recombinante Lc-13 cujo gene foi clonado de uma biblioteca de cDNA de L chagasi. Os esplenócitos destes animais foram fundidos com células SP 2-0 através de polietilenoglicol. Estes, juntamente com um AcMc já disponível, o 5A9H8, foram usados em um ensaio de imunohistoquímica utilizando fígado de hamster infectado com L chagasi parafinado fixado em formalina, testando-se diferentes protocolos de exposição de epitopos. Também com os mesmos AcMc foi realizado um ELISA de inibição a fim de quantificar amastigotas de /.e/s/?/nan/a.[RESULTADOS] Foram encontrados dois clones reativos em cada fusão; o 10B4/6 e o 10D8 na primeira e o 2C10D5 e o 11E8H7 na segunda. Os AcMc 10B4/6 e o 10D8 são ambos lgG1, reconhecem antígenos com peso molecular entre 47 e 57 kDa e são possivelmente idênticos. Foi encontrada marcação de amastigotas, em tecidos infectados, com os seguintes AcMc: 5A9H8, em seções pré-tratadas com calor, e o 10B4/6 e o 10D8, em seções pré-tratadas com pronase. No ELISA de inibição o AcMc 5A9H8 mostrou-se capaz de detectar no mínimo 10® parasitos por poço de placa de microtitulação. / The certainty of diagnosis in visceral leishmaniasis is only achieved through the direct demonstration of parasites in culture, implying in long procedures and in risks and/or pain for the patients. The objectives of this study is the production of anti-Leishmania chagasi monoclonal antibodies (mAb), standardization of an immunohistochemical assay for allowing the visualization of Leishmania amastigotes using the mAb, and finding out the potential of a mAb-based inhibition ELISA for quantifying Leishmania amastigotes. [MATERIAL & METHODS] BALB/c mice were immunized with L chagasi amastigote antigens or with a recombinant protein denominated Lc13, the gene of which was cloned from a L chagasi amastigote cDNA library. Splenocytes from these animals were obtained and fused with SP 2-0 myeloma cells by using polyethylene glycol. The mAb produced by the hybrid cells, together with an available mAb (5A9H8), were used in an immunohistochemical assay, using paraffin sections of infected hamster liver. Different protocols for the exposition of epitopes were evaluated. One of the mAb was also used in an inhibition ELISA to quantify Leishmania amastigotes. [RESULTS] Two anti-amastigote mAb-producing clones were obtained in each fusion, the 10B4/6 and the 10D8 mAb in the first and the 2C10D5 and the 11E8H7 mAb in the second. The 10B4/6 and the 10D8 mAb, obtained from the fusion of myeloma with splenocytes of an animal immunized with lysed amastigotes, were both lgG1, recognized the same antigens, with molecular weights ranging from 47 to 57 kDa, and were possibly identical, o 10B4/6 e o 10D8 na primeira e o 2C10D5 e o 11E8H7 na segunda. The following mAb stained amastigotes in infected tissues in an im mu noperoxidase reaction: the 5A9H8, using heat-treated sections, and the 10B4/6 and the 10D8, in pronase-treated sections. The 5A9H8 mAb detected a minimum of 10® parasites per well In the inhibition ELISA.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Classical swine fever virus (CSFV) is an enveloped, positive strand RNA virus, and member of the genus Pestivirus. It is a highly infectious and transmissible swine pathogen that threatens the global swine industry. The United States has been free of CSFV since 1977, however, monitoring the millions of domestic and feral pigs present in the US puts a significant strain on national surveillance efforts. There are no validated diagnostic techniques that can simultaneously sample multiple pigs (i.e. all pigs in a pen or barn). Similarly, there are no validated serological assays that can quickly test for CSFV without cross-reacting with other pestiviruses. The purpose of the first study was to establish a moderate CSFV-infectious model and determine how a single oral fluid sample from a pen of pigs can function as a diagnostic sample for detecting CSFV. Oral fluid (OF) and serum samples were collected from 10 pigs experimentally infected with CSFV Paderborn strain. Using RT-PCR, CSFV was detected in OF on 8 days post infection (dpi), and in the serum of one pig on 6 dpi. A single OF sample can, therefore, take the place of 10 serum samples to detect CSFV in a population. In a second study, monoclonal antibodies reactive to CSFV glycoproteins were generated in mice immunized with recombinant E2 and Erns antigens. Five E2-specific clones and two Erns-specific clones showed reactivity to CSFV-infected. Epitope mapping of the E2 clones showed that all reacted with the N-terminal portion of E2; a region highly variable among pestiviruses. Together with OF sampling, monoclonal antibodies can be used to develop new tools for improving CSF surveillance in large swine populations.

Orientador: Ticiana Sidorenko de Oliveira Capote / Resumo: A engenharia tecidual voltada ao tecido ósseo tem como objetivo proporcionar uma reparação que apresente propriedades físicas e biológicas similares ao osso natural com restabelecimento adequado das suas funções. Neste contexto, materiais aloplásticos baseados em biopolímeros como a fibroína da seda (FS) e celulose bacteriana (CB) têm proporcionado a síntese de excelentes biomateriais para reparação óssea. Esses biopolímeros são biocompatíveis, possuem características mecânicas interessantes, apresentam uma lenta taxa de degradação in vivo, além da capacidade de serem modificados quimicamente. Para conferir a estes biopolímeros uma melhor bioatividade com intuito de potencializar a eficácia local da reparação óssea, o objetivo do presente estudo consistiu em sintetizar uma membrana à base de FS e outra à base de CB, sendo cada uma associada à hidroxiapatita e anticorpo anti-proteína morfogenética óssea (anti-BMP-2). Para tanto, estas membranas foram caracterizadas físico-quimicamente e, para análise do potencial destas membranas na reparação óssea, foram realizados ensaios in vitro para investigação de citotoxicidade, genotoxicidade e mutagenicidade, além de expressão gênica desses materiais. As membranas à base de CB obtiveram resultados da caracterização físico-quimicamente mais satisfatórios em relação à FS e os testes seguintes foram realizados apenas com a membrana de CB. A membrana de CB associada à hidroxiapatita não demonstrou citotoxicidade, genotoxicidade e mutageni... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Tissue engineering related to the bone tissue aims to provide a repair that presents similar physical and biological properties to the natural bone with adequate restoration of its functions. In this context, alloplastic materials based on biopolymers such as silk fibroin (SF) and bacterial cellulose (BC) have provided the synthesis of excellent biomaterials for bone repair. These biopolymers are biocompatible, have interesting mechanical characteristics, have a slow rate of degradation in vivo, and the ability to be chemically modified. In order to give these biopolymers better bioactivity to potentiate the local effectiveness for bone repair, the aim of the present study was to synthesize a SF-based and BC-based membrane, each of them was associated with hydroxyapatite and antibody anti-bone morphogenetic protein (anti-BMP-2). The membranes with immobilized anti-BMP-2 are expected to be effective in capturing endogenous BMP-2, conferring a different therapeutic approach and potential use for bone tissue engineering. For this purpose, these membranes were physico-chemically characterized and, in order to analyze the potential of these membranes in bone repair, in vitro assays were performed to investigate cytotoxicity, genotoxicity, mutagenicity and gene expression of these materials. The BC-based membranes obtained physically-chemically characteristics more satisfactory than the SF, and the next tests were performed only on the BC membrane. The BC membrane associated with h... (Complete abstract click electronic access below) / Mestre

Submitted by Raquel Bester Liszbinski (raquelb.biomed@gmail.com) on 2018-06-12T15:27:51Z No. of bitstreams: 1 Dissertação Raquel Bester Liszbinski 04.2018 .pdf: 2684297 bytes, checksum: 39e14fcba58d472dfe21ab816c211c29 (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-06-13T16:59:06Z (GMT) No. of bitstreams: 1 liszbinski_rb_me_bot.pdf: 2684297 bytes, checksum: 39e14fcba58d472dfe21ab816c211c29 (MD5) / Made available in DSpace on 2018-06-13T16:59:06Z (GMT). No. of bitstreams: 1 liszbinski_rb_me_bot.pdf: 2684297 bytes, checksum: 39e14fcba58d472dfe21ab816c211c29 (MD5) Previous issue date: 2018-04-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O câncer colorretal (CCR) é o terceiro tipo mais prevalente de câncer no mundo, com frequentes complicações metastáticas muitas vezes associadas ao desenvolvimento de resistência adquirida aos fármacos. Uma das estratégias terapêuticas utilizadas no tratamento de pacientes que não respondem bem à quimioterapia convencional é a imunoterapia baseada na aplicação de anticorpos monoclonais cetuximab ou panitumumab, ambos dirigidos ao receptor do fator de crescimento epidérmico (EGFR). Em alguns casos observa-se associação da resistência aos anticorpos com a superexpressão de HER2, outro membro da família do EGFR. Assim, esta dissertação foi elaborada para testar a hipótese de que o uso conjunto de anticorpos anti-EGFR e anti-HER será capaz de reduzir o escape das células tumorais e que a associação com fármacos antitumorais tornará mais efetiva sua eliminação. Com esse propósito, nosso objetivo foi utilizar nanopartículas ouro (AuNP) para o transporte de 5-fluorouracil (5FU), endereçando esses nanocarreadores para as células de câncer colorretal recobrindo-os com anticorpos contra os receptores para fator de crescimento epidérmico EGFR (ErbB-1) e HER2 (ErbB-2). Os nanocarreadores foram testados in vitro quanto a sua interação por células de câncer colorretal HCT-116 e Caco-2 por citometria de fluxo, sua capacidade de induzir apoptose/necrose e seu efeito sobre a atividade proliferativa das células testadas. Nossos dados indicam que as AuNP produzidas e funcionalizadas com as moléculas de interesse possuem estrutura esférica, no entanto, algumas formulações apresentaram aglomeração devido a incorporação das moléculas orgânicas. Os ensaios biológicos mostraram que a AuNP 5FU EGFR teve efeito significativo na atividade citotóxica pela via apoptótica recente quando as células foram expostas à maior concentração testada, independentemente do tempo de exposição. Consequentemente, a proliferação celular foi cessada neste mesmo tratamentos e ambos os fenômenos foram observados de forma mais evidente na linhagem Caco-2, inclusive demonstrando efeito superior ao quimioterápico na forma livre. / Colorectal cancer (CRC) is the third most prevalent type of cancer in worldwide and is frequently associated with metastatic complications. Therapeutic strategies to treat those patients who do not respond to conventional chemotherapy is based on the application of monoclonal antibodies cetuximab or panitumumab, both directed targeting the epidermal growth factor receptor (EGFR). However, in some cases resistance is associated with an overexpression of HER2, another receptor belonging to the EGFR family. Therefore, this dissertation was designed to test the hypothesis that the combination of anti-EGFR and anti-HER antibodies will be able to reduce the escape of tumor cells, and that their association with antitumor drugs could improve the elimination of tumor cells. Then, our goal was to use gold nanoparticles (AuNP) for transporting 5-fluorouracil (5FU), addressing them to colorectal cancer cells by coating them with antibodies against the receptors of epidermal growth factor EGFR (ErbB-1) and HER2 (ErbB-2). Nanocarriers were tested in vitro by flow cytometry for their interaction and internalization by the colorectal cancer cell line HCT-116 and Caco-2, their ability to induce apoptosis/necrosis and stop cell proliferation. Our results show that AuNP produced and functionalized with the molecules of interest have spherical structure, however, some formulations showed agglomeration due to the incorporation of organic molecules. Biological assays showed that AuNP 5FU EGFR had a significant effect on cytotoxic activity by the recent apoptotic pathway when cells were exposed to the highest concentration tested independently of the time of exposure. As a result, cell proliferation was stopped at this same treatment and both phenomena were observed most clearly in the Caco-2 cell line, including demonstrating superior effect to chemotherapy in free form. / FAPESP: 2015/26729-2.

Made available in DSpace on 2014-10-09T12:52:27Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:01:38Z (GMT). No. of bitstreams: 0 / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Made available in DSpace on 2014-10-09T12:44:20Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:07:49Z (GMT). No. of bitstreams: 1 06880.pdf: 3809489 bytes, checksum: 1e6101f9ee96359cd04bad831acb93d8 (MD5) / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2016-06-22T14:36:15Z No. of bitstreams: 0 / Made available in DSpace on 2016-06-22T14:36:15Z (GMT). No. of bitstreams: 0 / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2015-03-26T13:46:40Z (GMT). No. of bitstreams: 1 01 - capa_abstract.pdf: 58716 bytes, checksum: 8427f1a81ed09f080394210a61912045 (MD5) Previous issue date: 2009-02-20 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / The Neospora caninum is a protozoan apicomplexan with implication in abortions in many countries. They are included in the economical losses, the abortament, the proportion of discarded cows and the replacement of new animals in the flock, the fall in the milk production, as well as in the fat production in the milk. It is known that the kits existent diagnoses in the healthy market of high cost and they are not produced at the national market, it is needed, to offer diagnosis alternatives reliable, and fast, with national technology. The proposal of the present work was the production of monoclonal antibodies (mAb) of high likeness against the Neospora caninum (sample Nc-1), for the use in immunodiagnostic tests as immunofluorescence, immunohistochemistry, ELISA and dot-ELISA. For the production of mAb the sonicated protozoan was used, originating from the culture in cells VERO and it was purified by filtration, it was used the tachyzoites for the immunization of the mice BALB/c, using as saponin adjuvant , what allowed the obtaining of polyclonal antibodies. The fusing of the esplenic cells of the immunized mice and the myelomas SP2/0 resulted in the obtaining of 72,4% of hybridomas secretors of antibodies anti-Nc-1. After the cloning for limiting dilution was obtained 78,2% of the clones secretors of antibodies anti-Nc- 1. After the recloning for limiting dilution, they were obtained 4 clones secretors of mAb of the class IgG. Obtained mAb IgG2a were capable to reveal the proteins of mass molecular 25 kDa and 70 kDa for "western blotting" to inhibit the multiplication of the taquyzoites in cells VERO. The monoclonal antibodies produced can be used in the development of more sensitive tests, in the epidemic studies of the neosporosis or they can be used in the identification of relevant proteins for the development of vaccines. / O Neospora caninum é um protozoário apicomplexa com implicação em abortamentos em muitos países. Estão inclusas nas perdas econômicas, os abortamentos, a proporção de vacas descartadas e a reposição de novos animais no rebanho, a queda na produção leiteira, bem como na produção de gordura no leite. Sabe-se que os kits diagnósticos existentes no mercado são de alto custo e não são produzidos no mercado nacional, necessita-se, oferecer alternativas de diagnóstico confiáveis, e rápidas, com tecnologia nacional. A proposta do presente trabalho foi a produção de anticorpos monoclonais (AcM) de alta afinidade contra o Neospora caninum (amostra Nc-1), para a utilização em testes de imunodiagnóstico como imunofluorescência, imunoistoquímica, ELISA e dot-ELISA. Para a produção dos AcM usou-se o protozoário sonicado, proveniente da cultura em células VERO e foi purificado por filtração, utilizaram-se os taquizoítos para a imunização dos camundongos BALB/c, usando como adjuvante a saponina, o que permitiu a obtenção de anticorpos policlonais. A fusão das células esplênicas, provenientes dos camundongos imunizados, com as células de mieloma SP2/0 resultou na obtenção de 72,4% de hibridomas secretores de anticorpos anti-Nc-1. Após a clonagem por diluição limitante obteve-se 78,2% dos clones secretores de anticorpos anti-Nc-1. Após a reclonagem por diluição limitante, foram obtidos 4 clones secretores de AcM da classe IgG. Os AcM IgG2a obtidos foram capazes de revelar as proteínas de massa molecular 25 kDa e 70 kDa por western blotting e inibirem a multiplicação dos taquizoítos em células VERO. Os anticorpos monoclonais produzidos poderão ser usados no desenvolvimento de testes mais sensíveis para estudos epidemiológicos da neosporose ou poderão ser utilizados na identificação de proteínas relevantes para o desenvolvimento de vacinas.

The overarching objective of this thesis was the development of an improved expression cell line for recombinant proteins, in which a transgene of interest can be inserted into a highly active gene locus using recombinase mediated cassette exchange. Random integration of transgenic DNA is a common route to achieve transgene expression. However, randomly integrated transgenes are susceptible to gene silencing over time, and do not show stable expression for extended periods in culture. Furthermore, every new cell clone generated requires regulatory approval. The improvement of expression strategies may significantly reduce the time required to bring novel recombinant protein products to the market. In order to identify a suitable expression locus for the integration of transgene expression cassettes, total protein samples were derived from the production cell lines HEK293 and CHO. Highly expressed proteins were isolated via 2D PAGE, and identified via peptide mass fingerprinting. Their promoter regions were then validated to express a recombinant transgene in HEK293 cells. The long term stability of these promoter regions was also assessed. Direct gene targeting of the highly active gene loci may or may not be possible in typical producer cell lines. Targeting of a murine homologue to these highly expressed CHO/HEK293 loci may be more efficient in a murine stem cell line. The transfer of a modified allele from HM1 murine embryonic stem cells, into a somatic cell line (HC11) was demonstrated in this thesis. These validated methods were then explored for the generation of viable HM1-HEK293 and HM1-CHO fusion hybrids. For these experiments, a fluorescence based fusion assay was generated, validated and used for in-situ monitoring of the cell fusion process. The random integration of transgenic DNA into mammalian genomes typically results in a highly unpredictable integration architecture. RMCE at such loci would be inefficient. However, a highly efficient RMCE reaction at (rare) single copy transgene integrations, may be possible under the correct conditions. RMCE at randomly integrated loci could therefore be more beneficial (for transgene expression) than random integration alone. This thesis explores this concept with the use of a randomly integrated RMCE platform, and subsequent selection of cell lines post RMCE attempts at these loci CRISPR/Cas9 technology was also applied to a highly expressed locus in HEK293 cells. A framework for successful direction of double strand breaks to a defined locus is demonstrated in this work. The methods used to achieve this can therefore be built upon for the homologous recombination of a transgenic cassette, into a highly expressed locus in HEK293 cells. Monoclonal antibodies have dominated the biologics market for over two decades, and mammalian expression systems are well suited to their production. The work in this thesis attempts to raise and verify antibody molecules against a potential tumour marker using hybridoma and phage display technologies.

Orientador: Ramon Kaneno / Resumo: O câncer colorretal (CCR) é o terceiro tipo mais prevalente de câncer no mundo, com frequentes complicações metastáticas muitas vezes associadas ao desenvolvimento de resistência adquirida aos fármacos. Uma das estratégias terapêuticas utilizadas no tratamento de pacientes que não respondem bem à quimioterapia convencional é a imunoterapia baseada na aplicação de anticorpos monoclonais cetuximab ou panitumumab, ambos dirigidos ao receptor do fator de crescimento epidérmico (EGFR). Em alguns casos observa-se associação da resistência aos anticorpos com a superexpressão de HER2, outro membro da família do EGFR. Assim, esta dissertação foi elaborada para testar a hipótese de que o uso conjunto de anticorpos anti-EGFR e anti-HER será capaz de reduzir o escape das células tumorais e que a associação com fármacos antitumorais tornará mais efetiva sua eliminação. Com esse propósito, nosso objetivo foi utilizar nanopartículas ouro (AuNP) para o transporte de 5-fluorouracil (5FU), endereçando esses nanocarreadores para as células de câncer colorretal recobrindo-os com anticorpos contra os receptores para fator de crescimento epidérmico EGFR (ErbB-1) e HER2 (ErbB-2). Os nanocarreadores foram testados in vitro quanto a sua interação por células de câncer colorretal HCT-116 e Caco-2 por citometria de fluxo, sua capacidade de induzir apoptose/necrose e seu efeito sobre a atividade proliferativa das células testadas. Nossos dados indicam que as AuNP produzidas e funcionalizadas com as molé... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Colorectal cancer (CRC) is the third most prevalent type of cancer in worldwide and is frequently associated with metastatic complications. Therapeutic strategies to treat those patients who do not respond to conventional chemotherapy is based on the application of monoclonal antibodies cetuximab or panitumumab, both directed targeting the epidermal growth factor receptor (EGFR). However, in some cases resistance is associated with an overexpression of HER2, another receptor belonging to the EGFR family. Therefore, this dissertation was designed to test the hypothesis that the combination of anti-EGFR and anti-HER antibodies will be able to reduce the escape of tumor cells, and that their association with antitumor drugs could improve the elimination of tumor cells. Then, our goal was to use gold nanoparticles (AuNP) for transporting 5-fluorouracil (5FU), addressing them to colorectal cancer cells by coating them with antibodies against the receptors of epidermal growth factor EGFR (ErbB-1) and HER2 (ErbB-2). Nanocarriers were tested in vitro by flow cytometry for their interaction and internalization by the colorectal cancer cell line HCT-116 and Caco-2, their ability to induce apoptosis/necrosis and stop cell proliferation. Our results show that AuNP produced and functionalized with the molecules of interest have spherical structure, however, some formulations showed agglomeration due to the incorporation of organic molecules. Biological assays showed that AuNP 5FU EGFR had... (Complete abstract click electronic access below) / Mestre