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Detecção e isolamento de anelovírus em suínos e cultivos celulares. / Detection and isolation of anelloviruses in pigs and in cell lineagesTeixeira, Thais Fumaco January 2012 (has links)
Estudos preliminares visando a identificação de possíveis agentes virais associados à síndrome multissistêmica do definhamento dos suínos (SMDS) revelaram uma possível associação inversa entre a presença de TTSuV1 e a ocorrência da SMDS. Com base neste achado, foi formulada a hipótese de que o TTSuV1 poderia ser capaz de inibir a multiplicação do PCV2, impedindo assim o desenvolvimento da SMDS. Buscando esclarecer esta questão, seria necessário desenvolver um sistema eficiente de replicação para este vírus, até o presente ainda não disponível. Em vista disso, foi desenvolvido um método de detecção de infecções por TTSuV em cultivos celulares para a avaliação de possíveis linhagens a serem potencialmente utilizadas para isolamento e multiplicação destes vírus. Genomas de TTSuVs foram detectados em células de linhagem de origem suína e não suína assim como em um dos lotes de tripsina. Os soros utilizados como suplemento para o meio de cultivo não apresentaram genomas de TTSuV. Desta forma, o lote de tripsina contaminado pode ser considerado uma importante fonte de contaminação, principalmente em células de origem não suína. Com o objetivo de avaliar uma possível associação entre os TTSuVs e a ocorrência da SMDS, a frequência de detecção e quantificação de genomas de TTSuV1 e TTSuV2 em tecidos e soros de suínos com e sem SMDS foram determinadas. A análise feita nos diferentes tecidos de suínos revelou uma aparente correlação inversa entre a presença do genoma de TTSuV1 e a ocorrência da SMDS. Quanto ao TTSuV2 em tecidos de suínos com e sem a SMDS, nenhuma diferença estatística foi observada. A distribuição do genoma de TTSuV1 e TTSuV2 nos diferentes tecidos examinados não revelou um órgão alvo específico. A frequência de detecção e a carga viral de TTSuV1 e 2 nas amostras de soro de suínos com e sem a SMDS não apresentaram diferença significativa. No entanto, a carga viral de TTSuV2 foi mais alta do que a carga viral de TTSuV1 nos soros de todos os grupos de animais estudados. Estes resultados indicam uma alta frequência de detecção de ambas as espécies de TTSuV em amostras de tecidos e soros de suínos com e sem a SMDS. / Preliminary studies aiming the identification of possible viral agents associated with the postweaning multisystemic wasting syndrome (PMWS) revealed a possible negative association between TTSuV1 and occurrence of PMWS. Based on this finding was hypothesized that TTSuV1 might be able to inhibit the PCV2 multiplication, preventing the development of PMWS. To better clarify this, would be require an efficient system of replication for this virus, which has not been reported in the literature. In view of this, a method for detection of TTSuV infections in cell culture was developed to assess possible cell lineages to be potentially used for virus isolation and multiplication. TTSuV genomes were detected in cell lineages of porcine and nonporcine origin as well as a batch of trypsin. Sera used as media supplement was not found to contain TTSuV genomes. Thus, the contaminated batch of trypsin can be considered an important source of contamination, especially in cells of non-porcine origin. In order to evaluate a possible association between the TTSuVs and the occurrence of PMWS, the frequency of detection and quantification of TTSuV1 and TTSuV2 genomes in tissues and sera from pigs with and without PMWS were determined. The analysis in the different tissues of pigs reveal an apparent inverse correlation between the frequency of detection of TTSuV1 genomes and the occurrence of PMWS. Regarding TTSuV2 in tissues of PMWS and non-PMWS-affected animals no significant differences was observed. The distribution of TTSuV1 and TTSuV2 genomes in tissues did not reveal any particular target organ. The frequency of detection and viral load of TTSuV1 and TTSuV2 in sera samples were no significant statistically among animals PMWS-affected and healthy pig. The mean of TTSuV2 viral load was significantly highest than TTSuV1 in sera of all groups studied. These results indicate a high frequency of detection of both TTSuV species in tissues and sera samples from PMWS-affected and healthy pig.
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Sintese de prolactina humana em celulas de ovario de hamster chines (CHO)SOARES, CARLOS R.J. 09 October 2014 (has links)
Made available in DSpace on 2014-10-09T12:43:49Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:10:05Z (GMT). No. of bitstreams: 1
06777.pdf: 5273885 bytes, checksum: b88f10c3d25adde0595b62adc866d4ee (MD5) / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP
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Desenvolvimento de matrizes poliméricas biodegradáveis à base de quitosana e possíveis blendas como sistemas de liberação controlada de fármacos / Development of biodegradable polymeric matrices based on chitosan and possible blend as controlled release systems for drugs.BATISTA, JORGE G. dos S. 08 April 2016 (has links)
Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2016-04-08T12:18:22Z
No. of bitstreams: 0 / Made available in DSpace on 2016-04-08T12:18:23Z (GMT). No. of bitstreams: 0 / De acordo com o conceito de sistemas de liberação controlada, o presente estudo foi baseado na utilização de polímeros hidrofílicos biocompatíveis, formadores de hidrogéis, para o desenvolvimento de matrizes na forma de filmes finos. Os polímeros utilizados para a formação das matrizes foram a quitosana proveniente das cascas de camarão, o amido de milho modificado e a poli(N-vinil-2-pirrolidona) - PVP. As matrizes foram reticuladas utilizando glutaraldeído. O fármaco escolhido para testar a capacidade de liberação dos dispositivos foi o anti-inflamatório não esteroidal (AINE) diclofenaco sódico. Para obtenção das matrizes com propriedades adequadas para essa finalidade, foram testadas misturas de quitosana-amido e quitosana-PVP. Após a triagem qualitativa, os dispositivos foram avaliados quanto à citotoxidade, intumescimento máximo, fração gel, parâmetros cinéticos associados à absorção de vapor de água e à capacidade de liberação de diclofenaco sódico in vitro. As formulações de quitosana-PVP foram as que apresentaram melhores propriedades para a aplicação proposta nesse estudo, se destacando a formulação A3, com alto percentual de liberação, boas propriedades de manuseio, poucos componentes na formulação diminuindo o potencial alergênico e aprovação no teste de citotoxicidade em células de camundongo (NCTC) pelo método de incorporação do vermelho neutro. / Dissertação (Mestrado em Tecnologia Nuclear) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
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Avaliação dos efeitos da radiação ionizante e do Resveratrol na cultura de células tumorais de pulmão / Evaluation of ionizing radiation and resveratrol effects in lung cancer cell cultureMORENO, CAROLINA dos S. 11 November 2016 (has links)
Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2016-11-11T17:21:57Z
No. of bitstreams: 0 / Made available in DSpace on 2016-11-11T17:21:57Z (GMT). No. of bitstreams: 0 / O carcinoma mucoepidermóide de pulmão, um tipo histológico que deriva das glândulas mucosas traqueobrônquicas, manifesta-se com sintomas obstrutivos e tende a comprometer a traqueia. Com finalidade curativa ou paliativa da doença, atualmente há uma forte tendência na oncologia em desenvolver estratégias terapêuticas que visam à administração de compostos com elevado potencial de otimizar o efeito do tratamento com a radiação ionizante, de modo a aumentar a morte de células tumorais e preservar íntegras as células dos tecidos sadios adjacentes. A intensa busca por tais estratégias evidenciou resultados promissores apresentados pelo composto denominado Resveratrol (3,4,5-trihidroxiestilbeno), tornando-o amplamente divulgado e alvo de intensas pesquisas. O principal objetivo do presente estudo foi determinar o efeito do resveratrol em cultura celular de carcinoma mucoepidermóide de pulmão exposta a diferentes doses de radiação ionizante. Para tal, os estudos de citotoxicidade utilizando o método de incorporação do vermelho neutro, e da determinação da dose letal 50 % (DL50) da radiação ionizante, foram realizados em cultura de células da linhagem NCI-H292 [H292] (ATCC® CRL-1848TM), CCIAL069. Com base nos resultados do IC50% (401,5 μM) e da DL50 (693 Gy) foram realizados o teste in vitro do micronúcleo e os ensaios para avaliar o efeito do resveratrol no ciclo celular, reparo da lesão no DNA e processo de lesão radioinduzida, necrose e apoptose celular. Os resultados evidenciaram que o resveratrol na concentração de 30 μM apresenta uma importante capacidade em promover danos às células NCI-H292 após 24 h da irradiação. / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
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Caracterização molecular e funcional de células de tumores adrenocorticais humanos. / Molecular and functional characterization of human adrenocortical cell cultures.Amanda Teixeira Rodrigues 14 August 2014 (has links)
O Adenoma adrenocortical é frequente em adultos, já o carcinoma é raro e agressivo. Mesmo com critérios padronizados, ainda há dificuldade para diferenciar esses tumores, sendo necessário o estudo de marcadores eficientes na detecção e diferenciação. Por serem raros e com diversas manifestações clínicas, culturas in vitro pode ser uma ferramenta para o estudo de processos que envolvem a doença. Foi realizada a caracterização molecular e funcional de culturas de células de tumores de pacientes. Resultados de PCR Array não mostraram um padrão que diferenciasse as culturas em função dos diagnósticos. Desta análise, 7 oncogenes apresentaram maior expressão e 9 supressores de tumor apresentaram baixa expressão nas culturas. WWOX, FHIT e TP73 foram validados por qPCR e a sugestiva interação entre esses fatores nos tumores adrenocorticais merecem futuras investigações. O potencial funcional das culturas T83-ACC, T36-REC e T7-ACA(P) foram evidenciados, e mostraram que podem ser bons modelos para estudo da ação de hormônios e seus mecanismos. / The adrenocortical adenoma is common in adults, since carcinoma is rare and aggressive. Even with standardized scores, it is still difficult to differentiate these tumors, the study of efficient markers in the detection and differentiation is necessary. Because they are rare and diverse clinical manifestations in vitro cultures can be a tool for the study of disease processes that involve. Molecular and functional characterization of cultured tumor cells of patients was conducted. PCR Array results did not show a pattern that differentiates cultures on the basis of diagnoses. This analysis showed higher expression 7 oncogenes and tumor suppressors 9 showed low expression in cultures. WWOX, FHIT and TP73 were validated by qPCR and suggestive interaction between these factors in adrenocortical tumors deserve further investigation. The functional potential of T83-ACC, T36-REC and T7-ACA(P) cell cultures were found, and shown confirm that they can be good models for studying the action of hormones and their mechanisms.
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Macroscopic modelling of hybridoma cell fed-batch cultures with overflow metabolism: model-based optimization and state estimationAmribt, Zakaria 23 June 2014 (has links)
Monoclonal antibodies (MAbs) have an expanding market for use in diagnostic and therapeutic applications. Industrial production of these biopharmaceuticals is usually achieved based on fed-batch cultures of mammalian cells in bioreactors (Chinese hamster ovary (CHO) and Hybridoma cells), which can express different kinds of recombinant proteins. In order to reach high cell densities in these bioreactors, it is necessary to carry out an optimization of their production processes. Hence, macroscopic model equations must be developed to describe cell growth, nutrient consumption and product generation. These models will be very useful for designing the bioprocess, for developing robust controllers and for optimizing its productivity.<p>This thesis presents a new kinetic model of hybridoma cell metabolism in fed batch culture and typical illustration of a systematic methodology for mathematical modelling, parameter estimation and model-based optimization and state estimation of bioprocesses. <p>In the first part, a macroscopic model that takes into account phenomena of overflow metabolism within glycolysis and glutaminolysis is proposed to simulate hybridoma HB-58 cell cultures. The model of central carbon metabolism is reduced to a set of macroscopic reactions. The macroscopic model describes three metabolism states: respiratory metabolism, overflow metabolism and critical metabolism. The model parameters and confidence intervals are obtained via a nonlinear least squares identification. It is validated with experimental data of fed-batch hybridoma cultures and successfully predicts the dynamics of cell growth and death, substrate consumption (glutamine and glucose) and metabolites production (lactate and ammonia). Based on a sensitivity analysis of the model outputs with respect to the parameters, a model reduction is proposed. <p>In the next step, the effort is directed to the maximization of biomass productivity in fed-batch cultures of hybridoma cells based on the overflow metabolism model. Optimal feeding rate, on the one hand, for a single feed stream containing both glucose and glutamine and, on the other hand, for two separate feed streams of glucose and glutamine are determined using a Nelder-Mead simplex optimization algorithm. Two different objective functions (performance criteria) are considered for optimization; the first criterion to be maximized is the biomass productivity obtained at the end of the fed-batch culture, the second criterion to be minimized is the difference between global substrate consumption and the maximum respiratory capacity.<p>The optimal multi exponential feed rate trajectory improves the biomass productivity by 10% as compared to the optimal single exponential feed rate. Moreover, this result is validated by the one obtained with the analytical approach in which glucose and glutamine are fed to the culture so as to control the hybridoma cells at the critical metabolism state, which allows maximizing the biomass productivity. The robustness analysis of optimal feeding profiles obtained with different optimization strategies is considered, first, with respect to parameter uncertainties and, finally, with respect to model structure errors.<p>Finally, the overflow metabolism model is used to develop an extended Kalman filter for online estimation of glucose and glutamine in hybridoma cell fed-batch cultures based on the considered available measurements (biomasses (on-line), lactate and ammonia (on-line or off-line)). The observability conditions are examined, and the performances are analysed with simulations of hybridoma cell fed-batch cultures. Glutamine estimation sensitivity is enforced by minimizing a cost function combining a usual least-squares criterion with a state estimation sensitivity criterion. <p> / Doctorat en Sciences de l'ingénieur / info:eu-repo/semantics/nonPublished
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Au delà des frontières du glioblastome : caractérisation de la zone péritumorale des glioblastomes / Beyond the frontiers of glioblastoma : multidisciplinary characterisation of glioblastoma's peritumoral brain zoneLemée, Jean-Michel 26 February 2015 (has links)
Le glioblastome (GB) est une tumeur hétérogène, agressive devant laquelle les possibilités thérapeutiques disponibles restent limitées. L’étude de la zone péritumorale macroscopiquement normale (ZMN) des GB est essentielle à la compréhension de ses mécanismes de progression et de récidive. Le premier objectif de ce travail de Thèse a été de comparer les données de transcriptomique et de protéomique issues de l’analyse de la zone tumorale des GB dans le cadre du Projet Gliome Grand Ouest. Le taux de concordance entre les 2 modalités est faible, retrouvant toutefois comme point commun une dysrégulation de la protéine légère des neurofilaments qui pourrait servir de biomarqueur potentiel des GB. Le deuxième objectif de ce travail de Thèse a été la caractérisation de la ZMN des GB. Nous avons mis en évidence que cette zone, dont l’aspect est similaire à première vue à celui du tissu cérébral sain, n’est pas une simple zone de transition entre le GB et le tissu cérébral sain. En effet, la ZMN est une entité spécifique possédant des caractéristiques qui lui sont propres, comme la présence d’un phénotype particulier de cellules tumorales infiltrantes et de cellules stromales et une sur’expression des protéines CRYAB et H3F3A. Ce travail de Thèse a aussi été l’occasion de développer de nouvelles techniques d’imagerie per-opératoire de la ZMN, afin d’évaluer la présence d’un contingent tumoral et ainsi optimiser la qualité de la résection chirurgicale. La caractérisation de cette ZMN nous permet de mieux appréhender son implication dans la tumorogenèse et la présence de caractéristiques spécifiques de cette zone ouvre la porte à la détection de biomarqueurs spécifiques, ainsi qu’au développement de thérapies ciblées. Ce travail de Thèse a été valorisé par 2 publications, 2 articles soumis et un brevet est en cours de dépôt et d’évaluation par un cabinet de brevet. / Glioblastoma (GB) is a heterogeneous andaggressive tumor, before which therapeutic options arelimited. The study of the macroscopically normalperitumoral brain zone (PBZ) of GB is essential tounderstand its mechanisms of progression andrecurrence.The first objective of this thesis work was tocompare the transcriptomic and proteomic data from theGB tumor area obtained through the “Grand Ouest”glioma Project. The concordance rate between the 2modalities is low. However, one of the common featureis the dysregulation of neurofilament light polypeptide,which could serve as a biomarker potential of GB.The second objective of this thesis was thecharacterization of the PBZ. We have shown that thisarea, similar at first glance to that of healthy braintissue, is not a simple transition area between the GBand healthy brain tissue but a specific entity withcharacteristics of its own. For example, the ZMNpresents a particular phenotype of infiltrating GB cellsand stromal cells and a surexpression of CRYAB andH3F3A proteins.This thesis work was also an opportunity todevelop new intraoperative imaging techniques of thePBZ, with the aim to assess the presence of a tumoralinfiltration and optimize the quality of the surgicalresection.The characterization of this PBZ allows us tobetter understand its involvement in tumorigenesis andthe presence of specific characteristics of this areaopens the door for the detection of specific biomarkersand the development of targeted therapies.This thesis work was led to 2 publications, 2articles submitted and a patent being evaluated andredacted by a patent office.
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Cytotoxicita vybraných naftochinonů na prostatických buněčných liniích / Cytotoxicity of selected naphthoquinones on prostatic cell culturesMondeková, Věra January 2013 (has links)
This master´s thesis discusses cytotoxicity of selected naphthoquinones on prostatic cell cultures. The introductory part is dedicated to general characteristic of naphthoquinones with focus on their cytotoxicity, testing of cytotoxicity and mechanisms of cytotoxicity. This part is followed by chapters about cytotoxicity, characteristics and biological activities of selected naphthoquinones; plumbagin and naphthazarin. The last part of this thesis’ theoretical section speaks about fluorescence microscopy and its use in research of naphthoquinones cytotoxicity. The practical part is dedicated to evaluation of cytotoxical tests’ results and to analysation of pictures of cells obtained by fluorescence microscope. At the end of thesis, all finding are summarized and put in the context.
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Physiological effects of conditioned medium and passage number on Spodoptera frugiperda Sf9 serum free culturesSvensson, Ingrid January 2005 (has links)
The aim of this study was to better understand the role of conditioned medium (CM) in Spodoptera frugiperda Sf9 insect cell proliferation and recombinant protein production using the baculovirus expression system. CM was found to stimulate cell proliferation. Addition of CM and 10 kDa CM filtrate to an Sf9 culture decreased the lagphase and the maximum cell density was reached earlier than for cultures in fresh medium. The positive effect of 10 kDa CM filtrate showed that CM contains at least one small growth promoting factor. The effect was not eliminated by trypsin treatment. Addition of CM or 10 kDa CM filtrate to Sf9 cultures was found to have a negative effect on the recombinant protein production. The effect was thought to be indirect and most probably via the impact of CM on cell physiology. CM was also found to contain proteinase activity. The proteinase was identified as Sf9 cathepsin L. A proform with a molecular mass about 49 kDa and two active forms at about 39 and 22 kDa were found. The role of cathepsin L in Sf9 cultures is not yet clear. However, the knowledge of the presence of this proteinase in CM can be of great value for improving product quality and yield. Further, CM was found to have other properties as well: a concentrated fraction of CM exhibited strong antibacterial activity towards Bacillus megaterium and a weaker activity towards Escherichia coli. B. megaterium lysed rapidly after incubation in the CM fraction. Repeated subculturing of Sf9 cells provoked a switch in growth kinetics. After 30-45 passages the cells started to proliferate earlier after inoculation and addition of CM had no longer a growth stimulating effect. However, CM still stimulated growth of a culture with low passage (LP) number (up to 45 passages). High passage cells (HP cells, over 100 passages) displayed a shorter lagphase than LP cells and the culture reached the maximum cell density 24-48 h earlier. Cell cycle analysis showed that the Sf9 cells were transiently synchronised in the G2/M phase 10 h after inoculation, before proliferation was initiated. This synchronisation was more pronounced for HP cells than for LP cells, which correlated to a higher recombinant protein production in baculovirus infected HP cells than in LP cells. Synchronisation of cells in G2/M by yeastolate-limitation before infection with baculoviruses suggested that the degree of synchronisation is connected to the cell density dependent decrease in recombinant protein production of Sf9 cultures. / QC 20101222
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Δ-9-Tetrahydrocannabinol: Effect on Macromolecular Synthesis in Human and Other Mammalian CellsBlevins, R. D., Regan, J. D. 01 June 1976 (has links)
The principal psychoactive component of marihuana is Δ-9-tetrahy-drocannabinol. This compound at 10-5 molar concentration in the medium of human cell cultures appeared to inhibit DNA, RNA, and protein synthesis by 50, 40, and 30% respectively, as measured by incorporation of radioactive precursors into acid-insoluble cell fractions in human diploid fibroblasts, human neuroblastoma cells, and mouse neuroblastoma cells. While Δ-9-tetrahydrocannabinol inhibited semiconservative DNA synthesis, it had no effect on DNA repair synthesis in human cells as assayed by the photolysis of 5-bromodeoxyuridine incorporation into DNA during repair after ultraviolet radiation damage. Δ-9-tetrahydrocannabinol also had no effect on rejoining of DNA single-strand breaks induced by γ-rays. The nonspecificity of the inhibition of macromolecular synthesis by Δ-9-THC suggested a possible interference with uptake of radioactive precursors. However, experimentation has shown that this depression of macromolecular synthesis cannot be accounted for by reduced transport of radioactive precursors into the cell because the rate of transport of these precursors into the cell is essentially the same in the presence or absence of Δ-9-THC. Pool sizes of macromolecular precursors as measured radioisotopically (3Hthymidine, 3H-uridine, 14C-leucine) appear to be reduced about 50%, and this reduced pool size could fully account for the reduced macromolecular synthesis seen in the presence of Δ-9-THC. We do not know what causes this apparent reduction of pool sizes in the presence of Δ-9-THC.
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