Made available in DSpace on 2014-08-20T13:32:47Z (GMT). No. of bitstreams: 1 dissertacao_neida_lucia_conrad.pdf: 1248896 bytes, checksum: effbf3a6777d64f323ea5206861ef2dd (MD5) Previous issue date: 2013-03-12 / The infectious bronchitis (IB) is an acute and highly contagious disease caused by a coronavirus that infects mainly cells of the respiratory and genitourinary of chicken. The economic importance of this disease results from decreased production, such as internal and external quality of eggs, reduced hatchability, feed efficiency and weight gain, increased mortality, condemnation of carcasses at slaughter, and also with drugs spending to suppress secondary bacterial infections. The diagnosis of IB is based on virus isolation, together with serology. The use of monoclonal antibodies (MAbs) has been collaborated to diagnosis of this type of disease, but their use are still limited because the MAbs used are imported, increasing the cost of such methodologies. This work reports the production, characterization and application of MAbs against the virus of infectious bronchitis (IBV). For the production of MAbs, Balb/c mice were immunized intraperitoneally with classic IBV (M41) or variant IBV. We generated two hybridomas secreting MAbs anti-IBV (IgM isotype) from the fusion of B-lymphocytes from a mouse immunized with the variant IBV and myeloma cells. Following the characterization by ELISA and Western blot, it was observed that MAbs recognize the IBV classic and variant, showing more intense reactions with IBV variant. The AcM 2 G4 seems to be specific for protein S1. The applicability of the MAbs was assessed using the immunohistochemistry technique on histological tissues of birds inoculated experimentally with classic IBV. These MAbs anti-IBV had the same pattern of label of the MAb used as positive control, labeling predominantly inflammatory cells. This result can be explained by the fact that in severe cases of infection with IBV, the antigen may be located also in inflammatory cells. It was concluded that the MAbs obtained have potential for use in immunodiagnostic for IB. / A bronquite infecciosa das galinhas (BI) é uma doença aguda, altamente contagiosa, causada por um Coronavírus que infecta principalmente células dos aparelhos respiratório e genito-urinário das galinhas. A importância econômica dessa doença decorre da diminuição na produção e qualidade interna e externa dos ovos, da redução da eclodibilidade, da diminuição da eficiência alimentar e do ganho de peso e do aumento da mortalidade e da condenação de carcaças ao abate, além dos gastos com medicamentos para debelar infecções bacterianas secundárias. A confirmação do diagnóstico de BI é baseada no isolamento viral, em conjunto com a sorologia. O uso de anticorpos monoclonais (AcMs) tem colaborado e facilitado o diagnóstico desse tipo de enfermidade, porém seu uso ainda é limitado, pois os AcMs utilizados são importados, elevando o custo de tais metodologias. Este trabalho relata a produção, caracterização e aplicação de AcMs contra o vírus da bronquite infecciosa das galinhas (VBI). Para a produção dos AcMs, camundongos Balb/c foram imunizados intraperitonealmente com o VBI clássico (M41) local ou com o variante. Foram gerados dois hibridomas secretores de AcMs anti-VBI do isotipo IgM a partir da fusão entre os linfócitos B de um camundongo imunizado com o VBI variante e células de mieloma. Foi observado, na caracterização por ELISA indireto e Western blot, que os AcMs reconhecem o VBI clássico e variante, sendo as reações com o VBI variante mais intensas. O AcM 2G4 parece ser específico para a proteína S1. A aplicabilidade dos AcMs foi avaliada através da técnica de imuno-histoquímica em tecidos de aves inoculadas experimentalmente com o VBI clássico. Os AcMs anti-VBI apresentaram o mesmo padrão de marcação que o AcM utilizado como controle positivo, ou seja, marcaram predominantemente células inflamatórias. Este resultado pode ser explicado pelo fato de que, em casos mais severos da infecção por VBI, o antígeno pode ser localizado também em células inflamatórias. Concluiu-se que os AcMs obtidos apresentam potencial para uso no imunodiagnóstico para BI.

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

No description available.

No description available.

Invariant natural killer T (iNKT) cells are a versatile subclass of T lymphocytes which recognize glycolipid antigens. iNKT cells are capable of rapidly producing a broad array of cytokines in response to stimulation; thus, they play an important role in the early regulation of a variety of immune responses. It was hypothesized that iNKT cells express functional Toll-like receptors (TLRs) and that stimulation of TLRs by their ligands modulates iNKT cells responses. In the first objective, it was revealed that upon stimulation with anti-CD3 monoclonal antibody and interferon (IFN)-α, expression of TLRs was enhanced in iNKT cells. Furthermore, stimulation of iNKT cells with TLR ligands led to a significant increase in the expression of several cytokines. In the second objective, the mechanisms behind the modulatory effects of the TLR9 ligand (CpG-ODN) on iNKT cells were determined. Altogether, these findings suggest a direct role for TLRs in iNKT cell activation. / Ontario Graduate Scholarship

Étant donné l'immunogénicité de l’antigène Dal et sa prévalence élevée (> 98% des chiens sont Dal-positifs), il peut être extrêmement difficile de trouver du sang compatible pour un patient Dal-négatif précédemment transfusé et ayant besoin d’une deuxième transfusion sanguine. De plus, l’accès aux réactifs pour le typage sanguin est actuellement limité, notamment parce qu'il dépend d’anticorps polyclonaux (AcP) produits à la suite de la sensibilisation de trois rares chiens Dal-négatifs identifiés sporadiquement au cours de la dernière décennie dans la colonie d'enseignement de la Faculté de médecine vétérinaire de l'Université de Montréal. Par conséquent, l’objective de cette étude était de produire et de caractériser un anticorps monoclonal murin (AcM) dirigé contre l’antigène canin Dal afin d’assurer la pérennité du typage sanguin Dal. Utilisant la technologie conventionnelle des hybridomes, 5 souris femelles BALB/c ont été immunisées par des injections intrapéritonéales répétées avec des concentrés de globules rouges canines lavés (GRc) Dal-positifs jusqu'à ce que le titrage d’anticorps soit suffisant (> 1 : 10000). Après la fusion de cellules spléniques avec des cellules de myélome, 573 surnageants ont été récoltés à partir du jour 12 post-fusion pour le dépistage avec la technique d'agglutination sur colonne de gel utilisant des GRc Dal-négatif et Dal-positif connus. Parmi 15 surnageants qui ont montré une réaction d’agglutination, un seul avait le patron souhaité (c'est-à-dire anti-Dal). Afin d’évaluer la spécificité et la sensibilité de l’AcM, le typage sanguin Dal de 62 échantillons de GRc a été réalisé en utilisant le AcM anti-Dal et deux AcP canins préalablement caractérisés: 45 échantillons Dal-positifs et 17 Dal-négatifs ont été identifiés avec une concordance de 100 % entre les réactifs (kappa = 1). L’AcM anti-Dal produit a été déterminé comme étant une IgG1. / Given the immunogenicity of the Dal antigen and its high prevalence (>98% of dogs are Dal-positive), it can be extremely difficult to find compatible blood for a previously transfused Dal-negative patient in need of a second blood transfusion. Moreover, access to blood typing reagents is currently limited, in part because it relies on polyclonal antibodies (PAb) produced following the sensitization of three rare Dal-negative dogs identified sporadically over the last decade in the teaching colony of the Faculty of Veterinary Medicine of the University of Montreal. Therefore, the objective of this study was to produce and characterize a murine monoclonal antibody (MAb) directed against the canine Dal antigen, to ensure perennity for Dal blood typing. Using conventional hybridoma technology, 5 BALB/c female mice were immunized by repeated intraperitoneal injections with Dal-positive washed canine red blood cell (cRBC) concentrates until antibody titer was sufficient (>1 : 10000). After fusion of splenic cells with myeloma cells, 573 supernatants were collected starting 12 days post-fusion for screening with the gel column agglutination technique using known Dal-negative and Dal-positive cRBC. Among 15 supernatants that showed an agglutination reaction, only one had the desired pattern (i.e., anti-Dal). To assess the specificity and sensitivity of the MAb, the Dal blood typing of 62 cRBC samples was performed using the anti-Dal MAb and two previously characterized canine PAb: 45 Dal-positive and 17 Dal-negative samples were identified with 100% agreement between reagents (kappa = 1). The anti-Dal MAb produced was determined to be IgG1.

Koffein (1,3,7-Trimethylxanthin) und Coprostanol (5beta-cholestan-3beta-ol) wurden im Berliner Oberflächenwasser nachgewiesen. Ihre Konzentrationen korrelierten mit dem Verunreinigungsgrad der Proben, was nahelegt, dass sie sich als Marker für menschliche Aktivität eignen. Bemerkenswerterweise wurde Koffein in jeder einzelnen Oberflächenwasserprobe oberhalb der Bestimmungsgrenze von 0,025 µg/L gefunden. Um Oberflächenwasserproben in größeren Serien zu untersuchen, war die Entwicklung zweier neuer Methoden erforderlich: ein Immunoassay, basierend auf einem monoklonalen Antikörper für Koffein und eine dispersive flüssig-flüssig Mikroextraktionsmethode (DLLME), gefolgt von Flüssigkeitschromatographie gekoppelt mit Tandem-Massenspektrometrie (LC-MS/MS) für Coprostanol. Der entwickelte Koffein-Immunoassay zeigt die beste je erhaltene Nachweisgrenze für Koffein (0,001 µg/L), erlaubt Hochdurchsatz-Analysen und erfordert keine Probenvorbereitung. Der Assay wurde auch erfolgreich für die Messung von Koffein in Getränken, Haarwaschmitteln, Koffeintabletten und menschlichem Speichel angewendet. Antikörper gegen Coprostanol sind nicht kommerziell erhältlich. Eine neue Strategie Anti-Coprostanol-Antikörper zu generieren wurde erarbeitet, die eine analoge Verbindung – Isolithocholsäure (ILA) – als Hapten verwendet, mit der eine Gruppe von Mäusen immunisiert wurde. Ein polyklonales Anti-ILA-Serum wurde produziert, welches Coprostanol bindet, aber die niedrige Affinität erlaubte nicht den Aufbau eines Immunoassays, der die Messung von Umweltkonzentrationen des Anayten (im Bereich ng/L) zulässt. Spezifische Anti-ILA-Immunglobuline G wurden auch in den Faeces der Mäuse gefunden. Coprostanol wurde in den Wasserproben durch die Verwendung einer neuentwickelten LC-MS/MS-Methode unter APCI-Ionisation (atmospheric pressure chemical ionisation) gemessen. Konzentrationen oberhalb von 0,1 µg/L wurden nach Voranreicherung der Probe mittels DLLME bestimmt. / Caffeine (1,3,7-trimethylxanthine) and coprostanol (5beta-cholestan-3beta-ol) were detected in samples of Berlin’s surface water. Their concentrations correlated with the contamination status of the samples, suggesting their usefulness as markers of human activity. Remarkably, caffeine concentrations were always well above the limit of quantitation of 0.025 µg/L. In order to screen surface water samples in larger series, the development of two novel methods was required: a monoclonal antibody-based immunoassay for caffeine and a dispersive liquid-liquid microextraction (DLLME) method, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) for coprostanol. The caffeine immunoassay developed shows the best analytical limit of detection (LOD) obtained so far for caffeine (0.001 µg/L), allows high-throughput analysis, and does not require sample pre-treatment. The assay was also successfully employed to measure caffeine in beverages, shampoos, caffeine tab-lets, and human saliva. Antibodies to coprostanol are not commercially available. A new strategy to generate anti-coprostanol antibodies was elaborated using an analogous com-pound as hapten – isolithocholic acid (ILA) – and immunizing a group of mice. A polyclonal anti-ILA serum was produced, which binds coprostanol but the low affinity did not permit setting up an immunoassay to measure environmental concentrations of the analyte (in the range of ng/L). Specific anti-ILA immunoglobulin G were also found in the faeces of the immunized mice. Coprostanol was quantified in the water samples using a newly developed LC-MS/MS method using atmospheric pressure chemical ionisation (APCI). Concentrations above 0.1 µg/L were determined after sample preconcentration using DLLME. This extraction method also proved to be successful for enrichment of coprostanol-related compounds such as cholesterol, cholestanol, cholestanone, ergosterol, and stigmasterol.