Academic literature on the topic 'Techniques in vitro – méthodes'
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Journal articles on the topic "Techniques in vitro – méthodes"
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Bezuidenhout, J. D. "L’importance des acquisitions récentes de la recherche sur la cowdriose." Revue d’élevage et de médecine vétérinaire des pays tropicaux 46, no. 1-2 (January 1, 1993): 101–8. http://dx.doi.org/10.19182/remvt.9344.
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Beaucoup des objectifs de la recherche sur la cowdriose, identifiés jusquà présent, ont été atteints pendant la décennie passée. Certains acquis, tels que la mise au point des sondes à ADN spécifiques pour Cowdria et l'atténuation de l'organisme, sont encore au stade expérimental, mais d'autres comme la culture in vitro, sont déjà des procédures bien établies dans nombres de laboratoires. Des techniques sérologiques sont plus généralement utilisées depuis que d'autres méthodes et d'autres sources d'antigènes sont disponibles. Néanmoins, des réactions croisées avec Ehrlichia continuent à compliquer l'interprétation des données épidémiologiques. Les études biochimiques pour identifier, isoler et caractériser les protéines antigéniques de Cowdria ont mis en évidence des protéines immunodominantes bien définies, qui pourraient convenir à des tests sérologiques.
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Komoin Oka, C., Philippe Truc, Zakaria Bengaly, Pierre Formenty, Gérard Duvallet, Francis Lauginie, J. P. Raath, A. E. N'Depo, and Yves Leforban. "Etude de la prévalence des infections à trypanosomes chez différentes espèces d'animaux sauvages du parc national de la Comoé en Côte d'Ivoire : résultats préliminaires sur la comparaison de trois méthodes de diagnostic." Revue d’élevage et de médecine vétérinaire des pays tropicaux 47, no. 2 (February 1, 1994): 189–94. http://dx.doi.org/10.19182/remvt.9108.
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Si de nombreuses études portent sur la trypanosomoses chez les animaux domestiques, peu de rcherches ont été effectuées sur la faune sauvage d'Afrique occidentale. Les résultats préliminaires sur la comparaison de trois méthodes de dépistages des trypanosomes : le frottis de sang, la détection des antigènes circulants par le technique ELISA et l'isolement in vitro des trypanosomes par le "kit for in vitro isolation" (KIVI), chez les animaux sauvages du parc national de la Comoé en Côte d'ivoire, ont permis de confirmer l'existence de cette infection chez ces animaux sans toutefois identifier de façon précise les espèces de trypanosomes en cause. Des investigations ultérieures permettront d'affiner l'identification des souches de parasites isolées par KIVI.
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De Champs, C., C. Martin, D. Caillaud, and J. M. Aiache. "Détection des anticorps anti-Micropolyspora faeni : comparaison de la méthode ELISA aux techniques in vitro classiques." Revue Française d'Allergologie et d'Immunologie Clinique 37, no. 4 (June 1997): 423–30. http://dx.doi.org/10.1016/s0335-7457(97)80069-x.
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HOUDEBINE, L. M. "Les manipulations génétiques : comment améliorer la croissance." INRAE Productions Animales 3, no. 3 (July 4, 1990): 207–14. http://dx.doi.org/10.20870/productions-animales.1990.3.3.4377.
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Les récents développements de la génétique moléculaire offrent désormais la possibilité d’isoler virtuellement n’importe quel gène, de le muter in vitro, de modifier ses éléments régulateurs, de le réintroduire dans des cellules ou des organismes entiers (transgénèse) et donc, de moduler à volonté une fonction physiologique donnée. Ces possibilités ont été et sont encore appliquées à la fonction de croissance. Ainsi, les gènes du GRF (Growth Releasing Factor), de la GH (Growth Hormone) et de l’IGF1 (Insulin-like Growth Factor) ont été introduits dans des embryons de divers animaux. Ces transgènes se sont exprimés et ont conduit à une accélération de la croissance et à une augmentation de la taille des animaux accompagnée d’un assez profond changement de leur métabolisme. L’incapacité de moduler finement l’expression des transgènes chez les animaux transgéniques se traduit par une sécrétion exacerbée des hormones qui, elle-même, induit une série de désordres physiologiques rendant les animaux transgéniques peu performants. La transgénèse, si elle est devenue une routine chez la souris dans un certain nombre de laboratoires, reste difficilement réalisable à grande échelle chez la plupart des animaux domestiques. Des études approfondies sur les mécanismes de contrôle de l’expression des transgènes et sur les méthodes de transgénèse doivent donc être menées à bien pour que les manipulations génétiques deviennent une réalité dans les techniques d’élevage.
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LOCATELLI, Y., and P. MERMILLOD. "Caractéristiques et maîtrise de la fonction de reproduction chez les cervidés." INRAE Productions Animales 18, no. 1 (March 14, 2005): 3–25. http://dx.doi.org/10.20870/productions-animales.2005.18.1.3505.
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Ces dernières années, l’élevage de cervidés s’est considérablement développé en Nouvelle-Zélande pour devenir une industrie profitable. Alors que quelques cervidés (daim d’Europe et cerf élaphe) font l’objet de domestication et d’élevage dans un but commercial, d’autres espèces et sous-espèces de cervidés sont menacées d’extinction dans le milieu naturel. Les travaux de recherches effectués afin de mieux caractériser et maîtriser la fonction de reproduction de ces ruminants sont présentés dans cette étude bibliographique.
Il apparaît qu’à l’instar de nos ruminants domestiques (ovins, caprins), les cervidés originaires des zones tempérées sont généralement caractérisés par un saisonnement marqué de leur fonction de reproduction. Ces variations saisonnières d’activité sexuelle sont dictées par les variations photopériodiques et permettent la naissance des jeunes en fin de printemps. Chez les cervidés, les différences entre les périodes d’activité et de repos sexuels semblent beaucoup plus marquées en comparaison de nos ruminants domestiques. La période d’activi-té sexuelle est variable d’une espèce à l’autre (été, automne ou début de l’hiver) mais très fixe pour une espèce donnée. La période de repos sexuel traduit des modifications importantes dans les sécrétions de gonadotrophines et se caractérise notamment par une aspermie complète chez le mâle. Chez la femelle, l’état d’anoestrus est profond et associé à une absence d’ovulation. La durée de gestation est également variable d’une espèce à l’autre mais est remarquablement fixe pour une espèce donnée.
Chez les cervidés originaires des zones subtropicales et selon les espèces, les variations d’activité sexuelle sont plus discrètes permettant une répartition des mises bas plus ou moins homogène au cours de l’année, y compris lorsque les animaux sont transportés sous des latitudes élevées.
Dans le cas des espèces de cervidés menacés d’extinction, l’utilisation des biotechnologies de la reproduction et des méthodes de procréation assistée pourrait, à terme, faciliter la réalisation des programmes conservatoires. Les techniques classiques de production in vivo d’embryons basées sur l’ovulation multiple, l’insémination artificielle et le transfert embryonnaire se sont avérées difficilement applicables aux cervidés. Aussi, les recherches portent actuellement sur le développement de techniques de production in vitro d’embryons.
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Jérôme, Simone, Françoise Yon-Cassat, Catherine Vernisson, Caroline Lopez, Michel Vajou, and Dominique Cotte. "Méthodes, techniques et outils." Documentaliste-Sciences de l'Information 45, no. 1 (2008): 14. http://dx.doi.org/10.3917/docsi.451.0014.
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Dassa, Michèle, Marie-Josèphe Pierrat, Adriana Lopez Uroz, Michèle Battisti, Serge Boulier, Dominique Cotte, Sylvie Dalbin, and Odile Giraud. "Méthodes, techniques et outils." Documentaliste-Sciences de l'Information 45, no. 2 (2008): 4. http://dx.doi.org/10.3917/docsi.452.0004.
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Amar, Muriel, Marc Maisonneuve, Claire Scopsi, Serge Boulier, Corinne Dupin, Charles-Henry Rey, Aline Auffret, Dominique Cotte, Sylvie Dalbin, and Odile Giraud. "Méthodes, techniques et outils." Documentaliste-Sciences de l'Information 45, no. 3 (2008): 14. http://dx.doi.org/10.3917/docsi.453.0014.
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Aubry, Sara, Thomas Chaimbault, Claire Scopsi, Dominique Cotte, Sylvie Dalbin, Odile Giraud, Loïc Lebigre, and Claudine Masse. "Méthodes techniques et outils." Documentaliste-Sciences de l'Information 45, no. 4 (2008): 12. http://dx.doi.org/10.3917/docsi.454.0012.
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Banat-Berger, Françoise, Dominique Aussant, and Stéphane Chaudiron. "Méthodes techniques et outils." Documentaliste-Sciences de l'Information 46, no. 1 (2009): 12. http://dx.doi.org/10.3917/docsi.461.0012.
Full textDissertations / Theses on the topic "Techniques in vitro – méthodes"
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Chrit, Linda. "Développement de méthodes microspectroscopiques pour l'évaluation in vitro et in vivo de l'hydratation du stratum corneum par des agents cosmetiques." Reims, 2006. http://www.theses.fr/2006REIMP205.
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La @peau est un organe complexe qui présente une forte hétérogénéité tant au niveau de sa composition moléculaire qu’au niveau de son organisation structurale. Ces variations dépendent du site anatomique mesuré mais aussi de la profondeur ou couche analysée. Celles-ci peuvent être affectées par des désordres cutanés liés à des pathologies ou encore à des facteurs environmentaux tels que l’exposition solaire, les variations climatiques, les traitements dermatologiques à visé médicamenteux ou cosmétiques. Ces traitements peuvent perturber les propriétés mécaniques de la peau. Ainsi, dans l’objectivation de la recherche cutanée et de la caractérisation de la peau, les méthodes non invasives sont particulièrement recherchées, induisant un essor considérable de l’utilisation de méthodes à spectroscopie vibrationnelle. Ces méthodes permettent une double approche à la fois in vitro et in vivo. Effectivement, le travail de cette thèse a consisté à développer un outil de criblage automatique permettant d'évaluer le pouvoir hydratant, in vitro, de nombreuses entités chimiques dans l’axe soin. Ce travail présenté dans cette thèse démontre ainsi l’apport des techniques de spectroscopie vibrationnelle, Raman et accessoirement infrarouge à l’étude et la compréhension des mécanismes d’action des agents hydratants au sein du stratum corneum. Le développement en parallèle d’une microsonde confocale Raman pour l’étude in vivo a permis d’évaluer les molécules d’intérêts retenues in vitro. Nous avons pu mettre en évidence le pouvoir hydratant de différentes entités chimiques non révélées par les méthodes conventionnelles. Ces méthodes, non invasives et non destructives, permettent l’investigation de la peau humaine dans son état naturel sans altérer sa morphologie, son intégrité et encore sa composition moléculaire, comme par exemple pour des études typologie. Tous ces travaux ont nécessité une approche statistique du traitement des différents spectres basée sur les méthodes d’analyse mixtes de la variance, de décomposition par la méthode des moindres carrées ou encore de classification hiérarchique (HCA)The @skin is a complex organ which has a high level of heterogeneity at the level its molecular and structural organisation. These variations depend on the depth and site of analysis. Such variations can be affected by subcutaneous disorder related to pathology or environmental factors such as solar exposure, climatic changes, and medical or cosmetic dermatological. These treatments can change the mechanical and optical properties of the skin. Thus, in order to study and characterise the properties of the skin objectively, non-invasive methods have been of particular interest recently, leading to a considerable growth in methods utilising vibrational spectroscopy. Vibrational spectroscopic methods permit characterisation of the Stratum corneum in vitro and in vivo. The work in this thesis was concerned with the construction of an automatic screening tool for the evaluation, in vitro, of the hydration potential of chemical species within the skin. The work presented in this thesis demonstrates that vibrational spectroscopic techniques (primarily Raman and secondarily Infra-Red imaging) have been successfully applied to the analysis and understanding of the mechanisms of hydration of the Stratum corneum by cosmetic agents. These results have also been extended to the development, in parallel, of a confocal Raman microprobe for the evaluation, in vivo, of the retention of molecules of interest in the skin. The hydration potential of different chemical species, which are not accessible by conventional methods, have been elucidated by the methods developed here. These non-invasive and non-destructive methods alow the analysis of the human skin in its natural state without altering its morphology, integrity and molecular composition during measurements of skin-typing, for example. The spectroscopic analysis presented herein required the use of statistical treatments based on mixed analysis of variance, spectral composition by minimum least square methods, hierarchical cluster analysis (HCA)
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Soullier, Noémie. "Evaluation des résultats de la fécondation in vitro : l'apport de l'imputation multiple." Paris 11, 2010. http://www.theses.fr/2010PA11T019.
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De, Conto Véronique. "Importance du microenvironnement dans les modèles cérébraux in vitro pour le criblage phénotypique." Thesis, Université de Lille (2018-2021), 2021. http://www.theses.fr/2021LILUS046.
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Environ 90% des candidats-médicaments échouent en phase clinique, pour des raisons d’efficacité ou de toxicité qui impliquent souvent le système nerveux central (SNC). Ce fort taux d’échec souligne un manque de pertinence des modèles expérimentaux utilisés en amont, dont les modèles in vitro de cellules humaines. En effet, ces derniers ne prennent pas en compte toute la complexité du SNC, où les neurones organisés en 3 dimensions (3D) interagissent avec leur microenvironnement composé de cellules, de facteurs solubles et des molécules de la matrice extracellulaire (MEC). Les objectifs de ce travail étaient i) d’étudier l’influence de ces trois composantes du microenvironnement sur les cellules neuronales dans des modèles cérébraux in vitro par imagerie cellulaire automatisée, et ii) de développer des modèles cérébraux in vitro plus pertinents pour évaluer les effets neurotoxiques ou thérapeutiques de molécules par criblage phénotypique, notamment dans le cadre de la maladie de Parkinson (MP).Dans un premier temps, la technologie BIOMIMESYS® Brain a été développée. Cette matrice à base d’acide hyaluronique permet de mimer la MEC et de cultiver des cellules cérébrales en 3D dans des plaques 96 puits. La sensibilité des cellules Luhmes, une lignée de neurones dopaminergiques, aux inducteurs de la MP a été étudiée : les cellules ont montré une sensibilité plus faible dans BIOMIMESYS® Brain qu’en 2 dimensions (2D). Cette différence a pu être expliquée par deux phénomènes : une rétention partielle des molécules toxiques dans la matrice, et un phénotype de neurone dopaminergique moins mature qu’en 2D. L’importance du microenvironnement cellulaire a ensuite été étudiée au travers d’une co-culture de cellules Luhmes et d’astrocytes primaires humains en 2D. Cette co-culture a ensuite été transposée dans la matrice BIOMIMESYS®, formant ainsi un modèle complexe incluant à la fois le microenvironnement glial et le microenvironnement matriciel.En parallèle, l’influence du microenvironnement moléculaire a été étudiée sur les cellules SH-SY5Y, une lignée cellulaire issue d’un neuroblastome, couramment utilisée pour évaluer la neurotoxicité de molécules. Dans cette étude, les 24 principaux milieux décrits dans la littérature pour différencier ces cellules en neurones ont été criblés. Les 3 conditions les plus différenciantes en matière de ralentissement de la prolifération cellulaire et de croissance des neurites ont été sélectionnées : l’acide rétinoïque, la staurosporine, et l’Adénosine Monophosphate cyclique (AMPc) associée à du supplément B21. L’expression de marqueurs protéiques neuronaux et la sensibilité des cellules à des composés de toxicités connues ont été mesurées, en 2D et en 3D dans BIOMIMESYS® Brain. La maturité neuronale et la sensibilité aux composés neurotoxiques différaient selon le milieu, en étant les plus hautes en milieu B21+AMPc. La culture en 3D modifiait aussi la réponse des cellules, avec une sensibilité plus faible comparée aux cellules cultivées en 2D.Cette thèse a mis en évidence que le microenvironnement des neurones, qui inclut la MEC, les cellules gliales et les facteurs solubles, modifie la réponse neuronale in vitro et devrait par conséquent être considéré avec attention dans la recherche académique comme industrielle, dès les étapes de criblage de nouveaux médicamentsAbout 90% of drug candidates fail in clinical trials, for efficacy- and toxicity-related reasons, which often involve the Central Nervous System (CNS). This high failure rate highlights a lack of relevance in experimental models used upstream, including human in vitro models. Indeed, they do not take into account the complexity of the CNS, in which neurons are organized in 3 dimensions (3D) and interact with their microenvironment, composed of cells, soluble factors and extracellular matrix (ECM). The objectives of this PhD were i) to study the influence of these three microenvironment components on neuronal cells in cerebral in vitro models by automatized cellular imaging, and ii) to develop more relevant cerebral in vitro models for phenotypic screening, to assess neurotoxic or therapeutic effects, in the frame of Parkinson’s Disease (PD).First, the BIOMIMESYS® Brain technology has been developed. This acid hyaluronic based-matrix allows the simulation of the ECM and a 3D culture of cerebral cells in 96-well plates. The sensitivity of Luhmes cells, a dopaminergic neuronal cell line, to PD inducers has been studied: the cells displayed a lower sensitivity in BIOMIMESYS® Brain compared to cells cultured in 2 dimensions (2D). This difference was explained by two phenomena: a partial retention of toxic molecules in the matrix, and a lower neuronal maturity compared to cells cultured in 2D.The importance of the cellular microenvironment has been studied through a co-culture of Luhmes cells and primary human astrocytes in 2D. This co-culture has then been transposed in BIOMIMESYS® matrix, to form a complex model including both the glial and the matricial microenvironments.In parallel, the influence of the molecular microenvironment has been studied on the SH-SY5Y cells, a cell line derived from a neuroblastoma, commonly used for neurotoxicity assessment. In this study, the 24 major differentiation media described in the literature to differentiate these cells into neurons have been screened. The 3 most differentiating conditions in terms of proliferation slowdown and neurite elongation have been selected: retinoic acid, staurosporine, and cyclic Adenosine Monophosphate (cAMP) combined to B21 supplement. The neuronal protein marker expression and the cell sensitivity to compounds of known-toxicity have been measured, in 2D and in 3D in BIOMIMESYS® Brain. Both maturity and sensitivity of these neurons varied according to the differentiation medium, and were higher in B21+cAMP. The 3D cell culture modified also the cell response, with a lower sensitivity of cells cultured in 2D.This PhD highlighted that the microenvironment of neurons, including the ECM, the glial cells and the soluble factors, can modify the neuronal response in vitro, and should thus be considered carefully in academic research and as early as possible in the drug discovery industrial process
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Aubert, L'Huillier Carole. "Evaluation du pouvoir bioadhésif de gels à base de techniques "in vitro" : étude de l'influence des facteurs de formulation - choix de la méthode." Paris 5, 1997. http://www.theses.fr/1997PA05P142.
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Mejlachowicz, Dan. "Développement de modèles in vitro et in vivo pour analyser la réponse aux radiations ionisantes du tissu thyroïdien normal humain." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL057.
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Le seul facteur étiologique démontré pour le cancer de la thyroïde est une exposition aux radiations ionisantes (RI) durant l’enfance. Les études épidémiologiques mesurent un risque significatif de développer un cancer suite à l’exposition pour une dose reçue à la thyroïde d’au minimum 50 mGy, le risque étant proportionnel à la dose. Ce risque est observé après une exposition externe à fortes doses/débits de dose (radiothérapie) et après une contamination à de plus faibles doses de radioisotopes de l’iode. Si des cancers radio-induits (R) se développent à des doses <50mGy, l’excès de cancers ne peut pas être mesuré par l’épidémiologie conventionnelle, d’autant plus que l’incidence des cancers spontanés de la thyroïde (S) augmente. En conséquence, il n’est pas possible de répondre aux interrogations sociétales sur les risques pour la santé d’exposition aux faibles doses délivrées lors d’examens d’imagerie médicale dans la région « tête et cou » chez l’enfant, ou reçues suite aux retombées des accidents de Tchernobyl et Fukushima, et aux essais nucléaires atmosphériques en Polynésie française.Pour répondre à ces interrogations, une meilleure connaissance de l’effet des RI en fonction de la dose et du débit de dose sur la thyroïde humaine est nécessaire. La carcinogenèse thyroïdienne présente des spécificités d’espèces : chez la souris, l’incidence de cancers de la thyroïde S et R est faible, et les quelques tumeurs R qui se développent sont principalement des cancers folliculaires alors qu’ils sont majoritairement papillaires chez l’Homme (S et R).Pour analyser la réponse aux RI du tissu thyroïdien humain normal, nous avons développé plusieurs approches à partir de biopsies de tissu normal provenant de patients ayant subi une thyroïdectomie : des cultures primaires de thyrocytes, des modèles 3D in vitro (thyrosphères, cultures organotypiques) et des xénogreffes chez la souris.Pour les modèles 3D, le maintien de la polarité, de la différenciation cellulaire, de la complexité tissulaire et de l’activité physiologique des cultures ont été contrôlés. Nous avons obtenu des thyrosphères (6 donneurs) organisées en follicules délimitant un lumen composé de thyroglobuline. Nous avons développé des cultures organotypiques en matrigel selon le protocole publié par Toda et al. (thyroïde de porc, 2002) (7 donneurs), et montré un choc hypoxique dans les tissus. Une oxygénation optimale du tissu a été obtenue par une culture en interface air-liquide (ALI) (3 donneurs). En culture ALI, les biopsies sécrétent l’hormone thyroïdienne T4 en réponse à la TSH (1 donneur, 1 semaine). Ce protocole a permis un maintien du tissu sur 4 semaines. Les xénogreffes devraient permettre une analyse à des temps plus tardifs, et ont déjà permis le maintien du tissu sur 5 semaines sur des souris SCID/beige (3 donneurs).En parallèle, nous avons comparé la prolifération, la survie et la cinétique d’induction/réparation des cassures après exposition aux RI de cultures primaires de thyrocytes de patients exposés (radiothérapie) pendant l’enfance (2 donneurs) ou non exposés (3 donneurs). Nous observons de façon reproductible que les thyrocytes exposés sont plus radiorésistants que les thyrocytes non exposés. Ces résultats suggèrent fortement l’existence d’une empreinte à long terme d’exposition aux RI dans le tissu normal, en accord avec l’identification de signatures moléculaires discriminant le tissu normal exposé et non exposé par C Ory et N Ugolin au LCE. Comme suggéré par l’équipe de C Dupuy, cette empreinte pourrait être due à la mise en place d’un stress oxydatif chronique dans la thyroïde suite à l’exposition aux RI.Les modèles développés pendant cette thèse seront essentiels pour comprendre les effets et les risques des RI à faibles et fortes doses, associées ou non aux perturbateurs endocriniens, sur la thyroïde humaine, ainsi que les premières étapes de la carcinogenèse et l’origine de cette empreinte d’exposition persistante sur le long termeThe only demonstrated etiologic factor for thyroid cancer is an exposure to ionizing radiation (IR) during childhood. Epidemiological studies can measure a significant risk to develop a cancer following thyroid doses above 50 mGy. This risk is observed after external exposure to high doses/dose rates (radiotherapy) and after contamination of iodine radioisotopes at lower doses.As the risk decreases with the dose, if radiation-induced cancers (R) develop after thyroid doses <50mGy, an excess of cancers cannot be measured by conventional epidemiology, especially because the incidence of sporadic cancers (S) increases. Consequently, it is not possible to answer the societal debates concerning the risk associated to low doses exposure, such as the risk for patients during medical diagnosis by imagery technics in the "head and neck" area especially for children, following Chernobyl and Fukushima accidents fallout, and following contamination after the atmospheric nuclear tests in French Polynesia.To answer the questionnning about the risk of low doses IR on human thyroid, a better knowledge of the exposure according to doses and dose rates is necessary,. Thyroid carcinogenesis presents specificities among species. Indeed, in mice, S or R thyroid cancers incidence is low, and the few R tumors developping are mainly follicular cancers whereas there are mainly papillary cancers in humans (S and R).In order to analyze the normal human thyroid tissue response to IR, we have developed several approaches using biopsies of non-pathological tissues from patients who have undergone thyroidectomy: primary thyrocyte cultures, in vitro 3D models (thyrospheres and organotypic cultures) and xenografts in mice.For 3D models, maintenance of polarity, cell differentiation, tissue complexity and physiological activity of the cultures were controlled. We obtained thyrospheres (6 donors) organized in follicles delimiting a lumen composed of thyroglobulin. We used the matrigel-based protocol described by Toda et al. (2002) for organotypic cultures of porcin tissue, and showed an hypoxic stress in the human tissue (7 donors). An optimal oxygenation of the tissue was obtained by air-liquid interface culture (3 donors). In ALI organotypic cultures, a secretion of free thyoid hormone T4 was observed (1 donor, at 1 week), and this protocol allowed the tissue maintenance over 4 weeks. Mouse xenografts permitted human tissue maintenance over a period of at least one year, we already succeed in maintaining the thyroid tissue over 5 weeks in SCID/beige mice (3 donors).In parallel, we compared the proliferation, the survival and the kinetics of DNA strand breaks induction/repair after exposure of thyrocytes from patients exposed (radiotherapy) during childhood (2 donors) or unexposed (3 donors), in primary cultures. We reproducibly observed that exposed thyrocytes are more radioresistant than unexposed thyrocytes. These results strongly suggest the existence of a long-term phenotypic signature of exposure to IR in normal thyroid tissue, consistent with the identification of molecular signatures discriminating exposed and unexposed normal thyroid tissue by C Ory and N Ugolin in the laboratory. As suggested by C Dupuy's team, this imprinting could be due to the development of a chronic oxidative stress following IR exposure in the thyroid.The models developed during this thesis will be essential to understand low and high doses IR effects and risks on the human thyroid. They will also be usefull to asses the effects of endocrine disruptors (ED) on human thyroid, alone or in cocktail and estimate if this ED exposure may modify the radiosensitivity of the thyroid. These models will be used to analyse the first steps on radiation-induced thyroid carcinogenesis as well as the origin of this persistent long-term exposure imprinting
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Ostrovidov, Serge. "Evaluation des activités antioxydantes de nouvelles molécules séléniées par méthode in vitro utilisant la culture cellulaire et la cytométrie de flux." Nancy 1, 1997. http://www.theses.fr/1997NAN19008.
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Les espèces oxygénées réactives (ROS) sont des métabolites normaux de la cellule. Cependant en trop grande quantité, ils deviennent néfastes pour l'hôte. Aussi afin de rechercher de nouvelles molécules séléniées ayant une activité antioxydante, nous avons créé un modèle expérimental basé sur la culture de cellules hybridomes Mark 3 et la mesure du taux de radicaux libres intracellulaires par cytométrie en flux à l'aide des sondes fluorescentes DHR 123 et DCFH-DA respectivement spécifiques de l'anion superoxyde (O2-) et des peroxydes (ROOH). Après une étude des effets d'un stress oxydant par H2O2 sur la prolifération cellulaire et sur la formation des ROS intracellulaires notre modèle expérimental a été validé par la mesure de l'activité antioxydante de la vitamine E, la mélatonine, le Ginkgo bibola (Tanakan ®) et la N-acétylcystéine. Nous avons alors effectué un "screening" antioxydant sur 34 composés de synthèse analogues de l'ellipticine et les avons classés selon la force de leur activité antioxydante. Enfin après sélection d'une de ces molécules pour ses propriétés antioxydantes, une étude plus approfondie de son activité a été effectuée afin de déterminer la nature des ROS préférentiellement éliminés par celle-ci ainsi que les compartiments cellulaires où elle agit. De plus il est à noter que ce modèle expérimental construit pour rechercher des activités antioxydantes peut aussi être utilisé pour rechercher des activités oxydantes ce qui peut être utile à la recherche d'anti-tumoraux.
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Biri, Venceslas. "Techniques d'animation dans les méthodes globales d'illumination." Université de Marne-la-Vallée, 2003. http://www.theses.fr/2003MARN0155.
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Dans cette thèse, nous abordons la problématique de l’animation dans le rendu. Celle-ci se découpe en deux grandes parties : rendu sans et avec milieux participants. Dans la première partie, après un survol des techniques d’illumination d’une scène, nous présentons un nouvel algorithme, fondé sur la radiosité et utilisant des bases de fonctions. Il prend profit de la cohérence temporelle pour calculer des animations deux à trois fois plus rapidement, et ce même lorsque la source de lumière se déplace. Dans la seconde partie, après une revue des différentes techniques de rendu de milieux participants , nous proposons un premier algorithme permettant de modéliser, de simuler et d’animer du brouillard non homogène en temps réel. Puis nous proposons un autre algorithme temps réel de rendu en simple diffusion, représentant les ombres surfaciques et volumiques. Finalement, nous tentons de généraliser notre précédent algorithme de radiosité dynamique dans le cas des milieux participantsIn this thesis, we focus on the problems of rendering in dynamic scene. It contains two main parts : one without participating medium and the other one with. In the first part, after an overview of rendering methods, we present a new algorithm, based on radiosity using higher order bases. It takes profits of temporal coherence to calculate animations twice to three times faster, even when light sources are moving. In the second part, after seeing the different algorithms that are able to render participating medium , we propose a first method allowing to model, simulate and animate non homogeneous fog in real time. Then, we propose an real time algorithm, in single scattering, representing surface and volume shadows. Finally, we try to generalise our preceding dynamic radiosity algorithm in the presence of the participating mediums
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Maire, Sylvain. "Quelques Techniques de Couplage entre Méthodes Numériques Déterministes et Méthodes de Monte-Carlo." Habilitation à diriger des recherches, Université du Sud Toulon Var, 2007. http://tel.archives-ouvertes.fr/tel-00579977.
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Les travaux présentes s'inscrivent dans le cadre de la réduction de variance pour les méthodes de Monte-Carlo et plus généralement dans l'optimisation de méthodes numériques à l'aide de couplage entre des méthodes déterministes et des méthodes probabilistes. Trois thèmes principaux seront abordés à l'aide de ces techniques: l'intégration numérique sur un hypercube, la résolution d' équations aux dérivées partielles linéaires et le calcul des éléments propres principaux (valeur propre et vecteur propre) de certains opérateurs linéaires.
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Lopez, Nestor. "Spécification formelle de systèmes complexes : méthodes et techniques." Paris, CNAM, 2002. http://www.theses.fr/2001CNAM0410.
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Nous proposons une methodologie de specification et de construction de systemes qui transforme la specification evenementielle d'un systeme en une specification pre-post, ce qui permet d'effectuer le processus de developpement a l'aide de techniques connues de derivation de programmes par raffinements et sans rompre le processus de raisonnement formel. Le role de la specification evenementielle est de decrire les interactions du systeme avec son environnement. Le resultat de la transformation est un module qui fournit les moyens de communication du systeme. Pour prendre en compte les aspects concurrents et distribues d'un systeme, nous proposons la notion de module partage. Nous utilisons des variables auxiliaires, non implantees, pour exprimer des proprietes abstraites et pour faire la preuve de correction des implantations effectuees. Pour repondre au besoin de reutilisation de modules existants, nous proposons une technique pour completer une specification a l'aide de variables auxiliaires afin de l'adapter a la solution de problemes particuliers, tout en preservant l'implantation fournie initialement.
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Trinh, Thi Huong. "Adapting recent statistical techniques to the study of nutrition in Vietnam." Thesis, Toulouse 1, 2018. http://www.theses.fr/2018TOU10010/document.
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L'objectif de cette thèse est d'adapter des méthodes récentes de statistique pour apporter une vision nouvelle de la transition nutritionnelle au Vietnam. Chapitre 1, nous faisons une brève introduction. Nous considérons que le Vietnam est une étude pilote sur le problème de la nutrition. Chapitre 2, nous revenons sur la question de l'estimation de la relation entre la prise de calories par personne et le revenu en utilisant six vagues de l'enquête VHLSS Survey sur la période 2004-2014. Nous adoptons plutôt la famille des modèles généralisés additifs (GAM) dans lesquels seul le revenu intervient de façon non linéaire. Nous comparons ces modèles avec une procédure récente. Les résultats mettent en relief une réponse forte de la prise de calories à un accroissement du revenu pour les foyers les plus pauvres. Chapitre 3, nous utilisons des méthodes de décomposition pour évaluer les déterminants des changements de consommation de macronutriments au Vietnam en utilisant les vagues 2004 et 2014. La méthode de décomposition récente proposée par Rothe (2015) a pour but de poursuivre la décomposition plus loin en décomposant l'effet de composition en trois composantes: la contribution directe de chaque covariable, plusieurs effets d'interaction d'ordre deux ou supérieur et un effet de la dépendance. Rothe utilise des copules pour modéliser les effets de dépendance. Chapitre 4, nous nous concentrons sur la composition de la diète en modélisant les proportions de protéines, de matières grasses et de glucides dans la prise moyenne de calories par personne. Nous utilisons des outils descriptifs pour montrer l'évolution des trois composantes au travers du temps et modélisons ensuite la consommation de macronutriments en fonction des caractéristiques des ménages avec des modèles de régression pour données de composition. Nous établissons la formule permettant le calcul des semi-elasticités de la consommation de macronutriments par rapport à la dépense totale de nourriture. Chapitre 5, nous nous penchons sur la relation entre les parts de macronutriments et l'indice de masse corporelle (IMC). Nous construisons un modèle de régression compositionnelle incluant un total pour expliquer les quantiles de l'indice de masse corporelle. Nous calculons ensuite les élasticités de l'IMC par rapport à chaque macronutriment. Notre travail est basé sur l'utilisation de la base de données de l'enquête GNS 2009-2010. Les résultats révèlent d'abord des effets significatifs de facteurs socio-économiquesThe objective of this thesis is to adapt recent statistical techniques and to bring new insights on the nutritional transition in Vietnam. Vietnam is a lower middle income country but it now faces the double burden of malnutrition characterized by the coexistence of undernutrition along with overweight and obesity, or diet-related noncommunicable diseases. Chapter 1 gives a brief introduction to this thesis. We consider Vietnam is a pilot case study about nutrition. Chapter 2, we revisit the issue of estimating the relationship between per capita calorie intake and income using six waves of the Vietnam Household Living Standard Survey over the period 2004-2014. Characterizing the response of calorie intake to income for the poorest households is a prerequisite for considering policies aimed at reducing starvation and correcting nutritional deficiencies. The classical log-log specification does not capture the nonlinearity of this relationship. We adopt rather various generalized additive models (GAM) specifications where only income is supposed to act in a nonlinear fashion and compare them with a recent procedure. The results highlight the strong response of calorie intake to an increase in income for the poorest households. A byproduct of the proposed methodology is the decomposition of the evolution of average calorie intake between the two waves into the part due to the change of population characteristics distributions and those coming from the change in calorie-income relationship, shedding new light on the nutritional transition in Vietnam. Chapter 3, we use decomposition methods to assess the determinants of changes in macronutrients consumption in Vietnam using the 2004 and 2014 waves. The common objective of decomposition methods is to decompose between-group differences in economic outcomes such as wage or income, into two components: a composition effect due to differences in observable covariates across groups, and a structure effect due to differences in the relationship that links the covariates to the considered outcome. The recent decomposition procedure proposed by Rothe (2015) aims at decomposing further the composition effect into three types of components: the direct contribution of each covariate, several two way and higher order interaction effects and a dependence. Rothe (2015) uses a parametric copula to model the dependence effects and we adapt this approach to the case of a mixture of continuous and discrete covariates. Chapter 4, we focus on food composition in terms of diet components. We consider modeling the proportions of protein, fat and carbohydrate in the average per capita calorie intake. We use descriptive tools, such as compositional biplots and ternary diagrams, to show the evolution of the three components over the years and then model macronutrients composition as a function of household characteristics, using compositional regression models. We derive the expression of the semi-elasticities of macronutrients shares with respect to food expenditure. We then compare the interpretations of these shares semi-elasticities to that of volumes of macronutrients and of total calorie intake obtained using classical linear models. Chapter 5, we focus on the relationship between macronutrient balances and body mass index. We develop a compositional regression model including a total at various quantile orders. We then compute the elasticities of BMI with respect to each macronutrient and to the total consumption. Our empirical research is based on the General Nutrition Survey 2009-2010. The results first reveal significant impacts of some socio--economics factors. All elasticities of BMI with respect to each macronutrient increase as BMI increases until a threshold (BMI=20) and then remain stable. Chapter 6, we briefly give our perspectives of future research in both mathematics and nutrition
Books on the topic "Techniques in vitro – méthodes"
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Anzieu, Didier. Les méthodes projectives. 8th ed. Paris: Presses universitaires de France, 1987.
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Boulton, Alan A., Glen B. Baker, and Alan N. Bateson. In Vitro Neurochemical Techniques. New Jersey: Humana Press, 1998. http://dx.doi.org/10.1385/0896035093.
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Jean-Paul, Crampon, and Labrune Gérard, eds. Histoire-géographie: Méthodes et techniques. Paris: Nathan technique, 1992.
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Daigneault, Armand. Méthodes et techniques du savoir-écrire. 3rd ed. Montréal, Qué: Guérin, 1989.
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Patel, Minesh M. In vitro techniques for evaluating mucoadhesion. Portsmouth: University of Portsmouth, School of Pharmacy and Biomedical Sciences, 2000.
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Rouessac, F. Analyse chimique: Méthodes et techniques instrumentales modernes. 2nd ed. Paris: Masson, 1994.
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Grais, Bernard. Techniques statistiques. 3rd ed. Paris: Dunod, 2003.
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Cocaud, Martine. Traiter des données historiques: Méthodes statistiques/techniques informatiques. Rennes: Presses universitaires de Rennes, 2001.
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Cellier, Jacques. Traiter des données historiques: Méthodes statistiques, techniques informatiques. Rennes: Presses universitaires de Rennes, 2001.
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New York (State). Legislature. Senate. Committee on Investigations, Taxation, and Government Operations. Dilemmas posed by human in vitro reproduction techniques. Albany, N.Y: The Senate, State of New York, 1998.
Find full textBook chapters on the topic "Techniques in vitro – méthodes"
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Weijers, Olga. "Techniques et méthodes d'enseignement." In L'enseignement des disciplines à la Faculté des arts (Paris et Oxford, XIIIe-XVe siècles), 337–44. Turnhout: Brepols Publishers, 1997. http://dx.doi.org/10.1484/m.sa-eb.4.00392.
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Harris, J. Robin, Geneviève Almouzni, Doris Kirschner, Daniela Dimitrova, Jeffrey A. Nickerson, Jean Underwood, Stefan Wagner, et al. "In Vitro Techniques." In Cell Biology Protocols, 201–378. Chichester, UK: John Wiley & Sons, Ltd, 2006. http://dx.doi.org/10.1002/0470033487.ch6.
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Slater, Robert J. "In Vitro Transcription." In Techniques in Molecular Biology, 203–27. Boston, MA: Springer US, 1987. http://dx.doi.org/10.1007/978-1-4615-9799-5_12.
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Ballanyi, Klaus. "In Vitro Preparations." In Modern Techniques in Neuroscience Research, 307–26. Berlin, Heidelberg: Springer Berlin Heidelberg, 1999. http://dx.doi.org/10.1007/978-3-642-58552-4_9.
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Bhattacharyya, Jutimala, Sankalp Singh, Madhab C. Das, and Kanna Jayaprakasan. "Embryo Transfer: Techniques and Troubleshooting." In In Vitro Fertilization, 735–49. Cham: Springer International Publishing, 2019. http://dx.doi.org/10.1007/978-3-319-43011-9_61.
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Knox, P. "Cellular and In Vitro Techniques." In The Future of Predictive Safety Evaluation, 171–80. Dordrecht: Springer Netherlands, 1986. http://dx.doi.org/10.1007/978-94-009-4139-7_13.
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Torres, Kenneth C. "In Vitro Propagation of Lilies." In Tissue Culture Techniques for Horticultural Crops, 85–91. Boston, MA: Springer US, 1989. http://dx.doi.org/10.1007/978-1-4615-9756-8_7.
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Valencia, C. Alexander, and Bradford Coffee. "In Vitro Amplification Methods in Molecular Diagnostics." In Modern Clinical Molecular Techniques, 49–66. New York, NY: Springer New York, 2012. http://dx.doi.org/10.1007/978-1-4614-2170-2_4.
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Zucco, Flavia, Isabella De Angelis, and Annalaura Stammati. "Cellular Models for In Vitro Toxicity Testing." In Animal Cell Culture Techniques, 395–422. Berlin, Heidelberg: Springer Berlin Heidelberg, 1998. http://dx.doi.org/10.1007/978-3-642-80412-0_21.
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O’Connor, Robert, Mary Heenan, Conor Duffy, and Martin Clynes. "Miniaturized In Vitro Methods in Toxicity Testing." In Animal Cell Culture Techniques, 423–33. Berlin, Heidelberg: Springer Berlin Heidelberg, 1998. http://dx.doi.org/10.1007/978-3-642-80412-0_22.
Full textConference papers on the topic "Techniques in vitro – méthodes"
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Blatter, C., and C. Raynal. "Méthodes d’analyse textuelle pour l’interprétation des REX humains, organisationnels et techniques." In Congrès Lambda Mu 19 de Maîtrise des Risques et Sûreté de Fonctionnement, Dijon, 21-23 Octobre 2014. IMdR, 2015. http://dx.doi.org/10.4267/2042/56070.
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Grasso, Rosaria, Rosalia Pellitteri, Francesco Musumeci, Rosaria V. Rapicavoli, Giovanni Sposito, Antonio Triglia, Agata Scordino, and Agata Campisi. "Delayed luminescence for in vitro study of mitochondrial dysfunctions in neurodegenerative diseases." In Novel Biophotonics Techniques and Applications, edited by Arjen Amelink and Seemantini K. Nadkarni. SPIE, 2019. http://dx.doi.org/10.1117/12.2526920.
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Bang, Hyun Jin, Seung Rag Lee, Byungyeon Kim, Kiri Lee, Minsuk Lee, Sung Jun Hong, Yong Shin, and Byung Jun Park. "A fiber based in vitro optical signal diagnosis technique for interspecies transmissibility." In Novel Biophotonics Techniques and Applications, edited by Arjen Amelink and Seemantini K. Nadkarni. SPIE, 2019. http://dx.doi.org/10.1117/12.2526937.
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Vasam, Mallikarjun, S. N. Sriharsha, and ShankarNayak Bhukya. "Composition, formulation and in-vitro evaluation studies of cefixime microspheres." In 2016 International Conference on Electrical, Electronics, and Optimization Techniques (ICEEOT). IEEE, 2016. http://dx.doi.org/10.1109/iceeot.2016.7755333.
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Batukaev, Abdulmalik. "IN VITRO BIOTECHNOLOGICAL TECHNIQUES FOR HEALTH IMPROVEMENT AND REPRODUCTION OF GRAPE." In 19th SGEM International Multidisciplinary Scientific GeoConference EXPO Proceedings. STEF92 Technology, 2019. http://dx.doi.org/10.5593/sgem2019/6.1/s25.094.
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Guo, Yiping, Shi Sheng, Wei Zhang, Michael C. Lun, Shih-Ming Tsai, Wei-Chun Chin, Roy Hoglund, and Changqing Li. "High energy photons excited photodynamic cancer therapy in vitro." In Optical Methods for Tumor Treatment and Detection: Mechanisms and Techniques in Photodynamic Therapy XXVII, edited by David H. Kessel and Tayyaba Hasan. SPIE, 2018. http://dx.doi.org/10.1117/12.2291252.
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"In-vitro Investigation of Air Plasma-Sprayed Hydroxyapatite Coatings by Diffraction Techniques." In Residual Stresses 10. Materials Research Forum LLC, 2016. http://dx.doi.org/10.21741/9781945291173-82.
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Zhu, Ruixue, Tatiana Avsievich, Alexey Popov, and Igor Meglinski. "Influence of interaction time on the red blood cell (dis)aggregation dynamics in vitro studied by optical tweezers." In Novel Biophotonics Techniques and Applications, edited by Arjen Amelink and Seemantini K. Nadkarni. SPIE, 2019. http://dx.doi.org/10.1117/12.2526778.
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Genina, Elina A., Alexey N. Bashkatov, Nina A. Lakodina, Igor D. Kosobutsky, Nina V. Bogomolova, Gregory B. Altshuler, and Valery V. Tuchin. "In-vitro study of penetration of magnetic particles into the human skin." In Optics and Optoelectronic Inspection and Control: Techniques, Applications, and Instruments, edited by Hong Liu and Qingming Luo. SPIE, 2000. http://dx.doi.org/10.1117/12.403936.
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Yasuno, Yoshiaki, Ibrahim Abd El-Sadek, Arata Miyazawa, Larina Tzu-Wei Shen, Thitiya Seesan, Lida Zhu, Daisuke Oida, et al. "Multi-functional optical coherence microscopy for in-vitro and ex-vivo tissue investigation." In Optical Coherence Imaging Techniques and Imaging in Scattering Media, edited by Maciej Wojtkowski, Yoshiaki Yasuno, and Benjamin J. Vakoc. SPIE, 2021. http://dx.doi.org/10.1117/12.2616034.
Full textReports on the topic "Techniques in vitro – méthodes"
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Ulukapi, Kamile, and Ayse Gul Nasircilar. Mutation Breeding Protocol for Development of Drought-tolerant Genotypes in Phaseolus vulgaris L. Using in-vitro Embryo Culture Techniques. "Prof. Marin Drinov" Publishing House of Bulgarian Academy of Sciences, November 2020. http://dx.doi.org/10.7546/crabs.2020.11.10.
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Leach, Roland M., Mark Pines, Carol V. Gay, and Shmuel Hurwitz. In vivo and in vitro Chondrocyte Metabolism in Relationship to the Developemnt of Tibial Dyschondroplasia in Broiler Chickens. United States Department of Agriculture, July 1993. http://dx.doi.org/10.32747/1993.7568090.bard.
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Skeletal deformities are a significant financial and welfare problem for the world poultry industry. Tibial dyschondroplasia (TD) is the most prevalent skeletal abnormality found in young broilers, turkeys and ducks. Tibial dyschondroplasia results from a perturbation of the sequence of events in the epiphyseal growth plate, the tissue responsible for longitudinal bone growth. The purpose of this investigation was to test the hypothesis that TD was the result of a failure of growth plate chondrocytes to differentiate and express the chemotactic molecules required for cartilage vascularization. In this investigation in situ hybridization and immunocytochemical techniques were used to study chondrocyte gene products associated with cartilage maturation and vascularization such as osteopontin, osteonectin, type X collagen, and alkaline phosphatase. All markers were present in the growth plate tissue anter or to the TD lesion but were greatly diminished in the TD lesion. Thus, rather than not acquiring the markers for hypertrophy, it appears that the growth plate chondrocytes reach a certain stage of hypertrophy and then de-differentiate into cells which resemble chondrocytes in the prehypertrophic zone. Similar patterns were observed in all TD tissues examined whether the lesions were spontaneous or induced by dietary treatments or genetic selection. The decrease in gene expression can at least be partially explained by the fact that many of the dysplastic chondrocytes show classic signs of apoptosis. These results provide an explanation for the observation that a variety of genes show reduced expression in the TD lesion when examined by in situ hybridization. This would suggest that future research should focus on the earliest detectable stages in the development of TD and examine endocrine and autocrine factors which cause chondrocytes to de-differentiate and undergo premature apoptosis.
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Ehrlich, Marcelo, John S. Parker, and Terence S. Dermody. Development of a Plasmid-Based Reverse Genetics System for the Bluetongue and Epizootic Hemorrhagic Disease Viruses to Allow a Comparative Characterization of the Function of the NS3 Viroporin in Viral Egress. United States Department of Agriculture, September 2013. http://dx.doi.org/10.32747/2013.7699840.bard.
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Project Title: "Development of a plasmid-based reverse genetics system for the Bluetongue and Epizootic Hemorrhagic Disease viruses to allow comparative characterization of the function of the NS3 viroporin in viral egress". Project details: No - IS-4192-09; Participants – Ehrlich M. (Tel Aviv University), Parker J.S. (Cornell University), DermodyT.S. (Vanderbilt University); Period - 2009-2013. Orbiviruses are insect-borne infectious agents of ruminants that cause diseases with considerable economical impact in Israel and the United States. The recent outbreaks of BTV in Europe and of Epizootic Hemorrhagic Disease Virus (EHDV) in Israel, underscore the need for: (i) a better comprehension of the infection process of orbiviruses, (ii) the identification of unique vs. common traits among different orbiviruses, (iii) the development of novel diagnosis and treatment techniques and approaches; all aimed at the achievement of more effective control and treatment measures. It is the context of these broad goals that the present project was carried out. To fulfill our long-term goal of identifying specific viral determinants of virulence, growth, and transmission of the orbiviruses, we proposed to: (i) develop reverse genetics systems for BTV and EHDV2-Ibaraki; and (ii) identify the molecular determinants of the NS3 nonstructural protein related to viroporin/viral egress activities. The first objective was pursued with a two-pronged approach: (i) development of a plasmid-based reverse genetics system for BTV-17, and (ii) development of an "in-vitro" transcription-based reverse genetics system for EHDV2-Ibaraki. Both approaches encountered technical problems that hampered their achievement. However, dissection of the possible causes of the failure to achieve viral spread of EHDV2-Ibaraki, following the transfection of in-vitro transcribed genomic segments of the virus, revealed a novel characteristic of EHDV2-Ibaraki infection: an uncharacteristically low fold increase in titer upon infection of different cell models. To address the function and regulation of NS3 we employed the following approaches: (i) development (together with Anima Cell Metrology) of a novel technique (based on the transfection of fluorescently-labeledtRNAs) that allows for the detection of the levels of synthesis of individual viral proteins (i.e. NS3) in single cells; (ii) development of a siRNA-mediated knockdown approach for the reduction in levels of expression of NS3 in EHDV2-Ibaraki infected cells; (iii) biochemical and microscopy-based analysis of the localization, levels and post-translational modifications of NS3 in infected cells. In addition, we identified the altered regulation and spatial compartmentalization of protein synthesis in cells infected with EHDV2-Ibaraki or the mammalian reovirus. In EHDV2-Ibaraki-infected cells such altered regulation in protein synthesis occurs in the context of a cell stress reponse that includes the induction of apoptosis, autophagy and activation of the stressrelated kinase c-Jun N-terminal Kinase (JNK). Interestingly, inhibition of such stress-related cellular processes diminishes the production of infectious virions, suggesting that EHDV usurps these responses for the benefit of efficient infection. Taken together, while the present project fell short of the generation of novel reverse genetics systems for orbiviruses, the development of novel experimental approaches and techniques, and their employment in the analysis of EHDV-infected cells, yielded novel insights in the interactions of orbiviruses with mammalian cells.
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Reisch, Bruce, Avichai Perl, Julie Kikkert, Ruth Ben-Arie, and Rachel Gollop. Use of Anti-Fungal Gene Synergisms for Improved Foliar and Fruit Disease Tolerance in Transgenic Grapes. United States Department of Agriculture, August 2002. http://dx.doi.org/10.32747/2002.7575292.bard.
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Original objectives . 1. Test anti-fungal gene products for activity against Uncinula necator, Aspergillus niger, Rhizopus stolonifer and Botrytis cinerea. 2. For Agrobacterium transformation, design appropriate vectors with gene combinations. 3. Use biolistic bombardment and Agrobacterium for transformation of important cultivars. 4. Characterize gene expression in transformants, as well as level of powdery mildew and Botrytis resistance in foliage of transformed plants. Background The production of new grape cultivars by conventional breeding is a complex and time-consuming process. Transferring individual traits via single genes into elite cultivars was proposed as a viable strategy, especially for vegetatively propagated crops such as grapevines. The availability of effective genetic transformation procedures, the existence of genes able to reduce pathogen stress, and improved in vitro culture methods for grapes, were combined to serve the objective of this proposal. Effective deployment of resistance genes would reduce production costs and increase crop quality, and several such genes and combinations were used in this project. Progress The efficacy of two-way combinations of Trichoderma endochitinase (CHIT42), synthetic peptide ESF12 and resveratrol upon the control of growth of Botrytis cinerea and Penicillium digitatum were evaluated in vitro. All pairwise interactions were additive but not synergistic. Per objective 2, suitable vectors with important gene combinations for Agrobacterium transformation were designed. In addition, multiple gene co-transformation by particle bombardment was also tested successfully. In New York, transformation work focused on cultivars Chardonnay and Merlot, while the technology in Israel was extended to 41B, R. 110, Prime, Italia, Gamay, Chardonnay and Velika. Transgenic plant production is summarized in the appendix. Among plants developed in Israel, endochitinase expression was assayed via the MuchT assay using material just 1-5 days after co-cultivation. Plants of cv. Sugraone carrying the gene coding for ESF12, a short anti-fungal lytic peptide under the control of the double 358 promoter, were produced. Leaf extracts of two plants showed inhibition zones that developed within 48 h indicating the inhibitory effect of the leaf extracts on the six species of bacteria. X fastidiosa, the causal organism of Pierce's disease, was very sensitive to leaf extracts from ESF12 transformed plants. Further work is needed to verify the agricultural utility of ESF12 transformants. In New York, some transformants were resistant to powdery mildew and Botrytis fruit rot. Major conclusions, solutions, achievements and implications The following scientific achievements resulted from this cooperative BARD project: 1. Development and improvement of embryogenesis and tissue culture manipulation in grape, while extending these procedures to several agriculturally important cultivars both in Israel and USA. 2. Development and improvement of novel transformation procedures while developing transformation techniques for grape and other recalcitrant species. 3. Production of transgenic grapevines, characterization of transformed vines while studying the expression patterns of a marker gene under the control of different promoter as the 35S CaMV in different part of the plants including flowers and fruits. 4. Expression of anti-fungal genes in grape: establishment of transgenic plants and evaluation of gene expression. Development of techniques to insert multiple genes. 5. Isolation of novel grape specific promoter to control the expression of future antimicrobial genes. It is of great importance to report that significant progress was made in not only the development of transgenic grapevines, but also in the evaluation of their potential for increased resistance to disease as compared with the non engineered cultivar. In several cases, increased disease resistance was observed. More research and development is still needed before a product can be commercialized, yet our project lays a framework for further investigations.
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Petitte, James, Hefzibah Eyal-Giladi, and Malka Ginsburg. The Study of Primordial Germ Cell Development as a Tool for Gene Transfer in Chickens. United States Department of Agriculture, October 1991. http://dx.doi.org/10.32747/1991.7561071.bard.
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The ability to introduce novel genetic material into the genome of commercial poultry has been impeded by a lack of kowledge regarding the origin in the early embryo of the target cell of interest, namely, the germ cell. Hence, this project investigated the emergence of primordial germ cells (PGCs) during the early development of the avian embryo to aid in efforts to produce transgenic poultry on a routine basis. The strategy was to introduce foreign DNA into the area of the unincubated embryo that is destined to give rise to the germ line. The objectives of this project were: 1) to identify and localize a subpopulation of cells in the early embryo which will give rise to PGCs, 2) to determine the best location and stage of development to transfer donor cells for efficient germline chimerism, and 3) to transfect donor cells to produce transgenic/germline chimeric embryos. We show that by using the monoclonal antibody SSEA-1 and by various cell culture techniques that germ cells appear to segregate from the somatic lineages at St. X., a process that is gradual and continues through St. XIV. Using microsurgical transplantation between quail and chick embryos, we demonstrated that the inner 1/3 of the area pellucida between states X-XII gives rise to about 2/3 of the germ cell population at the time of their residence in the germinal crescent. Because of the non-localized emergence of PGCs, attempts to introduce foreign DNA into clonal precursors of germ cells through liposome-mediated transfection yielded unacceptable levels of efficiency. However, through our investigation of germ cell origins, an in vitro model of germ cell differentiation was developed that could offer a means of determining the factors required for the long term culture of avian PGCs thereby providing a convenient means of manipulating the avian genome.
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Sessa, Guido, and Gregory Martin. role of FLS3 and BSK830 in pattern-triggered immunity in tomato. United States Department of Agriculture, January 2016. http://dx.doi.org/10.32747/2016.7604270.bard.
Full textAbstract:
Pattern-recognition receptors (PRRs) located on the plant cell surface initiate immune responses by perceiving conserved pathogen molecules known as pathogen-associated molecular patterns (PAMPs). PRRs typically function in multiprotein complexes that include transmembrane and cytoplasmickinases and contribute to the initiation and signaling of pattern-triggered immunity (PTI). An important challenge is to identify molecular components of PRR complexes and downstream signaling pathways, and to understand the molecular mechanisms that mediate their function. In research activities supported by BARD-4931, we studied the role of the FLAGELLIN SENSING 3 (FLS3) PRR in the response of tomato leaves to flagellin-derivedPAMPs and PTI. In addition, we investigated molecular properties of the tomato brassinosteroid signaling kinase 830 (BSK830) that physically interacts with FLS3 and is a candidate for acting in the FLS3 signaling pathway. Our investigation refers to the proposal original objectives that were to: 1) Investigate the role of FLS3 and its interacting proteins in PTI; 2) Investigate the role of BSK830 in PTI; 3) Examine molecular and phosphorylation dynamics of the FLS3-BSK830 interaction; 4) Examine the possible interaction of FLS3 and BSK830 with Pstand Xcveffectors. We used CRISPR/Cas9 techniques to develop plants carrying single or combined mutations in the FLS3 gene and in the paralogsFLS2.1 and FLS2.2 genes, which encode the receptor FLAGELLIN SENSING2 (FLS2), and analyzed their function in PTI. Domain swapping analysis of the FLS2 and FLS3 receptors revealed domains of the proteins responsible for PAMP detection and for the different ROS response initiated by flgII-28/FLS3 as compared to flg22/FLS2. In addition, in vitro kinase assays and point mutations analysis identified FLS2 and FLS3 domains required for kinase activity and ATP binding. In research activities on tomato BSK830, we found that it interacts with PRRs and with the co-receptor SERK3A and PAMP treatment affects part of these interactions. CRISPR/Cas9 bsk830 mutant plants displayed enhanced pathogen susceptibility and reduced ROS production upon PAMP treatment. In addition, BSK830 interacted with 8 Xanthomonastype III secreted effectors. Follow up analysis revealed that among these effectors XopAE is part of an operon, is translocated into plant cells, and displays E3 ubiquitinligase activity. Our investigation was also extended to other Arabidopsis and tomato BSK family members. Arabidopsis BSK5 localized to the plant cell periphery, interacted with receptor-like kinases, and it was phosphorylatedin vitro by the PEPR1 and EFRPRRs. bsk5 mutant plants displayed enhanced susceptibility to pathogens and were impaired in several, but not all, PAMP-induced responses. Conversely, BSK5 overexpression conferred enhanced disease resistance and caused stronger PTI responses. Genetic complementation suggested that proper localization, kinase activity, and phosphorylation by PRRs are critical for BSK5 function. BSK7 and BSK8 specifically interacted with the FLS2 PRR, their respective mutant plants were more susceptible to B. cinereaand displayed reduced flg22-induced responses. The tomato BSK Mai1 was found to interact with the M3KMAPKKK, which is involved in activation of cell death associated with effector-triggered immunity. Silencing of Mai1 in N. benthamianaplants compromised cell death induced by a specific class of immune receptors. In addition, co-expression of Mai1 and M3Kin leaves enhanced MAPKphosphorylation and cell death, suggesting that Mai1 acts as a molecular link between pathogen recognition and MAPK signaling. Finally, We identified the PP2C phosphatase Pic1 that acts as a negative regulator of PTI by interacting with and dephosphorylating the receptor-like cytoplasmickinase Pti1, which is a positive regulator of plant immunity. The results of this investigation shed new light on the molecular characteristics and interactions of components of the immune system of crop plants providing new knowledge and tools for development of novel strategies for disease control.
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Shpigel, Nahum, Raul Barletta, Ilan Rosenshine, and Marcelo Chaffer. Identification and characterization of Mycobacterium paratuberculosis virulence genes expressed in vivo by negative selection. United States Department of Agriculture, January 2004. http://dx.doi.org/10.32747/2004.7696510.bard.
Full textAbstract:
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of a severe inflammatory bowel disease (IBD) in ruminants, known as Johne’s disease or paratuberculosis. Johne’s disease is considered to be one of the most serious diseases affecting dairy cattle both in Israel and worldwide. Heavy economic losses are incurred by dairy farmers due to the severe effect of subclinical infection on milk production, fertility, lower disease resistance and early culling. Its influence in the United States alone is staggering, causing an estimated loss of $1.5 billion to the agriculture industry every year. Isolation of MAP from intestinal tissue and blood of Crohn's patients has lead to concern that it plays a potential pathogenic role in promoting human IDB including Crohn’s disease. There is great concern following the identification of the organism in animal products and shedding of the organism to the environment by subclinically infected animals. Little is known about the molecular basis for MAP virulence. The goal of the original proposed research was to identify MAP genes that are required for the critical stage of initial infection and colonization of ruminants’ intestine by MAP. We proposed to develop and use signature tag mutagenesis (STM) screen to find MAP genes that are specifically required for survival in ruminants upon experimental infection. This research projected was approved as one-year feasibility study to prove the ability of the research team to establish the animal model for mutant screening and alternative in-vitro cell systems. In Israel, neonatal goat kids were repeatedly inoculated with either one of the following organisms; MAP K-10 strain and three transposon mutants of K-10 which were produced and screened by the US PI. Six months after the commencement of inoculation we have necropsied the goats and taken multiple tissue samples from the jejunum, ileum and mesenteric lymph nodes. Both PCR and histopathology analysis indicated on efficient MAP colonization of all the inoculated animals. We have established several systems in the Israeli PI’s laboratory; these include using IS900 PCR for the identification of MAP and using HSP65-based PCR for the differentiation between MAV and MAP. We used Southern blot analysis for the differentiation among transposon mutants of K-10. In addition the Israeli PI has set up a panel of in-vitro screening systems for MAP mutants. These include assays to test adhesion, phagocytosis and survival of MAP to/within macrophages, assays that determine the rate of MAPinduced apoptosis of macrophages and MAP-induced NO production by macrophages, and assays testing the interference with T cell ã Interferon production and T cell proliferation by MAP infected macrophages (macrophage studies were done in BoMac and RAW cell lines, mouse peritoneal macrophages and bovine peripheral blood monocytes derived macrophages, respectively). All partners involved in this project feel that we are currently on track with this novel, highly challenging and ambitious research project. We have managed to establish the above described research systems that will clearly enable us to achieve the original proposed scientific objectives. We have proven ourselves as excellent collaborative groups with very high levels of complementary expertise. The Israeli groups were very fortunate to work with the US group and in a very short time period to master numerous techniques in the field of Mycobacterium research. The Israeli group has proven its ability to run this complicated animal model. This research, if continued, may elucidate new and basic aspects related to the pathogenesis MAP. In addition the work may identify new targets for vaccine and drug development. Considering the possibility that MAP might be a cause of human Crohn’s disease, better understanding of virulence mechanisms of this organism might also be of public health interest as well.
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Meidan, Rina, and Robert Milvae. Regulation of Bovine Corpus Luteum Function. United States Department of Agriculture, March 1995. http://dx.doi.org/10.32747/1995.7604935.bard.
Full textAbstract:
The main goal of this research plan was to elucidate regulatory mechanisms controlling the development, function of the bovine corpus luteum (CL). The CL contains two different sterodigenic cell types and therefore it was necessary to obtain pure cell population. A system was developed in which granulosa and theca interna cells, isolated from a preovulatory follicle, acquired characteristics typical of large (LL) and small (SL) luteal cells, respectively, as judged by several biochemical and morphological criteria. Experiments were conducted to determine the effects of granulosa cells removal on subsequent CL function, the results obtained support the concept that granulosa cells make a substaintial contribution to the output of progesterone by the cyclic CL but may have a limited role in determining the functional lifespan of the CL. This experimental model was also used to better understand the contribution of follicular granulosa cells to subsequent luteal SCC mRNA expression. The mitochondrial cytochrome side-chain cleavage enzyme (SCC), which converts cholesterol to pregnenolone, is the first and rate-limiting enzyme of the steroidogenic pathway. Experiments were conducted to characterize the gene expression of P450scc in bovine CL. Levels of P450scc mRNA were higher during mid-luteal phase than in either the early or late luteal phases. PGF 2a injection decreased luteal P450scc mRNA in a time-dependent manner; levels were significantly reduced by 2h after treatment. CLs obtained from heifers on day 8 of the estrous cycle which had granulosa cells removed had a 45% reduction in the levels of mRNA for SCC enzymes as well as a 78% reduction in the numbers of LL cells. To characterize SCC expression in each steroidogenic cell type we utilized pure cell populations. Upon luteinization, LL expressed 2-3 fold higher amounts of both SCC enzymes mRNAs than SL. Moreover, eight days after stimulant removal, LL retained their P4 production capacity, expressed P450scc mRNA and contained this protein. In our attempts to establish the in vitro luteinization model, we had to select the prevulatory and pre-gonadotropin surge follicles. The ratio of estradiol:P4 which is often used was unreliable since P4 levels are high in atretic follicles and also in preovulatory post-gonadotropin follicles. We have therefore examined whether oxytocin (OT) levels in follicular fluids could enhance our ability to correctly and easily define follicular status. Based on E2 and OT concentrations in follicular fluids we could more accurately identify follicles that are preovulatory and post gonadotropin surge. Next we studied OT biosynthesis in granulosa cells, cells which were incubated with forskolin contained stores of the precursor indicating that forskolin (which mimics gonadotropin action) is an effective stimulator of OT biosynthesis and release. While studying in vitro luteinization, we noticed that IGF-I induced effects were not identical to those induced by insulin despite the fact that megadoses of insulin were used. This was the first indication that the cells may secrete IGF binding protein(s) which regonize IGFs and not insulin. In a detailed study involving several techniques, we characterized the species of IGF binding proteins secreted by luteal cells. The effects of exogenous polyunsaturated fatty acids and arachidonic acid on the production of P4 and prostanoids by dispersed bovine luteal cells was examined. The addition of eicosapentaenoic acid and arachidonic acid resulted in a dose-dependent reduction in basal and LH-stimulated biosynthesis of P4 and PGI2 and an increase in production of PGF 2a and 5-HETE production. Indomethacin, an inhibitor of arachidonic acid metabolism via the production of 5-HETE was unaffected. Results of these experiments suggest that the inhibitory effect of arachidonic acid on the biosynthesis of luteal P4 is due to either a direct action of arachidonic acid, or its conversion to 5-HETE via the lipoxgenase pathway of metabolism. The detailed and important information gained by the two labs elucidated the mode of action of factors crucially important to the function of the bovine CL. The data indicate that follicular granulosa cells make a major contribution to numbers of large luteal cells, OT and basal P4 production, as well as the content of cytochrome P450 scc. Granulosa-derived large luteal cells have distinct features: when luteinized, the cell no longer possesses LH receptors, its cAMP response is diminished yet P4 synthesis is sustained. This may imply that maintenance of P4 (even in the absence of a Luteotropic signal) during critical periods such as pregnancy recognition, is dependent on the proper luteinization and function of the large luteal cell.
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Droby, Samir, Michael Wisniewski, Martin Goldway, Wojciech Janisiewicz, and Charles Wilson. Enhancement of Postharvest Biocontrol Activity of the Yeast Candida oleophila by Overexpression of Lytic Enzymes. United States Department of Agriculture, November 2003. http://dx.doi.org/10.32747/2003.7586481.bard.
Full textAbstract:
Enhancing the activity of biocontrol agents could be the most important factor in their success in controlling fruit disease and their ultimate acceptance in commercial disease management. Direct manipulation of a biocontrol agent resulting in enhancement of diseases control could be achieved by using recent advances in molecular biology techniques. The objectives of this project were to isolate genes from yeast species that were used as postharvest biocontrol agents against postharvest diseases and to determine their role in biocontrol efficacy. The emphasis was to be placed on the yeast, Candida oleophila, which was jointly discovered and developed in our laboratories, and commercialized as the product, Aspire. The general plan was to develop a transformation system for C . oleophila and either knockout or overexpress particular genes of interest. Additionally, biochemical characterization of the lytic peptides was conducted in the wild-type and transgenic isolates. In addition to developing a better understanding of the mode of action of the yeast biocontrol agents, it was also our intent to demonstrate the feasibility of enhancing biocontrol activity via genetic enhancement of yeast with genes known to code for proteins with antimicrobial activity. Major achievements are: 1) Characterization of extracellular lytic enzymes produced by the yeast biocontrol agent Candida oleophila; 2) Development of a transformation system for Candida oleophila; 3) Cloning and analysis of C.oleophila glucanase gene; 4) Overexpression of and knockout of C. oleophila glucanase gene and evaluating its role in the biocontrol activity of C. oleophila; 5) Characterization of defensin gene and its expression in the yeast Pichiapastoris; 6) Cloning and Analysis of Chitinase and Adhesin Genes; 7) Characterization of the rnase secreted by C . oleophila and its inhibitory activity against P. digitatum. This project has resulted in information that enhanced our understanding of the mode of action of the yeast C . oleophila. This was important step towards enhancing the biocontrol activity of the yeast. Fungal cell wall enzymes produced by the yeast antagonist were characterized. Different substrates were identified to enhance there production in vitro. Exo-b-1, 3 glucanase, chitinase and protease production was stimulated by the presence of cell-wall fragments of Penicillium digitatum in the growing medium, in addition to glucose. A transformation system developed was used to study the role of lytic enzymes in the biocontrol activity of the yeast antagonist and was essential for genetic manipulation of C . oleqphila. After cloning and characterization of the exo-glucanase gene from the yeast, the transformation system was efficiently used to study the role of the enzyme in the biocontrol activity by over-expressing or knocking out the activity of the enzyme. At the last phase of the research (still ongoing) the transformation system is being used to study the role of chitinase gene in the mode of action. Knockout and over expression experiments are underway.
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Schuster, Gadi, and David Stern. Integration of phosphorus and chloroplast mRNA metabolism through regulated ribonucleases. United States Department of Agriculture, August 2008. http://dx.doi.org/10.32747/2008.7695859.bard.
Full textAbstract:
New potential for engineering chloroplasts to express novel traits has stimulated research into relevant techniques and genetic processes, including plastid transformation and gene regulation. This proposal continued our long time BARD-funded collaboration research into mechanisms that influence chloroplast RNA accumulation, and thus gene expression. Previous work on cpRNA catabolism has elucidated a pathway initiated by endonucleolytic cleavage, followed by polyadenylation and exonucleolytic degradation. A major player in this process is the nucleus-encoded exoribonuclease/polymerasepolynucleotidephoshorylase (PNPase). Biochemical characterization of PNPase has revealed a modular structure that controls its RNA synthesis and degradation activities, which in turn are responsive to the phosphate (P) concentration. However, the in vivo roles and regulation of these opposing activities are poorly understood. The objectives of this project were to define how PNPase is controlled by P and nucleotides, using in vitro assays; To make use of both null and site-directed mutations in the PNPgene to study why PNPase appears to be required for photosynthesis; and to analyze plants defective in P sensing for effects on chloroplast gene expression, to address one aspect of how adaptation is integrated throughout the organism. Our new data show that P deprivation reduces cpRNA decay rates in vivo in a PNPasedependent manner, suggesting that PNPase is part of an organismal P limitation response chain that includes the chloroplast. As an essential component of macromolecules, P availability often limits plant growth, and particularly impacts photosynthesis. Although plants have evolved sophisticated scavenging mechanisms these have yet to be exploited, hence P is the most important fertilizer input for crop plants. cpRNA metabolism was found to be regulated by P concentrations through a global sensing pathway in which PNPase is a central player. In addition several additional discoveries were revealed during the course of this research program. The human mitochondria PNPase was explored and a possible role in maintaining mitochondria homeostasis was outlined. As polyadenylation was found to be a common mechanism that is present in almost all organisms, the few examples of organisms that metabolize RNA with no polyadenylation were analyzed and described. Our experiment shaded new insights into how nutrient stress signals affect yield by influencing photosynthesis and other chloroplast processes, suggesting strategies for improving agriculturally-important plants or plants with novel introduced traits. Our studies illuminated the poorly understood linkage of chloroplast gene expression to environmental influences other than light quality and quantity. Finely, our finding significantly advanced the knowledge about polyadenylation of RNA, the evolution of this process and its function in different organisms including bacteria, archaea, chloroplasts, mitochondria and the eukaryotic cell. These new insights into chloroplast gene regulation will ultimately support plant improvement for agriculture