Dissertations / Theses on the topic 'Toxin A and Toxin B'
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Fernandes, da Costa Sérgio Paulo. "Molecular and structural characterisation of epsilon toxin and necrotic enteritis toxin B : two pore-forming toxins from Clostridium perfringens." Thesis, University of Exeter, 2013. http://hdl.handle.net/10871/14608.
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Epsilon toxin (Etx) and necrotic enteritis toxin B (NetB) are two pore-forming toxins produced by C. perfringens. While Etx has been shown to be the key virulence factor for enterotoxemia in goats and sheep, NetB has been associated with the pathogenesis of avian necrotic enteritis (NE), a gastro-intestinal disease causing economic damage to the poultry industry worldwide. The crystal structure of Etx H149A (an Etx variant with 6x reduced toxicity relative to wild type toxin) was solved to 2.4 Å and showed that the H149A mutation in domain III does not affect organization of the receptor binding region in domain I. The Etx H149A structure also revealed a second putative glycan binding site in domain III. In addition, site-directed mutagenesis in domain I of Etx H149A affirmed the important role of tyrosine residues for toxin binding and demonstrated the capability of Etx H149A to be used as a platform for further receptor binding studies in the future. The crystal structure of the pore-form of NetB was solved to 3.9 Å and revealed high similarities to the Staphylococcus aureus α-hemolysin heptameric structure. However, in particular the region thought to interact with the target cell membrane showed some interesting divergence in amino acid composition. Site-directed mutagenesis within this domain significantly affected binding and toxicity of NetB to target cells. Mutagenesis within the β-sandwich domain of NetB revealed important amino acid residues for toxin oligomerisation and pore-formation. In order to test NetB toxoids as candidate vaccines, a NetB genetic toxoid and a formaldehyde NetB toxoid were used to immunise poultry in an in vivo NE disease model. Vaccination with any of the two antigens resulted in the induction of specific antibody responses against NetB and provided significant protection against disease.
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Balmforth, Matthew Royce. "Piggybacking on the cholera toxin : using cholera toxin B chain for the targeted delivery of proteins to motor neurones." Thesis, University of Leeds, 2017. http://etheses.whiterose.ac.uk/20115/.
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A significant unmet need exists for the delivery of biologics to the central nervous system for the treatment and understanding of neurodegenerative diseases. Naturally occurring toxoids such as the non-toxic B subunit of the cholera toxin have been considered as tools to meet this need. However, due to the complexity of tethering macromolecular drugs to toxins, and the inherent dangers of working with large quantities of recombinant toxin, no such route has been successfully exploited. Developing a method where toxoid and drug can be assembled immediately prior to administration could therefore be extremely useful. Using phage-display, two cholera toxin-binding antibody mimetics (Affimers) were identified that non-covalently associate with the non-GM1 binding face of the cholera toxin B subunit (CTB). The two unique interactions were characterised using a range of techniques to dissect the Affimer-CTB assembly process. Internalisation of the complex was demonstrated in tissue culture, and the system was used to deliver GFP to mammalian cells. Finally, the complex was shown to be successfully internalised into the motor neurones of the brainstem in a mouse model. A second route to modular assembly of a protein delivery system was also explored. By using a high affinity peptide staple, a cholergenoid-botulinum toxin chimera was produced that could be assembled in vitro and used to deliver the functional catalytic domain of botulinum neurotoxin to cultured neuronal cells.
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Flagler, Michael J. "Determination of the Molecular Basis for the Difference in Potency between Shiga Toxins 1 and 2." Cincinnati, Ohio : University of Cincinnati, 2010. http://rave.ohiolink.edu/etdc/view.cgi?acc_num=ucin1267131436.
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Thesis (Ph.D.)--University of Cincinnati, 2010.Advisor: Alison A. Weiss. Title from electronic thesis title page (viewed Apr. 26, 2010). Keywords: Shiga toxin; Shiga-like toxin; Verotoxin; Verocytotoxin; B-subunit; B-pentamer. Includes abstract. Includes bibliographical references.
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檀東煇 and T. F. Tan. "Elucidation of ganglioside binding domain in the B-subunit of cholera toxin." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2000. http://hub.hku.hk/bib/B31223448.
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Tan, T. F. "Elucidation of ganglioside binding domain in the B-subunit of cholera toxin." Hong Kong : University of Hong Kong, 2000. http://sunzi.lib.hku.hk/hkuto/record.jsp?B23636634.
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Batisse, Cornélie. "Targeting strategies using B-subunit of Shiga toxin : innovative drug-delivery systems." Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015USPCB220.
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Les stratégies thérapeutiques mises en place contre le cancer ont de nos jours besoin de nouveaux médicaments, à la fois plus actifs que ceux déjà existants et induisant moins d’effets secondaires. Ces nouvelles stratégies visent à cibler spécifiquement les cellules cancéreuses. Parmi ces stratégies, ces travaux de thèse concernent la vectorisation active, à l’aide d’un vecteur protéique dérivé de la toxine de Shiga, STxB. STxB reconnait spécifiquement son récepteur biologique Gb3, surexprimé à la surface des cellules cancéreuses humaines. Ce projet de recherche porte sur la conception et la synthèse de conjugués, combinant STxB et un agent cytotoxique. Le linker chimique, qui relie ces deux espèces, a été soigneusement conçu pour respecter les deux critères suivants : être suffisamment stable et néanmoins pouvoir être clivé pour libérer l’agent cytotoxique une fois les cellules cancéreuses atteintes. Un premier linker a été construit autour du motif mercaptoethanol, lié au vecteur STxB par une liaison disulfure. La libération de l’agent cytotoxique peut donc être initiée par un réducteur biologique comme le glutathion, puis par une étape d’auto-immolation. Ce linker a été appliqué à deux composés cytotoxiques très puissants, dérivés de l’auristatine, et a conduit à des résultats prometteurs in vitro. La labilité de la liaison ester à pH acide a également été mise à profit dans l’élaboration de deux linkers, conçus autour de motifs glutamate et thréoninate. L’utilisation d’un agent cytotoxique modérément puissant a été l’occasion de développer une stratégie de multivalence, consistant à augmenter la charge d’agents cytotoxiques sur STxB. Une autre option a été de considérer les nano-batônnets d’or comme une plate-forme nanométrique multimodale, capable de lier plusieurs milliers d’agents cytotoxiques et STxB. Enfin l’incorporation d’une séquence peptidique, connue pour être substrat d’une protéase, a donné lieu à une troisième étude, reposant sur un linker clivable plus sélectivement. Plusieurs linkers ont été étudiées, selon qu’ils libèrent l’agent cytotoxique sous sa forme native ou nonWe need new therapeutic strategies to treat cancerous patients by the discovery of new drugs that would be more active than those existing and especially assigning fewer side effects. These new therapies aim to specifically target cancer cells. Among the strategies for cancer targeting, we investigated drug-targeted strategies using a proteic carrier, STxB, derived from Shiga toxin. This protein recognizes specifically its biological receptor Gb3, which is over-expressed on human cancer cells. This work consisted in the design and synthesis of conjugates combining STxB and a cytotoxic drug. The chemical linker binding these two moieties was carefully designed in order to fit requirements of both stability and ability to trigger a drug-delivery. A first linker was designed around a mercaptoethanol core, able to be conjugated to STxB by a disulfide bond. This constitutes a drug-delivery trigger, activated by a biological reducing agent such as glutathion, and followed by a self-immolative step. Two highly potent conjugates of auristatin derivatives were obtained and showed promising results in vitro. The ester bonds lability in acidic pH was exploited for the design of two amino acid based linker. With the aim of increasing the ratio of drug on STxB, we investigated several multivalent linkers. Another option was to consider gold nanorods as a nanometric platform, able to carry thousands of drugs and STxB. The incorporation of a protease substrate to produce an enzyme-cleavable linker was investigated. Several spacers, which induced release of the drug under native form or under prodrug form, were designed and tested
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Lipscombe, Martin John. "Construction and characterisation of Escherichia coli heat-labile toxin B-subunit fusion proteins." Thesis, University of Warwick, 1991. http://wrap.warwick.ac.uk/108070/.
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A plasmid vector was constructed which allowed for the in-frame insertion of peptide-encoding sequences at the 3’ terminal of the Escherichia coli (E. coli) heat-labile enterotoxin B-subunit (LT-B) structural gene. Several synthetic oligonucleotides, encoding various B and T cell epitopes from other proteins, were ligated into this vector. The sequence across the junctions of these novel plasmid constructs was determined and found to be as predicted. The chimeric fusion proteins expressed by these constructs were characterised in vitro by SDS-PAGE, Western blotting and GM1-linked ELISA. All the fusion proteins were shown to behave like native LT-B in that they were transported to the periplasmic space when expressed in E. coli. In addition they formed pentamers which dissociated into their constituent monomers upon boiling. Furthermore, the pentameric forms were found to retain G,,,-binding properties as determined by G,,,-linked ELISA. Some of these plasmids, expressing LT-B fusion proteins containing T cell epitopes, were transferred into an aromatic-dependent attenuated strain of Salmonella typhlmurium SL1344, and these strains were used to inoculate mice. A weak serum antibody response to one of these epitopes was demonstrated. However, a consistent in vitro T cell response to these epitopes could not be detected. Another of the fusion proteins, termed LT-B69, was partially purified by ion-exchange chromatography and used to inoculate mice intranasally. Mice immunised in this way developed serum antibodies against LT-B and P.69 (an important Bordetella pertussis antigen). Additionally, LT-B-specific and P.69-specific antibody secreting cells could be detected in their lungs. There was some evidence to suggest that these mice were slightly protected against colonisation by B. pertussis after an aerosol challenge with live organisms, compared to the levels of colonisation in a control group.
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Schneider, Olivia Dawn. "An Analysis of the Effects of Pertussis Toxin on T Cell Signaling." University of Cincinnati / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1258667926.
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Péré-Védrenne, Christelle. "Etude de la Cytolethal Distending Toxin B des Hélicobacters dans l’inflammation et la carcinogenèse digestive." Thesis, Bordeaux, 2015. http://www.theses.fr/2015BORD0404/document.
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La démonstration du rôle de la CDT (« Cytolethal Distending Toxin ») de Helicobacterhepaticus dans le développement de l’hépatocarcinome murin fait de cette toxine un candidatpertinent dans l'activation de processus pro-cancéreux. Comme la toxine CagA de Helicobacterpylori, la sous-unité active CdtB de la CDT pourrait être une oncoprotéine. Nous avons étudié lerôle de la CdtB des Hélicobacters dans l’inflammation et la carcinogenèse digestive via unestratégie lentivirale d’expression constitutive ou conditionnelle de la CdtB ou de son mutant pourl’activité DNase. Nous avons réalisé une étude du transcriptome et montré que la CdtB deH. hepaticus induisait une réponse inflammatoire en surexprimant des cytokines, chimiokines,peptides antimicrobiens et en activant la voie du NF-κB des cellules épithéliales. La CdtB réguleégalement l’expression et la localisation nucléaire du facteur de transcription et oncogène MafB.Ces résultats ont été confirmés pour la CdtB de Helicobacter pullorum. Des expériencesd'infection des cellules avec des souches sauvages et mutées pour la CDT (deH. hepaticus & H. pullorum) ont permis de valider les résultats obtenus et de les attribuer à laCdtB et notamment à son activité DNase. Nous avons aussi développé un nouveau modèle dexénogreffes de cellules épithéliales inductibles pour l’expression de la CdtB de H. hepaticus.Dans ce modèle, la CdtB, en plus de ses effets déjà connus, retarde la croissance tumorale,induit l’apoptose, la sénescence et la surexpression du marqueur nucléaire de prolifération,Ki-67, suggérant la survie cellulaire. L’ensemble de ces résultats fournit de nouveaux argumentsen faveur du potentiel oncogénique de la CDTThe demonstration of the role of the Cytolethal Distending Toxin (CDT) of Helicobacter hepaticusin the development of hepatocarcinoma in mice, makes this toxin a relevant candidate in theactivation of precancerous processes. As in the case of the CagA toxin of Helicobacter pylori, theCdtB active subunit of CDT could be an oncoprotein. We studied the role of Helicobacter CdtB ininflammation and digestive carcinogenesis using a lentiviral strategy for constitutive or conditionalexpression of the CdtB subunit or its corresponding DNase mutant. We conducted a study of thetranscriptome and showed that CdtB induced an inflammatory response by overexpressingcytokines, chemokines, antimicrobial peptides and activating the NF-kB pathway in epithelialcells. The CdtB also regulated the expression and nuclear localization of the transcription factorand oncogene MafB. These results were confirmed for the CdtB of Helicobacter pullorum.Infection of cells with wild type strains and the corresponding CDT-mutant strains (of H. hepaticus& H. pullorum) were used to validate the results and to attribute the effects to the CdtB and, inparticular, to its DNase activity. We also developed a novel epithelial cell xenograft model toevaluate the inducible expression of H. hepaticus CdtB. In this model, the CdtB, in addition to itspreviously well-known effects, delayed tumor growth, induced apoptosis, senescence and theoverexpression of nuclear proliferation marker, Ki-67, suggesting cell survival. All of these resultsprovide new arguments in favor of the oncogenic potential of the CDT
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Kalluri, Anila. "EXPRESSION OF CHOLERA TOXIN B SUBUNIT-ROTAVIRUS NSP4 ENTEROTOXIN FUSION PROTEIN IN TRANSGENIC CHLOROPLASTS." Master's thesis, University of Central Florida, 2005. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/3069.
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Rotavirus, the major cause of life-threatening infantile gastroenteritis, is a member of the Reoviridae family and is considered to be the single most important cause of virus-based severe diarrheal illness in infants and young children particularly 6 months to 2 years of age in industrialized and developing countries. Infection in infants and young children is often accompanied by severe life threatening diarrhea, most commonly following primary infection. Diarrhea is the major cause of death among children around the world. Responsible for 4 to 6 million deaths per year according to the World Health Organization (WHO), diarrhea is especially dangerous for infants and young children. Globally, it is estimated that 1.4 billion episodes of diarrhea occur in children less than five years of age annually. In the United States alone, rotavirus causes more than 3 million cases of childhood diarrhea each year, leading to an estimated 55,000 to 100,000 hospitalizations and 20 to 100 deaths. And is a major cause of mortality for children in developing countries with approximately one million deaths annually. Rotaviruses belong to the family Reoviridae and are spherical 70-nm particles. The virus genome contains 11 segments of double-stranded RNA, each encoding a viral capsid or nonstructural protein. The identification of a rotavirus nonstructural protein gene (NSP4) encoding a peptide, which functions both as a viral enterotoxin and as a factor involved in the acquisition of host cell membrane during virus budding from cells, provides a new approach for mucosal immunization. Protein expression through chloroplast transformation system offers a number of advantages like high level of transgene expression, transgene containment via maternal inheritance, lack of gene silencing and position effect due to site specific gene integration and also the possibility of multi gene engineering in single transformation event. It is also an environmentally friendly approach due to effective gene containment and lack of transgene expression in pollen. To achieve an enhanced immune response to rotavirus infection, a fusion gene encoding the cholera toxin B subunit linked to rotavirus enterotoxin 90 aa protein (CTB-NSP490) was introduced into transgenic chloroplast and was transformed into chloroplast genome of Nicotiana tabacum by homologous recombination. The chloroplast integration of CTB-NSP4(90) fusion gene was confirmed in transgenic tobacco plants by PCR analysis. Southern blot analysis further confirmed site specific gene integration and homoplasmy. Immunoblot analysis of transformed chloroplast confirmed the expression of CTBNSP490 fusion protein both in monomeric and pentameric forms that retained the binding affinity to the enterocytes GM1 ganglioside receptor. Expression levels of CTB-NSP4 protein was quantified by GM1 ganglioside binding ELISA assay; mature leaves expressed CTB-NSP4 fusion protein to upto 2.45 % in total soluble protein, 100-400 fold higher than nuclear expression which was only 0.006%-0.026%. Antibody titration and virus challenge experiments will be performed in mice at Loma Linda University to evaluate the antigenic and protective properties of the chloroplast derived CTB-NSP4 fusion protein.M.S.
Department of Molecular Biology and Microbiology
Burnett College of Biomedical Sciences
Molecular Biology and Microbiology
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Maisey, Elizabeth Anne. "Botulinum toxin types A and B : isolation and biological characterisation of their polypeptide chains." Thesis, Imperial College London, 1990. http://hdl.handle.net/10044/1/46426.
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Eswaran, Jeyanthy. "Purification and characterisation of recombinant C. perfringens beta toxin from E. coli and B. subtilis." Thesis, University of Newcastle Upon Tyne, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341444.
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Murarasu, Thomas. "The Shiga Toxin B-Subunit : a Promising Scaffold for the Targeting of Tumor Specific Glycosphingolipids." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS512.
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Le cancer représente la second cause de décès au monde. Le développement de traitements innovants contre le cancer repose aujourd’hui sur l’identification de biomarqueurs des tumeurs et le développement de produits thérapeutiques capables de reconnaitre ces marqueurs de façon spécifique. Ces produits thérapeutiques de nouvelles générations ont le potentiel d’éliminer spécifiquement les cellules tumorales et donc de réduire les effets secondaires des traitements ainsi que les risques de rechute. Malheureusement, un certain nombre de patients ne peuvent bénéficier de ces traitements, du fait de l’absence de biomarqueurs connus à la surface de leur tumeur. Ce projet a ainsi pour ambition de développer de nouvelles thérapies ciblées en exploitant une nouvelle classe de biomarqueurs et ainsi de venir enrichir l’arsenal thérapeutique disponible pour le traitement des cancersCancer is the second cause of death worldwide. Recent advance in cancer treatments involved the identification of cancer biomarkers and the development of efficient therapeutic products able to specifically recognize them. This new class of products has the ability to specifically target tumor cells, with the major advantages to decrease or abolish treatments side effects and relapses of the disease. Unfortunately, a certain number of patients do not respond to those treatments lacking the expression of those biomarkers on their tumor. This project aims at developing new targeted therapies by exploiting a new class of cancer biomarkers, which would potentially extend the therapeutics options against cancer
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Johnson, Nicholas. "Construction of a novel epitope expression vector based on the B-subunit of the diphtheria toxin." Thesis, University of Southampton, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296057.
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Martin, Daniel Dalton. "Purification of Anthrax Toxin Protective Antigen Component and Characterization of its Binding Interaction with Bovine Kidney Cells." DigitalCommons@USU, 1986. https://digitalcommons.usu.edu/etd/4641.
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Protective antigen component of B. anthracis toxin was produced and purified to the >99% level. Toxin was purified from culture supernatant utilizing concentration and liquid chromatography techniques. Purity was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
The purified protective antigen retained biological and antigenic activity as evidenced respectively by lethality in Fischer 344 rats when injected in combination with lethal factor, and by positive results on the Ouchterlony double diffussion assay.
Radioiodinated protective antigen was used both in the in vivo and the in vitro experiments. In vivo distribution of labelled protective antigen was determined in Fischer 344 rats. Assay of organ tissues for labelled protective antigen aided in the decision to use Maden-Darby bovine kidney cells for the cell cultures in the protective antigen binding studies.
Protective antigen binding studies, all performed at 37°C, evaluated criteria for receptor existence. Labelled protective antigen was found to bind specifically and reversibly to Maden-Darby bovine kidney cells. Receptors proved to be saturable. Scatchard analysis showed a relatively high dissociation constant (KD= 17 X 10-9M) compared to other toxins in similar studies. This indicated moderately low affinity for protective antigen.
The receptor was also partially characterized. It was shown that cholera toxin subunit B blocked the binding of labelled protective antigen to Maden-Darby bovine kidney cells and that the protective antigen receptor was insensitive to trypsin treatment. Both of these observations suggest a ganglioside as the receptor for protective antigen.
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Rosselot, Andrew E. "Ontogeny of the intestinal circadian clock and its role in the response to Clostridium difficile toxin B." University of Cincinnati / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1573222475839068.
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Thorsell, Mikaela. "Evaluation of C. diff Quik Chek Complete® and comparison with GeneXpert to establish a new diagnostic algorithm." Thesis, Uppsala universitet, Institutionen för kvinnors och barns hälsa, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-390609.
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Clostridium difficile is the most common antibiotic related diarrhéa disease in Sweden. New recommendations from the Swedish public health authority and European Society of Clinical Microbiology and Infectious Diseases (ESCMID) had led to that a more advanced diagnostic algorithm is of priority. Hence this study, whose purpose was to investigate whether the performance of the rapid test C. diff Quik Chek Complete® could enable the introduction of a new diagnostic algorithm for detection of toxin-forming C. difficile in laboratory medicine in Sundsvall, according to these new recommendations. In the study 119 patient stool-samples were analysed with both GeneXpert and C. diff Quik Chek Complete® and these two combined fulfils these new recommendations of detecting toxin A and B from toxigenic C. difficile together with the enzyme Glutamate Dehydrogenase (GDH) which is produced by all C. difficile stems. The results shows that C. diff Quik Chek Complete® is well matched with GeneXpert and that most of the samples would come to be answered immediately after analysis with C. diff Quik Chek Complete®. The laboratory will save both time and money to establish C. diff Quik Chek Complete® in their algorithm for diagnosing C. difficile infection.
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Adam-Castrillo, David. "Local Administration of Botulinum Toxin Type-B in the External Anal Sphincter of Horses Produces Transient Reduction of Peak Anal Pressure." Thesis, Virginia Tech, 2003. http://hdl.handle.net/10919/33927.
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Toxins produced by the Gram-positive bacteria Clostridium botulinum cause transient chemodenervation of mammalian muscle. The toxin binds to specific proteins within cholinergic presynaptic nerve terminals which regulate the release of acetylcholine in the synaptic space resulting is loss of muscle activation and function.
Local injections with botulinum toxins are currently used in humans for the treatment of disorders that benefit from prolonged neuromuscular blockade such as strabismus, blepharospasm, focal dystonias, spasticity, tremors, and anal fissures. Injections with botulinum toxin type A into the internal or external anal sphincter cause relaxation of the anal canal and allow healing of chronic anal fissures.
Perineal lacerations in mares, which occur during foaling often dehisce after surgical repair due to the high pressure across the incision resulting from accumulation of feces in the rectum. We hypothesized local injections of Clostridium botulinum type B toxin into the external anal sphincter could cause a decrease in anal pressures, thus reducing the incidence of dehiscence if used before surgical repair of perineal laceration in mares.
The purpose of this project was to determine the effects of BTB injection in the external anal sphincter in normal horses. Our hypothesis was that local injection of BTB would result in transient reduction of anal tone without causing clinical side effects. Peak and resting anal sphincter pressures of horses were measured with a custom made rectal probe connected to a pressure transducer. Pressures were measured before treatment and after injection with Clostridium botulinum type B toxin (BTB) or saline. Dose titration with 500, 1000, 1500 and 2500 units of BTB was completed. The horses' physical changes, behavior, and anal pressure were recorded. Injection of 1000 units of BTB produced significant reduction in peak anal pressure from days 2 to 84 when compared to control animals (P<0.05). Maximal effect of the toxin was observed within the first 15 days after injections followed by a slow return to baseline over 168 days. Injection in the anal sphincter with 2500 units of BTB in one horse produced signs of depression, generalized weakness, and dysphagia for 14 days. Clinical side effects were not observed in horses after injections with 500, 1000, or 1500 units of BTB.
In summary, local injections of botulinum toxin type-B in the external anal sphincter of horses caused transient relaxation of the anus and reduction of peak anal pressures. Systemic side effects were observed in one horse, which suggested a narrow dosage range to avoid toxicity. Further research to test the effects of botulinum toxin in clinical cases is needed to determine the full potential of this treatment modality.Master of Science
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Gomez, Sheena Robin. "Investigation of pertussis toxin A- and B-subunit activities in acellular vaccines by enzymatic and carbohydrate-binding assays." Thesis, University of Glasgow, 2007. http://theses.gla.ac.uk/40959/.
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Pertussis toxin (PT) is a major virulence factor produced by Bordetella pertussis. In its detoxified form (PTd), it is an important component of acellular pertussis vaccines although some residual FT activity may be present because of the limitations of the detoxification processes used. The in vivo histamine sensitisation test (HIST) in mice is currently used for the safety testing of these vaccines to determine the level of their residual FT activity. However, an alternative test is needed because of large assay variability and ethical concerns with regard to animal usage. The main objective of this study was to search for an alternative test to the HIST. The ADP-ribosylation enzyme activity of FT is thought to be the major factor responsible for the histamine-sensitising activity detected in vivo. In the present study, the enzymatic activities in different acellular pertussis-based combination vaccine formulations were measured by a recently-developed ADF-ribosylation assay and compared with their reactivities in the HIST. The results indicated that different products showed differences in ADP-ribosylation activity and, these did not correlate with their reactivity in the HIST. FT has two functionally-distinct domains: the enzymatic A-protomer and the B- oligomer that facilitates host-cell binding and entry of FT into the cell. This dual biological function could explain why the residual enzyme activity of FT in vaccines did not fully reflect the in vivo reactivity observed by the HIST. Thus, refinement of the in vitro test to include a step which monitored the B-subunit activity of FT was attempted. A quantitative FT carbohydrate-binding assay using glycoproteins or defined oligosaccharides was developed. PT was found to bind preferentially to multiantennary N-glycans, with the highest binding towards the fully sialylated structures. In contrast, PTd lost the ability to bind to sialylated multiantennary structures but retained some capacity to bind to neutral multiantennary structures. Different vaccine preparations had different levels of PT binding activity as well as enzymatic activity. It was concluded that, although the enzymatic activity of PT plays a more important role in the death of mice in the HIST, a high binding activity of the B- subunit could increase the in vivo toxic effect by aiding the accessibility of the A- subunit to its cellular targets. A mathematical equation was devised to establish a preliminary relationship between the enzymatic, carbohydrate-binding and HIST assays in a product-dependent manner. Further studies with a larger number of vaccines are required for a more meaningful statistical analysis. However the methods form a sound basis for the future development of an alternative assay to the histamine challenge test. The in vitro assays could also be useful for investigating the mechanisms of PT detoxification. Comparisons of A- and B-subunit activities of purified PT and vaccine preparations of PTd indicated that both subunits are modified after chemical detoxification. Different vaccine products had different levels of enzymatic and binding activities and it was concluded that different detoxification procedures, as well as formulation factors, could contribute to this variation. A CHO cell clustering assay is used as an alternative in vitro test to the HIST for assessing residual PT activity at the bulk stage of vaccine production. In a parallel study to the above, comparative proteomics was used to gain insights into the mechanism of PT-induced CHO cell clustering with a view to developing a mechanistic-based alternative assay for the safety testing of pertussis-based combination vaccines. A proteomic map of CHO cells was established and PT-induced CHO cell clustering appeared to be a complex process involving subtle changes in various cellular functions, mainly related to intracellular transport, cell stress and the cell cycle. The information obtained will be useful for future studies into the possible mechanisms of the effect of PT on CHO cells.
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Ghei, M. "Effects of Botulinum toxin B on refractory detrusor overactivity : a randomised, double-blind, placebo controlled, cross over trial." Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1444297/.
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INTRODUCTION: Open, observational studies of intradetrusor injections of Botulinum toxin for detrusor overactivity have reported beneficial effects. This thesis reports the testing of the efficacy and safety of Botulinum toxin B (BTX-B) for treatment of the overactive bladder in a randomised, double-blind, placebo controlled cross-over trial.;METHOD: 20 patients, aged 18-80 years, with detrusor overactivity, and incompletely responsive to oral antimuscarinic agents, participated. They were injected with either placebo (20 mis normal saline) or botulinum toxin B (5000 IU diluted up to 20 mis) into the detrusor in a day case setting. After six weeks, the treatments were crossed over without washout. The primary outcome was the paired difference in change in the average voided volumes. Paired differences urinary frequency, incontinence episodes and in the quality of life (QOL), measured by the King's Health Questionnaire (KHQ), were the secondary outcome measures. On the occasion of each injection two biopsies were taken from the detrusor. These were processed and scrutinised for evidence of inflammatory responses to the injection.;RESULTS: The Wilcoxon Signed Ranks Test was used to test the paired difference in change between treatment phases. Statistically significant paired differences in the change in average voided volume, urinary frequency and episodes of incontinence between active treatment and placebo (Av Void Vol: 95% CI diff 16, 122 Z = -2.5 p=0.012 / Weekly Freq: 95% CI -21, -1 Z = -2.1, p=0.033 / Weekly incont: 95% CI -26, -7 Z=-3.3 p=0.001) along with significant paired differences in five domains of the KHQ were observed.;CONCLUSIONS: This double-blind, placebo-controlled, cross-over study provides evidence of efficacy of Botulinum Toxin B in the treatment of the overactive bladder. Autonomic side effects were observed in four of the patients. The short duration of action will presumably limit the use to patients who have experienced tachyphylaxis with (Botulinum toxin A) BTX-A.
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Rahn, Sarah Jane. "Allergy models and related assays to test the allergic qualities of Escherichia coli heat labile toxin subunit B." [Ames, Iowa : Iowa State University], 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:1468126.
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Ayache, Alexandra. "Amyloid-beta42 toxicity reduction in human neuroblastoma cells using cholera toxin b subunit-myelin basic protein expressed in chloroplasts." Honors in the Major Thesis, University of Central Florida, 2012. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/1535.
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Alzheimer's disease (AD) is an age progressive neurodegenerative brain disorder, affecting 37 million people worldwide. Cleavage of amyloid precursor protein by ?- and ?-secretase produces the amyloid-beta (A?) protein, which significantly contributes to AD pathogenesis. The A? aggregates, formed at the surface of neurons and intracellularly, cause neurotoxicity and decrease synaptic function. Inhibiting or degrading A? accumulation is a key goal for development of new AD treatments. Evidence shows that human Myelin Basic Protein (MBP) binds to and degrades A? thereby, preventing cytotoxicity. A potential method for oral drug delivery that will allow plant-derived bioencapsulated MBP to pass through intestinal epithelium and bypass denaturing stomach acidity is quite novel. Cholera Toxin B subunit (CTB), when fused with MBP, can serve as a vehicle for oral delivery of this chloroplast expressed therapeutic protein into the systemic circulation. Within chloroplast, CTB forms a pentameric structure that binds to GM1 ganglioside receptors, allowing receptor-mediated endocytosis. In order to investigate protein entry through neuronal GM1 receptors, we first created CTB fused to the green fluorescent protein (GFP). Incubation of this fusion protein with human neuroblastoma cells resulted in GFP entry into these cells whereas GFP alone was unable to enter. Similarly, co-incubation of CTB-MBP, via neuronal GM1 binding, allowed MBP to reduce neurotoxicity of A?42 treated cells by 37.1%. Delivery of CTB-MBP through GM1 receptor mediated binding should therefore facilitate oral administration, storage, heat stability and low cost AD treatment.ID: 031908400; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Accepted in partial fulfillment of the requirements for honors in the major in DEPT HERE.; Thesis (B.A.)--University of Central Florida, 2012.; Includes bibliographical references.
B.S.
Bachelors
Burnett School of Biomedical Sciences
Molecular Biology and Microbiology
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Aman, Abu Tholib. "Mutagenesis of a conserved loop in the B subunit of cholera toxin : identification of residues essential for toxicity and immunomodulation." Thesis, University of Bristol, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.311431.
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Dugyala, Raviprakash R. "Alteration of Key Cytokine Levels by Aflatoxin B1 and T-2 Toxin in Male CD-1 Mice." DigitalCommons@USU, 1995. https://digitalcommons.usu.edu/etd/4653.
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Aflatoxin B1 and T-2 toxin are mycotoxins, which produce their immunotoxic effects by affecting nonspecific and acquired immunity in different species. The mechanisms of their immunotoxicity are still obscure. Cytokines are the key signaling molecules during the immune response. In this study, expression of macrophage-produced cytokines Interleukin-lα (IL-lα), tumor necrosis factor (TNF), and IL-6, and lymphocyte-produced cytokines IL-2, interferon y (IFNy), and IL-3 was measured at the mRNA and protein levels, after in vitro activation with mitogens in AFB1-and T-2-toxin-exposed mice.
Significant changes in the organ weights, especially in the mice exposed to a high dose of T-2 toxin, and no effect in AFB1-exposed mice were observed.
ConA-induced production of IL-2, IFNy, and IL-3 mRNA and protein levels in AFB1-exposed mice showed a decrease in low dose groups (significant for IL-2 mRNA), but no change at other doses. However, in T-2-toxin-treated animals, there was a significant induction of IL-2 and IFNy mRNA in high and low doses and of IL-3 mRNA at the medium dose. The protein levels of IL-2 and IFNy did not follow the mRNA levels in high dose and the protein levels of IL-3 were significantly increased in medium and low doses.
LPS-induced IL-lα and TNF mRNA and protein levels in AFB1-exposed mice were suppressed at the high dose while mRNA levels of both cytokines were increased significantly in the low and medium doses. Low and medium doses of AFB1 also significantly decreased IL-lα protein levels and the high dose decreased IL-6 protein. In T-2 toxin-treated mice, no significant difference in mRNA levels of these cytokines was observed but a general pattern of significant suppression of their protein levels (except IL-lα at medium dose) showed that both toxins regulate the cytokine expression differently.
Based on the above discussed results and others, AFB1 may alter cell-mediated immunity by affecting the communication between macrophages and T lymphocytes through inhibiting the macrophage-producing cytokines. T-2 toxin-induced immunosuppression may be due not only to the inhibition of macrophage-producing cytokines, but also to the lack of effector cells to respond to the cytokines (IL-2, IFNy, and IL-3).
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Nityanandam, Ramya. "Expression and functional evaluation of exendin 4 fused to cholera toxin B subunit in tobacco chloroplast to treat type 2 diabetes." Master's thesis, University of Central Florida, 2011. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4815.
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The prevalence of type 2 diabetes has been steadily increasing around the globe. Glucagon like peptide (GLP-1), a powerful incretin increases insulin secretion in a glucose dependent manner. But GLP-1 is subjected to rapid enzymatic degradation (half-life: 2 min in circulation). The commercially available GLP-1 analog, exenatide has a longer half life with potent insulinotropic effects (about 2.4 hr) which requires cold storage and daily subcutaneous injections. In this study, exendin 4 (EX4), lizard derived GLP-1R agonist, was expressed as cholera toxin B subunit (CTB)-fusion protein in chloroplasts of tobacco to facilitate transmucosal delivery in the gut by utilizing the ability of CTB pentamer to bind the GM1 receptors on the intestinal epithelium and to bioencapsulate EX4 within plant cells to confer protection in the digestive system. The LAMD tobacco leaves were bombarded with chloroplast vectors expressing modified EX4. The transgene integration was confirmed by PCR analysis and Southern blot analysis. Densitometric analysis revealed expression level of the protein varied from 9-13% of the total leaf protein depending on the developmental stage and time of harvest. The pentameric structure and functionality of CTB-EX4 fusion protein was confirmed by CTB-GM1 binding assay. The effect of transplastomic protein on insulin secretion was tested in beta]-TC6, a mouse pancreatic cell line. The plant derived CTB-EX4, partially purified with anti-CTB antibody conjugated protein A beads, showed the increase of insulin ~ 2.5 fold increase when compared to untreated cells. The transplastomic protein showed a linear increase in insulin secretion comparable to the commercially available EX4. The current cost of treatment with EX4 varies between $1800-$2200, annually. Production of functional EX4 in plants should facilitate low cost orally deliverable form of this drug for treatment of type 2 diabetes.ID: 031001317; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Title from PDF title page (viewed March 26, 2013).; Thesis (M.S.)--University of Central Florida, 2011.; Includes bibliographical references (p. 35-40).
M.S.
Masters
Molecular Biology and Micro
Medicine
Biotechnology
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Bast, Darrin James. "Three biologically significant globotriasylceramide binding sites on the Verotoxin 1 B subunit, implications in toxin action, pathogenesis of disease and vaccine design." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ35106.pdf.
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Ando, Yoshikazu. "Inactivation of Rho GTPases with Clostridium difficile toxin B impairs centrosomal activation of Aurora-A in G2/M transition of HeLa cells." Kyoto University, 2008. http://hdl.handle.net/2433/135856.
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Aliprandini, Eduardo. "Obtenção de anticorpos monoclonais humanos antitetânicos." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-04122015-141425/.
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Anticorpos monoclonais (AcMos) para uso terapêutico correspondem a uma área importante na indústria de biofármacos, em especial os AcMos humanos, que apresentam menor probabilidade de elicitar imunogenicidade. O objetivo deste trabalho consistiu em obter AcMos humanos antitetânicos através da separação de linfócitos B produtores de anticorpos específicos utilizando o antígeno ou de plasmablastos. As células foram coletadas de doadores após vacinação e separadas por equipamento de cell sorter. As regiões variáveis dos anticorpos foram amplificadas e clonadas em vetores de expressão, que foram usados para transfectar transitoriamente células HEK293-F. O uso da toxina tetânica conjugada independentemente com dois marcadores, biotina e Alexa Fluor® 647, possibilitou a separação específica de linfócitos B produtores de AcMos antitetânicos, que foram avaliados por ELISA, western blotting e pela inibição da ligação da toxina ao gangliosídio GT1b. O ensaio in vivo mostrou proteção total dos animais contra a toxina tetânica quando três AcMos foram usados em conjunto.Monoclonal antibodies (mAbs) for therapeutic use correspond to a major area of the biopharmaceutical industry, especially human mAbs that are less prone to elicit immunogenicity. The objective of this work was to obtain anti-tetanus human mAbs through separation of memory B lymphocytes producing specific antibodies stained with the antigen or plasmablasts. Cells were collected from peripheral blood of donors after vaccination and separated through cell sorting. The variable regions of the antibodies were amplified and cloned in expression vectors for transient transfection of HEK293-F cells. The staining with the tetanus toxin labeled independently with two markers, biotin and Alexa Fluor® 647 allowed the separation of specific B lymphocytes producing anti-tetanus mAbs. The antibodies expressed were evaluated by ELISA, western blotting and the inhibition of the binding of the tetanus toxin to the ganglioside GT1b. The in vivo neutralization assay showed that a pool of three different mAbs were able to protect mice against the tetanus toxin.
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Fühner, Viola [Verfasser], Michael [Akademischer Betreuer] Hust, and Stefan [Akademischer Betreuer] Dübel. "Development of neutralizing and non-neutralizing antibodies targeting known and novel epitopes on Clostridioides difficile Toxin B / Viola Fühner ; Michael Hust, Stefan Dübel." Braunschweig : Technische Universität Braunschweig, 2020. http://d-nb.info/1203299036/34.
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Kapzan, Ruth [Verfasser], Gernot [Akademischer Betreuer] Längst, and Ralf [Akademischer Betreuer] Wagner. "Generierung neuer HIV-1 Impfstoffkandidaten zur Induktion breit neutralisierender Antikörper mit Cholera Toxin B als Trägerprotein / Ruth Kapzan. Betreuer: Gernot Längst ; Ralf Wagner." Regensburg : Universitätsbibliothek Regensburg, 2012. http://d-nb.info/1024198928/34.
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Fraser, Sylvia A. "The role of GM1-binding in mediating the immunomodulatory properties of the B subunits of cholera toxin and Escherichia coli heat-labile enterotoxin." Thesis, University of Bristol, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391162.
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Frazão, Renata. "Análise citoarquitetônica e imunoistoquímica de estruturas do sistema visual de macacos-prego (Cebus apella)." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/42/42131/tde-08092008-105313/.
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O estudo do sistema visual de macacos-prego representa importante questão devido ao aspecto evolutivo que a espécie apresenta. Foram utilizados cinco macacos-prego, 2 kg. Foi efetuda injeção intra-ocular de 100 ml de solução aquosa de toxina colérica subunidade B (CTb) a 1%, sendo a perfusão realizada 15 dias após a injeção intra-ocular. As retinas intactas e os encéfalos foram submetidos à procedimento de imunoistoquímica para análise. A caracterização da retina evidenciou dois tipos distintos de células bipolares, além disto, subunidades de receptores gabaérgicos co-localizam em retinas de macacos-prego, diferente dos resultados apresentados em outras espécies. As projeções retinianas foram observadas em todas as estruturas do sistema visual primário, óptico acéssório e de temporização circadiana, além de projeções para áreas adicionais. Os resultados evidenciam diferenças interespecíficas sugerindo que a extrapolação dos resultados adquiridos em diferentes espécies devam ser extrapolados com cautela.The diurnal habits and its complex SNC, make the tufted capuchin monkey an important subject for the study of the visual system. In the present study, five tufted capuchins received a single intraocular neuronal tracer subunit B of cholera toxin (CTb) injection and perfused 15 days later. The retina and brain were removed from the animals and processed with immunohistochemical techniques. The CTb analysis showed that the retina send projections to several structures, such as primary visual, optical accessory and circadian control systems. The immunohistochemical characterization also showed two different types of bipolar cells in the retina. These cells, differently from other species, were co-localized with gabaergic receptors. Overall our results showed several interspecies differences suggesting that comparison of the visual system between species must be undertaken with great caution.
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Nascimento, Dilzamar Veloso do. "Construção, clonagem e expressão do fragmento B da toxina diftérica de Corynebacterium diphtheriae (cepa PW- 8) em Mycobacterium bovis BCG sub-cepa Moreau." Universidade do Estado do Rio de Janeiro, 2014. http://www.bdtd.uerj.br/tde_busca/arquivo.php?codArquivo=9023.
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Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorA vacina anti-diftérica de uso corrente no Brasil (DTP), embora de alta eficácia na prevenção da difteria, está associada com episódios de toxicidade e reatogenicidade no recipiente vacinal, resultantes de proteínas residuais derivadas do processo de produção ou detoxificação. Estratégias para o desenvolvimento de vacinas menos reatogênicas e ao mesmo tempo mais eficazes e economicamente viáveis contra a difteria têm sido alvo de intensa investigação. A alternativa proposta por nosso grupo é a utilização da vacina contra a tuberculose (Mycobacterium bovis BCG sub-cepa Moreau), como vetor do gene que codifica o fragmento B da toxina diftérica (dtb) de 58,3 kDa. Neste trabalho o dtb foi clonado no vetor micobacteriano bifuncional (pUS977) de expressão citoplasmática e os clones recombinantes (pUS977dtbPW8), após a transformação do BCG, foram testados com relação a expressão do DTB em BCG e quanto a antigenicidade frente a anticorpos policlonais anti-toxóide diftérico por Immunobloting. A integridade do gene dtb e a identidade das sequências de DNA da construção plasmidial pUS977dtbPW8 foram confirmadas por sequenciamento de DNA e análise de similaridade. A imunogenicidade do BCGr pUS977dtbPW8 expressando o DTB foi investigada em camundongos BALB/c, os resultados obtidos revelaram uma soroconversão específica (IgG). A infectividade e atividade microbicida do BCGr pUS977dtbPW8 no ambiente intracelular foi avaliada através da infecção de linhagens de células de monócitos humano (THP-1), os dados obtidos indicaram que houve sobrevivência intracelular em até 12 dias. Nesse contexto, esplenócitos dos camundongos imunizados com 30 e 60 dias foram extraídos, mostrando que o BCGr pUS977dtbPW8 persistiu até 60 dias na ausência de pressão seletiva e a viabilidade celular não sofreu alteração significativa durante o período testado. Por outro lado, o BCGr pUS977dtbPW8, quando submetido a seis sub-cultivos consecutivos in vitro não apresentou diferença significativa na capacidade de expressar o DTB, demonstrando portanto a persistência da estabilidade funcional da linhagem recombinante. A estabilidade estrutural da construção pUS977dtbPW8 também foi avaliada por PCR confirmando a presença do gene dtb em colônias do BCGr pUS977dtbPW8 . Adicionalmente, foi possível avaliar preliminarmente in vitro a capacidade soroneutralizante dos soros de camundongos imunizados com BCGr pUS977dtbPW8 após 30 e 60 dias em células VERO. A ação citotóxica da toxina diftérica entre as diluições de 1/4 e 1/16 foram neutralizadas com o pool de soros imunes com 60 dias. Finalmente, em nosso estudo foi possível avaliar o potencial da vacina BCG como vetor de expressão de um antígeno de Corynebacterium diphtheriae in vitro e in vivo.
The diphtheria vaccine currently used in Brazil (DTP), despite its history of high efficacy in the prevention of diphtheria, is associated with episodes of toxicity and vaccine reactogenicity in the vaccinee, resulting from the presence in the vaccine of residual proteins derived from the production process or detoxification. Strategies for the development of new vaccines more effective and economically viable against diphtheria have been the subject of intense investigation. The alternative proposed by our group is the use of the vaccine against tuberculosis (Mycobacterium bovis BCG Moreau sub strain) as a vector for the gene that encodes the 58.3 kDa fragment B of the diphtheria toxin (DTB). In our project the dtb gene was cloned into the bifunctional vector pUS977 for cytoplasmic expression and recombinant BCG (rBCG) clones, selected after transformation of BCG, were tested for expression of the DTB polypeptide and antigenicity against polyclonal antibodies anti- diphtheria toxoid by immunoblotting. The integrity and identity of the DNA sequence encoding the dtb gene carried by the plasmid construct pUS977dtbPW8 was confirmed by DNA Sequencing and Analysis of Similarity. The immunogenicity of the rBCG expressing the DTB was investigated in BALB/c mice and the results revealed a specific seroconversion (IgG). Also, infectivity and microbicidal activity were analyzed in the intracellular environment by infecting human monocytes (THP-1 cell line) with rBCG. The data obtained indicated intracellular survival within 12 days. In this context, splenocytes collected from mice at days 30 and 60 after immunization were removed and assayed for live bacteria. The results showed that rBCG persisted viable up to 60 days in the absence of selective pressure and cell viable counts did not change significantly during testing. Additionally, the rBCG subjected to six consecutive sub-cultures in vitro showed no significant difference in the ability to express the DTB, thus demonstrating the functional stability of the recombinant vaccine. The structural stability of the construct pUS977dtbPW8 was also confirmed by PCR detection of the dtb gene in rBCG colonies. Also, it was possible to have a preliminary evaluation of the neutralizing capacity of sera from mice immunized with BCGr 30 and 60 days after immunization. The cytotoxic action of diphtheria toxin, between dilutions 1/ 4 and 1/16, was neutralized by mice sera in an in vitro assay using VERO cells. Finally, in our study it was possible to evaluate the potential of BCG as a vector for expression of an antigen of Corynebacterium diphtheriae in vitro and in vivo.
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Beyer, April Jean. "Evaluation of low dose exposure and immunogenicity of transgenic maize expressing the Escherichia coli heat-labile toxin B subunit when fed intermittently and daily." [Ames, Iowa : Iowa State University], 2007.
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Krabs, Isabel [Verfasser]. "Untersuchungen zur Suppression der NF-κB-Aktivierung [NF-kappa-B-Aktivierung] in Shiga-Toxin-produzierenden Escherichia coli (STEC) infizierten Säugerzellen / vorgelegt von Isabel Krabs." Gießen : DVG-Service, 2007. http://d-nb.info/988696908/34.
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Ong, Kong Wee. "Effects of a bacterial toxin on LMP specific CTL killing of EBV transformed B cells : the effect on systemic inflammatory response and clinical outcome." Thesis, University of Bristol, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.271912.
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Félix, Mellanie Karoline do Carmo. "Antígeno inativado de Clostridium Novyi tipo B em emulsão W/O: uma prova de conceito em camundongos Swiss visando o controle de necrose hepática de ruminantes." Universidade Federal do Tocantins, 2018. http://hdl.handle.net/11612/965.
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A bovinocultura brasileira possui grande ênfase no mercado nacional. Doenças que acometem rebanhos comprometem o mercado além de gerarem grandes prejuízos econômicos. O Clostridium novyi tipo B provoca necrose hepática em bovinos através da produção da alfa toxina, uma potente exotoxina que reduz a produtividade através de alterações como hemoglobinúria, redução do apetite, febre, letargia, diminuição da produção de leite e sangue nas fezes. Conter o microrganismo causador torna-se uma busca necessária tanto do ponto de vista econômico quanto social. Entretanto, o controle da doença ainda é realizado por vacinas formuladas com múltiplos antígenos. A emulsão pode ser uma alternativa promissora para a melhoria da adsorção de antígenos nas formulações vacinais. Camundongos da linhagem Swiss foram utilizados a fim de se avaliar aspectos clínicos e validar resultados referentes a composição de uma nova formulação vacinal contendo adjuvante Montanide ISA 61 VG e antígeno inativado de C. novyi. Os testes de caracterização e antigenicidade indicaram a presença da proteína alfa toxina na composição avaliada. A imunogenicidade do antígeno inativado em emulsão W/O (água/óleo) foi verificada e a proporção empregada (40/60) mostrou ser ideal no uso de múltiplos antígenos, apresentando inocuidade, estabilidade do produto, liberação controlada e estímulo da resposta imune. A determinação da concentração de antígeno foi averiguada pela relação antígeno ativo e inativado com soros de animais doentes, visto a eficácia vacinal de 40%. A adequação da concentração de alfa toxina inativada na emulsão mostrou ser necessária para atingir melhores valores de proteção animal. Análises de hemograma, bioquímicas e morfologia de fígado, baço e coxa contribuíram para elucidar os efeitos da emulsão e comprovar necrose hepática nos grupos não imunizados, além de sugerir avanços na adsorção de vacinas. Os resultados possibilitaram o estabelecimento de um modelo murino de infecção de C. novyi com futuras aplicações relacionadas à produção vacinal com múltiplos antígenos emulsificados para controle das clostridioses.Brazilian cattle breeding has great emphasis on the national market. Diseases that affect herds compromise the market as well as generate great economic losses. Clostridium novyi type B causes hepatic necrosis in cattle through the production of alpha toxin, a potent exotoxin that reduces productivity through changes such as hemoglobinuria, reduced appetite, fever, lethargy, decreased milk and stool production. Containing the causative micro-organism becomes a necessary quest both economically and socially. However, control of the disease is still performed by vaccines formulated with multiple antigens. The emulsion may be a promising alternative for the improvement of antigen adsorption in vaccine formulations. Swiss strain mice were used to evaluate clinical aspects and validate results regarding the composition of a new vaccine formulation containing Montanide ISA 61 VG adjuvant and C. novyi inactivated antigen. Characterization and antigenicity tests indicated the presence of the alpha toxin protein in the evaluated composition. The immunogenicity of antigen inactivated in W / O emulsion (water / oil) was verified and the ratio employed (40/60) showed to be ideal in the use of multiple antigens, presenting innocuousness, product stability, controlled release and stimulation of the immune response. The determination of the antigen concentration was investigated by the active antigen ratio and inactivated with sera from sick animals, since the vaccine efficacy was 40%. The suitability of the inactivated alpha toxin concentration in the emulsion was shown to be necessary to achieve better animal protection values. Hemogram, biochemical and liver, spleen and thigh morphology contributed to elucidate the effects of the emulsion and to verify hepatic necrosis in the nonimmunized groups, in addition to suggesting advances in the adsorption of vaccines. The results allowed the establishment of a murine model of C. novyi infection with future applications related to the vaccine production with multiple emulsified antigens to control clostridia.
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Chahboun, Siham. "Comparaison des régions variables des anticorps de macaques (Macaca fascicularis) et de l' Homme et leurs utilisation pour la neutralisation des toxines botuliques A et B." Thesis, Grenoble, 2013. http://www.theses.fr/2013GRENV022.
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Notre laboratoire a développé une stratégie d'isolement de fragments d'anticorps recombinants à partir de primates non humains (Macaca fascicularis) immunisés, en utilisant la technologie des phages. Dans le cadre de cette thèse, une comparaison des séquences d'anticorps de macaques (Macaca Mulatta) et d'anticorps humains a toutefois montré que les anticorps des deux espèces présentent des différences qui rendent souhaitable une étape d'humanisation des anticorps de macaques. Cette stratégie a été utilisée dans le cadre du projet Européen AntiBotABE (www.antibotabe.com) et l'étape de criblage a été adaptée pour isoler des scFv neutralisant de façon croisée les toxines botuliques BoNT/B des sous-types B1 et B2, en utilisant séquentiellement l'holotoxine BoNT/B1 et un fragment recombinant représentant la région C-terminale de la chaîne lourde de BoNT/B2. Le meilleur scFv ciblant les régions C-terminales des chaînes lourdes de BoNT/B1 et BoNT/B2, B2-7, a montré une bonne capacité de neutralisation de BoNT/B1 et BoNT/B2 dans le test ex vivo de paralysie hémidiaphragmatique. Les régions charpentes du scFv B2-7 ont un pourcentage d'identité élevé (80 %) avec leurs homologues humains. Des scFv neutralisant BoNT/A1 en ciblant sa chaîne légère ont aussi été isolés, dont le scFv le plus efficace, 2H8, induit une diminution de 50% de l'activité endopeptidasique à une concentration correspondant à un rapport molaire 2H8/BoNT/A1 de 64000. Les régions charpentes de 2H8 ont également un pourcentage d'identité élevée (88%) avec leurs homologues humains. La versatilité de cette stratégie en fait un outil permettant l'isolement de nombreux autres fragments d'anticorps à visée thérapeutiqueOur laboratory has developed a strategy to isolate recombinant antibody fragments technology from immunized non human primates (Macaca fascicularis) by phage display. In the course of the present thesis, a comparison between macaque (Macaca mulatta) and human antibody sequences has demonstrated that antibodies of the two species are different. This difference makes the humanization of macaque antibodies desirable. The strategy was used in the framework of the European AntiBotABE project, and the screening was adapted to isolate antibody fragments cross neutralizing the B1 and B2 subtypes of botulinum B neurotoxin, by using sequentially the holotoxin BoNT/B1 and a recombinant fragment representing the C-terminal region of the heavy chain of BoNTB2. The best scFv targeting the C-terminal region of BoNT/B1 and BoNTB2 heavy chains, B2-7, demonstrated a high capacity to neutralize BoNT/B1 and BoNT/B2 in the ex vivo hemidiaphragmatic assay. A high identity (80%) between the framework regions of B2-7 and their human homologs was observed. ScFvs neutralizing BoNT/A1 by targeting its light chain were also isolated and among them, the scFv 2H8 induced a decrease of 50% in the endopeptidase activity at a concentration corresponding to a molar ratio of 2H8/BoNT/A1 of 64000. A high identity (88%) between the framework regions of 2H8 and their human homologs was also observed. Our strategy can be used to isolate other therapeutic antibody fragments
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Moser, Rebekka. "IgG Antiköperbestimmung gegen Haemophilus influenza Typ b, Streptococcus pneumoniae Stereotypen 14 und 19F und Clostridium tetani Toxin in Nabelschnurseren und Seren von 0-6 Monate alten Säuglingen /." [S.l.] : [s.n.], 1998. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Price, Gregory A. "Immunogenicity of the Gonococcal Transferrin Binding Proteins." VCU Scholars Compass, 2005. http://scholarscompass.vcu.edu/etd_retro/76.
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The gonococcal transferrin binding proteins (Tbps) are two surface-exposed outer membrane proteins, TbpA and TbpB, which together function to remove and internalized iron from human transferrin. Iron is an essential nutrient to the gonococcus, without which it cannot survive. The Tbps have been established as virulence factors, demonstrating their importance in establishing infection. Both TbpA and TbpB are well conserved among gonococcal isolates, and have been considered potential vaccine targets. Vaccine studies with the closely related species Neisseria meningitidis, have demonstrated these proteins to be protective in murine challenge studies. Though the meningococcal Tbps have demonstrated promise, no similar gonococcal vaccine experiments have been conducted prior to the current studies. Here we demonstrate purification of recombinant TbpA and TbpB. These recombinant proteins were utilized to evaluate the human immune response to these proteins during natural infections, and their immunogenicity in murine vaccine studies. Our results demonstrate a paucity of antibodies elicited to these proteins during natural infections in serum and mucosal secretions from infected individuals. From this study we hypothesized the induction of both serum and genital antibodies to these proteins could serve to protect an individual from infection. To begin testing this hypothesis, we immunized mice both intranasally (IN) and subcutaneously (s.c.) with full-length Tbps in conjunction with the B subunit of cholera toxin (Ctb) as an adjuvant. We also performed another vaccine study using domains from both proteins in genetic fusions with Ctb and E. coli heat labile toxin IIb (LtbIIb). Both studies demonstrated that these antigens were immunogenic, as Tbp-specific antibodies were elicited in the serum and vaginal washes of female Balb/C mice. Intranasal immunization however was the only route with which we were able to elicit vaginal Tbp-specific IgA, and IgG, whereas subcutaneous immunization only elicited vaginal IgG. Furthermore, we found the full-length Tbps and the Ctb/LtbIIb chimeras were able to elicit bactericidal antibodies, which were also effective in killing heterologous gonococcal strains. This body of work comprises the first published study using the gonococcal transferrin binding proteins as vaccine antigens, and highlights their potential as vaccine antigens in the development of an efficacious gonococcal vaccine.
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Lica, Marta Anna [Verfasser], and Harald [Akademischer Betreuer] Genth. "Unterschiede in der biologischen Wirkung des Clostridium difficile Toxin B in proliferierenden und nicht-proliferierenden Zellen / Marta Anna Lica. Institut für Toxikologie der Medizinischen Hochschule Hannover. Betreuer: Harald Genth." Hannover : Bibliothek der Medizinischen Hochschule Hannover, 2012. http://d-nb.info/1023136376/34.
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Jacquier, Muriel. "Association covalente de C3b à la toxine tétanique : rôle dans l'apprêtement et la présentation de l'antigène aux lymphocytes T." Grenoble 1, 1992. http://www.theses.fr/1992GRE10200.
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Le travail presente constitue une premiere etape dans l'etude du role du ligand bifonctionnel c3b associe la toxine tetanique, sur la fonction d'appretement et de presentation de cet antigene par des clones b humains transformes par le virus d'epstein-barr, specifiques de la toxine tetanique, aux lymphocytes t. Dans une premiere partie, l'analyse en sds-page des fragments de c3b fixes sur les lymphocytes b indique que 60% de cette proteine est impliquee dans la formation de complexes covalents par pont disulfure avec des proteines membranaires et 40% dans des interactions non covalentes avec les recepteurs cellulaires du complement. L'implication de la cys1010 du thioester dans la formation des complexes covalents a ete confirmee apres alkylation de cet unique groupement sh libre par l'iodoacetamide. Lorsque ces cellules sont preincubees avec la toxine tetanique puis avec #1#2#5i-c3b, des complexes disulfure #1#2#5i-c3b-tt sont immunoprecipites a partir des lysats subcellulaires avec des antiserum contre c3 ou la tt. L'expression membranaire et la secretion dans les surnageants de culture d'une proteine reconnue par des anticorps anti-thioredoxine suggerent l'implication de cette enzyme dans la formation du disulfure. Dans une seconde partie, nous avons analyse le role de l'association covalente de c3b a la toxine tetanique, sur l'appretement et la presentation de la tt aux lymphocytes t, par ces clones b specifiques. Deux types de complexes c3b-tt ont ete utilises: des complexes disulfure ou des complexes ester prepares in vitro. L'analyse a ete faite en gel sds-page a partir de proteolysats obtenus apres endocytose de l'antigene in situ, ou par des fractions subcellulaires enrichies en lysosomes. Les resultats indiquent que les fragments de proteolyse de tt observes a partir de la tt ou de c3b-tt sont identiques. Des experiences de cinetiques revelent un effet retard de la proteolyse de la tt elle est associee a c3b. D'autre part, la presentation par des cellules b fixees de proteolysats de tt seule ou de c3b-tt obtenus a differents temps, montre que la production de peptides est maximale a 6 heures de traitement avec la tt, alors qu'elle est maintenue au bout de 24 heures avec les complexes. Ces derniers constituent un reservoir d'ag dans la cellule presentatrice et assurent une liberation graduelle de peptides immunogenes responsables d'une stimulation (10) de la proliferation des cellules th specifiques. Ces effets sont a relier a la stabilite de la liaison ester ou disulfure dans les compartiments subcellulaires, ainsi qu'au role chaperon de c3b. L'utilisation de cathepsines purifiees indique que cet effet protecteur de c3b resulte d'une susceptibilite differente pour les proteases intracellulaires de la tt seule ou associee a c3b
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Hickey, Ashley N. "Expression of CTB-proinsulin in transgenic chloroplasts." Honors in the Major Thesis, University of Central Florida, 2008. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/1088.
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This item is only available in print in the UCF Libraries. If this is your Honors Thesis, you can help us make it available online for use by researchers around the world by following the instructions on the distribution consent form at http://library.ucf.edu/Systems/DigitalInitiatives/DigitalCollections/InternetDistributionConsentAgreementForm.pdf You may also contact the project coordinator, Kerri Bottorff, at kerri.bottorff@ucf.edu for more information.Bachelors
Burnett College of Biomedical Sciences
Molecular and Microbiology
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Silva, Sebasti?o Franco da. "Centros subcorticais dos sistemas visual prim?rio e ?ptico acess?rio no moc? (kerodon rupestris): caracteriza??o pela proje??o retiniana e citoarquitetura." Universidade Federal do Rio Grande do Norte, 2009. http://repositorio.ufrn.br:8080/jspui/handle/123456789/17208.
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The primary and accessory optic systems comprise two set of retinorecipient neural clusters. In this study, these visual related centers in the rock cavy were evaluated by using the retinal innervations pattern and Nissl staining cytoarchigtecture. After unilateral intraocular injection of cholera toxin B subunit and immunohistochemical reaction of coronal and sagittal sections from the diencephalon and midbrain region of rock cavy. Three subcortical centres of primary visual system were identified, superior colliculus, lateral geniculate complex and pretectal complex. The lateral geniculate complex is formed by a series of nuclei receiving direct visual information from the retina, dorsal lateral geniculate nucleus, intergeniculate leaflet and ventral lateral geniculate nucleus. The pretectal complex is formed by series of pretectal nuclei, medial pretectal nucleus, olivary pretectal nucleus, posterior pretectal nucleus, nucleus of the optic tract and anterior pretectal nucleus. In the accessory optic system, retinal terminals were observed in the dorsal terminal, lateral terminal and medial terminal nuclei as well as in the interstitial nucleus of the superior fasciculus, posterior fibres. All retinorecipient nuclei received bilateral input, with a contralateral predominance. This is the first study of this nature in the rock cavy and the results are compared with the data obtained for other species. The investigation represents a contribution to the knowledge regarding the organization of visual optic systems in relation to the biology of species.
Os sistemas visual prim?rio e ?ptico acess?rio compreendem dois conjuntos de grupamentos neurais, que recebem proje??o direta da retina. Nesse estudo, estes dois sistemas foram avaliados com rela??o a citoarquitetura e padr?o de inerva??o retiniana, usando como tra?ador neural anter?grado, a subunidade B da toxina col?rica revelada por imunoistoqu?mica para detectar este tra?ador em terminais sobre grupamentos neuronais (alvos) no enc?falo do moc? (Kerodon rupestris), um roedor nativo do Nordeste Brasileiro. Os resultados permitiram identificar os componentes do sistema visual prim?rio o complexo geniculado lateral, o complexo pr?-tectal e o col?culo superior. O complexo geniculado lateral cont?m o n?cleo geniculado lateral dorsal, o n?cleo geniculado lateral ventral e o folheto intergeniculado. Todos recebem fibras da retina com predomin?ncia contralateral, menos intensa para o folheto intergeniculado. O complexo pr?-tectal cont?m os n?cleos pr?-tectal anterior, pr?-tectal medial, pr?-tectal posterior, olivar pr?-tectal e n?cleo do trato ?ptico, os quais recebem proje??o retiniana predominantemente contralateral. Do mesmo modo, o col?culo superior recebe fibras da retina contralateral nas camadas superficiais. Tamb?m foi identificado o sistema ?ptico acess?rio completo no moc?, constitu?do pelos n?cleos terminal medial, terminal lateral, terminal dorsal e intersticial do fasc?culo superior posterior. Esses n?cleos recebem inerva??o retiniana com forte predomin?ncia contralateral, sendo que o n?cleo terminal medial, embora preserve a predomin?ncia contralateral, exibe uma evidente inerva??o ipsolateral.
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Kuk, Chiu Ying. "Anthrax Lethal Toxin Is a Tumor Hemorragic Toxin." Thesis, Van Andel Research Institute, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10973827.
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Blood supply is crucial for tumor growth and metastasis. However, current anti-angiogenic therapy is not as effective as predicted, thus a better understanding of the tumor angiogenic process and new anti-angiogenic agent are urgently required. Anthrax lethal toxin (LeTx) has an anti-angiogenic effect on tumors. Tumors treated with LeTx are smaller, paler, and have lower mean vessel density compared to control treated tumors. Most interestingly, compared to current anti-angiogenic treatment, LeTx does not cause normalization of tumor vessels. Instead, tumors treated with LeTx have massive hemorrhages, pointing to a potential alternative mechanism to inhibit tumor angiogenesis. I hypothesize that instead of causing “normalization” of tumor vasculature, LeTx’s anti-angiogenic effects works in a manner similar to a hemorrhagic toxins. To test this hypothesis, I compared the effect of LeTx to snake venom metalloproteinase, a known hemorrhagic toxin, in tumor vasculature. Quantified by Nuance multispectral imaging system, both LeTx and SVMP caused an increase in tumor hemorrhage. Futher analysis of vasculature integrity using continued vessel length showed disruption of vessels by LeTx and SVMP. With these results, I conclude that the anti-angiogenic effects of LeTx are due to its hemorrhagic nature, and not due to normalization of tumor vasculature. Further understanding of LeTx mechanism can help design novel anti-angiogenic agent that compliments current therapy.
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Javid-Khojasteh, Vahideh. "Toxic Shock Syndrome Toxin-1 : detection of the toxin, anti-toxin antibodies and producer organisms in a paediatric burns unit." Thesis, University of Salford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365993.
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Bader, Carly. "The cytopathic activity of cholera toxin requires a threshold quantity of cytosolic toxin." Master's thesis, University of Central Florida, 2013. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/5762.
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Cholera toxin (CT), secreted from Vibrio cholerae, causes a massive fluid and electrolyte efflux in the small intestine that results in life-threatening diarrhea and dehydration which impacts 3-5 million people per year. CT is secreted into the intestinal lumen but acts within the cytosol of intestinal epithelial cells. CT is an AB5 toxin that has a catalytic A1 subunit and a cell binding B subunit. CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde transport. Much of the toxin is transported to the lysosomes for degradation, but a secondary pool of toxin is diverted to the Golgi apparatus and then to the ER. Here the A1 subunit detaches from the rest of the toxin and enters the cytosol. The disordered conformation of free CTA1 facilitates toxin export to the cytosol by activating a quality control mechanism known as ER-associated degradation. The return to a folded structure in the cytosol allows CTA1 to attain an active conformation for modification of its Gs? target through ADP-ribosylation. This modification locks the protein in an active state which stimulates adenylate cyclase and leads to elevated levels of cAMP. A chloride channel located in the apical enterocyte membrane opens in response to signaling events induced by these elevated cAMP levels. The osmotic movement of water into the intestinal lumen that results from the chloride efflux produces the characteristic profuse watery diarrhea that is seen in intoxicated individuals. The current model of intoxication proposes only one molecule of cytosolic toxin is required to affect host cells, making therapeutic treatment nearly impossible. However, based on emerging evidence, we hypothesize a threshold quantity of toxin must be present within the cytosol of the target cell in order to elicit a cytopathic effect. Using the method of surface plasmon resonance along with toxicity assays, I have, for the first time, directly measured the efficiency of toxin delivery to the cytosol and correlated the levels of cytosolic toxin to toxin activity. I have shown CTA1 delivery from the cell surface to the cytosol is an inefficient process with only 2.3 % of the surface bound CTA1 appearing in the cytosol after 2 hours of intoxication. I have also determined and a cytosolic quantity of more than approximately .05ng of cytosolic CTA1 must be reached in order to elicit a cytopathic effect. Furthermore, CTA1 must be continually delivered from the cell surface to the cytosol in order to overcome the constant proteasome-mediated clearance of cytosolic toxin. When toxin delivery to the cytosol was blocked, this allowed the host cell to de-activate Gs?, lower cAMP levels, and recover from intoxication. Our work thus indicates it is possible to treat cholera even after the onset of disease. These findings challenge the idea of irreversible cellular toxicity and open the possibility of post-intoxication treatment options.M.S.
Masters
Molecular Biology and Microbiology
Medicine
Biomedical Sciences; Biomedical Sciences
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Gao, Haifei. "Chemical biology approaches to study toxin clustering and lipids reorganization in Shiga toxin endocytosis." Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015USPCB147.
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La toxine bactérienne de Shiga se lie au glycosphingolipide (GSL) globotriaosylcéramide (Gb3) afin d’entrer par endocytose dans les cellules en utilisant une voie dépendante et indépendante de la clathrine. Dans la voie indépendante de la clathrine, la toxine de Shiga réorganise les lipides de la membrane de façon à imposer une contrainte mécanique sur la bicouche, conduisant ainsi à la formation de pic d’invagination d'endocytose profonds et étroits. Mécaniquement ce phénomène n’est pas encore compris, notamment il reste énigmatique, comment se traduisent les propriétés géométriques de l’agrégation des glycosphingolipides GSLS et de la toxine. Dans mon travail de thèse, via l’utilisation de la sous-unité B de la toxine de Shiga (STxB) comme un modèle, différentes espèces moléculaires de son récepteur Gb3 ont été synthétisés avec des structures délibérément choisis. Les études réalisées par imagerie de haute résolution et par la modélisation informatique ont permis d’élucider les contraintes mécano-chimique sous-jacente conduisant à une réorganisation efficace qui a pour résultat l’agrégation de la toxine et la réorganisation des lipides. En combinant des expériences de simulation sur ordinateur de dynamique des particules dissipatives (DPD) et des expériences sur des modèles de membranes cellulaires, nous avons fourni la preuve de l’induction d’une force de fluctuation-membrane, de type « force de Casimir », conduisant à l'agrégation des molécules de toxines associées à la membrane à des échelles de longueur mésoscoiques. Nous avons observé et mesuré, en outre la condensation lipidique induite par la toxine, quantitativement sur des monocouches de Langmuir en utilisant la réflectivité des rayons X (XR) et par la mesure de la diffraction des rayons X par incidence rasante (GIXD), fournissant ainsi une preuve directe de l'hypothèse que la toxine a le potentiel de réduire de façon asymétrique la surface moléculaire sur la partie membranaire exoplasmique, ce qui conduit à une déformation locale de la membrane. Durant ma thèse, nos efforts ont été consacrés à la réalisation de nouveaux glycosphinolipides (GSL) comme outils chimiques à visée biologique. Par ailleurs, une nouvelle stratégie de reconstitution de GSL fonctionnels sur la membrane cellulaire, basée sur une réaction de ligation de type « click » entre un glycosyl-cyclooctyne et un azido-sphingosine a été étudiée. Les résultats obtenus sur les cellules se sont avérés beaucoup moins efficace que ceux in vitro. Une poursuite de l'optimisation de cette méthodologie est actuellement en cours. Une sonde fluorescente du glycosphinolipide Gb3, marquée à l’Alexa Fluor 568 lui-même lié par l'intermédiaire d'un bras PEG-α à la position de la chaîne acyle, a été synthétisée. Cette sonde se lie à la STxB sur couche mince de TLC, mais pas sur des membranes modèles. D'autres améliorations sont discutéesBacterial Shiga toxins bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3) to enter cells by clathrin-dependent and independent endocytosis. In the clathrin-independent pathway, Shiga toxin reorganizes membrane lipids in a way such as to impose mechanical strain onto the bilayer, thus leading to the formation of deep and narrow endocytic pits. Mechanistically how this occurs is not yet understood, and notably how the geometric properties of toxin-GSLs complexes translate into function has remained enigmatic. In my thesis work, using the B-subunit of Shiga toxin (STxB) as a model, different molecular species of its receptor Gb3 have been synthesized with deliberately chosen structures, coupled with high resolution imaging and computational modeling, to understand the underlying mechano-chemical constraints leading to efficient toxin clustering and lipids reorganization. By combining dissipative particle dynamics (DPD) computer simulation and experiments on cell and model membranes, we provided evidence that a membrane fluctuation-induced force, termed Casimir-like force, drives the aggregation of tightly membrane-associated toxin molecules at mesoscopic length scales. Furthermore, toxin-induced lipid condensation was observed and measured quantitatively on Langmuir monolayers using X-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), thereby providing direct evidence for the hypothesis that the toxin has the potential to asymmetrically reduce the molecular area of the exoplasmic membrane leaflet, leading to local membrane deformation. During my PhD, effort was also invested to develop new GSL tools applied to the biological setting. A novel strategy based on the Cu-free click reaction between glycosyl-cyclooctyne and azido-sphingosine was designed with the goal to functionally incorporate GSLs into cellular membranes. Following the synthesis work, click reactions have been performed in solution and on cells. Compared to the former, results on cells were far less efficient. Further optimization is currently ongoing. A fluorescently labeled Gb3 probe with Alexa Fluor 568 coupled via a PEG linker to the α-position of the acyl chain, was synthesized, to which STxB bound on TLCs, but not on model membranes. Further improvements are discussed
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Karlsson, Sture. "Toxin production in Clostridium difficile /." Stockholm : Karolinska institutet, 2004. http://diss.kib.ki.se/2004/91-77349-812-2/.
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Strack, Julia [Verfasser], and Andreas [Akademischer Betreuer] Bechthold. "Osteoklastendifferenzierung durch Pasteurella multocida-Toxin." Freiburg : Universität, 2014. http://d-nb.info/1123480834/34.
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