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1
Pang, Ha Sang. "Identification of CD8+ T cell epitopes from HCA661 presented by HLA-A2 molecules /." View abstract or full-text, 2006. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202006%20PANG.
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2
張紀忠 and Jizhong Zhang. "Conformational antigenic determinants of the HEV CAPSID." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2000. http://hub.hku.hk/bib/B31241360.
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3
Zhang, Jizhong. "Conformational antigenic determinants of the HEV CAPSID /." Hong Kong : University of Hong Kong, 2000. http://sunzi.lib.hku.hk/hkuto/record.jsp?B2207918X.
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4
Beena, T. K. "Antigenic Determinants Of Chicken Riboflavin Carrier Protein: Structural And Functional Aspects." Thesis, Indian Institute of Science, 1994. https://etd.iisc.ac.in/handle/2005/141.
Full textAbstract:
Investigations detailed in this thesis constitute a part of the continuing programme of research undertaken in our laboratory on the riboflavin carrier protein (RCP) with particular reference to identification and synthesis of neutralizing antigenic determinants, design of relevant epitope mimetics with improved immunogenic characteristics and relationship between their secondary structures and immunological properties.
The riboflavin carrier protein is elaborated as a reproductive stratagem to ensure adequate vitamin deposition in the developing oocyte in the chickens. The protein is scrupulously conserved through evolution in terms of physico chemical and immunological characteristics from fish through birds to mammals, including primates. In rodents and subhuman primates immunization with the heteroantigen viz., chicken egg white RCP leads to functional neutralization of the endogenous maternal protein resulting in curtailment of early pregnancy. Thus, the crucial role of RCP in maintenance of pregnancy is established and the protein identified as a potential candidate vaccine for immimocontraception. Further studies with the reduced and carboxymethylated (RCM) RCP as the immunogen reveal that antibodies induced by RCM-RCP are equally effective in bioneutralization of the endogenous protein. So it can be surmised that the native folded structure of RCP is not obligatory for eliciting bioneutralizing antibodies. In an attempt to identify functionally relevant regions of the protein, a panel of monoclonal antibodies (MAbs) have been raised and characterized. One of the MAbs viz., 6J32Ci2 could bring about early fetal resorp-tion when injected to mice with confirmed pregnancies. These results prompted a detail molecular immunological approach to understand underlying mechanisms. The principal aims of the present investigations include: (1) identification of neutralizing epitopes; (2) synthesis of peptidyl sequences incorporating these determinants; (3) an understanding of the structure, antigenic and immunogenic characteristics of these peptides; (4) correlation of conformational and antigenic characteristics; (5) rational design and synthesis of peptide analogs with greater propensity to assume predicted secondary structures; (6) analysis of conformation dependency of peptide antigens and the importance of such conformation in generating an optimal B-cell response; (7) the efficacy of the antibodies elicited by these Peptide antigens in neutralizing endogenous protein with the ultimate aim of designing synthetic vaccines.
Chapter 1 of this thesis deals with a general introduction summarizing the current status of knowledge regarding the chemistry and biology of RCP as well as synthetic peptides as potential immunogens. Chapter 2 outlines details of the experimental procedures adopted. Chapter 3 describes the results of investigations on the C-terminal fragment (residues 200-219) of cRCR The main consideration in selecting this sequence for the design of a potential peptide-based vaccine relied on the epitopic specificity of the neutralizing MAb 6S2C12. Epitope mapping using the Pepscan method revealed that the monoclonal antibody recognizes a core sequence corresponding to residues 203-210 of the cRCP. A 21-residue synthetic peptide (C-21) comprising this epitope was synthesized and antibodies elicited to the peptide conjugated to two different carriers, namely diphtheria toxoid and purified protein derivative (PPD) for T-cell help. In both active and passive immunoneutralization experiments, the peptide specific neutralizing antibodies interfered with the biological function of the protein and hence either protected from pregnancy or caused early fetal resorption in rodents as well as in sub-human primates. The conforma-tional properties of the peptide in aqueous buffers were analyzed from circular dichroism which revealed the absence of any ordered structure in the native C-21 peptide. Theoretical predictions of secondary structure suggested a propensity for an t*-helical structure for this fragment in the native protein. Therefore, influence of the helix-promoting solvent, vizM 2,2,2,trifluroethanol (TFE) on the C-21 peptide was investigated. Addition of TFE resulted in spectral changes with negative bands at 208 and 222 nm and a positive band at 190 rim which are typical of an a-helix.
To gain more information on the conformational characteristics of this peptide, it was considered worthwhile to stabilize the native peptide in an a-helical conformation based on simple rational design principles. Towards this end, four analogs of the parent peptide were synthesized and helix stabilization was sought to be achieved by introducing either salt bridges or back-bone conformational constraints such as by incorporating a-amino isobutyric acid at appropriate positions. In all the analogs, the core sequence, recognised by the neutralizing MAb 6B2C12 was maintained intact to ensure induction of antibodies capable of recognizing the native protein. CD spectral analysis of the analog peptides indicated that all the engineered peptides had varying degrees of enhanced helicities as compared to the parent peptide. The immunogenicity of each analog was studied by to the relevant peptide-diphtheria toxoid conjugates and analyzing their reactivities with the native protein by direct and competitive ELISA. The results revealed that these engineered conformational analogs axe highly immunogenic eliciting high titers of anti-protein antibodies. The relative affinities of these antibodies to bind cRCP were investigated. The antibodies to peptide analogs had higher affinities for the native protein and a positive correlation was found between the helical content of the peptide antigen in question and the relative affinity of corresponding antibody. The antibodies directed to all the peptide analogs could block the function of RCP resulting in early embryonic resorption when administered to pregnant mice. An interesting pattern of immunological cross-reactivity has been observed with the native and designed peptides. Antibodies raised to constrained helical analogs could bind the C-21 peptide which is structurally flexible. In contrast, the antibodies raised to the flexible native peptide antigen were inefficient in recognizing the structured peptides. The ability of all the peptide antibody to bind the native protein has been interpreted in terms of a conformationally flexible C-terminus region in cRCP.
Chapter 4 details investigations on a 21-residue peptide (N- 21) from the N-terminiis (4-24) of the protein. Selection of this peptidyl sequence relied on theoretical prediction of potential sequential determinants on RCP other than at C-terminus as well as on the outcome of immunoneutralisation experiments using antibodies to egg yolk RCP which lacks the relevant C-terminal determinants. The structure of this peptide in solution was analyzed by two dimensional NMR and CD. NMR experiments revealed the presence of two structured regions in the peptide. Diagnostic nuclear Overhauser effects characteristics of reverse turns or short frayed helical segments over residues 3-9 and 18-21 of the peptide were obtained. CD spectra showed the presence of a strong, negative band at 204 nm over a wide range of solvent conditions, a feature which has been interpreted in terms of a "polyproline Il-like" segment encompassing residues 11-16 which corresponds to an interesting (X-Pro)^ repeat in the N-21 sequence.
Specific antibodies were generated to this peptide as a conjugate with diphtheria toxoid. Administration of the antipeptide antibodies could neutralize the protein in vivo as demonstrated by early embryonic loss in pregnant mice. In limited experiments the antipeptide antibodies showed propensity to protect bonnet monkeys from pregnancy over a few consecutive ovulatory cycles when titres are maintained elevated by periodic boosting. To address the relationship between peptide structures and antigenicity, epitope mapping of this antipeptide antibodies as well- as the polyclonal antibodies to native RCP was undertaken using the Pepscan method. The results reveal that antigenic regions correspond well to conformationally well-defined elements of structure with the polyproline II-like segment being a common antigenic determinant on both the peptide and the native protein. These observations are suggestive of the involvement of both the N and C-terminal regions of RCP in terms of its binding to putative plasma membrane receptors.
5
Beena, T. K. "Antigenic Determinants Of Chicken Riboflavin Carrier Protein: Structural And Functional Aspects." Thesis, Indian Institute of Science, 1994. http://hdl.handle.net/2005/141.
Full textAbstract:
Investigations detailed in this thesis constitute a part of the continuing programme of research undertaken in our laboratory on the riboflavin carrier protein (RCP) with particular reference to identification and synthesis of neutralizing antigenic determinants, design of relevant epitope mimetics with improved immunogenic characteristics and relationship between their secondary structures and immunological properties.
The riboflavin carrier protein is elaborated as a reproductive stratagem to ensure adequate vitamin deposition in the developing oocyte in the chickens. The protein is scrupulously conserved through evolution in terms of physico chemical and immunological characteristics from fish through birds to mammals, including primates. In rodents and subhuman primates immunization with the heteroantigen viz., chicken egg white RCP leads to functional neutralization of the endogenous maternal protein resulting in curtailment of early pregnancy. Thus, the crucial role of RCP in maintenance of pregnancy is established and the protein identified as a potential candidate vaccine for immimocontraception. Further studies with the reduced and carboxymethylated (RCM) RCP as the immunogen reveal that antibodies induced by RCM-RCP are equally effective in bioneutralization of the endogenous protein. So it can be surmised that the native folded structure of RCP is not obligatory for eliciting bioneutralizing antibodies. In an attempt to identify functionally relevant regions of the protein, a panel of monoclonal antibodies (MAbs) have been raised and characterized. One of the MAbs viz., 6J32Ci2 could bring about early fetal resorp-tion when injected to mice with confirmed pregnancies. These results prompted a detail molecular immunological approach to understand underlying mechanisms. The principal aims of the present investigations include: (1) identification of neutralizing epitopes; (2) synthesis of peptidyl sequences incorporating these determinants; (3) an understanding of the structure, antigenic and immunogenic characteristics of these peptides; (4) correlation of conformational and antigenic characteristics; (5) rational design and synthesis of peptide analogs with greater propensity to assume predicted secondary structures; (6) analysis of conformation dependency of peptide antigens and the importance of such conformation in generating an optimal B-cell response; (7) the efficacy of the antibodies elicited by these Peptide antigens in neutralizing endogenous protein with the ultimate aim of designing synthetic vaccines.
Chapter 1 of this thesis deals with a general introduction summarizing the current status of knowledge regarding the chemistry and biology of RCP as well as synthetic peptides as potential immunogens. Chapter 2 outlines details of the experimental procedures adopted. Chapter 3 describes the results of investigations on the C-terminal fragment (residues 200-219) of cRCR The main consideration in selecting this sequence for the design of a potential peptide-based vaccine relied on the epitopic specificity of the neutralizing MAb 6S2C12. Epitope mapping using the Pepscan method revealed that the monoclonal antibody recognizes a core sequence corresponding to residues 203-210 of the cRCP. A 21-residue synthetic peptide (C-21) comprising this epitope was synthesized and antibodies elicited to the peptide conjugated to two different carriers, namely diphtheria toxoid and purified protein derivative (PPD) for T-cell help. In both active and passive immunoneutralization experiments, the peptide specific neutralizing antibodies interfered with the biological function of the protein and hence either protected from pregnancy or caused early fetal resorption in rodents as well as in sub-human primates. The conforma-tional properties of the peptide in aqueous buffers were analyzed from circular dichroism which revealed the absence of any ordered structure in the native C-21 peptide. Theoretical predictions of secondary structure suggested a propensity for an t*-helical structure for this fragment in the native protein. Therefore, influence of the helix-promoting solvent, vizM 2,2,2,trifluroethanol (TFE) on the C-21 peptide was investigated. Addition of TFE resulted in spectral changes with negative bands at 208 and 222 nm and a positive band at 190 rim which are typical of an a-helix.
To gain more information on the conformational characteristics of this peptide, it was considered worthwhile to stabilize the native peptide in an a-helical conformation based on simple rational design principles. Towards this end, four analogs of the parent peptide were synthesized and helix stabilization was sought to be achieved by introducing either salt bridges or back-bone conformational constraints such as by incorporating a-amino isobutyric acid at appropriate positions. In all the analogs, the core sequence, recognised by the neutralizing MAb 6B2C12 was maintained intact to ensure induction of antibodies capable of recognizing the native protein. CD spectral analysis of the analog peptides indicated that all the engineered peptides had varying degrees of enhanced helicities as compared to the parent peptide. The immunogenicity of each analog was studied by to the relevant peptide-diphtheria toxoid conjugates and analyzing their reactivities with the native protein by direct and competitive ELISA. The results revealed that these engineered conformational analogs axe highly immunogenic eliciting high titers of anti-protein antibodies. The relative affinities of these antibodies to bind cRCP were investigated. The antibodies to peptide analogs had higher affinities for the native protein and a positive correlation was found between the helical content of the peptide antigen in question and the relative affinity of corresponding antibody. The antibodies directed to all the peptide analogs could block the function of RCP resulting in early embryonic resorption when administered to pregnant mice. An interesting pattern of immunological cross-reactivity has been observed with the native and designed peptides. Antibodies raised to constrained helical analogs could bind the C-21 peptide which is structurally flexible. In contrast, the antibodies raised to the flexible native peptide antigen were inefficient in recognizing the structured peptides. The ability of all the peptide antibody to bind the native protein has been interpreted in terms of a conformationally flexible C-terminus region in cRCP.
Chapter 4 details investigations on a 21-residue peptide (N- 21) from the N-terminiis (4-24) of the protein. Selection of this peptidyl sequence relied on theoretical prediction of potential sequential determinants on RCP other than at C-terminus as well as on the outcome of immunoneutralisation experiments using antibodies to egg yolk RCP which lacks the relevant C-terminal determinants. The structure of this peptide in solution was analyzed by two dimensional NMR and CD. NMR experiments revealed the presence of two structured regions in the peptide. Diagnostic nuclear Overhauser effects characteristics of reverse turns or short frayed helical segments over residues 3-9 and 18-21 of the peptide were obtained. CD spectra showed the presence of a strong, negative band at 204 nm over a wide range of solvent conditions, a feature which has been interpreted in terms of a "polyproline Il-like" segment encompassing residues 11-16 which corresponds to an interesting (X-Pro)^ repeat in the N-21 sequence.
Specific antibodies were generated to this peptide as a conjugate with diphtheria toxoid. Administration of the antipeptide antibodies could neutralize the protein in vivo as demonstrated by early embryonic loss in pregnant mice. In limited experiments the antipeptide antibodies showed propensity to protect bonnet monkeys from pregnancy over a few consecutive ovulatory cycles when titres are maintained elevated by periodic boosting. To address the relationship between peptide structures and antigenicity, epitope mapping of this antipeptide antibodies as well- as the polyclonal antibodies to native RCP was undertaken using the Pepscan method. The results reveal that antigenic regions correspond well to conformationally well-defined elements of structure with the polyproline II-like segment being a common antigenic determinant on both the peptide and the native protein. These observations are suggestive of the involvement of both the N and C-terminal regions of RCP in terms of its binding to putative plasma membrane receptors.
6
Choukri, Sam. "Selection of malaria-specific epitopes from random peptide libraries /." free to MU campus, to others for purchase, 1999. http://wwwlib.umi.com/cr/mo/fullcit?p9962513.
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Serafin, Ina Loretta. "Epitope mapping of the dengue 3 envelope protein." Thesis, Queensland University of Technology, 1999.
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8
Smyrnis, Elie Mario. "The generation of monoclonal antibodies to neural cell type-specific antigenic determinants." Thesis, University of British Columbia, 1988. http://hdl.handle.net/2429/28399.
Full textAbstract:
Somatic cell hybridization techniques were used to produce a panel of anti-neural cell monoclonal antibody-secreting murine hybridomas.
Whole living cultures of bovine oligodendrocytes were used to inject (intra-splenically and without adjuvants) Balb/C mice with a total of 2.0 to 2.5 x 10⁶ oligodendrocytes. Polyethylene glycol was subsequently used to fuse these sensitized splenic lymphocytes to the murine NS-1 myeloma cell line. After fusion, supernatants were screened immunocytochemically using murine neural cell cultures. Initial screening identified a total of 17 clones, each of which primarily labelled a surface antigen specific to galactocerebroside-positive oligodendrocytes; as demonstrated by double-immunostaining characterization. The monoclonal antibodies were determined to be of either the IgM class or IgG[sub 2b] subclass. Moreover, these antibodies recognized protein bands possessing an
apparent molecular weight of 29,000 and/or 59,000 Da on Western blots of homogenized bovine oligodendrocytes. This may, therefore, represent immunolabelling of a previously unrecognized surface constituent of oligodendrocytes.
Culture supernatants from a second separate fusion experiment involving an intrasplenic injection with 20 μg of SDS-PAGE subfractionated human newborn brain particulate fraction were tested using first, an ELISA, and subsequently, indirect immunofluorescence microscopy. Screening demonstrated 2 clones which produced antibodies immunoreactive to an astrocyte subclass (glial fibrillary acidic protein-positive cells) found only in cultures of newborn murine brain. These antibodies were determined to be of an IgM class and were shown to specifically reactivity with electroblots of a protein constituent of whole newborn brain particulate fraction having an apparent molecular weight of 50,000 Da.Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate
9
Grace, Christopher. "Diagnostically significant antigens of Treponema pallidum subsp. pallidum : identification, serological efficacy, and characterisation of the major antigenic determinants." Thesis, Open University, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.311861.
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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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Garrett, Joan Teresa. "Peptide-based B-cell epitope vaccines targeting HER-2/neu." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1189103626.
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Li, Ming 1957. "Generation of CD8+ T cell immunity with help from CD4+ T cells." Monash University, Dept. of Pathology and Immunology, 2002. http://arrow.monash.edu.au/hdl/1959.1/8476.
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Bertrand, Stephen. "Expression of pseudorabies virus and Rous associated virus antigenic determinants in Escherichia coli." Thesis, University of Ottawa (Canada), 1992. http://hdl.handle.net/10393/10670.
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Both pseudorabies virus and the various strains of avian sarcoma and leukosis viruses are economically important pathogens. In Canada, pseudorabies is an exotic virus which primarily infects swine. The disease is rapidly fatal in most species although adult pigs may become carriers. Because pseudorabies virus (PrV) is exotic to Canada, imported animals are rigorously tested for the presence of antibody titers to the virus. The leukosis caused by Rous associated virus (RAV) has a major impact on the health of poultry flocks. Different strains of the avian leukosis viruses cause damage of differing severity but the general characteristics of infection include decreases in both egg and meat production. There would be significant benefits if non-infectious, antigenic fractions from both of these viruses could be produced in bacterial cells. These proteins could be used in serum antibody testing to make the detection of both viruses considerably simpler than the currently used ELISA and virus neutralization tests. As well, the production of large quantities of RAV env protein could simplify studies of the mechanisms of host specificity and provide additional information about chicken cell receptors. I have attempted to produce an antigenically recognizable protein from both pseudo-rabies virus and Rous associated virus. Attempts were made to express such a protein from pseudorabies virus by cloning either cDNA or genomic DNA fragments into plasmid vectors followed by colony immunodetection using a polyclonal antibody against PrV prepared in pig. Although several recombinants produced positive immunological results, further examination showed that none contained PrV DNA sequences. Standard screening methods were modified and improved during the course of this work. Subsequently, other laboratories identified significant difficulties in using unpurified polyclonal antibodies prepared in swine which provided an explanation for the results I observed. A cDNA library constructed from RNA prepared from RAV-1 was screened using a probe prepared from env sequences in pSR-XD2, a plasmid containing much of the RSV sequence. Transformants containing RSV homologous DNA were identified. During purification of these clones and subsequent screenings many of the plasmids identified as containing RSV homologous sequence lost their inserts. Additional studies demonstrated that plasmids which exhibited the strongest homology with the RSV probe and which contained the longest inserts, lost these inserts at a high rate. A second cDNA cloning using a different bacterial host and a different viral strain (RAV-2) produced several positive clones, with insert lengths ranging from 250 by to 950 bp. These inserts were sequenced and compared to known RSV and RAV sequences. A high degree of homology was observed. The longest RAV-2 cDNA fragments included most of the env gp37 polypeptide coding region. In order to test for expression of an antigen, inserts from two independently isolated recombinants were subcloned into lambdagt11 in each of the three reading frames. A polyclonal antibody prepared against Rous sarcoma virus was used to test for the expression of any recognizable antigenic determinants. No antigen production was detected even though nucleic acid plaque hybridizations demonstrated that the subcloning had been successful in introducing RAV-2 sequences into lambdagt11. Possible explanations for this, as well as a discussion of the composition of the RAV-2 sequences and their placement on the RSV genetic map are considered.
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Harrison, Jacqueline Laura. "Presentation of foreign antigenic determinants at cell surface of enteric bacteria using the trat protein." Thesis, University of Southampton, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316441.
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Matthews, Leslie Jeanne. "Synthetic vaccines from peptide libraries : lessons from a model pathogen /." free to MU campus, to others for purchase, 1998. http://wwwlib.umi.com/cr/mo/fullcit?p9924906.
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Theiss, Patty M. "Mycoplasma fermentans : a minimalist parasite employing unique strategies generating high-frequency antigenic variation of surface lipoproteins /." free to MU campus, to others for purchase, 1996. http://wwwlib.umi.com/cr/mo/fullcit?p9720534.
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Vollaro, Cindy M. "Definition of a Cytotoxic T Lymphocyte Epitope of the Sin Nombre Hantavirus G2 Glycoprotein." Digital WPI, 1999. https://digitalcommons.wpi.edu/etd-theses/1063.
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"Sin Nombre virus is a hantavirus first recognized in New Mexico in 1993. This virus is responsible for causing Hantavirus Pulmonary Syndrome, an acute, life threatening illness characterized by pulmonary edema, capillary leaking, and extreme respiratory distress. CD8+ cytotoxic T-cell lines specific for Sin Nombre virus were isolated from the peripheral blood mononuclear cells (PBMC) of a donor (NM3) who was naturally infected with the Sin Nombre virus, and has survived hantavirus pulmonary syndrome (HPS). Cytotoxic T lymphocyte (CTL) assays showed that one of these cell lines, 10K, specifically recognizes a nine amino acid epitope, TAHGVGIIP (amino acids 664-672 of the precursor GPC protein), which is located in the G2 protein after cleavage. Another cell line, 10c27, specifically recognized an eight amino acid epitope, AHGVGIIP (amino acids 665-672 of the precursor GPC protein), located in the G2 protein after cleavage. Using polymerase chain reaction (PCR) and CTL assays, the recognition of these epitopes was shown to be restricted by the B35.01 Class 1 human leukocyte associated antigen (HLA) allele. This information will be useful in creating a vaccine for use in immunizing people against the Sin Nombre hantavirus, as well as elucidating the pathogenesis of this disease. "
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Servis, C. C. "The localization and identification of antigenic determinants involved in the regulation of immune responses to porcine lactate dehydrogenase B and IgGz myeloma protein." Thesis, Heriot-Watt University, 1986. http://hdl.handle.net/10399/1066.
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Ward, Chantelle Louise. "Antigenic variation in virulence determinants of Streptococcus zooepidemicus and Actinobacillus equuli involved in lower airway disease of the horse and strategies towards protective immunisation." Thesis, University of Portsmouth, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323327.
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Costa, Janina Z. "B cell epitopes in fish nodavirus." Thesis, University of Stirling, 2005. http://hdl.handle.net/1893/13240.
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Three epitope-mapping procedures were used to identify B-cell epitopes on Betanodaviruses: neutralisation escape mutant sequence analysis, phage display, and pepscan. Betanodaviruses have emerged as major pathogens of marine fish. These viruses are the aetiological agents of a disease referred to as viral nervous necrosis (VNN), which affects many species of fish that are economically valuable to the aquaculture industry. The identification of betanodavirus B-cell epitopes will facilitate the rational development of vaccines to counter VNN. A panel of mouse monoclonal antibodies (MAbs) was produced using hybridoma methodology for use in each of the epitope mapping procedures. These antibodies were characterised in Western blotting, ELISA, and virus neutralisation tests. Rabbit polyclonal sera, and serum samples from nodavirus-infected fish were also used for pepscan analyses. Attempts to produce betanodavirus neutralisation escape mutants, using plaque assay or limiting dilution based methods, were not successful. Two phage libraries expressing random peptides of seven (Ph.D.7™) or twelve (Ph.D.12™) amino acids in length as fusions to the coat protein were used to identify the ligands recognised by MAbs directed against betanodavirus. Neither of these phage libraries yielded conclusive results. Phage clones containing tandem inserts were obtained after MAb selection from library Ph.D.7™. Extensive screening and nucleotide sequence analysis of MAb-selected clones from library Ph.D.12™) failed to yield a consensus sequence. Pepscan analyses were performed using the recently developed suspension array technology (SAT). This was used to map the recognition sites of MAbs and serum samples onto a panel of overlapping synthetic peptides (12mers) that mimicked the betanodavirus coat protein. The results of pepscan analyses required careful interpretation due to the binding of antibodies and serum samples to multiple peptides. However, three regions of the nodavirus coat protein were identified as containing B-cell epitopes: amino acids 1-50, 141-162, and 181-212. These results are discussed in relation to previous studies of immune responses to betanodaviruses, and to the future development of betanodavirus vaccines and diagnostic reagents.
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Ogese, Monday. "Definition of antigenic determinants in drug hypersensitive patients : an integrated clinical, chemical and cellular approach to quantify and characterize the drug signals presented to T-Lymphocytes." Thesis, University of Liverpool, 2014. http://livrepository.liverpool.ac.uk/18733/.
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Idiosyncratic drug hypersensitivity remains a major challenge as it causes high morbidity and mortality. This is complicated by the multiple risk factors implicated and the inability to predict these reactions during the early stages of drug development. Thus, this study attempted to delineate the molecular pathomechanism(s) involved in sulfamethoxazole (SMX) hypersensitivity. The reactive metabolite, nitroso-SMX (SMX.NO) generated through the hepatic bioactivation of SMX has long been hypothesised as a major trigger of these reactions. SMX hypersensitivity has been used as a paradigm to study the role of drug metabolism in the activation of T-cells as the synthetic nitroso metabolite is available for functional studies. Metabolism of SMX in hepatic tissue has been extensively studied. CYP2C9 and Myeloperoxidase (MPO) are implicated in the formation of SMX.NO. However, it is unclear whether the SMX.NO generated in the liver migrates to the skin; the primary target in SMX hypersensitivity. It is possible that localised SMX metabolism by immune cells resident in the skin are implicated in the observed reactions. ELISA data revealed SMX metabolism in EBV-transformed B-cells used as antigen presenting cells (APCs). SMX-metabolism was significantly inhibited by methimazole. Furthermore, Western blotting and RT-PCR analyses suggested the presence of low concentrations of MPO in EBV-transformed B-cells. Interestingly, RT-PCR revealed mRNA expression of flavine containing monooxygenases (FMO1-5), TPO and LPO but the protein levels of these enzymes were not detected in immune cells. Subsequent experiments involved the generation and LC-MS/MS characterization of SMX.NO-modified MPO adducts. Although SMX.NO formed both the sulphinamide and N-hydroxysulfinamide adducts, drug specific T-cell clones failed to proliferate in response to drug-modified peptides. Since SMX.NO binds to multiple cellular proteins, it is assumed that peptides derived from the modified protein interact with a number of diverse HLA molecules to activate T-cells. However, the HLA molecules that interact with SMX.NO-modified peptides have not been defined. This study therefore examined the HLA molecules that present SMX.NO (derived peptides) to T-cells. T-cell clones (TCCs) were generated from 5 hypersensitive patients with cystic fibrosis. Fast growing TCCs from 2 SMX hypersensitive patients were used for HLA restriction studies. Drug-specific proliferative response, cytokine secretion and cytolytic markers were measured using [3H]-thymidine incorporation and ELIspot assays. Anti-human class I and class II (DR, DP, and DQ) antibodies were used to determine HLA restriction of drug-specific T-cell activation. APCs expressing similar or different HLAs were used to define the alleles involved in the presentation of SMX.NO-derived antigens to T-cells. A total of 1578 clones were tested for SMX.NO reactivity. Seventy-seven CD4+ clones were activated to proliferate and secrete IFN-ϒ, IL-5, IL-13 and granzyme-B by SMX.NO. Only one TCC was CD8+No cross reactivity with SMX was observed. The SMX.NO-specific response of clones was blocked with antibodies against MHC class II and HLA-DQ. Clones from 2 patients (Patient 1: HLA-DQB1*05:01:01G/ DQB1*06:03:01G; Patient 2: HLA-DQB1*02:01:01G/DQB1*02:01:01G) were used to define the DQ alleles involved in the presentation of SMX.NO derived antigens. SMX.NO-specific responses were detected with heterologous APCs expressing HLA-DQB1*05:01 (patient 1) and HLA-DQB1*02:01 (patient 2), but not other HLA-DQB1 alleles. Activation of PD-1 on T-cells is thought to inhibit antigen-specific T-cell priming and regulate T-cell differentiation. Thus, this study sought to measure the drug-specific activation of naïve T-cells after perturbation of PD-L1/PD-1 binding and investigate whether PD-1 signalling influences the differentiation of T-cells. Naive T-cells were co-cultured with monocyte-derived dendritic cells in the presence of SMX.NO for a period of 8 days (±PD-1/2 block) and T-cell priming investigated using readouts for proliferation and cytokine secretion. Priming of naïve T-cells against SMX.NO was found to be more effective when PD-L1 signalling was blocked. Drug-specific TCCs generated through priming and from hypersensitive patients were found to secrete IFN-γ, IL-5 and IL-13. More detailed analysis revealed two different cytokine signatures. Clones secreted either FasL/IL-22 or granzyme B. The FasL/IL22 secreting clones expressed the skin homing receptors CCR4, CCR10 and CLA and migrated in response to CCL17/CCL27. PD-1 was stably expressed at different levels on clones; however, PD-1 expression did not correlate with the strength of the antigen-specific proliferative response or the secretion of cytokines/cytolytic molecules. In conclusion, this study used a variety of in vitro assays to investigate the multiple factors involved in the pathomechanism of SMX hypersensitivity. A clear understanding of mechanisms of drug hypersensitivity will provide insights that aid drug design and reduce the frequency of such reactions.
22
Beasley, David Wayne Colin. "Identification of functional epitopes on dengue 1 environs." Thesis, Queensland University of Technology, 1999.
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23
Perikala, Satish Kumar. "Evolution of Epitope regions in HIV genome: Delineating Selective Forces acting on Conformational and Linear Epitopes." [Kent, Ohio] : Kent State University, 2010. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=kent1270735952.
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Thesis (M.S.)--Kent State University, 2010.Title from PDF t.p. (viewed Apr. 28, 2010). Advisor: Helen Piontkivska. Keywords: Conformational Epitopes; Linear Epitopes; HIV; Selective Forces; synonymous changes; nonsynonymous changes; Radical changes; Conservative changes. Includes bibliographical references (p. 81-96).
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Mickael, Claudia Silva. "Real-time RT-PCR analysis of two epitope regions encoded by the VP2 gene of infectious bursal disease viruses." Connect to this title online, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1116532082.
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Thesis (Ph. D.)--Ohio State University, 2005.Title from first page of PDF file. Document formatted into pages; contains xiii, 136 p.; also includes graphics (some col.) Includes bibliographical references (p. 120-136). Available online via OhioLINK's ETD Center
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Delpeyroux, Francis. "Insertions dans l'antigene de surface du virus de l'hepatite b : expression d'un epitope de neutralisation du poliovirus a la surface de particules de 22 nm." Paris 7, 1987. http://www.theses.fr/1987PA077198.
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Kawai, Jun. "Analyses of gene structures and antigen determinants of human class II major histocompatibility antigens." 京都大学 (Kyoto University), 1991. http://hdl.handle.net/2433/86431.
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Johnson, P. H. "#alpda#-L-fucosyltransferases involved in the biosynthesis of blood group determinants." Thesis, Open University, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.382460.
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Akpogheneta, Onome Joy. "Determinants of the longevity of antibody responses to Plasmodium falciparum antigens." Thesis, London School of Hygiene and Tropical Medicine (University of London), 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.438977.
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Layet, Corine. "Approche des bases structurales de l'antigenicite des antigenes hla de classe i." Aix-Marseille 2, 1986. http://www.theses.fr/1986AIX22046.
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Lynch, Marcus Phillip. "Evaluation of peptide based vaccines and inhibitors to prevent the onset of HTLV-1 associated diseases." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1164739126.
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Schecter, Robyn Lee. "A double determinant serum assay for detecting breast tumor associated antigen /." Thesis, McGill University, 1987. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=66270.
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Petitjean, Françoise. "Caracterisation a l'aide d'un anticorps monoclonal d'un antigene serospecifique de legionella pneumophila serogroupe 1." Paris 7, 1987. http://www.theses.fr/1987PA077058.
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Mentzer, Alexander. "Identification and characterisation of the genetic determinants of variable response to antigens from infectious agents." Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:702692ee-6971-4bc1-be8e-f6082a10cc92.
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Despite the success of vaccines in routine use worldwide, there are substantial challenges hampering our ability to develop vaccines against extant diseases including malaria and tuberculosis. Novel approaches are urgently required to help us understand immunological correlates of protection against disease and facilitate our understanding of the impact of human genetic variation on the success of diverse vaccines. To identify host genetic factors responsible for variation in antibody responses against vaccine antigens delivered routinely to infants worldwide I performed a genome-wide association study (GWAS) involving 2,499 infants recruited from three diverse sites across Africa. I identified strong genetic associations between variants in the class II major histocompatibility complex (MHC) locus and responses against five antigens: pertussis toxin (PT), filamentous haemagglutinin (FHA) and pertactin; diphtheria toxin (DT); and hepatitis B surface antigen. To characterise these associations at the gene and allelic level I developed a large, high-resolution (6-digit 'G') population-specific human leukocyte antigen (HLA) imputation reference panel including 697 individuals from the vaccine GWAS typed at 11 genes, highlighting the diversity of HLA across the African continent. Using this panel I imputed HLA into the remaining GWAS dataset to fine-map the associations to specific HLA alleles, amino acid and single nucleotide polymorphism sites; some of which were found to be African specific. I then used these HLA association findings observed with PT response to correlate, through genetics, this trait with susceptibility to whooping cough in an independently recruited and analysed set of cohorts from the UK. I further used these genetic correlations to demonstrate the relevance of levels of PT-specific circulating follicular helper T-cells and TRBV29-1 T-cell receptor gene expression levels in the development of this protective immune response against PT. By using HLA-peptide binding studies I also demonstrate the diversity of mechanisms that are involved in HLA-disease association, showing that the breadth and affinity of DT-peptide binding are increased with HLA-DRB1 alleles associated with increased DT antibody responses. Taken together, these data represent the first comprehensive genetic association study of multiple vaccine responses undertaken in African infants. These results highlight the importance of human genetics in modulating protective responses against vaccine antigens and demonstrate how such associations can be harnessed to understand biological mechanisms of protective efficacy in greater detail that may in turn facilitate future vaccine development.
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Alm, Richard A. "Molecular characterization of the haemolysin determinant of Vibrio cholerae O1 /." Title page, contents and abstract only, 1989. http://web4.library.adelaide.edu.au/theses/09PH/09pha444.pdf.
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Thesis (Ph. D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1990.Includes an appendix of author's previously published papers. Includes bibliographical references (leaves 123-160).
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Ringleb, Jennifer. "Identifikation antigener Determinanten des ZPB2-Proteins der Hauskatze und Charakterisierung ihrer kontrazeptiven und immunogenen Eigenschaften." Phd thesis, [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=974115282.
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STILLITANO, MARIA GIUSEPPINA. "Vettori virali influenzali contenenti determinanti antigenici di HIV-1 inducono immunità protettiva nei topi dopo singola immunizzazione per via mucosale." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2009. http://hdl.handle.net/2108/1091.
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Lo sviluppo di un vaccino efficace contro il virus dell’HIV-1 è una delle più importanti sfide che la Sanità pubblica sta affrontando in questi ultimi 20 anni. Una conoscenza ancora incompleta dei correlati di protezione e la variabilità genetica del virus HIV pone sostanziali impedimenti al raggiungimento di questo obiettivo. L’infezione dal virus HIV-1 si acquisisce prevalentemente attraverso la mucosa del tratto genito-rettale, pertanto, l’induzione di un’immunità mucosale rappresenta uno degli obiettivi primari nelle strategie che mirano a definire un efficace vaccino per il virus HIV-1. In particolare, un’immunizzazione in grado di attivare una risposta sia cellulare che umorale nelle mucose coinvolte nei processi di acquisizione del virus o dei linfonodi regionali può rappresentare una strategia efficace di prevenzione o controllo della replicazione e diffusione del virus dal sito di ingresso, ai tessuti linfoidi e al sangue.
Numerose osservazioni suggeriscono il ruolo importante che i linfociti T CD8+ svolgono nel contenimento dell’infezione con il virus HIV-1, tra queste l’evidenza dell’associazione temporale tra la comparsa di una risposta mediata dai linfociti T HIV-specifici in seguito all’infezione acuta e la riduzione della replicazione virale ad un livello stabile (set-point), l’associazione significativa di particolari alleli MHC I con la protezione dalla progressione della malattia, e l’aumento della replicazione virale in seguito alla deplezione dei linfociti T CD8+ in modelli di studio macachi/HIV-1.
Diverse strategie vaccinali nei confronti del virus HIV-1 si basano sull’impiego di protocolli di “prime-boost” in grado di indurre un aumento selettivo di linfociti T della memoria specifici per antigeni del virus HIV trasportati da vettori. Tra i diversi sistemi di trasporto e rilascio di antigeni, i vettori virali vivi ricombinanti sono ampiamente considerati, poiché hanno la capacità di attivare una forte risposta cellulare ed anticorpale nei confronti degli antigeni che esprimono. Il virus influenzale ricombinante, che esprime antigeni estranei provenienti dal virus HIV-1, è uno strumento promettente in tal senso.
Alla luce di tali premesse abbiamo ritenuto interessante valutare la capacità di un virus influenzale di tipo A che esprime un poliepitopo HIV fuso all’estremità N-terminale della emagglutinina (HA) matura, di indurre una risposta immunitaria cellulare e umorale in seguito all’infezione di topi BALB/c per via vaginale e di conferire protezione nei confronti di un’infezione secondaria con i virus Vaccinia ricombinanti che esprimono gli stessi antigeni. In particolare abbiamo generato un virus influenzale ricombinante dal fenotipo attenuato (WSN/CKG), che esprime il peptide cluster PCLUS3, derivato dalla glicoproteina gp120 di HIV-1, il peptide P18IIIB, epitopo per i linfociti T citotossici (CTL) derivato dal loop V3 della gp120 di HIV-1 IIIB, e un secondo epitopo CTL derivato dalla proteina Gag di HIV-1, nella regione N-terminale del virus A/WSN/CKG.
Mediante saggi ELISPOT, abbiamo osservato che una singola somministrazione per via vaginale con il virus WSN/CKG, induce nei topi una risposta immune a lungo termine mediata da linfociti T CD8+ antigene-specifici, nei linfonodi iliaci (ILN) drenanti la mucosa genito-rettale, e nella milza dei topi infettati, con un picco intorno al settimo giorno dopo l’infezione, che viene rapidamente richiamata nella milza in seguito ad un’infezione secondaria con il virus Vaccinia esprimente la proteina Env (vPE16) o Gag (vDK1). Tali risultati sono analoghi se i topi vengono immunizzati per via intranasale. Abbiamo poi analizzato mediante saggi ELISA la produzione di anticorpi antigene-specifici nel siero di topi ad un mese dall’infezione con il virus WSN/CKG e abbiamo osservato elevati livelli di IgG P18IIIB-specifiche nei topi che sono stati infettati sia per via intranasale che vaginale. L’immunità indotta attraverso l’infezione con il virus WSN/CKG è, inoltre, in grado di conferire protezione ai topi contro un’infezione secondaria con il virus Vaccinia ricombinante vPE16.
I dati che abbiamo ottenuto complessivamente dal nostro studio indicano che un’immunizzazione mucosale, ed in particolare, un’immunizzazione per via vaginale, con un virus influenzale ricombinante che esprime un poliepitopo derivante dal virus HIV-1 può indurre nei topi una risposta immunitaria antigene-specifica, protettiva e a lungo termine.The development of an efficacious HIV vaccine is one of the world’s greatest public-health challenges. The poor understanding of immune correlates of protection and the widespread genetic diversity of the virus pose substantial scientific hurdles. HIV infection is a mucosal acquired disease. Therefore, mucosal immune responses might function as a first line of defense against viral infection, and the development of vaccines against HIV-1 able to elicit mucosal immunity is a high priority. In particular, immunization targeting local mucosal surfaces or the regional lymph nodes to elicit both humoral and cellular specific immune responses may present a strategy for preventing or controlling HIV-1 replication. A number of clinical and experimental observations suggest that CD8+ T cells play an important role in the containment of HIV-1 infection. These include evidence of the temporal association between the appearance of HIV-specific CD8+ T cell responses following acute infection and the reduction in viral replication to set-point, the significant association of particular MHC class I alleles with protection from HIV-1 disease progression, and the increase in viral replication following depletion of CD8+ cells in the macaque model of AIDS virus infection. Several vaccine strategies depend on prime-boost protocols that produce a selective increase of memory T cells specific for the HIV antigen carried by vectors. Among the different antigen delivery systems, live recombinant viral vectors have the capacity of inducing strong cellular immune responses and can also prime antibody responses against expressed foreign antigens. In particular, recombinant influenza viruses engineered to express HIV-1 antigens represent promising tools to elicit both mucosal and systemic immune responses against HIV-1. Therefore, we generated a recombinant Influenza A virus (WSN/CKG) expressing the peptide cluster PCLUS3, derived from the gp120 of HIV-1, the P18IIIB cytotoxic T-lymphocyte (CTL) epitope derived from the V3 loop of HIV-1 IIIB gp120, and a second CTL epitope derived from Gag of HIV-1, fused to the N-terminal end of mature HA of A/WSN/33 virus. Then, we determined the capacity of WSN/CKG virus to induce antigen-specific mucosal and systemic immune responses upon intranasal or vaginal infection of progesterone-treated mice, and to provide protection against challenge with recombinant Vaccinia viruses, expressing Env (vPE16) or Gag (vDK1) proteins from HIV-1. We observed that a single vaginal inoculation of mice with WSN/CKG virus elicited antigen-specific CD8+ T cells, in the spleen and iliac lymph nodes (ILNs) draining the genitorectal mucosa, that peaked around day 7 postinfection, and that were rapidly recalled in the spleen upon intraperitoneal challenge with the recombinant Vaccinia viruses vPE16 and vDK1. These results were similar to those observed in mice primed intranasally with WSN/CKG virus. We therefore measured V3 loop-specific antibodies in serum samples of mice at 1 month post single immunization with WSN/CKG virus, and we observed significant levels of P18IIIB-specific IgG in mice receiving virus either by vaginal or intranasal route. Finally, we provide evidence that immune responses induced by WSN/CKG virus in the mucosal and systemic lymphoid compartments result in protection against systemic vPE16 virus challenge. Overall, these results indicate that mucosal immunization and, in particular, local vaginal immunization with recombinant Influenza viruses can provide protective and durable specific immune responses in mice.
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Taheri, Maryam. "Characterization of the structural determinants of two functions of human carcinoembryonic antigen (CEA) : intercellular adhesion and differentiation inhibition." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=36841.
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Human carcinoembryonic antigen (CEA) is a heavily glycosylated cell surface protein that belongs to the immunoglobulin superfamily (IgSF). It is overexpressed in a wide variety of human cancers. CEA functions in vitro, at least, as an intercellular adhesion molecule. It has also been shown that ectopic expression of CEA can block myogenic differentiation of rat L6 and mouse C2C12 myoblasts. Both the intercellular adhesion function of CEA as well as the inhibition of myogenic differentiation are dependent on homophilic binding of its extracellular domains. Previous studies have demonstrated that homophilic binding of CEA is mediated by double reciprocal bonds between the N and A3B3 domains of anti-parallel CEA molecules on apposed cell surfaces. Treatment of nonfusing L6 (CEA) with four fusion polypeptides representing the different domains of CEA showed that only N and A3B3 peptides were able to release the myogenic differentiation block. This information provoked studies designed to identify the precise subdomain (s) within the N domain that could be used to develop inhibitory agents to both disrupt the intercellular adhesion function and to release the myogenic differentiation block. Moreover, we show here that in addition to anti-parallel CEA-CEA interactions, parallel CEA-CEA interactions on the same cell surface are involved in the myogenic differentiation block. In this study, it is demonstrated by site directed mutagenesis, that at least three different subdomains, G30YSWYK, N42RQII, and Q 80NDTG, in the N-terminal domain are required for homophilic binding of CEA. Two cyclized peptides representing subdomains, N42RQII and Q80NDTG, were effective in blocking intercellular adhesion mediated by CEA. We also identified the binding epitope of an anti-CEA monoclonal antibody (A20), known to block homophilic binding of CEA, and showed that it bridges N42RQII and G30YSWYK. Furthermore, it was shown that G30YSWYK, N42RQII, and Q80NDTG are all involved in the diffe
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Pernollet, Martine. "Modification de l'antigène toxine tétanique par des radicaux libres oxygénés et par des protéines à activité peptidyl-prolyl cis-trans isomérase : influence sur sa présentation à des lymphocytes T spécifiques." Université Joseph Fourier (Grenoble ; 1971-2015), 1994. http://www.theses.fr/1994GRE10238.
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L'influence du radical libre oxygene, le radical hydroxyle (oh) et des proteines a activite peptidyl-prolyl cis-trans isomerase sur le traitement de l'antigene toxine tetanique par des cellules presentatrices d'antigenes (lymphocytes b) a ete etudiee. En ce qui concerne le radical hydroxyle, sa production par des cellules de type macrophagique a ete reproduite a l'aide d'un systeme chimique. La toxine tetanique traitee par le radical oh subit un changement de conformation, et des liaisons bityrosine intramoleculaires resistantes a la proteolyse sont formees. La toxine ainsi modifiee est plus resistante a la proteolyse in vitro par des fractions endosomales isolees des cellules presentatrices. Cette diminution de proteolyse est correlee a une meilleure presentation par des cellules presentatrices fixees, a des lymphocytes t lorsque cet antigene est pre-proteolyse in vitro par des fractions endosomales. Ainsi, le radical oh favorise la presentation des epitopes de la toxine en les protegeant contre une proteolyse trop importante. La production du radical oh par un autre systeme chimique a permis de montrer l'existence d'un site de fixation pour le zinc a l'interieur de la chaine legere de la toxine, au niveau d'un epitope t. En ce qui concerne les activites peptidyl-prolyl cis-trans isomerases (ppiase), celles-ci ont pu etre mises en evidence dans les fractions endosomales de cellules presentatrices. L'addition a ces fractions endosomales de proteines a activite ppiase telles que la cyclophiline ou la fkbp augmente la proteolyse de la toxine tetanique. Il reste a tester les consequences de cet effet sur la presentation de cette derniere a des lymphocytes t. Un autre point de ce travail a ete la mise en evidence de la phosphorylation in vitro de la cyclophiline par la proteine kinase c purifiee. L'etude des consequences de cette phosphorylation sur l'activite ppiase de la cyclophiline est en cours
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Petit, Edwige. "Application des anticorps monoclonaux a l'etude de la conformation de l'apo a-i a la surface des lipoproteines de haute densite." Toulouse 3, 1987. http://www.theses.fr/1987TOU30095.
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Sánchez, Arcila Juan Camilo. "Estudo de determinantes antigênicos para respostas imunes de células humanas em KMP-11 (Kinetoplastid membrane protein – 11) de Leishmania Amazonensis." reponame:Repositório Institucional da FIOCRUZ, 2010. https://www.arca.fiocruz.br/handle/icict/4103.
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CNPq e PEC-PG
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.
As leishmanioses formam um grupo de doenças antropozoonóticas, endêmicas em 88 países e presentes em quase todos os estados brasileiros. O desenvolvimento de uma vacina contra das leishmanioses é altamente desejável já que a terapia e as características biológicas e ecoepidemiólogicas dos parasitos e seus vetores associados não facilitam o controle da doença. Kinetoplastid Membrane Protein-11 (KMP-11) é uma molécula candidata a vacina contra as leishmanioses. Utilizando ferramentas in vitro e in silico, foi avaliada a antigenicidade de 13 peptídeos sintéticos abrangendo a sequência inteira de KMP-11. Para os estudos in vitro foram usadas células mononucleares de sangue periférico (PBMC) de pacientes com leishmaniose cutânea (LC) do estado do Rio de Janeiro. Estas células foram empregadas para testar a antigenicidade dos 13 peptídeos individualmente e da proteína integral KMP-11 recombinante, através de ensaios de ELISA e ELISPOT. Na dosagem de citocinas por ELISA observamos que a proteína KMP-11 recombinante estimulou respostas de citocinas quase sempre superiores às induzidas pelos peptídeos isolados. No que se refere a IFN-, dois dos 13 peptídeos (P9 e P10) estimularam níveis desta citocina significativamente (p<0,05) mais baixos do que os observados com a proteína inteira. Dez peptídeos (P4, P5, P6, P7, P8, P9, P10, P11, P12 e P13) apresentaram níveis de IL-10 e TNF-α significativamente inferiores aos observados com a proteína inteira. KMP-11 mostrou-se um potente indutor de IL-10 em PBMC de pacientes com LC, confirmando resultados anteriormente publicados, mas também foi capaz de induzir a produção de IFN-γ e altos níveis de TNF-α, em níveis superiores aos dos peptídeos estudados. Na avaliação da razão IFN-γ/IL-10 observou-se um acentuado contraste entre a maioria dos peptídeos e a proteína KMP-11. As respostas a 11 dos 13 peptídeos mostraram um claro viés de resposta de tipo 1 (IFN-γ>IL-10), a exceção dos peptídeos P1 e P10 (IFN-γ
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Shang, Lingling, and Lingling Shang. "Basic cultural determinants of recombinant protein yield in Nicotiana benthamiana used as a transient expression host for the flu vaccine antigen hemagglutinin H1." Doctoral thesis, Université Laval, 2019. http://hdl.handle.net/20.500.11794/37216.
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Les plantes sont des hôtes prometteurs pour la production de protéines recombinantes d’intérêt médical et de nombreuses études ont été réalisées au cours des années pour optimiser le taux d’expression des transgènes ou la maturation des protéines en systèmes végétaux. En comparaison, les connaissances demeurent limitées au sujet de l’influence des facteurs environnementaux et des pratiques culturales sur l’expression et le rendement en protéines recombinantes dans les plantes. Les pratiques culturales courantes en serriculture, si elles permettent en général une production importante de biomasse et des rendements élevés en produits horticoles, ne sont pas nécessairement bien adaptés à la production de protéines recombinantes dans un contexte de moléculture. Dans cette étude, nous avons étudié les effets d’un enrichissement en CO2 atmosphérique, d’une forte irradiance sur le couvert végétal, d’une fertigation riche en ammonium et d’une densité de plantation élevée sur la croissance, le développement et le rendement en protéine recombinante chez l’hôte d’expression Nicotiana benthamiana utilisé pour la production du principe actif d’un vaccin contre la grippe, l’hémagglutinine H1 du virus de l’influenza. En bref, nos données ont montré les effets positifs (1) d’un enrichissement en CO2, d’une forte luminosité, d’un éclairage intercalaire dans la canopée végétale ou d’une solution nutritive riche en ammonium sur la production de biomasse et le contenu en protéines dans la plante; et (2) d’un éclairage intercalaire et d’une forte densité culturale sur le rendement en protéine H1 par unité de surface en culture. En revanche, le rendement en H1 n’a pas été altéré, ou l’a été négativement, sous de fortes concentrations en CO2 atmosphérique, sous une forte luminosité au-dessus du couvert végétal ou par une solution nutritive riche en ammonium. En somme, nos données indiquent que les conditions de culture optimales pour la production de produits horticoles en conditions confinées peuvent ne pas être appropriées dans un contexte de moléculture où l’objectif ultime est le rendement en protéine recombinante, non pas la production de biomasse foliaire, la teneur en nutriments ou le rendement en fruits ou en fleurs.Les plantes sont des hôtes prometteurs pour la production de protéines recombinantes d’intérêt médical et de nombreuses études ont été réalisées au cours des années pour optimiser le taux d’expression des transgènes ou la maturation des protéines en systèmes végétaux. En comparaison, les connaissances demeurent limitées au sujet de l’influence des facteurs environnementaux et des pratiques culturales sur l’expression et le rendement en protéines recombinantes dans les plantes. Les pratiques culturales courantes en serriculture, si elles permettent en général une production importante de biomasse et des rendements élevés en produits horticoles, ne sont pas nécessairement bien adaptés à la production de protéines recombinantes dans un contexte de moléculture. Dans cette étude, nous avons étudié les effets d’un enrichissement en CO2 atmosphérique, d’une forte irradiance sur le couvert végétal, d’une fertigation riche en ammonium et d’une densité de plantation élevée sur la croissance, le développement et le rendement en protéine recombinante chez l’hôte d’expression Nicotiana benthamiana utilisé pour la production du principe actif d’un vaccin contre la grippe, l’hémagglutinine H1 du virus de l’influenza. En bref, nos données ont montré les effets positifs (1) d’un enrichissement en CO2, d’une forte luminosité, d’un éclairage intercalaire dans la canopée végétale ou d’une solution nutritive riche en ammonium sur la production de biomasse et le contenu en protéines dans la plante; et (2) d’un éclairage intercalaire et d’une forte densité culturale sur le rendement en protéine H1 par unité de surface en culture. En revanche, le rendement en H1 n’a pas été altéré, ou l’a été négativement, sous de fortes concentrations en CO2 atmosphérique, sous une forte luminosité au-dessus du couvert végétal ou par une solution nutritive riche en ammonium. En somme, nos données indiquent que les conditions de culture optimales pour la production de produits horticoles en conditions confinées peuvent ne pas être appropriées dans un contexte de moléculture où l’objectif ultime est le rendement en protéine recombinante, non pas la production de biomasse foliaire, la teneur en nutriments ou le rendement en fruits ou en fleurs.
Plants are promising hosts for the production of medically-useful recombinant protein sand numerous studies have been done over the years to optimize transgene expression rates and protein maturation processes in plant systems. By comparison, little is still known about the influence of basic environmental factors and cultural practices on the expression and yield of heterologous proteins in plants. Current cultural practices in greenhouse settings, that generally allow for an increased biomass or food/flower product yield, are not necessarily well suited to recombinant protein production in a molecular farming context. In this study, we investigated the effects of CO2 enrichment, supplemental lighting, ammonium fertigation and plant culture density on growth, development and recombinant protein yield of the protein expression host Nicotiana benthamiana used to express the flu vaccine antigen influenza virus hemagglutinin H1. In brief, our data showed (1) atmospheric CO2 enrichment, high-light irradiance, supplemental LED inter-lighting in the plant canopy and high-ammonium fertigation to enhanced leaf biomass production and endogenous protein content on a plant basis, and (2) LED inter-lighting or elevated plant density to increase recombinant protein yield on a whole-crop area basis. On the other hand, H1 content was not influenced or negatively affected by CO2 enrichment, high-light irradiance or high-ammonium supply on a leaf fresh weight basis. Overall, our findings indicate that the optimal cultural practices for the production of horticultural food products or ornementals in controlled environment settings may not be optimal in molecular farming settings, where the ultimate goal is recombinant protein yield and quality, not leaf biomass, nutrient content, fruit yield or flower quality.
Plants are promising hosts for the production of medically-useful recombinant protein sand numerous studies have been done over the years to optimize transgene expression rates and protein maturation processes in plant systems. By comparison, little is still known about the influence of basic environmental factors and cultural practices on the expression and yield of heterologous proteins in plants. Current cultural practices in greenhouse settings, that generally allow for an increased biomass or food/flower product yield, are not necessarily well suited to recombinant protein production in a molecular farming context. In this study, we investigated the effects of CO2 enrichment, supplemental lighting, ammonium fertigation and plant culture density on growth, development and recombinant protein yield of the protein expression host Nicotiana benthamiana used to express the flu vaccine antigen influenza virus hemagglutinin H1. In brief, our data showed (1) atmospheric CO2 enrichment, high-light irradiance, supplemental LED inter-lighting in the plant canopy and high-ammonium fertigation to enhanced leaf biomass production and endogenous protein content on a plant basis, and (2) LED inter-lighting or elevated plant density to increase recombinant protein yield on a whole-crop area basis. On the other hand, H1 content was not influenced or negatively affected by CO2 enrichment, high-light irradiance or high-ammonium supply on a leaf fresh weight basis. Overall, our findings indicate that the optimal cultural practices for the production of horticultural food products or ornementals in controlled environment settings may not be optimal in molecular farming settings, where the ultimate goal is recombinant protein yield and quality, not leaf biomass, nutrient content, fruit yield or flower quality.
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Franzke, Kati [Verfasser], and Ulrich [Akademischer Betreuer] Hahn. "Identifizierung antigener Determinanten der E-Proteine von Dengue-Viren zum Nachweis Dengue- sowie Serotyp-spezifischer Antikörper / Kati Franzke. Betreuer: Ulrich Hahn." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2015. http://d-nb.info/106893087X/34.
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Machado, Eleuza Rodrigues. "Estudo dos fatores determinantes do grau de infectividade de linhagens de Strongyloides venezuelensis e analise de antigenos heterologos no imunodiagnostico da estrongiloidiase humana." [s.n.], 2003. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317147.
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Orientadores: Marlene Tiduko Ueta, Lucia Helena FaccioliTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Neste estudo mostramos que camundongos infectados com as linhagens L-2 ou L-49 de Strongyloides venezuelensis induzem aumento de leucócitos totais, eosinófilos (EO) e células mononucleares (MO) no sangue, na cavidade peritoneal (LCP) e espaço broncoalveolar (LBA) de formas semelhantes. Ambas linhagens estimularam síntese de citocinas do padrão TH1 ou TH2 da resposta imune, com maior tendência para TH2. L-2 foi mais infectante que L-49, induzindo precocemente maior síntese de IL-4, IL-5 e IFNy e anticorpos. Houve correlação positiva entre indução de maior síntese de IL-4 e de IgG e maior expulsão do parasita pela L-2. Não houve correlação entre indução de maior síntese de IL-5 e maior proliferação e recrutamento de EO e MO. O tempo e a temperatura de manutenção das culturas influenciaram na infectividade do parasita. Com menor tempo de permanência das larvas nas culturas estas induziram resposta imune inata mais intensa no sangue e no LCP, com eliminação mais rápida dos parasitas. Larvas obtidas a temperatura mais alta induziram eosinofilia mais intensa e precoce no LCP, e sob temperatura mais baixa, a eosinofilia foi menor e tardia. Larvas mantidas por menor tempo nas culturas estimularam resposta imune celular capaz de expulsar mais rapidamente as fêmeas parasitas, e as mantidas por maior tempo e sob temperaturas mais altas induziram maior síntese de anticorpos e de IL-4 e IL-10. Larvas mantidas por menor tempo nas culturas induziram maior síntese de IL-12, e as de maior tempo, maior produção de IFN-y. O tempo e/ou temperatura não influenciaram a síntese de IL-5. Observamos que leucotrienos participam do aumento de leucócitos totais, EO e MO no sangue, no LCP e LBA na estrongiloidíase. A IL-5 também participa no aumento de leucócitos nesses compartimentos. IL-12 e IFN-y protegem o parasita contra as defesas do hospedeiro. Verificamos que as oito linhagens de S. venezuelensis apresentaram antigenicidade semelhante, detectada por imunofluorescência indireta, ELISA e "Immunoblot". O anticorpo IgG anti-S. stercotalis reconheceu a fração antigênica de peso molecular aparente de 45 kDa. Assim, antígenos destas linhagens podem ser usados no imunodiagnóstico da estrongiloidíase humana. Palavras chave: Strongyloides venezue/ensis, estrongiloidíase, leuc6citos totais, eosinofilia, células mononucleares, citocinas, anticorpos, leucotrienos, imunodiagnóstico
Abstract: In this study, we have shown that, in mice, L-2 and L-49 strains of S. venezuelensis induced an increase in the total number of leukocytes, eosinophils (EO) and mononuclear cells in blood, peritoneal cavity (LCP) and in the bronchoalveolar space (LBA), in a similar manner. Both strains stimulated synthesis of TH1 or TH2 type immune response cytokines, with a greater tendency to TH2. L-2 strain was more infective than L-49, inducing earlier synthesis of IL-4, IL-5 and IFN-y, and antibodies. In L-2, there was a direct correlation between greater production of IL-4 and IgG and expulsion of parasites, but no correlation to greater synthesis of IL-5 or proliferation and recruitment of EO and MO. Time and temperature had influence on parasite infectivity. Larvae that remained in the cultures for shorter periods of time had their infectivity affected, inducing a more intense innate immune response in blood and LCP, with rapid elimination of the parasites. Parasites kept in higher temperatures rapidly induced higher eosinophil levels in LCP, while for low temperatures, eosinophils appeared later and in lower numbers. When larvae were cultured for shorter periods of time, they stimulated a cellular immune response capable of quickly expelling female parasites, while those submitted to shorter periods and high temperatures induced a higher synthesis of antibodies, IL-4 and IL-10. Larvae kept in culture for short periods induced greater synthesis of IL-12, while longer periods lead to greater IFN-y production. Neither time nor temperature affected IL-5 synthesis. It was observed that, in strongyloidiasis, leukotrienes participate on the increase of total leukocyte numbers, EO, and MO in blood, LCP and LBA. IL-5 is also involved in the increase of leukocyte numbers in these compartments. 80th IL-12 and IFN-y protect the parasite against host defense. It was also verified, through indirect immunofluorescence, ELISA, and Immunoblot, that eight strains of S. venezuelensis presented similar antigenicity. The antibody anti-S. stercoralis IgG was able to recognize the antigenic fraction with apparent molecular weight of 45 kDa. Therefore, antigens to these strains may be used for the immunodiagnosis of human strongyloidiasis. Key words: S. venezue/ensis, strongyloidiasis, leukocytes, eosinophils, mononuclear cells, cytokines, antibody, leukotrienes, immunodiagnosis, mice
Doutorado
Doutor em Parasitologia
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Peri, C. "INVESTIGATING AND PREDICTING THE DETERMINANTS OF PROTEIN-PROTEIN INTERACTIONS THROUGH COMPUTATIONAL-STRUCTURAL BIOLOGY APPROACHES: IMPLICATIONS FOR STRUCTURAL VACCINOLOGY." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/243392.
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Clarifying the physico-chemical principles of protein-protein interactions is critically important to understand the relationships between biological structures and functions in all biochemical mechanisms. In this project we aim to develop, validate and apply new computational-theoretical methods to study and predict the binding regions of proteins starting from 3D structural information and from the analysis of the conformational and physico-chemical properties of the constituting amino acids. In particular, this project entails the integrated analysis of the energetic properties of different datasets of proteins solved at high resolution. In this context, we have focused on four main subjects with different, yet highly intertwined, objectives. The first subject will address the application of an energy-based computational predictor for the identification of possible antibody-binding surfaces (epitopes) of protein antigens from the pathogen Burkholderia pseudomallei, responsible for human melioidosis. The second will focus on the expansion of the same rationale, adapting the method towards different applications, and including as a novel functionality the prediction of MHC-II coupled epitopes to elicit the intervention of T helper cells. The third objective concerns the design and characterization of peptides and peptidomimetics to optimize the properties of the identified epitopes as better vaccine candidates. The fourth one will pursue the investigation of the energetic determinants of interacting proteins in a more general context (not limited to immunogenic epitopes), aiming at the identification of an energy-based property describing the interaction event at the atomistic level of resolution. This part of the project is aimed at the development of a computational tool based on such property to help improve the understanding of the determinants of protein interactions and help predict their binding interfaces and orientation. All four subjects have been investigated in the broad spectrum of activities of an academic consortium, devoted to the identification of antigens from B. pseudomallei showing sufficient immunogenic potential to be considered as components for a vaccine against the pathogen. The computational methods developed and tested within this framework have theoretical as well as practical implications, from the physico-chemical study and characterization of protein-protein interactions, to the design of biologically active molecules.
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Zinn-Justin, Sophie. "Etude structurale du site toxique et de deux sites antigéniques d'une toxine curaremimétique." Châtenay-Malabry, Ecole centrale de Paris, 1993. http://www.theses.fr/1993ECAP0326.
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Le but de l'étude présentée dans ce document est de montrer l'apport de la détermination de la structure tridimensionnelle en solution de la toxine extraite du venin du naja nigricollis a la compréhension des mécanismes biologiques dans lesquels la toxine est impliquée. Cette étude comprend trois parties. Dans une première partie, la résolution de la structure tridimensionnelle de la toxine (61 acides amines, 4 ponts disulfure) est présentée. Le repliement de la toxine consiste en 3 boucles principales stabilisées par 3 ponts disulfure et 1 boucle c-terminale de plus petite taille stabilisée par un 4eme pont disulfure. Cette structure est très proche de celle d'une autre toxine de serpent, l'erabutoxine B, telle qu'elle a été déterminée par radiocristallographie. En revanche, aucune des molécules d'eau observées dans le cristal d'erabutoxine B n'a pu être mis en évidence par RMN sur la toxine. L'analyse des vitesses d'échange des protons amide de la toxine a plusieurs températures a permis d'aborder l'étude de la dynamique de la toxine. Dans une deuxième partie, les sites antigéniques correspondant a deux anticorps neutralisant l'activité toxique de la toxine ont été identifies. La comparaison de ces sites avec le site toxique a permis d'élucider les événements moléculaires associes au mécanisme de neutralisation. Dans une dernière partie, la connaissance de la structure du site toxique a permis de tenter de reconstruire ce site au sein d'une plateforme structurale stable et de petite taille
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Jadal, Mohamed. "Etude biochimique et immunologique comparee des proteines du cytosquelette cortical des cilies ophryoscolecidae." Clermont-Ferrand 2, 1988. http://www.theses.fr/1988CLF21127.
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L e cortex somatique des cilies entodiniomorphes resulte de la superposition de 4 constituants principaux : la membrane plasmique, l'epiplasme, des rangees de microtubules et une couche filamenteuse plus interne, la limite ecto-endoplasmique (lee). Ces 3 derniers representent les elements squelettiques du cortex. La caracterisation biochimique et immunologique de l'epiplasme et de la lee a ete absorbee chez 4 especes. Des proteines ont ete identifiees, leur poids moleculaire determine chez les differents cilies examines. Les proprietes des proteines epiplasmiques ont ete definies : role structural, propriete de solubilisation, variation de pm entre les differentes especes, presence de domaines structureux homologues
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Bruyère, Thierry. "Etude immunologique et genetique d'une adhesine de streptococcus mutans." Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13044.
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Hartmann, Marie-Louise. "Application des anticorps monoclonaux a l'etude de quelques proteines de sous-unite 30s du ribosome d'e. Coli." Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13199.
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Nous avons prepare des anticorps monoclonaux diriges contre des proteines de la sous-unite 30s du ribosome d'e. Coli en vue de leur utilisation comme sondes fonctionnelles et structurales. Nous presentons d'abord une technique de fractionnement des proteines de la sous -unite 30s du ribosome d'e. Coli par chromatographie en phase inverse sur un systeme fplc. Puis, nous decrivons l'obtention et la caraterisation d'anticorps monoclonaux diriges contre quelques proteines de la sous-unite 30s: les proteines s1, s4, s12 et s17. Ces anticorps ont permis de montrer que des modifications des conditions ioniques dans lesquelles se trouvent la sous-unite 30s affectent l'accessibilite ou la conformation d'epitopes situes a la surface des proteines s4 (ou s17) et s12. Nous avons pu mettre en evidence des changements conformationnels discrets entre deux etats differents de la sous-unite 30s. Enfin, nous presentons l'obtention et la caracterisation d'anticorps monoclonaux diriges contre la proteine s1
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Alami, Harchali Asmae. "Détection par immunephelemetrie sur supports microparticulaires d'autoanticorps anti-thyroïde de spécificité épisodique définie : mise au point de la méthode et applications." Nancy 1, 1994. http://www.theses.fr/1994NAN10286.
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Ce travail a pour objectif d'adapter à la détection et à la caractérisation d'autoanticorps, la technique d'immunonephelemetrie sur supports microparticulaires. Le but est d'affiner le pouvoir discriminant de cette technique jusqu'à l'identification de la spécificité épitopique des anticorps décelés. Un premier groupe d'applications porte sur la détection des autoanticorps anti-thyroglobuline humaine. Un test de détection de ces autoanticorps anti-domaine antigénique deux de la molécule de thyroglobuline, a été développé. Il est basé sur l'aptitude des autoanticorps à inhiber les systèmes agglutinants thyroglobuline-anticorps monoclonaux anti-thyroglobuline ont ainsi été détectés et quantifiés chez tous les patients atteints de thyroïdite d'Hashimoto et dans plus du trois-quarts des patients présentant une maladie de basedow non traitée. Les résultats obtenus se comparent favorablement à ceux de l'immunofluorescence indirecte et de l'hemagglutination passive. Le système mis au point peut aussi se prêter au dosage de la thyroglobuline sérique par inhibition. La détection des autoanticorps anti-thyroperoxydase humaine est calquée dans son principe sur le protocole mis au point pour les anticorps anti-thyroglobuline. Ce test confirme la polyclonalité des autoanticorps anti-thyroperoxydase et leur réactivité préférentielle envers les épitopes localisés sur les domaines antigéniques a et b de la molécule de thyroperoxydase, épitopes impliques dans les maladies thyroïdiennes auto-immunes. Ces mêmes spécificités sont observées chez les patients atteints de thyroïdite d'Hashimoto et de maladie de basedow. Les données immunonephelemetriques sont en accord avec les résultats des autres moyens d'investigation et avec les autres éléments des dossiers cliniques
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BEAULANDE, MELANIE. "Etude de l'asparaginyl-arnt synthetase cytosolique humaine et son implication dans des reactions auto-immunitaires." Université Joseph Fourier (Grenoble), 2000. http://www.theses.fr/2000GRE10129.
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L'adnc codant pour l'asnrs cytosolique humaine (548 acides amines) a ete isole, sequence et exprime dans un systeme d'expression bacterien. La sequence proteique humaine obtenue est comparee a celles d'asnrs de d'autres organismes (procaryotes et eucaryotes) afin d'etudier la conservation ou non des differents domaines. Apres la mise au point d'un protocole de purification, la proteine humaine a pu etre utilisee pour des analyses biochimiques et immunologiques. La proteine humaine a ete utilisee pour des caracterisations biochimiques, en particulier pour sa specificite de reconnaissance pour le substrat arnt a s n de differents organismes. L'enzyme humaine montre une plus grande specificite pour l'arnt de foie de veau compare a l'arnt de levure de de bacterie. Ces etudes montrent aussi que la synthetase humaine fixe l'arnt bacterien mais n'est pas capable de l'aminoacyler. Le role des extensions n-terminales des synthetases eucaryotes reste encore tres contreverse. L'asnrs humaine est une enzyme libre ne rentrant pas dans la composition du complexe multi-enzymatique. L'analyse comparative de l'asnrs humaine et de quelques mutants suggere la presence d'interactions stabilisatrices entre l'extension n-terminale eucaryote et le substrat arnt. Un aspect particulier de l'asnrs humaine est d'etre specifiquement reconnue et neutralisee par un groupe d'auto-anticorps impliques dans le developpement de maladies auto-immunitaires (syndrome anti-synthetase). L'inhibition s'effectue lors de la deuxieme etape de la reaction d'aminoacylation via une augmentation d'affinite, induite par l'auto-anticorps, entre la proteine et son arnt. Deux epitopes ont ete localises au sein de l'asnrs humaine : un epitope reagissant en western-blot dans la partie n-terminale, et un epitope conformationnel immunodominant, responsable de la neutralisation d'activite, dans la partie catalytique de la proteine. Enfin, pour cloturer ce travail, des essais de cristallogenese de l'asnrs humaine entiere ont ete entrepris afin d'obtenir un complement structural capable de nous renseigner sur la partie immunogenique de l'asnrs et sur l'organisation structurale et fonctionnelle de l'extension n-terminale eucaryote. Les premiers cristaux ont recemment ete obtenus. Les conditions sont en cours d'amelioration.