Perlecan is an important basement membrane heparan sulfate (HS) proteoglycan that is essential for various cell signaling events involved in tissue development. Heparanase is a lysosomal enzyme involved in the turnover of HS. This project aimed to assist in researching the structure of HS on perlecan and how this structure changes with tissue development. This will be achieved by generating monoclonal antibodies that have an altered affinity for perlecan after heparanase treatment. Recombinant perlecan domain I was characterized by ELISA and western blotting and used as the antigen for two fusions. The first fusion was focused on the production of IgM the common subtype of anti-glycosaminoglycans antibodies. However, no clones were produced, which may have been due to the lack of feeder layers. In order to address this problem, the fibroblast cell line MRC-5 was used as a feeder layer in the second fusion. From this fusion, we obtained 216 positive cultures, which were screened against full length perlecan from endothelial cells. Of these, 26 cultures were tested against heparanase treated perlecan, and then 2 cultures were chosen for subcloning based on the different immunoreactivity between enzyme treated and nontreated perlecan. From the 2 chosen cultures, 13 sub clones were derived and 10 of them were adapted into a serum free culture environment. The 10 monoclonal antibodies displayed strong immunoreactivity with full length perlecan in ELISA and Western Blotting. When they were used as primary antibodies in Immunocytochemistry, they were able to recognize the native perlecan deposited by human chondrocytes. When the cells were incubated with heparanase, antibody 5D7-2E4 and 13E9-3G5 showed an increase in immunoreactivity while antibody 13E9-3B3 gave a decrease. These three antibodies will be the potential tools used in the future to study perlecan turnover in different cells and tissue. The remaining seven antibodies will also be very useful in the research of perlecan as they have been shown to bind to the protein core. In the future, it will be worth subcloning some of the frozen stored stocks of uncloned hybridomas, where there are potential opportunities to select antibodies, which will react with the carbohydrate chains on perlecan.

Submitted by Programa de Pós-graduação em Biotecnologia (mebiotec.ufba@gmail.com) on 2017-04-06T12:53:22Z No. of bitstreams: 1 Dissertação Final - Adriana Andrade.pdf: 2860120 bytes, checksum: 9c25748f97bf9e85c8b9bcc05e3f32d5 (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-06-29T14:41:03Z (GMT) No. of bitstreams: 1 Dissertação Final - Adriana Andrade.pdf: 2860120 bytes, checksum: 9c25748f97bf9e85c8b9bcc05e3f32d5 (MD5) / Made available in DSpace on 2017-06-29T14:41:03Z (GMT). No. of bitstreams: 1 Dissertação Final - Adriana Andrade.pdf: 2860120 bytes, checksum: 9c25748f97bf9e85c8b9bcc05e3f32d5 (MD5) / CAPES / O vírus da dengue (DENV) é um arbovírus pertencente à família Flaviviridae, gênero Flavivirus, apresentando quatro sorotipos denominados DENV-1, DENV-2, DENV-3 e DENV-4. No Brasil, a infecção pelo DENV-4 ressurgiu em 2010, após 31 anos, expondo a população ao alto risco de desenvolvimento da dengue grave, devido à co-circulação dos quatro sorotipos. Nesse contexto, os anticorpos monoclonais (AcM) apresentam-se como uma ferramenta importante devido à sua potencial aplicação como ferramenta biotecnológica. O objetivo deste trabalho foi a produção e caracterização de AcM contra DENV-4. A metodologia para a produção de AcM foi desenvolvida por Köhler e Milstein (1975). Resumidamente, os animais de experimentação foram hiperimunizados com antígeno viral DENV-4, obtidos a partir da multiplicação do vírus em células de cultura C6/36, e subsequente concentração e precipitação com polietilenoglicol 8000. A triagem dos sobrenadantes dos hibridomas para reatividade ao DENV-4 foi realizada pelo Ensaio Imunoenzimático (ELISA), e a caracterização dos AcM foi realizada pelas técnicas de ELISA, Dot-Blot, Imunofluorescência Indireta (IFI), Western-Blot. Um total de dez hibridomas foram obtidos e os AcM produzidos foram denominados A2, B1, B4, C11, D2, E4, F10, F12, H1, H12. Os AcM apresentaram diferentes perfis de reatividade frente aos diversos testes de detecção antigênica, demonstrando um reconhecimento direcionado principalmente para prM. Em conclusão, este estudo mostra o potencial de utilização dos AcM produzidos como insumo biotecnológico; sejam em estudos a respeito do vírus e sua patogênese, sejam em uma possível aplicação como ferramenta imunodiagnóstica. / Dengue virus (DV) is an arbovirus belonging to family Flaviviridae, genus Flavivirus, with four serotypes named DENV-1, DENV-2, DENV-3 and DENV-4. In Brazil, the infection by DENV-4 reemerged in 2010 after 31 years, exposing the population at high risk for developing severe diseases because of the co-circulation of the four serotypes. The monoclonal antibodies (AcM) are important tools due to its potential application in biotechnology. The aim of this work was to produce and to characterize of AcM against DENV-4. The methodology to produce AcM was developed by Köhler and Milstein (1975). Briefly, experimental animals were hyperimmunized with DENV-4 viral antigen obtained to virus multiplication in C6/36 culture cells and subsequent concentration and precipitation with polyethylene glycol 8000. ELISA test was performed to screen hybridoma supernatants for reactivity to DENV-4, and the characterization of AcM was performed by ELISA, Indirect Immunofluorescence (IFI), Dot-Blot and Western-Blot techniques. A total of ten hybridomas were obtained producing AcM named A2, B1, B4, C11, D2, E4, F10, F12, H1 and H12. AcM showed different reactivity profiles in the tests, showing a dominant recognition to prM. In conclusion, this study shows the potential use of AcM produced as biotechnology tool; to study the virus and its pathogenesis, or a possible application in immunodiagnostic assays.

Orientador: Wirla Maria da Silva Cunha Tamashiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-05T10:09:32Z (GMT). No. of bitstreams: 1 Scuro_LorenSemionatto_M.pdf: 980651 bytes, checksum: f54e323b2050cb528e7e829af26086ad (MD5) Previous issue date: 2005 / Resumo: O objetivo do presente trabalho foi a obtenção e caracterização de anticorpos monoclonais contra a IL6 humana recombinante (hr) para serem empregados em ensaios imunoenzimáticos do tipo ELISA (Enzyme linked immunosorbent assay) de detecção da IL6 humana nativa, presente em fluídos biológicos de pacientes portadores de quadros onde os níveis de IL6 encontram-se elevados, ou então produzida por monócitos humanos e murinos ativados in vitro. Dois grupos de hibridomas (1A6 e 3B1) secretores de anticorpos monoclonais anti-IL6 foram obtidos pela fusão de células de mieloma da linhagem SP2 Ag14/0 com esplenócitos de camundongos BALB/c, previamente imunizados com a IL6 humana recombinante. Esses hibridomas foram selecionados com base em sua reatividade com a hrIL6, através de ensaios do tipo ELISA indireto. As imunoglobulinas (Igs) monoclonais produzidas pelos hibridomas dos dois grupos são do isotipo IgG1 Kappa e foram purificadas de líquidos ascíticos e dos sobrenadantes de cultura por cromatografia de afinidade. As proteínas purificadas foram conjugadas com biotina para uso em ensaios de ELISA de captura da hrIL6, de modo a se identificar um ou mais pares de anticorpos adequados a esse tipo de teste, bem como para definir a sensibilidade de detecção da citocina. O par de anticorpos monoclonais 3B1E4 x 1A6F10-biotinilado se mostrou mais promissor nos ensaios de ELISA, detectando a citocina recombinante entre 8 e 512 ng/mL, liberando densidades óticas mais elevadas. Todos os anticorpos monoclonais (AcMos) anti IL6 estudados foram capazes de neutralizar a atividade biológica da citocina em ensaios empregando o hibridoma B13.9, uma célula dependente de IL-6 para seu crescimento. Finalmente, o par de hibridomas anti IL6 3B1E4 e 1A6F10 foi estudado quanto às suas principais características de cultivo: crescimento, produção in vitro dos anticorpos, consumo de glicose e produção de lactato e amônia. O seqüênciamento da porção N-terminal do par 3B1E4 e 1A6F10 revelou que as cadeias leves dos dois anticorpos apresentam seqüência idêntica de aminoácidos. Porém, a análise dos dez resíduos de aminoácidos presentes na região variável das cadeias pesadas resultou em seqüências completamente distintas nos dois monoclonais, sendo um forte indício de diferença nos sítios de ligação ao antígeno dos anticorpos estudados / Abstract: In the present study we have developed monoclonal antibodies against human recombinant (hr) IL6, for the use in ELISA assays to detect the human native IL6 present in biological fluid of patients or in supernatant of activated human and rodent monocytes. Two families of hibridomas (1A6 and 3B1) secreting MAb against-IL6 were obtained from the fusion of mieloma cells from SP2 Ag14/0 lineage with spleen cells from BALB/c mice, previously immunized with human recombinant IL6. Those hibridomas were selected on the basis of their reactivity with the hrIL6, through an ELISA indirect assay. The monoclonal immunoglobulins (Igs) produced by these hibridomas are from IgG1 Kappa isotype and were purified from ascitic fluid and culture supernatant by affinity chromatography. The purified proteins from the ascitic fluid were conjugated with biotin for the use in ELISA hrIL6 capture assay, to identify one or more pairs of antibodies appropriated to this kind of test, as well to define the sensibility of cytokine detection. The pair of MAbs 3B1E4 x 1A6F10-biotinilates was shown promising in ELISA assays, detecting the recombinant cytokine between 8 and 512 ng/mL, liberating elevated optical densities. All of the a-IL6 MAbs studied were capable to neutralize the biological activity of the cytokine in attempt employing the hibridoma B13.9 IL-6 dependent. Finally, the pair of hibridomas a-IL6 3B1E4 and 1A6F10 was studied as regards his main cultivation characteristics: growth, MAb production in vitro, lactate and ammonia production and glucose consumption. The N-Terminal portion sequence of 3B1E4 and 1A6F10 revealed that both light-chains present identical amino acid sequence. However, the analysis of the ten amino acid residues present in variable region of heavy-chains resulted in completely distinct sequences in both antibodies, being a strong indication of difference in their ability to recognize the antigen / Mestrado / Imunologia / Mestre em Genética e Biologia Molecular

Colorectal cancer (CRC) is one of the most common forms of cancer in Western nations, it is however uncommon in sub-Saharan Africa. In South Africa there is an approximate ten-fold lower incidence of CRC in black patients compared to Caucasian patients. This could be due to differences in lifestyles and environment that exist between the various population groups. Underlying molecular events could also account for the difference in susceptibility to colorectal cancer. Mutations in the Peutz-Jeghers syndrome (PJS) gene, STK11, predispose to amongst others colorectal cancer. To examine the role of this gene in South African patients with CRC, DNA from 208 tumours (104 black patients, 104 Caucasian patients) was screened for STK11 mutations via PCR-SSCP analysis. In total 8 novel missense mutations, one of which was germline, were identified in seven tumours (~3.4% 7/208) from 5 black and two Caucasian patients. One tumour from a Caucasian patient was found to be a compound heterozygote. Peutz-Jeghers syndrome was thus diagnosed in 0.96% (1/104) of black patients via a germ line mutation. Thus 4.8% (5/104) of tumours from black patients and 1.9% (2/104) of tumours from Caucasian patients harbour STK11 missense mutations. In addition, 3 synonymous and 5 intronic mutations were detected in a further 73 tumours from black patients, whereas only 3 synonymous and 5 intronic mutations were detected in 25 tumours from Caucasian patients. The present study is the sixth to suggest that somatic mutation of the STK11 gene in sporadic colorectal cancer of Caucasians is an infrequent event. However, this is only the second study of a non-Western population to show somatic mutations in sporadic cases of CRC. Furthermore with regard to the anatomic site of tumours with somatic missense mutations, the present study found that for black patients 7.69% (2/26) of the left-sided tumours, 2% (1/50) of rectal tumours and 4.54% (1/22) of right-sided tumours harboured mutations. Thus the frequency of missense mutations of left-sided CRC tumours compared to right-sided tumours was not significantly elevated (÷2-test, 1df, p = 0.881) in the black population. This study represents the first investigation into the role of the STK11 gene in putative sporadic cases of CRC from both black and Caucasian South African patients. The observed mutations clearly show that mutations of the STK11 gene are infrequent in the CRCs of the South African Caucasian population, and more frequent in the South African black population. This may be a reflection of the differences in lifestyle and incidence of CRC in the different populations. / Dissertation (MSc (Human Genetics))--University of Pretoria, 2006. / Genetics / unrestricted

A digoxina é um medicamento usado para tratar distúrbios cardíacos, com janela terapêutica muito estreita. Para combater seu efeito tóxico, fragmentos Fab do anticorpo policlonal anti-digoxina estão disponíveis comercialmente. Nosso objetivo foi a obtenção de variantes de fragmentos Fab do anticorpo monoclonal anti-digoxina usando a tecnologia phage display, que permite gerar fragmentos de anticorpos de alta afinidade e especificidade. Uma biblioteca combinatória de fragmentos Fab anti-digoxina foi construída no vetor pComb3X a partir do RNA total do hibridoma anti-digoxina. Seis clones foram isolados, todos com sequência idêntica na cadeia pesada. A cadeia leve apresentou 2 clones idênticos, um pseudogene e um clone com um aminoácido distinto no CDR2. Quatro clones apresentando variações na sequência do framework1 da cadeia leve foram expressos como fragmentos Fab solúveis. Todos apresentaram ligação à digoxina-BSA por ELISA e Western blotting. A ligação específica do anticorpo também foi confirmada pelo BIAcore, que permitiu ranqueamento entre os clones. / Digoxin is a pharmaceutical used in the control of cardiac dysfunction. Its therapeutic window is very narrow. To counteract the toxic effect, polyclonal anti-digoxin Fab fragments are commercially available. Our goal was to obtain variants of monoclonal anti-digoxin Fab fragments by phage display technology, which allows the generation of high affinity and specificity antibody fragments. Anti-digoxin Fab fragments combinatorial library was constructed into pComb3X vector from total RNA of anti-digoxin hybridoma. Six clones were isolated and the heavy chain presented the same sequence. For the light chain, 2 clones were identical, one was a pseudogene and other one presented a distinct amino acid in the CDR2. Four clones presenting variations in the framework 1 were induced to express soluble Fab fragments, all positive for anti-digoxin binding in ELISA assays and Western blotting. The specific binding of the antibody was further confirmed by BIAcore, which allowed ranking of the clones.

O diagnóstico da infecção por ETEC é baseada na detecção dos seus principais fatores de virulência, as toxinas termo-lábil e termo-estável, através de métodos de biologia molecular ou imunossorológicos. A tecnologia de anticorpos recombinantes permite a obtenção de moléculas com baixo custo, afinidades e especificidades desejáveis, através da clonagem dos domínios variáveis das cadeias leve e pesada da imunoglobulina, fusionados a um ligante flexível que permite a correta interação entre os domínios e a preservação do sítio de ligação ao antígeno. Este estudo teve como objetivo a construção de um fragmento variável em cadeia única (scFv), a partir de hibridomas produtores de anticorpo monoclonal anti-LT, seguida da sua produção em células bacterianas. Um fragmento variável em cadeia única com 723 pb foi obtido, sendo expresso em cepa de Escherichia coli como uma proteína com peso molecular aparente de 30 kDa. Após purificação em cromatografia de afinidade a Ni2+ e renaturação, o scFvLT foi capaz de reconhecer a toxina LT por ELISA de captura e Immunodot, porém não foi capaz de reconhecer as subunidades da toxina LT, por Immunoblotting, nem de neutralizar sua atividade citopática em células adrenais Y1. / Heat-labile (LT) and heat-stable (ST) toxins are the main ETEC\'s virulence factors and infection diagnosis is based on their detection by molecular biology or immunoserological methods. The advances on antibody biotechnology provide alternatives to obtain low cost antibodies with desirable affinities and specificities by cloning immunoglobulin\'s heavy and light variable domains (HV and LV) as a single-chain fusion interspaced by a flexible linker, which allowing the correct interaction between the domains and preserving the antigen-binding site. In this study we aimed the construction of a scFv upon hybridoma cells that produce an anti-LT monoclonal antibody following its bacterial production. A single-chain fragment variable with 723 bp was obtained and expressed as a protein with apparent molecular weight of 30 kDa in Escherichia coli strain. The scFvLT recombinant antibody was submitted to metal affinity chromatography and refolding and was tested by immunoenzimatic assays. Refolded scFvLT was able to recognized LT toxin by capture ELISA and Immunodot. However, scFvLT wasnt able to recognize LT toxin subunits by Immunoblotting neither neutralize its activity in Y1 adrenal cells.

Bovine herpesvirus type 1 (BoHV-1) is an important pathogen of cattle and causes significant economic losses to livestock industry. Monoclonal antibodies (mAbs) represent useful tools for diagnostic and research purposes. Most mAbs produced against BoHV-1 are directed to glycoprotein gC (gC), an abundant and immunodominant envelope antigen. In the present study, antigens of a BoHV-1 strain defective in the gene coding for glycoprotein C was used to immunize BALB/c mice to produce mAbs with other protein specificities. After fusion and selection of 54 hybridomas resistant to the selective medium HAT, three hybridomas secreting mAbs directed to BoHV-1 antigens were obtained (1F1, 2H4, 4D7). The mAbs belong to the IgG2a isotype and reacted in an indirect fluorescent antibody assay (IFA) and indirect immunoperoxidase staining (IPX) of BoHV-1-infected cells at dilutions up to 1:640 (culture supernatant) and 1:20,000 (ascitic fluid). The three mAbs tested reacted with 14 isolates of respiratory and/or genital disease and with 17 isolates of neurological disease and showed variable level of neutralizing activity against the most of these isolates. The protein specificity of the mAbs could not be determined, because none of them reacted with viral proteins in western immunoblot. On the other hand, the three mAbs reacted in IFA with viral antigens of BoHV-1 and BoHV-5 mutant strains defective on gC, gE, gI and US9 genes, demonstrating that they are directed against other viral antigens. Because the high reaction titer and the wide range of reactivity, these mAbs have potential use in diagnostics techniques. Also, these mAbs may be useful to map conserved neutralizing epitopes in the envelope glycoproteins / O herpesvírus bovino tipo 1 (BoHV-1) é um dos principais patógenos de bovinos e causa perdas significativas para a bovinocultura. Anticorpos monoclonais (AcMs) se constituem em importantes ferramentas para o diagnóstico e pesquisa em diversos aspectos da biologia desse agente. A maioria dos AcMs produzidos contra o BoHV-1 são direcionados contra a glicoproteína C (gC), um antígeno abundante e imunodominante do envelope viral. No presente trabalho, antígenos de uma cepa do BoHV-1 defectiva no gene da gC foram utilizados para a imunização de camundongos visando a produção de AcMs com outras especificidades protéicas. Após fusão e seleção de 54 clones resistentes ao meio seletivo HAT, foram obtidos três hibridomas secretores de AcMs contra antígenos do BoHV-1 (1F1, 2H4, 4D7). Os AcMs pertenceram ao isotipo IgG2a e reagiram em diluições de até 1:640 (sobrenadante de cultivo) e 1:20.000 (fluído ascítico) em testes de imunofluorescência indireta (IFA) e imunoperoxidase indireta (IPX). Os três AcMs reagiram na IFA com 14 isolados de doença respiratória e/ou genital e com 17 isolados de doença neurológica, e apresentaram atividade neutralizante em níveis variáveis contra a grande maioria desses isolados. A especificidade protéica dos AcMs não foi determinada, pois nenhum deles reagiu com proteínas virais na técnica de Western blot. Por outro lado, os três AcMs reagiram na IFA contra antígenos virais de cepas do BoHV-1 e BoHV-5 com deleção dos genes que codificam as proteínas gC, gE, gI e US9, demonstrando que são direcionados contra outros antígenos virais. Pelo seu alto título de reação e pelo amplo espectro de reatividade, esses AcMs possuem aplicação potencial em técnicas diagnósticas. Esses AcMs também podem ser úteis para o mapeamento de epitopos neutralizantes conservados nas glicoproteínas do envelope

O diagnóstico da infecção por ETEC é baseada na detecção dos seus principais fatores de virulência, as toxinas termo-lábil e termo-estável, através de métodos de biologia molecular ou imunossorológicos. A tecnologia de anticorpos recombinantes permite a obtenção de moléculas com baixo custo, afinidades e especificidades desejáveis, através da clonagem dos domínios variáveis das cadeias leve e pesada da imunoglobulina, fusionados a um ligante flexível que permite a correta interação entre os domínios e a preservação do sítio de ligação ao antígeno. Este estudo teve como objetivo a construção de um fragmento variável em cadeia única (scFv), a partir de hibridomas produtores de anticorpo monoclonal anti-LT, seguida da sua produção em células bacterianas. Um fragmento variável em cadeia única com 723 pb foi obtido, sendo expresso em cepa de Escherichia coli como uma proteína com peso molecular aparente de 30 kDa. Após purificação em cromatografia de afinidade a Ni2+ e renaturação, o scFvLT foi capaz de reconhecer a toxina LT por ELISA de captura e Immunodot, porém não foi capaz de reconhecer as subunidades da toxina LT, por Immunoblotting, nem de neutralizar sua atividade citopática em células adrenais Y1. / Heat-labile (LT) and heat-stable (ST) toxins are the main ETEC\'s virulence factors and infection diagnosis is based on their detection by molecular biology or immunoserological methods. The advances on antibody biotechnology provide alternatives to obtain low cost antibodies with desirable affinities and specificities by cloning immunoglobulin\'s heavy and light variable domains (HV and LV) as a single-chain fusion interspaced by a flexible linker, which allowing the correct interaction between the domains and preserving the antigen-binding site. In this study we aimed the construction of a scFv upon hybridoma cells that produce an anti-LT monoclonal antibody following its bacterial production. A single-chain fragment variable with 723 bp was obtained and expressed as a protein with apparent molecular weight of 30 kDa in Escherichia coli strain. The scFvLT recombinant antibody was submitted to metal affinity chromatography and refolding and was tested by immunoenzimatic assays. Refolded scFvLT was able to recognized LT toxin by capture ELISA and Immunodot. However, scFvLT wasnt able to recognize LT toxin subunits by Immunoblotting neither neutralize its activity in Y1 adrenal cells.

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

High grade aggressive lymphomas have high mortality. By their nature, more than 40% of patients die from these diseases even with the improved treatment strategies currently available for oncology patients. The characteristic feature is that they are functionally heterogeneous and therefore have different biological and molecular signatures which make it difficult for all groups to respond to same line of treatment. Based on the above, I set out to look at the impact of viral and cellular gene products on these groups of diseases: In chapter 3 I developed monoclonal antibodies against HERV‐K10. I subsequently investigated their expressions in aggressive lymphomas including Diffuse Large B‐cell lymphoma, Hodgkin’s lymphoma and Primary CNS lymphomas. I showed HERV‐K10 is expressed in cell lines of aggressive lymphomas, but not in paraffin‐embedded tissues. In chapter 4 I showed that the expression of ATM using immune‐histochemistry techniques in aggressive lymphomas does offer a guide to prognosis and treatment. Nearly 30% of Diffuse Large B‐cell lymphomas express ATM, 55% of Hodgkin’s lymphomas and more than 80% of Primary CNS lymphomas. I also showed there is a correlation of ATM expression and EBV‐driven aggressive lymphomas and that this has a poor prognostic significance. Chapter 5 analysed the results obtained by generating, validating and evaluating data base of DLBCL and PCNSL from a retrospective cohort over a 17‐year period. The results confirmed that prognostic indicators including ATM, S1PR2, Autotaxin and EBV using immuno‐histochemistry techniques help with categorising aggressive lymphomas into different prognostic groups and does influence future management. In summary, my results showed there is a critical place for immuno‐histochemistry techniques in convincingly helping understand the expressions of viral and cellular gene products in aggressive lymphomas and in contributing positively to their management.