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1
Lawrence, W. A. "Microinjection of tobacco protoplasts." Thesis, University of East Anglia, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.372559.
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Martanto, Wijaya. "Microinjection Into Skin Using Microneedles." Diss., Georgia Institute of Technology, 2005. http://hdl.handle.net/1853/11645.
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The development of microneedles that penetrate the skin barrier, but are small enough not to stimulate nerves, has the potential to deliver drugs across skin in a painless way. Controlled injection by convective flow into skin using hollow microneedles, however, has remained a challenge. To address this challenge, the goals of this study were (i) to provide experimental measurements coupled with numerical simulations to quantitatively describe fluid mechanics of flow within microneedles over a range of experimental conditions and needle geometries, (ii) to demonstrate and study the effects of diffusion-based delivery of insulin to diabetic rats in vivo using solid and hollow microneedles and (iii) to determine the effect of experimental parameters on microinfusion through hollow microneedles into skin to optimize drug delivery protocols and identify rate-limiting barriers to flow.
Experimentally, we quantified the relationship between pressure drop and flow rate through microneedles as a function of fluid viscosity and microneedle length, diameter, and cone half-angle. Microneedle tip diameter and taper angle were the primary controlling parameters for flow through conically tapered microneedles as shown by numerical simulations. Flow rates over a range of 1.4 56 l/s were achieved through microneedles (in the absence of skin) with pressure drops in the range of 4.6 196.5 kPa.
This work also studied the use of solid and hollow microneedle arrays to insert into the skin of diabetic animals for transdermal delivery of insulin. Blood glucose levels dropped by as much as 80% in diabetic rats in vivo. Larger drops in blood glucose level and larger plasma insulin concentrations were shown due to higher donor solution insulin concentration, shorter microneedles insertion time and fewer repeated insertions.
The final scope of this work was to determine the effect of microneedle geometry and infusion protocols on microinfusion flow rate into skin in vitro. Infusion flow rates ranged from 21 to 1130 l/h was demonstrated using glass microneedles. The presence of a bevel at the microneedle tip, larger retraction distance and insertion depth, larger infusion pressure and the presence of hyaluronidase led to larger infusion flow rates.
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Bou, malhab Nada. "Moulage par microinjection des polymères semi-cristallins." Phd thesis, Paris, ENSAM, 2012. http://pastel.archives-ouvertes.fr/pastel-00831028.
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La miniaturisation des pièces est une étape importante pour la progression de la microtechnologie dans plusieurs domaines (connectique, médical, optique, microsystèmes mécaniques). Pour cela, le moulage par microinjection, semble être la solution clé pour la production à grande échelle de micro-composants de polymères. Pour les polymères semi-cristallins, la cristallisation, sous fort taux de cisaillement et sous des vitesses de refroidissement élevées (about 100 K/s), induit des morphologies et des propriétés spécifiques. Elle prend donc une importance considérable dans le processus de microinjection par rapport au moulage par injection classique où les épaisseurs injectées sont généralement supérieures à 1 mm. Ces microstructures ont une grande influence sur les propriétés mécaniques du produit final. La prédiction de ces propriétés à partir de la description de la microstructure est un défi technique et scientifique. Durant cette thèse, deux polymères semi-cristallins ont été microinjectés, le polyéthylène haute densité et le polyamide 12. Les analyses obtenues par la microscopie otiques montrent que les morphologies cristallines varient entre les micro- et les macro-pièces. Tandis que la morphologie de 'peau-cœur' est présente dans les macropièces, les micropièces présentent une morphologie plutôt particulière. Les analyses combinées de diffusion et de diffraction des rayons X (SAXS et WAXS) avec un microfaisceau synchrotron, nous ont permis de déterminer la microstructure induite par le processus de microinjection dans toute l'épaisseur des pièces. Nous avons constaté que la morphologie et les orientations cristallines induites sont très dépendantes des conditions d'injection ou de microinjection. Une diminution de l'épaisseur, de la vitesse et de la température du moule, augmente l'orientation cristalline en limitant la relaxation des chaînes de polymères.
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Ward, Kenneth Glenn. "Microinjection and regeneration of tobacco and potato protoplasts." Thesis, University of Cambridge, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.303971.
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Silhol, Michelle. "La microinjection dans les cellules somatiques : effet d'agents antiviraux." Montpellier 2, 1987. http://www.theses.fr/1987MON20232.
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Silhol, Michelle. "La Microinjection dans les cellules somatiques effet d'agents antiviraux /." Grenoble 2 : ANRT, 1987. http://catalogue.bnf.fr/ark:/12148/cb37609920n.
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Wang, Guang Wei. "Position and force control for piezo-driven microinjection system." Thesis, University of Macau, 2018. http://umaclib3.umac.mo/record=b3951592.
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Kinoshita, Masato, and Kenjiro Ozato. "Cytoplasmic microinjection of DNA into fertilized medaka (Oryzias latipes) eggs." Laboratory of Freshwater Fish Stocks Bioscience Center Nagoya University, 1995. http://hdl.handle.net/2237/13806.
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McKenna, John E. (John Erwin). "Analgesic effects of lidocaine microinjection into the rat dentate gyrus." Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=59653.
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Previous studies in our laboratory have indicated that anesthetic block of neural activity at discrete sites within the limbic system, including the lateral hypothalamus and anterior cingulum bundle, causes a significant long-lasting analgesia during the formalin test. In this experiment, the local anesthetic lidocaine was microinjected into the dentate region of the hippocampus, an important limbic structure presumed to subserve the affective-motivational aspects of pain. The dentate gyrus is strategically situated at a point of convergence of widespread polysensory cortical input to the hippocampus, to allow modulation of cortical signals before they diverge into numerous limbic circuits. The results indicate that anesthetic block of the anterior region of the dentate gyrus produces analgesia in the rat during the formalin test. The analgesia produced by this procedure became apparent 30 minutes after regional block contralateral to the site of injury and persisted for the duration of the test period. These data provide further evidence that limbic forebrain structures are involved in pain and analgesia.
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Ladjal, Hamid. "Développement d'un simulateur haptique pour la cacaractérisation et la microinjection cellulaires." Thesis, Orléans, 2010. http://www.theses.fr/2010ORLE2019/document.
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L'objectif fondamental de cette thèse est de développer et de mettre en oeuvre un outil interactif desimulation des techniques de micromanipulation biologiques de cellules. Au moyen de cet outil, l'opérateurpourra se former, s'entraîner et améliorer sa maîtrise en développant une gestuelle proche de celle exécutéeen réalité. La conception d'un tel environnement de simulation en temps-réel nécessite de trouver uncompromis entre le réalisme des modèles de comportement biomécanique utilisés, la précision et la stabilitédes algorithmes des méthodes de résolution et de rendu haptique utilisées ainsi que la vitesse de calcul. Lamodélisation mécanique retenue repose sur l'utilisation du modèle hyperélastique de St Venant-Kirchhoff etune formulation dynamique explicite éléments-finis du type masses-tenseurs. Le bien-fondé de cettemodélisation est vérifié sur des essais de microindentation par Microscopie à Force Atomique (AFM) decellules souches embryonnaires de souris et de microinjection d'ovocytes. Nous avons développé etimplémenté des modèles d'interaction en temps-réel qui s'articulent autour de la détection et la gestionrapide des collisions entre outil/cellule.La synthèse du rendu haptique fourni à l'opérateur est également proposée par l'intermédiaire d'un couplagevirtuel. Pour chaque application, nous avons justifié nos choix méthodologiques et Algorithmiques qui sontguidés par les contraintes de "réalisme+précision" "temps-réel". Les différents modèles proposés ont étéintégrés dans le simulateur SIMIC que nous avons développé pendant cette thèse. Ce dernier est dédié à lasimulation interactive pour l'aide à l'apprentissage du geste de microinjection et de nanoindentationcellulaireThe fundamental objective of this thesis is to develop and implementing an interactive simulation techniquesfor micromanipulation biological cells. Using this tool, the operator can form, train and improve its control bydeveloping a gesture similar to that performed in reality. The design of such a simulation environment in realtime requires a compromise between the realism of biomechanical models used the accuracy and stability ofalgorithms and solution methods used haptic rendering and computational speed. Modeling Mechanicalrestraint involves the use of hyperelastic model of St Venant-Kirchhoff formulation and explicit dynamic finiteelement-type mass tensors. The validity of this model is tested on microindentation tests by Atomic ForceMicroscopy (AFM) of mouse embryonic stem cells and microinjection of oocytes. We have developed andimplemented models of real-time interaction that revolve around the detection and management of rapidcollisions between tool / cell.The synthesis of the haptic feedback provided to the operator is also available through a virtual coupling. Foreach application, we have justified our methodological choices and Algorithms that are guided by theconstraints of realism + precision "" real time ". The various proposed models have been integrated into thesimulator SIMIC that we developed during this thesis. This is dedicated to interactive simulation to supportlearning of gesture microinjection and cell nanoindentation
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Aten, Quentin Theodore. "Design and Testing of a Pumpless Microelectromechanical System Nanoinjector." BYU ScholarsArchive, 2008. https://scholarsarchive.byu.edu/etd/1926.
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A deeper understanding of human development and disease is made possible partly through the study of genetically modified model organisms, such as the common mouse (Mus musculus). By genetically modifying such model organisms, scientists can activate, deactivate, or highlight particular characteristics. A genetically modified animal is generated by adding exogenous (foreign) genetic material to one or more embryonic cells at their earliest stages of development. Frequently, this exogenous genetic material consists of specially engineered DNA, which is introduced into a fertilized egg cell (zygote). When successfully introduced into the zygote, the exogenous DNA will be incorporated into the cell's own genome, and the animal that develops from the zygote will exhibit the genetic modification in all of its cells. The current devices and methods for generating genetically modified animals are inefficient, and/or difficult to use. The most common and efficient method for inserting new DNA into zygotes is by directly injecting a DNA solution through a tiny glass tube into the cell in a process called microinjection. Unfortunately, microinjection is quite inefficient (success rates are commonly between 1 and 5%), but often it is the only method for inserting DNA into eggs, zygotes, or early stage embryos. This thesis presents the design and testing of a micrometer sale, pumpless microelectromechanical system (MEMS) nanoinjector. Rather than use pumps and capillaries, the nanoinjector employs electrostatic charges to attract and repel DNA onto and off of the surface of a solid lance. The nanoinjector also includes a mechanical system for constraining the target cells during injection. Initial testing indicates the nanoinjector does not decrease cell viability, and it has a very high initial success rate (up to 90%). With the addition of an on-chip actuator, the nanoinjector could be packaged as an inexpensive, fully automated system, enabling efficient, high volume genetic modification of developing animals. Such a device would greatly increase the ease and speed of generating the model organisms needed to study such critical diseases such as Alzheimer's disease, cancer, and diabetes.
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Cabrera, Eusebio Duarte. "Evaluation of the Capabilities of Microinjection Molding to Produce Deformable Membrane Mirrors." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1268093770.
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Mnekbi, Djebali Cheima. "Rhéologie des polymères fondus à hauts taux de cisaillement : application à la microinjection." Phd thesis, Ecole Nationale Supérieure des Mines de Paris, 2012. http://pastel.archives-ouvertes.fr/pastel-00820185.
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La rhéologie à hauts taux de cisaillement pour deux polymères, le PEHD semi-cristallin et le PMMA amorphe a été étudiée. Des outils de rhéométrie classique, un rhéomètre plan-plan en mode dynamique, et un rhéomètre capillaire, ont été utilisés dans des conditions extrêmes (avec des filières pour la rhéométrie capillaire de diamètres allant jusqu'à 0,3 mm) mais les dépouillements de ces résultats ont été fait suivant les hypothèses conventionnelles en négligeant les instabilités et les phénomènes physiques qui interviennent lors de ces écoulements.Nous avons par la suite développé un modèle mathématique de l'écoulement dans un capillaire pour rendre compte de l'importance des différents phénomènes physiques qui peuvent avoir lieu dans des écoulements extrêmes, à savoir l'échauffement et la piezodépendance de la viscosité, la compressibilité et le glissement à la paroi. Les résultats du modèle développé ont été comparés avec les résultats expérimentaux.Nous avons aidé au développement d'une presse de microinjection originale et nous l'avons testée avec un moule de plaque instrumenté d'épaisseur allant jusqu'à 0,2 mm. Nous avons montré qu'il était possible de réaliser des pièces de qualité ce qui est avéré par des mesures de pression, vitesse et de température bien reproductibles. Nous avons exploité les données rhéologiques expérimentales dans la modélisation de la phase de remplissage avec le logiciel de calcul Rem3D. Des corrélations entre les mesures expérimentales et les calculs ont été réalisées en comparant l'évolution des pressions dans le système d'alimentation et dans l'empreinte.
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Dunlap-Brown, Marya. "The In Vitro Transgene Expression and In Vivo Transgene Integration of Condensed DNA Injected into the Cytoplasm of Murine Zygotes." Thesis, Virginia Tech, 2010. http://hdl.handle.net/10919/33743.
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Pronuclear stage murine embryos received electrical stimulation in 5, 10, or 20 µs pulse lengths, and 0, 100, 200, 250, 300 or 400 voltages. Minimal embryo development occurred with 400 V. Irreversible electroporation occurred in embryos electroporated for 5 µs pulse length at 100 and 400 V, 10 µs pulse length at 400 V, and 20 µs pulse length at 100, 250, 300 and 400 V. Electroporated embryos that underwent reversible electroporation received 100 V for 5 µs, 400 V for 10 µs, and 250 for 20 µs and had similar development (P > 0.05) between the best and worst developed groups.
Enhanced green fluorescent protein on a cytomegalovirus promoter (CMV-EGFP) was condensed with MgCl2 and injected into the cytoplasm of murine zygotes at three concentrations (100, 425 and 625 µg/ml). Zygotes injected with the highest concentration had the highest percentages of fluorescing embryos (44%), fluorescing morula and blastocysts (16.7%), and the lowest percentage of mosaicism after 4 d in culture. Five PCR analyses of tail DNA gave conflicting results between 33.3% positive in two or more analyses to 2.8% positive in all five analyses. Southern Analysis detected 2.8% transgenesis. Cytoplasmic injection of linear CMV-EGFP (625 µg/ml in water) was 3.7% transgenic. Pronuclear injections produced 7.9% transgenesis.
This research identified a range of reversible electroporation that could easily be verified in vitro with a selectable dye or marker protein and applied in transgenic as well as preclinical treatment models of research. Furthermore this research identifies the benefits and disadvantages of using Mg2+ in DNA condensation and injection buffers.Master of Science
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Sparks, Amy Elizabeth Thuemmel. "Bovine embryo microinjection, culture, microsurgery, and DNA analysis by the polymerase chain reaction technique." Diss., This resource online, 1992. http://scholar.lib.vt.edu/theses/available/etd-06062008-170630/.
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Krisher, Rebecca L. "Gene injection in the bovine : effect of time of microinjection and nuclear transfer technologies /." Diss., This resource online, 1994. http://scholar.lib.vt.edu/theses/available/etd-06062008-164748/.
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Schmotzer, Carolyn Anne. "Assessment of Murine Embryo Development Following Electroporation and Microinjection of a Green Fluorescent Protein DNA Construct." Thesis, Virginia Tech, 2001. http://hdl.handle.net/10919/34369.
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Transgenic techniques have rapidly evolved in recent years. However, the efficiency of these techniques to produce viable offspring is still disappointingly low. The purpose of this study was to assess in vitro development, transgene expression, and integration following pronuclear or cytoplasmic microinjection of condensed or linear green fluorescent protein DNA into murine embryos using electroporation. In experiment 1, the effect of embryo orientation (group or linear) within the electroporation chamber on development was evaluated using zygotes which received one pulse duration (10 msec), and one of two voltages (250 or 400 V). Zygotes that received 400 V had the lowest development score (Group, 2.06 ? 0.12; Linear, 1.97 ? 0.13), irrespective of orientation. Embryos that received 250 V had the highest development of the voltage treated groups (Group 3.42 ? 0.12; Linear 3.32 ? 0.12), irrespective of orientation, and development was lower than the control embryos (Control 4.28 ? 0.12; Mannitol control 4.36 ? 0.18). In experiment 2, the efficiency of utilization of the prepared enhanced green fluorescent protein (EGFP) construct as a visual marker of protein expression was evaluated using pronuclear microinjection. Embryo development and fluorescence were evaluated following pronuclear injection of EGFP at a concentration of 3 μg/ml and compared to an uninjected control. Embryos injected with the EGFP had lower development scores (3.85 ± 0.15) than uninjected control embryos (5.72 ± 0.2). Of the embryos injected, 32.4% fluoresced due to expression of EGFP. Experiment 3 evaluated the effect of combining cytoplasmic injection of EGFP (425 μg/ml) with electroporation at 250 V on EGFP expression. The non-manipulated control embryos had significantly higher (P < 0.01) 4 d development scores (5.57 ± 0.11) than manipulated control embryos (4.6 ± 0.18), where the injection needle was inserted into the cytoplasm and no DNA was injected. Combining cytoplasmic DNA injection and electroporation caused a significant (P < 0.01) decrease in development scores, irrespective of DNA construct, when compared to embryos injected with a DNA construct alone. The mechanical effects of needle insertion combined with electroporation were not significantly different (P > 0.05) from embryos injected with DNA alone, irrespective of construct injected. Cytoplasmic injection of condensed DNA (0.38%), linear DNA (0.38%), and condensed DNA combined with electroporation (0.36%) resulted in one fluorescent embryo respectively. Cytoplasmic injection of linear DNA when combined with electroporation (3.57%) resulted in 13 fluorescent embryos. Pronuclear injection of the prepared EGFP construct results in lower development than control embryos. Electrical stimulation of zygotes reduces early embryo development. However, low amounts of electrical stimulation may allow for enhancement of gene integration in transgenic embryos.Master of Science
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Wilson, Aubrey Marie Mueller. "Transgene Delivery via Microelectromechanical Systems." BYU ScholarsArchive, 2012. https://scholarsarchive.byu.edu/etd/3936.
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The invention of pronuclear microinjection initiated the field of transgenic research. Over 30 years later microinjection remains the most straight-forward and most commonly used transgene delivery option. In this work we address the current progress of microelectromechanical systems (MEMS) used as transgenic delivery mechanisms. The nanoinjector is a specially designed MEMS device which uses electrostatic charge to manipulate transgene molecules. The process of nanoinjection was designed as an alternative to microinjection which causes less damage to developing embryos, improves embryo survival, birth rates, and overall efficiency of injections. In vivo testing of nanoinjection demonstrates it is both safe and effective. Additionally nanoinjection has the potential to make transgenesis via yeast artificial chromosomes more practical as the nanoinjector may prevent shearing of the YAC molecules. A second nanoinjection protocol termed intracellular electroporetic nanoinjcetion (IEN) was designed to allow for cytoplasmic injections. Cytoplasmic injections are faster and easier than pronuclear injection and do not require the pronuclei to be visible; yet previous attempts to develop cytoplasmic injection have met with limited success. In IEN injections the nanoinjector is used to place transgenic molecules in the cytoplasm. The transgenes are then propelled through the cytoplasm and electroporated into the pronucleus using electrical pulses. Electroporation of whole embryos has not resulted in transgenic animals, but the MEMS device allows localized electroporation to occur within the cytoplasm, giving transgene access to the pronucleus before degradation can occur. In this report we describe the principles which allow for localized electroporation of the pronuclei including: the location of the pronuclei between 21-28 hours post-hCG treatment, modeling data predicting the voltages needed for localized electroporation of pronuclei, and data on the movement of transgenic DNA based on the voltages delivered by IEN. We further report results of an IEN versus microinjection comparative study in which IEN produced transgenic pups with viability, transgene integration, and expression rates statistically comparable to microinjection. The ability to perform injections without visualizing or puncturing the pronuclei will widely benefit transgenic research, and will be particularly advantageous for the production of transgenic animals with embryos exhibiting reduced pronuclear visibility.
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Cumberland, Peter F. T. "Species-specificity of transferrin as investigated in the rat conceptus using an improved microinjection technique." Thesis, University of Leicester, 1991. http://hdl.handle.net/2381/34316.
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Transferrin, a widespread vertebrate protein, is responsible, along with its specific receptor, for all of the iron transported into the cell. Despite its ubiquity and structural similarity across species, heterologous molecules display different binding affinities for human cells. In general transferrin binds more avidly to the homologous receptor whatever the species. The impetus for the first part of this thesis came from these observations, coupled with research, which showed that rat conceptuses grown in human serum were anaemic and malformed, conditions rectified by supplementation with rat but not human transferrin. The second part of the thesis concerns the development of an intravitelline injection system to by-pass the rat visceral yolk sac which surrounds the embryo in its amniotic sac; in the human the yolk sac is situated externally to both. Due to this arrangement and also because of its known degradative abilities, the rat yolk sac presents toxicologists with a problem when using the rat model in teratological studies because they cannot be sure what quantity of a compound or even what metabolite the rat embryo is receiving. Thus if the extraembryonic membranes could be by-passed it would be possible to perform experiments which might provide more information regarding acute toxicity and help in the accurate assessment of teratological risk. This improved injection method allows such experiments to be carried out in a reproducible manner with greater accuracy and with more discrete quantities than was possible with the earlier system. The two parts have been drawn together in experiments which compare the uptake of human and rat transferrin in embryos cultured in serum containing, and injected with, these two molecules. Over all, the results indicate that the rat yolk sac has an affinity three times greater for rat transferrin than it does for human transferrin. Furthermore, it appears that the embryo cannot differentiate between the two molecules and that it probably does not possess a specific transferrin receptor. The practical aspects of this technique in relation to experimental embryology and acute reproductive toxicology have been explored in chapter 6 of this thesis. From the point of view of acute toxicity testing the results have been promising enough to elicit industrial sponsorship for developing the procedure further.
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Gibbons, John R. "Ultrasound-guided transvaginal follicular aspiration to provide a source of bovine oocytes for gene microinjection." Thesis, This resource online, 1994. http://scholar.lib.vt.edu/theses/available/etd-12162009-020346/.
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Weissenborn, Ruth. "Functional roles of the rat nucleus accumbens : further investigations using microinjection, lesion and electrochemical techniques." Thesis, University of St Andrews, 1993. http://hdl.handle.net/10023/14699.
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The nucleus accumbens (N.Acc.) has been ascribed an important role in mediating locomotor activity and exploration, as well as more complex processes such as reinforcement, reward and the acquisition of displacement activities. Previous investigations of N.Acc. functions have primarily been based on pharmacological manipulations of activity of one of the main neurotransmitters in the N.Acc., dopamine (DA), either through administration of dopaminergic agonists or antagonists or through depletion of DA terminal fields in the N.Acc. In the present thesis, the functional role of the N.Acc. in a number of different forms of behaviour has been investigated further using specific, fibre-sparing excitotoxic lesions of intrinsic neurones, intra-accumbens injections of DA and in vivo electrochemical measurements of extracellular levels of DA in the N.Acc. Excitotoxic lesions in the N.Acc. were found to enhance spontaneous locomotion and exploratory behaviours while leaving intact the locomotor- stimulating effects of an indirect dopaminergic agonist, displacement drinking in response to intermittent food-reinforcement (SIP) and amphetamine-induced conditioned place preference (CPP). Thus, fibre-sparing excitotoxic lesions induced a pattern of behaviour distinct from that observed following terminal depletion in the N.Acc. Further, microinjection and in vivo electrochemical experiments showed no direct relationship between DA activity in the N.Acc. and SIP. Overall, these results are discussed in terms of a theoretical model proposing that the N.Acc. may function as an interface between sensory input and locomotor output and that inhibitory activity in the N.Acc. is needed to channel activity levels appropriately in response to cortical input about the direction of change. It is suggested that rather than viewing it as a unitary structure with specific functions, the N.Acc. should be considered as a heterogeneous part of the striatal complex with a number of distinct subsystems that exist within a complex framework of interactive processes, where changes in one structure can only be understood by taking into account other, related structures.
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Ng, Shuk-ming Sandy. "A study on the production of transgenic mice by pronuclear microinjection and by sperm incorporation of immunoglobulin genes /." [Hong Kong : University of Hong Kong], 1992. http://sunzi.lib.hku.hk/hkuto/record.jsp?B13215905.
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El-Taleb, Ahmed Salem. "Investigation of Mold Design and Process Parameters in Microinjection Molding to Fabricate a Deformable Membrane Mirror." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1376610533.
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Yehya, Alaa. "New insights into Brain-derived Neurotrophic Factor Dual Signaling : imbalance implications in mechanisms of neuroprotection and neurotoxicity." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTS058.
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Le « Brain-Derived Neurotrophic Factor » (BDNF) est la neurotrophine la plus abondante et la plus répandue dans le cerveau humain. De nombreuses études se sont intéressées à son rôle dans la survie neuronale, la croissance et la plasticité synaptique. La signalisation BDNF est dépendante de deux récepteurs, le récepteur tyrosine kinase (TrkB) et le récepteur neurotrophine p75 (p75NTR). Il est bien établi que le rôle trophique du BDNF est assuré via son récepteur de haute-affinité TrkB, alors que la forme précurseur proBDNF active p75NTR vers la voie d'apoptose. Cette double signalisation est physiologiquement contrôlée par un équilibre entre les différentes voies. Les résultats obtenus à partir des études cliniques et des modèles animaux suggèrent un rôle de la signalisation BDNF dans les tauopathies, caractérisées par l'existence de dépôts intracérébraux de protéine tau, une caractéristique commune à certaines maladies neurodégénératives, notamment la maladie d'Alzheimer (MA). Cependant, aucune investigation n'a été menée jusqu'à présent sur les modifications que pouvaient induire les tauopathies dans la signalisation BDNF et si une dérégulation de l'expression du BDNF pouvait affecter ses propres récepteurs TrkB et p75NTR.Dans ce travail de thèse, nous avons utilisé une lignée de poisson-zèbre transgénique portant la mutation humaine TAUP301L retrouvée notamment dans le démence fronto-temporale. Nous avons mesuré l'expression de BDNF et de ses deux récepteurs au niveau transcriptionnel et protéique. Nous n'avons observé aucune modification des taux d'expression de BDNF et de TrkB, en revanche, nous avons noté une augmentation significative de p75NTR. A l'aide de la même lignée transgénique, nous avons induit une baisse d'expression de BDNF via la micro-injection de morpholinos. De manière remarquable, la baisse d'expression de BDNF affecte de façon différentielle TrkB et p75NTR. En effet, nous avons observé une diminution de l'expression de TrkB et parallèlement une augmentation de p75NTR. De plus, la baisse d'expression de BDNF aggrave la neurotoxicité associée au développement de la tauopathie ce qui se traduit par une augmentation de la mort neuronale et de l'hyperphosphorylation de tau, cette dernière étant concommittante à une activation de la Glycogen Synthétase Kinase 3 beta (GSK3beta).Une diminution de l'effet neuroprotecteur de BDNF à travers un déséquilibre de ces récepteurs de signalisation a été également montré en étudiant le rôle de BDNF au cours du développement de la ligne latérale postérieure (PLL). Ce système est considéré comme un modèle d'étude particulièrement pertinent pour évaluer différents processus biologiques comme la migration cellulaire collective ou la régénération cellulaire. Nous avons détecté l'expression de BDNF dans plusieurs structures de la PLL. La diminution d'expression de BDNF conduit à un défaut de migration du primordium de la PLL, associé à une augmentation de la mort cellulaire. De plus, nous avons observé une réduction de la prolifération cellulaire et un défaut de repousse axonale du nerf, ce qui conduit à des anomalies de régénération à la fois du nerf de la PLL et des cellules ciliées. Nos résultats suggèrent que le BDNF joue un rôle essentiel au cours du développement de la PLL et démontrent la pertinence du système de la ligne latérale en tant que modèle d'étude des fonctions de BDNF.En conclusion, notre étude représente la première analyse du rôle in vivo de BDNF et de ses 2 récepteurs de signalisation. Nous avons ainsi montré les répercussions d'une dérégulation des voies de signalisation du BDNF. Un équilibre entre ces deux voies est essentiel pour le développement et la survie cellulaire, ce qui fait de BDNF non seulement une cible thérapeutique potentielle, mais également une neurotrophine clé pouvant activer plusieurs circuits de signalisation, potentialisant ainsi son rôle protecteurBrain-derived neurotrophic factor (BDNF) is the most abundant secreted and widely distributed neurotrophin in human brain. It has been extensively studied for its role in neuronal survival, growth and synaptic plasticity. BDNF signaling mediated through tryosine receptor kinase B (TrkB) and p75NTR neurotrophin receptor (p75NTR). It is well established that BDNF beneficial actions are mediated by it is high-affinity TrkB, whereas pro-BDNF activates p75NTR towards apoptosis. This diverse dual signaling is normally under a tight balance regulation. Based on clinical and animal studies, it has been suggested that BDNF signaling is involved in tauopathy, which is a pathological hallmark in several neurodegenerative diseases, including Alzheimer's disease (AD). However, what changes tauopathy may induce on BDNF signaling, and whether BDNF deregulation could affect its two signaling receptors (TrkB, p75NTR), and eventually tauopathy pathogenesis, have not been investigated. In this study we used a transgenic zebrafish line for human Tau-P301L tauopathy, and measured transcriptional and protein levels of BDNF and of its two signaling receptors. We found no modification of BDNF and TrkB expression levels, but a significant up-regulation of p75NTR. We then used the same transgenic line to generate BDNF knockdown using morpholino microinjection technique. Interestingly, BDNF knockdown differentially affects TrkB and p75NTR; we observed a reduction of TrkB expression and an increase in p75NTR expression. In addition, BDNF knockdown aggravates tauopathy-associated toxicity; we found an increase in neuronal cell death and tau hyperphosphorylation, the latter was accompanied by an activation of tau glycogen synthase kinase 3beta (GSK3beta). Attenuation of BDNF neuroprotective effects through imbalance of its signaling receptors was further highlighted through studying BDNF role in the development of zebrafish posterior lateral line system (PLL). This system has recently emerged as a powerful tool to study several dynamic biological processes, including collective cell migration and nerve/hair cells regeneration. We detected BDNF expression in different PLL components. BDNF knockdown led to an impairment of the PLL primordium migration due to concomitant increase in cell death rate. In addition, reduced cell proliferation and defect in axonal re-growth were observed , which led to major defects of PLL nerve/hair cells regeneration, respectively. These findings suggest that BDNF has an essential role in PLL development, but more important they introduce PLL as research model to study BDNF functions. This is the first study to provide a detailed in vivo analysis of BDNF and its two signaling receptors. Our findings highlight several implications of BDNF signaling deregulation. Balanced signaling clearly has essential roles in survival and development, in addition to being a therapeutic target, BDNF can itself activate diverse molecular pathways, thus setting up a potential circuitry that could enhance its protective role
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吳淑明 and Shuk-ming Sandy Ng. "A study on the production of transgenic mice by pronuclear microinjection and by sperm incorporation of immunoglobulin genes." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1992. http://hub.hku.hk/bib/B31210521.
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Versavaud, Sophie. "Mise en forme des thermoplastiques chargés de nanotubes de carbone : application à la microinjection de Polyamide 12." Phd thesis, Ecole nationale supérieure d'arts et métiers - ENSAM, 2012. http://pastel.archives-ouvertes.fr/pastel-00866487.
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L'addition de nanotubes de carbone multiparois (MWNT) dans une matrice de polyamide 12 (PA 12), électriquement isolante, permet d'augmenter les propriétés électriques vers un comportement conducteur. Cette modification est influencée par l'arrangement des MWNT en chemins de conduction qui permettent le transfert des charges électriques entre deux électrodes. La conductivité électrique des nanocomposites isotropes atteint une valeur asymptote (~10-2 S.m-1) pour des teneurs supérieures à 1,2% en masse (seuil de percolation électrique). En microinjection, les nanocomposites sont soumis à des taux de cisaillement très élevés (~104 s-1) et des gradients de températures extrêmes, qui conditionnent fortement la microstructure et les propriétés électriques de pièces mises en forme par ce procédé. Cette thèse a eu pour but d'expliquer l'influence de la vitesse de cisaillement (0,02 s-1 - 1 s-1) et la vitesse de refroidissement (3 °C.min-1) sur l'évolution des propriétés électriques du nanocomposite PA12/MWNT. L'analyse de ces propriétés a permis de déduire, à l'état fondu, l'évolution de l'arrangement de MWNT dans cette fenêtre de conditions. Dans les pièces microinjectées, nous constatons une perte complète du comportement conducteur dans la direction normale au plan d'écoulement et une chute de la conductivité dans les directions d'injection et transverse. Ces faits suggèrent alors un arrangement en forme d'agrégats faiblement orientés dans le plan d'écoulement, qui est corroboré par la très large distribution d'orientation déterminée par l'analyse en spectroscopie Raman des pièces micro-injectées. Lors du procédé de microinjection, les agrégats de MWNT seraient alors cassés dans des agrégats plus petits, mais fortement déconnectés les uns des autres, expliquant ainsi la chutedes propriétés électriques mais aussi l'observation d'une microstructure quasi isotrope à l'échelle macro et micro.
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Herbert, Danielle. "Studies of assisted reproduction in the spotted grass frog Limnodynastes tasmaniensis: ovulation, early development and microinjection (ICSI)." Thesis, The University of Newcastle, 2004.
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Assisted Reproductive Technologies (ART) offer a wide range of techniques that have the potential to augment efforts to conserve and manage endangered amphibians and improve wild and captive population numbers. Gametes and tissues of species nearing endangered or extinct status can be cryopreserved and stored in gene banks, to provide material that can be utilised in the future as ART methods are refined. The Spotted Grass Frog, Limnodynastes tasmaniensis, is an abundant amphibian species in South-Eastern Australia of the family Myobatrachidae, that is suitable for the development of ART systems that can be applied to the threatened and endangered myobatrachid and other amphibian species native to Australia. The aim of this study was to advance the understanding of ovulation, fertilisation and embryo nic development of Lim. tasmaniensis and in vitro manipulations of reproduction and development for use in the development of advanced ART procedures such as intracytoplasmic spermatozoon injection (ICSI), androgenesis and nuclear transfer.
Ovulation in amphibians can be induced by protocols utilising natural or synthetic hormones. All protocols tested on Lim. tasmaniensis in this study required two injections and the most effective protocols continued to require a first injection of pituitary extracts to induce ovulation. The second injection was, however, successfully replaced by synthetic chorionic gonadotrophin at a threshold dosage of 100 iu and halved the number of cane toads required to source the pituitaries. A combination of LHRH and Pimozide offered a less effective protocol, that did not require the use of pituitary extracts, and avoided the risk of pathogen transfer associated with unsterilised pituitary extracts.
Unfertilised eggs of Lim. tasmaniensis were exposed to media of various osmolalities to determine media effects on eggs and their surrounding jelly layers that might impact on egg viability and fertilisability. Osmolality had no effect upon the egg diameter, however, rapid swelling of the jelly layers occurred within 15 minutes of exposure to various media treatments and plateaued from 30-90 minutes without further expansion. Swelling of the jelly layers was increased in hypotonic media (2.5% SAR, H2O) and minimised in the isotonic media (100% SAR). The optimal conditions for the culture of Lim. tasmaniensis eggs were identified as a holding media of 100% SAR, followed by a medium change to 2.5% SAR at insemination. This sequence of media minimised the rate of swelling of the jelly layers prior to contact with the spermatozoa, and maximised the activation of spermatozoa and eggs throughout fertilisation and embryonic development.
Embryos of Lim. tasmaniensis were cultured at four temperatures (13 C, 17 C, 23 C and 29 C), to determine the effect of temperature on cleavage and embryonic development rates. Embryonic development progressed through a sequence of stages that were not altered by changes in temperature. However cleavage rates were affected by changes in temperature as compared with normal embryonic growth at 23 C. Embryonic development was suspended at the lowest temperature (13 C) while embryonic viability was maintained. A moderate decrease in temperature (17 C) slowed cleavage, while the highest temperature (29 C) increased the cleavage rate, but decreased the embryo survival. Rates of embryonic development can be manipulated by changes in temperature and this method can be used to source blastomeres of a specific size/stage at a predetermined age or halt cleavage at specific stages for embryos or embryo derived cells to be included in ART procedures.
This study produced the first report of the application of Intracytoplasmic Spermatozoon Injection (ICSI) in an Australian amphibian. Eggs that were activated by microinjection with a single spermatozoon (n=50) formed more deep, but abnormal, cleavage furrows post-injection (18/50, 36%), than surface changes (12/50, 24%). This result is in contrast to eggs injected without a spermatozoon (n=42), where the majority of eggs displayed limited surface changes (36/42, 86%), and few deep, abnormal furrows (3/42, 7%). Three advanced embryos (3/50, 6%) were produced by ICSI that developed to various stages within the culture system. Technical difficulties were encountered that prevented the generation of any metamorphs from ICSI tadpoles. Nevertheless, when these blocks to ICSI are overcome, the ICSI procedure will be both directly useful as an ART procedure in its own right, and the associated refinement of micromanipulation procedures will assist in the development of other ART procedures in Lim. tasmaniensis. A greater understanding of basic reproductive and developmental biology in Lim. tasmaniensis would greatly facilitate refinement of fertilisation by ICSI.
Assisted Reproductive Technologies, in conjunction with gene banks may in the future regenerate extinct amphibian species, and assist in the recovery of declining amphibian populations nationally and worldwide.
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Teichert, Gregory Herlin. "Design and Testing of a Biological Microelectromechanical System for the Injection of Thousands of Cells Simultaneously." BYU ScholarsArchive, 2012. https://scholarsarchive.byu.edu/etd/3366.
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The ability to inject DNA and other foreign particles into cells, both germ cells (e.g. to produce transgenic animals) and somatic cells (e.g. for gene therapy), is a powerful tool in genetic research. Nanoinjection is a method of DNA delivery that combines mechanical and electrical methods. It has proven to have higher cell viability than traditional microinjection, resulting in higher integration per injected embryo. The nanoinjection process can be performed on thousands of cells simultaneously using an array of microneedles that is inserted into a monolayer of cells. This thesis describes the needle array design requirements and the fabrication process used to meet them. The process uses unpassivated and passivated deep reactive ion etching (DRIE) to create needles with a constant diameter shaft and a pointed tip. The needle diameter and height are about 1 µm and 8 µm, respectively. A buckling analysis and physical testing show that the needles can withstand the force required to penetrate the cells. The chip is attached to a plastic suspension with a counter electrode and electrical connections to a voltage source. The suspension's motion is defined by two compliant orthoplanar springs that have been vertically and rotationally offset for added stability. The base of the suspension is designed to exactly fit in the bottom of a cell culture dish, where the needle array can be pushed into the cell monolayer. Injection protocol was created and followed to perform tests with needle insertion only, voltage application only, and the full nanoinjection process. The average cell viability for the full injection process was 98.2% compared to an average control viability of 99.5%. Zero volt injections with a high concentration of propidium iodide, a cell impermeable dye with two positive charges, resulted in dye uptake from diffusion, proving that the needles are penetrating the cells. Tests comparing injections with and without voltage had high variability in dye uptake. Therefore, glass cover slips were placed in the culture dishes to provide more consistent injection conditions. This reduced variation in zero voltage tests. It is recommended that this procedure be followed for performing injections with voltage.
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Tebourbi-Chikhaoui, Lamia. "Le spermatozoi͏̈de fécondant peut-il transmettre le cytomégalovirus à l'embryon ? : étude expérimentale chez la souris." Paris 7, 2001. http://www.theses.fr/2001PA077149.
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Regnier, Stéphanie Caumes Éric. "Infections cutanées à mycobactéries atypiques après mésothérapie 16 cas /." Créteil : Université de Paris-Val-de-Marne, 2009. http://doxa.scd.univ-paris12.fr:80/theses/th0510898.pdf.
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Garst, Amy S. "In-vitro developmental potential of bovine oocytes obtained by transvaginal follicular aspiration as related to their morphological quality and after microinjection of DNA." Thesis, This resource online, 1996. http://scholar.lib.vt.edu/theses/available/etd-08292008-063300/.
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Ziyyat, Ahmed. "Séparation de spermatides rondes par cytométrie en flux et expression génique durant le développement préimplantatoire après microinjection de la spermatide dans l'ovocyte de souris." Paris 11, 2000. http://www.theses.fr/2000PA11T062.
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La spermatogenèse peut être anormalement altérée à tous les stades, conduisant à l'azoospermie et à l'infertilité. La microinjection de spermatides (ROSI) peut pallier cette déficience en spermatozoïdes. Pour déterminer la faisabilité de la ROSI et ses conséquences sur le développement, nous avons mis au point une technique de séparation des spermatides chez la souris et l'homme : la cytométrie en flux couplée à un tri cellulaire. La population triée est homogène et exprime les marqueurs spécifiques de la spermatide. Cette technique ne permet pas d'isoler des spermatides dans les éjaculats d'hommes atteints d'azoospermie non obstructive. La PCR inverse a établi leur présence dans 22% des cas seulement, et leur déficience en protamine 2. Nous avons mis au point au laboratoire, la microinjection de spermatozoïdes et de spermatides dans l'ovocyte de souris. Les taux de fécondation et de développement jusqu'au stade 4-cellules sont similaires dans les deux cas. Dans les embryons conçus par microinjection de spermatides, la transcription de la protamine 2 est réprimée dès le stade pronoyau, alors que les ARNm d'Ubely et Ubelx sont présents jusqu'au stade 2- cellules. Les messagers de HSP70. 1 et Smcy sont détectés au stade 2-cellules, en phase avec l'activation du génome zygotique. Ces résultats suggèrent que des mécanismes régulateurs inhibent la transcription inapropriée de certains gènes dans les embryons de souris obtenus par microinjection de spermatides.
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Meyer, Lena. "The Francisella pathogenicity island : its role in type VI secretion and intracellular infection." Doctoral thesis, Umeå universitet, Institutionen för klinisk mikrobiologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-101321.
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Intracellular bacteria have developed various mechanisms to enter and persist in host cells and, at the same time, to evade the host immune response. One such pathogen is Francisella tularensis, the etiological agent of tularemia. After phagocytosis, this Gram-negative bacterium quickly escapes from the phagocytic compartment and replicates in the host cell cytosol. For this mode of infection, several components of the Francisella pathogenicity island (FPI) are critical. Interestingly, some FPI proteins share homology to components of Type VI Secretion Systems (T6SSs), but their assembly and functionality remains to be shown in Francisella.The thesis focused on the characterization of several of these FPI components; more specifically, how they contribute to the infection cycle as well as their possible role in the putative T6SS. We identified three unique mutants, ΔiglG, ΔiglI and ΔpdpE, which to various degrees were able to escape the phagosomal compartment, replicate in the host cytosol and cause host cell cytotoxicity. In contrast, ΔiglE as well as mutants within the conserved core components of T6SSs, VgrG and DotU, were defective for all of these processes. In the case of IglE, which is a lipoprotein and localized to the outer membrane of the bacterial cell wall, residues within its N-terminus were identified to be important for IglE function. Consistent with a suggested role as a trimeric membrane puncturing device, VgrG was found to form multimers. DotU stabilized the inner membrane protein IcmF, in agreement with its function as a core T6SS component. The functionality of the secretion system was shown by the translocation of several FPI proteins into the cytosol of infected macrophages, among them IglE, IglC and VgrG, of which IglE was the most prominently secreted protein. At the same time, the secretion was dependent on the core components VgrG, DotU but also on IglG. Although we and others have shown the importance of FPI proteins for the escape of F. tularensis, it has been difficult to assess their role in the subsequent replication, since mutants that fail to escape never reach the growth-permissive cytosol. For this reason, selected FPI mutants were microinjected into the cytosol of different cell types and their growth compared to their replication upon normal uptake. Our data suggest that not only the metabolic adaptation to the cytosolic compartment is important for the replication of intracytosolic bacteria, but also the mechanism of their uptake as well as the permissiveness of the cytosolic compartment per se.
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Babenko, Maksims. "A Study of Heat Transfer at the Cavity-Polymer Interface in Microinjection Moulding. The effects of processing conditions, cavity surface roughness and polymer physical properties on the heat transfer coefficient." Thesis, University of Bradford, 2015. http://hdl.handle.net/10454/14745.
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This thesis investigates the cooling behaviour of polymers during the microinjection moulding process. The work included bespoke experimental mould design and manufacturing, material characterisation, infra-red temperature measurements, cooling analysis and cooling prediction using commercial simulation software. To measure surface temperature of the polymers, compounding of polypropylene and polystyrene with carbon black masterbatch was performed to make materials opaque for the IR camera. The effects of addition of carbon black masterbatch were analysed using differential scanning calorimetry and Fourier transform infrared spectroscopy. Sapphire windows formed part of the mould wall and allowed thermal measurements using an IR camera. They were laser machined on their inside surfaces to generate a range of finishes and structures. Their topographies were analysed using laser confocal microscope. The surface energy of sapphire windows was measured and compared to typical mould steel, employing a contact angle measurement technique and calculated using Owens-Wendt theory. A heating chamber was designed and manufactured to study spreading of polymer melts on sapphire and steel substrates. A design of experiments approach was taken to investigate the influence of surface finish and the main processing parameters on polymer cooling during microinjection moulding. Cooling curves were obtained over an area of 1.92 by 1.92 mm of the sapphire window. These experiments were conducted on the Battenfeld Microsystem 50 microinjection moulding machine. A simulation study of polymer cooling during the microinjection moulding process was performed using Moldflow software. Particular interest was paid to the effect of the values of the interfacial heat transfer coefficient (HTC) on the simulated cooling predictions. Predicted temperature curves were compared to experimentally obtained temperature distributions, to obtain HTC values valid for the material and processing parameters.
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Plichon-Gaub, Marie-Pierre. "Etude des mecanismes de regulation de la transcription de genes sous controle hormonal : regulation des genes conalbumine et ovalbumine." Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13047.
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Vernet, Muriel. "Specificite transcriptionnelle et demarrage de l'activite du genome chez les mammiferes." Paris 6, 1992. http://www.theses.fr/1992PA066357.
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Chez la souris, le genome zygotique est active au stade 2-cellules. A ce stade, les embryons expriment de l'adn micro-injecte et utilisent efficacement certains promoteurs dans des essais a court terme. Cette capacite apparait a la fin du stade 1-cellule, et est contemporaine, dans ces ufs, de l'activation mineure du genome. Avant ce stade l'arn micro-injecte est efficacement traduit. Il existerait apres la fecondation, un blocage transcriptionnel, leve independamment du clivage de l'uf et de la formation du noyau zygotique mais, dependant de l'age de l'uf. Une etude similaire menee chez le lapin a montre l'existence d'un meme type de blocage. La micro-injection du gene lacz sous controle de differents promoteurs a permis de degager des caracteristiques de la specificite transcriptionnelle des embryons (souris et lapin). Ces caracteristiques transcriptionnelles ont ete aussi observees dans les ovocytes et au stade 4-cellules chez la souris; elles rappellent celles des cellules ec et es. L'expression du gene lacz dirige par le promoteur du vih-1 a ete comparee au stade 2-cellules, en expression transitoire et a l'etat integre (souris transgenique). L'expression detectee en expression transitoire n'est plus observee chez l'embryon transgenique, sauf apres activation par les uv et/ou la mise en culture. Dans ce cas l'expression est contemporaine de l'activation mineure du genome
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Giordano-Santini, Rosina. "Développement d’un nouveau marqueur de transgénèse pour la transformation de nématodes." Thesis, Bordeaux 2, 2011. http://www.theses.fr/2011BOR21814/document.
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La construction d’animaux transgéniques est une technique clef qui a permis l’étude de nombreux aspects de la biologie du nématode Caenorhabditis elegans. Les animaux transgéniques peuvent être construits soit en injectant l’ADN exogène dans les gonades syncitiales de l’hermaphrodite adulte, soit en bombardant une population de vers avec des microbilles enrobées d’ADN. Dans les deux cas, l’utilisation de marqueurs génétiques est indispensable pour l’identification des individus transgéniques et la maintenance des lignées. Nous avons développé un vecteur d’expression pour les nématodes contenant le gène de résistance à la néomycine (neo), qui fonctionne comme marqueur génétique. Le gène neo confère la résistance au G-418, un antibiotique qui inhibe la synthèse de protéines chez les eucaryotes et qui est létal pour les nématodes sauvages. Nous avons montré que le marqueur neo est un marqueur génétique très puissant qui permet l’identification rapide des animaux transgéniques et qui permet l’enrichissement des populations transgéniques en présence de l’antibiotique, facilitant ainsi la maintenance des lignées. Ce système ne nécessite aucun contexte génétique particulier pour fonctionner et est donc compatible avec des lignées receveuses mutantes, ainsi que des lignées transgéniques ayant été transformées avec d’autres marqueurs génétiques. De plus, le gène neo est sous le contrôle du promoteur du gène de C. elegans rps-27, codant pour une protéine ribosomale dont la séquence est hautement conservée entre les nématodes. Nous avons utilisé ce gène comme marqueur génétique pour la transgénèse de l’espèce Caenorhabditis briggsae, ce qui suggère que le système neo pourrait aussi être utilisé pour d’autres espèces de la famille Caenorhabditis. Finalement, nous avons aussi montré que le système neo peut être utilisé dans le contexte des techniques d’ingénierie génétique basées sur le transposon Mos1. En conclusion, la sélection en présence de G-418 offre des nouvelles possibilités d’expériences pour la transgénèse de C. elegans et d’autres espèces proches. Les avantages du système neo devraient ainsi contribuer à développer des techniques de transgénèse du ver plus flexibles et efficacesThe generation of transgenic animals has been instrumental to study many biological aspects of Caenorhabditis elegans biology. Transgenic animals can be obtained by either microinjection of the exogenous DNA into the syncitial gonad of the hermaphrodite or by bombardment of a population of worms with DNA coated microparticles. Both techniques rely on the use of genetic markers to facilitate the recovery of transformed animals and the maintenance of transgenic lines. We developed a nematode expression vector carrying the neomycin resistance gene (neo) as a selection marker. This gene confers resistance to G-418, an antibiotic that normally inhibits protein synthesis in eukaryotes and is lethal for wild-type nematodes. We showed that the neo marker is a potent tool that allows a clear-cut selection of transgenic animals and hands-off maintenance of non-integrated populations on G-418 plates. This system does not imply any prerequisite on the original genotype of the recipient strain and can therefore be used on mutants lines as well as transgenic strains obtained with common markers. Moreover, we placed the neo gene under the control of the C. elegans rps-27 promoter, a highly conserved ribosomal protein throughout the nematode phylogeny. We were able to provide resistance to Caenorhabditis briggsae using this vector; this likely indicates that neo can be used in any species from the Caenorhabditis family. Finally, we demonstrated that this powerful selection system can be used in the context of Mos1 transposon excision-repair methods. Therefore, the neo system offers a wide range of new possibilities for transgenesis both in C. elegans and in other related species. We therefore believe that the benefits of the neo system should contribute to the development of more flexible and efficient techniques for nematode transgenesis
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Turner, Joel G. "Human topoisomerase II alpha nuclear export is mediated by two Crm-1 dependent nuclear export signals." [Tampa, Fla.] : University of South Florida, 2004. http://purl.fcla.edu/fcla/etd/SFE0000258.
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SILVA, Carliane Rebeca Coelho da. "Seleção de eventos transformados de algodão resistente a insetos por meios moleculares e de imunodetecção." Universidade Federal Rural de Pernambuco, 2014. http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/4692.
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Various educational institutions and research throughout the world have devoted efforts in the discovery of new insecticidal proteins and their genes for the control of agricultural pests. Studies have shown the potential of some molecules in insect control as the Bacillus thuringiensis Bt toxins, protease inhibitors and alpha-amylase and lectins. Genes coding for these proteins have been stably integrated into the genome of transgenic plants giving them resistance to pests, such as cultures of tobacco, tomato, potato, corn and cotton with Bt resistance to lepidopteran which are widely marketed in various countries. Several studies have confirmed the economy and the ecological benefit of these cultivars in international agribusiness. In cotton field lepidopteran and coleoptera orders are the worldwide major economic problems. Embrapa develops research for the control of cotton pests in order to reduce production costs, of these, 20-30% are for pest control. Despite the important results obtained so far chemical control has still been the best answer although more costly in financial and more aggressive aspect in environmental aspect. The cry1Ia gene derived from Bacillus thuringiensis strain S1451 may be responsible for this type of control. Insecticidal activity of this gene was tested aiming at the analysis of the recombinant protein for the boll weevil (Anthonomus grandis) and Spodoptera frugiperda caterpillar. Indicating that this gene is very promising for use in molecular area aimed at obtaining cultivate transgenic cotton resistant to these two important pests. In previous work this gene was inserted into three commercial cotton cultivars by microinjection technique via ovary drip. At that moment the cultivars received two different buildings, composed of a minimum linear cassette mlc (≈ 3 Kb) and a circular complete construction ccc (≈ 15 Kb), both containing the gene cry1Ia. The integration of the gene and protein expression was analyzed by PCR of genomic DNA, semi-quantitative RT-PCR, Southern blot and ELISA. Over 1,800 transgenes were tested and the full integration of (1 copy) gene was detected by Southern blot assay and found in only a single event called T0 34 which was derived from the cultivar BRS 293 in mlc treatment, suggesting a genotype-dependent trend. The expression of Cry1Ia protein in T0 34, estimated by ELISA was similar to the commercial event Bollgard (Monsanto, USA). The transmission of the transgene to T1 progeny was demonstrated by PCR analysis and no pleiotropic effects were observed in these plants were phenotypically normal, fertile flowers and with abundant production of seeds. In entomological aspect of military caterpillar larvae were used in feeding bioassays with leaves of T0 events resulting from BRS 293. These same plants were also analyzed for expression of Cry1Ia protein via ELISA. A total of 48 plants were only T0 34 selected in both tests, the mortality rate of 89% and a high concentration of the toxic protein in the leaves. Dried flower buds of this plant were provided to larvae of the boll weevil to study immunodetection via optical microscopy, using midgut tissues of the insect. It was found that this protein in flower buds specifically bound to the antibody used to demonstrate that this event is very promising for further studies of improvement of resistance to two important pest of the cotton crop.
Várias instituições de ensino e pesquisa, em todo o mundo têm devotado esforços na descoberta de novas proteínas inseticidas e dos respectivos genes para o controle de pragas agrícolas. Trabalhos têm evidenciado o potencial de algumas moléculas no controle de insetos como as toxinas Bt do Bacillus thuringiensis, inibidores de proteases e de alfa-amilases e lectinas. Genes codificantes para essas proteínas têm sido estavelmente integrados ao genoma de plantas transgênicas conferindo-lhes resistência a pragas, como é o caso das culturas de tabaco, tomate, batata, milho e algodão Bt com resistência a lepidópteros que já são amplamente comercializadas em vários países. Vários estudos comprovam a economia e o benefício ecológico dessas cultivares no agronegócio internacional. Na lavoura algodoeira as pragas das ordens lepidóptera e coleóptera são os principais problemas econômicos em nível mundial. A Embrapa desenvolve pesquisas voltadas para o controle de pragas do algodoeiro com o intuito de reduzir os custos de produção, destes, 20 a 30% são para controle das pragas. Apesar dos importantes resultados obtidos até o momento o controle químico ainda tem sido o de melhor resposta embora mais oneroso no aspecto financeiro e mais agressivo no aspecto ambiental. O gene cry1Ia derivado da estirpe de Bacillus thuringiensis S1451 pode ser responsável por este tipo de controle. A atividade inseticida deste gene foi testada visando a análise da proteína recombinante para o bicudo do algodoeiro (Anthonomus grandis) e para a lagarta Spodoptera frugiperda. Indicando que este gene é bastante promissor para uso na área molecular visando obtenção de cultivar transgênica de algodão resistente a estas duas importantes pragas. Em um trabalho anterior esse gene foi inserido em três cultivares comerciais de algodão pela técnica de microinjeção via ovary drip. Nesse momento as cultivares receberam duas diferentes construções, compostas por um cassete linear mínimo mlc (≈ 3 Kb) e uma construção circular completa ccc (≈ 15 Kb), ambas contendo o gene cry1Ia. A integração do gene e expressão de proteína foram analisadas por PCR de DNA genômico, RT-PCR semi-quantitativo, Southern blot e ELISA. Mais de 1.800 transgenes foram testados e a integração do gene completo (1 cópia) foi detectada pelo ensaio de Southern blot e encontrada em apenas um único evento denominado T0 34 que foi derivado da cultivar BRS 293 no tratamento mlc, sugerindo uma tendência genótipo-dependente. A expressão da proteína Cry1Ia no evento T0 34, estimada por ELISA, foi semelhante a Bollgard comercial (Monsanto, EUA). A transmissão do transgene para progênies T1 foi demonstrada por análise de PCR e nenhum efeito pleiotrópico foi verificado nessas plantas que foram fenotipicamente normais, com flores férteis e produção de sementes abundantes. No aspecto entomológico larvas da lagarta militar foram utilizadas em bioensaios de alimentação com folhas dos eventos T0 resultantes da BRS 293. Essas mesmas plantas também foram analisadas quanto a expressão da proteína Cry1Ia, via ELISA. De um total de 48 plantas, apenas a T0 34 foi selecionada em ambos os ensaios, com taxa de mortalidade de 89% e elevada concentração da proteína toxica nas folhas. Botões florais desidratados dessa planta foram fornecidos a larvas do bicudo do algodoeiro para estudo de imunodetecção via microscopia ótica, utilizando-se tecidos do intestino médio do inseto. Verificou-se que a proteína presente nos botões florais se ligaram especificamente ao anticorpo utilizado demonstrando que esse evento é muito promissor para posteriores estudos de melhoramento visando resistência a duas importantes pragas da lavoura algodoeira.
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Chung, Jihye. "Analysis of characteristic differentiation processes at the single cell level." Kyoto University, 2016. http://hdl.handle.net/2433/215585.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第19759号
農博第2155号
新制||農||1039(附属図書館)
学位論文||H28||N4975(農学部図書室)
32795
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 植田 充美, 教授 宮川 恒, 教授 栗原 達夫
学位規則第4条第1項該当
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Cozzi, Jean. "Apport de la micro-injection à l'étude du pouvoir fécondant et du genome du spermatozoide humain." Université Joseph Fourier (Grenoble), 1995. http://www.theses.fr/1995GRE10027.
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La technique de micro-injection des spermatozoides humains sous la pellucide (suzi) des ovocytes de hamster est developpee afin d'etudier le pouvoir fecondant des spermatozoides humains et de proceder a l'analyse de leur caryotype. L'analyse de l'interaction gametique apres micro-injection de plusieurs milliers d'ovocytes dans differentes conditions environnementales a mis en evidence la relation existant entre la dynamique de la reaction acrosomiale et la capacite des spermatozoides a fusionner avec l'ovocyte. D'autre part la selection de sous populations spermatiques presentant differents etats acrosomiques et pouvoirs fecondants a ete obtenue par centrifugation sur gradient de percoll. Une technique originale d'induction de la reaction acrosomiale par l'ionophore calcique a23187 est proposee pour apprecier le pouvoir fusiogene des spermatozoides humains apres micro-injection. Le pouvoir fusiogene des spermatozoides humains micro-injectes sous la pellucide a ete compare dans les modeles heterospecifique et homospecifique. La valeur predictive de la suzi-hamster pour la suzi humaine est discutee. La maitrise des parametres de la fecondation apres suzi-hamster a permis de realiser l'analyse des spermatozoides micro-injectes. Chez deux sujets temoins, 72 metaphases interpretables de spermatozoides ont ete obtenus. Cependant la suzi se revele etre une technique moins performante pour l'analyse cytogenetique des spermatozoides humains que la technique conventionnelle d'insemination des ovocytes depellucides. Elle semble donc difficilement applicable aux sujets dont la spermatogenese est deficiente. De facon concomitante, l'analyse cytogenetique des spermatozoides d'un patient porteur d'un syndrome de klinefelter en mosaique (46,xy/47,xxy) a ete realisee par la technique conventionnelle d'insemination in vitro des ovocytes depellucides de hamster. 543 caryotypes spermatiques ont ete obtenus. Un taux significativement augmente de spermatozoides 24,xy a ete mis en evidence, suggerant que les cellules germinales de la lignee 47,xxy seraient aptes a franchir toutes les etapes de la meiose
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Chicheportiche, Yves. "Identification et caractérisation d'antigènes de l'appareil de Golgi et autres membranes lisses par les anticorps monoclonaux : études préliminaires par microinjection du rôle de ces antigènes dans les mécanismes de sécrétion." Montpellier 2, 1987. http://www.theses.fr/1987MON20046.
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Des anticorps monoclonaux ont ete produits contre des proteines de l'appareil de golgi et autres membranes lisses. Les antigenes ont ete caracterises par des techniques biochimiques et leur localisation intracellulaire determinee par immunocytochimie en microscopie electronique. Une phosphorylation calcium-dependante a ete decrite pour une proteine associee au golgi. Une etude de l'effet sur la secretion de l'injection dans le cytoplasme des cellules des anticorps anti-golgi a ete abordee
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Chicheportiche, Yves. "Identification et caractérisation d'antigènes de l'appareil de Golgi et autres membranes lissés par les anticorps monoclonaux études préliminaires par microinjection du rôle de ces antigènes dans les mécanismes de secrétion /." Grenoble 2 : ANRT, 1987. http://catalogue.bnf.fr/ark:/12148/cb376039388.
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Guedon, Gérard. "Diadenosine tetraphosphate : synthese et relations avec le choc thermique." Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13040.
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Astinotti, Daniel. "Regulation de la transcription du gene de l'ovalbumine de l'oviducte de poule par les hormones steroides." Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13023.
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La regulation du gene ovalbumine se fait essentiellement au niveau transcriptionnel a l'aide de construction reumbinents exprimant les genes marqueurs (antigene t du sv40, ou chloramphenicol acetyltransferase) sous controle de sequences pronostics du gene ovalbumine, il a ete trouve une unite de regulation hoemonale negative fonctionnant de facon unidirectionnelle (inhibition en presences d'antagonistes des hormones steroides de sa propre promoteur, repression du promoteur du gene ovalbumine en absence d'hormone steroide) et un motif 5'-gctcaca-3' implique dans une regulation portive du promoteur du gene de l'ovalbumine par les oestrogenes a leur recepteur. Ces deux elements hormonaux dependants fonctionnellement liees, l'un induisant, l'autre inhibant l'expression du promoteur du gene ovalbumine, constituent un excellent modele pour etudies la regulation transcriptionnelle chez les eucaryote superieurs
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Douglas, Mark William. "Retrograde Cellular Transport of Herpes Simplex Virus: Interactions between Viral and Motor Proteins." Thesis, The University of Sydney, 2005. http://hdl.handle.net/2123/628.
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Herpes simplex virus type 1 (HSV-1) is a common human pathogen that establishes life-long latent infection in sensory neurones. This makes it potentially useful as a gene therapy vector to target neuronal cells. HSV-1 enters cells by membrane fusion, the viral envelope and most tegument proteins dissociate, and the capsid is transported to the cell nucleus to establish infection. There is increasing evidence that the retrograde transport of HSV-1 along sensory axons is mediated by cytoplasmic dynein, but the viral and cellular proteins involved are not known. Cytoplasmic dynein is the major molecular motor involved in minus-end-directed cellular transport along microtubules. It is a large complex molecule, with heavy chains providing motility, while intermediate and light chains are involved in specific cargo binding. A library of HSV-1 capsid and tegument structural genes was constructed and tested for interaction with dynein subunits in a yeast two-hybrid system. A strong interaction was demonstrated between the HSV-1 outer capsid protein VP26 (UL35), as well as the tegument protein VP11/12 (UL46), with the homologous 14 kDa dynein light chains rp3 and Tctex1. In vitro pull-down assays confirmed binding of VP26 to rp3, Tctex1 and cytoplasmic dynein complexes. Recombinant HSV-1 capsids +/- VP26 were used in similar pull-down assays. Only VP26+ capsids bound to rp3. Recombinant HSV-1 capsids were microinjected into living cells and incubated at 37ºC. After 1 h capsids were observed to co-localise with rp3, Tctex1 and microtubules. After 2 or 4 h VP26+ capsids had moved closer to the cell nucleus, while VP26- capsids remained in a random distribution. Our results suggest that the HSV-1 outer capsid protein VP26 mediates binding of incoming capsids to the retrograde motor cytoplasmic dynein during cellular infection, through interactions with dynein light chains. It is hoped that these findings will help in the development of a synthetic viral vector, which may allow targeted gene therapy in patients with neurological diseases.
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Douglas, Mark William. "Retrograde Cellular Transport of Herpes Simplex Virus: Interactions between Viral and Motor Proteins." University of Sydney. Westmead Millennium Institute, 2005. http://hdl.handle.net/2123/628.
Full textAbstract:
Herpes simplex virus type 1 (HSV-1) is a common human pathogen that establishes life-long latent infection in sensory neurones. This makes it potentially useful as a gene therapy vector to target neuronal cells. HSV-1 enters cells by membrane fusion, the viral envelope and most tegument proteins dissociate, and the capsid is transported to the cell nucleus to establish infection. There is increasing evidence that the retrograde transport of HSV-1 along sensory axons is mediated by cytoplasmic dynein, but the viral and cellular proteins involved are not known. Cytoplasmic dynein is the major molecular motor involved in minus-end-directed cellular transport along microtubules. It is a large complex molecule, with heavy chains providing motility, while intermediate and light chains are involved in specific cargo binding. A library of HSV-1 capsid and tegument structural genes was constructed and tested for interaction with dynein subunits in a yeast two-hybrid system. A strong interaction was demonstrated between the HSV-1 outer capsid protein VP26 (UL35), as well as the tegument protein VP11/12 (UL46), with the homologous 14 kDa dynein light chains rp3 and Tctex1. In vitro pull-down assays confirmed binding of VP26 to rp3, Tctex1 and cytoplasmic dynein complexes. Recombinant HSV-1 capsids +/- VP26 were used in similar pull-down assays. Only VP26+ capsids bound to rp3. Recombinant HSV-1 capsids were microinjected into living cells and incubated at 37�C. After 1 h capsids were observed to co-localise with rp3, Tctex1 and microtubules. After 2 or 4 h VP26+ capsids had moved closer to the cell nucleus, while VP26- capsids remained in a random distribution. Our results suggest that the HSV-1 outer capsid protein VP26 mediates binding of incoming capsids to the retrograde motor cytoplasmic dynein during cellular infection, through interactions with dynein light chains. It is hoped that these findings will help in the development of a synthetic viral vector, which may allow targeted gene therapy in patients with neurological diseases.
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Hajdu, Melissa Anne. "Effect of culture conditions, donor source, and injection site on in vitro development of deoxyribonucleic acid microinjected porcine zygotes." Thesis, This resource online, 1993. http://scholar.lib.vt.edu/theses/available/etd-12172008-063219/.
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GILSON, GEORGES. "Le diadenosine tetraphosphate : etudes immunochimiques et analyse de son role dans les mecanismes de reponse au stress et de reparation du dna." Université Louis Pasteur (Strasbourg) (1971-2008), 1988. http://www.theses.fr/1988STR13110.
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Guinn, Jessie Jr. "Assessment of the Integrative Roles of the Intergeniculate Leaflet in Circadian Timing and Reward Pathways." Kent State University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=kent1320094118.
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