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Colazet, Magali. "Génération de nanobodies pour des applications en immunothérapie." Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0013.
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L’immunothérapie est une approche thérapeutique qui consiste à restaurer les fonctions anti-tumorales du système immunitaire pour qu’il soit à nouveau capable d’éliminer les cellules cancéreuses. Pour cela, différentes stratégies sont étudiées : l’une d’entre elles consiste à cibler des récepteurs inhibiteurs présents à la surface des cellules immunitaires infiltrées dans les tumeurs de façon à réactiver leurs fonctions. L’entreprise Innate Pharma développe des anticorps monoclonaux capables de restaurer les fonctions immunitaires de cellules effectrices de l’immunité innée. Le but de la collaboration était de générer des sdAbs (single-domain Antibodies), fragments d’anticorps dérivés de camélidés, capables de bloquer des interactions de type « immune checkpoint ». Ces anticorps à domaine unique sont notamment capables de lier des épitopes inaccessibles aux anticorps conventionnels tels que des cavités. Dans ce manuscrit, les résultats concernant deux de ces projets sont exposés : la modulation de l’axe inhibiteur SIRPα/CD47 et le blocage de l’interaction réalisée entre les récepteurs Siglec-7/-9 et leurs ligands sialylés. Lors de ces travaux, plusieurs sdAbs ciblant les récepteurs d’intérêt ont été sélectionnés grâce à la technologie du phage display. Ces molécules monovalentes ont été caractérisées puis clonées en différents formats multivalents de façon à améliorer leur affinité par effet d’avidité, et ainsi potentialiser leur efficacité de blocage. Enfin, plusieurs tests fonctionnels ont été réalisés pour évaluer l’effet de ces molécules sur les fonctions effectrices de différentes cellules du système immunitaireImmunotherapy is a therapeutic approach which consists in restoring anti-tumoral functions of the immune system for eliminating cancer cells. For this, several strategies are developed: one of them is to target inhibitory receptors at the surface of effector cells in order to reactivate their functions in the tumor microenvironment.The company Innate pharma develops monoclonal antibodies able to restor immune functions of innate effector cells. The aim of the collaboration was to generate single-domain Antibodies (sdAbs), antibody fragments derived from camelids, which have the capacity of blocking interactions such as immune checkpoints. These sdAbs have several useful characteristics in terms of stability, production and especially in epitope binding. Indeed, because of their small size, they are able to bind on epitopes which are not accessible to conventional antibodies.In this manuscript, the results of two projets are reported: the modulation of the inhibitory axis SIRPα/CD47 and the blocking of the interaction between the receptors Siglec-7/-9 and their sialylated ligands. In these studies, several sdAbs targeting the receptors of interest were isolated by selection using phage display technology. These monovalent molecules were characterized to determine their specificity and estimate their binding and blocking capacities. Best candidates were cloned into several multivalent formats to optimize their affinity by avidity effect and to potent their blocking efficacy. Finally, several functional assays were performed to evaluate the efficacy of these multivalent constructions to restore immune functions of several effector cells
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Burbidge, Owen David. "Developing nanobodies to stabilise the tumour suppressor protein p16INK4a." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/288375.
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The tumour suppressor protein p16INK4a (p16) is a cyclin-dependent kinase (CDK) inhibitor that plays a key role in the regulation of the cell cycle by controlling the progression of cells through the G1 to S phase transition. Dysregulation of the protein through deletion, silencing or mutation of the gene encoding p16 is implicated in a range of different cancers including melanoma, cervical and oesophageal to name a few. p16 is composed of four ankyrin repeats and it has a very low thermodynamic and kinetic stability and rapidly unfolds even in the absence of denaturants. This low stability means that the protein is highly vulnerable to point mutations, which can result in functional inactivation through a range of different mechanisms such as deletion of key binding contacts, disruption of secondary or tertiary structure and consequent destabilisation leading to unfolding or aggregation. Heavy-chain antibodies are a unique form of antibody devoid of light chains found in the serum of the Camelid family (camels and llamas). Despite the absence of light chains, heavy-chain antibodies have evolved to complement traditional antibodies and retain the full binding capacity seen in canonical IgG antibodies. The single variable domain, known as a nanobody, is, at 15 kDa, the smallest antigen binding fragment, a tenth the size of a standard IgG antibody. The small size and relative ease of production, coupled with an unusually high stability, makes nanobodies useful tools as biological reagents, crystallography chaperones and therapeutics. The research contained within this PhD looks at the development of nanobodies to target p16. By leveraging the high stability of selected nanobodies, the aim was to obtain binders that could stabilise and reactivate a range of unstable cancer-associated mutants. The initial stages of the project focused on generating and optimising the expression and purification of p16 constructs prior to immunisation of animals to raise nanobodies. A high-throughput approach was taken to generate forty-five different p16 constructs with a range of different solubility and purification tags. These constructs were assessed in a multi-factorial expression screen, which resulted in the identification of a p16 construct with a ten-fold improvement in soluble expression levels compared with previous studies. A range of biophysical techniques, including circular dichroism and chemical denaturation, were performed to characterise this protein fully prior to immunisation. The second part of this project utilised a phage display library of two immune nanobody libraries generated against p16 and a p16 variant stabilised by previously published second-site mutations. This process yielded a large number of diverse nanobodies. Biophysical characterisation of these nanobodies was first performed, and they were found to have a range of chemical and thermal stabilities. Assays were then developed to test the ability of the nanobodies to stabilise p16. Two nanobodies were found to dramatically stabilise wild-type p16, with an increase in stability of approximately 44 % and 60 %, respectively. Furthermore, these nanobodies were also able to stabilise a subset of cancer-associated point mutants. Although there are NMR structures of p16, as well as a crystal structure of p16 bound to CDK6, the resolution of is very low, most likely due to the high backbone flexibility of p16. The last part of the project aimed to obtain a higher-resolution structure of p16 by using the two stabilising nanobodies as crystallisation chaperones. The more stabilising of the two nanobodies resulted in crystals that diffracted to a resolution of less than 2 $\AA$, a significant improvement compared with the previously published structure. In conclusion, a number of nanobodies were generated against tumour-associated p16 and shown to be capable of stabilising p16, allowing structure determination to high resolution and restoration of the stability of cancer-associated mutants to wild-type levels. In the project, a range of different approaches for nanobody production were explored, and these will be important for future applications. Moreover, the crystal structure of the p16-nanobody complex showed that the nanobody binds on the opposite face of p16, to the face involved in binding to CDKs; thus, this nanobody could potentially be exploited as a pharmacological chaperone to stabilise and restore the activity of cancer-associated mutant p16 in the cell.
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Cutolo, Pasquale. "Etude de l'interaction structurelle et fonctionnelle entre la chimiokine CXCL12 et ses récepteurs : CXCR4 et ACKR3/CXCR7." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS550/document.
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L'axe formé par la chimiokine CXCL12 et son récepteur CXCR4 est conservé chez les vertébrés où il joue un rôle important dans l'embryogenèse et la vie adulte, régule de nombreux processus des réponses immunitaires grâce à ses fonctions dans la migration cellulaire, la survie et la prolifération.En outre, cet axe est impliqué dans les processus pathologiques tels que les cancers (croissance et métastase) et immunodéficiences ainsi que des dysfonctionnements (par exemple l'expression dérégulée, polymorphismes ou mutations) et est également détourné par certains agents pathogènes (par exemple le virus de l'immunodéficience humaine, virus du papillome humain).Un grand groupe de travail est consacré à cette paire comme cible thérapeutique, mais seulement un composé (à savoir Plérixafor) a atteint l'approbation pour une utilisation clinique faisant le potentiel de cet axe comme cible de médicament encore inexploré.Bien que cet axe est l'objet d'un grand intérêt, des questions demeurent quant aux déterminants structurels impliqués dans l'interaction CXCL12/CXCR4.Cependant, la structure récemment résolue par diffraction de CXCR4 a donné quelque indice au sujet de ces questions, et au delà, la possible stoichiométrie entre CXCL12 et CXCR4.Plusieurs éléments de preuve appuient le concept que les formes CXCR4 homo- et hétéro- oligomères (qui peut contribuer à la diversité des fonctions de récepteur), telles que la structure de diffraction, le gain de fonction d'un récepteur CXCR4 mutant responsable du syndrome WHIM et la modulation allostérique des fonctions de CXCR4 par CXCR7 (ACKR3), le second récepteur de CXCL12. La possibilité de former des oligomères ouvre de nombreuses questions en matière de CXCL12 et ses interactions avec CXCR4 et CXCR7/ACKR3. La stoichiométrie de cette interaction reste une question ouverte, comme le récepteur est capable de former des oligomères avec le même récepteur ou autre récepteurs, en particulier CXCR7/ACKR3. Ce récepteur, connu comme scavenger, n'a pas de structure résolue et son mécanisme d'interaction avec CXCL12 reste inconnu.Afin d'étudier les interactions CXCL12/CXCR4/CXCR7, nous avons appliqué plusieurs techniques de modélisation moléculaire tels que peptid-peptide docking et simulations de dynamique moléculaire.Objets du projet ont étés : la résolution des possibles formes stoichiométriques de l'interaction CXCR4/CXCL12 (modélisation moléculaire, docking et dynamique); la modélisation de la structure du récepteur CXCR7/ACKR3 et son interaction avec CXCL12 (homology modeling), avec caractérisation des domaines et des résidus clef de l'activation des pathways de signalisation en aval du récepteur (mutants CXCR7/ACKR3); l'étude et la caractérisation de nouveaux outils innovants pour la détection de l'oligomerisation de ces récepteurs en conditions endogènes. (Nanobodies, HTRF)Les résultats du premier objectif ont été publiés en janvier 2016 : PMID 26813575.La modélisation de CXCR7/ACKR3 nous a permit de générer plusieurs mutants du récepteur pour tester nos hypothèses sur l’activation.Les nanobodies caractérisés pour CXCR4 seront utilisé dans une deuxième étude pour l’identification des formes oligomériques du récepteur sur tissus et cellulesThe axis formed by the chemokine CXCL12 and its receptor CXCR4 is conserved in vertebrates where it plays an important role in embryogenesis and adult life, regulates many processes of immune responses through its functions in cell migration, survival and proliferation.In addition, this axis is involved in pathological processes such as cancers (growth and metastasis) and immune deficiencies and malfunctions (eg deregulated expression, mutations or polymorphisms) and is also hijacked by certain pathogens (eg HIV, human papilloma virus).A large working group is dedicated to this pair as a therapeutic target, but only a compound (ie Plerixafor) achieved approval for clinical use by the potential of this area as a drug target unexplored.Although this axis is the subject of great interest, questions remain about the structural determinants involved in CXCL12 / CXCR4 interaction.However, the recently resolved diffraction structure of CXCR4 gave some clue about these questions, and beyond possible stoichiometry between CXCL12 and CXCR4.Several lines of evidence support the concept that forms CXCR4 homo- and hetero-oligomers (which can contribute to the diversity of the receptor functions), as shown in the diffraction structure, the gain function of a mutant CXCR4 receptor responsible for the syndrome WHIM and allosteric modulation of CXCR4 functions by CXCR7 (ACKR3), the second receptor of the chemokine CXCL12. The ability to form oligomers opens many issues of CXCL12 and its interaction with CXCR4 and CXCR7 / ACKR3.The stoichiometry of this interaction still remains an open question, as the receptor is capable to form oligomers with the same receptor or other receptors, particularly CXCR7 / ACKR3. This receptor, known as scavenger, has not solved structure and the mechanism of interaction with CXCL12 is unknown.To study the interactions CXCL12 / CXCR4 / CXCR7, we applied several molecular modeling techniques such as peptide-peptide docking and molecular dynamics simulations.Objectives of this project were: the resolution of the different stoichiometric forms for the interaction of CXCR4 and CXCL12 (molecular modeling, docking and dynamic); modeling the CXCR7 / ACKR3 receptor structure and its interaction with CXCL12 (homology modeling), with the characterization of domains and residues key in the activation of downstream signaling pathways of the receptor (CXCR7 / ACKR3 mutants); the study and characterization of new innovative tools for the detection of oligomerization of these receptors in endogenous conditions. (Nanobodies, HTRF)The results of the first objective were published in January 2016: PMID 26813575.Modeling of CXCR7 / ACKR3 allowed us to generate several mutants of the receptor to test our hypothesis about the activation pathways.Nanobodies were fully characterized for CXCR4 to be used in a second study to identify oligomeric forms of the receptor in tissues and cells
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Keller, Laura. "Conception de nano-anticorps conformationnels comme nouveaux outils d'étude de l'activité des GTPases de la sous-famille RHOA." Thesis, Toulouse 3, 2017. http://www.theses.fr/2017TOU30005/document.
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Les GTPases de la sous famille RHOA participent à la régulation de nombreuses voies de signalisation qui contrôlent la dynamique du cytosquelette cellulaire et une grande diversité de fonctions telles que la prolifération, la division, la migration et la polarité cellulaires. Ce sont de véritables interrupteurs moléculaires qui, en réponse à un stimulus, changent de conformation tridimensionnelle pour activer leurs protéines effectrices cibles. Elles existent donc sous deux formes, une forme inactive liant le GDP et une forme active, liant le GTP. La proportion de forme active est extrêmement régulée au niveau spatial et temporel dans une cellule et représente moins de 10% de sa totalité. Depuis près de 20 ans, le seul outil disponible pour étudier leur activation est constitué par le domaine de liaison d'un effecteur, le RBD. Peu stable, faiblement soluble et peu adaptable, de nouveaux outils sont nécessaires afin de mieux comprendre la fine régulation de ces protéines. Les anticorps à simple domaine, VHH ou nanobodies, sont caractérisés par leur stabilité, solubilité, haut rendement de production et versatilité de fonctionnalisation. A partir d'une nouvelle banque d'anticorps à simple domaine optimisée pour la production d'intracorps, nous avons isolés différents clones capables de reconnaître in vitro et de bloquer in cellulo la forme active de ces protéines. L'un de ces clones permettra le développement d'un nouvel outil de mesure de l'activité de ces protéines in vitro tandis qu'un autre, in cellulo, permettra de mieux comprendre la régulation spatiale et temporelle des protéines endogènesRHOA small GTPase belongs to a subfamily acting as a molecular switch activating major signaling pathways that regulate cytoskeletal dynamics and a variety of cellular responses such as cell cycle progression, cytokinesis, migration and polarity. RHOA activity resides in a few percent of GTP loaded protein, which is finely tuned by a crosstalk between regulators of the GTPase cycle. Manipulating a single RHO at the expression level often induces imbalance in the activity of other RHO GTPases, suggesting that more specific tools targeting these active pools are needed to decipher RHOA functions in time and space. We decided to use single domain antibodies, also known as VHH or nanobodies, as a new tool for studying RHOA activation. We produced and screened a novel fully synthetic phage display library of humanized nanobodies (NaLi-H1) to develop conformational sensors of the GTP loaded active conformation of RHO subfamily. We obtained several high affinity nanobodies against RHOA's active form which we characterized as RHO active antibodies in vitro and RHO signaling blocking intrabodies in cellulo. These new tools will facilitate and improve our current knowledge of this peculiar protein subfamily and will be a paradigm for the study of other RHO related small GTPases
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Plagmann, Ingo [Verfasser]. "Entwicklung von Strategien zur Dimerisierung von Single-Domain Antikörpern (Nanobodies) sowie zu ihrer Produktion in transgenen Pflanzen (anhand eines Tumor Nekrose Faktor neutralisierenden Nanobodies) / Ingo Plagmann." Kiel : Universitätsbibliothek Kiel, 2013. http://d-nb.info/1044891831/34.
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Patris, Stéphanie. "Développement d'immunoessais associés aux électrodes sérigraphiées: des particules superparamagnétiques aux nanobodies." Doctoral thesis, Universite Libre de Bruxelles, 2014. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209208.
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Cette thèse a pour vocation de contribuer au développement de différents immunocapteurs ampérométriques associés aux électrodes sérigraphiées (SPE). Les immunocapteurs sont des dispositifs simples associant un anticorps ou un antigène qui assurent la sélectivité à un transducteur (ici une SPE) ;ce dernier transforme la liaison anticorps/antigène en un signal mesurable (ici ampérométrique).Le travail est divisé en deux volets principaux.
Le premier est consacré à la mise en œuvre de différents modèles d’immunocapteurs ampérométriques pour le dosage d’anticorps anti-tetani. La vaccination contre le tétanos est généralisée, toutefois pour maintenir un taux d’anticorps suffisant, il est indispensable d’administrer un rappel tous les 10 ans. Ce schéma vaccinal n’est pas toujours respecté, ce qui a pour conséquence qu’une partie de la population n’est plus protégée. Afin de déterminer le statut immunitaire du patient, il est indispensable de pouvoir déterminer le taux d’anticorps. Les immunocapteurs ampérométriques répondent à cet objectif. Plusieurs stratégies d’immobilisation de l’anatoxine tétanique (antigène) sur une SPE ont été mises en œuvre et comparées. L’une d’elles repose sur l’utilisation de microparticules superparamagnétiques pour la réaction immunologique et d’une SPE rendue magnétique par un support aimanté pour la mesure. D’autres reposent sur l’immobilisation de l’antigène et les réactions immunologiques directement à la surface de la SPE. L’utilisation de plans d’expérience, pour l’optimisation des immunoessais sur SPE est également exploitée dans ce travail. Les immunocapteurs développés ont permis de doser les anticorps anti-tetani dans le sérum de cobaye en dessous des valeurs considérées comme protectrices.
Dans le second volet, un immunocapteur basé sur l’utilisation de nanobodies® (NB) a été mis au point. Nous avons qualifié ce type d’immunocapteur original de nanoimmunocapteur. Le récepteur de facteur de croissance épidermique humain (HER2) a été utilisé comme cible. La protéine HER2 est considérée comme un biomarqueur important car sa surexpression provoque un type agressif de cancer du sein. Les NB sont des fragments à domaine unique dérivés d'anticorps à chaînes lourdes de camélidés. La stratégie de dosage immunologique en sandwich développée a tiré profit de la petite taille des NB pour la détection du marqueur électroactif d’oxydoréduction. La stabilité élevée des NB immobilisés a permis une durée de stockage des SPE modifiées supérieure à 3 semaines. De très courtes durées d'incubation étaient suffisantes pour obtenir une réponse satisfaisante. Le nanoimmunoessai a permis de déterminer le taux d’HER2 dopé dans des lysats cellulaires.
Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished
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Andersson, Klara. "Characterization of nsP-specific nanobodies targeting Chikungunya and Semliki Forest Virus." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-414971.
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Viral infections are constantly increasing and impose a large threat to the public health. Alphaviruses are responsible for several animal and human diseases and have a large medical importance with few treatments available today. Alphaviruses are small, spherical single stranded RNA viruses, and are most often transmitted by mosquito vectors. Alphaviruses contains a domain of nonstructural proteins that compose the replication machinery. The domain is crucial for viral replication to occur and is therefore an interesting target for antiviral therapy. With the focus on Chikungunya and Semliki Forest Virus this work investigates the events in the cells on molecular level during infections. To do this a panel of Camelid derived single domain antibodies are developed to target the nonstructural proteins of Chikungunya and Semliki Forest Virus. Binding of the produced nanobodies to the viral proteins was investigated by biochemical methods including immunoprecipitations, western blot, and ELISA. Cell lines that express nsP-specific nanobodies in the cytosol were employed for infection- and plaque assays with Semliki Forest Virus in order to determine the antiviral potential of the new nanobodies. Three of the nanobodies proved to bind two different nonstructural proteins of the viruses, providing opportunities for further investigations and a possible use of these nanobodies to identify viral vulnerabilities that could be exploited for antiviral intervention.
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Guilliams, Tim Thomas. "Nanobodies as tools to gain insights into [alpha]-synuclein misfolding in Parkinson's disease." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608094.
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Broster, Christine. "Caractérisation et Ciblage de Protéines Essentielles via l'utilisation de nanobodies chez Trypanosoma brucei." Thesis, Bordeaux, 2019. http://www.theses.fr/2019BORD0158.
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Les parasites de la classe des Kinetoplastidae, comprenant notamment les trypanosomes et les leishmanies, sont responsables pour plusieurs maladies d’importance socio-économique et de santé publique. La maladie du sommeil, la maladie de Chagas et la leishmaniose, classées comme maladies tropicales négligées (NTD) par l’Organisation mondiale de la santé (OMS) et la Surra, reportée par l’Organisation pour l’alimentation et l’agriculture, des Nations Unies (FAO). La Trypanosomiase Animale Africain sub-saharienne entraîne la mort de 3 millions bovins par an accompagné d'une perte annuelle de l'économie de 4,5 milliards de dollars américains. La leishmaniose cutanée, une maladie zoonose, présente 1,5 millions de nouveaux cas chaque année.Trypanosoma brucei (T. brucei) est un ancien eucaryote, utilisé comme organisme modèle dans le laboratoire pour l’étude des cils et des flagelles. Le remodelage du cytosquelette des trypanosomes est essentiel pour la morphologie cellulaire, le positionnement et la division des organites. L’étude des protéines essentielles du cytosquelette permet de mieux comprendre les processus cellulaires. Ces protéines pourraient également constituer des cibles potentielles pour des traitements thérapeutiques. Les trypanosomes échappent au système immunitaire de l’hôte en modifiant périodiquement les antigènes de présent à leur surface. En effet ces antigènes de surface sont endocytés, ainsi que les anticorps de l’hôte qui y sont attachés, au niveau d’une structure appelée la poche flagellaire (FP). TbBILBO1 est une protéine structurelle du collier de la poche flagellaire (FPC), essentielle à la biogenèse du FPC et à la survie du parasite. En raison du rôle majeur de la protéine TbBILBO1 dans le parasite, des partenaires de TbBILBO1 ont été recherchés.Dans ce travail, j’ai pu caractériser une nouvelle protéine essentielle du cytoskelette, la protéine FPC6, partenaire de TbBILBO1, qui se situe au niveau du complexe FPC/Complexe du Hook de T. brucei. L’ARN interférence de FPC6 conduit à une mort rapide des formes sanguines des trypanosomes, accompagnée d’un blocage de l’endocytose. Ensuite, j’ai produit un nanobody (Nb48), dirigé contre TbBILBO1, dans le système d’expression bactérien. Je l’ai également exprimé dans les lignées de trypanosomes. Le Nb48 reconnait TbBILBO1 sur les trypanosomes fixés par immunofluorescence et dans les extraits totaux de protéines dénaturées. L’analyse par résonance plasmonique de surface (SPR) a confirmé une haute affinité du Nb48 pour TbBILBO1. L’expression de Nb48 dans le parasite T. brucei en tant qu’intrabody demontrant que ce nanobody pouvait être exprimé de manière fonctionnelle, capable de reconnaitre spécifiquement sa cible protéique, TbBILBO1, intra-cellulaire et de bloquer sa fonction conduit à un effet trypanocide rapide. Ces études ouvrant ainsi la voie pour de nouvelles utilisations potentielles thérapeutiques dans le traitement des trypanosomiasesKinetoplastid parasites, including trypanosomes and leishmania, are responsible for several diseases of socio-economic and public health importance worldwide. These include the Neglected Tropical Diseases: Sleeping Sickness, Chagas disease and Leishmaniasis, as classified by the World Health Organisation (WHO) and the global wasting disease of animals, Surra, as reported by the Food and Agricultural Organisation of the United Nations (FAO). Animal African Trypanosomiais (AAT) causes the death of 3 million cattle per year in sub-Saharan Africa, with an annual loss of 4.5 billion US dollars to the African economy. Cutaneaous leishmaniasis is a zoonotic disease, with 1.5 million new cases reported globally each year.Trypanosoma brucei is an ancient, early diverging eukaryote, used as a model organism in the laboratory for studying eukaryotic cilia and flagella. Remodelling of the trypanosome cytoskeleton is essential for cell morphology, organelle positioning and division. Study of essential proteins of the cytoskeleton provides insight into intracellular processes and could provide potential targets for therapeutic interventions. Trypanosomes evade the host immune system by periodically changing their external surface coat, which is endocytosed, along with any attached host antibodies, via a structure called the flagellar pocket. TbBILBO1 is a structural protein of the Flagellar Pocket Collar (FPC) that is essential for FPC biogenesis and parasite survival. Due to the importance of TbBILBO1 for the parasite, protein partners were investigated.In my thesis, I describe, firstly, the characterisation of a novel and essential cytoskeletal protein, FPC6, of the FPC/Hook complex of T. brucei; FPC6 is a partner of TbBILBO1. RNAi Knock-down of FPC6 protein leads to rapid cell death in the blood-stream form of the parasite accompanied with a block in endocytosis. Secondly, I describe the purification and intracellular expression of a nanobody (Nb48), raised against TbBILBO1. The purified Nb is able to identify TbBILBO1 in fixed trypanosomes and denatured protein. Surface Plasmon Resonance analysis confirmed a high affinity of Nb48 to TbBILBO1. Expression of Nb48 as an intrabody in T. brucei, reveals that it binds precisely to its target, TbBILBO1 and leads to rapid cell death. Further exploration of the potential uses of this trypanocidal nanobody is warranted
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Sola, Colom Mireia [Verfasser]. "Nucleoporin-binding nanobodies that either track or trap uclear pore complex assembly / Mireia Sola Colom." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2021. http://nbn-resolving.de/urn:nbn:de:gbv:7-21.11130/00-1735-0000-0008-57CA-3-3.
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Ackerer, Léa. "Les Nanobodies, un nouvel outil de diagnostic de la maladie du court-noué de la vigne." Thesis, Strasbourg, 2016. http://www.theses.fr/2016STRAJ103/document.
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La maladie du court-noué est principalement causée en Europe par le Grapevine fanleaf virus (GFLV) et l’Arabis mosaic virus (ArMV). Le principal moyen de lutte contre sa dispersion consiste à certifier l’absence de ces virus dans les vignes commercialisées par des méthodes sérologiques tels que le DAS-ELISA. De par leurs propriétés biophysique et structurale exceptionnelles, les Nanobodies (Nb) issus des domaines variables d’immunoglobulines (Ig) composées uniquement de chaînes lourdes, se distinguent avantageusement des Ig conventionnelles. L’objectif majeur de ma thèse était d’établir un test de diagnostic du court-noué à base de Nb. À partir de deux collections de Nb contre le GFLV et l’ArMV, j’ai identifié des Nb reconnaissant un large spectre d’isolats viraux. Leur fusion à une protéine fluorescente ou à la phosphatase alcaline a conduit à l’obtention de réactifs de détection performants du GFLV et de l’ArMV par DAS-ELISA. La structure atomique d’un complexe Nb/GFLV résolue à 2.8 Å par cryomicroscopie électronique a permis de cartographier l’épitope en surface du virus et a révélé une couverture maximale de la particule virale par le Nb. La comparaison des tests Nb à des réactifs sérologiques commerciaux a révélé leur supériorité en terme de sensibilité et de spécificité, ouvrant ainsi la voie à la commercialisation d’un nouveau test de diagnostic des virus du court-noué de la vigneThe grapevine fanleaf disease is mainly caused in Europe by the Grapevine fanleaf virus (GFLV) and the Arabis mosaic virus (ArMV). The principal mean to limit their spread, is to certify their absence in marketed grapevines by serological methods such as DAS-ELISA. Their unique biophysical and structural properties make the variable domains of heavy chain-only immunoglobulin, called Nanobodies (Nb) a real asset for the development of a diagnostic test against fanleaf disease viruses. I identified Nb able to detect a broad spectrum of viral isolates from two Nb collections against GFLV and ArMV. Their fusion to a fluorescent protein or to a bacterial alkaline phosphatase resulted in the production of efficient DAS-ELISA detection reagents. The atomic structure of a Nb/GFLV complex was solved at 2.8 Å by cryoelectron microscopy, allowing the precise mapping of the viral epitope. This result showed a maximum coverage of the viral particle by the Nb, leading to a maximal signal in DAS-ELISA. The full Nb tests against GFLV and ArMV were compared to commercial reagents and showed the superiority of the former in both sensitivity and specificity, opening the way for the development and commercialization of a new type of serological kits for the detection of grapevine viruses
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Iacenda, Domenica [Verfasser], and Tim [Akademischer Betreuer] Magnus. "Pharmakologische Charakterisierung von P2X7-blockierenden Nanobodies in vitro und in vivo / Domenica Iacenda ; Betreuer: Tim Magnus." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2018. http://d-nb.info/1173323090/34.
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Bergmann, Philine [Verfasser], and Friedrich [Akademischer Betreuer] Koch-Nolte. "Selektion und Charakterisierung von P2X4-spezifischen monoklonalen Antikörpern und Nanobodies / Philine Bergmann ; Betreuer: Friedrich Koch-Nolte." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2016. http://d-nb.info/1116605090/34.
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Bergmann, Philine Verfasser], and Friedrich [Akademischer Betreuer] [Koch-Nolte. "Selektion und Charakterisierung von P2X4-spezifischen monoklonalen Antikörpern und Nanobodies / Philine Bergmann ; Betreuer: Friedrich Koch-Nolte." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2016. http://nbn-resolving.de/urn:nbn:de:gbv:18-81003.
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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 pénétrantThe 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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Maier, Julia [Verfasser], and Ulrich [Akademischer Betreuer] Rothbauer. "Novel Nanobodies and Chromobodies to Study Biomarkers of Epithelial-Mesenchymal Transition (EMT) / Julia Maier ; Betreuer: Ulrich Rothbauer." Tübingen : Universitätsbibliothek Tübingen, 2015. http://d-nb.info/1198119926/34.
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Eichhoff, Anna Marei [Verfasser], and Friedrich [Akademischer Betreuer] Nolte. "Nanobodies als Werkzeuge zur Optimierung Adeno-assoziierter Viren für die Gen- und Tumortherapie / Anna Marei Eichhoff ; Betreuer: Friedrich Nolte." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2018. http://d-nb.info/116153024X/34.
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Hemmer, Caroline. "Développement et utilisation de nanobodies dirigés contre le Grapevine fanleaf virus (GFLV) en lutte antivirale et comme biocapteur in planta." Thesis, Strasbourg, 2015. http://www.theses.fr/2015STRAJ085/document.
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Par leur stabilité, leur petite taille et leur nature monomérique, les domaines variables des immunoglobulines à chaînes lourdes, ou Nanobodies (Nb), sont incontournables en diagnostic et recherche médicale. Pourtant, leur utilisation en agro-biotechnologies demeure confidentielle.Dans l'idée de les utiliser pour étudier et combattre le Grapevine fanleaf virus (GFLV), responsable de la maladie du court-noué très préjudiciable à l'économie viticole mondiale, j'ai produit une collection de Nb spécifiques du GFLV.Fusionné à une protéine fluorescente et exprimé en plante de façon stable, un de ces Nb (alors appelé Chromobody, Cb) a conféré une haute résistance au GFLV inoculé mécaniquement ou transmis par nématodes.Le potentiel du Cb comme biocapteur a été validé par le suivi in vivo d’un isolat contournant la résistance mais toujours reconnu par le Cb. La structure du complexe Nb/GFLV a été résolue à 2,8 Å et révèle la zone occupée par le Nb à la surface de la capside.Ces résultats ouvrent des perspectives innovantes pour la compréhension du cycle infectieux d'un phytovirus et l'élaboration de nouvelles stratégies de lutte antiviraleDue to their small size, high stability and strict monomeric nature, Nanobodies (Nbs) deriving from camelids heavy chain only antibodies have proven very valuable as diagnostic and therapeutic tools. However their use in agro biotechnology remains limited.In order to apply Nbs to the study and the control of grapevine fanleaf degeneration, I produced acollection of Nbs against Grapevine fanleaf virus (GFLV), the causal agent of this devastating disease worldwide.When fused to a fluorescent protein and stably expressed in plants, one of these Nbs (calledChromobody, Cb) conferred high resistance to GFLV, whether inoculated mechanically or by vector-mediated transmission.The identification of an isolate overcoming the resistance but still bound by the Cb allowed real-time tracking of the infection showing the high potential of Cbs as biosensors.The cryoEM structure of the Nb/GFLV complex was obtained at 2,8 Å and provides a clear picture of the footprint of the Nb on the surface of the GFLV capsid.These results pave the way for the innovative use of Nbs to unravel viral life cycle and to counter viral diseases
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Medeiros, Luan Merida de. "Produção de fragmentos de anticorpos VHH contra toxinas de Bothrops jararacussu em biorreator por Escherichia coli HB 2151." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/3/3137/tde-05112018-142057/.
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Em caso de envenenamento ofídico, o tratamento no Brasil hoje é realizado pela administração de soros geralmente produzidos por equinos, que apresentam eficácia limitada: são úteis para os efeitos sistêmicos, mas não inibem efetivamente a evolução dos danos locais, podem causar reações adversas e apresentam alto custo de produção. De acordo com a Organização Mundial da Saúde (OMS), trata-se de uma doença negligenciada pelas autoridades científicas mundiais. O presente projeto, em parceria com o Instituto de Pesquisas em Patologias Tropicais da Fundação Oswaldo Cruz - Rondônia, propõe a produção por Escherichia coli de fragmentos de anticorpos de cadeia pesada de camelídeos, denominados VHH, contra as toxinas do veneno de Bothrops jararacussu, utilizando biorreator. Neste trabalho há interesse em produzir VHH, através da otimização do crescimento desta E. coli. A cinética do crescimento bacteriano foi realizada em shaker orbital sob diferentes condições, variando tamanho do frasco, rotação do shaker, composição do meio de cultura e concentração de substrato; e em biorreatores, alternando meios de cultura e modo de operação do reator (descontínuo e descontínuo alimentado), alterando a vazão de alimentação (linear e exponencial) O processo cinético é fortemente limitado pela formação de acetato, por condições auxotróficas da célula e pela transferência de oxigênio. Nos ensaios em frascos agitados, uma melhor condição de crescimento foi obtida utilizando frascos de 1 L, sob rotação a 270 rpm e 5,0 g/L de glicose. Nos ensaios em reator, quando operados em batelada obtiveram-se cerca 5,5 g/L de células finais, contra 9,3 g/L de células em batelada alimentada com vazão constante. Um maior crescimento foi ainda obtido em um reator de 2 L em regime de batelada alimentada exponencialmente. O biorreator varia a agitação do meio e mantém um nível pré-definido de oxigênio dissolvido, evitando a limitação de oxigênio e controlando a oferta de glicose para o crescimento celular. Neste processo, atingimos 25,6 g/L de células e 0,35 g/L de proteína total após purificação, utilizando meio M9 suplementado.Nowadays in Brazil, the treatment for snakebite poisoning is carried out by the administration of horse sera, which have limited effectiveness: they are useful for systemic effects, but do not effectively inhibit local damage, cause adverse reactions, and present high production costs. According to the WHO, this disease is neglected by the world`s scientific authorities. This project, in partnership with the Institute for Research in Tropical Diseases of Oswaldo Cruz Foundation - Rondonia, proposes the production of heavy chain antibody fragments from camelids, called VHH, using Escherichia coli, to be used against the toxins from the Bothrops jararacussu poison, using a bioreactor. This work is interested in producing VHH through the use of E. coli. The kinetics of bacterial growth were performed in orbital shaker under different conditions, varying vial size, shaker rotation, composition of the culture medium and substrate concentration; and bioreactors, alternating culture media and operation mode of reactor (batch and fed-batch), changing feeding rate (linear and exponential). The kinetic process is statically bound to acetate formation, auxotrophic conditions of the cell and oxygen transfer. In assays in shaken flasks, an output of 270 rpm and 5.0 g/L of glucose. In reactor runs, when operated in a batch 5.5 g/L of final cells were obtained, against 9.3 g/L of final cells in fed-batch with constant flowrate. A larger value was obtained in an exponentially fed-batch reactor of 2 L. The bioreactor varies the agitation of oxygen and controls glucose addition for cell growth. In this process, 25.6 g/L cells and 0.35 g/L total protein after purification were reached, using supplemented M9 medium.
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Kent, Lisa. "Targeting the N-myc oncoprotein using nanobody technology." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/278020.
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The myc family of oncogenic transcription factors, which includes c-myc, N-myc and L-myc, control major cellular processes such as proliferation and differentiation by integrating upstream signals and orchestrating global gene transcription. They do this largely through dimerising with Max, which together bind to enhancer (E)-box elements in DNA. Myc proteins function similarly but differ in potency and tissue distribution. For instance, N-myc is expressed predominantly during development in undifferentiated cells of the nervous system, whereas c-myc is ubiquitously expressed in all proliferating cells. Myc proteins, when deregulated, are major drivers of tumourigenesis. Myc deregulation occurs in up to 70% of all human cancers and is often associated with the most aggressive forms. For example, MYCN, the gene encoding N-myc, is amplified in 20-30% of neuroblastomas, and amplification strongly correlates with advanced stage and poor prognosis. Myc proteins are therefore considered “most wanted” targets for cancer therapy, but have long been considered undruggable mainly due to challenges in nuclear drug delivery and physically targeting myc directly given that it is a largely disordered protein that lacks discernible clefts and pockets for small molecules to inhabit. Furthermore, c-myc is important in normal tissue maintenance so the effect of its inhibition in humans is difficult to predict. However, recent in vivo studies showed that systemic myc inhibition (using the peptide pan myc inhibitor Omomyc) has mild and reversible side effects and induces tumour regression. This has alleviated concerns about the side effects that myc inhibition might have, and reinforced the promise of myc as a powerful drug target. However, the translation of Omomyc into the clinic has been hindered by poor cellular delivery. In fact, no direct myc inhibitor has yet been approved, indicating that novel approaches are needed. Moreover, inhibitors in development tend to inhibit all myc family proteins. An inhibitor that could specifically target N-myc might improve safety through bypassing c-myc inhibition. This could be used for the treatment of N-myc-driven cancers such as MYCN-amplified neuroblastoma. Nanobodies, camelid-derived single-domain antibodies, are a relatively new drug class. Whilst some are already in clinical trials for a wide range of diseases, these are specific for cell-surface or extracellular targets. However, their properties also make them ideal for use as intracellular antibodies or ‘intrabodies’. For example, they are small (just 12-15 kDa) and highly soluble due to naturally occurring hydrophobic to hydrophilic amino acid substitutions. Their small size and convex shape makes them advantageous in capturing structures in intrinsically disordered proteins and allows them to reach hidden epitopes not accessible to conventional antibodies, which could improve biological activity. Importantly, nanobodies retain the high specificities and affinities of conventional antibodies. Their small, single-domain nature also means they can be engineered with ease to modify aspects of their localisation and/or function. For example, they can be coupled to carrier molecules to facilitate cellular entry, and a nuclear localisation signal (NLS) can be added to drive them into the nucleus. Also, it was recently shown that an F-box domain could also be incorporated into nanobodies to recruit degradation machinery to its antigen, which depletes the antigen from cells via the proteasomal degradation pathway. Due to their highly advantageous properties, nanobodies raised against N-myc might overcome the barriers to targeting N-myc, providing potent and specific means of directly inhibiting N-myc therapeutically, which has not yet been achieved. In this thesis, nine unique nanobodies were raised against N-myc. These included three against the basic helix-loop-helix leucine zipper (bHLH-LZ) domain where Max dimerises, and six against the transactivation domain where numerous regulatory and cofactor proteins bind, such as the E3 ubiquitin ligase Skp2. Nanobodies against the transactivation domain were more specific for N-myc and were shown to inhibit its Skp-2-mediated ubiquitylation. This could provide novel means of eradicating tumours based on a study showing that inhibition of ubiquitylation at this domain triggers a transcriptional ‘switch’ that induces a non-canonical target gene Egr1, leading to p53-independent apoptosis. A nanobody against the bHLH-LZ (Nb C2) was shown to bind both N- and c-myc to similar magnitudes. Its affinity for N-myc bHLH-LZ was superior to that of the small molecule myc inhibitor 10058-F4, which prolongs survival in a MYCN-dependent mouse model of high-risk neuroblastoma. Nb C2 spontaneously transduced cell membranes and its coupling to a novel small molecule carrier (SMoC) enhanced its cellular uptake. Furthermore, the addition of a NLS increased its nuclear localisation. Preliminary experiments showed that Nb C2 might slow proliferation and induce apoptosis in cancer cell lines expressing c-myc, suggesting that Nb C2 might also be effective against cancers characterised by deregulated c-myc. Taken together, data generated in this thesis have revealed intriguing findings that provide a basis for the development of these nanobodies for the treatment of N-myc- and c-myc-driven cancers.
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Well, Lennart [Verfasser], and Friedrich [Akademischer Betreuer] Koch-Nolte. "Vergleich von Nanobodies und konventionellen Antikörpern für die molekulare in vivo Bildgebung von T-Zellen / Lennart Well. Betreuer: Friedrich Koch-Nolte." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2015. http://d-nb.info/1068930853/34.
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Gebura, Myroslav [Verfasser], Dirk [Akademischer Betreuer] Görlich, Jürgen [Gutachter] Wienands, and Markus [Gutachter] Bohnsack. "Nanobodies as new tools for studying large cargo transport and lamina organization / Myroslav Gebura ; Gutachter: Jürgen Wienands, Markus Bohnsack ; Betreuer: Dirk Görlich." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2018. http://d-nb.info/1161942297/34.
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Lenz, Alexander Verfasser], and Gerhard [Akademischer Betreuer] [Adam. "Molecular imaging of tumors with nanobodies and antibodies : Timing and dosage are crucial factors for improved in vivo detection / Alexander Lenz ; Betreuer: Gerhard Adam." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2016. http://nbn-resolving.de/urn:nbn:de:gbv:18-81731.
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Kunick, Valentin Helmut Verfasser], and Gerhard [Akademischer Betreuer] [Adam. "Vergleichende Analyse von Nanobodies und monoklonalen Antikörpern gegen humanes CD38 für die NIRF-Bildgebung von Lymphomen in vivo / Valentin Helmut Kunick ; Betreuer: Gerhard Adam." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2020. http://nbn-resolving.de/urn:nbn:de:gbv:18-105726.
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Lenz, Alexander [Verfasser], and Gerhard [Akademischer Betreuer] Adam. "Molecular imaging of tumors with nanobodies and antibodies : Timing and dosage are crucial factors for improved in vivo detection / Alexander Lenz ; Betreuer: Gerhard Adam." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2016. http://d-nb.info/1119319978/34.
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Kunick, Valentin Helmut [Verfasser], and Gerhard [Akademischer Betreuer] Adam. "Vergleichende Analyse von Nanobodies und monoklonalen Antikörpern gegen humanes CD38 für die NIRF-Bildgebung von Lymphomen in vivo / Valentin Helmut Kunick ; Betreuer: Gerhard Adam." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2020. http://d-nb.info/1214811957/34.
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Pinto, Espinoza Carolina [Verfasser], and Friedrich [Akademischer Betreuer] Koch-Nolte. "Optimization of nanobodies for in vivo targeting of P2X7 ion channel on brain microglia and kidney T cells / Carolina Pinto Espinoza ; Betreuer: Friedrich Koch-Nolte." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2019. http://d-nb.info/119028572X/34.
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Pinto, Espinoza Carolina Verfasser], and Friedrich [Akademischer Betreuer] [Koch-Nolte. "Optimization of nanobodies for in vivo targeting of P2X7 ion channel on brain microglia and kidney T cells / Carolina Pinto Espinoza ; Betreuer: Friedrich Koch-Nolte." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2019. http://nbn-resolving.de/urn:nbn:de:gbv:18-97989.
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Boyé, Kevin. "Implication de CXCR3 dans la progression tumorale : une nouvelle cible thérapeutique." Thesis, Bordeaux, 2016. http://www.theses.fr/2016BORD0315/document.
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CXCR3 appartient à la famille des récepteurs couplés aux protéines G. Avec ses ligands, les chimiokines CXC, CXCR3 régule diverses fonctions biologiques et participe à de nombreux processus comme l’angiogenèse, l’inflammation et le cancer. La complexité de CXCR3 provient de son épissage alternatif qui conduit à des isoformes distinctes. CXCR3-A est reconnu pour promouvoir la prolifération, la survie et la migration cellulaire tandis que CXCR3-B induit des signaux inhibiteurs de la croissance cellulaire.Le modèle cellulaire U87, dérivé d’un glioblastome humain, a été utilisé afin d’étudier les mécanismes moléculaires régulant l'activité et le trafic des isoformes de CXCR3 dans les cellules tumorales. CXCR3 est le récepteur fonctionnel de l’activité angiostatique de CXCL4 et son variant CXCL4L1. En fonction de leur état d'oligomérisation, ces deux chimiokines ont des interactions préférentielles avec les isoformes de CXCR3. L’activation de CXCR3-A conduit à un important changement conformationnel et induit des voies de signalisation pro-migratoires. L’étude du trafic souligne l’importance de la clathrine et du réseau Trans-Golgi dans l’internalisation et le recyclage de CXCR3-A. Pour la première fois, LRP-1 a été identifié comme nouveau partenaire de CXCR3-A. LRP1 n’est pas seulement reconnu comme un récepteur de l’endocytose mais également comme une protéine de la signalisation. LRP1 interagit avec CXCR3-A au niveau extracellulaire et régule sa conformation, son trafic et son activité pro-tumorale.L'utilisation de modèles cellulaires d'adénocarcinome pancréatique a permis de caractériser CXCL4L1 comme facteur pro-tumoral, via l’activation de CXCR3-A dans les cellules tumorales. CXCL4L1 apparait pour la première fois comme un biomarqueur important dans la progression du cancer pancréatique.Dans les différents modèles, les signalisations chimiokines CXC/CXCR3-A induisent une augmentation des propriétés invasives tumorales. Au niveau moléculaire, l’association de CXCR3 à diverses protéines (ligands et partenaires) est essentielle pour réguler les fonctions biologiques de la cellule tumorale.Les nanoparticules sont désormais connues comme une nouvelle génération d'anticorps thérapeutiques présentant de nombreux avantages par rapport aux anticorps conventionnels. Ainsi, le développement de nanoparticules associées à des inhibiteurs de CXCR3 apparaît comme une nouvelle stratégie thérapeutique anti-tumorale prometteuseCXCR3 belongs to the G-protein-coupled receptors family. With its ligands, the CXC chemokines, CXCR3 regulates several biological functions and plays important roles in angiogenesis, inflammation and cancer. The interaction with CXCR3 is rather complex due to the presence of distinct spliced isoforms. CXCR3-A is known to promote cell proliferation, survival, and migration while CXCR3-B leads to cell growth inhibition.The human glioblastoma cell model, U87, was used to study the molecular mechanisms regulating the activity and trafficking of CXCR3 isoforms in tumor cells. CXCR3 has been reported as the functional receptor for the angiostatic activity of CXCL4 and its variant CXCL4L1. Depending on their oligomerization status, these two chemokines present preferential interaction with CXCR3 isoforms. Activation of CXCR3-A leads to an important conformational change and induces pro-migratory signaling pathways. Studies on the vesicular trafficking highlight the importance of the clathrin and the Trans-Golgi network for both internalization and recycling of CXCR3-A. For the first time, LRP-1 is identified as a new partner of CXCR3-A. LRP1 is not only recognized as an endocytic receptor but also as a signaling protein. LRP1 interacts with CXCR3-A via its extracellular α subunit and regulates CXCR3-A conformation, trafficking and pro-tumoral activity.Pancreatic ductal adenocarcinoma cell models were used to characterize CXCL4L1 as a pro-tumoral factor that activates CXCR3-A in tumor cells. For the first time, CXCL4L1 appears as an important biomarker for pancreatic cancer progression.In the different cell models, signaling pathways of CXC chemokine/CXCR3-A lead to an increase in tumor invasive properties. At the molecular level, the association of CXCR3 with various proteins (ligands and partners) is essential to regulate tumor cell biological functions.The nanoparticles are now known as a new generation of therapeutic antibodies with many advantages over conventional antibodies. Thus, the development of nanoparticles associated to CXCR3 inhibitors appears as a new promising pharmacological targeted strategy to treat cancer
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Maidorn, Manuel [Verfasser], Dávila Luis Felipe [Akademischer Betreuer] Opazo, Silvio O. [Gutachter] Rizzoli, Peter [Gutachter] Rehling, and Mikael [Gutachter] Simons. "Development of Nanobodies to Image Synaptic Proteins in Super-Resolution Microscopy / Manuel Maidorn ; Gutachter: Silvio O. Rizzoli, Peter Rehling, Mikael Simons ; Betreuer: Luis Felipe Opazo Dávila." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2018. http://d-nb.info/1169396461/34.
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Hartmann, Lucie. "Développement et caractérisation d'outils immunologiques dirigés contre des récepteurs membranaires d'intérêt thérapeutique." Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ017/document.
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Les Récepteurs Couplés aux Protéines G (RCPG) constituent la plus grande famille de protéines membranaires chez l’Homme, et leur implication dans un grand nombre de processus physiologiques justifie pleinement l’intérêt de leur étude. Les anticorps spécifiques de ces récepteurs sont des outils polyvalents à haute valeur ajoutée, qui restent toutefois encore trop rarement disponibles, notamment en raison des difficultés techniques posées par leur génération. Ce manuscrit présente la mise au point d’une méthode d’immunisation alternative et innovante, mettant en jeu des particules virales recombinantes dérivées du Virus de la Forêt de Semliki (SFV) codant pour le récepteur d’intérêt. Appliquée au récepteur de l’adénosine A2A humain, l’immunisation permet d’engendrer la surexpression de celui-ci à la surface des cellules de l’animal infecté, et de provoquer l’apparition d’une réponse immunitaire. Cette approche permet d’une part de générer un sérum polyclonal de souris spécifique au récepteur, et ouvre donc une nouvelle voie pour l’obtention d’anticorps monoclonaux murins. Elle semble d’autre part prometteuse pour la génération de nanobodiesG Protein Coupled Receptors (GPCRs) constitute the largest membrane protein family represented in the human genome. Their involvement in a wide number of biological processes fully supports their study. GPCR-targeting antibodies are versatile and valuable tools, which remain scarcely available, chiefly because their generation is a challenging process. This thesis presents an alternative and innovative strategy in which recombinant Semliki Forest Virus (SFV) particles coding for the receptor of interest are used as immunogens. When applied to the human version of the Adenosine A2A receptor, this method enables to cause the receptor’s overexpression at the surface of the infected animal cells, which generates an immune response. This strategy enables to raise receptor-specific mouse polyclonal serum. It opens a new path towards the generation of monoclonal mouse antibodies. Additionally, it seems to also be a promising approach to develop nanobodies
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Dudeffant, Clémence. "Développement et évaluation d'agents de contraste pour détecter la maladie d'Alzheimer par IRM : coloration par le Gadolinium et nanocorps ciblant les protéines Aβ et Tau." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS281/document.
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L’imagerie par résonnance magnétique (IRM) est devenue un outil incontournable en recherche biomédicale. Le développement d’outils de neuroimagerie pour le petit animal est primordial pour valider des hypothèses biologiques et tester l’efficacité de nouvelles thérapies. Dans le contexte de la maladie d’Alzheimer (MA), l’utilisation d’IRM à haut champ magnétique permet l’imagerie des plaques amyloïdes, signature moléculaire de cette pathologie. Cependant, seule une faible proportion de ces lésions sont détectées et aucune méthode de neuroimagerie ne permet actuellement d’imager les dégénérescences neurofibrillaires, deuxième signature microscopique de la MA. Dans cette thèse, deux méthodes complémentaires, utilisant différents agents de contraste à base de Gadolinium (Gd), ont été étudiées pour améliorer la détection des plaques amyloïdes par IRM. La première partie de cette thèse s’est intéressée à la méthode du « Gd- staining ». Une étude comparative réalisée sur différents modèles murins d’amyloïdose et sur des primates non-humains, a permis de mieux comprendre les mécanismes impliqués dans la détection des plaques amyloïdes grâce à cette technique. Également, nous avons montré pour la première fois qu’il est possible d’imager les lésions Aβ humaines, en post mortem, par Gadolinium-staining. La deuxième partie de cette thèse s’est intéressée au développement d’agents de contraste vectorisés, ciblant spécifiquement les lésions de la MA. Ces molécules seraient capables de traverser spontanément la barrière hémato-encéphalique, principale limite au développement de molécules à visée cérébrale, et permettraient de cibler spécifiquement les deux lésions caractéristiques de la MA. Ces propriétés font de ces molécules de outils potentiels intéressants pour l’imagerie cérébrale de la MAMagnetic resonance imaging (MRI) has become a key tool for both clinical and preclinical research. Developing innovative neuroimaging techniques specifically designed for small animal models is therefore crucial to validate biological hypotheses and screen new drugs. Amyloid plaques and neurofibrillary tangles are the major lesions characterizing Alzheimer’s disease (AD) and intensive research has been carried out in order to enable the accurate detection of amyloid plaques using high-field MRI. However, only a small portion of plaques are spontaneously detected by MRI and no method is so far available for neurofibrillary tangles imaging. Here, we develop two contrast-enhanced MRI techniques to improve amyloid plaques detection using high-field MRI. The first part of this work focuses on Gadolinium-Staining for amyloid plaques detection by MRI. We performed a comparative study between mice model of amyloi-dosis and non-human primate models, which al-lowed us to gain a better understanding of the origin of the contrast induced by amyloid plaques when performing contrast-enhanced MRI. We also showed for the first time that Gd-stained MRI is able to detect amyloid plaques in human-AD brain tis-sues. The second part of this manuscript describes two novel single-domain antibodies (VHHs or nano-bodies) that are able to specifically recognize and bind amyloid plaques or neurofibrillary tangles. The detection of intracerebral targets with imaging probes is challenging due to the non-permissive nature of the blood-brain barrier. Interestingly, VHH exhibiting a basic isoelectric point are able to transmigrate across the blood-brain barrier, thus making them promising tools for in vivo imaging of AD’s lesion at high-field MR
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Gulati, Sahil Gulati. "Modulating G Protein-Coupled Receptor Signaling Pathways with Selective Chemical- and Protein-Based Effector Molecules." Case Western Reserve University School of Graduate Studies / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1530642105672697.
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Duhoo, Yoan. "Etudes moléculaires et structurales des complexes membranaires au coeur du système de sécrétion de type IX." Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0294.
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Les maladies parodontales sont causées par une infection bactérienne touchant les tissus entourant les dents, appelés parodonte. L’inflammation aggravée du parodonte peut conduire au déchaussement ou à la chute des dents. Porphyromonas gingivalis est une bactérie anaérobique qui sécrète des toxines appelées gingipaïnes, considérées comme le facteur de virulence majeur. Le système de sécrétion de type IX (T9SS) a été récemment mis en évidence exclusivement chez les membres de la famille des bacteroidetes. Chez P. gingivalis, ce système et directement relié à la sécrétion des gingipaïnes est donc sa pathogénicité. Des études ont montré que plus d’une quinzaine de protéines sont impliquées dans l’assemblage, la fonction et la régulation de ce système de sécrétion. Parmi ces protéines, PorK, PorL, PorM, PorN forment un complexe membranaire au cœur de la machinerie de sécrétion, enchâssé dans les deux membranes bactériennes. L’objectif de ce travail de thèse a été de mettre en place une méthodologie d’extraction et de solubilisation du complexe membranaire PorK-L-M-N afin d’étudier sa structure moléculaire par des méthodes de biochimie intégrative. Trois sous-complexes ont été étudiés successivement. Le complexe de membrane externe PorK-N, le complexe de membrane externe étendu PorK-N-M et le complexe de membrane interne PorL-M. Les résultats obtenus montrent que le complexe de membrane externe PorK-N présente une structure en forme d’anneau de 50nm de diamètre et le complexe de membrane interne PorL-M possède une structure étendue avec une base sphérique de 25nm. Ces résultats valident une méthodologie qui pourra par la suite être utilisée pour d'autres études du T9SSPeriodontal diseases are caused by a bacterial infection affecting the tissues surrounding the teeth, called periodontal. The aggravated inflammation of the periodontium may lead to loosening or falling of the teeth. Porphyromonas gingivalis is an anaerobic bacterium able to secrete toxins called gingipains, considered as the major virulence factor. First called PorSS, the type IX secretion system (T9SS) was recently found exclusively in members of bacteroidetes. In P. gingivalis this system is directly related to the secretion of gingipains and is therefore its pathogenicity. Studies have shown that more than fifteen proteins are involved in the assembly, function and regulation of this secretory system. Among these proteins PorK, PorL, PorM, PorN form a membrane core complex, the central part of the secretory machinery embedded in the two bacterial membranes. The objective of this thesis was to set up a methodology of extraction and solubilization of the PorK-L-M-N membrane complex in order to study its molecular structure by integrative biochemistry methods. Three sub-complexes have been studied successively. PorK-N the outer membrane complex, PorK-N-M extended outer membrane complex and PorL-M the inner membrane complex. The results show that the PorK-N outer membrane complex has a ring-shaped structure of 50nm in diameter, confirming published results, and the PorL-M inner membrane complex has an extended structure of 25nm with a spherical base. These results validate the established methodology that can subsequently be used to continue the structural study of T9SS
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Schumacher, Dominik. "Site-specific functionalization of antigen binding proteins for cellular delivery, imaging and target modulation." Doctoral thesis, Humboldt-Universität zu Berlin, 2017. http://dx.doi.org/10.18452/18547.
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Antikörper und Antigen-bindende Proteine, die an Fluorophore, Tracer und Wirkstoffe konjugiert sind, sind einzigartige Moleküle, welche die Entwicklung wertvoller diagnostischer und therapeutischer Werkzeuge ermöglichen. Allerdings ist der Konjugationsschritt sehr anspruchsvoll und trotz intensiver Forschung noch immer ein bedeutender Engpass. Zusätzlich sind Antigen-bindende Proteine oftmals nicht dazu in der Lage, die Zellmembran zu durchdringen und im Zellinneren nicht funktionsfähig. Daher ist ihre Verwendung auf extrazelluläre Targets beschränkt, was eine bedeutende Anzahl wichtiger Antigene vernachlässigt. Beide Limitierungen bilden Kernaspekte dieser Arbeit. Mit Tub-tag labeling wurde ein neuartiges und vielseitiges Verfahren für die ortsspezifische Funktionalisierung von Biomolekülen und Antigen-bindenden Proteinen entwickelt, und so die Palette der Proteinfunktionalisierungen bedeutend erweitert. Tub-tag wurde erfolgreich für die ortsspezifische Funktionalisierung verschiedener Proteine und Antigen-bindender Nanobodies angewendet, die für konfokale Mikroskopie, Proteinanreicherung und hochauflösende Mikroskopie eingesetzt wurden. In einem weiteren Projekt wurden zellpermeable Antigen-bindende Nanobodies hergestellt und somit das schon lange Zeit bestehende Ziel, intrazelluläre Targets durch in vitro funktionalisierte Antigen-bindende Proteine zu visualisieren und manipulieren, erreicht. Hierzu wurden zwei verschiedene Nanobodies an ihrem C-Terminus cyclischen zellpenetrierenden Peptiden unter Verwendung von Expressed Protein Ligation funktionalisiert. Diese Peptide ermöglichten die Endozytose-unabhängige Aufnahme der Nanobodies mit sofortiger Bioverfügbarkeit. Mit Tub-tag labeling und der Synthese von zellpermeablen Nanobodies konnten wichtige Bottlenecks im Bereich der Proteinfunktionalisierung und Antikörperforschung adressiert werden und neue Tools für die biochemische und zellbiologische Forschung entwickelt werden.Antibodies and antigen binding proteins conjugated to fluorophores, tracers and drugs are powerful molecules that enabled the development of valuable diagnostic and therapeutic tools. However, the conjugation itself is highly challenging and despite intense research efforts remains a severe bottleneck. In addition to that, antibodies and antigen binding proteins are often not functional within cellular environments and unable to penetrate the cellular membrane. Therefore, their use is limited to extracellular targets leaving out a vast number of important antigens. Both limitations are core aspects of the presented thesis. With Tub-tag labeling, a novel and versatile method for the site-specific functionalization of biomolecules and antigen binding proteins was developed expanding the toolbox of protein functionalization. The method is based on the microtubule enzyme tubulin tyrosine ligase. Tub-tag labeling was successfully applied for the site-specific functionalization of different proteins including antigen binding nanobodies which enabled confocal microscopy, protein enrichment and super-resolution microscopy. In addition to that, cell permeable antigen binding nanobodies have been generated constituting a long thought goal of tracking and manipulating intracellular targets by in vitro functionalized antigen binding proteins. To achieve this goal, two different nanobodies were functionalized at their C-terminus with linear and cyclic cell-penetrating peptides using expressed protein ligation. These peptides triggered the endocytosis independent uptake of the nanobodies with immediate bioavailability. Taken together, Tub-tag labeling and the generation of cell-permeable antigen binding nanobodies strongly add to the functionalization of antibodies and their use in biochemistry, cell biology and beyond.
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Abdallah, Bassim Violla. "Structural and functional analysis on GacS homodimeric histidine kinase reveals a non-canonical autokinase activity and new insights into the heterodimer partnership with RetS." Thesis, Aix-Marseille, 2020. http://www.theses.fr/2020AIXM0256.
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Pseudomonas aeruginosa (PA) est un pathogène qui infecte particulièrement les patients atteints de la mucoviscidose. PA provoque des infections aiguës et chroniques et alterne entre des modes de vie planctonique et sédentaire. Cette transition est principalement régulée par les systèmes à deux composants (TCS). Durant ma thèse, je me suis focalisée sur le TCS GacS/GacA, impliqué dans un réseau de signalisation multikinase. GacS est une histidine kinase (HK) et GacA est son régulateur de réponse (RR). La région cytoplasmique de GacS est composée des domaines HAMP, S-Hélice, H1, D1 et H2. Des tests fonctionnels ont montré que la région HAMP/S-Hélice est nécessaire pour la transduction du signal et contribue à son activité. L’analyse structurale a montré une boucle de fixation de l’ATP structurée contrairement à ce qui est observé dans d'autres HKs. De plus, un nouveau domaine (ND) a été identifié et s’est révélé essentiel pour assurer une activité autokinase. Nous avons proposé que le ND contribue à des changements conformationnels pour placer GacS dans une configuration active. Par ailleurs, RetS HK inhibe GacS par des interactions directes. Des tests de spectrométrie de masse native et des tests d’interaction ont montré que le domaine HAMP est nécessaire pour assurer l'interaction entre ces HKs. De plus, des anticorps à domaine unique contre GacS nous ont permis d’identifier des sites potentiels d'inhibition de la voie GacS/GacA et de la formation de biofilm. Dans cette étude, nous avons dévoilé une activité autokinase non canonique de GacS et de nouvelles perspectives sur son interaction avec RetS qui contribuent au processus décisionnel pendant l'infection par PAPseudomonas aeruginosa (PA) is a pathogen that particularly infects patients with cystic fibrosis. PA causes acute and chronic infections and can switch from a planktonic to a sedentary lifestyle. This transition is mainly coordinated by Two-Component Systems (TCS). During my thesis, I focused on GacS/GacA TCS, a master regulator of the acute-to-chronic transition. In fact, GacS is a membrane-bound histidine kinase (HK) and GacA is the response regulator (RR). GacS cytoplasmic region is composed by the HAMP, S-Helix coiled-coil region. This helical part is followed by the H1, the D1 and H2 domains. GacS/GacA TCS is involved in a multikinase regulatory network and its activity is modulated by three HKs. Mutagenesis and functional assays showed that the HAMP and S-Helix orchestrate the signal transduction prior to its autokinase activity. In addition, GacS presented a folded ATP lid in contrast to what is observed in other HKs. Furthermore, we unveiled a new domain (ND) in GacS sequence. Functional assays showed that the ND domain is essential to insure a full autokinase activity of GacS. We proposed that this domain contributes in conformational rearrangements in order to shift GacS to its active state. RetS HK is known to inhibit GacS/GacA via direct interactions. Native mass spectrometry and microscale thermophoresis assays showed that the HAMP domain is essential for this interaction. Furthermore, nanobodies generated against the cytoplasmic region of GacS revealed potential sites of inhibition of GacS activity. In this study, we unveiled a non-canonical autokinase activity in GacS and new insights into its interaction with RetS that underlie the decision-making of PA
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Maidorn, Manuel. "Development of Nanobodies to Image Synaptic Proteins in Super-Resolution Microscopy." Doctoral thesis, 2017. http://hdl.handle.net/11858/00-1735-0000-002E-E4D1-D.
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Machado, Maria Eugénia Freire Torres Alves. "Vectored immunoprophylaxis to produce anti-BACE1 nanobodies for treatment of Alzheimer's disease." Master's thesis, 2019. http://hdl.handle.net/10451/40536.
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Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2019Atualmente, a Doença de Alzheimer e a principal causa de demência, afetando mundialmente cerca de 46 milhões de pessoas. A incessante procura de uma cura para o Alzheimer, de terapias que impeçam a propagação da doença ou até mesmo de biomarcadores para o diagnóstico prematuro da doença, está longe de acabar. No entanto, os custos relacionados com esta doença serão incomportáveis nas próximas gerações. Em 1906, Alois Alzheimer descreveu pela primeira vez uma doença que numa fase celular foi caracterizada pela formação de emaranhados neurofibrilhares e pela deposição de placas amiloides no cérebro, posteriormente ocorre perda neuronal, distrofia dos astrócitos e neurónios e alterações vasculares, nesta fase, os pacientes apresentam declínio cognitivo e por fim demência. A maioria dos casos de Alzheimer são esporádicos e surgem em pacientes com idade avançada. No entanto, existem casos hereditários e de early onset, que ocorrem devido a mutações em genes que codificam para as presinilinas (PSEN) e para a amyloid precursor protein (APP), proteínas que estão envolvidas na via amiloidogénica, e que favorecem a formação das espécies de amiloide-b (Ab) toxicas. A hipótese de cascata amiloidogénica propõe que as placas amiloide, os péptidos Ab que as constituem, e o seu metabolismo, têm um papel fundamental no desenvolvimento da doença de Alzheimer. Consequentemente, mutações nas PSEN e em APP, enzima e substrato da via amiloidogénica, são causadoras da doença, e são frequentemente manipuladas para criar modelos de Alzheimer in vitro e in vivo. O modelo de cascata amiloidogénica, por ter apenas em conta o papel da deposição das placas amiloides ao nível dos neurónios, exclui outros elementos, como a neuro inflamação e a disrupção da barreira hematoencefálica (BBB), que também apresentam uma função fundamental no desenvolvimento do Alzheimer. A enzima BACE1 (b-site amyloid precursor protein cleaving enzyme 1) é a responsável pelo processamento da APP pela via amiloidogénica, deste modo, tem sido um dos principais alvos de investigação para terapias de Alzheimer. Inibidores de BACE1 têm sido um êxito em estudos in vitro, sendo que muitas moléculas com potencial terapêutico avançarem para ensaios clínicos, no entanto a maioria dos ensaios não foram bem sucedidos e foram posteriormente descontinuados. Uma alternativa às moléculas inibidoras de BACE1 e, então, a administração de anticorpos anti-BACE1, que por serem mais estáveis e apresentarem uma maior especificidade ao substrato, têm um elevado interesse terapêutico. No entanto, até estas moléculas apresentam desvantagens, tais como ineficiência em atravessar a BBB e um tempo de vida curto. O desenvolvimento de terapia génica baseada em vetores virais permite o transporte de genes de que codificam para anticorpos, ultrapassando as desvantagens dos anticorpos. Recentemente, a terapia génica baseada em vetores virais emergiu como um método eficiente de expressão de genes com potencial terapêutico, através de técnicas moleculares. Este método, dependendo da origem do vetor, tem duas subdivisões. A terapia génica não viral, que consiste na administração de moléculas de origem não viral, tal como polímeros catiónicos, lípidos catiónicos, entre outros. Estes métodos apesar de baratos, não são eficientes para o tratamento de doenças do sistema nervoso central (CNS). Por outro lado existe a terapia génica viral , em que variados tipos de vetores têm sido desenvolvidos, sendo os mais estudados os adenovírus, lentivírus e vírus adeno-associados (AAV), sendo este último mais eficientes. AAV são vírus não replicativos e não patogénicos, que contêm um genoma de 4,7 kb de DNA de cadeia simples. Os vetores AAV não integram o genoma, e são produzidos através da remoção dos genes virais que são flanqueados pelas duas regiões terminais invertidas (ITR), sendo posteriormente substituídos por uma cassete de expressão génica que contém o gene de interesse. Existem 11 serotipos naturais e cerca de 100 variações destes, sendo este determinado pela sequência de aminoácidos da capside, sendo que o tropismo de cada serotipo depende da capside. O serotipo 9 e o serotipo PHP.B, uma variação do serotipo 9, apresentam uma grande afinidade para células do sistema nervoso, sendo que o último e bastante eficiente a atravessar a BBB e a transduzir células nervosas. O genoma destes vetores pode adquirir duas conformações, uma de cadeia simples (ss), outra de cadeia complementar (sc), a segunda e formada devido a uma mutação em um dos ITR, que confere à molécula de DNA uma conformação de cadeia dupla, pronta a ser transcrita pela maquinaria da célula hospedeira, sendo que ambos permitem uma expressão contínua do transgene. A conformação sc permite uma expressão do transgene mais rápida e mais eficiente, apesar a capacidade de transporte do vetor diminuir para 2,5 kb. O desenvolvimento e otimização destes mecanismos permitem ao vetor atravessar a BBB, eficientemente transduzir neurónios e aumentar a expressão de moléculas terapêuticas, tais como anticorpos ou nanoanticorpos altamentes eficientes. Esta técnica e chamada de vectored immunoprophylaxis (VIP). VIP consiste na imunização passiva de anticorpos através de terapia génica baseada em vírus, sendo que as células transduzidas expressam o anticorpo codificado no transgene, estas proteínas têm uma expressão endógena e continuada, permitindo que apenas uma administração do vetor tenha um efeito prolongado. Os anticorpos monoclonais, apesar de apresentarem resultados positivos, têm-se provado ineficientes após administração de vetores que contêm a sua informação genética no genoma, contrariamente aos nanoanticorpos. Os nanoanticorpos são produzidos por camelídeos e são pequenas moléculas com alta estabilidade, baixa imugenogeneicidade e alta afinidade para pequenos antigénios. Os nanoanticorpos, devido ao seu tamanho, são facilmente introduzidos no genoma de um vetor scAAV, que apresenta uma conformação que aumenta a expressão do transgene contido no seu genoma. Este trabalho focou-se no desenvolvimento de uma nova terapia VIP baseada em vírus AAV. O vetor administrado continha no seu genoma o transgene que codifica para um nanoanticorpo com atividade anti-BACE1 (B9), anteriormente validado in vitro. Após administração do vetor que continha o gene que codifica para o nanoanticorpo B9, a diminuição do conteúdo Ab foi detetável em duas das três estirpes em estudo, APPDutch e APPNL-G-F, no entanto não foi detetável a alteração nos níveis de Ab na estirpe APP/PS1, possivelmente devido a agressividade do modelo na deposição das espécies Ab. A otimização do vetor e fundamental para aumentar a expressão do transgene e para aumentar o efeito terapêutico do nanoanticorpo de modo a que haja uma diminuição dos níveis de Ab. Deste modo, avaliou-se a eficiência de um vetor com uma conformação scAAV, sendo expectável que aumente a expressão do transgene. Os conteúdos de Ab entre os grupos injetados com ssAAV e o scAAV permaneceram semelhantes aos do grupo injetado com o vetor controlo, possivelmente devido à baixa concentração de Ab nos cérebros dos ratos APPDutch na idade analisada. Posteriormente, a expressão do nanoanticorpo após injeção com as duas conformações, ssAVV e scAAV, foi comparada. No entanto e contrariamente ao expectável, a conformação ssAAV conferiu uma expressão mais acentuada do que a conformação scAAV, deste modo concluímos que os benefícios que advêm da conformação sc não compensam a perda dos elementos regulatórios presentes no vetor com a conformação ss. A administração de vetores capazes de transportar um nanoanticorpo com efeitos terapêuticos e de elevado interesse não só para tratamento do Alzheimer mas também para o tratamento de outras doenças do CNS. A administração de B9 por via de um AAV vector, ainda precisa de otimização, no entanto apresenta um grande potencial terapêutico.
Alzheimer’s disease (AD) is the main cause of dementia, a disease characterized by an impairment in memory, language, and cognition. It is estimated to affect around 46 million people worldwide. The economic costs of AD will soon be unbearable. Therefore, efforts to find a treatment to halt or even slow the progression of the disease have become a priority for the scientific community. The symptoms of AD are cognitive impairment and progressive neurodegeneration. At the cellular level, the abnormal deposition of amyloid-b (Ab) species is thought to be the trigger of the disease, although there are cellular stressors that also play a role in the progression of AD. b-site amyloid precursor protein cleaving enzyme 1 (BACE1) is responsible for 80% of Ab production in the brain. Hence, strategies such as small molecules inhibitors or antibodies targeting BACE1 have been generated. However, limitations such as cross-reactivity with BACE2 and poor blood-brain-barrier (BBB) crossing respectively, have limited their success. In this context, gene therapy has emerged as an alternative approach for the treatment of neurodegenerative diseases like AD. Therapies for central nervous system (CNS) based on the use of adeno-associated virus (AAV) are being widely developed, as they have the advantages of providing a higher specificity to the target, being safe, infect several cells types and provide long-term expression of any given therapeutic transgene. Thus, strategies such as passive immunization by AAV-mediated delivery of antibody-encoding genes (vectored immunoprophylaxis: VIP) may be an ideal alternative to treat not only AD, but also a wide spectrum of neurodegenerative disorders. Here, a novel AAV-based therapeutic strategy is evaluated, aiming to deliver an anti-BACE1 nanobody that efficiently decreases the levels of Ab in the CNS after systemic delivery. Therefore, we validated the expression of the nanobody in three AD mouse strains: APPDutch, APPNL-G-F and APP/PS1. The activity of the nanobody was evaluated indirectly by measuring Ab content in tissue extracts. A significant reduction in Ab was observed in APPDutch and APPNL-G-F. However, the Ab content did not change in the APP/PS1 strain, most probably due to the severity of amyloidosis, characteristic of this model. In this thesis, we attempted an optimization of the AAV vector to boost the nanobody transgene expression by using a self-complementary (sc) AAV conformation.
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Gebura, Myroslav. "Nanobodies as new tools for studying large cargo transport and lamina organization." Doctoral thesis, 2017. http://hdl.handle.net/11858/00-1735-0000-002E-E42F-E.
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Morgenstern, Travis James. "Development of next-generation voltage-gated calcium channel inhibitors using engineered nanobodies." Thesis, 2021. https://doi.org/10.7916/d8-4xfc-0y25.
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High-voltage activated calcium channels underlie many critical functions in excitable cells and their dysfunction has been implicated in a myriad of cardiovascular and neurological diseases. These channels are multimeric protein complexes composed of α1, β, and α2δ subunits; currently, all calcium channel blockers target either the pore-forming α1 or extracellular-facing α2δ auxiliary subunit. These pharmacological agents have been invaluable in delineating the individual function of each subunit within excitable cells that express multiple calcium channels. Yet, no current tool allows similar pharmacological dissection of individual cytosolic β subunits, preventing our understanding of how distinct β subunits affect the function of calcium channel complexes. Further, small-molecule calcium channel blockers are highly-valued therapeutics for certain conditions, yet their propensity for off-target effects precludes their use in other diseases. In certain applications, genetically-encoded calcium channel blockers may enable channel inhibition with greater tissue-precision and versatility than is achievable with small molecules.
Previous work that found the family of RGK proteins powerfully inhibits high-voltage activated calcium channels in part via an association with the β subunit. However, the myriad functions of RGK proteins limit the utility of this approach. In this work, we circumvent this issue by isolating single-domain antibodies (nanobodies) that target the auxiliary CaVβ subunit. We then paired these nanobodies with the powerful enzymatic activity of the HECT domain E3 ubiquitin ligase Nedd4L, to selectively target the calcium channel for ubiquitination. We found this strategy effectively eliminated functional calcium channels from the surface of HEK293 cells, myocytes, and DRG neurons. This modular design permitted us to characterize a pan-β inhibitor (CaV-aβlator) in chapter 2 while refining the approach with a β1-selective channel inhibitor in chapter 3. In chapter 4 I demonstrate that it is possible to hijack the endogenous ubiquitin machinery of the cell by creating Divas: divalent nanobodies that are capable of recruiting endogenous Nedd4L to regulate the calcium channel. Finally, we demonstrate the potential for these genetically-encoded calcium inhibitors to be employed as therapeutic agents by targeting CaV-aβlator to sensory neurons in order to reduce the onset of neuropathic pain. Altogether, this work lays the foundation for nanobody-based genetically-encoded calcium channel inhibitors that have the potential to achieve superior precision in regards to molecular and tissue specificity.
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De, Luca AJ. "Development of synthetic immunological tools for Tasmanian devil research." Thesis, 2022. https://eprints.utas.edu.au/47589/1/De_Luca_whole_thesis.pdf.
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The Tasmanian devil (Sarcophilus harrisii) is the world’s largest extant carnivorous marsupial. The wild population of devils has been decimated by two transmissible cancers, collectively named Devil Facial Tumour Disease (DFTD). Transmissible cancers are exceedingly rare and only three are found in mammalian species. The occurrence of two of these transmissible cancers in a single species raises a number of questions about the devil’s immune system and the pathogenesis of these diseases. Understanding the Tasmanian devil immune system and how it responds to these transmissible tumours is imperative for the continued conservation of this species. This research may also provide insight into the tumour - host immune system relationship in other cancers including human disease.
Development of immune checkpoint immunotherapies and cancer vaccines have provided more targeted cancer therapies that are safer for patients and are in many cases more effective than traditional chemotherapy or radiotherapy. The understanding of immune system function and disease pathogeneses enhances the development of safe, effective, and rational therapies for disease.
One of the major problems facing the field of wild immunology is the reagent gap. This refers to the lack of research tools and reagents available that specifically and reliably bind to non-human, non-model species targets. While thousands of antibodies are commercially available that bind to rodent or human targets, there are less than 30 that bind to Tasmanian devil targets, many of which have had to be developed in-house or custom ordered and are not commercially available.
The development of specific, affordable, and reliable research tools will allow investigation into DFT pathogenesis, development of therapies, and analysis of therapy efficacy to be faster, more reliable, and more reproducible. A digital database of hybridoma derived anti-Tasmanian devil checkpoint molecule antibody sequences was designed and will provide a resource for the development of several novel antibodies and other affinity reagents for use in DFTD research, diagnoses, and therapies.
Producing synthetic affinity reagents from known sequences is faster and cheaper than conventional antibodies without compromising specificity or affinity. Devilisation of hybridoma derived antibodies involves hybridising the mouse derived variable regions with devil derived constant regions. Compared to mouse derived antibodies, devilised reagents are less immunogenic and more appropriate for functional immune research and eventually, therapeutic applications. This thesis presents a method of producing anti-PDL1 devilised recombinant antibodies using a mammalian cell-based protein expression system.
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Neto, Alexandre Nunes. "Gene deletion and nanobodies strategies to target S100B and improve recovery in an ex vivo demyelinating model." Master's thesis, 2020. http://hdl.handle.net/10316/93997.
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Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e TecnologiaMultiple sclerosis (MS) is a chronic neurodegenerative and autoimmune disease, characterized by inflammation and demyelination of the central nervous system (CNS). Altogether, these processes produce focal lesions in specific CNS regions, and depending on the affected region, different symptoms will appear, leading to an increase in both motor and cognitive disability, dependent on disease progression. Despite the enormous efforts done to unravel the mechanisms underlying MS pathology, the complex onset phenomenon might be due to an interplay of different factors, hampering the development of new therapeutics. The pursuit to found new biomarkers is extremely crucial, in order to find new different ways to tackle this disease. S100B is an inflammatory molecule that has been described as a major player in MS pathology. At physiological levels, S100B promotes differentiation and maturation of oligodendrocytes, required for the correct myelination of the CNS. Nevertheless, S100B has been found at high levels, in both cerebrospinal fluid and serum of MS patients at the diagnosis stage, as well in the active demyelinating lesions. This abnormal S100B overexpression was also detected in ex vivo demyelinating models, leading to microglia and astrocyte activation, exacerbating the neurodegenerative process. Recently, our group demonstrated that through S100B neutralization, a lower degree of demyelination, reactive gliosis, and also inflammation were obtained.First, in the present thesis we went further to analyse S100B neurotoxic effects using cerebellar organotypic slice cultures from three different mice. S100B heterozygotic and knockout mice demonstrated a decrease in demyelination as well as in inflammation, when compared to wild type mice, evidencing the neurotoxic effect of S100B when overexpressed. Afterwards, we tried to neutralize S100B using three different nanobodies. These small molecules possess several advantages such as extreme specificity to recognize unique epitopes, higher affinity, stability, and solubility than commonly used antibodies, as well as reduced immunogenicity. Interestingly, in the presence of these nanobodies, we observed a reduced degree of demyelination as well as a diminished inflammatory environment. Overall, our results emphasize the already described pejorative roles of S100B on MS pathophysiology, highlighting that S100B neutralization by the use of nanobodies, might be a promising therapeutic possibility regarding demyelinating and inflammatory diseases as is the case of MS.
A Esclerose Múltipla (EM) é uma doença crónica, neurodegenerativa e autoimune, caraterizada por processos inflamatórios e desmielinizantes no sistema nervoso central (SNC). Devido a estes processos, começam por aparecer lesões focais em certas regiões do SNC e, dependendo da região, podem surgir diferentes sintomas, resultando no aumento da incapacidade motora e cognitiva à medida que a doença evolui. Apesar dos inúmeros avanços a nível do conhecimento dos mecanismos subjacentes à complexa dinâmica patofisiológica desta doença, a mesma possui uma causalidade multifatorial que torna difícil o desenvolvimento de novas terapêuticas. Neste sentido, a procura de novos biomarcadores e alvos terapêuticos tem sido essencial para o desenvolvimento de novas estratégias para combater a EM. Tomando partido destes, vários avanços têm sido feitos na área da EM, como é o caso da molécula S100B. Em níveis fisiológicos, esta proteína promove a correta diferenciação e maturação de oligodendrócitos, necessários para a correta mielinização do SNC. No entanto, foram encontrados elevados níveis da proteína S100B tanto no soro como no líquido cefalorraquidiano de doentes com EM em fase de diagnóstico, estando também presente nas lesões ativas no SNC. Esta elevação de S100B é também detetada em modelos ex vivo de desmielinização, tendo sido demostrado que leva à ativação tanto de astrócitos como de células da microglia, potenciando o processo neurodegenerativo. Recentemente, o nosso grupo demonstrou que a neutralização desta proteína resultava num menor grau de desmielinização, de reatividade glial e consequentemente numa redução da resposta inflamatória. Desta forma na presente dissertação analisámos em primeiro lugar o efeito neurotóxico desta proteína, através da utilização de culturas organotípicas de cerebelo, provenientes de três linhagens de murganho, nos quais o gene para a S100B está alterado. Assim, através da comparação de culturas provenientes de murganhos expressando a S100B wild type, com as de heterozigóticos e ainda de homozigóticos com S100B knockout, foi possível notar uma diminuição tanto da desmielinização como da inflamação. Após a comprovação dos efeitos pejorativos desta proteína, procedemos à sua neutralização recorrendo a três Nanocorpos diferentes dirigidos para a S100B. A possibilidade do uso de Nanocorpos específicos para a S100B despertou a nossa curiosidade, devido às várias características vantajosas destas nanomoléculas, tal como a sua alta capacidade para reconhecer epítopos específicos, a sua maior estabilidade e solubilidade tal como uma imunogenicidade reduzida. A utilização dos Nanocorpos não só levou a uma diminuição da desmielinização, como também a um decréscimo do processo inflamatório. Desta maneira, estes resultados vêm reforçar o papel da S100B na patogénese desta doença e demonstrar que a sua neutralização, pelo uso de Nanocorpos específicos para esta proteína, constitui uma nova possibilidade terapêutica para doenças desmielinizantes inflamatórias como a EM.
Outro - (GMSI–Merck) Targeting multiple sclerosis immune- and psycho-pathophysiology by modulation of neuroinflammation Besides the typical central nervous system pathogenesis of multiple sclerosis (MS) (e.g. demyelination, axonal degeneration, inflammation and immune cell activation), greater clinical attention is now drawn to concomitant psychopathology of patients with MS, which hinders the ability to cope with the associated disability. Importantly, inflammation has also been linked with the psychopathology of MS, suggesting that targeting inflammation may effectively reduce both pathogenesis and psychopathology. Recently, the pro-inflammatory protein S100B has been suggested as a biomarker of central nervous system damage. Interestingly, we showed that S100B is increased in the cerebrospinal fluid/serum of patients with MS at diagnosis and highly expressed in active post-mortem MS lesions. We further demonstrated that neutralization of S100B in an ex vivo demyelinating model prevented not only the induced demyelination but also the axonal injury and inflammatory response. Most interestingly, increased S100B has been also associated with several psychiatric and mood disorders. In our study, we aim to use a holistic approach, looking at data from both patients with MS and animal models to clarify how neuroinflammation, using S100B as a biomarker, may be involved in the emergence of the psychopathological symptoms of MS and whether we may prevent them by targeting S100B. Ultimately, we aim to translate the results of the present project to benefit patients, using personalized medicine to reduce both MS-related central nervous system damage and psychiatric comorbidity.
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Santos, Sofia Jorge Calvet de Magalhaes Fernandes dos. "Uncovering the molecular mechanisms of Ataxin-3 interaction with nanobodies: clues to discover new approaches to treat Machado-Joseph Disease." Master's thesis, 2021. https://hdl.handle.net/10216/136716.
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Kirchhofer, Axel [Verfasser]. "Structural and functional analysis of RIG-I like helicases : modulating spectral properties of the green fluorescent protein with nanobodies / Axel Kirchhofer." 2009. http://d-nb.info/999625969/34.
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Gomes, Ana Rita Coelho. "Design of a Novel Phage Display Vector to Improve Enrichment of Phages with Antigen-Specific Nanobodies and to Identify the Nanobody Clones of Highest Affinity." Master's thesis, 2019. http://hdl.handle.net/10362/89178.
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Although phage display-based enrichments became a standard procedure, they still suffer from some drawbacks. Nonspecific phage binding limits the enrichment that can be achieved per se-lection round and therefore, in most cases, at least three or four rounds are required to identify the antigen binding nanobodies (Nbs) from the library. Moreover, the release of the phages captured via their antigen-specific Nb on the immobilized antigen is accomplished most often, by a pH shock that will also release the a-specific absorbed phages, thereby generating an unwanted back-ground. However, the biggest shortcoming is the difficulty to identify the Nb of best binding affinity. We now have to ferment and purify all individual Nb clones to homogeneity and measure their affinity parameters one after the other. This is very tedious, certainly, as the identified Nbs often share a high degree of amino acid sequence identity, which makes it impossible to predict the one having the best affinity. The objective of this thesis aimed to modify the phage display vector so that one round of selection will be sufficient, while the affinity of all antigen-positive Nbs can be compared immediately after ELISA.
Firstly, a vector containing the Calmodulin Binding Peptide (CBP) tag was created, by substitut-ing the hemagglutinin (HA) tag present in a pMECS-GG plasmid by the CBP tag. Moreover, 6 Nbs with well-known and variable kinetic binding rates were also inserted into the vector pMECS-CBP.
Next, the vector was used in a phage display setting, where two mini libraries comprising the 6 Nbs were made. One library had the conventional pMECS vector while the other was made using the pMECS-CBP vector. Although an enrichment of 1000 times was achieved using pMECS, pMECS-CBP failed to show any enrichment, supporting the hypothesis that Nb-CBP encountered serious expression problems. To investigate the validity of this hypothesis, periplasmic expression of Nb-CBP was performed and compared with that of the Nb without the tag. While the Nb yielded almost 4 mg per liter of culture media, the Nb-CBP could only be obtained at 0.63 mg per liter culture. Moreover, the Nb-CBP couldn’t be detected on a western blot. Several methods were used, such as adding lysozyme to improve the periplasmic extraction step, expressing 5 different colonies to check if there was a homogeneity of expression between them. In all meth-ods, the amount produced always remained below 1 mg per liter and Nb-CBP protein couldn’t be detected in any step of expression by Coomassie stained SDS-PAGE or western blot.
Additionally, a high yield expressing-protein (SIRPα) was cloned into the CBP vector to identify whether the expression problem was caused by the peptide tag itself or by the Nb-CBP combina-tion. Even though SIRPα was obtained at a yield of 8.7 mg/L of culture when expressed from a vector not containing the CBP, its expressed yield using the CBP vector, dropped to 0.7 mg/L of culture, indicating that the CBP tag is incompatible with good periplasmic expression.
Lastly, a nucleotide alignment of our CBP tag with a previously reported one, revealed differences in two Arginine codons, two Alanine codons and one Lysine codon. Moreover, our sequence employed two codons, AGA and CGG, rarely used in E. coli, which might be linked to the re-duced expression levels that we observed during this thesis.
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Cristóvão, Joana Margarida Lopes da Silva. "The calcium binding S100B protein as a new modulator of amyloid-β peptide aggregation." Doctoral thesis, 2019. http://hdl.handle.net/10451/42537.
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Amyloid aggregation and chronic neuroinflammation are pathological hallmarks in Alzheimer’s disease. Interestingly, important neuronal components such as pro-inflammatory cytokines and transition metal ion levels are also consistently deregulated in AD. S100B is the most abundant pro-inflammatory cytokine in the brain and acts as an alarmin in AD, being upregulated and around amyloid plaques. In this PhD thesis we aimed at targeting the role of S100B over extracellular aggregation of amyloid-β at early stages of AD and at understanding the influence of metal ions over these regulatory processes. We found that S100B acts as a new modulator of Aβ aggregation and toxicity and exhibits some features similar to molecular chaperones. This regulatory action is activated by calcium or zinc-binding to S100B as by its quaternary state. We developed single-domain antibodies targeting S100B that increase the suppressor effect of S100B over the onset and progression of Aβ aggregation. Additionally, we also designed and synthetized S100Bbased peptides that can induce S100B oligomerization as a mean to control S100B levels in the brain. Moreover, we explored the potential effects of S100B in the synaptic cleft by performing assays in primary hippocampal neurons where S100B chelate zinc ions from the medium, contributing to changes in the postsynaptic scaffold Shank proteins, proteins essential to the maintenance of synapse plasticity. We generate new scientific and technological knowledge with potential applicability in human health, as S100B-based molecules can be used as a new druggable target to slow down AD progression and lead to diseasemodifying therapies.
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Sograte, Idrissi Shama. "Optimization of tools for multiplexed super resolution imaging of the synapse." Thesis, 2019. http://hdl.handle.net/21.11130/00-1735-0000-0005-140A-A.
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