Dissertations / Theses on the topic 'Antibody recognition'
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Bristow, Richard G. W. "Antibody recognition of HIV-1 glycoproteins." Thesis, Open University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315370.
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Davies, Julian. "Antibody VH domains as small recognition units." Thesis, University of Cambridge, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263483.
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Robakiewicz, Stefania. "Minimal structural glyco-epitope for antibody recognition." Thesis, Lille 1, 2020. http://www.theses.fr/2020LIL1S101.
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L'importance biologique de la glycosylation pour la santé et la maladie est largement reconnue. Les structures tronquées à terminaison mannose consistant en 1 à 3 résidus de mannose, deux N-acétylglucosamines et un nombre variable de fragments fucose sont appelées paucimannose. Les N-glycanes paucimannosidiques sont abondamment exprimés dans les plantes et les invertébrés. Cependant, chez les vertébrés, leur présence est limitée à certaines conditions pathophysiologiques, telles que le cancer, les troubles immunitaires, les infections et l'inflammation, et chez les individus en bonne santé, ils ne sont détectables qu'en quantités infimes. Il a été démontré que le Mannitou, un anticorps monoclonal murin, reconnaît spécifiquement les glycoépitopes de paucimannose.Une tentative de caractérisation de la structure de Mannitou IgM a été faite en appliquant des techniques de modélisation d'homologie, de microscopie cryoélectronique et de cristallisation. L'anticorps complet de Mannitou a été généré en utilisant la technologie des hybridomes. Le Fab recombinant de Mannitou a été exprimé avec succès de manière transitoire dans des cellules HEK293T. La spécificité de liaison de Mannitou envers différents N-glycanes de paucimannose ont été élucidées par une combinaison de méthodes expérimentales. Le criblage par microréseau a révélé que le glyco-épitope minimal était Man2GlcNAc2. À son tour, Man3GlcNAc2 a manifesté l'une des interactions les plus fortes avec l'anticorps Mannitou. Des études de reconnaissance moléculaire, utilisant des mesures de résonance plasmonique de surface et une calorimétrie de titrage isotherme, ont établi une affinité de liaison micromolaire de l'anticorps Mannitou envers le glycane Man3GlcNAc2. La cartographie de l'épitope de liaison par résonance magnétique nucléaire de transfert de saturation a démontré que Manα1-3 est le principal résidu impliqué dans la reconnaissance des anticorps de Mannitou. La régulation positive des N-glycanes paucimannosidiques dans des conditions physiopathologiques fait de l'anticorps Mannitou un outil diagnostique et thérapeutique prometteur.Pour déterminer la structure minimale des glycanes requise pour mimer l'activité antigénique du polysaccharide MenX natif, des études de résonance plasmonique de surface ont été réalisées. Les expériences ont consisté à étudier les interactions de liaison entre un anticorps anti-MenX et des oligosaccharides capsulaires du sérogroupe X de Neisseria meningitides de différentes longueurs. Les résultats suggèrent que la portion minimale de saccharide capable d'assurer une protection contre les infections à MenX pourrait être DP5, ce qui en fait un candidat prometteur pour le développement de vaccinsThe biological importance of glycosylation in health and disease is broadly acknowledged. The truncated, mannose-terminating structures consisting of 1–3 mannose residues, two N-acetylglucosamines, and a variable number of fucose moieties are termed paucimannose. Paucimannosidic N-glycans are abundantly expressed in plants and invertebrates. However, in vertebrates their presence is restricted to some pathophysiological conditions, such as cancer, immune disorders, infections, and inflammation, and in healthy individuals, they are detectable only in trace amounts. Mannitou, a murine monoclonal antibody, has been demonstrated to specifically recognise paucimannose glycoepitopes. An attempt to characterise Mannitou IgM structure was made by applying homology modelling, cryo-electron microscopy, and crystallisation techniques. Full-length Mannitou antibody has been generated using hybridoma technology. Recombinant Mannitou Fab has been successfully transiently expressed in HEK293T cells. The binding specificity of Mannitou towards different paucimannose N-glycans have been unravelled by a combination of experimental methods. The microarray screening revealed the minimal glyco-epitope to be Man2GlcNAc2. In turn, Man3GlcNAc2 manifested one of the strongest interactions with Mannitou antibody. Molecular recognition studies, employing surface plasmon resonance measurements and isothermal titration calorimetry, established a micromolar binding affinity of Manniotu antibody towards Man3GlcNAc2 glycan (Kd = ~50 μM). The mapping of the binding epitope by saturation transfer difference nuclear magnetic resonance demonstrated Manα1-3 as the main residue involved in Mannitou antibody recognition. The upregulation of paucimannosidic N-glycans in pathophysiological conditions makes Mannitou antibody a promising diagnostic and therapeutic tool.For determining the minimal carbohydrate structure required for mimicking the antigenic activity of the native MenX polysaccharide, surface plasmon resonance studies were performed. The experiments involved studying the binding interactions between an anti-MenX antibody and Neisseria meningitides serogroup X capsular oligosaccharides of different length. The results suggest that the minimal saccharide portion capable of ensuring protection against MenX infections may be DP5, making it a promising candidate for vaccine development
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Scherer, Erin M. "Antibody recognition of a protein epitope close to a membrane : a novel solution." Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510216.
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Webster, Duncan F. "Characterisation of human hepatic cytochrome P450 : comparison of metabolic markers and antibody recognition." Thesis, University of Aberdeen, 1997. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU530008.
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A cDNA library was produced in lambdagt11 from an induced human liver. Twenty-one clones were isolated. All clones were identified by two of the four antibodies used in the screening process. The phage insert was recovered by PCR with two specific primers. The PCR product was ligated into M13 vectors and sequenced. The DNA was a CYP2A sequence with highest identity to CYP2A6 but contained a point deletion mutation. Coumarin metabolism was measured in microsomal preparations of fifteen livers and was inhibited by three of the antibodies used in screening. RP1 monoclonal gave partial inhibition (60%) at ratios of 100:1. Two other antibodies IIB1 polyclonal and RP3 (monoclonal) were highly inhibitory at low ratios. IIB1 gave a 90% inhibition at 20:1 ratio, RP3 was 95% inhibitory at 6:1 ratio. O-dealkylation of alkoxyresorufins was determined for sixteen livers. BROD activity was partially inhibited by IIB1 polyclonal (60% at a 20:1 ratio). This supports the possibility that BROD is not a marker for CYP2B activity in humans and that BROD may be primarily metabolised by CYP4A. The results were compared with fifty-seven separate sets consisting of metabolism and antibody immunoquantification data. There were strong correlations between BROD activity and several CYP3A marker activities and protein levels identified by a polyclonal antibody that recognises P4503A. The metabolism of indomethacin was measured for fifteen livers. One of the metabolites of indomethacin, desmethylindomethacin, was produced by all livers. The metabolism could not be inhibited with antibodies as they interfered with the reaction in a non-specific manner. Comparisons with other metabolism in the same livers showed that indomethacin is probably a CYP3A substrate. The indomethacin method was modified ibuprofen and data was collected for fourteen livers. Two metabolites, I and II, were seen for each liver in 1:2 ratio respectively. These both correlated highly with tolbutamide metabolism.
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Raina, Monika. "Development of an impedimetric biosensor using a non-antibody based biological recognition molecule." Thesis, University of Leeds, 2013. http://etheses.whiterose.ac.uk/6844/.
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The molecular recognition layer generally immobilised on the active interface of a biosensor is one of the key factors in governing the biosensor’s performance, and in particular its sensitivity and selectivity. The aim of this thesis was to investigate a novel non-immunoglobulin-based recognition molecule as the capture molecule for electrochemical biosensors with the aim to improve sensitivity and specificity of label-free biosensing. To understand the characteristics of the biomolecular layer of a biosensor formed from the non-immunoglobulin-based recognition molecule, the Adhiron scaffold developed at the University of Leeds was used as the model system. The Adhiron scaffold consists of one α-helix, four β-sheets, and three variable regions. The three variable regions comprise two surface-exposed loops and the N-terminus of the protein. Adhiron-based binders against a well-characterised antibody, the anti-myc tag antibody, were selected using phage-display and used as a model system for this study. The phage-display library was constructed by inserting randomised peptides into the three variable regions of the Adhiron scaffold. The best performing binder, selected from the ten Adhiron myc binders panned from a phage display screen against polyclonal anti-myc tag antibodies, myc binder 2, was chosen as the biological recognition molecules for the development of an electrochemical impedimetric biosensor. Cloning of the Adhiron binders in pET-11(a) expression vector, optimisation of expression and purification of the binders, was carried out and the binders were obtained in soluble form. Adhiron myc binder 2, which showed the best binding against monoclonal anti-myc tag antibodies, showed a high thermal stability of 85º C, with well-defined α-helical and β-sheet structures. This binder was thoroughly characterised further before being used as a recognition molecule of an electrochemical biosensor. An electrochemical Adhiron-based myc binder 2 sensor was fabricated to detect monoclonal anti-myc tag antibodies over a range of concentrations. The Adhiron myc binder 2 based EIS biosensor comprised a highly sensitive insulating layer formed by a self-assembled monolayer of carboxylic acid terminated alkylthiol-PEG onto which Adhiron myc binder 2 was grafted. The sensing mechanism was based on the change in phase of the electrochemical impedance measured at 0.1 Hz observed upon binding of monoclonal anti-myc tag antibodies onto the sensor. Monoclonal anti-myc tag antibodies were detected down to a concentration of less than 100 pM, over a range from 0.1–200 nM anti-myc tag antibodies. These findings demonstrated for the first time the successful use of Adhiron-based antibodymimetics as recognition molecules in label-free biosensors. To improve the sensitivity of the Adhiron-based electrochemical biosensor, and potentially to modulate the sensitivity in situ, the performance of the sensor at different electrochemical DC-biases was investigated. The sensitivity of the sensor was observed to increase with increasing DC bias applied to the sensor surface. This sensitivity modulation was demonstrated to be reversible, therefore opening up a range of opportunities for future label-free biosensor architectures.
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Topping, Katherine P. "Structural studies on serotype-specific opsonic antibody recognition of protective streptococcal M protein epitopes." Thesis, University of Newcastle Upon Tyne, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294877.
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Koh, W. W. L. "Characterisation of subtype C HIV-I envelope glycoproteins and their recognition by llama antibody fragments." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/18999/.
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Subtype C HIV-1 is currently responsible for the majority of new infections in the world, particularly in parts of Africa where the adult prevalence rate is as high as 15%. In the absence of a viable vaccine in the near future, the study of new neutralising antibodies that can inhibit virus entry is urgently needed. To understand the subtype C HIV-1 envelopes, the env gene was cloned directly from 15 patient plasma samples obtained from a few countries in Africa and in the UK, and 18 replication-competent chimeric viruses were created. These envelopes were then characterised and compared with other envelopes in standard reference panels. We then exploited the unique properties of llama heavy-chain antibodies to create antibody fragments (VHH) that can recognise HIV-1 envelopes and prevent infection. Four VHH that recognise a conformation dependent epitope on gp41 were isolated from a llama that was immunised with recombinant gp140 derived from a subtype B’/C isolate after panning of the phage libraries on recombinant gp41. These VHH were more potent in neutralising subtype C isolates than subtype B isolates. Based on the success of an earlier study on VHH that recognise an epitope overlapping the CD4 binding site on gp120, a novel strategy was used to isolate variants of the VHH to create a family-specific VHH library. Thirty-one new VHH were characterised and grouped according to their neutralisation breadth against 3 subtype C viruses. The neutralisation breadth of the VHH correlated with its dissociation rate with gp120, and was found to be dependent on 3 amino acid residues in the third complementarity determining region of the VHH. These VHH may have further use in applications such as HIV-1 microbicides development and immunogen design through reverse immunology.
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Naqid, Ibrahim. "Investigation of antibody-based immune recognition of infections with Salmonella enterica serovars Typhimurium and Enteritidis." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/33141/.
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Salmonellosis causes significant economic losses to the pig and poultry industries. Pigs and chickens are also a significant source of Salmonella for humans, usually transmitted through the consumption of Salmonella contaminated chicken and pork products. Predominantly, Salmonella enterica serovar Typhimurium and Enteritidis remain a global health problem. Probiotics and prebiotics have been previously used as alternatives to antibiotic treatments in the protection against enteropathogens including S. Typhimurium. Here, we determined the effects of probiotic, prebiotic and synbiotic diets on humoral immune responses to oral S. Typhimurium challenge of pigs. The inclusion of probiotic Lactobacillus plantarum in the diet of piglets enhanced serum IgG, and IgM (p <0.001), and IgA (p <0.01) responses to S. Typhimurium infection. Similarly, inclusion of prebiotic lactulose in the diet increased serum levels of IgG and IgM (p <0.01) responses to pathogen, but not IgA levels. Inclusion of both feed additives as a synbiotic diet also significantly increased the level of IgG responses (p <0.05) to S. Typhimurium, but no differences were seen in the levels of IgM and IgA responses. However a significant interaction of the pre and probiotics was observed when considering the immune responses to S. Typhimurium (IgM P=0.004; IgG and IgA, P<0.001 for interaction). These data support the use of L. plantarum or lactulose as strategies to contribute to the protection of weaned piglets from zoonotic bacterial pathogens, but caution must be taken when combining dietary supplements as combinations can interact. The mapping of antibody-based immune responses to Salmonella enterica infections for identifying epitopes/mimotopes has an important role in the development of both novel serological diagnostic assays and vaccines. Serological assays often underpin disease surveillance programs and are also required for the differentation of infected from vaccinated animals (DIVA) to allow the full implementation of vaccines alongside such surveillance. Here, panning of phage display peptide libraries coupled with Next Generation Sequencing was applied to the mapping of B-cell responses to Salmonella infections in both pigs and chickens. IgG from 12 pigs infected with S. Typhimurium were probed in parallel and compared to the equivalent IgG from the same pigs prior to infection. Seventy-seven peptide were enriched against IgG from multiple infected pigs, thirty-one peptides were synthesised and tested in ELISA and twelve peptides were highly discriminatory for pure IgG from infected pigs (P<0.05). Similarly, IgY from chickens infected with different Salmonella serovars were probed in order to identify mimotopes specific for S. Enteritidis infection. Twenty-nine peptides were enriched against IgY from multiple infected chickens, and then synthesised and tested in ELISA assays, tweleve of them were highly discriminatory for IgY following S. Enteritidis infections (p<0.05). The technology was also used to identify multiple peptides that were specifically bound by IgY from Salmonella infected chickens compared to a live attenuated vaccine and a killed vaccine. Twenty-five and thirty-six peptides for attenuated and inactivated vaccines, respectively, were identified as being specifically enriched in multiple infected chickens. Twenty and twenty-six of the most discriminatory peptides for live and killed vaccines, respectively, were applied in multi-peptide diagnostic assays that diagnosed infection with 100% sensitivity and specificity. The results demostrate that the identified peptides can be used to design serological DIVA tests with established inactivated and attenuated vaccines. Overall, the described next generation phage display (NGPD) technology repeatedly identified panels of epitopes/mimotopes recognised by multiple animals with a particular infection, providing an extremely efficient method to map host polyclonal antibody responses to S.Typhimurium and S. Enteritidis infections.
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Grinstead, Jeffrey Scott. "Structural immunology of humoral and cellular recognition of a MUC1 breast cancer antigen /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/8180.
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Ting, Joy Holtvluwer. "Molecular ecology of mate recognition in the harpacticoid copepod Tigriopus : antibody production, protein purification, and fitness consequences." Diss., Georgia Institute of Technology, 2001. http://hdl.handle.net/1853/25202.
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Wang, Hongsheng. "Natural antibody recognition, signaling and surveillance in v-Ha-ras- and PKC-ß1-overexpressing 10T1/2 fibroblasts." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape17/PQDD_0003/NQ32033.pdf.
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Madigan, Judith. "Antibody and T-Cell recognition of MHC- and mimicking tissue-peptides in autoimmune disease, particularly ankylosing spondylitis." Thesis, King's College London (University of London), 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.444151.
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Al, Qaraghuli Mohammed. "Investigating the antibody recognition of different hapten classes using a combination of phage display and protein modelling." Thesis, University of Aberdeen, 2014. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=214816.
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Easton, Donna Meredith, and n/a. "Functional and Antigenic Characterisation of the Moraxella catarrhalis protein M35." University of Canberra. n/a, 2008. http://erl.canberra.edu.au./public/adt-AUC20081217.083105.
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This thesis reports the characterisation of a novel outer membrane protein (OMP)
from M. catarrhalis, designated M35, with a molecular mass of 36.1 kDa. This protein is
structurally homologous to classic Gram-negative porins, such as OMP C from E. coli and
OMP K36 from K. pneumoniae, with a predicted structure of 8 surface loops connecting 16
antiparallel -sheets. Comparison of the DNA sequences of the M35 genes from 18 diverse
clinical isolates showed that the gene was highly conserved (99.6-100 % of nucleotides)
with only one isolate (ID78LN266) having base variations that resulted in amino acid
substitutions.
A single amino acid mutation in the 3rd external loop of M35 in isolate ID78LN266
significantly affected antibody recognition, indicating that loop 3 contains an immunodominant
B-cell epitope. The reduction in antibody-binding to M35 from ID78LN266
was similar to that caused by complete removal of loop 3. Since loop 3 folds into the porin
channel in the classic structure, the antibody specificity to loop 3 was hypothesised to be
a potential mechanism for evasion of host immune responses targeted to M35, potentially
explaining the high degree of conservation across isolates.
A series of recombinant proteins were constructed to analyse the binding to M35 of
antibodies specificity for loop 3 or the remainder of the protein. It was found that loop 3-
specific antibodies were not able to bind to M35 on the surface of M. catarrhalis and
that this corresponds both with a lack of ability to enhance opsonophagocytosis in vitro
and bacterial clearance in vivo. Additionally, antibodies raised against a version of M35
lacking loop 3 and M35 from the variant isolate ID78LN266 were both no less effective
than the full consensus M35 by both these measures. It therefore appears that while the
majority of antibodies raised against M35 are specific for loop 3 these antibodies do not
mediate anti-M. catarrhalis actions.
Two deletion mutant strains of M. catarrhalis that do not contain the outer membrane
protein M35 were created by insertional inactivation of the M35 gene. Growth comparisons
between these mutant strains and their wildtype parent strains initially led to the
hypothesis that M35 is necessary for efficient glutamic acid uptake by M. catarrhalis,
however this hypothesis was later shown to be incorrect. Efficient uptake of glutamic acid
seemed to be mediated by a novel 40 kDa protein that was up-regulated in the deletion
mutant strains, presumably to compensate for the lack of M35. M35 was also found to
be essential for in vivo survival of M. catarrhalis in the nasal cavities of mice, indicating
that it is an essential functional protein for colonisation of the mucosal surface.
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Rogers, Todd H. "Receptor recognition and response of dendritic cells to biomaterials." Diss., Georgia Institute of Technology, 2010. http://hdl.handle.net/1853/37107.
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The goal of the work presented was to further understand how both the body and dendritic cells (DCs) interact and respond to biomaterials through receptor-mediated mechanisms. The role of Toll-like receptor 4 (TLR4) was investigated in the host response to biomaterials, and it was found that TLR4-deficient mice (in comparison to wild-type) had a delayed acute inflammatory response as seen through an altered adherent leukocyte profile on implanted polymer discs. However, following a 2 week implantation, the response was resolved potentially through compensatory receptors. Therefore, TLR4 may aid in the initial response to a biomaterial through recognition of 'danger signal' molecules. An investigation into the role of TLR4 in the response of DCs to biomaterials was investigated using murine bone marrow-derived DCs (BMDC), and PLGA film or microparticle treatment of BMDCs resulted in TLR4-dependent signs of slight maturation in non/loosely adherent BMDCs. However, further investigation into BMDC populations within the culture system revealed that non/loosely adherent BMDCs took on an activated/mature phenotype while adherent BMDCs appeared to be less mature and more responsive to both LPS and biomaterial stimuli. Therefore, it was concluded that investigations into the responsiveness of BMDCs to stimuli in the future analyze both adherent and non/loosely adherent populations. Lastly, the role of integrin-mediated adhesion in biomaterial-induced DC maturation was investigated. Gene expression analysis revealed that PLGA treatment of human DCs increased adhesion molecule expression (including β1 and β2 integrin subunits), LPS treatment reduced adhesion molecule expression and agarose treatment did not alter their expression. Antibody blocking techniques pinpointed the role of β2 integrins (and not β1 integrins) in both the adhesion of DCs to TCPS or PLGA substrates and the regulation of a DC maturation marker (CD86). β2 (and not β1) was found co-localized with F-actin in podosomes of DCs adhering to PLGA, and the direct interaction of β2 (and not β1) to PLGA substrate was confirmed through crosslinking and immunofluorescence studies. Therefore, DCs utilized β2 integrins for both adhesion and maintenance of immunomodulatory status. This aids the field of tissue engineering and vaccine design by further developing the criteria for biomaterial-influenced immunomodulation.
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Writer, Michele. "Molecular analysis of the recognition of the tumour associated antigen CD55 by the mouse monoclonal antibody 791T/36." Thesis, University of Nottingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342476.
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Meng, Guangxun. "Cellular recognition of microbial patterns through toll-like receptor (TLR) 2 analysis of molecular requirements and monoclonal antibody mediated blockage /." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=973389672.
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Diestel, Uschi [Verfasser], and Yves A. [Akademischer Betreuer] Muller. "Structural Basis for TGF-β-Receptor Interaction and Antibody Recognition of HCMV Envelope Protein gB / Uschi Diestel. Gutachter: Yves A. Muller." Erlangen : Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 2014. http://d-nb.info/1075832683/34.
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Otali, Dennis. "The combined effect of formalin fixation and individual steps in tissue processing on immunorecognition." Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2008r/otali.pdf.
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Panagiotopoulou, Maria. "Organic-inorganic composite materials for specific recognition and optical detection of environmental, food and biomedical analytes." Thesis, Compiègne, 2016. http://www.theses.fr/2016COMP2315/document.
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Cette thèse décrit l'état de l'art des sondes et nanoparticules fluorescents traditionnels utilisés en imagerie de fluorescence ainsi que le développement de nouveaux nanomatériaux à base de polymère à empreinte moléculaire, aussi dénommé ‘anticorps plastique’, pour le ciblage et la bioimagerie. En biologie et en médecine, il y a un besoin constant de diagnostiquer diverses maladies pour leur éventuel traitement et prévention. Une distribution anormale et un taux élévé de glycosylation (e.g. acides hyaluronique et sialique) à la surface ou dans les cellules sont indicateurs d’une infection ou d’un cancer. Généralement, l’imagerie par fluorescence permet de visualiser, localiser et quantifier les biomarqueurs de pathologie mais à l’heure actuelle, il n’existe pas d’outil analytique fiable pour cibler spécifiquement les molécules de glycosylation car les anticorps et les lectines vendus dans le commerce ont une faible affinité et sélectivité vis-à-vis de ces cibles. Dans ce contexte, les polymères à empreintes moléculaires (MIPs) pourraient apporter une solution. Les MIPs sont des récepteurs synthétiques possédant des affinités et sélectivités comparables à ceux des anticorps, mais exhibant une stabilité physique, thermique et chimique bien plus accrue. De plus, leur fabrication est peu coûteuse et ne nécessite pas de tuer des animaux comme pour l’obtention des anticorps biologiques. Dans cette thèse, nous avons optimisé et synthétisé des MIPs biocompatibles pour leur utilisation en bioimagerie afin de détecter et quantifier l’acide hyaluronique et l’acide sialique sur les cellules et les tissus de peau humaine. L’acide glucuronique, une composante de l’acide hyaluronique et l’acide N-acétylneuraminique, l’acide sialique le plus commun, ont été utilisés comme molécules ‘patron’, générant des MIPs très sélectifs envers leur cible en milieu aqueux. Deux types de nanoparticules de MIPs fluorescents ont été synthétisés: (1) en incorporant un colorant rhodamine polymérisable dans la solution de pré-polymérisation et (2) en encapsulant des boîtes quantiques InP/ZnS générant ainsi des MIPs de type cœur-coquille. Pour cela, nous avons adopté une stratégie innovante qui consiste à synthétiser les coquilles de MIPs directement autour des boîtes quantiques en utilisant l’énergie de l’onde fluorescente émise par l’excitation des points quantiques, pour initier la polymérisation. Un protocole d'immunocoloration standard a ensuite été optimisé afin d’imager des kératinocytes humains fixés et vivants ainsi que des tissus de peau, par microscopie à épifluorescence et confocale. Les résultats étaient similaires à ceux obtenus par la méthode de référence utilisant une protéine biotinylée reconnaissant l'acide hyaluronique. L'imagerie multiplex en combinant deux MIPs couplés à deux couleurs de boîtes quantiques et l’imagerie des cellules cancéreuses ont également été démontrées. Bien que les MIPs n’étaient pas cytotoxiques aux concentrations utilisées pour la bioimagerie, la toxicité des différentes composantes du MIP pourrait être un frein à leur utilisation dans le domaine biomédical. Afin de rendre ces MIPs plus ‘inoffensifs’, nous avons supprimé l’amorceur de polymérisation, une molécule considérée comme toxique. Les MIPs ont été synthétisés en employant des monomères qui s’auto-initient sous l’effet de l’UV ou de la chaleur. La spécificité et la sélectivité des MIPs obtenus étaient similaires à ceux préparés avec des amorceurs. En conclusion, cette thèse décrit la première utilisation des MIPs comme anticorps synthétique pour la bioimagerie de fluorescence. Ce travail ouvre la voie à de nouvelles applications en détection, diagnostique et thérapie par des MIPsThis thesis describes the state of the art in nanomaterials-based targeted bioimaging and introduces molecularly imprinted polymers, also termed ‘plastic antibodies’ as novel biorecognition agents for labeling and imaging of cells and tissues. In fundamental biology and medical diagnostics, there is a constant need to localize and quantify specific molecular targets. Abnormal glycosylation levels or distributions of hyaluronan or sialic acids on cells are indicators of infection or malignancy. In general, bioimaging with fluorescent probes enables the localization and qualitative or quantitative determination of these pathological biomarkers. However, no reliable tools for the recognition of glycosylation sites on proteins exist, because the commercially available antibodies or lectins have poor affinity and selectivity for these targets. In this context, tailor-made molecularly imprinted polymers (MIPs) are promising synthetic receptor materials since they present a series of advantages over their natural counterparts such as the ease and low cost of preparation and their physical and chemical stability. Thus, MIPs could provide a robust and specific imaging tool for revealing the location/distribution, time of appearance and structure of glycosylation sites on/in cells, which would lead to a better insight of the tremendously diverse biological processes in which these molecules are involved. Herein, we describe the synthesis of water-compatible MIPs for the molecular imaging of hyaluronan and sialylation sites on cells and tissues. Since molecular imprinting of entire biomacromolecules like oligosaccharides is challenging, we opted for what is commonly called the ‘epitope approach’, which was inspired by nature. The monosaccharides, glucuronic acid and N-acetylneuraminic acid were imprinted, and the resulting MIPs were able to bind these molecules when present and accessible on the terminal unit of hyaluronan and sialylation sites. Fluorescent MIPs were synthesized as rhodamine-labeled nanoparticles and as MIP-coated InP/ZnS core-shell quantum dot (QD) particles. For the coating of the QDs, a novel versatile solubilization and functionalization strategy was proposed, which consists of creating polymer shells directly on QDs by photopolymerization using the particles as individual internal light sources. A standard immunostaining protocol was then successfully adapted for the application of the fluorescently labeled MIPs to image fixed and living human keratinocytes and skin tissues, by epifluorescence and confocal fluorescence microscopy. The results were comparable to those obtained with a reference method where staining was done with a biotinylated hyaluronic acid binding protein. Multiplexed and cancer cell imaging were also performed, demonstrating the potential of molecularly imprinted polymers as a versatile biolabeling and bioimaging tool. Although the MIPs were not cytotoxic at the concentrations used for bioimaging, in order to render them generally applicable in biomedicine, where toxicity of the polymerization precursors is a matter of concern, we suppressed the initiator, a toxic chemical. Initiator-free MIPs were thus synthesized by using monomers that can self-initiate under UV irradiation or heat. The specificity and selectivity of the obtained MIPs were as good as the ones prepared with initiators. In conclusion, we have demonstrated for the first time the great potential of MIPs as synthetic antibody mimics for bioimaging. The possibility to associate other functionalities such as QDs and additionally attach drugs to the same material appears rather straightforward due to the synthetic polymeric nature of MIPs, which paves the way to new potential applications in theranostics
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Joel, Smita. "ENGINEERING PROTEINS WITH UNIQUE CHARACTERISTICS FOR DIAGNOSTICS AND BIOSENSORS." UKnowledge, 2011. http://uknowledge.uky.edu/gradschool_diss/180.
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Proteins possess a broad range of structural and functional properties and, therefore, can be employed in a variety of biomedical applications. While a good number of protein-based biosensing systems and biosensors for target analytes have been developed, the search for versatile, highly sensitive and selective sensors with long term stability able to provide fast detection of target analytes continues to be a challenge. To that end, we now report the design and development of modified proteins with tailored characteristics and their further utilization in the development of biosensing systems.
We take advantage of binding proteins that undergo a change in conformation upon binding to their respective target ligand analytes for the development of highly selective biosensing systems. The first class of binding proteins that was explored for this purpose was antibodies. A non-canonical site in the variable region of a monoclonal antibody was tagged with a fluorescent probe to sense the binding of analyte to its corresponding antigen-binding site. The strategy employed for designing antibodysensing molecules is universal as it can be employed for sensing any biomolecule of interest provided that there is an available antibody against the target ligand analyte.
In a second strategy, we utilized designer glucose recognition proteins (GRPs) that were prepared by incorporation of unnatural amino acids in the glucose/galactose binding protein (GBP) of Escherichia coli and its truncated fragments. By taking advantage of the global incorporation method, we were able to fine-tune the binding affinity and thermal stability of the proteins, thus, allowing for the development of a reagentless fluorescence based fiber optic glucose biosensor capable of monitoring glucose in the hypoglycemic, normal, and hyperglycemic range, as well as in the hypothermic and hyperthermic temperature range.
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Lamendour, Lucille. "Modulation des fonctions des cellules dendritiques humaines par des fragments d'anticorps." Thesis, Tours, 2018. http://www.theses.fr/2018TOUR3303/document.
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Le système immunitaire protège un organisme du développement de pathogènes et participe activement au maintien de la tolérance immunitaire. Les cellules dendritiques (DC) sont des cellules spécialisées dans l’équilibre pro et anti-inflammatoire de la réponse immunitaire. Les DC jouent un rôle important dans de nombreux contextes pathologiques notamment la transplantation d’organes, en oncologie et dans les pathologies inflammatoires. Elles sont modulables grâce à divers facteurs, intrinsèques et extrinsèques. Parce qu’elles sont capables d’induire une réponse tolérogène, ces cellules représentent des cibles intéressantes pour moduler la réponse immunitaire dans le contexte de la transplantation d’organes et des pathologies inflammatoires. Certains agents pathogènes utilisent des mécanismes d’échappement au système immunitaire en favorisant l’induction d’une tolérance immunitaire. Cette modulation est réalisée par le ciblage des récepteurs de reconnaissance des pathogènes (PRR) sur la présence des DC, induisant la synthèse d’une cytokine antiinflammatoire IL-10, un des inducteurs de la tolérance immunitaire. Notre stratégie a été de construire un anticorps bispécifique ciblant deux PRR différents à partir d’une banque d’anticorps anti-PRR. Notre travail montre que cet anticorps bispécifique est capable d’orienter les DC vers d’un profil tolérogène. Cet anticorps bispécifique induit un phénotype de DC semi-mature avec un profil de sécrétion pro-tolérogène avec de l’IL-10 et peu de cytokines inflammatoires. Le profil de tolérance immunitaire induite par ces cellules reste à explorer. Nos travaux ouvrent de perspectives intéressantes sur l’association des PRR en vue d’obtenir la modulation des cellules de l’immunitéThe immune system protects an organism from the development of pathogens and actively participates in maintaining immune tolerance. Dendritic cells (DC) are specialized cells in the balance and anti-inflammatory immune response. DC play an important role in many pathological contexts, including organ transplantation, oncology and inflammatory diseases. Various factors, both intrinsic and extrinsic, can modulate. Because they are capable to inducing a tolerogenic response, these cells represent interesting targets for the immune response in the context of organ transplantation and in inflammatory pathologies. Some pathogens use mechanisms of escape to the immune system by promoting the induction of immune tolerance. This modulation is achieved by targeting the pathogen recognition receptors (PRRs) present on the surface of DC, inducing the synthesis of an anti-inflammatory cytokine IL-10, one of the main inducers of immune tolerance. Our strategy was to construct a bispecific antibody targeting two different PPRs from an anti-PRR antibody library. Our work shows that this bispecific antibody is able to direct the DC to a pro-tolerogenic profile. This bispecific antibody induces a semi-mature DC phenotype with a secretion profile of pro-tolerogenic cytokines such as IL-10 and few inflammatory cytokines. The immune tolerance profile of these DC remains to be explored. Our work opens interesting perspectives on the association of PRRs in order to obtain the modulation of the cells of the immunity
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Martí, Fernández Iris [Verfasser], and Wolfang [Akademischer Betreuer] Enard. "Antibodies to myelin oligodendrocyte glycoprotein (MOG): Analysis of the impact of the glycosylation site of MOG for recognition of human autoantibodies and dissection of effector functions of the anti-MOG monoclonal antibody 8-18C5 / Iris Martí Fernández ; Betreuer: Wolfang Enard." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2020. http://d-nb.info/1227840047/34.
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Belot, Laura. "Etude structurale et fonctionnelle de la glycoprotéine des Rhabdovirus Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein Structural and cellular biology of rhabdovirus entry Monomeric Intermediates Formed by Vesiculovirus Glycoprotein during Its Low-pH-induced Structural Transition." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS522.
Full textAbstract:
Les Rhabdovirus sont des virus enveloppés à ARN simple brin de polarité négative. Ils possèdent une unique protéine transmembranaire à leur surface, la glycoprotéine G. G est impliquée dans les étapes précoces du cycle viral. G lie dans un premier temps un récepteur cellulaire, menant à l’endocytose du virus. Ensuite, G orchestre la fusion membranaire entre les membranes virale et endosomale. Cette fusion membranaire permet la libération du génome viral dans le cytoplasme de la cellule infectée.Le récepteur des lipoprotéines de basse densité (LDLR) est le récepteur principal du VSV. L’ectodomaine du LDLR est composé d’un grand site de liaison au ligand constitué de domaines riches en cystéines (CR1 à CR7).Afin de comprendre les bases moléculaires de l’interaction entre G et son récepteur, nous avons réalisé des tests de fixation de G aux différents domaines CR du LDLR. Cela a révélé que seuls les domaines CR2 et CR3 pouvaient lier G. Lorsque CR2 et CR3 sont incubés avec VSV dans l’inoculum, ils protègent les cellules de l’infection par VSV. Nous avons cristallisé G en complexe avec chacun de ces domaines CR. Les structures révèlent que le site de liaison de CR2 et de CR3 sur G sont identiques, et que les mêmes résidus sur G sont impliqués dans la liaison des deux domaines CR. Des cellules HAP-1 dans lesquelles le gène codant pour le LDLR a été invalidé, sont toujours sensibles à l’infection par VSV. Ceci confirme que VSV peut utiliser d’autres récepteurs que le LDLR pour entrer dans les cellules. Cependant la mutation des résidus de G qui sont clefs dans l’interaction avec les domaines CR du LDLR abolissent l’infectiosité de VSV aussi bien dans les cellules de mammifères que dans les cellules d’insectes. Ceci indique que les seuls récepteurs de VSV dans ces cellules sont des membres de la famille du LDLR et que VSV G a spécifiquement évolué pour interagir avec leur domaines CR. Par ailleurs nous avons montré que les G dont les résidus clef pour l’interaction avec les domaines CR sont mutés, ont conservé leur activité de fusion. Ces travaux montrent que les activités de reconnaissance du récepteur et de fusion de G peuvent être découplées. Ceci ouvre la possibilité de développer des glycoprotéines dérivées de G au tropisme modifié.Chez les Rhabdovirus, G est la seule cible des anticorps neutralisants. A ce jour il n’existe aucune structure de G de Rhabdovirus en complexe avec un anticorps. L’anticorps 8G5F11 neutralise plusieurs génotypes du genre Vésiculovirus dont VSV. Nous avons montré que les FAB 8G5F11 empêchent l’infection des cellules par VSV Indiana d’une part et qu’ils reconnaissent la forme pré- et post-fusion de VSV G avec une stœchiométrie G : FAB de 1 : 1 d’autre part. Nous avons mis au point les conditions d’observation du complexe G-FAB en cryo-microscopie électronique, ce qui permet d’envisager à terme une résolution de la structure du complexe G-FAB.Enfin, nous avons débuté une étude visant à caractériser les glycoprotéines d’autres Rhabdovirus du genre Lyssavirus. Pour cela, nous avons produit et purifié les ectodomaines de G du virus de la rage (RABV), du virus Mokola (MOKV) et du virus de la chauve-souris ouest-caucasienne (WCBV). Ces G ont été caractérisées par microscopie électronique à différents pH. Les mesures effectuées sur G à pH 8 sont compatibles avec celles attendues pour un monomère de G pré-fusion. A pH 6, les ectodomaines observés pourraient correspondre à un intermédiaire monomérique allongé apparaissant tardivement lors de la transition structurale. Ces résultats pourraient être prochainement validés par l’obtention de la structure cristallographique de l’ectodomaine de G MOKVRhabdoviruses are single stranded RNA enveloped viruses. They own a unique glycoprotein G anchored on the viral membrane. G is involved in the early stages of the viral cycle. At first G binds a cellular receptor, leading to the virus endocytosis. Then G orchestrates the membrane fusion between the viral and endosomal membranes. Membrane fusion allows the release of the viral genome into the cytoplasm of the infected cell.The low-density lipoprotein receptor (LDLR) is the main receptor of VSV. The ectodomain of the LDLR is composed of a large ligand binding domain constituted of cysteine-rich domains (CR1 to CR7).In order to understand the molecular basis of the interaction between G and its receptor, we performed binding test of G with all the CR domains of the LDLR. This revealed that only CR2 and CR3 domains could bind G. When CR2 and CR3 are present in the VSV inoculum, they protect the cells from VSV infection. We crystallized G in complex with each of these CR domains. The structures reveal that CR2 and CR3 binding sites on G are identical, and that the same residues on G are involved in the binding of the two CR domains. HAP-1 cells in which the gene encoding the LDLR has been invalidated are still susceptible to VSV infection. This confirms that VSV can use other receptors than the LDLR itself to enter the cells. However, mutation of G residues that are key in the interaction with the CR domains of the LDLR abolish the infectivity of VSV in mammalian and insect cells. This indicates that the only VSV receptors in these cells are members of the LDLR family and that VSV G has specifically evolved to interact with their CR domains. Moreover, we have shown that G mutated for key residues involved in the interaction with CR domains, are still able to induce fusion. This work shows that receptor recognition and G fusion activities can be decoupled. This paves the way to develop glycoproteins derived from G with modified tropism.In Rhabdoviruses, G is the only target of neutralizing antibodies. There is no structure of a Rhabdovirus G in complex with an antibody. The 8G5F11 antibody neutralizes several genotypes of the genus Vesiculovirus including VSV. We have shown that FAB 8G5F11 prevents infection of cells by VSV Indiana on the one hand and that they recognize the pre- and post-fusion form of VSV G with a 1: 1 G: FAB stoichiometry on the other hand. We have developed the observation conditions of the G-FAB complex in cryo-electron microscopy, which could be useful to obtain the structure of the G-FAB complex.We also started a study to characterize the glycoproteins of other Rhabdoviruses of the Lyssavirus genus. We produced and purified G ectodomains of rabies virus (RABV), Mokola virus (MOKV) and West Caucasian bat virus (WCBV). We characterize these G by electron microscopy at different pHs. The measurements made on G at pH 8 are compatible with those expected for a pre-fusion monomer of G. At pH 6, these ectodomains could correspond to a monomeric late intermediate in the structural transition. Obtaining the crystallographic structure of the G ectodomain of MOKV could validate these results
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Chauvet, Margaux. "Étude de la modulation, par l'hémoglobine S, de la présentation des antigènes plasmodiaux à la surface du globule rouge infecté par Plasmodium falciparum, et de la réponse immunitaire contre le paludisme Impact of hemoglobin S trait on cell surface antibody recognition of Plasmodium falciparum infected erythrocytes in pregnancy-associated malaria." Thesis, Sorbonne Paris Cité, 2019. http://www.theses.fr/2019USPCB037.
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Le paludisme est une maladie tropicale résultant de l'infection par le parasite Plasmodium falciparum transmis par piqûre de moustique. Les symptômes du paludisme résultent du développement de P. falciparum dans les globules rouges (GR). Depuis des siècles, le paludisme a exercé des pressions sur le génome humain, sélectionnant des mutations conférant une protection contre les formes sévères de la maladie. C'est le cas de la mutation du gène de l'hémoglobine (HbA), composant principal des GRs. La forme mutée du gène produit une hémoglobine anormale (HbS). Contrairement à la drépanocytose (HbSS), le portage hétérozygote (trait drépanocytaire) de cette mutation (HbAS) est asymptomatique. Les porteurs sains HbAS développent moins de symptômes graves du paludisme. Aujourd'hui, les mécanismes responsables de cette protection restent partiellement élucidés. Lors de son développement, P. falciparum modifie la membrane et le cytosquelette du GR afin d'exposer des protéines parasitaires à la surface de l'hématie. Parmi ces protéines, l'adhésine majeure parasitaire "P. falciparum erythrocyte membrane protein 1" (PfEMP1), se lie aux récepteurs endothéliaux, entraînant la cytoadhérence et la séquestration des GRs infectés (iGRs). Cette cytoadhérence permet d'éviter le passage et la clairance splénique des iGRs. Des études ont montré que les iGRs HbAS cytoadhèreraient moins, en association avec une présentation anormale de PfEMP1. Cette thèse porte sur l'étude des mécanismes de résistance du trait drépanocytaire contre le paludisme à P. falciparum. Le premier projet de cette thèse porte sur le phosphoprotéome des membranes de GRs HbAA et HbAS infectés par P. falciparum. Les protéines parasitaires exposées à la surface du GR interagissent avec des protéines érythrocytaires impliquées dans l'ancrage de la membrane du GR au cytosquelette. Il s'agit des protéines du complexe Ankyrine-R et du complexe jonctionnel. Le stress oxydant généré par le trait drépanocytaire et par l'invasion parasitaire perturbe l'équilibre kinase/phosphatase dans la cellule, pouvant entraîner des modulations de la phosphorylation des protéines, interférer dans les interactions protéiques et par conséquent dans la présentation des antigènes parasitaires. Des extraits membranaires de GRs HbAA et HbAS infectés ont été produits et analysés en spectrométrie de masse et en Western-Blot. Cette étude a montré que le trait drépanocytaire modulait la phosphorylation des protéines érythrocytaires de la membrane du iGR (transporteurs membranaires et protéines du cytosquelette majoritairement), mais aussi celle de protéines parasitaires. Le deuxième projet porte sur la réponse anticorps anti-VAR2CSA dans le cadre du paludisme gestationnel selon le portage de l'HbS. Le paludisme gestationnel est une des formes sévères du paludisme, due à la cytoadhérence des iGRs dans le placenta. Cette cytoadhérence résulte de l'interaction d'un PfEMP1 particulier, VAR2CSA, à la chondroïtine sulfate A des syncytiotrophoblastes. 159 plasmas de femmes HbAA et HbAS Béninoises, collectés à l'accouchement, ont été utilisés pour mesurer leur capacité de reconnaissance de VAR2CSA à la surface de GRs HbAA et HbAS infectés. La reconnaissance immune des iGRs HbAS par les plasmas provenant des mères HbAS est significativement plus faible que celle des iGRs HbAA par les plasmas des mères HbAA. Par ailleurs, d'autres maladies génétiques affectant le GR peuvent influencer la réponse en anticorps spécifiques aux GRs parasités. Les co-portages du déficit en G6PD et de l'alpha-thalassémie avec l'HbS ont ainsi été évalués pour ce groupe d'étude. Respectivement, 26,7% et 51,7% des femmes étaient porteuses du déficit G6PD ou de l'alpha-thalassémie. Ces données soulignent l'importance de considérer simultanément les différents désordres érythrocytaires existant parmi la population considérée, pour étudier les mécanismes protecteurs conférés par le portage d'HbS contre le paludismeMalaria is a tropical disease resulting from infection by the parasite Plasmodium falciparum transmitted by mosquito bite. The symptoms of malaria are caused by the development of P. falciparum in red blood cells (RBCs). For centuries, malaria has put pressure on the human genome, having selected mutations conferring protection against severe forms of the disease. This is the case of the mutation of the hemoglobin gene (HbA), the principal constituent of RBCs. The mutated form of the gene produces abnormal hemoglobin (HbS). In contrast to sickle cell disease (HbSS), the heterozygous carriage (sickle cell trait) of this mutation (HbAS) is asymptomatic. Healthy HbAS carriers are protected from severe symptoms of malaria. Today, the mechanisms responsible for this protection remain partially understood. During its intra-erythrocytic development, P. falciparum modifies the RBC membrane and cytoskeleton to expose parasite proteins at the surface of the erythrocyte. Among these proteins, the major parasitic adhesin, "P. falciparum erythrocyte membrane protein 1" (PfEMP1), binds to endothelial receptors, resulting in cytoadherence and sequestration of infected RBCs. This cytoadherence permits the infected RBCs to avoid splenic clearance. Studies have shown that infected HbAS RBCs have a reduced cytoadherence, in association with an abnormal PfEMP1 display. This PhD project attempts to decipher the mechanisms of resistance conferred by the sickle cell trait against P. falciparum malaria. The first part of this project considers the phosphoproteome of the infected HbAA and HbAS red cell membranes. Parasitic proteins exposed on the surface of RBCs interact with erythrocyte proteins involved in the anchorage of the cytoskeleton to the erythrocyte membrane. These human proteins belong to the Ankyrin-R and the junctional complexes. The oxidative stress generated by sickle cell trait, and by parasite invasion, disrupts the kinase / phosphatase balance, leading to modulation of protein phosphorylation. As protein interactions could be regulated by their state of phosphorylation, this modulation may interfere in parasite antigens' display. Thus, protein membrane extracts of infected HbAA and HbAS RBCs were produced and analyzed by mass spectrometry and Western-Blot. This study showed that the sickle cell trait modulated the phosphorylation of erythrocyte proteins of the infected RBCs (membrane transporters and cytoskeletal proteins mainly), but also that of parasite proteins. The second part of the project deals with the anti-VAR2CSA antibody response in the context of pregnancy-associated malaria according to the heterozygous carriage of hemoglobin S. Placental malaria is one of the severe forms of malaria, resulting from the cytoadherence of infected RBCs in the placenta. This cytoadherence results from the interaction of a particular PfEMP1, VAR2CSA, with chondroitin sulfate A expressed on syncytiotrophoblasts. 159 plasma samples of HbAA and HbAS Beninese women, collected at delivery, were used to measure their ability to recognize VAR2CSA on the surface of infected HbAA and HbAS RBCs. Immune recognition of infected HbAS RBCs by plasma from HbAS mothers is significantly lower than the immune recognition of infected HbAA RBCs by HbAA mothers' plasma. In addition, other genetic diseases affecting RBCs may influence the antibody response to parasitized red blood cells. Co-carriage of G6PD deficiency and alpha-thalassemia with HbS were assessed for this study group. G6PD deficiency and alpha-thalassemia were present in, respectively, 26.7% and 51.7% of the women. These data underline the importance of simultaneously considering the different erythrocyte disorders present at high prevalence among the population considered, in order to study the protective mechanisms conferred by the carriage of HbS against malaria
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Santoro, Lyse. "Appretement et présentation d'un anticorps monoclonal murin par une lignée monocytaire ou lymphocytaire B humaine : influence de la liaison covalente entre anticorps et fragment C3b du complément." Grenoble 1, 1994. http://www.theses.fr/1994GRE10126.
Full textAbstract:
La proteine c3 du complement influence l'elaboration de la reponse immune specifique dirigee contre un antigene defini. L'etude presentee dans cette these contribue a demontrer que le fragment c3b du complement, en se fixant de facon covalente a un antigene d'origine exogene, module l'appretement de l'antigene par une cellule presentatrice de l'antigene. Des donnees bibliographiques recentes concernant l'appretement d'antigenes, le fragment c3b et son implication dans la reponse immune specifique sont presentees dans un chapitre d'introduction. L'etude experimentale decrite a ete realisee en utilisant des anticorps monoclonaux murins comme antigenes et des cellules monocytaires ou lymphocytaires b humaines comme cellules presentatrices ; des complexes covalents anticorps monoclonaux-c3b ont ete produits et caracterises. Les resultats obtenus sont exposes dans trois chapitres. Dans un premier chapitre, des experiences montrent que la presentation d'anticorps monoclonaux murins a des cellules t humaines specifiques de ces anticorps est modulee lorsqu'ils sont complexes au fragment c3b. Puis certaines des principales etapes de l'appretement des anticorps utilises sont caracterisees dans des cellules monocytaires u937 ou lymphocytaires b humaines non specifiques de l'antigene (fixation a des recepteurs membranaires, internalisation, transit intracellulaire, modifications biochimiques) ; enfin, l'influence de la liaison covalente entre anticorps et c3b sur ces differentes etapes est mise en evidence. Des hypotheses sont proposees concernant un role chaperon de c3b
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Parker, Matthew J. "Protein recognition of clinically-relevant carbohydrates." Thesis, 2015. http://hdl.handle.net/1828/6264.
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A diverse array of proteins has evolved to detect and affect carbohydrate structures, thereby performing critical roles in important biological events. Carbohydrate recognition usually employs a high degree of precision, as discriminating between two carbohydrate structures can depend on a single hydrogen bond or the configuration of a hydroxyl group. My work has focused on the molecular recognition of carbohydrate antigens by two biologically important classes of carbohydrate-binding proteins: antibodies and lectins. Single crystal x-ray diffraction has been employed to study the IgG2a antibody LPT3-1 and the lectins Griffonia simplicifolia 1-A4 (GSI-A4) and Lathyrus odoratus lectin (LOdL). LPT3-1 targets the conserved inner core structure of lipooligosaccharide from Neisseria meningitidis, the leading cause of meningitis and septicaemia. Structural characterization of LPT3-1 with an inner core fragment demonstrates how this antibody achieves selective cross-reactivity to variants of the inner core and provides insight that could support the development of a broadly protective N. meningitidis vaccine. Legume lectin GSI-A4 displays specificity towards the terminal galactose and N-acetyl-D-galactosamine of carbohydrates, yet the closely related lectin GSI-B4 will only recognize a terminal galactose. The structures of GSI-A4 co-crystallized with two different carbohydrates reveals the mechanism by which GSI-A4 displays this cross-reactivity, which allows for specific recognition of two important tumour-associated carbohydrate antigens. LOdL is a member of the Mannose/Glucose legume lectin family that can recognize an array of clinically significant antigens including abnormal glycosylation patterns on gp120 of HIV. Characterization of LOdL in complex with glucose at high resolution provides a putative primary sequence and molecular level insight into the molecular recognition displayed by this lectin. Structural data indicates LOdL is cross-reactive with the related glucose epimer mannose, and would display a similar if not identical affinity for glucose and mannose, enabling cross-reactivity with oligosaccharides displaying a terminal mannose. The similarity in sequence and primary recognition between LOdL and Pisum sativum lectin (PSL) suggests that LOdL also shares oligosaccharide specificity with PSL and similarly could demonstrate anti-HIV activity. Overall, the structural characterization of these three carbohydrate-binding proteins reveals mechanisms by which antibodies and lectins can employ selective cross-reactivity to discriminate among clinically-relevant carbohydrate structures.Graduate
mj3parker@gmail.com
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Cobaugh, Christian Wessel 1971. "Single scaffold antibody libraries created with high rates of mutagenesis or diversity focused for peptide recognition." 2007. http://hdl.handle.net/2152/15990.
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This dissertation describes several strategies used to create diversity in non-immune antibody libraries. Two of the strategies were used to create two separate peptide focused libraries. Both of these strategies used to create these antigen-class focused libraries used a single scaffold antibody gene that contained diversity only in the variable heavy region. The scaffold antibody gene one of the libraries, the M:anti-pep library, was chosen based on hypervariable loop canonical structures that are characteristic of other anti-peptide antibodies. Additionally, all of the contact residues of this antibody are commonly used contact residues in other anti-peptide antibodies. These positions and others were varied to incorporate the natural diversity of other anti-peptide antibodies. The second library, the Hu:anti-pep, is based on a widely used, unique combination of human germline antibody segments that express well in bacterial expression. Positions were chosen for variation based on their usage as contact residues in both anti-peptide and anti-protein antibodies. The diversity was less focused than with the M:anti-pep library, incorporating all 20 amino acids at "high usage" positions and only four amino acids at "low usage" positions. Both libraries were validated by phage display selections against the peptide angiotensin (AT) and neuropeptide Y (NPY). The M:anti-pep library yielded specific antibodies to both peptides with dissociation constants as low as 14 nM against AT and 18 nM against NPY. The Hu:anti-pep library yielded specific clones with higher dissociation constants: 49 nM against NPY and 11 [mu]M against AT. The final strategy used to introduce diversity is widely used for affinity maturation of low affinity, previously selected antibodies. Extremely high rates of mutagenesis (2.2% of the gene to 2.7%) were used to create two libraries of the anti-digoxin antibody 26-10. The libraries had been screened by others in an attempt to examine the effects of highrates of mutagenesis on the directed evolution of an antibody. A total of 91 isolated clones from both libraries were sequenced. Several consensus mutations were identified near the CDRH3 in the isolated clones, indicating that they had a positive, selectable effect. This study confirmed that high-error rate antibody libraries contain more active clones than expected. Combinations of the selected consensus mutations from these libraries provide moderate enhancements to the kinetics and expression of the wild-type antibody in a non-synergistic manner.text
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Wolf, Cornel [Verfasser]. "Structured antibody surfaces for bio-recognition and a label-free detection of bacteria / Cornel Wolf." 2010. http://d-nb.info/1009999826/34.
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Agnew, Heather Dawn. "Rapid Construction of Protein Capture Agents with Chemically Designed Stability and Antibody-Like Recognition Properties." Thesis, 2010. https://thesis.library.caltech.edu/5583/11/Thesis.pdf.
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This thesis describes technologies for the rapid and scalable production of high-affinity, high-specificity protein capture agents which possess the affinities and specificities of antibodies, but also exhibit improved chemical, biochemical, and physical stability. I will discuss how the chemical flexibility of comprehensive, one-bead-one-compound (OBOC) libraries of oligopeptides may be combined with iterative in situ click chemistry to select multi-ligand capture agents. Large OBOC libraries form the basis of individual peptide ligands, and also permit chemically designed stability through the incorporation of artificial (azide or acetylene) and non-natural amino acid building blocks. The in situ click chemistry method then utilizes the target protein as the catalyst, or template, for assembling its own biligand via formation of a 1,2,3-triazole linkage between two individual ligands (azide and acetylene). This process can be repeated to produce triligands, tetraligands, and other higher-order multi-ligands with an accompanying increase in affinity and specificity through cooperative interactions. Once found, multi-ligand capture agents can be produced in gram amounts via conventional synthetic methods such as the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). This is a general and robust strategy for the inexpensive, high-throughput construction of protein capture agents that can be exploited to detect protein biomarkers in multi-parameter clinical diagnostic assays.
While high-affinity protein capture agents represent a significant technology advance, they are just one component of what is necessary for highly multiplexed measurements of protein biomarkers. It is also important to develop or optimize the actual assay platforms that can enable sensitive multi-parameter protein measurements using these capture agents. Silicon nanowire (SiNW) nanoelectronic sensors can provide quantitative, label-free multi-parameter measurements of protein biomarkers in real time. However, SiNW sensors can be challenging to deploy because unprotected Si forms a native oxide layer that can significantly reduce the detection sensitivity of the nanowire sensors via dielectric shielding. Another technical challenge is the development of chemistries which allow for the selective encoding of nanowire surfaces with the capture agents. To overcome these challenges, the final part of this thesis presents a general method to functionalize organic and biological molecules on highly passivated Si(111) surfaces with minimal surface oxidation.
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Yu-Ming, Wang. "Specific Recognition Force, Dissociation and Thermodynamics of Single-pair Antibody-Antigen Interaction Using Atomic Force Microscopy." 2006. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0001-1110200612471100.
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Wang, Yu-Ming, and 王裕銘. "Specific Recognition Force, Dissociation and Thermodynamics of Single-pair Antibody-Antigen Interaction Using Atomic Force Microscopy." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/32594189490753914708.
Full textAbstract:
博士國立臺灣大學
應用力學研究所
95
Molecular recognition and intermolecular binding are essential for implementing many biochemical and biological processes in living organisms. To further understand the molecular binding mechanisms, this study used atomic force microscopy as a force-based senor for investigation of the force strength between antibody and antigen complex in a single pair level with varied physiological (pH and temperature) and physical (loading rate) conditions. The results provided direct evidence of unbinding force, dissociation rate and thermodynamic parameters that explained intermolecular behavior of human IgG1/anti-human IgG1 complex and glucagon/anti-glucagon IgG complexes, respectively. Mean measured forces of human IgG1 and its specific antibody system with pH-varied liquid environments showed a sharp decrease with a decrease of pH value (acidic environment), and a gradual decrease with an increase of pH value (alkaline environment) from a reference level at neutrality. This could have corresponded to the pH-induced change in conformational change and outer functional groups of amino acids which are protonated. As a result of change in pH environment of human IgG1/anti-human IgG1 complex, surface protonated properties and conformation weakened intermolecular force. Molecular dynamic behavior and free energy change were also contributed to a high probability of bonds breaking and a low magnitude of energy barrier when molecules were immersed in acidic or alkaline solution. Temperature-dependent unbinding force experiments were also carried out. The results showed that the unbinding forces decreased with an increase of environmental temperatures. This could be largely due to temperature-induced conformational change in volume expansion and strong Brownian motion. As a result, interaction forces decreased. Estimated dynamic behavior and thermodynamic parameters also showed weak interactions under high temperatures. This could have corresponded to looser molecular structure and weaker intermolecular interaction in high temperature, thereby increasing entropy and enthalpy. The interaction between glucagon and anti-glucagon IgG with pH- varied liquid environment exhibited weak interaction force, low energy barriers and high dissociation rates under acidic and alkaline solutions. This indicated that molecular interactions turned out weak forces when pH values increased or decreased away from neutrality. Force measurement as a function of temperature exhibited a nearly linear decrease of force strength with an increase of temperature. This could have been attributed to molecular charge-free conformational changes, resulting in incomplete binding. The thermodynamic enthalpy and entropy for interaction showed an increase with increasing temperature. Specific interactions of human IgG1/anti-human IgG1 pairs and glucagon/anti-glucagon IgG pairs have been successfully investigated by the atomic force microscope. With the use of an extended Bell and Evans model, the molecular dynamic behavior, free energy change and thermodynamic parameters can be obtained by varying physiological (pH and temperature) and physical (loading rate) conditions. The results provided directly evidence to explain the biological interactions.
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Wang, Hongsheng. "Natural antibody recognition, signaling and surveillance in v-Ha-ras- and PKC-B1-overexpressing 10T1/2 fibroblasts." 1998. http://hdl.handle.net/1993/1574.
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Extensive evidence supports a role for polyclonal serum natural antibody (NAb) acting as a mediator of natural resistance against tumors in mice. However, little is known about its mechanisms of action or about the phenotype of susceptible cells. C3H 10T${1\over 2}$ fibroblasts overexpressing an activated ras oncogene or a PKC-$\beta$1 gene increased their NAb binding capacities, identifying PKC, an integral signaling molecule of normal cellular activation, as a key regulator of NAb binding structures. This, coupled with corresponding decreases in expression of membrane PKC-$\alpha$ and NAb binding in resting confluent 10T${1\over 2}$ cells raised the possibility that, in general, cells activated through PKC are NAb sensitive. In addition, NAb interaction with 10T${1\over 2}$ variants initiated a signal transduction mechanism including activation of PKC, shedding of cell surface molecules and bound NAb, a reduction in phosphotyrosine levels of a membrane-associated 60 KDa molecule, and, over time, the inhibition of DNA synthesis. Together with the increased in vivo elimination of the high NAb binding PKC-$\beta$1-overexpressing cells and the beneficial effect of passive syngeneic NAb in the rejection of syngeneic tumors injected s.c. and i.v. in both xid-bearing B cell deficient and B cell normal mouse models, the data argued that NAb not only participates in tumor surveillance of preneoplasia and neoplasia but contributes to homeostasis of the organism.
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Kuo, Ting Yu, and 郭庭佑. "A study of antibody X in the recognition of Helicobacter pylori neutrophil-activating protein as a new antigen." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/15235655246083217830.
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碩士國立清華大學
分子與細胞生物研究所
104
Helicobacter pylori (H. pylori) is a major pathogen involved in gastritis, peptic ulcer disease, and gastric cancer. Helicobacter pylori neutrophil-activating protein (HP-NAP) is an important virulence factor of H. pylori. The inflammation of the gastric mucosa caused by H. pylori infection might be resulted from the cytokines and reactive oxygen species (ROS) produced by HP-NAP-stimulated human leukocytes. Thus, H. pylori-induced inflammation of the gastric mucosa could be attenuated by blocking the activity of HP-NAP. Here, I found that antibody X not only detected their target protein but also detected recombinant HP-NAP. By western-blot, enzyme linked immunosorbent assay (ELISA) and native western-blot analyses, the antibody X detects denatured and native form recombinant HP-NAP of H. pylori 26695 strain. To determine the epitope sequence of the antibody X on HP-NAP, HP-NAP mutants were generated by using the modified PCR-based site-directed mutagenesis method and then purified by one-step DEAE anion-exchange chromatography. The antibody X is able to recognize HP-NAP through a new set of epitope sequence which is different from the original epitope of antibody X. The epitope sequence is conserved in all H. pylori strains. The non-identical amino acid residues which nearby the epitope sequence of HP-NAP in various H. pylori strains were then subjected to site-directed mutagenesis. I found that the antibody X could detect these mutated HP-NAP, indicating that antibody X is able to detect HP-NAP of various H. pylori strains. Furthermore, antibody X is able to inhibit HP-NAP-stimulated ROS production by human neutrophils. Thus, antibody X is able to detect HP-NAP and block its activity through the new epitope sequence of HP-NAP.
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Meng, Guangxun [Verfasser]. "Cellular recognition of microbial patterns through toll-like receptor (TLR) 2 : analysis of molecular requirements and monoclonal antibody mediated blockage / Guangxun Meng." 2004. http://d-nb.info/973389672/34.
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