Dissertations / Theses on the topic 'Nanobody'
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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.
Full textHoff, Merle [Verfasser]. "Kombinatorische Analyse von Nanobody-markierten Epitopen zur Proteinbestimmung / Merle Hoff." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2021. http://d-nb.info/122862383X/34.
Full textTaylor, Edward John Robert. "Synthesis and characterisation of peptide-based probes for quantitative multicolour STORM imaging." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/284553.
Full textCAPALDO, PIETRO. "Capacitance immunosensors for the early detection of circulating cancer biomarkers." Doctoral thesis, Università degli Studi di Trieste, 2016. http://hdl.handle.net/11368/2908095.
Full textPeyrassol, Xavier. "Développement et caractérisation d’anticorps de camélidés dirigés contre des récepteurs couplés aux protéines G et leur utilisation dans des approches structurales." Doctoral thesis, Universite Libre de Bruxelles, 2018. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/270870.
Full textDoctorat en Sciences biomédicales et pharmaceutiques (Médecine)
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Hajj, Sleiman Nawal. "Approche par nanobody pour capturer les interactomes de complexes protéiques dimériques en contexte cellulaire vivant." Electronic Thesis or Diss., Lyon, École normale supérieure, 2024. http://www.theses.fr/2024ENSL0041.
Full textCell fate and fitness depend on the protein content, and in particular on the interaction networks (also called interactomes) connecting the different proteins. Proteins have the general property to engage in diverse and occasionally overlapping macromolecular assemblies, each serving distinct purposes. Therefore, identifying protein-protein interactions (PPIs) and linking them to complexes is a crucial yet challenging issue in biology. This issue was at the core of my PhD work. The first part of my work was dedicated to the improvement of an existing method for capturing novel PPIs in the context of defined biological functions. This work was established with ERK1, which is a key downstream regulator of several signaling pathways involved in many different cancers. The new tools were tested in the context of two different inhibitory molecules to capture drug-sensitive interactions of ERK1 in human HEK293T cells. One such interaction was confirmed at the functional and molecular levels, by using an original imaging strategy to access the PPI dynamics in live cells. The second part of my PhD work was dedicated to the establishment of a pioneer methodology to capture endogenous PPIs established by a specific dimeric protein complex in human live cells. This methodology couples Bimolecular Fluorescence Complementation (BiFC) and proximity biotin labelling technologies. More specifically, it is based on a GFP-nanobody directed toward the BiFC complex and fused to the TurboID biotin ligase. Tools were established to map TAZ/14-3-3 and TAZ/TEAD complexes interactome, which translate the activity of the Hippo signaling pathway in the cytoplasm and nucleus, respectively. Our approach allowed capturing specific interactomes of the two dimeric protein complexes and identifying a novel key regulator of TAZ/14-3-3 complexes in a cancer cell context. Collectively, my PhD work introduced two complementary methodologies for deciphering PPI networks in the context of specific biological functions or in the context of a specific protein complex in human live cells. These approaches provide a novel dimension for understanding protein functions and the underlying interactomes in normal or pathological cell contexts
Nordeen, Sarah Ann. "A nanobody suite for yeast scaffold nucleoporins provides details of the Y complex structure and nuclear pore complex assembly." Thesis, Massachusetts Institute of Technology, 2020. https://hdl.handle.net/1721.1/127138.
Full textCataloged from the official PDF of thesis.
Includes bibliographical references.
Nuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups, that can be classified into three categories: 1. Stably associated scaffolding nups, 2. Peripheral nups, and 3. Phenylalanine-glycine (FG) repeat containing nups that form the permeability barrier of the NPC. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 complex. Working in S. cerevisiae to study the assembly of these two essential subcomplexes, we developed a suite of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. The nanobodies bind their targets specifically and with high affinity, albeit with varying kinetics. We mapped the epitope of eight members of the nanobody library via crystal structures of nup-nanobody co-complexes.
Nuclear pore complexes (NPCs) are the main conduits for molecular exchange across the nuclear envelope. The NPC is a modular assembly of ~500 individual proteins, called nucleoporins or nups, that can be classified into three categories: 1. Stably associated scaffolding nups, 2. Peripheral nups, and 3. Phenylalanine-glycine (FG) repeat containing nups that form the permeability barrier of the NPC. Most scaffolding nups are organized in two multimeric subcomplexes, the Nup84 or Y complex and the Nic96 complex. Working in S. cerevisiae to study the assembly of these two essential subcomplexes, we developed a suite of twelve nanobodies that recognize seven constituent nucleoporins of the Y and Nic96 complexes. The nanobodies bind their targets specifically and with high affinity, albeit with varying kinetics. We mapped the epitope of eight members of the nanobody library via crystal structures of nup-nanobody co-complexes.
In two cases, the nanobodies facilitated the crystallization of novel nup structures, namely the full-length Nup84-Nup133 [alpha]-helical domain structure and the Nup133 [beta]-propeller domain structure. Together these two structures completely characterize the S. cerevisiae Y complex molecular assembly. Further, the Nup133 [beta]-propeller domain contains a structurally conserved amphipathic lipid packing sensor (ALPS) motif thought to anchor the Y complex to the nuclear envelope, which we confirmed by liposome interaction studies. An additional nanobody facilitated the structure of Nic96 at an improved resolution, revealing previously missing helices. In addition to the utility of these nanobodies for in vitro characterization of NPC assemblies, we also show that expression of nanobody-fluorescent protein fusions reveals details of the NPC assembly in their native, in vivo environment, and possibly of NPC heterogeneity within the nuclear envelope.
Overall, this suite of nanobodies provides a unique and versatile toolkit for the study of the NPC.
by Sarah Ann Nordeen.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biology
Baum, Natalie [Verfasser]. "Targeting the EGF-receptor and the CD38/NADase in solid and hematological malignancies with nanobody-based heavy chain antibodies and AAV vectors / Natalie Baum." Hamburg : Staats- und Universitätsbibliothek Hamburg Carl von Ossietzky, 2020. http://d-nb.info/1241743088/34.
Full textFleetwood, Filippa. "Bacterial display systems for engineering of affinity proteins." Doctoral thesis, KTH, Proteinteknologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-156420.
Full textQC 20141203
Duquénois, Isoline. "Modification du tropisme de la glycoprotéine du virus de la stomatite vésiculaire : ciblage de récepteurs d'intérêt." Electronic Thesis or Diss., université Paris-Saclay, 2024. http://www.theses.fr/2024UPASL098.
Full textThe transfer of vesicles containing cargos toward cells of interest remains a challenge for targeted therapy. Viral fusion glycoproteins having the property of receptor recognition and fusion activity constitute promising tools for this kind of approach. VSV glycoprotein (G) is the most used viral glycoprotein to pseudotype lentiviruses in gene therapy. However, we encounter limits to the use of G: G cellular receptors (from LDLR family) are ubiquitous and expressed at the surface of non-target cells. The work of the team on VSVG/LDLR structure enabled us to identify G mutants that no longer bind the LDLR without affecting its fusion activity. This uncoupling between the recognition of the receptor and the fusion capacity opened up the possibility of retargeting G towards receptors of interest. A chimeric glycoprotein fused with a nanobody directed against the mCherry protein, in N-terminal, of G has been constructed. The insertion of a nanobody in G is deleterious for its activity. Using experimental evolution, we identified two mutations on G enhancing the chimera folding. Remarkably, these mutations improve the folding of chimeric Gs, regardless of the sequence of the nanobody inserted in amino-terminal. Pseudotyped viruses (both VSV and lentiviruses) with these chimeric Gs at their surface show 10 times higher titers with these mutations of optimisation. We then constructed chimeric Gs with several nanobodies targeting the receptor HER2. We introduced the mutations abolishing LDLR recognition in these Gs. Viruses pseudotyped with these glycoproteins only infected cells expressing HER2. We therefore identified G mutations conferring a new tropism of G thanks to the N-terminal insertion of a nanobody. All this work opens the way to personalised targeted therapies
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.
Full textDue 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
HASSAN, AMAL. "FROM PROTEIN STRUCTURE TO DRUG DESIGN (DISCOVERY): TARGETING THE ION CHANNEL ASIC1 AND A PATHOGENIC VARIANT OF HUMAN GELSOLIN." Doctoral thesis, Università degli Studi di Milano, 2019. http://hdl.handle.net/2434/629877.
Full textKnowledge of the three-dimensional structure of therapeutically relevant proteins paves the way for novel strategies in pharmacological research (such as the structure-based drug design (SBDD) method) and establishes the foundations for structural bioinformatics. In this context, during my PhD Thesis, two therapeutically relevant proteins have been studied. First, a membrane protein, Acid Sensing Ion Channel (ASIC) isoform 1, a validated target in neurodegenerative disorders, was selected. Previous studies showed that diminazene aceturate (DA) is a potent small-molecule inhibitor of ASIC channels. Here, several DA analogues were screened by molecular docking and the best binders were tested in cell-based assays to further assess their efficacy. In order to determine the inhibitory capability of the synthesized analogues in vitro on the purified protein, the expression of ASIC1 was undertaken, using different organisms of expression. The protein purification was performed in a high-throughput approach in order to recover enough protein for crystallization, with the final aim of studying the mechanism of action of DA analogs, and support the design of new, isoform-selective and brain-penetrant drugs. Secondly, the soluble protein Gelsolin (GSN), responsible for a familial degenerative disease (AGel amyloidosis) was studied. Aim of this project was to understand the impact of the D187N mutation on GSN structure and its propensity to aberrant aggregation and/or degradation. D187N GSN mutant was the first identified in man, but its crystal structure had until now eluded any characterization. Conversely, a nanobody (Nb11) was shown to protect GSN from aberrant proteolysis, but its mechanism of protection remained unclear. Here, the structure of the Nb11:D187N complex was solved at 1.9 Å resolution, enabling the characterization of the Nb11action mechanism. The structural data were complemented with biophysical and biochemical characterisations. These studies were then extended to two recently identified pathological variants of GSN (G167R and N184K).
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.
Full textBroster, Christine. "Caractérisation et Ciblage de Protéines Essentielles via l'utilisation de nanobodies chez Trypanosoma brucei." Thesis, Bordeaux, 2019. http://www.theses.fr/2019BORD0158.
Full textKinetoplastid 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
Fedor, Yoann. "Nouveau biomarqueur en temps réel de cassures double-brin de l'ADN et génotoxicité de la cytolethal distensing toxin." Toulouse 3, 2012. http://thesesups.ups-tlse.fr/2029/.
Full textHuman DNA is constantly damaged by endogenous (cellular metabolism) or exogenous (radiations, food contaminants) sources. Among these lesions, DNA double-strand breaks (DSB) are the most cytotoxic. To survive to these lesions, a cellular pathway is in charge for the detection and the signaling of DSB. This pathway involves recruitment and post-translationnal modifications of several proteins around the DSB site (like the phosphorylation of a H2A histone variant called H2AX). This signalization pathway elicits cellular checkpoints in order to stop proliferation, and stimulates DSB repair systems in order to restore DNA initial integrity. An error-prone repair of DSB can lead to base additions/deletions, or chromosomal aberrations that can induce cancer. In order to understand genotoxicity, it is important to elucidate causes and mechanisms responsible for DSB formation and to follow their management by the cell. Techniques allowing DSB formation analysis (immunofluorescence, pulse-field gel electrophoresis, neutral COMET assay. . . ) exist, but can only show DNA state for a given point. During the first part of my thesis work, I created a new tool to detect and follow DSB formation in real time, in human cells. This tool rely on nanobody technology, which are miniatures antibodies produced by camelidae species and some sharks. An intracellular nanobody directed against phosphorylated H2AX (gammaH2AX) has been expressed, and seems to relocate to microirradiation-induced DSB. In order to build this tool, anti-gammaH2AX peptides were designed to immunize a llama, and nanobodies coding sequences were isolated/cloned and gathered as a library. Nanobodies specific for gammaH2AX were selected by phage display. Fused to a fluorophore these nanobodies were expressed in human cells in order to analyze their relocalization to DSB in real time. The second part of my phD shed a new light on the mechanism of action of a bacterial génotoxine causing cancers in mouse models: the Cytolethal Distending Toxin (CDT). This toxin is secreted by commensal and pathogenous bacteria, translocate into the nucleus of targeted cells and induces DSB. CDT mechanism of action was previously described as those of a nuclease inducing DSB. But my work demonstrated for lower doses (equivalent to lethal dose 50), that CDT induced first single-strand breaks leading to double-strand breaks through DNA replication. Moreover, homologous recombination repair of these DSB is crucial in order for cells exposed to CDT to survive. In conclusion, thanks to my thesis work, I developed a new tool to analyze real time dynamic of DSB in human cells in one hand. And in another hand, my work shed a new light on the mechanism of action of CDT genotoxicity, a toxin displaying cancer hazard in mammalians. Contributions brought by this work are discussed here
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.
Full textPansieri, Jonathan. "Les amyloïdoses : détection à l'aide de nanoparticules et propriétés optiques originales Multimodal imaging Gd-nanoparticles functionalized with Pittsburgh compound B or a nanobody for amyloid plaques targeting Gd-nanoparticles functionalization with specific peptides for ß-amyloid plaques targeting." Thesis, Université Grenoble Alpes (ComUE), 2017. http://www.theses.fr/2017GREAY105.
Full textAmyloidosis are diseases characterised by self-agregation of misfolded proteins in fibrillary forms, called amyloid fibrils. They are associated with many diseases, and their early diagnosis remains a clinical challenge. In this work, we show the targeting and the detection of amyloid fibrils, from in vitro to in vivo experiments in mice models, useful for early diagnostic of amyloidosis. For that purpose, we use multimodal nanoparticles, further functionalized with specific molecules against amyloid fibrils. This multimodality for imaging (fluorescence, MRI, PET) represent a breakthrough in modern medicine, to add structural and functional informations. After negative toxicity tests on various cell lines, these nanoparticles have been tested on three different amyloid fibrils, formed with amyloid β(1-42) peptide (Alzheimer’s disease), amylin (type II diabetes mellitus), or mutated Val30Met transthyretin (familial polyneuropathy). As it is shown by spectroscopy and surface plasmon resonance experiments, nanoparticles grafted with generic targeters (Pittsburgh compound B or a nanobody) target the three amyloid fibrils, with good affinity, whereas nanoparticles vectorised with peptides show specific targeting for amyloid β(1-42) or Val30Met mutated transthyretin, but with lower affinity. The targeting by nanoparticles have been confirmed ex vivo on pathological tissues with each amyloid burden, thanks to fluorescence microscopy. Generic nanoparticles have been injected in Alzheimer’s mice model, and the monitoring in vivo by IRM and post-mortem by optical microscopy supposed a targeting of amyloid β(1-42) aggregates in brain. Furthermore, we demonstrate the possibility to detect amyloid fibrils without labelling. Spectroscopic measurements show original, and specific optical properties of amyloid fibrils, around the UV-visible and the near infra-red regions. Interestingly, these properties are also observed on pathological tissues by ex vivo fluorescence microscopy, and in vivo ongoing analysis by photoacoustic imagery and real-time imaging seems to be very promising. By multimodality of non-toxic grafted nanoparticles or by intrinsic properties of amyloid fibrils suggesting completely non-invasive tests, these two strategies can be useful for early diagnostic of amyloidoses in humans
Chen, Wei-Hsin Chen. "Localised dosing and nanodetection using a novel scanning ion conductance microscope and its application to Alzheimer's disease." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/279090.
Full textPatris, 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.
Full textLe 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
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Nidhi, Vagisha. "Radiotactic colloids : towards the Decontamination Nanobots." Electronic Thesis or Diss., université Paris-Saclay, 2024. http://www.theses.fr/2024UPASF052.
Full textTraditional methods of decontamination face significant challenges, such as difficulty in accessing complex or confined spaces, high amount of waste, etc. There is still a need for the development of new methods to reach complex geometries with effective decontamination processes. While macro-robots have been useful in large-scale decontamination tasks, their size limits their ability to navigate in intricate environments. Micro or nano robots, on the other hand, can traverse small, complex spaces and target specific contamination sites, making them more suitable for detailed decontamination work. In this context, this thesis studies the capacities of micro/nanoparticles to move towards contaminated spot in complex geometries, by mimicing the chemotaxism guided by H₂O₂ (product of water radiolysis). To this end, the large-scale synthesis and mobility of active colloids, in particular Janus particles is described. A set of different assemblies of gold particles on silica (isotropic or Janus assembly, discrete nanoparticles or continuous gold layer) were prepared, characterized and compared. Their movements were monitored in different environments. A key part of this work was the developement of a microfluidic device capable of generating stable hydrogen peroxide gradients. This device was essential to study the directionnal orientation of the different particles. This work showed that silica-gold assemblies could move autonomously towards a source of H₂O₂, which could make them effective for targeting radioactive contamination sites. We have also shown that isotropic assemblies, which are simpler to prepare, can also exhibit directional movement
Payan, Hugo. "Réseaux de protéines associés aux récepteurs de la sérotonine : rôle et options thérapeutiques pour la Maladie d'Alzheimer." Thesis, Montpellier, 2020. http://www.theses.fr/2020MONTT012.
Full textAlzheimer’s Disease is the most common form of dementia worldwide and has become a major public health problem. This pathology is characterized by the presence of two main features in the brain: neurofibrillary tangles (NFTs) composed of hyperphosphorylated Tau and amyloid plaques, dense aggregates of hydrophobic β-amyloid peptide (Aβ). Aβ peptide results from the amyloidogenic degradation of the Amyloid Precursor Protein (APP) by β- and ɣ-secretases. On the other hand, the non-amyloidogenic proteolysis of APP, within the Aβ sequence by an α-secretase (mainly ADAM10), releases the extracellular fragment of APP (sAPPα), which is neurotrophic and neuroprotective. The 5-HT4 receptor (5-HT4R) is a G protein coupled receptor (GPCR) which interacts with APP and ADAM10, modulates their trafficking and maturation, and which pharmacological activation promotes the non-amyloidogenic cleavage of APP. The 5-HT6 receptor (5-HT6R) is another serotoninergic receptor which pharmacological modulation induces pro-cognitive effects. Thus, 5-HT4R and 5 HT6R constitute interesting therapeutic targets in the context of Alzheimer’s Disease. Nevertheless, the molecular and cellular mechanisms underlying these effects linked to 5-HT4R expression and activation remain elusive. This thesis aimed, in a first part, to develop a screening technique to isolate nanobodies (single domain antibody fragment from camelids) directed against 5-HT4R and 5 HT6R, by using TR FRET and flow cytometry approaches. In a second part, the use of a proteomic approach combining affinity purification of 5-HT4R partner proteins followed by their systematic identification by mass spectrometry (AP-MS) lead to the identification of potential partner proteins associated with 5-HT4R in HEK-293N cells. Amongst proteins recruited by 5 HT4Rs, we identified IRS4 (Insulin Receptor Substrate 4) protein. IRS4 protein is a transducer of the insulin signaling pathway. Given that a dysregulation of the insulin signaling pathway is one of the major events involved in Alzheimer’s Disease development, we decided to focus on this partner. We validated the interaction by Western blotting and showed a colocalization of endogenous IRS4 with 5 HT4R in HEK 293 cells. Then, functional studies showed that IRS4 downregulation induced 5 HT4R overexpression in HEK-293 cells, associated with an increase in the canonical signaling of the receptor through its Gs protein pathway.These results suggest a modulation of 5-HT4R trafficking and of its associated complex including APP and ADAM10 by IRS4 protein. This thesis work reveals for the first time a functional link between 5-HT4R signaling and insulin signaling pathway via IRS4 protein which could be of interest in the context of Alzheimer’s disease. It also establishes the preliminary basis for a fast identification of nanobodies targeting 5-HT4 and 5-HT6 receptors for which immunohistological tools are lacking to explore their underlying mechanisms
AMATO, PAOLO. "Swarm-Intelligence Strategy for Diagnosis of Endogenous Diseases by Nanobots." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2013. http://hdl.handle.net/10281/41950.
Full textDe, pascali Francesco. "Allosteric modulation of follicle stimulating hormone receptor and GPR54 : new tools to study signalling." Thesis, Tours, 2019. http://www.theses.fr/2019TOUR4030.
Full textGPR54 and FSHR regulate reproduction by acting on the hypothalamus-pituitary-gonads (HPG) axis. Acting in the hypothalamus, GPR54 is an upstream regulator of the axis whereas FSHR controls gametogenesis in both sexes. They represents two major pharmacological targets for the treatment of fertility-related problems. Both GPR54 and FSHR belong to the G protein-coupled receptor (GPCR) superfamilly. GPR54 preferentially activates the Gαq/PLC/Ca2+ pathway whereas FSHR mainly activates the Gαs/PKA/cAMP pathway. Both receptors recruit and activate β-arrestins. Increasing number pharmacological profiles have been reported to act on GPCR. Indeed biased ligands capable of preferentially eliciting a subset of the full signalling repertoire, compared to the endogenous ligand are discovered at a high rate. Orthosteric and allosteric ligands can both induce biased signalling by stabilizing specific receptor conformations. Therapeutically, biased ligand have demonstrated the potential to avoid side effects while still activating the signalling pathways leading to therapeutic effects. Moreover, allosteric ligands allow positive or negative modulation of a receptor while keeping the temporal information provided by the endogenous ligand. Until recently, such diverse and valuable pharmacological tools were not available for FSHR and GPR54. The aim of this thesis was to identify allosteric ligands at the FSHR and GPR54 and to characterize their biased signalling. In the first section, we pharmacological characterized a panel of low molecular weight ligands, recently reported to allosterically activate the FSHR and belonging to two chemical classes. We profiled their actions on different signalling pathways in living HEK293 cells expressing different biosensors. We demonstrated each of these compounds induced biased signalling at the FSHR compared to FSH. Using different cell models, we confirmed that system bias is a crucial confounding factor in bias determination. We also identified limit cases in which the operational model did not allow to calculated bias factors. In parallel, we characterized two novel compounds belonging to chemical classes that were not yet reported to activate FSHR. We demonstrated that they were allosteric and that their biased profiles were distinct from the compounds characterized in the first study. In second section of the thesis, we selected and pharmacological characterized nanobodies targeting GPR54 and FSHR. We identified a nanobody that behaved as a positive allosteric modulator (PAM) at the GPR54. We also identified a nanobody against FSHR. This nanobody displayed striking biased properties as it was negative allosteric modulator (NAM) for cAMP production but PAM for β-arrestin 2 recruitment. In the last section of the thesis, we attempted to develop nanobody-drug conjugates (NDC) by linking our nanobodies to agonists - either kisspeptin or one of the low molecular weight agonist of the FSHR - through a flexible linker. Though we did not have time to achieve a proof of concept for NDC, we believe that such hybrid compound could represent at minimum a promising research tools. As a whole, this thesis provides novel pharmacological tools that should allow deciphering the relative contributions of the different transduction mechanisms operating at the FSHR and GPR54, in vivo, in the reproductive function. This work also opens possible avenues for future therapeutic strategies in the control of reproduction in farm animals and in reproductive medicine
Hughes, William L. "Synthesis and characterization of zinc oxide nanostructures for piezoelectric applications." Diss., Available online, Georgia Institute of Technology, 2006, 2006. http://etd.gatech.edu/theses/available/etd-08232006-155547/.
Full textWang, Zhong Lin, Committee Chair ; Wong, C.P., Committee Member ; Summers, Christopher J., Committee Member ; Degertekin, F. Levent, Committee Member ; Bottomley, Lawrence A., Committee Member.
Vachon, Lauren Marie. "Glow: A Novel." Kent State University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=kent1374695902.
Full textHoff, Merle. "Kombinatorische Analyse von Nanobody-markierten Epitopen zur Proteinbestimmung." Doctoral thesis, 2021. http://hdl.handle.net/21.11130/00-1735-0000-0005-1565-2.
Full textRomano, Sofia Pereira Constantino. "Novel anti-nucleolin antibodies for targeted cancer therapy." Doctoral thesis, 2018. http://hdl.handle.net/10316/79730.
Full textCancer is nowadays the second leading cause of death and current therapeutic approaches still reveal ineffective in several cases. Therefore, there is a need to develop more efficacious therapeutic agents, especially for subtypes of cancer where targeted therapies are still missing, and, as such, unmet medical needs are evident. Limited drug penetration into tumors limits the efficacy of therapies targeting cancer cells. One of the strategies to overcome this problem is upon targeting the more accessible tumor vasculature. In this context, nucleolin arises as a target of extreme relevance, as it is overexpressed at the surface of cancer and angiogenic endothelial cells thus enabling a dual cellular targeting strategy. Antibodies have been extensively studied and some have been approved for the treatment of different types of tumors. Antibodies act by several mechanisms, from direct cell death to immune-mediated mechanisms (through the Fc region of the antibody). The latter include antibody-dependent cell-mediated cytotoxicity (ADCC), which plays a central role in the clinical efficacy of some antibodies. However, the high molecular weight of these proteins (≈150 kDa) limits effective penetration in solid tumors. Therefore, smaller units for antigen recognition have been developed. Nanobodies or VHHs are the variable domains of the heavy chains from camelid antibodies that lack light chains, presenting small size (≈15 kDa), high affinity to their antigens and reduced immunogenicity in humans. In addition, they are also versatile platforms for the development of different antibody formats. The main aim of this project was to develop and characterize new nanobody-based antibody formats against nucleolin. These consisted of (i) nanobodies generated by a grafting strategy using a nucleolin-binding sequence and (ii) an anti-nucleolin nanobody- Fc antibody, aiming at exploring immune mechanisms, namely ADCC. In addition, this ii work aimed also at exploring the use of nanobodies as bispecific proteins, where nucleolin and tumor necrosis factor alpha (TNF-α) were selected as models of target proteins. An anti-TNF-α VHH was used as a scaffold for grafting F3 peptide-derived nucleolin-binding sequences onto either complementarity determining region 1 (CDR1) or 3 (CDR3), the two most relevant antigen-binding regions. The grafted sequences consisted on a 10-amino acid sequence, with or without flanking linkers at each end, aiming at improving CDR flexibility. The generated nanobodies revealed binding to both purified nucleolin and nucleolin-overexpressing cells, as well as to the original target, TNF-α. These nanobodies presented cytotoxic effects in the micromolar range against these nucleolin-overexpressing cells. Grafting of the F3 peptide-derived sequence onto CDR3 enabled improved binding and cytotoxicity relative to grafting of the same sequence onto CDR1. However, the presence of flanking linkers in the grafted sequence did not alter the binding and cytotoxicity patterns. These results led to the selection of the nanobody presenting the F3 peptidederived sequence (without linkers) in CDR3 to generate a nanobody-Fc, upon fusion to the Fc region of a human IgG1. This anti-nucleolin nanobody-Fc antibody presented increased cytotoxic effects (in the nanomolar range) and also capacity of triggering a nucleolin-mediated ADCC effect against a cancer cell line. In conclusion, in this work, new nucleolin-targeting entities with cytotoxic activity against nucleolin-overexpressing cells were developed. In addition, the nanobodies presented bispecific properties, as evidenced by their binding to two different antigens. Notably, the nanobody-Fc antibody presented nucleolin-mediated ADCC capacity, which had not been described for this target yet. Therefore, the antibodies here described might contribute for the development of novel therapies targeting nucleolin.
O cancro é atualmente a segunda principal causa de morte e as abordagens terapêuticas atuais ainda se revelam ineficazes em muitos casos. Assim, há necessidade de desenvolver agentes terapêuticos mais eficazes, principalmente para subtipos de cancro para os quais não existem terapias direcionadas e para os quais são evidentes as necessidades médicas não satisfeitas. A limitada penetração de fármacos nos tumores compromete a eficácia das terapias direcionadas para as células cancerígenas. Uma das estratégias para ultrapassar este problema é o direcionamento da terapia para a vasculatura tumoral, que se encontra mais acessível. Neste contexto, a nucleolina surge como um alvo de extrema relevância, uma vez que é sobre-expressa à superfície de células cancerígenas e células endoteliais angiogénicas, permitindo uma estratégia de direcionamento multicelular. Os anticorpos têm sido extensivamente estudados e alguns foram aprovados para o tratamento de diferentes tipos de tumores. Os anticorpos atuam através de diversos mecanismos, desde morte celular direta a mecanismos imunitários (através da região Fc do anticorpo). Nestes últimos, inclui-se a citotoxicidade celular dependente de anticorpos (ADCC), que desempenha um papel central na eficácia clínica de alguns destes agentes terapêuticos. No entanto, o elevado peso molecular destas proteínas (≈150 kDa) limita a penetração eficaz em tumores sólidos. Consequentemente, foram desenvolvidas unidades mais pequenas de reconhecimento de antigénio. Os nanobodies ou VHH são os domínios variáveis das cadeias pesadas dos anticorpos de camelídeos sem cadeias leves, apresentando tamanho reduzido (≈15 kDa), elevada afinidade para os seus antigénios e imunogenicidade reduzida em humanos. Além iv disso, os nanobodies são também plataformas versáteis para o desenvolvimento de diferentes formatos de anticorpos. O principal objetivo deste projeto foi o desenvolvimento e caracterização de novos formatos de anticorpos baseados em nanobodies contra a nucleolina. Estes formatos consistiram em (i) nanobodies obtidos por uma estratégia de inserção de um peptídeo de 10 de aminoácidos, derivado de um outro (designado por F3) com demonstrada ligação à nucleolina sobre-expressa à superfície de células cancerígenas e (ii) um anticorpo nanobody-Fc anti-nucleolina, com o objetivo de explorar mecanismos imunitários, nomeadamente ADCC. Além disso, este trabalho teve também o objetivo de explorar o uso de nanobodies como proteínas bi-específicas, tendo a nucleolina e o fator de necrose tumoral alfa (TNF-α) sido selecionados como modelos de proteínas alvo. Um nanobody anti-TNF-α foi usado como base para inserção de sequências derivadas do péptido F3 na região determinante de complementaridade 1 (CDR1) ou 3 (CDR3), as duas regiões mais importantes na ligação ao antigénio. As sequências inseridas consistiram na referida sequência de 10 aminoácidos, com ou sem linkers em cada extremidade, os quais visavam aumentar a flexibilidade da CDR. Os nanobodies gerados apresentaram ligação a nucleolina purificada e a células que sobre-expressam nucleolina, assim como ao alvo original, TNF-α. Estes nanobodies apresentaram efeitos citotóxicos na ordem dos micromolar contra células que sobre-expressam nucleolina. A inserção da sequência derivada do péptido F3 em CDR3, resultou em ligação e citotoxicidade superiores às observadas com a inserção da mesma sequência em CDR1. Contudo, a presença de linkers nas extremidades da sequência inserida não alterou os padrões de ligação e citotoxicidade. Estes resultados levaram à seleção do nanobody com a sequência derivada do péptido F3 no CDR3 (sem linkers) para gerar um nanobody-Fc, após fusão a uma região Fc de uma IgG1 humana. Este anticorpo nanobody-Fc anti-nucleolina apresentou efeitos citótoxicos mais pronunciados (na ordem dos nanomolar) e também a v capacidade de promover um efeito de ADCC mediado por nucleolina, contra uma linha celular de cancro. Em conclusão, neste trabalho foram desenvolvidas entidades direcionadas para a nucleolina com atividade citotóxica contra células que sobre-expressam nucleolina. Além disso, os nanobodies apresentaram propriedades bi-específicas, como evidenciado pela sua ligação a dois antigénios diferentes. De notar que o anticorpo nanobody-Fc apresentou capacidade de ADCC mediada por nucleolina, um efeito que ainda não tinha sido descrito para este alvo. Desta forma, os anticorpos apresentados neste trabalho poderão contribuir para o desenvolvimento de novas terapias direcionadas para a nucleolina sobre-expressa em tumores.
Mittelmeier, Lucas. "Multiplexe optische und Rasterkraftmikroskopie für biomedizinische Bildgebung." Doctoral thesis, 2020. http://hdl.handle.net/21.11130/00-1735-0000-0005-13FB-B.
Full textGomes, 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.
Full textHouserová, Jana. "Studium interakce receptoru NKp46 s adhesinem Epa1." Master's thesis, 2020. http://www.nusl.cz/ntk/nusl-434858.
Full textMaidorn, 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.
Full textJeon, Suekyoung. "Analyzing UNC-50/GMH1 dependent membrane trafficking in yeast and C. elegans." Doctoral thesis, 2014. http://hdl.handle.net/11858/00-1735-0000-0028-864B-5.
Full textSograte, 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.
Full textCmunt, Denis. "Příprava fúzních domén lidských imunoreceptorů pro jejich využití v imunoterapii." Master's thesis, 2019. http://www.nusl.cz/ntk/nusl-403988.
Full textKrüwel, Thomas. "In vivo imaging of the voltage-gated potassium channel Kv10.1 utilizing SPECT in combination with radiolabeled antibodies." Doctoral thesis, 2015. http://hdl.handle.net/11858/00-1735-0000-0028-8635-6.
Full textMajumder, Puja. "Structural and Functional Investigation of a Multi-drug Efflux Transporter QacA." Thesis, 2020. https://etd.iisc.ac.in/handle/2005/5103.
Full textalsaiari, shahad. "NANOBOTS Smart Systems to Improve Therapeutics Delivery." Diss., 2018. http://hdl.handle.net/10754/630004.
Full textSchrodi, Inken [Verfasser]. "In-vivo-Analyse von Biokompatibilität und Gewebeintegration des synthetischen Knochenersatzstoffes NanoBone / vorgelegt von Inken Schrodi." 2009. http://d-nb.info/1006874437/34.
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