Redox regulation in innate immune signaling:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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2016
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| Beschreibung: | VIII, 168 Blätter Illustrationen, Diagramme |
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| 100 | 1 | |a Virreira Winter, Sebastian |d 1986- |e Verfasser |0 (DE-588)1125539577 |4 aut | |
| 245 | 1 | 0 | |a Redox regulation in innate immune signaling |c von Diplom-Molekularbiomediziner Sebatsian Virreira Winter |
| 264 | 1 | |a Berlin |c 2016 | |
| 300 | |a VIII, 168 Blätter |b Illustrationen, Diagramme | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Humboldt-Universität zu Berlin |d 2016 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 776 | 0 | 8 | |i Reproduziert als |a Virreira Winter, Sebastian |t Redox regulation in innate immune signaling |d Ketsch bei Mannheim : Mikroform Dissertation, 2016 |h 2 Mikrofiches |w (DE-604)BV044313848 |
| 856 | 4 | 2 | |m DNB Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029449661&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 999 | |a oai:aleph.bib-bvb.de:BVB01-029449661 | ||
Datensatz im Suchindex
| _version_ | 1804177054230380544 |
|---|---|
| adam_text | TABLE OF CONTENTS
1 IN T R O D U C T IO N
....................................................................................................................................
1
1.1 REACTIVE OXYGEN SPECIES, REACTIVE NITROGEN SPECIES, AND REDOX
SIGNALING
............................................
1
1.1.1 GENERAL ASPECTS OF REACTIVE OXYGEN AND NITROGEN SPECIES
..............................................................
1
1.1.2 THE ANTIMICROBIAL FUNCTION OF REACTIVE OXYGEN AND NITROGEN
SPECIES.............................................1
1.1.3 REDOX REGULATION BY CYSTEINE
OXIDATION...........................................................................................
2
1.1.4 THE CELLULAR ANTIOXIDANT
SYSTEM........................................................................................................5
1.1.5 SPECIFICITY IN REDOX
SIGNALING.............................................................................................................6
1.2 GENETICALLY ENCODED REDOX AND PH
SENSORS................................................................................................7
1.2.1 CONVENTIONAL METHODS TO DETECT REACTIVE OXYGEN AND NITROGEN
SPECIES........................................7
1.2.2 PRINCIPLES OF GENETICALLY ENCODED REDOX AND PH SENSORS
................................................................
7
1.2.3 CHARACTERISTICS OF
ROGFPS..................................................................................................................
8
1.2.4 GRXL-ROGFP2 AND
ROGFP2-ORPL........................................................................................................9
1.2.5 IMAGING OF ROGFP2-BASED REDOX
SENSORS.........................................................................................
9
1.3 INNATE
IMMUNITY..............................................................................................................................................11
1.3.1 MACROPHAGES, MONOCYTES, AND
NEUTROPHILS..................................................................................
11
1.3.2 TOLL-LIKE
RECEPTORS...........................................................................................................................
12
1.3.3 IL-LSS AND
INFLAMMASOMES...............................................................................................................
13
1.4 PSEUDOMONAS
AERUGINOSA.............................................................................................................................
16
1.4.1 P. AERUGINOSA: A GRAM-NEGATIVE
PATHOGEN.....................................................................................
16
1.4.2 PYOCYANIN AND OTHER
PHENAZINES....................................................................................................16
1.4.3 INFLAMMASOME ACTIVATION BY P.
AERUGINOSA...................................................................................
17
1.5 HIGH-RESOLUTION MASS SPECTROMETRY-BASED PROTEIN
ANALYSIS...................................................................
18
1.5.1 PRINCIPLES OF MASS
SPECTROMETRY.....................................................................................................18
1.5.2 SHOTGUN
PROTEOMICS........................................................................................................................
18
1.5.3 QUANTITATIVE
MS..............................................................................................................................
20
1.5.4 REDOX
PROTEOMICS...........................................................................................................................
21
1.6 AIM OF THIS
THESIS............................................................................................................................................
22
2 RESULTS
...........
......................................................................................................................................
23
2.1 SPATIOTEMPORAL ANALYSIS OF REDOX CHANGES IN
MACROPHAGES..................................................................23
2.1.1 DEVELOPMENT OF AN IN VITRO SYSTEM TO STUDY GLUTATHIONE OXIDATION
IN MACROPHAGES
...................
23
2.1.2 AUTOFLUORESCENCE CORRECTION IMPROVES THE RATIOMETRIC ANALYSIS OF
GRXL-ROGFP2
.......................
26
2.1.3 GRXL-ROGFP2 AND CM-H2-DCFDA HAVE COMPARABLE SENSITIVITIES TOWARDS
H20 2 IN MACROPHAGES 29
2.1.4 GLUTATHIONE IS OXIDIZED UPON PYROPTOSIS FOLLOWING NLRP3
INFLAMMASOME ACTIVATION
.................
31
2.1.5 TLR ACTIVATION TRANSIENTLY OXIDIZES MITOCHONDRIAL GLUTATHIONE
.....................................................
37
2.1.6 TLR ACTIVATION RESULTS IN A TRANSIENT PH INCREASE IN
MACROPHAGES................................................ 39
2.1.7 ROS/RNS RARELY LEAK FROM PHAGOSOMES IN BMDMS
......................................................................
41
2.1.8 ACTIVATED PLB-985 CELLS OXIDIZE INTRACELLULAR GLUTATHIONE
UBIQUITOUSLY........................................42
2.2 EFFECTS OF PYOCYANIN ON INFLAMMASOME ACTIVATION IN
MACROPHAGES....................................................45
2.2.1 PYOCYANIN INHIBITS THE NLRP3 INFLAMMASOME IN V ITRO
..................................................................
45
2.2.2 PYOCYANIN DOES NOT BLOCK OTHER INFLAMMASOMES OR CLASSICAL
CYTOKINES.......................................47
2.2.3 SPECIFIC PHENAZINES INDUCE INTRACELLULAR ROS/RNS AND BLOCK
NLRP3............................................49
2.2.4 PYOCYANIN ACTS INDEPENDENT OF TRANSLATION AND UPSTREAM OF SPECK
FORMATION
...........................
52
2.2.5 PYOCYANIN IN SUPERNATANTS OF PA14 INHIBITS NLRP3 ACTIVATION IN V
ITRO .........................................54
2.2.6 PYOCYANIN INHIBITS THE NLRP3 INFLAMMASOME VIA ROS/RNS
.........................................................
55
2.3 OXINOME ANALYSIS - PROTEOMIC DETECTION OF REVERSIBLE CYSTEINE
OXIDATION.......................................57
2.3.1 A NEW STRATEGY TO IDENTIFY OXIDIZED CYSTEINES VIA MASS
SPECTROMETRY.........................................57
2.3.2 DEVELOPMENT OF SSL1 - AN MS-CLEAVABLE LINKER WITH A SYMMETRIC
SULFOXIDE
...............................
61
2.3.3 DEVELOPMENT AND CHARACTERIZATION OF THE ASYMMETRIC SULFOXIDE
LINKER ASL1
............................
65
2.3.4 ASL1 DOES NOT INTERFERE WITH MS ANALYSIS AND IS EFFICIENTLY
CLEAVED IN AIF SCANS
........................
70
2.3.5 OPTIMIZATION OF THE ACQUISITION AND ANALYSIS OF ASLL-COUPLED
PEPTIDES......................................76
2.3.6 OXINOME ANALYSIS TO IDENTIFY TARGETS OF ANTIOXIDANT
ENZYMES...................................................... 80
3
DISCUSSION.......................................................................................................................................
85
3.1
SUMMARY...........................................................................................................................................................85
3.2 SPATIOTEMPORAL ANALYSIS OF REDOX CHANGES IN MACROPHAGES
.................................................................
86
3.2.1 A NOVEL IN VITRO SYSTEM TO STUDY REDOX CHANGES IN
MACROPHAGES.................................................86
3.2.2 PYROPTOSIS PRECEDES INTRACELLULAR OXIDATION UPON NLRP3
ACTIVATION............................................88
3.2.3 TLRS MODULATE MITOCHONDRIAL ROS PRODUCTION AND THE INTRACELLULAR
PH......................................90
3.2.4 ROS/RNS OFTEN ESCAPE FROM PHAGOSOMES IN NEUTROPHILS BUT NOT
MACROPHAGES
........................
92
3.3 EFFECTS OF PYOCYANIN ON INFLAMMASOME ACTIVATION IN MACROPHAGES
...................................................
95
3.3.1 PYOCYANIN INTERFERES WITH ACTIVATION OF THE NLRP3
INFLAMMASOME..............................................95
3.3.2 REDOX REGULATION OF
NLRP3.............................................................................................................96
3.3.3 DOES P. AERUGINOSA USE PYOCYANIN TO EVADE IMMUNE
RECOGNITION?.............................................98
3.4 OXINOME ANALYSIS - PROTEOMIC DETECTION OF REVERSIBLE CYSTEINE
OXIDATION
101
3.4.1 THE REQUIREMENT FOR IMPROVED REDOX PROTEOMICS APPROACHES
..................................................
101
3.4.2 SSL1 AND ASL1: TWO MS-CLEAVABLE ENRICHMENT
TAGS....................................................................101
3.4.3 OXINOME ANALYSIS TO IDENTIFY TARGETS OF THIOREDOXIN IN HEK 293T
CELLS
........................................
104
3.5 CONCLUSION AND
OUTLOOK.............................................................................................................................
109
4 MATERIALS A N D M E T H O D S
.....................................................................................................I
L L
4.1
MATERIALS.....................................................................
I L L
4.1.1
PLASMIDS.........................................................................................................................................
I L L
4.1.2 ANTIBODIES USED FOR WESTERN BLOT
ANALYSIS..................................................................................
112
4.1.3
BUFFERS/MEDIA................................................................................................................................112
4.1.4
CELLS................................................................................................................................................
112
4.1.5 M
ICE...............................................................................................................................................
113
4.2
METHODS..........................................................................................................................................................114
4.2.1 CELL BIOLOGICAL
METHODS..................................................................................................................114
4.2.1.1 COLLECTION OF 1929-CONDITIONED M EDIUM
...............................................................................
114
4.2.1.2 CELL
CULTURE...............................................................................................................................
114
4.2.1.3 GENERATION OF BONE MARROW-DERIVED MACROPHAGES (BMDMS)
.............................................
114
4.2.1.4 RETROVIRUS
PRODUCTION.............................................................................................................
115
4.2.1.5 LENTIVIRUS
PRODUCTION...............................................................................................................115
4.2.1.6 VIRAL TRANSDUCTION OF BMDMS AND
PLB-985...........................................................................
116
4.2.1.7 IMAGING OF GRXL-ROGFP2/ ROGFP2-ORPL, AND
SYPHER.............................................................116
4.2.1.8 OXIDATIVE BURST ANALYSIS WITH FC
OXYBURST...........................................................................
117
4.2.1.9 CALIBRATION OF
SYPHER..............................................................................................................
117
4.2.1.10 ROS/RNS DETECTION BY CM-H2-DCFDA AND
DHE..................................................................118
4.2.1.11 STIMULATION OF CELLS WITH PYO, 1-HP, PCA OR
PHZ.................................................................118
4.2.1.12 DETERMINATION OF LDH CONTENT
...........................................................................................
119
4.2.1.13 TNF-A SECRETION UPON EPS STIMULATION AND PYO TREATM ENT
...............................................
119
4.2.1.14 ACTIVATION OF NLRC4 AND AIM2
INFLAMMASOMES.................................................................119
4.2.1.15 CHX INHIBITION OF
TRANSLATION...............................................................................................120
4.2.1.16 SPECK FORMATION ASSAY FOLLOWING PYO TREATM ENT
...............................................................
120
4.2.1.17 ASC DIMERIZATION
ASSAY........................................................................................................121
4.2.1.18 NLRP3 ACTIVATION ASSAY WITH PA14
SUPERNATANTS................................................................121
4.2.1.19 SCAVENGING OF ROS/RNS INDUCED BY PYO, H20 2, OR
PEROXYNITRITE.......................................121
4.2.1.20 COUPLING OF ASL1 OR 1AM TO HEK 293T OR HELA CELLS
..........................................................
122
4.2.1.21 GENERATION OF HEK 293T CELLS WITH GENETIC DEFICIENCIES BY
CRISPR/CAS9
.........................
122
4.2.1.22 DIFFERENTIAL ALKYLATION AND ENRICHMENT OF REVERSIBLY OXIDIZED
PEPTIDES WITH ASL1
..........
123
4.2.1.23 PREPARATION OF SAMPLES FOR WHOLE PROTEOME ANALYSIS
124
4.2.2 MOLECULAR BIOLOGY & BIOCHEMICAL
ASSAYS.....................................................................................
124
4.2.2.1 CLONING OF VIRAL
CONSTRUCTS.....................................................................................................
124
4.2.3 IMAGE
ANALYSIS................................................................................................................................125
4.2.3.1
ELISA.......................................................................................................................................126
4.2.3.2 WESTERN BLOT
ANALYSIS............................................................................................................
127
4.2.3.3 SYNTHESIS OF SSL1 AND
ASL1....................................................................................................
127
4.2.3.4 COUPLING OF ASL1 TO
BSA........................................................................................................
128
4.2.3.5 CLONING OF
GRNAS...................................................................................................................
128
4.2.4 MASS
SPECTROMETRY.......................................................................................................................
128
4.2.4.1
LC-MS/MS...............................................................................................................................
128
4.2.4.2 MS DATA
ANALYSIS....................................................................................................................
129
4.2.4.3 STATIC SPRAY INJECTION
EXPERIMENTS.......................................................................................
130
4.3
SEQUENCES.............................................................................
4.3.1 PRIMER
SEQUENCES....................................................
4.3.2 OLIGO SEQUENCES FOR LIGATION-INDEPENDENT CLONING
131
131
132
5 REFERENCES
133
6 ABBREVIATIONS
165
7 A P P E N D IX
...........................................................................................................................................167
ACKNOWLEDGEMENTS....................................................................................................................................167
SELBSTSTAENDIGKEITSERKLAERUNG.......................................................................................................................
168
|
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| spelling | Virreira Winter, Sebastian 1986- Verfasser (DE-588)1125539577 aut Redox regulation in innate immune signaling von Diplom-Molekularbiomediziner Sebatsian Virreira Winter Berlin 2016 VIII, 168 Blätter Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier Dissertation Humboldt-Universität zu Berlin 2016 (DE-588)4113937-9 Hochschulschrift gnd-content Reproduziert als Virreira Winter, Sebastian Redox regulation in innate immune signaling Ketsch bei Mannheim : Mikroform Dissertation, 2016 2 Mikrofiches (DE-604)BV044313848 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029449661&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Virreira Winter, Sebastian 1986- Redox regulation in innate immune signaling |
| subject_GND | (DE-588)4113937-9 |
| title | Redox regulation in innate immune signaling |
| title_auth | Redox regulation in innate immune signaling |
| title_exact_search | Redox regulation in innate immune signaling |
| title_full | Redox regulation in innate immune signaling von Diplom-Molekularbiomediziner Sebatsian Virreira Winter |
| title_fullStr | Redox regulation in innate immune signaling von Diplom-Molekularbiomediziner Sebatsian Virreira Winter |
| title_full_unstemmed | Redox regulation in innate immune signaling von Diplom-Molekularbiomediziner Sebatsian Virreira Winter |
| title_short | Redox regulation in innate immune signaling |
| title_sort | redox regulation in innate immune signaling |
| topic_facet | Hochschulschrift |
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