{"id":{"repo_id":"aachen","oai_identifier":"oai:publications.rwth-aachen.de:51872"},"canonical_url":"https://search.dev.ndltd.org/etd/aachen/oai:publications.rwth-aachen.de:51872","repository":{"repo_id":"aachen","name":"RWTH Aachen University","base_url":"https://publications.rwth-aachen.de/oai2d"},"display":{"title":"Identifizierung regulatorischer Proteindomänen der zytolytischen Eigenschaften des humanen P2X7-Rezeptors","abstract":"The P2X7-receptor is a ligand-gated ion channel of the P2X-purigenic-receptor group. In different of the other receptors of the P2X-family the P2X7-receptor is able to build both an ion channel and a cytolytic-pore which leads to membrane-blebbing. The aim of this MD-thesis was to develop a method which provides evidence whether the activation of selected P2X7-receptor-mutants with agonists like ATP or the more potent BzATP mediates a cytolytic membrane pore. As cell-system we choose COS-7-cells because of the lack of an endogenous P2X7-Receptor. As the cytolytic pore is permeable for cations up to a molecular mass of 900 Da, its possible to detect the cytolytic pore through fluorescence-microscopy of diffused 320 Da Ethidium-bromid cations which intercalates with the cellular DNA. DIC-microscopy accorded as an easier way to detect membrane-blebbing with the same explanatory power. After addition of the agonists a Zeiss-cell-observer system acquires over a period of 30 minutes automatically a series of pictures. After 30 minutes of activation there were no further changes of the membrane-integrity detectable.The co-transfection of an EGFP-cDNA accorded to an advantageous way to differ between transfected from un-transfected cells. Furthermore with the co-transfection of the EGFP-cDNA it was possible to localize changings of plasmamembrane at an early state. To valdidate the developed method, I investigated COS7-cells expressing the wildtype-P2X7-cDNA and 264.7-RAW-macrophages which expresses an endogenous P2X7-receptor. Furthermore I investigated the previous published E496A-Polymorphismem. According to the literature the activation of transfected COS7-cells couldnt mediate membrane-blebbing. The expression of the investigated P2X7-receptor-mutants was verified by western blot. After developing a suitable assay to show the cytolytic pore-formation, some hP2X7-receptor mutants were cloned and investigated. An up to now unpublished glycin-arginin-polymorphism at position 441 leads to a extended activation period with BzATP. Remarkable is the great number of cystein-residues in the cytosolic localized c-terminal endodomain. The exchange of the 362 cystein-residue also leads to a extended activation period and the membrane-blebs are formed less than the wildtype. Its possible that the high amount of cystein-residues is responsible for the species-dependent difference of the cytolytic pore-formation kinetic. The truncation of the last 12 amino acids resulted to a receptor that looses pore forming abilities. If the tyrosine motif 400YSVF403 can also affect pore forming abilities is not investigated finally, because of weak expression of the 400YSVF403-P2X7-receptor in the COS7-cell line. Finally these experiments show that a lot of amino acids take influence of the hP2X7-receptor pore forming abilities.","abstract_html":"The P2X7-receptor is a ligand-gated ion channel of the P2X-purigenic-receptor group. In different of the other receptors of the P2X-family the P2X7-receptor is able to build both an ion channel and a cytolytic-pore which leads to membrane-blebbing. The aim of this MD-thesis was to develop a method which provides evidence whether the activation of selected P2X7-receptor-mutants with agonists like ATP or the more potent BzATP mediates a cytolytic membrane pore. As cell-system we choose COS-7-cells because of the lack of an endogenous P2X7-Receptor. As the cytolytic pore is permeable for cations up to a molecular mass of 900 Da, its possible to detect the cytolytic pore through fluorescence-microscopy of diffused 320 Da Ethidium-bromid cations which intercalates with the cellular DNA. DIC-microscopy accorded as an easier way to detect membrane-blebbing with the same explanatory power. After addition of the agonists a Zeiss-cell-observer system acquires over a period of 30 minutes automatically a series of pictures. After 30 minutes of activation there were no further changes of the membrane-integrity detectable.The co-transfection of an EGFP-cDNA accorded to an advantageous way to differ between transfected from un-transfected cells. Furthermore with the co-transfection of the EGFP-cDNA it was possible to localize changings of plasmamembrane at an early state. To valdidate the developed method, I investigated COS7-cells expressing the wildtype-P2X7-cDNA and 264.7-RAW-macrophages which expresses an endogenous P2X7-receptor. Furthermore I investigated the previous published E496A-Polymorphismem. According to the literature the activation of transfected COS7-cells couldnt mediate membrane-blebbing. The expression of the investigated P2X7-receptor-mutants was verified by western blot. After developing a suitable assay to show the cytolytic pore-formation, some hP2X7-receptor mutants were cloned and investigated. An up to now unpublished glycin-arginin-polymorphism at position 441 leads to a extended activation period with BzATP. Remarkable is the great number of cystein-residues in the cytosolic localized c-terminal endodomain. The exchange of the 362 cystein-residue also leads to a extended activation period and the membrane-blebs are formed less than the wildtype. Its possible that the high amount of cystein-residues is responsible for the species-dependent difference of the cytolytic pore-formation kinetic. The truncation of the last 12 amino acids resulted to a receptor that looses pore forming abilities. If the tyrosine motif 400YSVF403 can also affect pore forming abilities is not investigated finally, because of weak expression of the 400YSVF403-P2X7-receptor in the COS7-cell line. Finally these experiments show that a lot of amino acids take influence of the hP2X7-receptor pore forming abilities.","abstract_has_math":false,"creators":["Schmitz, Stephan"],"institution":"Publikationsserver der RWTH Aachen University","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Schmalzing, Günther"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2007,"date_issued":"2007","date_published":"2007","updated_at":"2026-07-30T19:40:42Z","subjects":["info:eu-repo/classification/ddc/610","Medizin","Ionenkanal","P2X7","ionchannel","ATP","BzATP","GFP"],"languages":["ger"],"rights":["info:eu-repo/semantics/openAccess"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-114121%22"],"render_values":[{"text":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-114121%22","href":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-114121%22","code":true}]}]},"links":{"outbound_url":"https://publications.rwth-aachen.de/record/51872","outbound_label":"Repository record","outbound_source":"dc:identifier"},"source_record":{"url":"https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A51872","prefix":"oai_dc"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Schmalzing, Günther"]},{"key":"dc:creator","label":"Author","values":["Schmitz, Stephan"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:coverage","label":"Dc Coverage","values":["DE"]},{"key":"dc:date","label":"Dc Date","values":["2007"]},{"key":"dc:publisher","label":"Institution","values":["Publikationsserver der RWTH Aachen University"]},{"key":"dc:relation","label":"Dc Relation","values":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-18529"]},{"key":"dc:type","label":"Dc Type","values":["info:eu-repo/semantics/doctoralThesis","info:eu-repo/semantics/publishedVersion"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["info:eu-repo/classification/ddc/610","Medizin","Ionenkanal","P2X7","ionchannel","ATP","BzATP","GFP"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["ger"]},{"key":"dc:rights","label":"Dc Rights","values":["info:eu-repo/semantics/openAccess"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/record/51872","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-114121%22"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["The P2X7-receptor is a ligand-gated ion channel of the P2X-purigenic-receptor group. In different of the other receptors of the P2X-family the P2X7-receptor is able to build both an ion channel and a cytolytic-pore which leads to membrane-blebbing. The aim of this MD-thesis was to develop a method which provides evidence whether the activation of selected P2X7-receptor-mutants with agonists like ATP or the more potent BzATP mediates a cytolytic membrane pore. As cell-system we choose COS-7-cells because of the lack of an endogenous P2X7-Receptor. As the cytolytic pore is permeable for cations up to a molecular mass of 900 Da, its possible to detect the cytolytic pore through fluorescence-microscopy of diffused 320 Da Ethidium-bromid cations which intercalates with the cellular DNA. DIC-microscopy accorded as an easier way to detect membrane-blebbing with the same explanatory power. After addition of the agonists a Zeiss-cell-observer system acquires over a period of 30 minutes automatically a series of pictures. After 30 minutes of activation there were no further changes of the membrane-integrity detectable.The co-transfection of an EGFP-cDNA accorded to an advantageous way to differ between transfected from un-transfected cells. Furthermore with the co-transfection of the EGFP-cDNA it was possible to localize changings of plasmamembrane at an early state. To valdidate the developed method, I investigated COS7-cells expressing the wildtype-P2X7-cDNA and 264.7-RAW-macrophages which expresses an endogenous P2X7-receptor. Furthermore I investigated the previous published E496A-Polymorphismem. According to the literature the activation of transfected COS7-cells couldnt mediate membrane-blebbing. The expression of the investigated P2X7-receptor-mutants was verified by western blot. After developing a suitable assay to show the cytolytic pore-formation, some hP2X7-receptor mutants were cloned and investigated. An up to now unpublished glycin-arginin-polymorphism at position 441 leads to a extended activation period with BzATP. Remarkable is the great number of cystein-residues in the cytosolic localized c-terminal endodomain. The exchange of the 362 cystein-residue also leads to a extended activation period and the membrane-blebs are formed less than the wildtype. Its possible that the high amount of cystein-residues is responsible for the species-dependent difference of the cytolytic pore-formation kinetic. The truncation of the last 12 amino acids resulted to a receptor that looses pore forming abilities. If the tyrosine motif 400YSVF403 can also affect pore forming abilities is not investigated finally, because of weak expression of the 400YSVF403-P2X7-receptor in the COS7-cell line. Finally these experiments show that a lot of amino acids take influence of the hP2X7-receptor pore forming abilities."]},{"key":"dc:source","label":"Dc Source","values":["Aachen : Publikationsserver der RWTH Aachen University 72 S. : Ill., graph. Darst. (2007). = Aachen, Techn. Hochsch., Diss., 2007"]},{"key":"dc:title","label":"Title","values":["Identifizierung regulatorischer Proteindomänen der zytolytischen Eigenschaften des humanen P2X7-Rezeptors"]}]}],"canonical_facts":{"dc:contributor":["Schmalzing, Günther"],"dc:coverage":["DE"],"dc:creator":["Schmitz, Stephan"],"dc:date":["2007"],"dc:description":["The P2X7-receptor is a ligand-gated ion channel of the P2X-purigenic-receptor group. In different of the other receptors of the P2X-family the P2X7-receptor is able to build both an ion channel and a cytolytic-pore which leads to membrane-blebbing. The aim of this MD-thesis was to develop a method which provides evidence whether the activation of selected P2X7-receptor-mutants with agonists like ATP or the more potent BzATP mediates a cytolytic membrane pore. As cell-system we choose COS-7-cells because of the lack of an endogenous P2X7-Receptor. As the cytolytic pore is permeable for cations up to a molecular mass of 900 Da, its possible to detect the cytolytic pore through fluorescence-microscopy of diffused 320 Da Ethidium-bromid cations which intercalates with the cellular DNA. DIC-microscopy accorded as an easier way to detect membrane-blebbing with the same explanatory power. After addition of the agonists a Zeiss-cell-observer system acquires over a period of 30 minutes automatically a series of pictures. After 30 minutes of activation there were no further changes of the membrane-integrity detectable.The co-transfection of an EGFP-cDNA accorded to an advantageous way to differ between transfected from un-transfected cells. Furthermore with the co-transfection of the EGFP-cDNA it was possible to localize changings of plasmamembrane at an early state. To valdidate the developed method, I investigated COS7-cells expressing the wildtype-P2X7-cDNA and 264.7-RAW-macrophages which expresses an endogenous P2X7-receptor. Furthermore I investigated the previous published E496A-Polymorphismem. According to the literature the activation of transfected COS7-cells couldnt mediate membrane-blebbing. The expression of the investigated P2X7-receptor-mutants was verified by western blot. After developing a suitable assay to show the cytolytic pore-formation, some hP2X7-receptor mutants were cloned and investigated. An up to now unpublished glycin-arginin-polymorphism at position 441 leads to a extended activation period with BzATP. Remarkable is the great number of cystein-residues in the cytosolic localized c-terminal endodomain. The exchange of the 362 cystein-residue also leads to a extended activation period and the membrane-blebs are formed less than the wildtype. Its possible that the high amount of cystein-residues is responsible for the species-dependent difference of the cytolytic pore-formation kinetic. The truncation of the last 12 amino acids resulted to a receptor that looses pore forming abilities. If the tyrosine motif 400YSVF403 can also affect pore forming abilities is not investigated finally, because of weak expression of the 400YSVF403-P2X7-receptor in the COS7-cell line. Finally these experiments show that a lot of amino acids take influence of the hP2X7-receptor pore forming abilities."],"dc:identifier":["https://publications.rwth-aachen.de/record/51872","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-114121%22"],"dc:language":["ger"],"dc:publisher":["Publikationsserver der RWTH Aachen University"],"dc:relation":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-18529"],"dc:rights":["info:eu-repo/semantics/openAccess"],"dc:source":["Aachen : Publikationsserver der RWTH Aachen University 72 S. : Ill., graph. Darst. (2007). = Aachen, Techn. 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