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De, Leon Ellen Jane Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Engineering antibodies against complex platelet antigens using phage display technology." Awarded by:University of New South Wales, 2007. http://handle.unsw.edu.au/1959.4/37009.
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Platelets are small anucleate cell fragments found in blood whose physiological role is important in maintaining haemostasis. In vivo, platelet surface glycoproteins mediate the mechanistic roles of platelets, and polymorphic changes to these glycoproteins have been observed to have significant effects on the platelet cellular function and such changes may include over-expression, under-expression and antigenicity of the protein. Human platelet antigens (HPA) are a result of polymorphic differences in platelet surface glycoproteins which have been found to be variably expressed in the population. Foetal maternal alloimmune thrombocytopaenia (FMAIT) is a condition that is observed in the unborn foetus and neonates due to HPA incompatibility between the mother and the foetus. HPA incompatibility accounts for a majority of severe thrombocytopaenic cases in neonates, and delayed diagnosis and treatment of such a condition often lead to intracranial haemorrhage. The risk in neonates diagnosed with FMAIT becomes increasingly significant in cases where intra-uterine (during pregnancy) platelet transfusion is the only effective therapeutic option. There are currently no antenatal screening programs for this condition, and laboratory diagnosis of FMAIT relies on the detection of maternal alloantibodies and parental HPA typing. For these reasons a significant amount of research is currently being invested into the isolation of recombinant antibodies with specific reactivity against FMAIT-related platelet antigens. Stable and specific recombinant platelet antibodies have great potential as a diagnostic agent in antenatal screening and broad-scale HPA typing of blood donors for platelet transfusion. Further characterisation of the isolated antibody may lead to a possible therapeutic agent. Studies by previous researchers have shown that the traditional methods (ie. Mouse monoclonal and EBV transformation) of obtaining monoclonal antibodies against FMAIT-related antigens have proven unsuccessful. The continuing progress in the discipline of phage display has produced several novel antibodies against self and non-self antigens. A further advantage in the application of phage display technology for the isolation of novel antibodies is the easy transition from bacterial to mammalian expression for the characterisation of glycosylated antibodies. The main focus of this project was to create and isolate a recombinant human anti-HPA-3a antibody using phage display for its possible application as a therapeutic or diagnostic agent.
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Nangola, Sawitree. "The interference of human immunodeficiency virus assembly and maturation by ankyrin repeat proteins." Thesis, Paris 11, 2011. http://www.theses.fr/2011PA112044.
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Le but de ce travail est de découvrir des nouvelles protéines suceptibles d'interférer avec le cycle vital du virus HIV. De par leur repliement, les protéines à motifs ankyrines peuvent constituer une ossature protéique trés bien adaptée à cet objectif. Plusieurs interacteurs spécifiques de la protéine MA-CA du HIV ont été sélectionnées par exposition sur phage à partir d'une bibliothèque de variants d'ankyrines. Trois protéines isolées ont été produites à partir de clones ayant une forte activité de liaison. Le meilleur interacteur protéique (1D4) interagit avec un épitope situié sur le domaine CA. La constante de dissociation entre 1D4 et la protéine HACA a été déterminée par Calorimétrie de Titrage Isotherme (ou ITC) et est égale à 0,45M. La protéine 1D4 n'a pas d'effet détectable sur la maturation virale suivie par une technique ELISA de dosage de la Protease du HIV. En revanche, cette protéine interfere avec l'assemblage viral dans des cellules supT1 qui exprime de façon stable la protéine 1D4 sous forme myristoylée. Ce resultat ouvre une perspective d'appoche pour interferer avec le cycle vital du HIV<br>Presently, the standard regimen for antiretroviral treatment is highly active antiretroviral therapy (HAART). However, this strategy inherits the well-known side effects and is prone to promote the HIV drug-resistant strains. As a consequence, gene therapy has been introduced as an alternative approach. In this study, we aimed to discover the novel protein-based agents for intervening viral replication by gene targeting procedure. Regarding the efficient folding dynamic in cytoplasm, ankyrin repeat protein was considered to be a candidate scaffold. Several engineered ankyrin binders specific to HIV MA-CA domain were successfully retrieved from the ankyrin-displayed phage library. Three positive clones with high binding activity by ELISA were selected for further analyzing their binding property in soluble form. The best binder, 1D4, recognized its epitope located on CA domain as shown by Western immunobloting and ELISA. The affinity of 1D4 against H6MA-CA was 0.45 μM with one to two moles of target molecule determined by isothermal titration calorimetry (ITC). Although 1D4 exhibited no effect on viral maturation as verified by an ELISA based HIV protease assay technique, it disturbed the viral assembly process in Sup-T1 cells which stably expressed the myristoylated 1D4. This finding has provided a concrete prospect for HIV life cycle interruption by stem cell gene therapy in the future
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Rosander, Anna. "Novel applications of shotgun phage display /." Uppsala : Dept. of Microbiology, Swedish Univ. of Agricultural Sciences, 2004. http://epsilon.slu.se/a450.pdf.
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Holmes, David Ian Roderick. "Phage display of chymotrypsin inhibitor II." Thesis, Imperial College London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283414.
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Smith, Mathew Wayne. "Phage display and experimental brain therapeutics." Thesis, Cardiff University, 2009. http://orca.cf.ac.uk/55853/.
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Phage display, a powerful polypeptide display technology, affords the rapid identification of peptides and proteins that interact with a target of interest The aims of the project were the phage display identification of peptides that interact with a druggable target in a brain disorder (glioblastoma multiforme) and the identification of peptides that serve as targeting vectors for brain delivery. Validation studies were undertaken to qualify the use of a cyclic 7-mer peptide phage library against targets including streptavidin and paracetamol chosen as examples of a large complex and small simple molecule, respectively. With the aim of identifying peptide phages that bind to the luminal surface of brain micro vasculature, a primary in-vitro porcine model of the blood-brain barrier (BBB) comprising primary brain capillary endothelial cells was established and characterised. An in-vivo phage display was undertaken in the rat with the aim of identifying peptide sequences that mediated translocation across the BBB into brain grey matter. A 7-mer cyclic peptide was identified with sequence AC-SYTSSTM-CGGGS that enhanced the uptake of phages into brain grey matter by 4-fold compared to control wild-type phages. This peptide may serve as a novel targeting vector for the delivery of a therapeutic cargo to the brain. Caveolin-1 was identified as a potential new therapeutic target in in-vitro models of grade IV astrocytomas (glioblastoma multiforme), with siRNA knockdown of caveolin-1 associated with reduced glioma cell proliferation and invasiveness. With the caveolin-1 scaffolding domain (aa 81-101 in the caveolin-1 protein) as a target, an in-vitro peptide phage selection was undertaken and identified a series of peptides that bind the scaffolding domain with high affinity. These peptides will serve as a template for the development of low molecular weight peptidomimetics that inhibit caveolin-1 function. In conclusion, the studies in this thesis have demonstrated the utility of phage display in experimental therapeutics of brain disorders.
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Drever, Matthew. "Generating microcystin antibodies by phage display." Thesis, University of Aberdeen, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445137.
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A naïve human antibody fragment phage display library was screened against conjugated microcystin-LR (MCLR). Phage antibodies were eluted with free MCLR to promote the isolation of free antigen binders. Isolated antibody fragments were cloned into a soluble expression vector and single-chain antibody (scAb) expressed in a bacterial host. All scAbs bound free antigen in an indirect competition ELISA and levels of sensitivity were determined. The most responsive clone 3A8 had an 800-fold increase in sensitivity to MCLR than previously documented scAbs and was able to detect levels below guidelines for MCLR in drinking water set by the World Health Organisation (WHO) at 1 μg/L. Despite its sensitivity to MCLR, full characterisation revealed low levels of cross-reactivity with other microcystin variants. <i>In vitro</i> affinity maturation was employed to increase cross-reactivity to other microcystin variants in scab 3A8. A chain-shuffled library was constructed where a naïve V<sub>L</sub> repertoire was shuffled with clone 3A8 V<sub>H</sub> chain. The library was screened and isolated phage antibodies cloned and expressed as scAbs. Chain-shuffled clone CSB-D9 displayed a 50-fold increase in cross-reactivity for a single microcystin variant and was capable of detecting all variants available below the guideline levels of 1 μg/L. The highly cross-reactive scab was used for the determination of total microcystin content in cyanobacterial extracts and results showed good correlation with HPLC quantification. Immunoaffinity chromatography utilising scab CSB-D9 was used for the concentration of trace amount of microcystin from large volumes of water, out-performing columns generated with anti-microcystin whole antibody molecules. The ability of scab CSB-D9 to protect cultured hepatocytes against microcystin-induced apoptosis was also determined. Results indicated a possible therapeutic application for human antibody fragments isolated from phage display libraries.
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Meyer, Scott C. "Non-Covalent Selection Methodologies Utilizing Phage Display." Diss., The University of Arizona, 2007. http://hdl.handle.net/10150/194059.
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In nature, non-covalent interactions are as important and dynamic as they are elusive. As such, the study of non-covalent interactions both in vivo and in vitro has proven to be challenging. Given the potential benefits of elucidating protein-protein, ligand-receptor, and other biologically relevant interactions, the development of methodologies for the study of non-covalent interactions is an attractive goal.Biologically encoded protein and peptide libraries that connect the genotypic information with the expressed phenotype have emerged in recent years as powerful methods for studying non-covalent interactions. One of the quintessential platforms for the creation of such libraries is phage display. In phage display, the connection between genetic information and the corresponding protein allows for the iterative isolation and amplification of library members that possess a desired function. Hence, an in vitro selection can be used to isolate epitopes that bind to desired targets or display specific attributes.We have sought to develop novel phage display methodologies that have the potential to expand the scope of this in vitro selection platform. Specifically, we developed a method for the non-covalent attachment of a small molecule ligand to a cyclic peptide library. This system localizes the phage display library to the ligand binding site, thus allowing for the translation of the selected cyclic peptides to a covalently tethered bivalent inhibitor.The first class of biological molecules that we chose to target with our methodology is the biologically and therapeutically important class of enzymes called protein kinases. In the first demonstration of this strategy, we were able to isolate cyclic peptide ligands for the model kinase PKA (cAMP-dependent protein kinase), which were subsequently translated to a bivalent inhibitor. This inhibitor showed both increased affinity and selectivity for PKA in relation to other protein kinases.In a separate project, we sought to develop a method for the isolation of small molecule-responsive mutants of a well-characterized protein scaffold from a phage display library. During these investigations, we discovered interesting homologous single-point mutations of the protein that resulted in large spherical oligomers that may mimic species relevant to the study of protein misfolding diseases such as Alzheimer's.
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Bonnert, Timothy Peter. "Strategies for making antibodies by phage display." Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294402.
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Ching, Ana Tung Ching. "Identificação de adesinas bacterianas por phage display." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-05022013-084410/.
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A leptospirose é uma zoonose de importância mundial causada por bactérias do gênero Leptospira. No Brasil, a maioria dos casos é causada por L. interrogans sorovar Copenhageni. O objetivo destre trabalho foi identificar adesinas de leptospira pela técnica de Phage display. Bibliotecas com fragmentos genômicos resultaram na idendificação de ligantes de leptospira com afinidade por tecidos de hamster. Uma varredura dessas bibliotecas contra heparan sulfato proteoglicano (HSPG) identificou como ligantes as proteínas LigA e LigB. Proteínas recombinantes foram produzidas e submetidas à ligação às células de mamíferos e aos componentes de matriz extracelular. LigB recombinante foi capaz de se ligar ao HSPG, à heparina e às células de mamíferos. HSPG e heparina foram capazes de reduzir significativamente a interação dessa proteína com as células. Estes resultados evidenciam o papel de proteínas da leptospira na sua interação com o hospedeiro e ilustram a possibilidade do uso da técnica de phage display para identificar possíveis adesinas.<br>Leptospirosis is a worldwide important zoonosis caused by bacteria of the genus Leptospira. In Brazil, most cases is caused by L. interrogans serovar Copenhageni. Our goal was to identify leptospiras adhesins by phage display technique. Libraries of genomic fragments resulted in the identification of ligands with affinity for leptospiras hamster tissues. Screening these libraries against heparan sulfate proteoglycan (HSPG) identified the proteins LigA and LigB. Recombinant proteins were produced and subjected to binding to mammalian cells and extracellular matrix components. LigB recombinant was able to bind to HSPG, heparin and mammalian cells. HSPG and heparin were able to significantly reduce the interaction of this protein with cells. These results highlight the role of leptospiras proteins in its interaction with the host and illustrate the possibility of the use of phage display technique to identify potential adhesins.
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Gomes, Margarida. "Développement de bibliothèques de protéines artificielles permettant la création d’outils de reconnaissance moléculaire innovants." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS030.
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Le travail de thèse présente une approche innovante pour la construction d’une bibliothèque de protéines basées sur l’ossature protéique. L’objectif est de générer une source de biodiversité artificielle permettant la création de nouvelles capacités d’interaction avec des cibles d’intérêts. Une banque, basée sur une ossature protéique bactérienne avait déjà été construite dans l’équipe, mais elle nécessitait d’être optimisée. L’étape initiale a été d’explorer les raisons de l’instabilité des protéines de la banque de première génération, ceci par des approches d’étude de la structure in silico suivie d’une stratégie de mutagenèse dirigée. Des positions déstabilisantes existant dans la première banque ont donc été remplacées dans la banque de deuxième génération. La deuxième étape a eu pour objectif de diminuer le nombre de positions diversifiées et de simplifier le schéma de diversification des variants de la banque. Puis un procédé de filtration et de shuffling de ces variants a été mis au point pour augmenter la proportion de séquences codantes correctes. Une nouvelle stratégie de filtration basée sur la technique d’exposition sur phage a été élaborée, en exploitant le fait que la protéine matrice de la banque, l'ossature protéique a un partenaire biologique capable d’interagir sur la zone « constante », non modifiée par le schéma de diversification. Ainsi les variants de la banque exposés dans une conformation correcte à la surface des phages ont pu être capturés par ce partenaire. Ensuite, les séquences correspondant à ces variants ont été recombinées entre elles pour recréer une plus grande diversité utile. Une bibliothèque optimisée composée de 2.8 x 108 protéines indépendantes a ainsi été obtenue. Cette nouvelle banque optimisée a permis de sélectionner par Phage display, des interacteurs contre plusieurs cibles de structures différentes. Ces nouveaux interrupteurs sont spécifiques de leurs cibles et présentent des affinités de l’ordre du μM. Une approche de séquençage à haut débit a également été entreprise pour réaliser une analyse plus approfondie des séquences de cette bibliothèque et de notre processus de sélection. Cette approche nous a apporté une nouvelle dimension pour la caractérisation des banques construites au laboratoire notamment concernant la diversité réelle de ces banques. Pour le suivi de sélections, nous avons appréhendé le séquençage haut débit comme un moyen d’identifier les interacteurs spécifiques d’une cible par l’analyse exhaustive des séquences issues des sélections. L’objectif est ici de mettre au point un protocole utilisant l’approche NGS pour identifier les interacteurs spécifiques isolées par Phage Display<br>Here new methods to build a library of artificial proteins based on a new protein framework have been developed. The objective is to generate a source of artificial biodiversity allowing the creation of new interaction capacities with various specific targets. A first-generation library was previously built with this scaffold, but it needed to be optimized. The first step was to explore the reasons of the instability of the first generation proteins library through an in silico approaches followed by a site-directed mutagenesis strategy. The second step was to reduce the number of diversified positions and simplify the randomization scheme of the variants of the library. Then a method of filtering and shuffling of the variants of the bank was elaborated. To increase the proportion of correct coding sequences, a new filtration strategy based on the phage display technique has been developed, exploiting the fact that the scaffold of the librarie. This scaffold has a particularly interesting biological partner able to interact on the "constant" zone. This filtration made it possible to recover a set of well-folded clones. Then, DNA sequences corresponding to these clones were recombined with each other to recreate a greater useful diversity. An optimized library of 2.8 x 108 independent proteins was obtained. This new optimized library has enabled us to select, by a phage display approach, binders against several targets of different structures. These new binders are specific for their targets and have affinities in the μM range. A high throughput sequencing (NGS) approach was also undertaken to further analyse the library sequences and the selection process. This approach offers a new dimension for the characterization of the library built in the laboratory, especially concerning it actual diversity. To follow the selections, we have considered the NGS as a way to identify the target-specific binders through exhaustive analysis of the sequences obtained from selections. The objective here is to develop a general protocol using the NGS approach to identify specific binders isolated by Phage Display
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Wright, Michael John. "Anti-tumour chelating recombinant antibodies by phage display." Thesis, Imperial College London, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.405798.
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Madeja, Benjamin [Verfasser]. "Phage Display Screening for Cement Systems / Benjamin Madeja." Konstanz : KOPS Universität Konstanz, 2018. http://d-nb.info/1202440800/34.
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Dueñas, Marta. "Phage display and bacterial expression of antibody fragments." Lund : Dept. of Immunotechnology, Lund University, 1995. http://catalog.hathitrust.org/api/volumes/oclc/38164515.html.
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Gomes, Carlos Henrique Rodrigues. "Construção de uma bibilioteca de anticorpos ScFv dirigidos contra o fator de crescimento vascular (VEGF)." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-09082013-093807/.
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Angiogênese é a formação de novos vasos sanguíneos a partir de vasos já existentes e é importante em processos fisiológicos, que em adultos é restrita ao reparo tecidual e ao ciclo reprodutivo feminino. Entretanto, doenças, como câncer ou retinopatias, induzem a formação da angiogênese patológica, necessária para a progressão destas patologias. Anticorpos monoclonais constituem uma das classes de biofármacos que mais cresce e com impacto importante nas doenças dependentes de angiogênese. Entre as diversas metodologias para a identificação de anticorpos monoclonais contra alvos terapêuticos, está o phage display. Por causa do fator de crescimento endotelial vascular (VEGF) ser o principal fator responsável pela formação de novos vasos, o principal biofármaco anti-angiogênico disponível na clínica atualmente é um anticorpo monoclonal (bevacizumab) direcionadas contra o VEGF. Embora terapias anti-VEGF sejam eficazes, ainda não são ideais devido a efeitos colaterais indesejados e a resistência medicamentosa. Novas alternativas são necessárias a fim de aperfeiçoar as terapias angiogênicas. O objetivo do nosso estudo é identificar novos alvos moleculares e desenvolver novos agentes terapêuticos para doenças dependentes de angiogênese. Para atingirmos nossa meta escolhemos o sistema de phage display para selecionarmos anticorpos com propriedades angiogênicas. Uma biblioteca de de anticorpos foi desenvolvida em nosso laboratório, dirigida contra a molécula VEGF, em particular uma de suas isoformas. Os animais imunizados desenvolveram anticorpos específicos, detectados por ELISA e Western-blot. A amplificação do pool de genes das cadeias leve e pesada de imunoglobulinas foi realizada para produzir os fragmentos single-chain (ScFv) que foram então clonados no vetor para a construção da biblioteca. A biblioteca de display de anticorpos ScFv será, portanto, analisada em plataformas angiogênicas para isolar anticorpos específicos contra isoformas de VEGF e novos marcadores moleculares de superfície celular expressos por células endoteliais ativadas.<br>Angiogenesis is the formation of new blood vessels from pre-existing ones and is an important physiological process, which in adults is mostly restricted to wound healing or the female reproductive cycle. However, different illnesses, such as cancer or retinopathies, induce the formation of pathological angiogenesis, necessary for disease progression. Monoclonal antibodies are one of the fastest growing class of biopharmaceuticals with important implications in angiogenesis dependent diseases. Among various methods for the identification of monoclonal antibodies against therapeutic targets is phage display technology. Because the vascular endothelial growth factor (VEGF) is the main molecular factor responsible for the formation of new blood vessels, the major anti-angiogenic drug available in the clinic today is a monoclonal antibody (bevacizumab) directed against VEGF. However, although anti-VEGF therapies are effective, they are not yet ideal due to undesirable side effects and drug resistance. Novel alternatives are necessary to improve on angiogenic therapies. The aim of our study is to identify novel molecular targets and to develop new therapeutic agents for angiogenic dependent diseases. To achieve our goal we have chosen the phage display system in order to select for antibodies with angiogenic properties. An antibody phage library has been developed in our laboratory, directed against VEGF molecule, particularly one of it isoforms. The animals were immunized and developed specific antibodies, detected by ELISA and Western-blot. Amplification of the pool of light and heavy chain Ig genes was performed to produce the single chain (ScFv) fragments for library construction. The ScFv antibody display libraries will be then screened in angiogenic settings to isolate antibodies against specific VEGF isoforms and novel cell surface molecular markers expressed by activated human endothelial cells
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Mathonet, Pascale. "Création d'un site allostérique dans la beta-lactamase TEM-1 par évolution dirigée." Université catholique de Louvain, 2004. http://edoc.bib.ucl.ac.be:81/ETD-db/collection/available/BelnUcetd-04282004-121335/.
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L'objectif de ce travail est d'introduire, par ingénierie génétique, un site allostérique dans une enzyme pour moduler son activité catalytique par la liaison d'un ligand choisi. Ces enzymes pourraient servir de senseurs moléculaires pour la détection, voire le dosage du ligand. Nous avons décidé de créer ces sites allostériques dans la beta-lactamase TEM-1 d'E. coli, une enzyme dégradant les antibiotiques de type pénicilline, en réalisant l'ingénierie de trois boucles de l'enzyme. Celles-ci sont proches l'une de l'autre dans l'espace et pourraient servir d'épitope discontinu pour la reconnaissance par un anticorps ou de paratope, à savoir le site de liaison d'un anticorps, pour la reconnaissance d'autres protéines ou de petites molécules. Notre hypothèse de travail est que ces beta-lactamases chimériques partageront certaines similitudes avec les anticorps de camélidés dont le site de reconnaissance n'est constitué que de trois boucles. Comme nous ne savions pas a priori quelles sont les séquences à introduire pour lier la molécule cible, nous avons introduit des séquences dégénérées pour créer une grande banque de mutants et sélectionner dans cette banque les clones ayant une affinité pour le ligand d'intérêt. La partie la plus difficile de ce projet fut la création de la collection d'enzymes mutantes contenant trois boucles dégénérées. La dégénérescence de la boucle 1, située à proximité du site actif, a été réalisée en remplaçant aléatoirement trois résidus. Dans la boucle 2, un peptide aléatoire de cinq, six ou sept résidus a été inséré entre deux cystéines et dans la boucle 3, une insertion de six résidus aléatoires a été réalisée. En pratique, l'ingénierie des boucles 1 et 2 a été effectuée dans la même construction pour créer trois banques (trois tailles d'insert différentes dans la boucle 2). D'autre part, nous disposions déjà de la banque contenant l'insertion dans la boucle 3. Ces banques de première génération ont ensuite subit une sélection in vivo pour ne garder que les clones possédant une certaine activité catalytique, par culture des bactéries infectées sur un milieu contenant de l'ampicilline. Cette étape nous a permis d'avoir un contrôle sur la qualité des banques créées car tous les mutants d'insertions retenus ont obligatoirement une structure tertiaire. Nous avons également montré que la présence de deux résidus cystéines est nécessaire de part et d'autre de l'insert dans la boucle 2 pour maintenir une certaine activité enzymatique. D'autre part, la boucle 1 ne semble pas tolérer d'insertion peptidique et ne permet que la mutagenèse par remplacement. Les banques de première génération ont ensuite été combinées pour créer une banque de seconde génération contenant les trois boucles dégénérées. Cette construction hiérarchisée a permis, avec un bon rendement, la création d'une banque combinatoire de grande taille et de bonne qualité. Grâce à la technique d'expression en surface de phage, cette banque a ensuite été soumise à une sélection in vitro afin d'isoler les clones ayant acquis de l'affinité pour des molécules cibles. Ces ligands sont un ion métallique, le Ni(II), et deux petites molécules organiques, la kanamycine et la sulfanilamide, pour lesquelles une protéine liante serait intéressante comme outil de détection. En ce qui concerne les clones sélectionnés avec le nickel, tous les clones testés contiennent des résidus histidine et montrent une modulation de l'activité en présence du métal, que ce soit une activation ou une inhibition de celle-ci. La meilleure activation a été obtenue pour le clone Ni-5-Amp20-#11, qui voit son activité doublée lorsqu'il est complexé au nickel, tandis que la meilleure inhibition a été obtenue pour le clone Ni-5-#2 avec une diminution de 77 % de l'activité. Quant à la meilleure affinité, elle a été obtenue pour le clone Ni-5-#12 avec une constante de dissociation de 7.10-5 M. Les autres valeurs de Kd varient entre 10-4 M et 10-3 M. Parmi les clones issus de la sélection avec la kanamycine, nous avons trouvé un mutant dont l'activité catalytique est augmentée de 44 % en présence du ligand. Sa constante de dissociation est de 6,6.10-4 M. Quant à la sélection avec la sulfanilamide, elle ne nous a pas permis à ce jour d'isoler des clones ayant acquis une affinité détectable pour cette molécule. Ces résultats nous laissent espérer que la banque construite contient potentiellement des clones ayant la capacité de lier une variété de cibles différentes. Si c'est le cas, ces enzymes chimériques possèdent des avantages sur les anticorps conjugués habituellement utilisés en immuno-détection, car la fonction de reconnaissance et la fonction enzymatique sont portées par la même molécule. Ces protéines pourraient donc être produites facilement et à plus faible coût. De plus, la modulation d'activité permet en principe de détecter la présence du ligand cible en phase homogène.
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Murarasu, Thomas. "The Shiga Toxin B-Subunit : a Promising Scaffold for the Targeting of Tumor Specific Glycosphingolipids." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS512.
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Le cancer représente la second cause de décès au monde. Le développement de traitements innovants contre le cancer repose aujourd’hui sur l’identification de biomarqueurs des tumeurs et le développement de produits thérapeutiques capables de reconnaitre ces marqueurs de façon spécifique. Ces produits thérapeutiques de nouvelles générations ont le potentiel d’éliminer spécifiquement les cellules tumorales et donc de réduire les effets secondaires des traitements ainsi que les risques de rechute. Malheureusement, un certain nombre de patients ne peuvent bénéficier de ces traitements, du fait de l’absence de biomarqueurs connus à la surface de leur tumeur. Ce projet a ainsi pour ambition de développer de nouvelles thérapies ciblées en exploitant une nouvelle classe de biomarqueurs et ainsi de venir enrichir l’arsenal thérapeutique disponible pour le traitement des cancers<br>Cancer is the second cause of death worldwide. Recent advance in cancer treatments involved the identification of cancer biomarkers and the development of efficient therapeutic products able to specifically recognize them. This new class of products has the ability to specifically target tumor cells, with the major advantages to decrease or abolish treatments side effects and relapses of the disease. Unfortunately, a certain number of patients do not respond to those treatments lacking the expression of those biomarkers on their tumor. This project aims at developing new targeted therapies by exploiting a new class of cancer biomarkers, which would potentially extend the therapeutics options against cancer
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Wilson, Daniel Ross. "Multivalent pIII phage display libraries, selected issues and applications." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq25187.pdf.
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Yeoh, Tsin Inn Louisa. "Single-chain antibody engineering against mecoprop using phage display." Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429362.
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Bell, Matthew John. "Phage display of a cDNA library from Arabidopsis thaliana." Thesis, Coventry University, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.393652.
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Chen, Kuan Ming. "Submillisecond-response blue phase liquid crystals for display applications." Doctoral diss., University of Central Florida, 2012. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/5158.
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With exploding growth of information exchanges between people, display has become indispensable in our daily lives. After decades of intensive research and development in materials and devices, and massive investment in manufacturing technologies, liquid crystal display (LCD) has overcome various obstacles and achieved the performance we need, such as wide viewing angle, high contrast ratio, and high resolution, etc. These excellent performances make LCD prevailed in every perspective. Recently, with the demands of energy conservation, a greener LCD with lower power consumption is desired. In order to achieve this goal, new energy-effective driving methods, such as field sequential color display, have been proposed. However, in order to suppress color breakup the LC response time should be faster than 1 ms. To overcome this challenge, various fast-response liquid crystal modes, such as thin cell gap, low viscosity materials, overdrive and undershoot voltages, polymer stabilization, and ferroelectric liquid crystal, are under active investigations. Among these approaches, blue phase liquid crystal (BPLC) shows a greater potential with less fabrication limitations. In this dissertation, the feasibility of polymer-stabilized blue phase liquid crystal for display applications is explored starting from the building blocks of the material system, polymer-stabilization processes, test cell preparations, electro-optical (EO) properties, to suggested approaches for further improvements. Because of the nature of blue phase liquid crystals, delicate balance among system components is critically important. Besides the properties of each composition, the preparation process also dictates the EO performance of the self-assembled nano-structured BPLC composite. After the preparation of test cells, EO properties for display applications are investigated and results described. Approaches for further improvements of the EO properties are also suggested in the final part of this dissertation.<br>ID: 031001409; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Title from PDF title page (viewed June 12, 2013).; Thesis (Ph.D.)--University of Central Florida, 2012.; Includes bibliographical references (p. 82-89).<br>Ph.D.<br>Doctorate<br>Optics and Photonics<br>Optics and Photonics<br>Optics
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Kelly, Michael A. "Developing Peptide Probes for Membrane Lipids via Phage Display:." Thesis, Boston College, 2020. http://hdl.handle.net/2345/bc-ir:108919.
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Thesis advisor: Jianmin Gao<br>Lipid reporters are key signaling molecules in a number of biological processes ranging from apoptosis in mammalian cells to novel resistance mechanisms in pathogenic bacteria. Developing probes to target these lipids is a worthy endeavor, especially when better reporters could mean lives saved. This is particularly true considering new antibiotic resistant pathogens emerge every year with evolving lipid compositions. To combat these pathogens and prevent a potential global pandemic, it is imperative to continue the development of novel and innovative probes/drugs to meet this daunting challenge. To fulfill this demand, we must continue to establish new strategies, enhance current technologies and advance scientific understanding. Only by pushing the boundaries of what is currently possible will we remain one step ahead of these diseases. Diseases like mcr-1 positive bacteria, first documented in 2016, remain largely uncontested. Herein, we seek to expand the available probes specific to key lipid reporters for phosphatidylserine, lysyl-phosphatidylglycerol, and phosphoethanolamine lipid A. Cyclic phage libraries were first utilized to target phosphatidylserine, ultimately producing weak binders. Refining our phage display libraries to include reversible covalent warheads allowed for the identification of more potent lipid reporters. In doing so, we have created the tools necessary to interrogate the unique resistance mechanisms expressed by these drug-resistant pathogens. A strong correlation was observed between peptides binding mcr-1 positive strains, LPS modification on the surface of these bacteria, and level of colistin resistance. To our knowledge, these peptides are the only probes capable of demonstrating this correlation. We surmise that the methods discussed here will pave the way for better diagnostic tools for these resistant pathogens. A recurring method of resistance among gram-positive and gram-negative bacteria has been to decorate their surface with positive amines to repel cationic antimicrobial peptides. As such, our current APBA library and the libraries in development in the Gao lab would be ideally suited to target these and other undiscovered resistance mechanisms<br>Thesis (PhD) — Boston College, 2020<br>Submitted to: Boston College. Graduate School of Arts and Sciences<br>Discipline: Chemistry
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Filipovic, Nedim. "Phage display to identify functional resistance mutations to Rigosertib." Scholarship @ Claremont, 2017. http://scholarship.claremont.edu/cmc_theses/1475.
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In vitro protein selection has had major impacts in the field of protein engineering. Traditional screens assay individual proteins for specific function. Selection, however, analyzes a pool of mutants and yields the best variants. Phage display, a successful selection technique, also provides a reliable link between variant phenotype and genotype. It can also be coupled with high throughput sequencing to map protein mutations; potentially highlighting vital mutations in variants. We propose to apply this technique to cancer therapy. RAF, a serine/threonine kinase, is critical for cell regulation in mammals. RAF can be activated by oncogenic RAS, found in over 30% of cancers, to drive cancer proliferation. Rigosertib, a benzyl styryl sulfone in phase III clinical trials for myelodysplastic syndrome (MDS), is an inhibitor of the RAS binding domain (RBD) in RAF. Phage display can be used to select RAF mutants for RAS binding affinity, in the presence of Rigosertib. High-throughput sequencing of these variants can provide a means of anticipating, and mapping resistance to this anti-cancer drug.
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Johansson, Louise. "Proteomic peptide phage display of syntenin-1 and scribble." Thesis, Uppsala universitet, Institutionen för kemi - BMC, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-255887.
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Scott, Nathan. "Anti-tetanus toxin chelating recombinant antibodies by phage display." Thesis, Imperial College London, 2008. http://hdl.handle.net/10044/1/4638.
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Chelating Recombinant Antibodies (CRAbs) are a form of high affinity tandem single chain antibody (scFv) with two component scFvs which target non‐overlapping epitopes on the same antigen molecule. The optimised inter‐scFv linker allows simultaneous engagement of the scFvs resulting in a synergistic improvement in affinity. High affinity is a desirable characteristic in therapeutic antibodies since it strongly correlates with improved potency. Building upon previous work, this thesis describes the development of a phage‐display based approach toward isolating CRAbs with both the optimal scFv pairing and inter‐scFv linker using tetanus toxin as a model antigen. This would circumvent the need for structural data and complement the traditional approaches used to enhance antibody affinity. Fifteen scFvs specific for the C‐terminal sub‐domain of the tetanus toxin heavy chain (TT‐Hc) were characterised in terms of overall and soluble expression levels, sequence and bioinformatics features, phage‐display propensity and toxin neutralisation potency. Seven clones were identified for further analysis by affinity measurements, oligomerisation analysis and quantitative toxin neutralisation capacity. These characteristics varied significantly between clones including the affinity (KD) which varied, from 10nM to over 1000nM. ScFv characterisation revealed a strong positive correlation between phage display propensity and overall expression levels as well as a strong correlation between clone affinity and potency. Multiple scFv pairings hypothetically capable of chelation were identified by competition binding analysis and many of these clones exhibited cooperative binding effects when added in combination to TT‐Hc. A tandem‐scFv library was constructed linking seven of the characterised scFv clones (C1, C2, C4, J2, J4, N4 and N5) in all 49 possible combinations and permutations each with 7 inter‐scFv linker lengths. The inter‐scFv linkers harboured some randomised codons giving a total diversity of 5x107 clones. The library contained a mixture of hypothetically chelating and competing scFv pairings. Carefully optimised affinity driven phage display which included off‐rate selection enriched the anti‐TT‐Hc tandem‐scFv library to up to 90% chelating clones from under 50% after just two‐rounds of selection with a single clone, C4‐N4, dominating around 30% of the output. This clone was classified as hypothetically chelating from the scFv competition binding experiments. Despite some modest biases in the constructed library, the results clearly indicated that phage display can enrich for an optimal and chelating scFv pairing. This approach for isolating CRAbs will allow for the facile isolation of very high affinity antibodies against any target of interest given a sufficiently diverse panel of scFvs circumventing the need for prior scFv characterisation or elucidation of antibody‐antigen structural data.
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Tomini, Khaled. "The isolation of novel leptin variants using phage display." Thesis, University of Sheffield, 2015. http://etheses.whiterose.ac.uk/12363/.
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Leptin is a polypeptide hormone with important roles in a number of physiological pathways. Most significantly leptin acts a major regulator of energy metabolism and defects in hormone signalling form the basis of a number of human conditions, including morbid obesity, cardiovascular disease, and diabetes. Leptin is also an important immunomodulatory hormone and may contribute to a range of inflammatory diseases. For these reasons there is significant interest in the development of novel forms of leptin as possible therapeutic agents. Research into therapeutic applications of leptin has focused on attempts to engineer the leptin molecule to introduce properties such as improved affinity for the corresponding receptor, increased protein stability, and the development of both agonists and antagonists. These efforts have been largely based on rational engineering of the hormone based on structural studies and site-directed mutagenesis. Phage display of proteins and guided enrichment by selective biopanning is a powerful technique that allows the sampling of very large populations of protein variants. In this study I expressed functional leptin on the surface of filamentous phage and used this technique to synthesise a library of random mutant leptins in the form of a phage library. A selective procedure was developed based on immobilised leptin receptor and a competitive binding strategy to select novel variants from the leptin mutant library with increased receptor affinity. The mutants were characterised and leptin proteins expressed and purified. The recombinant leptins were analysed for receptor stimulating activity and receptor affinity determined in real-time studies using biointerferometry. A novel leptin mutant was recovered with increased receptor affinity and agonist activity. This study established an approach for further phage based studies of leptin and other polypeptide hormones.
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Magalhães, Leila da Silva. "Importância do domínio extracelular do receptor tirosina quinase Tie1 na angiogênese." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-11042017-081713/.
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Tie1 é um receptor tirosina quinase expresso em células endoteliais importante em angiogênese, formação de vasos sanguíneos a partir de vasos pré-existentes. Este receptor pertence a uma família pequena composta por apenas dois membros (Tie1 e Tie2) para os quais angiopoietinas foram identificadas como ligantes de Tie2. No entanto, Tie1 continua a ser um receptor órfão, sem ligantes identificados até o momento. Sendo assim, é difícil compreender completamente as propriedades biológicas de Tie1 e seus mecanismos moleculares em angiogênese sem um ligante identificado. Entretanto, como sugerido através de estudos de deleção gênica, este receptor é uma molécula essencial na angiogênese, apresentando um papel importante no desenvolvimento da vascularização da retina e desenvolvimento de tumores. O nosso objetivo foi estudar a participação do domínio extracelular de Tie1 na neovascularização e, no processo, identificar possíveis ligantes para este receptor. Através da tecnologia de phage display, identificamos um peptídeo específico e seletivo para Tie1, sugerindo a existência de um sítio de ligação único neste receptor. Mostramos que este peptídeo é capaz de inibir a proliferação de células endoteliais induzida por Ang1, um ligante bem caracterizado de Tie2 que também modula a atividade de Tie1. Além disso, também mostramos que este peptídeo inibe a angiogênese in vivo num modelo animal bastante relevante para estudo de doenças humanas, o modelo da retinopatia induzida por oxigênio. Uma vez que este peptídeo liga-se a um sítio único e seletivo para Tie1, hipotetizamos que o mesmo poderia mimetizar possíveis ligantes naturais deste receptor. Para identificá-los, proteínas com mimetopo cruzado com este peptídeo foram identificadas em extrato proteico de diferentes linhagens celulares. Tais proteínas são possíveis candidatos a interação com Tie1. Em resumo, demonstramos que o domínio extracelular de Tie1 é importante para a angiogênese patológica e identificamos proteínas como possíveis ligantes deste receptor, o que poderá contribuir para um melhor entendimento da participação de Tie1 na formação de vasos. O peptídeo aqui identificado poderá ser ainda uma ferramenta útil para o desenvolvimento de novas terapias anti-angiogênicas com importantes aplicações à saúde humana.<br>Tie1 is a tyrosine kinase receptor expressed by endothelial cells and important in angiogenesis, the formation of new blood vessels from pre-existing ones. This receptor belongs to a small family of receptors composed of two members only (Tie1 and Tie2) to which angiopoietins have been identified as ligands for Tie2. On the other hand, Tie1 is still an orphan receptor with no ligand identified to date. Thus, it is difficult to assess Tie1 mechanism of action in neovascularization without a known ligand. Nevertheless, gene deletion studies have shown that Tie1 is essential in angiogenesis, and plays an important role in retinal and tumoral vascularization. The aim of our study was to evaluate the participation of Tie1 extracellular domain in angiogenesis, and in the process, to identify putative ligands for this receptor. Utilizing phage display, we have identified and characterized a Tie1 specific and selective ligand peptide, which suggests the existence of a binding site unique to this receptor and not shared by other family members. We show that this peptide prevents endothelial cells proliferation, induced by angiopoetin-1, a ligand for Tie2 but which also modulates Tie1 activity. Using a well-accepted mouse model for human diseases, the oxygen induced retinopathy model, we show that this peptide inhibits angiogenesis in vivo. Since this peptide maps to a unique binding site in Tie1, we hypothesized that it might mimic a natural ligand for this receptor. To identify them, proteins with cross reactive epitopes with an anti-peptide sera were identified by proteomic approaches. These proteins are thus possible ligands for Tie1. In summary, we have shown that Tie1 extracellular domain is important in angiogenesis and we have identified putative ligand for this receptor, which might contribute to a better understanding of the molecular mechanisms associated with Tie1 in blood vessel formation. The peptide here characterized may also be an important tool for the development of novel anti-angiogenesis therapeutic approaches for disesase with an angiogenic component.
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Lima, Swiany Silveira. "Identificação de adesinas de Leptospira interrogans por shotgun phage display." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-02052013-095417/.
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Em Leptospira interrogans algumas proteínas com capacidade de ligação aos componentes de matriz extracelular foram identificadas e, em sua maioria, são fatores de virulência. Phage display é considerada uma técnica poderosa na identificação de novos ligantes, inclusive de moléculas adesinas, importantes no primeiro estágio de infecção do hospedeiro. A técnica de shotgun phage display foi utilizada visando à obtenção de ligantes à células de mamíferos. Quatro bibliotecas, por inserção de fragmentos aleatórios obtidos por sonicação do DNA de L. interrogans nos fagomídeos pG8SAET (BBT1 e BBT2) e pG3DSS (BBT5 e BBT6), foram construídas. As bibliotecas BBT1 e BBT5 contém insertos maiores e as BBT2 e BBT6 contém insertos menores, com tamanhos médios de 1500 pb e 350 pb, respectivamente. Após ensaio de panning da BBT5 contra células de mamíferos e soro fetal bovino, as sequências de clones selecionados foram analisadas quanto a orientação correta e se a fusão estava em fase com a proteína pIII. As proteínas codificadas pelos genes LIC11719, LIC10769, LIC13143 e LIC12976 foram selecionadas com estas características. Os genes que codificam a LIC12976, LIC10768, LIC10769 e LIC13418, tiveram sua conservação avaliada em diferentes sorovares da espécie patogênica L. interrogans e no sorovar Patoc da espécie de vida livre L. biflexa. As proteínas LIC12976 (selecionada pela técnica de phage display) e LIC13418 (selecionada por ferramentas de bioinformática) tiveram suas sequências amplificadas por PCR, clonadas em pGEM T easy, subclonadas em vetor de expressão pAE e expressas na fração celular correspondente ao corpúsculo de inclusão em E. coli BL21 (DE3) Star pLysS e E. coli BL21 SI, respectivamente. Após renaturação e purificação destas proteínas por cromatografia de afinidade a metal bivalente, um grupo de cinco animais BALB/c fêmeas foi imunizado. Ambas as proteínas se mostraram imunogênicas com títulos dos soros policlonais 1:256000 e 1:512000, respectivamente. Em ensaio de Western Blot os soros foram específicos no reconhecimento das proteínas recombinantes e as proteínas nativas foram verificadas em extratos de sorovares patogênicos de L. interrogans. Em ensaios de adesão, as proteínas recombinantes aderiram às células A31, LLC-PK1 e Vero e especificamente à laminina. Em ensaios de interferência em células usando laminina houve um aumento da adesão das proteínas recombinantes, o que pode ser explicado pela ligação da laminina às células e uma maior ligação das LICs estudadas. Em ensaio de localização celular usando imunofluorescência e microscopia eletrônica, foi observado que ambas as proteínas se encontram na superfície da L. interrogans. No experimento de desafio animal, a LIC12976 e a LIC13418 não se mostraram protetoras. Este trabalho contribuiu para a identificação das novas adesinas LIC13418 e LIC12976 que podem participar da virulência de leptospiras patogênicas envolvendo a primeira etapa da infecção na interação patógeno-hospedeiro<br>In Leptospira interrogans, proteins capable to bind to extracellular matrix components have been identified and most of them are important virulence factors. Phage display is a powerful technique to identify new ligands, including adhesin molecules that are important in the first stage of host infection. A shotgun phage display technique was used in order to obtain cell ligands. Four libraries were constructed by inserting random fragments obtained by sonication of L. interrogans DNA into phagemids pG8SAET (BBT1 and BBT2) and pG3DSS (BBT5 and BBT6). The libraries BBT1 and BBT5 contain larger inserts and BBT2 and BBT6 contain smaller inserts, with 1500 bp and 350 bp average sizes, respectively. After panning of BBT5 against mammalian cells and bovine fetal serum, the sequences of selected clones were analyzed for correct orientation and fusion with pIII protein. The proteins encoded by genes LIC11719, LIC10769, LIC13143 and LIC12976 were selected. The genes LIC12976, LIC10768, LIC10769 and LIC13418 were evaluated for their conservation in different pathogenic serovars of L. interrogans and free-living L. biflexa serovar Patoc. Proteins LIC12976 (selected by phage display technique) and also LIC13418 that was selected by bioinformatic tools, were amplified by PCR, cloned into pGEM T easy, subcloned into expression vector pAE and expressed in cellular fraction corresponding to the inclusion body in E. coli BL21 (DE3) Star pLysS and E. coli BL21 SI, respectively. After protein renaturation protocol and purification by affinity chromatography, a group of five BALB/c mice was immunized with the purified proteins. Both proteins were shown to be immunogenic with 1:256000 and 1:512000 polyclonal sera titers, respectively. In Western blot the sera were specific to recognize recombinant proteins and native proteins were detected in pathogenic L. interrogans serovars extracts. In binding assays, recombinant proteins bind to A31, LLC-PK1 and Vero cells and specifically to laminin. In interference cell assay using laminin there was an increase of recombinant protein bindings, which can be explained by the laminin binding to cells and further binding of the recombinant LICs. In cellular localization assay using immunofluorescence and electron microscopy, it was observed that both are surface proteins of L. interrogans. In the animal challenge, the LIC12976 and LIC13418 were not protective. As a whole, this work contributed to the identification of LIC12976 and LIC13418 as new adhesins and they can participate in the virulence of pathogenic Leptospira in the first stage of host pathogen interaction.
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Kioshima, Érika Seki [UNIFESP]. "Caracterização de marcadores de virulência em Paracoccidioides brasiliensis." Universidade Federal de São Paulo (UNIFESP), 2009. http://repositorio.unifesp.br/handle/11600/9171.
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Previous issue date: 2009-06-24<br>Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)<br>A paracoccidioidomicose (PCM) é uma doença sistêmica de caráter granulomatoso, prevalente na América do Sul, causada pelo fungo termodimórfico Paracoccidioides brasiliensis. O fungo apresenta estrutura complexa de proteínas e glicoproteínas e dentre esses componentes, alguns estão envolvidos na patogenicidade da doença. A recuperação da virulência de isolados por meio de passagem in vivo foi realizada com sucesso em nosso laboratório. Os isolados Pb18 atenuado e virulento foram analisados sob diversos ângulos para confirmação dessa modificação. Os resultados de curva de sobrevida, recuperação de CFU de órgão e histologia, mostraram claramente as diferenças no padrão de patogenicidade desses dois isolados. Outras características também foram avaliadas como morfometria, padrão de crescimento e ultraestrutura celular. Análises de expressão gênica diferencial apontaram um perfil de regulação positiva para genes relacionados ao metabolismo de proteínas, de lipídeos e aminoácidos. Algumas moléculas anteriormente descritas como putativos fatores de virulência foram moduladas positivamante, entre as quais podemos citar a calmodulina, proteína kex-like e hsp70. Entretanto, ainda pouco se sabe sobre estes fatores virulência para maioria dos fungos dimórficos, entre eles o P.brasiliensis. Várias técnicas têm sido utilizadas, sem sucesso, para caracterização destas moléculas. Utilizando a metodologia de Phage display, foram selecionados três fagos que se ligam preferencialmente ao isolado Pb18 virulento. Por meio de ensaio de ligação, o fago p04 foi capaz de distinguir graus de virulência de outros quatro isolados. As imagens obtidas por microscopia confocal mostraram que o pep04, acoplado a 6-FAM, foi internalizado somente por leveduras do isolado virulento. Imagens seriadas indicam marcação do meio intracelular, frequentemente associada ou co-localizada à marcação por DAPI. Estes resultados demonstraram que ambos, pep04 e p04, podem ser considerados biomarcadores de virulência na PCM. Para avaliar as consequências das interações destes biomarcadores com células fúngicas, foram realizados ensaios in vitro e in vivo. O fago p04 foi suficiente para impedir a implantação e disseminação do fungo. Além disso, foi capaz de reduzir o número de CFUs recuperadas de animais tratados com este fago, quando comparado ao controle (fago sem inserto). Experimentos in vitro utilizando o pep04 demostraram a atividade fungicida deste peptídeo contra, apenas, o isolado virulento. Desta foram, estes biomarcadores poderão ser utilizados tanto no diagnóstico, quanto na pratica clínica como adjuvante terapêutico.<br>Paracoccidioidomycosis (PCM) is a human systemic granulomatous disease, prevalent in South America, caused by a thermodimorphic fungus, Paracoccidioides brasiliensis. This fungus presents complex antigenic structure and some of these components have been related with its pathogenicity, of which little is known. The virulence recovery of isolates by passage in vivo was performed in our laboratory. Attenuated and virulent Pb18 isolates were analyzed from various angles to confirm this change. The results of the survival curve, the number of CFUs and histology, showed clear differences in pathogenicity pattern of these isolates. Other features were also evaluated as morphology, growth curve and cell ultrastructure. Analysis of differential gene expression profile showed positive regulation genes related to metabolisms of proteins, lipids and amino acids. Some molecules, previously described as putative virulence factors, were positive regulated, among which calmodulin, kex-like protein and Hsp70. However, the number of defined virulence factors for dimorphic fungal pathogens, up to now, is relatively small. Several techniques have unsuccessfully been employed to characterize these elusive antigenic structures. Using phage display methodology, three peptide-displaying phages that bound preferentially to virulent isolates of P. brasiliensis were selected. By binding assay, p04 phage distinguished predefined degrees of virulence of isolates. Using confocal microscopy, the homologue synthetic peptide (pep04), labeled with 6-FAM, was internalized by only virulent isolate yeast cells. Sequential optical section imaging indicated that the labeling was within the intracellular milieu and frequently close or overlapping DAPI staining. These results showed that both, phage p04 and pep04, can be considered as biomarkers of virulence in PCM since both bound to virulent P. brasiliensis. To evaluate the consequences of interactions between the biomarkers and fungal cells, in vitro and in vivo experiments were performed. The latter demonstrated that p04 phage was sufficient to prevent the implantation of the fungus in the lungs and its migration to spleen and liver. In addition, this phage reduced colony-forming units in the lungs of mice infected with P. brasiliensis as compared to controls. In vitro experiments showed that pep04 exhibited fungicidal activity only against virulent P. brasiliensis, leaving unaltered the growth of the attenuated isolate. Therefore, these biomarkers may be useful tools for prognosis in PCM and may be possibly used in the routine clinical practice as therapeutic drug adjuvants.<br>TEDE<br>BV UNIFESP: Teses e dissertações
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Minter, Ralph. "Development of antibody technology to identify natural killer cell surface antigens in Xenopus laevis." Thesis, Durham University, 1999. http://etheses.dur.ac.uk/4598/.
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Natural killer (NK)-like lymphocytes have recently been identified in thymectomised (Tx) Xenopus which are capable of spontaneous cytotoxicity towards the MHC- deficient, allogeneic thymus tumour cell line B(_3)B(_7). This Thesis describes attempts to raise antibodies to Xenopus NK cell surface antigens by phage display and hybridoma technology. The phage display technique was optimised for raising antibodies to novel, cellular antigens in a trial run using the Xenopus thymus tumour cell line B(_3)B(_7). Having isolated a phage antibody which was shown by flow cytometry to bind B(_3)B(_7) cells, the technique was then used to try and raise antibodies to Xenopus NK cells. Isolation of an NIC-specific phage antibody was not achieved but phage antibody XL-6 was raised, which bound an antigen on Xenopus lymphocytes. Phage antibody XL-6, and soluble scFv derived from this, were able to identify a putative mature T cell population in the thymus and may be specific for an amphibian homologue of the mammalian leukocyte common antigen CD45. Hybridoma technology was used to isolate three monoclonal antibodies, 1F8, 4D4 and 1G5, which were shown by flow cytometric analysis to identify a putative NK cell population in control and Tx Xenopus. Following immunomagnetic purification, 1F8- positive spleen cells from control and Tx animals were shown to kill the MHC- deficient tumour target B(_3)B(_7), confirming that this antibody was specific for Xenopus NK cells. Western blotting experiments showed that 1F8, 4D4 and 1G5 identified a doublet of protein bands at 72 and 74 kilodaltons in Xenopus gut lymphoid lysates. Initial attempts to isolate cDNA encoding a Xenopus NK cell surface antigen through immunoscreening a xenopus gut cDNA expression library with antibody 1G5 were unsuccessful as was an attempt to clone a Xenopus homologue of the mammalian NK receptor NKR-Pl by PGR.
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Kwiatkowski, Eric. "Towards a novel group B meningococcal vaccine : peptide mimicry of capsular polysaccharide epitopes." Thesis, University of Nottingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.324533.
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Li, Yan. "High-efficiency Blue Phase Liquid Crystal Displays." Doctoral diss., University of Central Florida, 2012. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/5399.
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Blue phase liquid crystals (BPLCs) have a delicate lattice structure existing between chiral nematic and isotropic phases, with a stable temperature range of about 2 K. But due to short coherent length, these self-assembled nano-structured BPLCs have a fast response time. In the past three decades, the application of BPLC has been rather limited because of its narrow temperature range. In 2002, Kikuchi et al. developed a polymer stabilization method to extend the blue-phase temperature range to more than 60 K. This opens a new gateway for display and photonic applications. In this dissertation, I investigate the material properties of polymer-stabilized BPLCs. According the Gerber's model, the Kerr constant of a BPLC is linearly proportional to the dielectric anisotropy of the LC host. Therefore, in the frequency domain, the relaxation of the Kerr constant follows the same trend as the dielectric relaxation of the host LC. I have carried out experiments to validate the theoretical predictions, and proposed a model called extended Cole-Cole model to describe the relaxation of the Kerr constant. On the other hand, because of the linear relationship, the Kerr constant should have the same sign as the dielectric anisotropy of the LC host; that is, a positive or negative Kerr constant results from positive or negative host LCs, respectively. BPLCs with a positive Kerr constant have been studied extensively, but there has been no study on negative polymer-stabilized BPLCs. Therefore, I have prepared a BPLC mixture using a negative dielectric anisotropy LC host and investigated its electro-optic properties. I have demonstrated that indeed the induced birefringence and Kerr constant are of negative sign. Due to the fast response time of BPLCs, color sequential display is made possible without color breakup. By removing the spatial color filters, the optical efficiency and resolution density are both tripled. With other advantages such as alignment free and wide viewing angle, polymer-stabilized BPLC is emerging as a promising candidate for next-generation displays. However, the optical efficiency of the BPLC cell is relatively low and the operating voltage is quite high using conventional in-plane-switching electrodes. I have proposed several device structures for improving the optical efficiency of transmissive BPLC cells. Significant improvement in transmittance is achieved by using enhanced protrusion electrodes, and a 100% transmittance is achievable using complementary enhanced protrusion electrode structure. For a conventional transmissive blue phase LCD, although it has superb performances indoor, when exposed to strong sunlight the displayed images could be washed out, leading to a degraded contrast ratio and readability. To overcome the sunlight readability problem, a common approach is to adaptively boost the backlight intensity, but the tradeoff is in the increased power consumption. Here, I have proposed a transflective blue phase LCD where the backlight is turned on in dark surroundings while ambient light is used to illuminate the displayed images in bright surroundings. Therefore, a good contrast ratio is preserved even for a strong ambient. I have proposed two transflective blue phase LCD structures, both of which have single cell gap, single gamma driving, reasonably wide view angle, low power consumption, and high optical efficiency. Among all the 3D technologies, integral imaging is an attractive approach due to its high efficiency and real image depth. However, the optimum observation distance should be adjusted as the displayed image depth changes. This requires a fast focal length change of an adaptive lens array. BPLC adaptive lenses are a good candidate because of their intrinsic fast response time. I have proposed several BPLC lens structures which are polarization independent and exhibit a parabolic phase profile in addition to fast response time. To meet the low power consumption requirement set by Energy Star, high optical efficiency is among the top lists of next-generation LCDs. In this dissertation, I have demonstrated some new device structures for improving the optical efficiency of a polymer-stabilized BPLC transmissive display and proposed sunlight readable transflective blue-phase LCDs by utilizing ambient light to reduce the power consumption. Moreover, we have proposed several blue-phase LC adaptive lenses for high efficiency 3D displays.<br>Ph.D.<br>Doctorate<br>Optics and Photonics<br>Optics and Photonics<br>Optics
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Rao, Linghui. "Low Voltage Blue Phase Liquid Crystal Displays." Doctoral diss., University of Central Florida, 2012. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/5448.
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From cell phones, laptops, desktops, TVs, to projectors, high reliability LCDs have become indispensable in our daily life. Tremendous progress in liquid crystal displays (LCDs) has been made after decades of extensive research and development in materials, device configurations and manufacturing technology. Nowadays, the most critical issue on viewing angle has been solved using multidomain structures and optical film compensation. Slow response time has been improved to 2-5 ms with low viscosity LC material, overdrive and undershoot voltage, and thin cell gap approach. Moving image blur has been significantly reduced by impulse driving and frame insertion. Contrast ratio in excess of one million-to-1 has been achieved through local dimming of the segmented LED backlight. The color gamut would exceed 100% of the NTSC (National Television System Committee), if RGB LEDs are used. Besides these technological advances, the cost has been reduced dramatically by investing in advanced manufacturing technologies. Polymer-stabilized blue phase liquid crystal displays (BPLCDs) based on Kerr effect is emerging as a potential next-generation display technology. In comparison to conventional nematic devices, the polymer-stabilized BPLCDs exhibit following attractive features: (1) submillisecond response time, (2) no need for molecular alignment layers, (3) optically isotropic dark state when sandwiched between crossed polarizers, and (4) transmittance is insensitive to cell gap when the in-plane electrodes are employed. However, aside from these great potentials, there are still some tough technical issues remain to be addressed. The major challenges are: 1) the operating voltage is still too high (~50 Volts vs. 5 Volts for conventional nematic LCDs), and the transmittance is relatively low (~65% vs. 85% for nematic LCDs), 2) the hysteresis effect and residual birefringence effect are still noticeable, 3) the mesogenic temperature range is still not wide enough for practical applications (?40 oC to 80 oC), and 4) the ionic impurities in these polymer-stabilized nano-structured LC composites could degrade the voltage holding ratio, which causes image sticking. In this dissertation, the BPLC materials are studied and the new BPLC device structures are designed to optimize display performances. From material aspect, the electro-optical properties of blue phase liquid crystals are studied based on Kerr effect. Temperature effects on polymer-stabilized blue phase or optically isotropic liquid crystal displays are investigated through the measurement of voltage dependent transmittance under different temperatures. The physical models for the temperature dependency of Kerr constant, induced birefringence and response time in BPLCs are first proposed and experimentally validated. In addition, we have demonstrated a polymer-stabilized BPLC mixture with a large Kerr constant K~13.7 nm/V2 at 20 oC at 633 nm. These models would set useful guidelines for optimizing material performances. From devices side, the basic operation principle of blue phase LCD is introduced. A numerical model is developed to simulate the electro-optic properties of blue phase LCDs based on in-plane-switching (IPS) structure. Detailed electrode dimension effect, distribution of induced birefringence, cell gap effect, correlation between operation voltage and Kerr constant, and wavelength dispersion are investigated. Viewing angle is another important parameter. We have optimized the device configurations according to the device physics studied. With proper new device designs, the operating voltage is decreased dramatically from around 50 Volts to below 10 Volts with a reasonably high transmittance (~70%) which enables the BPLCDs to be addressed by amorphous silicon thin-film transistors (TFTs). Moreover, weak wavelength dispersion, samll color shift, and low hysteresis BPLCDs are achieved after their root causes being unveiled. Optimization of device configurations plays a critical role to the widespread applications of BPLCDs. In addition to displays, blue phase liquid crystals can also be used for photonic applications, such as light modulator, phase grating, adaptive lens and photonic crystals. We will introduce the application of blue phase liquid crystal as a modulator to realize a viewing angle controllable display. The viewing angle can be tuned continuously and precisely with a fast response time. The detailed design and performance are also presented in this dissertation.<br>Ph.D.<br>Doctorate<br>Optics and Photonics<br>Optics and Photonics<br>Optics
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Abidi-Azzouz, Naïma. "Élaboration d'Intrabodies ciblant l'organisation conformationnelle du complexe de reverse transcription de VIH-1." Thesis, Montpellier 2, 2013. http://www.theses.fr/2013MON20050/document.
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Les traitements actuels dirigés contre le VIH ne sont que partiellement efficaces en raison de l'apparition de mutations qui confèrent au virus une grande capacité de résistance aux antirétroviraux existants. Un moyen d'améliorer la lutte contre le virus consiste par conséquent à trouver de nouvelles stratégies d'inhibition. Le complexe de reverse transcription est une des principales cibles pour le développement de traitement anti-SIDA, il catalyse une étape obligatoire du cycle de réplication du virus. Cependant, l'ensemble des inhibiteurs de la transcriptase inverse sont limités par l'apparition rapide de souches résistances. Dans ce contexte, mes travaux de thèse ont permis de développer des inhibiteurs ciblant spécifiquement la reverse transcriptase (RT) du VIH-1, basée sur des fragments d'anticorps dérivés des anticorps chaînes lourdes de dromadaire appelés VHHs ou encore Nanobodies. Associé à une stratégie de vectorisation non invasive basée sur l'utilisation de peptides vecteurs pénétrants, les Nanobodies ont été délivré efficacement dans les cellules et par conséquent ils présentent tous une forte activité antivirale de l'ordre du nanomolaire. L'étude du mécanisme d'action du Nanobody leader NbRT20 montre qu'il agit en tant qu'inhibiteur conformationnel. Il interagit avec la forme intermédiaire inactive de la RT et empêche la mobilité du sous-domaine thumb requis pour le positionnement correct de la matrice/amorce sur la RT et inhibant l'incorporation des nucléotides dans la chaîne d'ADN naissante déstabilisant l'enzyme dans une conformation inactive, non processive. Pris ensemble, ces résultats montrent que la plate-forme Nanobody peut être très efficace pour générer des intracorps extrêmement puissants et sélectifs pour neutraliser la RT et la réplication virale.Mots clés : HIV-1, RT, Nanobodies, peptide vecteur pénétrant<br>The rapid emergence of drug-resistant viruses against all approved HIV clinical drugs together with inaccessible latent virus reservoirs and side effects of currently used compounds have limited the potency of existing anti-HIV-1 therapeutics. Therefore, there is a critical need for new safer drugs, active against resistant viral strains. Reverse transcriptase (RT) plays an essential role in the replication of human immunodeficiency virus type 1 (HIV-1) and remains a primary target of anti-HIV-1 drugs. To develop specific HIV inhibitors, we have elaborated a new strategy based on short antibody fragments derived from the unique Heavy-chain antibodies present in Camelidae called Nanobodies that targets RT-activation. The immunization of dromedaries with RT has lead to the isolation of a panel of Nanobodies that tightly bind the two subunits of RT and inhibit its DNA-dependent DNA polymerase activity at nanomolar range. From that screen we have elaborated an intrabody (cell penetrating anti-RT Nanobody) NbRT20 that constitutes a potential interesting anti-HIV compound.We demonstrated that NbRT20 inhibits RT polymerase activity and exhibiting a potent antiviral activity with a subnanomolar IC50. NbRT20 binds the thumb subdomain and restricts its flexibility and mobility resulting in an inactive/non processive dimeric conformation of the enzyme. From a mechanistic point of view, we have showed that NbRT20 is a conformational inhibitor. it prevents proper binding of primer/template and of dNTP and destabilizes the enzyme in an inactive/non processive dimeric conformation.Taken together, these results demonstrated that, the Nanobody platform may be highly effective at generating extremely potent and selective intrabody to neutralize RT and HIV proliferation.Key words: HIV-1, RT, Nanobodies, cell penetrating peptide
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Gadzekpo, Isaac Kwabena. "Selection of Phage Displaying Peptides Specific for Staphylococcus Aureus." Youngstown State University / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1619516455248529.
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Glökler, Jörn Felix. "Identifikation und Charakterisierung von Metallchelat-bindenden Peptiden mittels Phage-Display." [S.l. : s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=958911894.
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Zwick, Michael Bruce. "Applications and characterization of peptides derived from phage-display libraries." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0028/NQ51943.pdf.
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Starzomski, Mark Stephen. "Characterization and applications of the phase-modulating liquid crystal display." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape8/PQDD_0016/MQ48289.pdf.
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Weber, Patric. "Display of trypanosomal antigens on the surface of filamentous phage /." [S.l.] : [s.n.], 1994. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Draghici, Alex-Bogdan. "Sind Antikörper gegen fusionsrelevante HIV-Fragmente mittels Phage Display generierbar?" Göttingen Sierke, 2008. http://d-nb.info/992286891/04.
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Sang, Sheila J. "The Use of Phage Display to Identify Specific Peptide Ligands." Youngstown State University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1409923241.
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Joulie, Michaël. "Recherche de nouvelles protéines humaines se liant à l'ADN méthylé." Thesis, Paris 11, 2011. http://www.theses.fr/2011PA112157/document.
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L'épigénétique est un composant essentiel du fonctionnement des génomes eucaryotes. Les divers phénomènes épigénétiques modifient l’état chromatinien et participent à la plasticité du génome, mais aussi au maintien de son identité fonctionnelle à travers les générations cellulaires. Parmi ces processus, la méthylation de l’ADN joue un rôle fondamental dans la régulation de l’expression des gènes.Chez les mammifères, la méthylation de l'ADN est associée à la répression transcriptionnelle, et elle remplit au moins trois fonctions essentielles. Premièrement, elle permet de réprimer les séquences répétées afin de préserver l’intégrité du génome. Deuxièmement, la méthylation contrôle l’expression des gènes soumis à l’empreinte parentale, qui sont des régulateurs cruciaux du développement et de la vie adulte. Enfin, la méthylation permet de réprimer certains gènes tissu-spécifiques dans les organes où ils doivent être silencieux. En plus de ces rôles physiologiques, la méthylation est liée au cancer. En effet, des patrons de méthylation anormaux sont fréquemment observés dans les cellules tumorales, et ces anomalies participent à la transformation cellulaire par plusieurs mécanismes.La méthylation exerce ces effets par l'intermédiaire de protéines dédiées, qui reconnaissent spécifiquement l'ADN méthylé et contrôlent la transcription en modulant la chromatine. Trois familles de protéines liant l'ADN méthylé sont connues chez les mammifères, et elles totalisent entre elles neuf membres. De nombreux arguments suggèrent que cette liste est encore incomplète, et que des protéines humaines liant l'ADN méthylé restent à découvrir. Dans cette optique, nous avons opté pour deux types d’approches distinctes, une approche basée sur la littérature et une approche génétique. L’étude des protéines candidates ne nous a pas permis d’identifier de nouvelles protéines liant l’ADN méthylé et l’approche génétique par phage display a révélé deux protéines intéressantes, CHD3 et HMGB1 qui doivent désormais être validées par des approches in vivo et in vitro.Par ailleurs, nous avons entrepris l’étude de la régulation des éléments répétés par la protéine Zbtb4 chez la souris. Les expériences préliminaires indiquent une possible régulation des satellites mineurs par Zbtb4. Le rôle de cette régulation sera, par la suite, approfondi<br>Epigenetic phenomena are key contributors to the function of eukaryotic genomes. These processes act on chromatin, and they are used to render the genome dynamic, but also stable throughout successive rounds of cell division. Among epigenetic processes, DNA methylation is especially well known for its role in the regulation of gene expression.In mammals, DNA methylation is strongly correlated with transcriptional repression, and fulfills at least three essential roles. First, it maintains repeated sequences transcriptionally silenced, thus ensuring the stability of the genome. Second, it is responsible for the proper regulation of parentally imprinted genes, which are crucial regulators of embryonic development and adult life. Finally, DNA methylation ensures that some tissue-specific genes are kept inactive in the organs in which they should be repressed. Besides these roles in the physiology of normal cells, DNA methylation has strong links to cancer. Indeed the pattern of DNA methylation on the genome is frequently altered in cancer cells, and these anomalies contribute to transformation by several mechanisms.DNA methylation does not control transcription directly, but instead acts via a set of dedicated proteins that specifically recognize methylated DNA and repress transcription by acting at the chromatin level. At present, three families of such proteins, totalling 9 members altogether, are known in humans. However, several lines of evidence suggest that the list is not exhaustive, and that other human proteins that bind methylated DNA remain to be found. This was the goal of the current project.To this end, we opted for two distinct types approaches, an approach based on literature and a genetic approach. The study of candidate proteins does not allow us to identify new methylated DNA binding proteins and the genetic approach by phage display revealed two proteins of interest, HMGB1 and CHD3 that must now be validated by in vivo and in vitro approaches.Furthermore, we studied the regulation of DNA repeats by Zbtb4 in mice. Preliminary results show a regulation of minor satellites by Zbtb4. The role of this regulation will be analyse further in the future
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Even, Klervi. "Développement d' outils innovants pour le diagnostic et la découverte de cibles dans le cancer du sein." Thesis, Aix-Marseille, 2012. http://www.theses.fr/2012AIXM4019/document.
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Au cours de sa vie, 1 femme sur 9 sera atteinte du cancer du sein, 1 sur 27 sera emportée par cette maladie et 10 à 15 % des patientes développeront des métastases dans les trois années suivant le diagnostic. Le diagnostic précis et personnalisé du cancer du sein ainsi que l'évaluation de son potentiel métastatique est donc un enjeu majeur. Une analyse plus précise des caractéristiques moléculaires d'une tumeur primaire devrait conduire à une médecine personnalisée, un traitement et un suivi plus efficace. La détection de biomarqueurs sériques serait un moyen de diagnostiquer un cancer métastatique. Dans le but de découvrir de nouveaux marqueurs, l'analyse protéomique d'échantillons de patient a un fort potentiel mais souffre de limitations techniques, incluant le manque d'anticorps stables reconnaissant des marqueurs tumoraux d'intérêt. Par l'utilisation de fragments d'anticorps aux propriétés remarquables nommé single domain antibody (sdAb), et grâce à la mise au point d'une stratégie innovante de phages display, ce travail apporte d'importantes réponses en termes de disponibilité d'anticorps, d'analyse spécifique d'échantillon et de découverte de nouvelles cibles. Nous avons élaboré une stratégie permettant la découverte de biomarqueurs et l'isolement des anticorps correspondants. Après la construction de banques de sdAb à partir de lamas immunisés par des biopsies, une nouvelles stratégie de sélection in vitro par phage display, la sélection masquée, nous a permis d'isoler des anticorps spécifiques du cancer du sein<br>In a lifetime, 1 in 9 women will develop breast cancer, 1 of 27 will be swept away by the disease and from 10 to 15% of patients will develop metastases within three years of diagnosis. Accurate and personalized diagnosis of breast cancer and the detection of its metastatic potential is a major challenge. It is essential to develop new analytical methods allowing an effective monitoring of breast cancer. A closer analysis of the molecular characteristics of a primary tumor should lead to more effective personalized medicine, treatment and monitoring. The efficient detection of serum biomarkers would be a way to diagnose metastatic cancer and to modify treatment based on these results. Toward this goal, the proteomic analysis of patient samples has great potential but suffers from technical limitations, including the lack of a wide variety of antibodies and tumor marker. By the use of innovative antibody fragments with remarkable properties named single domain antibody (sdAb), and through the development of a new innovative strategy of phage display, this work provides important answers in terms of availability of antibody, specific proteomic analysis of sample and new target discovery. We have developed a strategy allowing the simultaneous discovery of new biomarkers and the isolation of corresponding antibodies. After the construction of sdAb libraries from llamas immunized with biopsies, and using a new in vitro selection strategy by phage display named masked selection, we have isolated breast cancer-specific antibodies
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Ferreira, Fabiana Lauretti. "Identificação e avaliação de novas adesinas em Leptospira interrogans por shotgun phage display." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-15122015-091903/.
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Leptospirose é uma doença infecciosa emergente cujos agentes etiológicos são espécies patogênicas do gênero Leptospira. Leptospiras patogênicas possuem inúmeros genes específicos codificando proteínas com funções desconhecidas, sugerindo que as leptospiras apresentam fatores de virulência únicos. Adesinas bacterianas são importantes fatores de virulência e, assim, a identificação de adesinas conservadas em espécies patogênicas de Leptospira pela construção de bibliotecas genômicas expostas na superfície de bacteriófagos (shotgun phage display), seguida por seleção em células e/ou componentes da matriz extracelular (biopanning), pode revelar novos antígenos e alvos para o tratamento e prevenção da leptospirose. Bibliotecas foram construídas com o DNA genômico de L. interrogans fragmentado e o fagomídeo pG8SAET, sendo testadas algumas abordagens para clonagem como a ligação entre extremidades cegas (blunt-end) e técnicas baseadas em ligação entre extremidades coesivas, incluindo a obtenção de ORESTES e a utilização de adaptadores em grampo. Apesar de serem encontradas algumas limitações, a clonagem por ligação blunt-end se mostrou a mais eficiente para a construção de bibliotecas, sendo adotada para a construção de três bibliotecas em maior escala. A seleção de novas possíveis adesinas a partir das bibliotecas construídas foi realizada em células eucarióticas através da metodologia BRASIL. A primeira biblioteca (BBT1) exibiu 106 clones totais, a partir da qual foram selecionados quatro proteínas em fase apenas com a proteína VIII do fago (pVIII). No entanto, nenhuma delas seria exposta por programas de predição na bactéria. Outras duas bibliotecas foram construídas (BBT2 e BBT3), as quais obtiveram um número ideal de clones para uma ampla cobertura do genoma (>2x107 clones). Por apresentar maior proporção de clones válidos, a BBT2 foi utilizada para a seleção de adesinas, resultando em onze clones em fase com pVIII e/ou sequência sinal do fago. Análises por programas de predição revelaram três proteínas hipotéticas, denominadas LepA962, LepA069 e LepA388, as quais poderiam estar expostas ou ser secretadas pela bactéria, sugerindo uma possível função de adesina. O estudo da proteína LepA388 levou ao reconhecimento de outras doze proteínas semelhantes e pertencentes a uma família paráloga contendo um domínio denominado DUF_61, motivo de função desconhecida presente em proteínas compartilhadas somente entre as espécies patogênicas mais virulentas de Leptospira. Por esta razão, a proteína LepA388 foi a mais estudada. A clonagem de três porções da proteína (LepA388P, LepA388NR e LepA388F) para expressão heteróloga resultou em proteínas recombinantes insolúveis e, considerando a riqueza em resíduos de cisteína presente em sua estrutura, não foi possível renaturá-las adequadamente. Diante dos obstáculos encontrados, apenas a porção contendo a sequência apresentada pelo fago (LepA388P) foi utilizada para obtenção de antissoros em camundongos, os quais apresentaram altos títulos, demonstrando a alta imunogenicidade da proteína LepA388P. O reconhecimento de proteínas nativas da família paráloga DUF_61 em extratos de diferentes sorovares de Leptospira não foi observado, assim como sua expressão in vitro a partir de bactérias em diferentes condições de cultivo. Estudos adicionais sobre a expressão in vivo e funções dos membros desta família são necessários para uma compreensão mais ampla de seu papel na biologia de leptospiras e, possivelmente, na patogênese da leptospirose.<br>Leptospirosis is an emerging infectious disease whose etiologic agents are pathogenic species of the genus Leptospira. Pathogenic leptospires have countless specific genes encoding proteins with unknown functions, suggesting that leptospires have unique virulence factors. Bacterial adhesins are important virulence factors and so the identification of conserved adhesins in pathogenic Leptospira species from shotgun phage display libraries, followed by selection (biopanning) in cells and/or extracellular matrix components, can reveal new antigens and strategies for leptospirosis treatment and prevention. Libraries were constructed using fragmented genomic DNA from L. interrogans and pG8SAET phagemid vector. Cloning approaches included blunt-end ligation and techniques based in cohesive-end ligation, such as ORESTES strategy and hairpin linkers. Despite some limitations, cloning by blunt-end ligation was the most efficient for library construction, being adopted for the construction of three libraries on a larger scale. Selection of new possible adhesins was performed by biopanning of the libraries in eukaryotic cells through BRASIL methodology. The first library called BBT1 exhibited approximately 106 total clones, and its biopanning resulted in four proteins fused to phage protein VIII, but none of them were expected to be exposed by the bacteria. Other libraries were built (BBT2 and BBT3) which reached the expected number of clones to obtain a larger genome representation (> 2x107 clones). Since it showed the highest proportion of positive clones, BBT2 was selected to perform a second biopanning, resulting in eleven proteins fused to phage protein VIII and/or signal peptide. In silico analysis revealed three hypothetical proteins, named LepA962, LepA069 and LepA388, that would be exposed or secreted by the bacteria, suggesting a possible adhesin function. The study of LepA388 protein led to the recognition of twelve other similar proteins belonging to a paralogous family that contains a domain called DUF_61, domain of unknown function that is present in proteins shared only among the most virulent pathogenic species of Leptospira. For this reason, the LepA388 protein was the most studied. The cloning of three portions of the protein (LepA388P, LepA388NR and LepA388F) for heterologous expression resulted in insoluble recombinant proteins, and given the presence of many cysteine residues in its structure, it was not possible to renature them appropriately. In face of the imposed obstacles, only the portion containing the sequence presented by the bacteriophage (LepA388P) was used to obtain antisera in mice, which showed high titers, demonstrating the high immunogenicity of the protein LepA388P. Recognition of native DUF_61 paralogous family proteins in extracts from distinct Leptospira serovars was not observed, as well as its in vitro expression from bacteria cultured in different conditions. Additional studies on the in vivo expression and functions of members of this family are needed for a broader understanding of their role in leptospiral biology and possibly in the pathogenesis of leptospirosis.
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Revilliod, Claude. "Photo-thermochimie : mécanisme et cinétique d'une réaction de méthanation en phase gaz-solide /." [S.l.] : [s.n.], 1991. http://library.epfl.ch/theses/?display=detail&nr=956.
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Stoiber, Johannes. "Hysteresis effects during martensitic phase transformations in Cu-Zn-Al shape memory alloys /." [S.l.] : [s.n.], 1993. http://library.epfl.ch/theses/?display=detail&nr=1115.
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Hald, Rikke. "Generation and characterisation of a naive human antibody phage display library : a resource for clinically relevant reagents /." Cph. : Department of Pharmacology, The Danish University of Pharmaceutical Sciences, 2004. http://www.dfh.dk/phd/defences/rikkehald.htm.
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Moutel, Sandrine. "Sélection et amélioration d'anticorps recombinants, applications à la recherche fondamentale et à l'immunothérapie." Paris 6, 2009. http://www.theses.fr/2009PA066520.
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Les anticorps (Ac) monoclonaux sont maintenant des outils couramment utilisés aussi bien pour le diagnostic que pour la recherche fondamentale dans l’étude ou la purification des protéines. Avec le développement de l’ingénierie génétique des Ac, il a été possible de cloner des régions variables d’anticorps humains et d’obtenir des banques de grande diversité. Au laboratoire, un investissement particulier a été fait ces dernières années pour adapter les méthodes de sélection d'anticorps recombinants (rAc) aux questions de la biologie cellulaire. La technique du phage display que nous utilisons pour cribler notre banque de scFv, nous a permis d’obtenir de nombreux anticorps notamment contre des échantillons complexes comme des membranes intactes de Golgi ou encore des rAc sensibles aux changements conformationnels indispensables pour nos recherches. Ces scFv ont été sélectionnés rapidement à moindre frais et sans avoir recours au passage par l’animal. Malgré le nombre incroyable de publications décrivant des scFv ou d’autres formats d’rAc, ils n’ont toujours pas réussi à s’imposer comme véritables outils alternatifs aux Ac naturels dans les laboratoires de recherche académiques. Nous avons donc développé notre propre système afin de simplifier et d’améliorer la production d’rAc. Nous avons choisi de les remettre dans un contexte d’Immunoglobuline en leur ajoutant une portion Fc pour les dimériser et de les produire dans des cellules de mammifères. Nous avons choisi le format appelé minibody, il diffère de la structure d’une IgG classique par l’abscence des domaines CH1 et Ckappa, permettant de facilement manipuler l’rAc qui reste sous forme monocaténaire. Pour tous les scFv obtenus au laboratoire, le format minbody devient très robuste. Outre la dimérisation, ce système est versatile, on a pu aisément remplacer le Fc humain par un Fc de lapin ou un Fc de souris. Nous avons aussi développé une nouvelle méthode de sélection d'rAc totalement in vitro. Cette méthode de sélection in vitro d’rAc offre tout d’abord l’avantage d’obtenir des Ac difficiles voire impossibles à sélectionner par immunisation d’animaux , avec des quantités d’Ag de l’orde du µg. L’Ag peut être une protéine très conservée au cours de l’évolution, toxique, être sous une forme native ou dans une conformation d’intérêt. Cette méthode nous a permis de préparer les Ag cibles sans passer par l'expression et la purification chez E. Coli. Deux cribles ont été réalisés avec succès, un contre la GFP pour démontrer la faisabilité du système et un en collaboration avec l’équipe de Philippe Benaroch contre Tsg101. Pour ces études, nous avons développé des méthodes permettant d'obtenir des outils uniques, dont nous étendons maintenant les applications, notamment au domaine thérapeutique. En effet, un avantage lié à ces techniques de sélection in vitro est le fait de disposer simultanément de la séquence nucléotidique codant pour ces rAc entièrement humains. Dans le cadre d’un travail collaboratif, nous nous sommes intéressés aux applications thérapeutiques qui peuvent découler de telles molécules. Le but du projet est d’évaluer l’efficacité de notre rAc chimérique anti-Tn en immunothérapie passive in vivo dans des modèles précliniques de souris greffées par des lignées épithéliales tumorales et d’évaluer la potentialité de son utilisation chez l’homme. C’est un très bon marqueur diagnostic en histologie et aussi pronostic car son expression est corrélée avec le grade de la tumeur Dans tous les tissus Tn est masqué, et il est présent sur la membrane cellulaire dans la majorité des carcinomes (90%), ce qui en fait une cible de choix pour l'immunothérapie. .
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Coulon, Stéphane. "Amélioration de la Spécificité et de l'Affinité de Fragments d'Anticorps Anti-stéroïde par Mutagenèse et Phage-display." Paris 7, 2001. http://www.theses.fr/2001PA077130.
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Castel, Guillaume. "Recherche de peptides à activités antivirales ciblant le complexe de réplication/transcription des Mononegavirales." Paris 6, 2009. http://www.theses.fr/2009PA066021.
Full textAbstract:
Le complexe RNP assure la transcription/réplication du génome des Mononegavirales. Afin d’identifier des peptides inhibiteurs ce complexe a été ciblé par 2 approches « cognitive » ou « aléatoire». Approche « cognitive » : L’extrémité Nt de la protéine P est capitale dans ce complexe interagissant avec les protéines N et L. Le potentiel antiviral de 3 peptides (P42, P57, P60) couvrant les 42, 57 et 60 aa Nt de P a été évalué. P42 et P60 interagissent avec L et N et la transfection de leur ADNc permet d’inhiber fortement un miniréplicon ou une infection rabiques. Ils ont été synthétisé et leur effet inhibiteur, confirmé sur cellules infectées. Approche par « phage display » : 3 banques de peptides présentés à la surface de phages M13 ont été construites et soumises à des cycles de sélection sur des matrices N-ARN de Mononegavirales. Les peptides isolés ont été séquencés et l’émergence de motifs spécifiques a permis d’élaborer une sous-banque, à son tour criblée contre la cible N-ARN pour isoler des peptides de meilleure affinité. Leur capacité inhibitrice a été testée sur miniréplicon et sur infection virale. Les niveaux d’inhibition modérés sont en voie d’amélioration
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Chahboun, Siham. "Comparaison des régions variables des anticorps de macaques (Macaca fascicularis) et de l' Homme et leurs utilisation pour la neutralisation des toxines botuliques A et B." Thesis, Grenoble, 2013. http://www.theses.fr/2013GRENV022.
Full textAbstract:
Notre laboratoire a développé une stratégie d'isolement de fragments d'anticorps recombinants à partir de primates non humains (Macaca fascicularis) immunisés, en utilisant la technologie des phages. Dans le cadre de cette thèse, une comparaison des séquences d'anticorps de macaques (Macaca Mulatta) et d'anticorps humains a toutefois montré que les anticorps des deux espèces présentent des différences qui rendent souhaitable une étape d'humanisation des anticorps de macaques. Cette stratégie a été utilisée dans le cadre du projet Européen AntiBotABE (www.antibotabe.com) et l'étape de criblage a été adaptée pour isoler des scFv neutralisant de façon croisée les toxines botuliques BoNT/B des sous-types B1 et B2, en utilisant séquentiellement l'holotoxine BoNT/B1 et un fragment recombinant représentant la région C-terminale de la chaîne lourde de BoNT/B2. Le meilleur scFv ciblant les régions C-terminales des chaînes lourdes de BoNT/B1 et BoNT/B2, B2-7, a montré une bonne capacité de neutralisation de BoNT/B1 et BoNT/B2 dans le test ex vivo de paralysie hémidiaphragmatique. Les régions charpentes du scFv B2-7 ont un pourcentage d'identité élevé (80 %) avec leurs homologues humains. Des scFv neutralisant BoNT/A1 en ciblant sa chaîne légère ont aussi été isolés, dont le scFv le plus efficace, 2H8, induit une diminution de 50% de l'activité endopeptidasique à une concentration correspondant à un rapport molaire 2H8/BoNT/A1 de 64000. Les régions charpentes de 2H8 ont également un pourcentage d'identité élevée (88%) avec leurs homologues humains. La versatilité de cette stratégie en fait un outil permettant l'isolement de nombreux autres fragments d'anticorps à visée thérapeutique<br>Our laboratory has developed a strategy to isolate recombinant antibody fragments technology from immunized non human primates (Macaca fascicularis) by phage display. In the course of the present thesis, a comparison between macaque (Macaca mulatta) and human antibody sequences has demonstrated that antibodies of the two species are different. This difference makes the humanization of macaque antibodies desirable. The strategy was used in the framework of the European AntiBotABE project, and the screening was adapted to isolate antibody fragments cross neutralizing the B1 and B2 subtypes of botulinum B neurotoxin, by using sequentially the holotoxin BoNT/B1 and a recombinant fragment representing the C-terminal region of the heavy chain of BoNTB2. The best scFv targeting the C-terminal region of BoNT/B1 and BoNTB2 heavy chains, B2-7, demonstrated a high capacity to neutralize BoNT/B1 and BoNT/B2 in the ex vivo hemidiaphragmatic assay. A high identity (80%) between the framework regions of B2-7 and their human homologs was observed. ScFvs neutralizing BoNT/A1 by targeting its light chain were also isolated and among them, the scFv 2H8 induced a decrease of 50% in the endopeptidase activity at a concentration corresponding to a molar ratio of 2H8/BoNT/A1 of 64000. A high identity (88%) between the framework regions of 2H8 and their human homologs was also observed. Our strategy can be used to isolate other therapeutic antibody fragments