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Ajili, Widad. "Étude des processus de biominéralisation de la nacre chez l'ormeau européen haliotis tuberculata." Thesis, Sorbonne université, 2018. http://www.theses.fr/2018SORUS160.
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La nacre présente dans la coquille de certains mollusques est un matériau fascinant aux propriétés physiques (mécaniques et optiques) remarquable. Ce matériau biologique de nature composite organique et minérale présente un architecture complexe dont la structure et les mécanismes de formation ne sont pas encore parfaitement compris. Dans ces travaux, en prenant comme modèle d’étude comme modèle l’ormeau Européen Haliotis tuberculata, nous avons cherché à contribuer au savoir autour de ce matériau en axant notre étude sur trois aspects en particulier : i) l’étude ultrastructurale de la phase minérale et plus particulièrement des environnements minéraux désordonnés, ii) l’étude de l’interface organominérale dans la nacre adulte en formation ainsi que dans la coquille larvaire et iii) l’étude des premiers biominéraux déposés dans la coquille larvaire aux très jeune stades de développement. Pour réaliser cette étude nous avons eu recours à un couplage de techniques avancées spectroscopiques (Résonnance Magnétique Nucléaire à l’Etat Solide, Microscopie en transmission à balayage de rayons X et Spectroscopie des pertes d'énergie) couplé à des techniques de pointes de microscopie électronique (Microscopie électronique à transmission à haute résolution et Microscope électronique à balayage à effet de champ) et de préparation d’échantillon (Abrasion ionique focalisée) et de biologie moléculaire (Immunohistochimie, Microscopie optique et électronique corrélative)The mother-of-pearl found in the shell of certain mollusks is a fascinating material with remarkable physical (mechanical and optical) properties. This biological material of organic and mineral composite nature presents a complex architecture whose structure and mechanisms of formation are not yet perfectly understood. In this work, taking as a model study the European abalone Haliotis tuberculata, we sought to contribute to the knowledge around this material by focusing our study on three aspects in particular: i) the ultrastructural study of the phase mineral and more particularly disordered mineral environments, ii) the study of the organo-minerale interface in the adult nacre in formation as well as in the larval shell and iii) the study of the first biominerals deposited in the larval shell at very young stages of development. To carry out this study, we used a coupling of advanced spectroscopic techniques (Solid-State Nuclear Magnetic Resonance, Scanning transmission X-ray microscopy and Electron energy loss spectroscopy) coupled to advanced electron microscopy techniques (High-Resolution Transmission Electronic Microscopies and Field Emission Gun Scanning Electronic Microscopy) and preparation of sample (Focused Ion beam) and molecular biology (immunohistochemistry, Correlative Light and Electronic Microscopy)
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Djerbi, Soraya. "Cellulose synthases in Populus- identification, expression analyses and in vitro synthesis." Doctoral thesis, Stockholm, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-414.
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Gagnepain, Anne. "Fécondation in vitro : synthèse clinique, biologique, épidémiologique, législative et éthique ; méta-analyse des essais thérapeutiques." Montpellier 1, 1989. http://www.theses.fr/1989MON11258.
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Johnston, Julie Catherine. "In vitro translation of cucumber necrosis virus RNA." Thesis, University of British Columbia, 1989. http://hdl.handle.net/2429/28969.
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The in vitro translation products directed by cucumber necrosis virus (CNV) RNA were analyzed in both rabbit reticulocyte lysate and wheat germ extract cell-free translation systems. In rabbit reticulocyte lysates, one major protein of ca. 33 Mr was produced. In wheat germ extracts, four proteins of ca. 41, 33, 21 and 20 Mr were produced. Hybrid-arrested translation (HART) studies using synthetic CNV antisense RNA corresponding to the entire CNV genome demonstrated that the four major proteins synthesized from CNV virion RNA in wheat germ extracts are virus-specific translation products. The genomic locations of the CNV in vitro translation products were determined using a number of experimental approaches including: (1) HART using antisense RNA corresponding to selected regions of the CNV genome; (2) in vitro translation of synthetic messenger-sense CNV transcripts; (3) immunoprecipitation of in vitro translation products with CNV polyclonal antisera and (4) in vitro translation of size-fractionated CNV virion RNA. Together, these experiments demonstrated that the ca. 33 Mr protein is derived from the 5' proximal coding region, the ca. 41 Mr protein is derived from an internal coding region, and that at least one but probably both of the ca. 20 and 21 Mr proteins are derived from the 3' terminal coding region(s) of the CNV genome. In addition, immunoprecipitation experiments provided further evidence that the ca. 41 Mr protein is the viral coat protein. The size, number, and genomic locations of the CNV in vitro translation products reported here are in agreement with those predicted from nucleotide sequence data (Rochon & Tremaine, 1989). The natural template for the expression of downstream cistrons in the CNV genome was investigated by in vitro translation of sucrose fractionated CNV virion RNA as well as in vitro translation of messenger-sense synthetic transcripts. These studies indicate that in vitro, both subgenomic and genomic-length CNV RNA molecules may act as templates for the synthesis of the ca. 41,21 and 20 Mr proteins as well as the ca. 33 Mr protein.Land and Food Systems, Faculty of
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El, Badaoui Houriya. "Comparaison entre différentes techniques de culture in vitro de Solanum paludosum Moric. Pour la production de solamargine, glycoalcaloi͏̈de principal." Toulouse, INPT, 1993. http://www.theses.fr/1993INPT015A.
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Ce travail porte sur un arbuste tropical: solanum paludosum moric pour la production en glycoalcaloides qui entrent dans l'hemisynthese des hormones steroidiques. Les fruits issus de plantes obtenues par culture in vitro produisent autant de solamargine que les fruits des plantes meres d'origine. Les teneurs sont de 2%. La production des glycoalcaloides par la culture de cals et des cellules desorganisees est faible. Les teneurs sont environ de 0,015%. En revanche la culture de parties aeriennes multiples a permis d'obtenir 0,05% de solamargine alors que les parties aeriennes des plantes entieres ne produisent pas ces glycoalcaloides. La mise au point de la composition des macroelements du milieu de culture in vitro adapte aux besoins de la plante apres analyse minerale a permis d'augmenter 3 fois la production de la biomasse des parties aeriennes multiples et 2 fois la production de la solamargine par ces cultures. La culture des racines in vitro a ete tentee. La biomasse necessaire au dosage de solamargine s'est averee insuffisante ceci confirme les resultats obtenus par d'autres auteurs sur des especes ligneuses comme l'est solanum paludosum
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Marshall, Jonathon J. A. "Regulation of insulin gene expression in rat islets of Langerhans." Thesis, University of Leicester, 1986. http://hdl.handle.net/2381/35103.
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The rate of glucose stimulated in vitro (pro)insulin synthesis in islets of Langerhans isolated from fed male rats was determined. Increases in the glucose concentration of the incubation medium, over the physiological range (2 to 20 mM), stimulated the rates of both (pro)insulin and total protein synthesis during a two hour incubation. This stimulation was preferential for (pro)insulin synthesis and had a sigmoidal dose response curve with a concentration threshold of between 2.5 mM and 5.0 mM and a maximal rate at 20 mM glucose. Inhibition of islet RNA synthesis by.;ctinomycin D depressedthe rate of total protein synthesis within 30 minutes of the application of a 20 mM glucose stimulus. A specific inhibition of (pro)insulin synthesis by actinomycin D, that occured 60 minutes after the application of a 20 mM glucose stimulus, was thought to reflect the inhibition of glucose stimulated preproinsulin mRNA synthesis. The effect of glucose on islet preproinsulin mRNA content during in vitro incubations was also determined. To achieve this a dot blot hybridization assay for preproinsulin mRNA was 3 2 developed which used a P-labeled cloned human insulin gene as the hybridization probe. The assay proved to be sensitive enough to detect preproinsulin mRNA in as few as fifty islets. Incubation of islets, for 2 hours, at 2.5 mM glucose had no effect on islet preproinsulin mRNA content but incubation at 20 mM glucose increased islet preproinsulin mRNA content. Actinomycin D had no effect on this pattern of glucose stimulation and it is suggested that the degradation of preproinsulin mRNA is in some way dependent on RNA synthesis and that its inhibition by glucose plays an important role in the regulation of islet preproinsulin mRNA content.
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Di, Stasio Benoît. "Etude de nouveaux photosensibilisants pour des applications en Thérapie Photodynamique." Thesis, Vandoeuvre-les-Nancy, INPL, 2006. http://www.theses.fr/2006INPL069N/document.
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Les dérivés de porphyrines sont impliqués dans de nombreux processus d'oxydoréduction. Ces composés conduisent à de nombreuses applications, dont la Thérapie Photodynamique (PDT). Il existe deux générations de dérivés de porphyrines qui sont actuellement remplacés par des composés de 3ème génération, plus actifs et entraînant moins d'effets secondaires. Ces composés sont capables de reconnaître spécifiquement et directement (par adressage) ou indirectement (par vectorisation) les cellules cancéreuses. Nous avons orienté notre travail vers la synthèse et l'évaluation biologique de composés tétrapyrroliques associés à des modules tels que des sucres ou des motifs peptidiques de type -RGD- qui permettent une reconnaissance spécifique des cellules cancéreuses, via les lectines ou les intégrines, respectivement. De plus, dans le cadre d'un programme européen Cost-Chemistry intitulé "New molecular systems with therapeutic applications in photodynamic therapy of cancer and microbial infections", nous avons étudié les propriétés photophysiques de nombreux photosensibilisants synthétisés par une équipe roumaineDerived of porphyrins are tetrapyrrolic macrocycles involved in several redox processes. These compounds are used for different biological applications, like photodynamic therapy (PDT). Many teams in the world seek to synthesize compounds able to directly recognize specifically and (by targeting) or indirectly (by vectorization) cancer cells. These compounds are known as of 3rd generation. We are involved in the synthesis and the biological evaluation of tetrapyrrolic compounds associated to recognition and/or transport agents such as sugars or RGD-like peptide sequences. These moieties allow a specific recognition of the cancerous cells, via the lectins for the sugar moieties and the integrins for the RGD type moieties, respectively. Within the framework of an European Cost-Chemistry program entitled "New mo/ecu/ar systems with therapeutic applications in photodynamic therapy of cancer and microbia/ infections", we also studied the photophysical properties of photosensitizers synthesized by a Rumanian team
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Maake, Takalani Whitney. "Development of an actinobacteria based in vitro transcription and translation systems." University of the Western Cape, 2015. http://hdl.handle.net/11394/4750.
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>Magister Scientiae - MScHeterologous metagenomic screening strategies have relied largely on the construction of DNA libraries and screening in Escherichia coli to access novel enzymes. There is an increased demand for the identification of novel lignocellulose degrading enzymes with enhanced biochemical properties which are suitable for applications in industrial processes; biofuels being one of them. The use of heterologous gene expression in function based metagenomic studies has resulted in the discovery of enormous novel bioactive compounds. However, there are limitations associated with using E. coli as a heterologous host which does not allow transcription and translation of all genes in the metagenome. E. coli can only express 40% of the environmental DNA because of promoter recognition, codon usage, and host toxicity of gene products. Therefore alternative strategies for expressing or producing novel enzymes are needed, which can also be employed in metagenomic gene discovery. In vitro protein synthesis is an important tool in molecular biology and used to obtain proteins from genes for functional and expression studies. These systems may hold the key to unlock more of the potential in metagenomic DNA. The broader aim of the study is to develop non- E. coli based cell-free protein synthesis systems to further the metagenomics screening. In this study, Rhodococcus erythropolis H8 was evaluated for its suitability in cell-free expression. Crude extracts containing the macromolecular components (70S or 80S ribosomes, tRNAs, initiation, elongation and termination factors) fromR. erythropolis were prepared using existing crude extract based cell-free protein synthesis (CFPS) protocols. Three genes were selected and used as templates for synthesis: cell11, xp12 and acetyl xylan esterase (axe10), all previously isolated from metagenomic libraries screened inE. coli. As judged by zymograms and enzyme assays, all enzymes were successfully expressedfrom their native promoters and in recombinants clones using the PtipA promoter, and wereactive. Furthermore, the amounts of XP12 protein produced using pFos-XP_12 was 1.2mg/mlfrom E. coli and 1.67mg/ml from R. erythropolis CFPS, showing that the R. erythropolismachinery was more efficient in the expression of XP12 than the E. coli machinery. To the best of our knowledge this is the first demonstration of a cell-free expression using an actinomycete.
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Mould, A. P. "The aggregation properties of type I procollagen in vitro." Thesis, University of Manchester, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.377670.
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Ahmed, El Sayed Neveen. "Study of RNA synthesis of hepatitis C virus in vitro and in cells of hepatocarcinoma." Thesis, Bordeaux 2, 2011. http://www.theses.fr/2011BOR21868/document.
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La polymérase NS5B du virus de l’hépatite C (VHC) porte une activité ARN polymérase ARN-dépendante essentielle pour la réplication de l'ARN génomique viral. Cette réplication implique la synthèse d'un intermédiaire de réplication de polarité négative. In vitro et probablement in vivo, la NS5B initie la synthèse d'ARN par un mécanisme de novo qui nécessite des interactions spécifiques entre la polymérase virale et des éléments des ARN viraux. Dans une première partie nous avons étudié le rôle du GTP et d’un domaine C-terminal nommé linker de la polymérase. Nos résultats démontrent que des concentrations élevées de GTP sont nécessaires pour la transition de l'initiation à l'élongation de la synthèse de l'ARN. Des mutations dans le linker à la position 556 ne modifient pas la concentration de GTP nécessaire pour la transition. Toutefois, l'initiation de la synthèse d'ARN est augmentée par la mutation S556K. Une analyse structurale menée en parallèle suggère une implication directe du linker dans l'initiation de novo de la synthèse de l'ARN. Dans les deuxièmes et troisièmes parties, nous avons étudié le rôle de motifs ARN dans la traduction et la synthèse de l’'ARN du VHC. Nous avons démontré que la tige boucle SL-E1 formée par la région entre les nt 177 et 222 de l'extrémité 3' de l’ARN (-) est importante pour la synthèse d'ARN in vitro par la NS5B recombinante et dans les cellules Huh7 exprimant le complexe de réplication (RC) du VHC. SL-E1 est impliquée dans l’initiation de la synthèse d’ARN, au moins in vitro. Nous avons également étudié le rôle des tiges boucles SLV et SLVI du gène core. Nos données n'ont pas montré de rôle évident de ces séquences ou de leur complément dans la synthèse de l'ARN in vitro par la NS5B recombinante et en culture cellulaire par le RC du VHC. Nous avons confirmé leur effet négatif sur la traduction IRES dépendante par interaction ARN-ARN longue distance entre SL-VI et le 5'UTR et démontré que le miR122 ne peut pas empêcher cet interaction. Par contre, la présence de SL-VI prévient l’inhibition de la traduction induite par l’interaction entre le domaine III de l’IRES et la tige boucle 5BSL3.2 en 3’ du génome. Ces résultats démontrent la complexité des interactions ARN/ARN et ARN/protéines dans la régulation de la réplication viraleThe hepatitis C virus (HCV) NS5B protein displays a RNA-dependent RNA polymerase activity essential for replication of the viral RNA genome. This replication involves the synthesis of a replication intermediate of negative polarity. In vitro and likely in vivo, the NS5B initiates RNA synthesis by a de novo mechanism which requires specific interactions between the polymerase and viral RNA elements. In the first part of results, we described a combined structural and functional analysis of HCV-NS5B to study the role of a C-terminal segment (termed linker) and of GTP in RNA synthesis. Our results demonstrated that high GTP concentrations are necessary for the transition from the initiation to the elongation of RNA synthesis, and that linker mutations at position S556 did not modify the GTP requirement of NS5B for this transition. However, the initiation of RNA synthesis was greatly enhanced by a S556K mutation. These results together with a structural analysis point to the direct involvement of the linker in the de novo initiation of RNA synthesis. In the second and third parts of results, we studied the role of RNA elements in RNA synthesis. We demonstrated that the SL-E1 stem–loop formed by nucleotides 177–222 from the 3’-end of the HCV (-) RNA is important for RNA synthesis both in vitro by the recombinant NS5B and in Huh7 cells by HCV replication complex (RC). We also showed that SL-E1 is involved in initiation of RNA synthesis, at least in vitro. Then we studied the role of other viral RNA elements in core coding sequences (SLV and SLVI stem loops) and the involvement of the microRNA miR122 in RNA translation and RNA synthesis. For SLV and SLVI, our data did not show any clear role of these core-coding sequences or of their complement in the (-) RNA in RNA synthesis both in vitro by the recombinant NS5B and in cell culture by HCV-RC. We confirmed their negative effect on HCV-IRES translation through long range RNA-RNA interaction between SL-VI sequences and the 5’UTR and demonstrated that miR122 cannot disrupted this interaction and switches the region to an open conformation. Conversely, our data indicated that the SL-VI domain can counteract the negative effect of the interaction between the domain III of IRES and the 5BSL3.2 stem loop localized at the 3’end of the genome. These results point to the complexity of RNA/RNA and RNA/proteins interactions in the HCV replication cycle
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Wieczorek, Florence. "Les oximes dans la recherche d'inhibiteurs de la glucosamine-6P Synthase : Synthèses, études physico-chimiques et évaluations in vitro." Paris 11, 2010. http://www.theses.fr/2010PA112092.
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La Glucosamine-6P synthase (GlmS) catalyse l’étape limitante de la biosynthèse des hexosamines. Sa surexpression est à l'origine des complications liées au diabète et son inhibition constitue donc une nouvelle piste thérapeutique. Au cours de ce travail, nous avons développé des outils pour la conception de nouveaux inhibiteurs de la GlmS selon une stratégie par assemblage de fragments. Une synthèse en parallèle d'O-aryloxyamines et d'éthers d'oxime de sucres phosphates a d’abord été mise au point. Un test permettant l’analyse simultanée par spectrométrie de masse des deux activités de l’enzyme (glutaminase et synthase) a ensuite été développé. La stéréospécificité de l’interaction des deux stéréoisomères des éthers d’oxime avec la protéine a enfin été étudiée par RMN en utilisant la technique de transfert de saturation (STD), ce qui a permis de mettre en évidence l’existence de deux sites de fixation distincts, spécifiques de chacun des isomèresThe hexosamine biosynthetic pathway is closely associated with the side effects of diabetes. The conversion of fructose-6P (Fru6P) into glucosamine-6P (GlcN6P), the rate-limiting step in this pathway, is catalyzed by glucosamine-6P synthase (GlmS). This enzyme is hence considered as a potential therapeutic target for the treatment of vascular complications linked to diabetes. During this project, we set up the necessary tools for the discovery of new specific GlmS inhibitors using a fragment-based strategy. Therefore, O-aryloxyamines and oxime ethers were prepared using parallel synthesis. A mass spectrometry assay allowing simultaneous analysis of glutaminase and synthase enzyme activities was developed. Finally, the study of the interaction between GlmS and oxime ether isomers by Saturation Transfer Difference NMR revealed for the first time the existence of two separate binding sites specific of each isomer
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Gillespie, Charles Stewart. "Myelin membrane protein biosynthesis : an in vitro study." Thesis, University of Stirling, 1988. http://hdl.handle.net/1893/22868.
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The sites of biosynthesis and incorporation of the abundant CNS myelin proteins 2' , 3' -cyclic nucleotide-3'-phosphodiesterase (CNPase) and P2 protein into the growing myelin membrane were investigated. Cell-free translation systems programmed with mRNA from rat brain, rabbit spinal cord, free and bound polysomes and purified myelin demonstrated conclusively that both CNPase and P2 are synthesized on free polysomes like the myelin basic proteins (MBPs) but unlike the proteolipid protein (PLP), the major intrinsic membrane protein of CNS myelin, which is known to be synthesized at the oligodendrocyte endoplasmic reticulum on bound polysomes (Colman et al., 1982) . These observations were supported by labelling studies on rats in vivo during the period of maximal myelin deposition. Newly synthesized CNPase associated with the myelin membrane very rapidly after labelling (~2 minutes) and this is consistent with the view that there is only a brief delay between synthesis and incorporation into their target membrane for extrinsic-type plasma membrane proteins. An RNA fraction isolated from purified CNS myelin was not enriched in mRNAs coding for CNPase and P2 but a considerable enrichment of mRNAs coding for MBPs was observed. This phenomenon has important implications for the cell biology of myelination since it suggests that although MBPs, CNPase and P2 are all basic extrinsic membrane proteins, and synthesized on free polysomes, different mechanisms for their transport to the myelin membrane exist. The addition of dog pancreatic microsomes (DPM) during translation showed no membrane association for CNPase however, at least 50% of MBPs were observed to non-specifically associate with these membranes. When newly synthesized MBP and P2 were incubated post-translationally with DPM or rabbit spinal cord myelin P2 only associated with myelin whereas MBP showed an equal affinity for both types of membranes. The segregation of MBP free polysomes at the myelin membrane during synthesis ensures that the nascent MBP polypeptides associate with the correct membrane. Recent evidence has shown that the free polysome-mRNA complex is bound to the cytoskeleton during protein synthesis. After extensive characterization of the purified rat brain oligodendrocyte and myelin-associated cytoskeletons it was shown that the synthesis of MBPs and CNPase only occurs from mRNA that is associated with the cytoskeleton and not when it is part of the cytoplasmic mRNA pool. Lipid analysis of the purified rat brain myelin-associated cytoskeleton revealed the presence of tightly bound lipid with a considerable enrichment of cerebroside and sphingomyelin (the latter at the expense of phosphatidylethanolamine). These studies on the cytoskeletal involvement in myelinogenesis suggest that extrinsic CNS myelin proteins are synthesized on the cytoskeleton and that post-translational cytoskeletal transport of these proteins to the growing myelin membrane may take place.
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Cohen, Potier de Courcy Anita. "Synthèse et évaluation antiparasitaire de nouveaux dérivés du thiazole et apparentés." Thesis, Aix-Marseille, 2012. http://www.theses.fr/2012AIXM5503.
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L'objectif de ce travail consiste en la synthèse et l'évaluation antiparasitaire in vitro de nouveaux dérivés du thiazole et apparentés. Plusieurs stratégies de synthèse visant à une pharmacomodulation en séries mono- et polycycliques ont été étudiées : en série 2-méthyl-5-nitrothiazole, la pharmacomodulation anti-Trichomonas de la position 4 par stratégie SRN1 n'a pas permis d'améliorer l'activité déjà démontrée en série 2-méthyl-5-nitroimidazole, mais a conduit à des dérivés à activité antiproliférative in vitro, spécifique de la lignée HepG2. En série 4-arylsulfonylméthyl-2-méthylthiazole, la pharmacomodulation de la position 5, par couplage de Suzuki-Miyaura d'une part, et par arylation directe et réaction de Knoevenagel intramoléculaire d'autre part, a conduit à des dérivés mono- et polycycliques dont certains ont démontré une activité antiplasmodiale in vitro encourageante. En série 5H-thiazolo[3,2-a]pyrimidin-5-one, la réaction de double couplage de Suzuki-Miyaura a révélé l'importance du groupement phényle en position 6 pour l'activité antiplasmodiale de ces dérivés. Enfin, l'évaluation biologique in vitro de thiéno[3,2-d]pyrimidin-4(3H)-ones a permis de caractériser le pharmacophore responsable de l'activité antiplasmodiale significative de cette série. Les résultats préliminaires encourageants d'une étude mécanistique antiplasmodiale présentent l'inhibition spécifique des kinases plasmodiales comme un mécanisme d'action potentiel de ces composésThe objective of this work consists of the synthesis and the antiparasitic in vitro evaluation of new thiazole derivatives and related structures. Several synthetic strategies aiming at the pharmacomodulation on mono- and polycyclic series have been studied: in 2-methyl-5-nitrothiazole series, the anti-Trichomonas pharmacomodulation on position 4 by SRN1 strategy did not improve the activity previously demonstrated in 2-methyl-5-nitroimidazole series, but led to derivatives displaying a selective in vitro antiproliferative activity toward the HepG2 cell line. In 4-arylsulfonylmethyl-2-methylthiazole series, the pharmacomodulation on position 5, by Suzuki-Miyaura cross-coupling reaction on the one hand, and by direct arylation and intramolecular Knoevenagel reaction on the other hand, led to mono- and polycyclic derivatives among which some displayed an encouraging in vitro antiplasmodial activity. In 5H-thiazolo[3,2-a]pyrimidin-5-one series, a double Suzuki-Miyaura cross-coupling reaction revealed that the phenyl group on position 6 contributes to the antiplasmodial effect of this series. Finally, the in vitro biological evaluation of the thieno[3,2-d]pyrimidin-4(3H)-one scaffold let to characterize the pharmacophore responsible for the significant antiplasmodial activity. Some preliminary encouraging results regarding a mechanistic antiplasmodial study show the specific inhibition of plasmodial kinases, as a potential mechanism of action of some of these compounds
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Paradis, Renée. "Synthèse et essais in vitro de dérivés du paclitaxel." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0016/NQ47585.pdf.
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Brière, Francine. "Régulation de la synthèse des immunoglobulines A, in vitro." Lyon 1, 1989. http://www.theses.fr/1989LYO1T162.
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Stojanovic, Vesna. "The development of reconstituted translation system for peptidomimetic mRNA display synthesis." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/3001.
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The generation of high affinity, selective, and in vivo-stable peptide-based drugs is currently a major challenge in the field of drug development. Technologies exist that permit the generation of a vast diversity of chemical and conformational space and an example of such a technology is mRNA display, which utilizes protein translation machinery to produce a wide array of polypeptides starting from a combinatorial library of mRNA templates. The intention of this research was to bridge mRNA display to a reconstituted translation system using protein synthesis using recombinant elements (PURE) system for a new drug discovery platform. We hypothesized that it is possible to generate mRNA-peptidomimetic fusions using reconstituted translation system and chemo-enzymatically charged tRNAs, to incorporate unnatural amino acids into mRNA-peptidomimetic fusions.
Upon demonstating that the reconstituted system was functional, we have synthesized hexapeptide fusion products containing four alanine residues and one biocytin residue. Fusions were assayed using urea-PAGE in the presence of streptavidin which allowed for unambiguous evaluation of the full length fusion fraction. It was determined that overall more fusion product was generated with template that codes for biocytin early in the coding sequence, but that the percent of biocytin-containing product stays similar regardless of the biocytin place in the coding region. We have also found that the change in template untranslated region length does not improve incorporation of biocytin in dipeptide fusions within the tested range.
Finally, after first unsuccessful attempts to make sarcosine hexapeptide fusions, we investigated the effect of magnesium ion concentration on the translation reaction. As a result of four series of experiments performed involving both alanine and sarcosine fusion synthesis in parallel, we concluded that an increase in magnesium concentration from 5 mM to 20 mM coincided with enabling of the reconstituted system in making hexapeptide fusions with sarcosine in a significantly high number of cases.
This research work arises from the need to enable a new drug discovery tool that will allow both synthesis and affinity maturation of peptide-based compounds. It represents our pioneering efforts to develop a new technology and ultimately help bring to existence compounds of significant therapeutic value.
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Nies, Silke Marie. "Tryptophanabhängige Synthese von indolhaltigen Pigmenten bei verschiedenen humanpathogenen Asco- und Basidiomyceten." Giessen : VVB Laufersweiler, 2006. http://geb.uni-giessen.de/geb/volltexte/2006/3839/index.html.
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Rochd, Mohamed. "Etude de la biosynthèse des saponines et des stérols produits par la plante et par des tissus cultivés in vitro de Gypsophila paniculata L." Toulouse 3, 1990. http://www.theses.fr/1990TOU30096.
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Des tissus de g. Paniculata cultives in vitro produisent des saponines et cette production est parallele a la croissance des cellules en milieu non renouvele. L'incorporation de la radio-activite fournie sous forme d'acetate de na 2-#1#4c, dans les saponines et les sterols est rapide et reste constante pendant au moins 72 heures. La repartition de la radioactivite entre les sterols et les saponines est sensiblement equivalente. L'addition de gypsogenine 3,0-glucuronide exogene a une concentration de 16 mg/l a des cultures in vitro de g. Paniculata, ne perturbe pas leur croissance et a pour effet de reduire la synthese des saponines endogenes, sans perturber celle des sterols. Le squalene s'accumule momentanement sous l'action du gypsogenine 3,0-glucuronide. L'etude de la localisation de la biosynthese des saponines dans la plante montre que les racines sont le lieu de synthese et d'accumulation de ces molecules, alors que les sterols sont synthetises dans les differentes parties de la plante. Les tiges et les feuilles ne contiennent pas de saponines. Seules les fleurs, dans la partie aerienne de la plante, en contiennent une faible quantite. La teneur en saponines dans les racines est la plus elevee au printemps avant la floraison et la plus faible en ete en pleine floraison. La teneur dans les racines augmente avec l'age de la plante, au cours des cycles annuels successifs
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Xiao, Zhe. "Biosynthetic studies of tetrodotoxin and its anticancer activities assessment in vitro." HKBU Institutional Repository, 2014. https://repository.hkbu.edu.hk/etd_oa/56.
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In this study, the synthesis of TTX by three species of TTX-producing bacteria (Vibrio alginolyticus, Microbacterium arabinogalactanolyticum and Serratia marcescens) was conducted in a 10-L fermentor under the same controlled fermentation conditions for each of a period of 60 hours. The bacterial growth curves were monitored and the TTX synthesized in the culture medium was determined by HPLC. The TTX biosynthesis was found limited at the microgram level per L of culture medium with toxicities 14.7 MU (mouse unit) and 13.0 MU per mL in the partially purified culture medium of V. alginolyticus and M. arabinogalactanolyticum respectively by mouse bioassay. In the studies on SW480 and SW620 colorectal carcinoma cell lines, the expression, distribution, invasion and proliferation of voltage-gated sodium channels (VGSCs) were investigated by MTT assay (24-48 hours) and wound healing assay (0-120 hours). The different subtypes of VGSCs were expressed by semiquantitative RT-PCR and the locations of Nav1.5 and Nav 1.7 were detected by immunofluorescence microscopy. In the MTT assay, 40μmol/L of TTX showed significant inhibitory effect on both cell lines, with maximum inhibition rate, 33% and 40%, in SW480 and SW620 respectively. In the wound-healing assay, the inhibitory rate of 80μmol/L of TTX on SW480 reached 22% after 120 hours, compared with 30% in the control group. Moreover, VGSCs were highly expressed in both SW480 and SW620, with the main subtypes of Nav1.5 and Nav1.7 located on the cell surface, which might increase the metastatic rate of the cell lines. Keywords: Tetrodotoxin (TTX), Bacterial synthesis, Anticancer, VGSCs
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Scott, Linda A. "Arginine deprivation and cancer in vitro and in vivo investigations." Thesis, University of Aberdeen, 1999. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU532695.
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A number of amino acid deprivations have been tested in our laboratory, for the selective eradication of tumour cells in vitro. Withdrawal of the essential amino acid, L-arginine, produced the greatest differential effect on cell proliferation. Normal cells ceased to proliferate and remained viable in G, of the cell cycle, while tumour cells attempted to proliferate in conditions unfavourable for growth, resulting in rapid cell death. Of the six tumour cell lines studied here, four displayed the latter response, while the other two responded in an apparently similar manner to normal cells. Most tumour cells cannot arrest in G1 and are therefore defective in G1 cell cycle control (particularly at the R-point). Analysis of a normal cell line, and the two tumour cell lines that survived arginine deprivation, revealed that cdk4 was downregulated, and the cells were found to possess functional p53. The other four tumour cell lines had dysfunctional p53 and did not downregulate cdk4 upon arginine withdrawal, or relied upon cdk6 for pRb phosphorylation. Arginine is required for histone synthesis during S phase. Histone synthesis in the absence of arginine was compared in a normal and a tumour cell line. Normal fibroblasts synthesise histones to support previously initiated DNA synthesis for the first 24 h of arginine deprivation until the cells reach the R-point. However, HeLa cells cannot synthesise adequate amounts of histone proteins, despite continued DNA synthesis and this is to their detriment. A novel cancer therapy has been developed which exploits the differential response of normal and tumour cells to arginine deprivation. Extracorporeal dialysis was used to reduce blood arginine levels in normal and tumour-bearing dogs. Arginine was successfully reduced to <10 M within the first 12 h of dialysis and this low level was maintained for up to 5 days, but arginine was not reduced for a long enough period of time to see significant tumour regression.
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Nobes, Geoffrey A. R. "In vitro enzymatic synthesis and degradation of polyhydroxyalkanoates." Thesis, McGill University, 1997. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37549.
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Studies of the use of enzymes to both produce and degrade polyhydroxyalkanotes (PHAs) under in vitro conditions are presented in this thesis. PHAs were produced using a lipase-catalyzed ring-opening polymerization of the corresponding lactones. Poly(3-hydroxybutyrate), PHB, was obtained in up to 89% yield with a degree of polymerization of 3--12. Polymerization of other lactones, including beta-propiolactone, gamma-butyrolactone, and e -caprolactone, also yielded the corresponding polyesters in good yields with degrees of polymerization up to 25. MALDI-TOF mass spectroscopy was used to characterize the polymer products. The growth and kinetics of PHB granules produced in an in vitro polymerization were studied using TEM and CRYO-TEM in conjunction with image analysis. The in vitro reaction was confirmed to be a pseudoemulsion polymerization. The average granule diameter and volume increased with reaction time while the number of granules fell throughout the reaction due to coalescence. Basic kinetic parameters including KM, Vmax, and the rate constants of polymerization were determined and compared to those obtained for the in vivo biosynthesis of poly(3-hydroxybutyrate).With the aim of improved understanding of the mechanism of depolymerase action on water insoluble crystalline PHAs, folded chain lamellar single crystals of PHAs were partially degraded with PHA depolymerases and examined using TEM. Enzymatically degraded single crystals of bacterial PHB were found to be splintered by PHB-depolymerase A from Pseudomonas lemoignei parallel to their long axes into a needle-like morphology. These results support an "edge attack" model for the degradation of PHB single crystals and suggest that PHB-depolymerase A has both endo and exo activity. A further study was performed using single crystals of a number of PHAs which were partially degraded with depolymerases from Pseudomonas lemoignei and examined by TEM. In contrast to previous results with single crystals of bacterial PHB, the predominant effect observed with all crystals was a significant narrowing of the lamellae. This suggests an edge attack mechanism which because of lateral disorder of the crystals (caused by the introduction of valerate or repeat units of opposite stereochemistry) leads to a narrowing of the crystalline lamellae as opposed to the splintering effect previously observed.
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Nobes, Geoffrey A. R. "In vitro enzymatic synthesis and degradation of polyhydroxyalkanoates." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0017/NQ44533.pdf.
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Furdas, Silviya Dimitrova [Verfasser], and Manfred [Akademischer Betreuer] Jung. "Histon-Acetyltransferase-Hemmstoffe: Synthese und In-vitro-Charakterisierung." Freiburg : Universität, 2012. http://d-nb.info/1123469253/34.
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Rodier, Stéphane. "Synthèse d'analogues d'acétogénines d'annonaceae et d'inhibiteurs de topoisomérases à visée anticancéreuse." Poitiers, 1999. http://www.theses.fr/1999POIT2283.
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Malgre les progres realises, grace a l'amelioration de la prevention, du depistage et de l'efficacite des traitements au cours des vingt cinq dernieres annees, le cancer est aujourd'hui la deuxieme cause pathologique de mortalite. Le developpement de medicaments plus efficaces est donc particulierement important. Ceci implique la mise au point de methodes generales de synthese totale ou d'hemisynthese, dans le but d'etablir une relation structure-activite. Ainsi, deux familles d'agents cytotoxiques, les 5,12-naphtacenequinones et les acetogenines d'annonaceae, ont ete etudiees. Les 5,12-naphtacenequinones qui sont des quinones tetracycliques, sont des composes peu decrits dans la litterature. Cependant, certains ont montre une activite biologique importante : ces composes possedent une capacite d'inhibition vis-a-vis des topoisomerases i et ii, enzymes essentielles a la replication de l'adn. Apres un rappel des proprietes et des mecanismes d'action des topoisomerases, la synthese d'analogues d'une naphtacenequinone isolee recemment, la saintopine, a ete etudiee. Une deuxieme partie concerne la synthese d'analogues d'acetogenines d'annonaceae, qui representent une famille originale de produits naturels. L'interet pour cette famille de produit est motive par leur potentiel biologique (cytotoxique, antitumoral, pesticide,). Cependant, des tests in vivo ont montre que leur activite est fortement limitee par leur toxicite. Afin d'augmenter la selectivite de ces molecules, la synthese de lactones et d'analogues d'acetogenines a ete realisee. Des tests biologiques (cytotoxicite, effet sur le cycle cellulaire) ont ete effectues sur ces deux familles d'agents cytotoxiques et ont permis d'etablir les premieres relations structure-activite.
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Landis, Daniel Marc. "In vitro evolution of 5-fluorouracil resistant thymidylate synthases for cancer gene therapy /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/6329.
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Liautaud, Jacques. "Effet des antisécrétoires gastriques sur la prolifération des hépatocytes humains in vitro/ par Jacques Liautard." Montpellier 1, 1992. http://www.theses.fr/1992MON11157.
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Santai, Catherine Theresa. "In vitro Condensation of Mixed-Stranded DNA." Diss., Georgia Institute of Technology, 2006. http://hdl.handle.net/1853/14043.
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DNA condensation is the process in which an anionic polymer in combination with condensing agents undergoes a drastic reduction in volume and collapses into ordered structures. Double-stranded DNA has a uniform helical secondary structure, whereas single-stranded DNA is complex and adopts numerous different conformations. Novel mixed-stranded DNA molecules, with defined regions of both single-stranded and double-stranded secondary structures attached to one another in the same molecule, were created in this body of work. Mixed-stranded DNA was designed to be intermediate between its parent secondary structures in order to discover if mixed-stranded DNA will find a balance in terms of condensation properties as well. Mixed-stranded DNA was found to condense into minimally aggregated, globular particles in the presence of low mM concentrations of divalent transition metals in aqueous solvent at room temperature, a property not observed for either pure dsDNA or ssDNA. A model is presented to describe how mixed-stranded DNA -Mn2+, -Ni2+, and -Cd2+ condensates with the observed properties are produced. Multivalent-induced condensation of mixed-stranded DNA is also characterized and found to involve an unusual rod-like morphology in order to accommodate the secondary structures condensing independent of one another at different concentrations of multivalent cations. The attachment of a ss region to an otherwise ds molecule was found to greatly influence condensation properties of the entire molecule.
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Licari, Frank G. "A Programmable Pulse Generator for In-Vitro Neurophysiologic Experiments." University of Toledo / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1179278911.
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Ahadi, Sara. "Ribosomal Synthesis of N-methylated peptides." VCU Scholars Compass, 2010. http://scholarscompass.vcu.edu/etd/134.
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Natural peptide products isolated from various organisms often contain N-methylated backbones. Such a modification of backbone of the peptide changes its conformational rigidity. This modification improves the biological properties of the peptide, such as improved target affinity, proteolytic stability or membrane permeability. Therefore synthesis of N-methylated peptide libraries is valuable in screening for drug-like peptides suitable for therapeutic uses. Protein synthesis using recombinant elements (PURE) and Flexizyme were used in order to reassign specific codons to N-methyl amino acids. mRNA-dependent translation system enable us to make our desired peptides with N-methyl amino acids. This technology is a convenient tool for the construction of N-methyl peptide libraries. Using Flexizyme in order to make library of N-methyl peptides requires significant amount of tRNA. Therefore developing a simple and rapid method for purification of specific tRNA from fully modified E. coli total tRNA would be advantageous Here we reported a new technique in purification of individual tRNAs using fluorous affinity tag. From total tRNA, desired tRNA could be charged with related amino acid and tagged with fluorous molecule through reductive amination.
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Wang, Jinfan. "In Vitro Kinetics of Ribosomal Incorporation of Unnatural Amino Acids." Doctoral thesis, Uppsala universitet, Struktur- och molekylärbiologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-282023.
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Ribosomal incorporation of unnatural amino acids (AAs) into peptides or proteins has found broad applications in studying translation mechanism, discovering potential therapeutics, and probing protein structure and function. However, such applications are generally limited by the low incorporation efficiencies of the unnatural AAs. With in vitro kinetics studies using a purified E. coli translation system, we found that the natural N-alkyl AA carrier, tRNAPro, could hasten the incorporation of N-methyl AAs. Also, the incorporation rate increased remarkably with increasing pH in the range of 7 to 8.5, suggesting the rate was limited by peptidyl transfer, not accommodation. Competition experiments revealed that several futile cycles of delivery and rejection of the A site N-methyl AA-tRNA were required per peptide bond formation, and the incorporation yield could be increased by using a higher Mg2+ concentration. Kinetics of ribosomal polymerization, using AA-tRNA substrates prepared from the standard N-NVOC-AA-pdCpA chemoenzymatic ligation method, clarified that the inefficiency of incorporation was due to the penultimate dC. This dC prompted faster peptidyl-tRNA drop-off, leading to loss of processivities along consecutive incorporations. Circumventing the penultimate dC by using our N-NVOC-AA-pCpA chemoenzymatic ligation or the flexizyme charging method to prepare the AA-tRNA substrates was able to improve the efficiencies of ribosomal consecutive incorporations of unnatural AAs. By studying the translation steps after aminoacylation of tRNAPyl, the favored carrier for unnatural AAs in vivo, we demonstrated surprisingly slow biphasic kinetics of tRNAPyl-mediated amber suppression in vitro. The fast phase amplitude increased with increasing EF-Tu concentration, allowing measurement of Kd of EF-Tu binding. Results revealed ~25-fold weaker EF-Tu binding affinity of the tRNAPyl body than that of E. coli tRNAPhe. The fast phase rate was ~30-fold slower than that of native substrates, and this rate was limited by the ~10-fold less efficient AA-tRNAPyl:EF-Tu:GTP ternary complex binding to the ribosome. The incorporation was so slow that termination by RF2 mis-reading of the amber codon became a significant competing reaction. The processivity was unexpectedly impaired as ~40% of the dipeptidyl-tRNAPyl could not be elongated to tripeptide. This new overall understanding opens a window of improving unnatural AA incorporation both in vitro and in vivo.
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Prainer, Bianca-Cristine. "Tryptamin-Derivate als 5-HT4-Rezeptorliganden : Synthese und In-vitro-Pharmakologie." kostenfrei, 2008. http://www.opus-bayern.de/uni-regensburg/volltexte/2009/1045/.
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Webb, Vera Ann B. "In vivo in vitro synthesis of ribosomal RNA in bacillus subtilis." Thesis, University of British Columbia, 1988. http://hdl.handle.net/2429/29448.
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The work presented explored the in vivo and in vitro synthesis of ribosomal RNA in the Gram positive, spore-forming bacterium Bacillus subtilis. The investigation began with a study of rRNA synthesis in B. subtilis during steady state growth and under nutritional shift-up conditions. The percent of transcription which is ribosomal RNA was measured by hybridization of pulse labeled RNA to a specific DNA probe carrying the 3' end of the 23S RNA gene. The fractional rate of ribosomal RNA synthesis increased with cellular growth rate, and showed a rapid increase after a nutritional shift up. RNA synthesis during infection with an amber mutant of bacteriophage SP01 was also examined. Infected cells continued to synthesize rRNA at the preinfection rate, but could not respond to media enrichment by increasing the percent rRNA-synthesis. The latter study suggested the existence of a specific RNA polymerase that transcribed ribosomal RNA genes.
The conclusions from the in vivo study led to an analysis of rRNA transcription in vitro. The isolation of the putative ribosomal RNA specific RNA polymerase was attempted by affinity chromatography on cellulose complexed with plasmid DNA containing the promoter region of the B. subtilis rrnB rRNA operon, and by sedimentation through a glycerol gradient. No difference in activity profile was observed when transcription
activity at the rRNA tandem promoters was compared to activity at a non-ribosomal promoter. Since in vivo analysis of the control of rRNA synthesis in Escherichia coli suggested that regulation occurs at the level of transcription initiation, in vitro transcription initiation at the B. subtilis rRNA promoters was investigated using the single round transcription assay. Initial rates of transcription were different at each of the two tandem promoters of the B. subtilis rrnB operon: the upstream promoter, PI, initiated slowly, while the downstream promoter, P2, initiated faster. In addition, transcription initiation at the two promoters appeared to be linked. The formation of a heparin resistant complex at the PI promoter affected the stability of the heparin resistant complex formed at the P2 promoter. The kinetics of transcription initiation at the tandem rRNA promoters were examined using the tau plot analysis. RNA polymerase had a high affinity for both rRNA promoters, but the rate of initiation at these promoters was relatively slow when compared to non-ribosomal promoters. Finally, transcription initiation on two artificial tandem promoter constructs was compared with initiation on the native tandem promoter construct. In general, PI was shown to have a positive effect on transcription from downstream promoters, but had specific effects on different promoters.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Kim, Yong-Keun. "Amphiphile Polymere als Transfektionssysteme Synthese und In-vitro-Gentransfer /." [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=96383150X.
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Kotaras, Peter J. "Synthesis and secretion of rat pineal proteins in-vitro /." Title page, contents and abstract only, 1992. http://web4.library.adelaide.edu.au/theses/09PH/09phk871.pdf.
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Poisson, Jessica. "Synthesis and In Vitro Applications of Ice Recrystallization Inhibitors." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39466.
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Recent advances in the clinical diagnosis and treatment of diseases using cell transplantation have emphasized the urgent need to cryopreserve many types of cells. In transfusion medicine, red blood cell (RBC) transfusions are used to treat anemia and inherited blood disorders, replace blood lost during or after surgery and treat accident victims and mass casualty events. In regenerative medicine, mesenchymal stem cell (MSC) therapy offers promising treatment for tissue injury and immune disorders. Current cryoprotective agents (CPAs) utilized for RBCs and MSCs are 40% glycerol and 10% dimethyl sulfoxide (DMSO), respectively. Although glycerol is required for successful cryopreservation of RBCs, it must be removed from RBCs post-thaw using costly and time-consuming deglycerolization procedures to avoid intravascular hemolysis. Unfortunately, while DMSO prevents cell damage and increases post-thaw MSC viability and recovery, recent reports have suggested that MSCs cryopreserved in DMSO display compromised function post-thaw. As a result, improvements to the current cryopreservation protocols such as reducing post-thaw RBC processing times and improving MSC function post-thaw are necessary in order to meet the increasing demands of emerging cellular therapies. Ice recrystallization has been identified as a significant contributor to cellular injury and death during cryopreservation. Consequently, the ability to inhibit ice recrystallization is a very desirable property for an effective CPA, unlike the conventional CPAs such as DMSO and glycerol that function via a different mechanism and do not control or inhibit ice recrystallization. Over the past few years, our laboratory has reported several different classes of small molecules capable of inhibiting ice recrystallization such as lysine-based surfactants, non-ionic carbohydrate-based amphiphiles (alkyl and aryl aldonamides) and O-linked alkyl and aryl glycosides. The use of these small molecule ice recrystallization inhibitors (IRIs) as novel CPAs has become an important strategy to improve cell viability and function post-thaw. With the overall goal to identify highly effective inhibitors of ice recrystallization, the first part of this thesis examines the IRI activity of three diverse classes of small molecules including carbohydrate-based surfactants bearing an azobenzene moiety, fluorinated aryl glycosides and phosphate sugars. While the majority of the carbohydrate-based surfactants and fluorinated aryl glycosides were not effective inhibitors of ice recrystallization, this work revealed that monosaccharides possessing a phosphate group could be effective IRIs. Our laboratory has previously demonstrated that small molecule IRIs β-PMP-Glc and β-pBrPh-Glc can protect human RBCs from cellular injury during freezing using reduced concentrations of glycerol (15% w/v). This was significant as reducing the concentration of glycerol can drastically decrease deglycerolization times. Consequently, structure- function studies were conducted on β-PMP-Glc and β-pBrPh-Glc to elucidate key structural features that further enhance their IRI activity and may increase their cryoprotective ability. In particular, the influence of an azido moiety on the IRI activity of β-PMP-Glc and β-pBrPh-Glc was investigated and it was determined that the position of the azide substituent on the pyranose ring is crucial for effective inhibition of ice recrystallization. Furthermore, the presence of an azido group at C-3 was found to increase the IRI activity of β-PMP-Glc and β-pBrPh-Glc. Despite the discovery that β-PMP-Glc and β-pBrPh-Glc are beneficial additives for the freezing of RBCs, a significant amount of cellular damage occurred during deglycerolization, resulting in very low cell recoveries. Thus, IRI active azido aryl glucosides were explored for their cryopreservation potential in RBCs to determine whether they could function as effective additives that reduce cellular damage post-thaw and improve cell recovery. One of the most significant results of this thesis is the discovery that azido aryl glucosides can successfully cryopreserve RBCs in the presence of 15% glycerol with significantly improved cell recovery. This thesis also explores the use of small molecule IRIs to improve the cryopreservation of MSCs. In particular, the addition of an N-aryl-aldonamide (2FA) to the standard 10% DMSO solution was found to enhance the proliferative capacity of MSCs post-thaw. Lastly, the ability of small molecule IRIs to cross the cell membrane and behave as permeating CPAs was evaluated in two different cell models, RBCs and human umbilical vein endothelial cells (HUVECs). These studies demonstrated that small molecule IRIs are capable of permeating the cell membrane and controlling intracellular ice recrystallization.
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Brunet, Aurelie Claude Laure. "Synthesis and in vitro applications of fluorescent imaging agents." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/9659.
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Fluorescent imaging technologies that offer new ways to visualise and quantify fluorescently labelled molecules are increasing, necessitating the development of fluorescent molecules that can efficiently and specifically label targets in vitro and in vivo. The first aim of this thesis was the study of human neutrophil elastase. Human neutrophil elastase is an important enzyme in the regulation of inflammation but if over expressed can become part of the cause of inflammation itself. To elucidate this dual function and have a greater understanding of this enzyme, an imaging probe for neutrophil elastase was designed. Firstly, the syntheses of fluorescently labelled three branched dendron core structures were optimised, and studied in neutrophils. The selected core structure was functionalised with an elastase specific peptide sequence and fluorescently labelled. The probe was specifically cleaved by neutrophil elastase in an enzymatic assay and in the presence of activated neutrophils (Chapter 1). Fluorescein and rhodamine are dyes that are readily available, are affordable and have convenient wavelengths for microscopy and flow cytometry. Carboxyfluorescein diacetate N-succinimidyl ester (CFDA-SE) is a commonly used fluorescein derivative, widely used in cell proliferation assay. It is mainly used as a mixture of isomers and its synthesis is not reported. Herein a short and simple synthesis of the two individual isomers of carboxyfluorescein diacetate N-succinimidyl ester as well as the equivalent rhodamine variation (carboxytetraethylrhodamine N-succinimidyl ester) is reported (Chapter 2). The labelling properties of these probes were studied in proliferation assays on mouse and human T lymphocytes. Finally, the nuclear penetration of the dendron structure combined with nuclear localisation sequences (NLS) was investigated. Attachment of nuclear localisation sequences to the probe in the presence of fluorescein demonstrated successful entry into the nucleus in human alveolar adenocarcinoma cell line (A549) (Chapter 3).
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Tonnaire, Thierry. "Conception et synthese d'inhibiteurs contraints de la protease du vih-1 (doctorat : pharmacochimie)." Paris 11, 1999. http://www.theses.fr/1999PA114843.
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Remy, Jean-Serge. "Synthese et utilisation in vitro de nouveaux vecteurs de transfert de genes." Strasbourg 1, 1994. http://www.theses.fr/1994STR15039.
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Luciani-Sentier, Nathalie. "Optimisation d'un système de traduction in vitro : rôle des molécules oxydantes sur la synthèse protéique acellulaire." Nancy 1, 1994. http://www.theses.fr/1994NAN10340.
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Ce travail a été réalisé dans le but d'optimiser le système de traduction in vitro de germe de blé. L'arrêt prématuré de la synthèse protéique dans un tel système est lié à une limitation en oxygène du milieu réactionnel, puisque l'augmentation de la surface d'échange entre milieu de traduction et atmosphère augmente la synthèse protéique. La quantité de protéines synthétisées ainsi que la vitesse de traduction sont augmentées lorsqu'on enrichit le milieu en oxygène. Lorsqu'on ajoute du peroxyde d'hydrogène au milieu de traduction, on observe le même effet qu'avec l'oxygène, on peut donc penser que la stimulation de la synthèse protéique observée est liée aux propriétés oxydantes communes aux deux molécules. Lorsque les molécules oxydantes participent à des réactions d'oxydoréduction, des radicaux libres sont formés. Ces radicaux libres, et principalement l'anion superoxyde, sont impliqués dans la traduction in vitro, hypothèse renforcée par l'inhibition de la synthèse protéique en présence d'agents chélateurs de fer, ainsi que par l'effet positif d'un ajout de système générateur de radicaux libres et par l'effet négatif de celui d'enzymes bloquant les radicaux libres
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Ring, Christine. "Optimization of in vitro transcription/translation conditions for in vitro compartmentalization studies and synthesis of 4-fluorohistidine." VCU Scholars Compass, 2017. http://scholarscompass.vcu.edu/etd/4807.
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Genetic code expansion allows the incorporation of non-canonical amino acids with a variety of new functional groups: fluorescent amino acids,1-3 azides,4-6 alkynes,5-10 and photocrosslinkers.4,11,12 This incorporation requires the evolution of new tRNA/aminoacyl tRNA sythetase pairs. Traditionally screenings of novel tRNA/aminoacyl tRNA synthetase pairs have been done in vivo. While these in vivo screenings have proven robust, they are limited in multiple ways: non-canonical amino acids (ncAAs) must be nontoxic and bioavailable. Furthermore, library size is limited by transformation efficiency. Lastly, in vivo screenings require substantial amounts of the target ncAA, which is often not available in large masses. In vitro screenings bypass these limitations: toxicity and bioavailibilty are no longer concerns. Library size can be expanded by several orders of magnitude as we are no longer limited by transformation efficiency. Lastly, because in vitro transcription/translation reactions are routinely conducted on the μL scale, ncAA usage can be minimized. We set out to use in vitro compartmentalization to further expand the code. In an in vitro compartmentalization screening, the water droplets in a water-in-oil emulsion serve as separate reaction chambers in which individual library members are transcribed and translated. Here we report optimization of S30 transcription/translation reactions. Optimizations include cell lysis method, reaction temperature, template amount, and T7 RNA polymerase amounts. Yields remained low and we transistioned into the use of PURExpress.
Fluorohistidines are isosteric with histidine, but not isoelectronic.13 This change in environment results in a reduction of pKa. We set out to synthesize 4-fluorohistidine to use as a pH probe in several target proteins. A synthesis of 4-fluorohistidine was published in 1973.14,15 We were able to improve upon this synthesis by reducing cost and improving yield of a key step in the reaction. Next, small peptides with polyhistidine tags were translated in vitro using our 4-fluorohistidine. We are calling this polyhistidine tag incorporating 4-fluorohistidine our “hexafluorohistag.” Because of the reduced pKa of the 4-fluorohistidine, the hexafluorohistag showed affinity to Nickel-NTA resin even at reduced pH. This allowed for the purification of hexafluorohistagged peptides in the presence of traditional polyhistidine-tagged peptides.
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Sweeney, Scott Francis 1977. "Towards well-defined gold nanomaterials via diafiltration and aptamer mediated synthesis." Thesis, University of Oregon, 2007. http://hdl.handle.net/1794/6240.
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xvii, 203 p.Gold nanoparticles have garnered recent attention due to their intriguing size- and shape-dependent properties. Routine access to well-defined gold nanoparticle samples in terms of core diameter, shape, peripheral functionality and purity is required in order to carry out fundamental studies of their properties and to utilize these properties in future applications. For this reason, the development of methods for preparing well-defined gold nanoparticle samples remains an area of active research in materials science. In this dissertation, two methods, diafiltration and aptamer mediated synthesis, are explored as possible routes towards well-defined gold nanoparticle samples. It is shown that diafiltration has considerable potential for the efficient and convenient purification and size separation of water-soluble nanoparticles. The suitability of diafiltration for (i) the purification of water-soluble gold nanoparticles, (ii) the separation of a bimodal distribution of nanoparticles into fractions, (iii) the fractionation of a polydisperse sample and (iv) the isolation of [rimers from monomers and aggregates is studied. NMR, thermogravimetric analysis (TGA), and X-ray photoelectron spectroscopy (XPS) measurements demonstrate that diafiltration produces highly pure nanoparticles. UV-visible spectroscopic and transmission electron microscopic analyses show that diafiltration offers the ability to separate nanoparticles of disparate core size, including linked nanoparticles. These results demonstrate the applicability of diafiltration for the rapid and green preparation of high-purity gold nanoparticle samples and the size separation of heterogeneous nanoparticle samples. In the second half of the dissertation, the identification of materials specific aptamers and their use to synthesize shaped gold nanoparticles is explored. The use of in vitro selection for identifying materials specific peptide and oligonucleotide aptamers is reviewed, outlining the specific requirements of in vitro selection for materials and the ways in which the field can be advanced. A promising new technique, in vitro selection on surfaces (ISOS), is developed and the discovery using ISOS of RNA aptamers that bind to evaporated gold is discussed. Analysis of the isolated gold binding RNA aptamers indicates that they are highly structured with single-stranded polyadenosine binding motifs. These aptamers, and similarly isolated peptide aptamers, are briefly explored for their ability to synthesize gold nanoparticles. This dissertation contains both previously published and unpublished co-authored material.
Adviser: James E. Hutchison
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Bruce, Jeremy Charles. "Enzymatic studies on IPN amidohydrolase and IPN synthase in vitro." Thesis, University of Oxford, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.306693.
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Xynos, Ioannis D. "Bioactive glasses for the in vitro synthesis of bone tissue." Thesis, Imperial College London, 2001. http://hdl.handle.net/10044/1/11494.
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Boularot, Adrien. "La peptide déformylase, une cible pour de nouveaux agents antibiotiques : conception d'inhibiteurs et étude fonctionnelle des effets biologiques in vitro et in vivo." Paris 11, 2005. http://www.theses.fr/2005PA112239.
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La résistance des bactéries aux antibiotiques est devenue un problème de santé publique qui touche la quasi-totalité des agents antibactériens. Une des priorités pour traiter ce problème est la recherche d'antibiotiques innovants, agissant sur de nouvelles cibles. A ce titre, le peptide déformylase (PDF) qui est présente chez toutes les bactéries répond à ce critère de sélection. Cependant, une PDF humaine a été isolée au laboratoire récemment et démontrée comme se localisant dans la mitochondrie. Le principe de précaution impose que cette forme ne puisse en aucun cas être inhibée. Au laboratoire, notre approche de la problématique nous a donc amenés à penser que des nouvelles têtes de séries seraient plus efficaces et surtout sélectives des formes bactériennes. Mettant à profit les propriétés de chimie de coordination des PDFs, des séries de molécules possédant une fonction coordinante du métal ont été synthétisée. D'autre part des squelettes possédant soit une chaîne pseudopeptidique respectant les conditions de reconnaissance par l'enzyme, soit un hétérocycle y ont été associés afin d'augmenter la sélectivité de ces composés. Ces molécules ont été testée in vitro soit par des tests d'inhibition enzymatiques, soit par la technique d'empreinte RMN. Les composés s'avérant les plus efficaces in vitro ont été testés in vivo sur des cultures bactériennes. Leur action spécifique in vivo sur la PDF a été mesurée et les effets antibiotiques déterminés sur des bactéries modèles Gram-positives et Gram-négatives. Enfin, nos composés ont été démontrés comme sélectifs et agissant comme de potentiels antibiotiques ciblant spécifiquement les PDFs bactériennes et non la forme humaineThe emergence of bacteria' resistance to antibiotics is becoming a major public health issue which involves quite all-antibacterial agents. One of the priorities to solve this problem is the search of innovative antibacterial agents active against novel targets. Hence the peptide deformylase (PDF), a metalloprotease present in all bacteria, fulfils this selection criterion. However, human PDF has been recently isolated and shown to be located in the mitochondria. This identification could impose major reservations as to the possible medical use of PDF's inhibitors since human form must not be inhibited. New series' leader compounds would therefore appear to be more efficient and selective against bacterial forms. Taking advantage of the chelating properties of PDFs, series of molecules with a one or two-coordinate metal-binding group were synthesized. Moreover backbones with either a pseudo-peptidic chain recognized by the enzyme, or a heterocycle were used to increase the selectivity of compounds. About sixty molecules were tested in vitro for their affinity towards PDF and their capacity to inhibit it, by either enzymatic inhibition tests or MNR spectroscopic experiments. Finally, the selectivity of these compounds was clearly shown as they act as potential antibiotics targeting specifically bacterial PDFs and not the human form. After the in vitro assessments of the inhibition potency, the most efficient compounds were tested in vivo on bacterial cultures. Their specificity in vivo against PDF has been shown, measured and their antibiotic effects have been determined on Gram-positives and Gram-negatives bacteria in different genetic contexts
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Tendjoun, Victor. "Synthese d'aminoacides et derives precurseurs d'hydantoines et de piperazine-2,5-diones spiranniques potentiellement immunomodulateurs (doctorat : sciences pharmaceutiques)." Besançon, 1997. http://www.theses.fr/1997BESA3516.
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Leyva, Lida. "Complexes cycloruthénés à activité anticancéreuse : Synthèse, activité et toxicité." Université Louis Pasteur (Strasbourg) (1971-2008), 2007. http://www.theses.fr/2007STR13159.
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Pontikis, Renée. "Synthèses de la colchicine marquée au carbone 14 : contribution à l'étude in vitro de sa toxicité." Paris 11, 1986. http://www.theses.fr/1986PA112300.
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En vue d’une meilleure compréhension des phénomènes toxiques de la colchicine, nous avons entrepris l’étude « in vitro » de son métabolisme et de son éventuelle fixation covalente aux protéines hépatiques, ceci à l’aide de produits radiomarqués. Compte tenu de la complexité de la molécule de la colchicine il nous a semblé préférable d’introduire l’isotope dans le squelette. Nous avons donc élaboré un schéma réactionnel qui permettait d’accéder à la colchicine marquée sur le carbone en position 7. Afin de vérifier la fiabilité du schéma envisage et de préciser les conditions expérimentales pour chacune des 17 étapes, une synthèse en « traceur » a tout d’abord été réalisée. Par la synthèse à haute radioactivité spécifique (AS=55 mCi=2035 MBq/mmol) la (+/-) colchicine ¹⁴C-7 a été préparée avec un rendement de 2,5% à partir de 1700 mCi de carbonate de baryum ¹⁴C. Conjointement, la colchicine (méthoxyle-1¹⁴C) a été préparée par une méthode de marquage rapide sur un groupement latéral. Une étude préliminaire du métabolisme « in vitro », à l’aide de cette molécule radio marquée et de microsomes de foie de rats, a permis de mettre en évidence la fixation covalente de la colchicine aux protéines hépatiques ; sa dépendance vis-à-vis des monooxygénases à cytochrome P-450 a été confirmée. L’analyse des extraits organiques de ces incubats, par CLHP, permet de confirmer la présence de deux métabolites déjà décrits par SCHONHARTING : les déméthyl-2 et 3 colchicines. Trois nouveaux métabolites sont observés mais nous ne disposons pas de quantités pondérales suffisantes pour leur analyse structurale. En vue de l’élaboration d’un dosage de ce médicament par radio-immunologie nous avons synthétisé trois haptènes de la colchicine, dont les modifications portent sur les différents cycles, puis effectué leur conjugaison au sérum albumine bovine. La molécule de colchicine marquée au carbone 14 dans le noyau nous permettra d’approfondir les études métaboliques abordées lors de ce travail.
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Chan, Yiu-ming, and 陳耀明. "The chemistry and in vitro cytotoxicity study of manganese oxide nanostructures." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2007. http://hub.hku.hk/bib/B39557121.
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Gaceur, Meriem. "Nanosondes bimodales pour l'imagerie médicale par résonance magnétique et par fluorescence optique : synthèse, caractérisation et évaluation de leur toxicité in vitro." Paris 7, 2012. http://www.theses.fr/2012PA077020.
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L'utilisation de matériaux semi-conducteurs est aujourd'hui en plein essor dans le domaine des nanobio technologies, notamment pour l'imagerie médicale. En particulier, les particules luminescentes magnétiquement diluées semblent être très prometteuses en tant que nanosondes bimodales en imagerie par résonance magnétique (IRM) et l'imagerie par fluorescence optique (EFO). Ce travail de thèse présente, dans un premier temps, l'optimisation du protocole de synthèse en milieu polyol de nanoparticules de type Zn₁₋MxS (x < 0,4) paramagnétiques et luminescentes. Puis, nous nous sommes intéressés à la préparation d'une solution aqueuse colloïdale de ces nanoparticules après leur fonctionnalisation par l'acide mercaptoacétique. Par ailleurs, en plus des caractérisations magnétiques effectuées sur les poudres, les résultats IRM effectués sur les colloïdes ainsi préparés confirment le comportement paramagnétique des particules de Zn₁₋xJMxS avec des valeurs de relaxivité longitudinale ri, mesurées à l'ambiante et à 3. 0 T, de 20 et 74 mM⁻¹. S⁻¹ pour une teneur respective en Mn²⁺ de 10 et 30 %. De plus, ces colloïdes émettent dans le visible (bleu) après une excitation à 405 nm. Pour permettre une éventuelle application de ces systèmes comme sondes, l'évaluation des risques de cyto- et génotoxicité des particules est indispensable. Elle a été réalisée sur des cellules ovariennes de hamster chinois (CHO) et a montré l'absence d'effets cyto- et génotoxiques avérés dans la gamme de concentrations étudiées (1 - 100 ug/mL) sur ces cellules. Ces résultats forts prometteurs, nous encouragent à considérer sérieusement les nano-objets préparés comme nanosondes bimodales pour l'imagerie duale par IRM et IFOSemi-conductive nanomaterials have become of great interest lately in biotechnology area especially in medical imaging. Typically, magnetically diluted luminescent nanoparticles offer a real profit as potentially being bimodal probes in magnetic resonance imaging (RMI) and optical fluorescence imaging (OFI). The present work, firstly deals with the optimization through a polyol process of the synthesis of paramagnetic and fluorescent Zn!. XMnxS (x < 0,4) nanoparticles, secondly their functionalization with mercapto-acetic acid and eventually the preparation of their aqueous based colloids. The magnetic characterization and MRI measurements performed on post functionalized nanoparticles respectively before and after their dispersion as stable colloids both confirm the paramagnetic behavior of Zni_xMnxS (x < 0,4) nanomaterials. Indeed, regarding MRI, longitudinal relaxivity r₁ at room temperature and at 3. 0 T is of 20 and 74 mM⁻¹. S⁻¹ for a Mn²+ amount of respectively 10 and 30 %. Moreover, these colloids emit in the visible light range (blue) when excited at 405 nm. The use of these probes in any possible medical application is not conceivable unless their cyto- and genotoxicities are evaluated. Therefore, a serious study was carried out on chinese hamster ovarian cells (CHO) and evidenced the absence of any cyto- or genotoxic effects on these latter in the range of the nanoparticle studied concentrations (1-100 Hg/mL). These results make us seriously consider the ZnMnS hybrids as potential bimodal probes for dual MRI and OFI imaging
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Jenning, Stefan. "Ondansetron-analoge 5-HT 3 -Rezeptorliganden : Synthese, Stereochemie und in-vitro-Pharmakologie." kostenfrei, 2009. http://www.opus-bayern.de/uni-regensburg/volltexte/2009/972/.
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