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|a urn:nbn:de:hbz:6-04239528707
|2 urn
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|a eng
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|a 570 Biowissenschaften; Biologie
|2 23
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|a Becker, Miriam
|u FB 13: Biologie
|0 http://d-nb.info/gnd/1117167852
|4 aut
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|a Universitäts- und Landesbibliothek Münster
|0 http://d-nb.info/gnd/5091030-9
|4 own
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|a Rate-limiting steps for human papillomavirus 16 internalization into host cells
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|a [Electronic ed.]
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|c 2016
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|b Universitäts- und Landesbibliothek Münster
|c 2016-10-28
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|a Table of figures ................................................................................................................. 4 -- Summary .......................................................................................................................... 5 -- Zusammenfassung ............................................................................................................ 7 -- 1 Introduction ................................................................................................................ 9 -- 1.1 Viruses ............................................................................................................................ 9 -- 1.2 Virus entry .................................................................................................................... 10 -- 1.2.1 Binding ............................................................................................................................. 10 -- 1.2.2 Internalization .................................................................................................................. 12 -- 1.2.3 Intracellular trafficking, membrane penetration and viral uncoating ............................. 13 -- 1.2.4 Nuclear import ................................................................................................................. 14 -- 1.2.5 Kinetics of virus entry ...................................................................................................... 15 -- 1.3 Human papillomaviruses ............................................................................................... 15 -- 1.3.1 The HPV life cycle ............................................................................................................. 16 -- 1.3.2 Virus particles in HPV research ........................................................................................ 17 -- 1.3.3 Structure .......................................................................................................................... 18 -- 1.4 Host cell entry of Human Papillomaviruses .................................................................... 19 -- 1.4.1 Primary binding ................................................................................................................ 19 -- 1.4.2 Structural modifications................................................................................................... 19 -- 1.4.3 Secondary receptor interaction ....................................................................................... 21 -- 1.4.4 Internalization and signaling ............................................................................................ 23 -- 1.4.5 Endosomal trafficking and nuclear import ...................................................................... 24 -- 1.4.6 Internalization kinetics ..................................................................................................... 25 -- 1.5 Model and Aims ............................................................................................................ 27 -- 2 Materials and methods ............................................................................................. 29 -- 2.1 Material ........................................................................................................................ 29 -- 2.1.1 Cell lines ........................................................................................................................... 29 -- 2.1.2 Growth media .................................................................................................................. 30 -- 2.1.3 Specialized media ............................................................................................................. 31 -- 2.1.4 Cell culture reagents ........................................................................................................ 31 -- 2.1.5 Plasmids ........................................................................................................................... 33 -- 2.1.6 siRNA ................................................................................................................................ 33 -- 2.1.7 Virus production and labeling reagents ........................................................................... 35 -- 2.1.8 Virus stocks ...................................................................................................................... 35 -- 2.1.9 Inhibitors .......................................................................................................................... 36 -- 2.1.10 Antibodies ...................................................................................................................... 36 -- 2.1.11 Primer ............................................................................................................................ 38 -- 2.1.12 qRT-PCR kits ................................................................................................................... 38 -- 2.1.13 Staining reagents ........................................................................................................... 38 -- 2.1.14 Buffers and solutions ..................................................................................................... 39 -- 2.2 Methods ....................................................................................................................... 42 -- 2.2.1 Cell culture methods ........................................................................................................ 42 -- 2.2.2 Virus production and labelling ......................................................................................... 43 -- 2.2.3 Raft-derived HPV16 production ....................................................................................... 45 -- 2.2.4 Infection assays and small molecule inhibitor treatments .............................................. 48 -- 2.2.5 Immunofluorescence staining and microscopic imaging of virus particles, raft tissues and cellular proteins ..................................................................................................................... 53 -- 2.2.6 Biochemical methods ....................................................................................................... 56 -- 3 Results ...................................................................................................................... 58 -- 3.1 Furin precleaved HPV16 is a valid tool to study HPV16 uptake kinetics ................................ 58 -- 3.2 FPC-HPV16 uses the same endocytic route as HPV16 ............................................................ 62 -- 3.3 Furin-precleavage of HPV16 particles is rate limiting for uptake ........................................... 66 -- 3.4 KLK8 cleavage of HPV16 is no rate-limiting step during HPV16 internalization ..................... 69 -- 3.6 Cyclophilin activity has only minor impact on the kinetics of HPV16 uptake......................... 72 -- 3.7 Towards raft-derived HPV16 as a tool to study HPV16 endocytosis ...................................... 74 -- 3.8 Internalization of ECM-bound FPC-HPV16 is faster and more synchronous after cell cycle synchronization ................................................................................................................................. 80 -- 3.9 The accessibility of the secondary receptor for virus interaction is limited ........................... 82 -- 3.10 RNAi confirms the importance of potential secondary receptor candidates ...................... 85 -- 3.11 Low incidence of co-localization of HPV16 with CD151 and EGFR ....................................... 89 -- 3.12 Super resolution imaging reveals localization of HPV16 to CD151 patches ........................ 92 -- 4 Discussion ................................................................................................................. 96 -- 4.1 The role of structural modifications on infectious internalization of HPV16 ......................... 97 -- 4.2 Interaction with HSPGs is not limited ..................................................................................... 97 -- 4.3 KLK8 cleavage is not rate limiting for infectious internalization ............................................ 98 -- 4.4 L2 N-terminal exposure is rate limited ................................................................................. 100 -- 4.5 L2 N-terminal cleavage by furin is rate limiting .................................................................... 102 -- 4.6 What are possible additional rate-limiting steps during HPV16 entry? ............................... 103 -- 4.7 Why does HPV16 enter host cells by a slow and asynchronous mechanism? ..................... 108 -- 4.8 Concluding remarks .............................................................................................................. 109 -- 5 References ........................................................................................................................ 110 -- 6 Abbreviations ................................................................................................................... 135 -- 8 Publications and presentations ........................................................................................ 139 -- 9 Acknowledgments ............................................................................................................ 140 -- 10 Curriculum Vitae ............................................................................................................ 141.
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|a free access
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|a Eine Infektion mit dem humanen Papillomvirus 16 (HPV16) zählt zu den Hauptursachen für die Entwicklung von Gebärmutterhalskarzinomen. HPV16 nutzt einen neuartigen Endozytoseweg für den Eintritt in Wirtszellen. Dafür ist es essentiell, dass HPV16 zunächst Strukturveränderungen in der Virushülle erfährt. Diese ermöglichen die anschließende Interaktion mit einem Sekundärrezeptor, welcher zur Aufnahme in die Zelle führt. Die Endozytose von HPV16 verläuft mit Halbwertszeiten von 10-12 Stunden in die Wirtszellen außerordentlich langsam und asynchron. Diese Arbeit untersucht den Einfluss der strukturellen Veränderungen auf die Halbwertszeiten der Endozytose. Hierfür wurden strukturell veränderte Viruspartikel verwendet, mit deren Hilfe gezeigt werden konnte, dass die Prozessierung des Partikels mit Cyclophilinen und Furin für nur ein Drittel der langsamen und asynchronen Aufnahmezeit verantwortlich war. Ein weiterer geschwindigkeitsbestimmender Schritt war möglicherweise die Interaktion mit dem Sekundärrezeptor von HPV16.
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|a specialized
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|a InC 1.0
|u https://rightsstatements.org/vocab/InC/1.0/
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|a Papillomviren
|a HPV16
|a Endozytose
|a strukturelle Modifikationen
|a Internalisierungskinetik
|a Furin
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|2 DRIVER Types
|a Dissertation/Habilitation
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|2 DCMI Types
|a Text
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|a Schelhaas, Mario
|0 http://d-nb.info/gnd/128928107
|4 ths
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|3 Zum Volltext
|q text/html
|u https://nbn-resolving.de/urn:nbn:de:hbz:6-04239528707
|u urn:nbn:de:hbz:6-04239528707
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|3 Zum Volltext
|q application/pdf
|u https://repositorium.uni-muenster.de/document/miami/7231639f-0517-43db-97e5-a7a00d604b52/diss_becker_miriam.pdf
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