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Biotechnology is defined as the use of biological techniques to engineer or manufacture a product. Development and optimization of systems in biotechnology have witnessed extraordinary advances over the last few decades. It can be further enhanced through combination with micro/nanotechnology, enabling de vices miniaturization to the microscale that leads to a rapid and cost effective analysis using microfluidic systems. Microfluidic systems offer high parallelization and high throughput screening assays. This thesis aims to expand biotechnology in two main areas: (i) preparation of a suitable expression system for production of human Interleukin-7 (hIL-7) in insect cells, and (ii) fabrication of microfluidic systems for integration with biotechnology for high throughput cell screening assays using insect and yeast cells. Human IL-7 has multiple immune-enhancing properties, which make it an ideal candidate for immunotherapy in a variety of clinical situations. Currently, there is no convenient and cost effective method for producing hIL-7. We present the production of hIL-7 in insect cells for the first time. We used a baculovirus expression vector system (BEVS) and a non-lytic system to produce hIL-7 in insect cells. In addition, we investigated large scale production of hIL-7 using various bioreactors. The resulting insect cells produce hIL-7 at different rates. To select highly productive single cells, the presently existing methods are time consuming, labor intensive and have low throughput capacity. In addition, these methods need special equipment for operation, as well as large amounts of chemicals. In order to rapidly select cells that produce high amounts of hIL-7, we designed and fabricated a new microfluidic system based on a polyethylene glycol (PEG) microwell array and a track etched membrane. Using this system, single cells can be selected on the basis of their protein secretion rate after a few hours only wit / La biotechnologie est définie comme l'utilisation de techniques biologiques pour développer ou fabriquer un produit. Le développement et l'optimisation de systèmes dans la biotechnologie ont été témoins d'avances extraordinaires pendant les dernières dix années. Ils peuvent être davantage améliorés par la combinaison avec la micro/nanotechnology, qui nous permet de miniaturiser des artifices à la microéchelle qui cause une analyse rapide, efficace et à un bon prix en utilisant les systèmes microfluidiques. Les systèmes microfluidiques offrent une haute parallélisassion et un haut débit de criblage. Cette thèse a pour but de développer la biotechnologie dans deux régions principales : (i) la préparation d'un système d'expression convenable pour la production d'Interleukine-7 humaine (hIL-7) dans les cellules d'insectes et (ii) la fabrication de systèmes microfluidiques, intégrant la biotechnologie, pour le criblage à haut débit de cellules en utilisant des cellules de levure et d'insecte.L'IL-7 humaine a des propriétés activatrices du système immunitaire, qui le font un candidat idéal à l'immunothérapie dans une variété de cas cliniques. Actuellement, il n'y a aucune méthode convenable et peu coûteuse pour produire hIL-7Nous présentons la production de hIL-7 dans les cellules d'insectes pour la première fois. Nous avons utilisé un système de vecteurs d'expression baculovirus (BEVS) et un système non-lytique pour produire hIL-7 dans des cellules d'insectes. En plus nous avons enquêté la production grande échelle de hIL-7 en utilisant différents bioréacteurs.Les cellules d'insectes produites génèrent l'hIL-7 à différents taux. Les méthodes existantes pour choisir les cellules extrêmement productives sont très lentes avec une faible capacité de traitement en plus d'exiger de l'équipement spécifique pour l'opération, aussi bien que de grandes quantités de produits c

The mechanism of action of erythropoietin (EPO) and the effects of different cyclic nucleotides on hemoglobin synthesis have been studied using a serum-free system of fetal calf liver cells. dbcAMP, dbcGMP and cCMP stimulated hemoglobin synthesis at different concentrations, and their effects were quite different depending on the type of hemoglobin being synthesized. The analysis of (alpha) and (gamma) chains by high pressure liquid chromatography (HPLC) indicated that both the (alpha) and (gamma) globin chains are stimulated by the three cyclic nucleotides. Furthermore, Step III sheep plasma EPO stimulated the synthesis of cGMP in fetal calf liver cells. On the other hand, only cGMP stimulated the nuclear RNA polymerases measured at low ionic strength in the presence of methyl-isobutyl-xanthine (MIX), an inhibitor of phosphodiesterase. These results indicate that cGMP could be one of the second messengers of EPO on the stimulation of RNA synthesis and probably hemoglobin synthesis. / EPO also stimulated the RNA polymerase activities of fetal calf liver cells after one hour of incubation in vitro. This stimulation seems to be mediated by some factors present in the cytoplasm of treated cells, because nuclei incubated with this cytoplasmic preparation had a higher RNA polymerase activity than those incubated with cytoplasmic preparations of control cell cultures. The nature of these cytoplasmic factors are heterogenous, because the type of RNA polymerase stimulated is dependent of the method used in its preparation.

No description available.

No description available.

Regulation of eucaryotic protein synthesis was studied by three different approaches. One of these studies focused on the regulation of mRNA translation in poliovirus infected cells. Evidence indicated that proteolysis of the p220 component of the CBP complex was not sufficient to obtain complete inhibition of host cell protein synthesis after poliovirus infection. This suggested the existence of an additional viral function in order to achieve the full inhibition of cellular mRNA translation observed after infection. / The effect of viral infection on host protein synthesis and, in particular, on mRNA association with the cytoskeleton was also investigated. Results indicated that (1) all mRNAs studied, appeared to associate with the cytoskeleton fraction during protein synthesis, irrespective of their 5$ sp prime$ and 3$ sp prime$ terminal structures; (2) although the association of mRNA with the cytoskeletal framework appears to be required, it was not found to be sufficient for protein synthesis to occur; (3) the shut-off of host protein synthesis during poliovirus but not vesicular stomatitis virus infection correlated with the release of host mRNAs from the cytoskeleton. / Finally, the regulation of protein synthesis during mitosis was studied. The addition of CBP complex to cell-free extracts was shown to stimulate endogenous mRNA translation in mitotic but not interphase extracts. It was found that the 24k-CBP from mitotic cells was considerably less phosphorylated than the interphase 24k-CBP. Moreover, its ability to recognize the cap structure of mRNA, as judged by cross-linking assays, was decreased in mitotic extracts. Evidence indicated that the extent of phosphorylation of the 24k-CBP did not directly affect its cap binding activity. Possible mechanisms for the regulation of the 24k-CBP activity during mitosis have been suggested.

The objective of this study was to examine the inhibition of protein synthesis in murine P388 cells by sodium cyanate (NaOCN). The characterization of amino acid transport systems present in P388 cells provided a basis for some of these studies. NaOCN had no effect on the kinetics of amino acid transport systems L, A, ASC, N, or y$ sp+.$ The decrease in protein synthesis seen after NaOCN treatment was not secondary to alterations in amino acid metabolism or changes in nucleotide pools. Significant reductions in DNA and RNA synthesis were observed in P388 cells from NaOCN pretreated mice. No effect with NaOCN was observed on total cellular RNA. NaOCN does not appear to affect the P388 cell ribosomal machinery, tRNA or protein synthesis elongation processes. The formation of the 48 S initiation complex was significantly inhibited by NaOCN. These results suggest that the decrease in protein synthesis observed with NaOCN in P388 cells is due to alterations in mRNA synthesis and/or the inhibition of the early stages of protein synthesis initiation.

In the rat, basement membranes are thin, resilient layers which support epithelia and endothelia and form a covering for muscle fibers, fat cells and the nervous system. By immunostaining for light microscopy with peroxidase methods, all examined basement membranes were shown to contain type IV and V collagen, laminin, heparan sulfate proteoglycan and fibronectin. Immunostaining for electron microscopy revealed that the five substances are localized in the basal lamina part of the basement membrane. / Reichert's membrane, a multilayered basement membrane, was shown by immunostaining to contain type IV collagen and laminin. To examine the origin of these two substances, the endodermal cells associated with Reichert's membrane were immunostained and displayed reactivity in rough endoplasmic reticulum, Golgi saccules and secretory granules, a result suggesting that the two substances are secreted along the usual pathway of protein synthesis. The endothelial cells in the incisor tooth showed similar cytoplasmic immunostaining, but only at early stages of development.

Cognition shapes the interactions of an animal with its environment. Species vary greatly in all aspects of cognition, and studying the relationship between this variation and ecology is crucial for understanding how intelligence has evolved. In this thesis, I approach questions about the ecology and evolution of cognition that are often ignored because cognition is difficult to quantify. I use innovation rate as an operational measure and residual brain size as a correlate of general cognition in birds. I first examine the link between cognition and ecology through comparative analyses of the relationships between residual brain size, innovativeness, and measures of ecological generalism across a broad sample of the avian phylogeny, and within a single clade (Icteridae). I find that innovation is positively correlated with habitat breadth but not diet generalism, and that neither measure of generalism is associated with residual brain size. Although residual brain size and innovation rate are strongly correlated with one another, they each appear to have different relationships to a species' ecology. Further analysis finds that the relationship between innovativeness and residual brain size is driven by innovations that involve the use of novel foraging techniques and not the ingestion of new food items. Comparative studies use the traits of extant species to infer their evolutionary history, but can only speculate on the forces driving changes in a trait. The latter half of my thesis focuses on these underlying forces and behavioural mechanisms. Using a game theory model, I show that unpredictable food availability might drive both behavioural flexibility and sociality, two traits strongly associated with cognitive complexity. Finally, I focus on innovativeness at the intraspecific level and examine foraging innovation in a large-brained grackle species, Quiscalus lugubris. I find that this gregarious species is slower to innovate when conspecifics are nearby, and that individuals differ in their ability to solve novel problems. I use these differences to describe the process of innovation, and show that novelty responses, attention, persistence, and flexibility are all important factors underlying individual variation in the ability to innovate. / La cognition dirige les interactions d'un animal avec son environnement. Les espèces varient énormément dans tous les aspects de la cognition, et étudier les relations entre ces variations, l'écologie et l'évolution est crucial pour comprendre comment l'intelligence a évolué. Dans cette thèse, j'aborde les questions de l'écologie et l'évolution de la cognition souvent ignorées, la cognition étant difficile à évaluer quantitativement. J'ai utilisé le taux d'innovation comme une mesure opérationnelle, et la taille résiduelle du cerveau comme un corrélat de la capacité cognitive générale des oiseaux. J'ai d'abord examiné le lien entre la cognition et l'écologie en procédant à des analyses comparatives des relations entre la taille résiduelle du cerveau, la capacité innovatrice, et des mesures du généralisme écologique à travers un large échantillon de la phylogénie aviaire, et dans un clade unique (Icteridae). J'ai trouvé que la capacité innovatrice est corrélée positivement avec l'étendue de l'habitat, mais non avec le régime généraliste, et qu'aucune mesure du généralisme n'est associée avec la taille résiduelle du cerveau. Bien que la taille résiduelle du cerveau et le taux d'innovation soient fortement corrélés entre eux, chacun d'eux semble avoir des relations différentes avec l'écologie de l'espèce. Une analyse plus poussée a montré que la relation entre la capacité innovatrice et la taille résiduelle du cerveau est déterminée par les innovations impliquant l'utilisation de nouvelles techniques d'approvisionnement, et non l'ingestion de nouveaux types d'aliments. Les études comparatives utilisent les traits d'espèces existantes pour en déduire leur évolution, mais peuvent seulement spéculer sur les forces conduisant les changements d'un trait. La seconde moitié de ma thèse se concentre sur ces forces sous-jacentes et les mécanismes comportementaux. En utilisant un modèle de théorie des jeux, j'ai montré que l'imprévisibilité de la disponibilité alimentaire peut mener tant à la flexibilité comportementale qu'à la socialité, deux traits fortement associés à la complexité cognitive. Enfin, je me suis concentrée sur la capacité innovatrice au niveau interindividuel et j'ai étudié l'innovation lors de l'approvisionnement chez le Quiscale merle, Quiscalus lugubris, une espèce possédant un gros cerveau. J'ai trouvé que cette espèce grégaire est moins rapide à innover lorsque des congénères sont à proximité, et que les individus diffèrent dans leur capacité à résoudre de nouveaux problèmes. J'ai utilisé ces différences pour décrire le processus de l'innovation, et montré que la réponse à la nouveauté, l'attention, la persistance et la flexibilité sont d' importants facteurs sous-jacents de la variation interindividuelle de la capacité à innover.