Investigation of the biochemistry and function of neutrophil serine protease 4 (NSP4):
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Gießen
VVB Laufersweiler Verl.
2014
|
| Ausgabe: | 1. Aufl. |
| Schriftenreihe: | Edition scientifique
|
| Schlagworte: | |
| Online-Zugang: | https://nbn-resolving.org/urn:nbn:de:bvb:19-167536 kostenfrei Inhaltsverzeichnis |
| Beschreibung: | 136 S. Ill., graph. Darst. |
| ISBN: | 9783835961289 |
Internformat
MARC
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| 020 | |a 9783835961289 |c Pb. : EUR 29.80 (DE), EUR 30.70 (AT), sfr 40.90 (freier Pr.) |9 978-3-8359-6128-9 | ||
| 035 | |a (OCoLC)879390994 | ||
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| 100 | 1 | |a Perera, Natascha Cynthia |d 1983- |e Verfasser |0 (DE-588)1049861302 |4 aut | |
| 245 | 1 | 0 | |a Investigation of the biochemistry and function of neutrophil serine protease 4 (NSP4) |c Natascha Cynthia Perera |
| 250 | |a 1. Aufl. | ||
| 264 | 1 | |a Gießen |b VVB Laufersweiler Verl. |c 2014 | |
| 300 | |a 136 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 490 | 0 | |a Edition scientifique | |
| 502 | |a Zugl.: München, Ludwig-Maximilians-Univ., Diss., 2013 | ||
| 650 | 0 | 7 | |a Neutrophiler Granulozyt |0 (DE-588)4171673-5 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Serinproteinasen |0 (DE-588)4181042-9 |2 gnd |9 rswk-swf |
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 689 | 0 | 0 | |a Neutrophiler Granulozyt |0 (DE-588)4171673-5 |D s |
| 689 | 0 | 1 | |a Serinproteinasen |0 (DE-588)4181042-9 |D s |
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Datensatz im Suchindex
| _version_ | 1804152106297327616 |
|---|---|
| adam_text | TABLE OF CONTENTS
1 INTRODUCTION 1
1.1 NEUTROPHIL GRANULOCYTES 1
L.L.L FORMATION OF GRANULES AND OF GRANULE PROTEINS I
1.2 NEUTROPHIL IMMUNE RESPONSE 2
1.3 NEUTROPHIL SERINE PROTEASES (NSPS) 3
1.3.1 BIOSYNTHESIS 3
1.3.2 ROLE OF NSPS 3
1.3.3 NSPS IN HUMAN DISEASES 4
1.4 CLASS 6 SERINE PROTEASES 5
1.5 NEUTROPHIL SERINE PROTEASE 4 (NSP4) 6
1.6 PROTEASE SPECIFICITY 6
1.6.1 THE SCHECHTER AND BERGER NOMENCLATURE 6
1.6.2 PROTEOLYTIC SPECIFICITIES OF NE, CG AND PR3 7
1.7 INHIBITORS OF NEUTROPHIL SERINE PROTEASES 7
1.7.1 SERPINS 9
1.8 PROTEINASE-ACTIVATED RECEPTOR-2 (PAR-2) 10
1.9 NSP4-DEFICIENT MOUSE MODEL 10
1.10 OBJECTIVES 12
2 MATERIALS AND METHODS 13
2.1 MATERIALS 13
2.1.1 CHEMICALS AND CONSUMABLES 13
2.1.2 OLIGONUCLEOTIDES 13
2.1.3 PLASMIDS 14
2.1.4 CELL LINES 15
HTTP://D-NB.INFO/1048196070
TABLE OF CONTENTS
2.1.5 RECOMBINANT AND PURIFIED PROTEINS IS
2.1.6 ANTIBODIES 16
2.1.7 SYNTHETIC SUBSTRATES 16
2.1.8 MOUSE STRAIN 16
2.1.9 LABORATORY EQUIPMENT 17
2.2 MOLECULAR BIOLOGICAL METHODS 18
2.2.1 PREPARATION OF COMPETENT BACTERIA WITH CACH 18
2.2.2 TRANSFORMATION OF COMPETENT BACTERIA BY HEAT SHOCK 18
2.2.3 PLASMID DNA PURIFICATION FROM E. COLI 19
2.2.4 POLYMERASE CHAIN REACTION (PCR) 19
2.2.5 FORMATION OF OLIGODUPLEXES 21
2.2.6 RESTRICTION DIGESTS 21
2.2.7 DEPHOSPHORYLATION OF VECTOR DNA 22
2.2.8 AGAROSE GEL ELECTROPHORESIS 22
2.2.9 DNA PURIFICATION 22
2.2.10 DETERMINATION OF DNA CONCENTRATION 23
2.2.11 LIGATION OF DNA 23
2.2.12 SEQUENCE ANALYSIS OF DNA 23
2.2.13 EXPRESSION CONSTRUCTS 24
2.3 RECOMBINANT PROTEIN EXPRESSION 26
2.3.1 EXPRESSION OF S-NSP4 IN FLP-LN*-293 26
2.3.2 EXPRESSION OF CXLPL*, S-MNSP4 AND ALL NSP4 CONSTRUCTS IN PTT5 IN
HEK 293E 28
2.4 CELL BIOLOGICAL METHODS 29
2.4.1 DETERMINATION OF CELL COUNT AND VIABILITY 29
2.4.2 ANALYSIS OF HUMAN PMN 30
2.4.3 CALCIUM SIGNALING IN HACAT AND HT1080 32
2.5 PROTEIN ANALYSIS 33
2.5.1 PURIFICATION OF RECOMBINANT PROTEINS 33
2.5.2 PROCESSING OF S-, NGS-NSP4 AND S-MNSP4 BY ENTEROKINASE 33
TABLE OF CONTENTS
2.5.3 CONVERSION OF NSP4 PRECURSOR BY DIPEPTIDYL PEPTIDASE I 34
2.5.4 DETERMINATION OF PROTEIN CONCENTRATION 34
2.5.5 SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS
PAGE) 35
2.5.6 PROTEIN DETECTION 36
2.5.7 GDMAN SEQUENCING 37
2.5.8 MEASUREMENT OF ENZYMATIC ACTIVITY 37
2.5.9 PROTEOMIC IDENTIFICATION OF PROTEASE CLEAVAGE SITES (PICS) 37
2.5.10 INHIBITION TESTS 39
2.6 IMMUNOLOGICAL METHODS 41
2.6.1 WESTERN BLOT 41
2.6.2 IMMUNOHISTOCHEMISTRY 42
2.7 MOUSE MODEL ANALYSIS . . . 42
2.7.1 ISOLATION OF GENOMIC DNA FROM MOUSE TAILS 43
2.7.2 GENOTYPING OF MICE 43
2.7.3 ISOLATION OF BONE MARROW CELLS 44
2.7.4 REVERSE TRANSCRIPTASE (RT) PCR 44
2.7.5 ISOLATION OF NEUTROPHILS 45
2.7.6 DETERMINATION OF NEUTROPHIL OXIDATIVE BURST 46
3 RESULTS - 47
3.1 PRODUCTION OF NSP4 47
3.1.1 IMPROVED PRODUCTION IN FLP-IN*-293 48
3.1.2 PRODUCTION IN HEK 293E 49
3.1.3 OTHER RECOMBINANT EXPRESSION SYSTEMS 51
3.1.4 CONVERSION OF S- OR NGS-NSP4 TO MATURE NSP4 BY ENTEROKINASE (EK).
51
3.2 CHARACTERIZATION OF NSP4 IN HUMAN NEUTROPHILS 52
3.2.1 NSP4 EXPRESSION IS RESTRICTED TO THE MYELOMONOCYTE CELL LINEAGE
.... 52
3.2.2 THE AMOUNT OF NSP4 IS 20-FOLD LOWER THAN THAT OF CG IN NEUTROPHILS
. . 52
3.2.3 NSP4 IS RELEASED AFTER NEUTROPHIL ACTIVATION 54
3.2.4 NSP4 LOCALIZES TO THE AZUROPHIL GRANULES OF NEUTROPHILS 55
3.2.5 PROCESSING OF NSP4 PRECURSOR BY DPPI 57
III
TABLE OF CONTENTS
3.2.6 PATIENTS WITH PAPILLON-LEFEVRE SYNDROME (PLS) LACK NSP4 IN THEIR
NEUTRO
PHILS 57
3.3 FUNCTIONAL ANALYSIS OF NSP4 58
3.3.1 PROTEOLYTIC SPECIFICITY OF NSP4 58
3.3.2 CHEMICAL INHIBITORS OF NSP4 61
3.3.3 NATURAL INHIBITORS OF NSP4 63
3.3.4 POSITIONAL SPECIFICITY PROFILING OF NSP4 65
3.3.5 PRODUCTION OF AN NSP4 REACTIVE SERPIN FORMING COVALENT COMPLEXES
(ALPL*) 69
3.3.6 DETECTION OF ACTIVE NSP4 IN NEUTROPHIL LYSATE AND RELEASED BY
NEUTROPHILS 72
3.4 IS PAR-2 A POTENTIAL NATURAL SUBSTRATE OF NSP4? 72
3.4.1 PAR-2 FRET 73
3.4.2 NSP4 TRIGGERED CALCIUM SIGNALING IN HACAT, BUT NOT IN HTI080 ....
73
3.4.3 PAR-2 BUT NOT PAR-1 AGONISTS PREVENTED NSP4 INDUCED CALCIUM SIGNA
LING IN HACAT CELLS 76
3.5 NSP4 IN THE MOUSE 78
3.5.1 PRODUCTION OF MNSP4 79
3.5.2 SPECIFICITY COMPARISON BETWEEN HUMAN AND MOUSE NSP4 80
3.5.3 ANALYSIS OF MNSP4 MRNA IN WT AND NSP4-/- MICE 80
3.5.4 PRODUCTION OF REACTIVE*
OXYGEN SPECIES (ROS) WAS NOT IMPAIRED IN NSP4-/-
MICE 81
3.5.5 NEUTROPHIL ACTIVATION BY IMMUNE COMPLEXES WAS NOT IMPAIRED IN
NSP4-/-
MICE 82
4 DISCUSSION 85
4.1 NSP4, A NOVEL NEUTROPHIL PROTEASE HIGHLY CONSERVED IN EVOLUTION 85
4.2 NSP4, AN UNUSUAL SERINE PROTEASE WITH ARGININE SPECIFICITY 86
4.2.1 SI POCKET SPECIFICITY 86
4.2.2 THE EXTENDED SPECIFICITY OF NSP4 . 86
4.3 DETECTION OF ACTIVE NSP4 88
4.4 PAR-2 AS A POTENTIAL NSP4 SUBSTRATE 89
IV
TABLE OF CONTENTS
4.5 THE NSP4-DEFICIENT MOUSE MODEL 90
4.6 THE FUNCTION OF NSP4 91
4.7 CONCLUSIONS 92
5 BIBLIOGRAPHY 93
6 ABBREVIATIONS 103
7 APPENDIX 107
7.1 VECTOR MAPS 107
7.1.1 PTT5 107
7.1.2 PFASTBACL 108
7.2 LIST OF PRIMERS 109
7.3 DNA AND PROTEIN SEQUENCES 110
7.3.1 S-NSP4 (HUMAN) 110
7.3.2 NGS-NSP4 (HUMAN) ILL
7.3.3 NSP4 PRECURSOR (HUMAN) 112
7.3.4 S-MNSP4 (MOUSE) 113
7.3.5 OCLPI* 114
7.4 LIST OF SYNTHETIC PROTEASE SUBSTRATES 115
7.5 LISTS OF NSP4 CLEAVAGE SITES IN PICS LIBRARIES 120
7.5.1 CHYMOTRYPSIN 121
7.5.2 GLUC 123
7.5.3 TRYPSIN 127
8 CURRICULUM VITAE 133
9 PUBLICATIONS AND MEETINGS 135
9.1 PUBLICATIONS IN PEER-REVIEWED JOURNALS 135
9.1.1 CONFERENCE ABSTRACT 135
9.2 PRESENTATIONS AT INTERNATIONAL CONFERENCES 136
9.2.1 ORAL PRESENTATIONS 136
9.2.2 POSTER PRESENTATIONS 136
EIDESSTATTLICHE ERKLARUNG 137
V
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| any_adam_object | 1 |
| author | Perera, Natascha Cynthia 1983- |
| author_GND | (DE-588)1049861302 |
| author_facet | Perera, Natascha Cynthia 1983- |
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| ctrlnum | (OCoLC)879390994 (DE-599)DNB1048196070 |
| dewey-full | 572.76 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 572 - Biochemistry |
| dewey-raw | 572.76 |
| dewey-search | 572.76 |
| dewey-sort | 3572.76 |
| dewey-tens | 570 - Biology |
| discipline | Biologie Agrar-/Forst-/Ernährungs-/Haushaltswissenschaft / Gartenbau |
| edition | 1. Aufl. |
| format | Thesis Book |
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| indexdate | 2024-07-10T01:05:26Z |
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| isbn | 9783835961289 |
| language | English |
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| spelling | Perera, Natascha Cynthia 1983- Verfasser (DE-588)1049861302 aut Investigation of the biochemistry and function of neutrophil serine protease 4 (NSP4) Natascha Cynthia Perera 1. Aufl. Gießen VVB Laufersweiler Verl. 2014 136 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Edition scientifique Zugl.: München, Ludwig-Maximilians-Univ., Diss., 2013 Neutrophiler Granulozyt (DE-588)4171673-5 gnd rswk-swf Serinproteinasen (DE-588)4181042-9 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Neutrophiler Granulozyt (DE-588)4171673-5 s Serinproteinasen (DE-588)4181042-9 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:19-167536 https://nbn-resolving.org/urn:nbn:de:bvb:19-167536 Resolving-System http://edoc.ub.uni-muenchen.de/16753 Verlag kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027235568&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Perera, Natascha Cynthia 1983- Investigation of the biochemistry and function of neutrophil serine protease 4 (NSP4) Neutrophiler Granulozyt (DE-588)4171673-5 gnd Serinproteinasen (DE-588)4181042-9 gnd |
| subject_GND | (DE-588)4171673-5 (DE-588)4181042-9 (DE-588)4113937-9 |
| title | Investigation of the biochemistry and function of neutrophil serine protease 4 (NSP4) |
| title_auth | Investigation of the biochemistry and function of neutrophil serine protease 4 (NSP4) |
| title_exact_search | Investigation of the biochemistry and function of neutrophil serine protease 4 (NSP4) |
| title_full | Investigation of the biochemistry and function of neutrophil serine protease 4 (NSP4) Natascha Cynthia Perera |
| title_fullStr | Investigation of the biochemistry and function of neutrophil serine protease 4 (NSP4) Natascha Cynthia Perera |
| title_full_unstemmed | Investigation of the biochemistry and function of neutrophil serine protease 4 (NSP4) Natascha Cynthia Perera |
| title_short | Investigation of the biochemistry and function of neutrophil serine protease 4 (NSP4) |
| title_sort | investigation of the biochemistry and function of neutrophil serine protease 4 nsp4 |
| topic | Neutrophiler Granulozyt (DE-588)4171673-5 gnd Serinproteinasen (DE-588)4181042-9 gnd |
| topic_facet | Neutrophiler Granulozyt Serinproteinasen Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:19-167536 http://edoc.ub.uni-muenchen.de/16753 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027235568&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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