Peroxiredoxin-mediated mitochondrial H2O2 signalling:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Heidelberg
2018
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| Online-Zugang: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Beschreibung: | Im Titel ist "2" tiefgestellt |
| Beschreibung: | IX, 105 Seiten Illustrationen, Diagramme 30 cm |
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| 100 | 1 | |a Gomes Barata, Ana |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Peroxiredoxin-mediated mitochondrial H2O2 signalling |c presented by Ana Gomes Barata, M.Sc. |
| 264 | 1 | |a Heidelberg |c 2018 | |
| 300 | |a IX, 105 Seiten |b Illustrationen, Diagramme |c 30 cm | ||
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| 502 | |b Dissertation |c Ruperto-Carola University of Heidelberg |d 2018 | ||
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Datensatz im Suchindex
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|---|---|
| adam_text | TABLE OF CONTENTS
FREQUENTLY USED
ABBREVIATIONS......................................................................................
VIII
1 INTRODUCTION 1
1.1. HYDROGEN P E RO X ID E
..................................................................................................1
1.1.1. REACTIVE OXYGEN SP ECIES
...................................................................................
1
1.1.2. 0 2 PRODUCTION BY THE M TE T C
.......................................................................
2
1.1.3. H 20 2 D E G RA D A TIO N
............................................................................................
3
1.1.4. DETECTION AND QUANTIFICATION OF H20 2
...........................................................
5
1.2. H 20 2-MEDIATED
SIGNALLING............................................................................................7
1.2.1. PEROXIREDOXINS AS MEDIATORS OF H20 2-BASED
SIGNALLING.................................9
1.3. MITOCHONDRIAL SIG N ALLIN G
..........................................................................................
11
1.3.1. M
ITOCHONDRIA...................................................................................................11
1.3.2. ANTEROGRADE RE G U LA TIO N
.................................................................................
12
1.3.3. RETROGRADE SIG N A LLIN G
....................................................................................
13
1.3.4. MITOCHONDRIAL H 20 2 SIGNALLING
.....................................................................
13
1.4. P38 MAPK SIGNALLING PATHW
AY.................................................................................
14
1.4.1. THE CANONICAL MAPK SIGNALLING P ATH W AY
....................................................14
1.4.2. MEKKL M
AP3K................................................................................................15
1.4.3. ASK 1 REDOX
REGULATION....................................................................................
16
1.5. O BJECTIV
E....................................................................................................................
16
2 MATERIALS 19
2.1. LABORATORY
EQUIPMENT................................................................................................19
2.2. CHEMICALS AND BIOCHEM
ICALS....................................................................................20
2.2.1.
ENZYMES...........................................................................................................
21
2.2.2.
ANTIBODIES........................................................................................................
22
2.2.3.
KITS....................................................................................................................
23
2.3. PLASMIDS SYNTHETIC GENES AND OLIGONUCLEOTIDES
.....................................................
23
2.4. SOLUTIONS, GELS AND M E D IA
..........................................................................................31
2.5. BIOLOGICAL M
ATERIAL.....................................................................................................
33
2.6. SOFTWARE
33
3 METHODS 35
3.1. MOLECULAR B IO LO G Y
......................................................................................................
35
3.1.1. CHEMICALLY COMPETENT E. C O LI
........................................................................
35
3.1.2. E. COLI TRAN SFO RM ATIO N
....................................................................................
35
3.1.3. ISOLATION OF PLASMID D N A
..............................................................................
36
3.1.4. CLONING OF PROTEIN EXPRESSION V E C TO RS
..........................................................36
3.1.5. PURIFICATION OF GENOMIC D N A
........................................................................
38
3.1.6. PURIFICATION OF TOTAL R N A
..............................................................................
39
3.1.7. SYNTHESIS OF DS R N A
.......................................................................................
39
3.2. D. MELANOGASTER CELL
CULTURE.......................................................................................
40
3.2.1. M A IN TEN A N CE
...................................................................................................
40
3.2.2. CRYOCONSERVATION AND RECONSTITUTION OF CE
LLS..............................................41
3.2.3. TREATMENT WITH DSRN A
S.................................................................................
41
3.2.4. TRANSFECTION OF PROTEIN-EXPRESSING V E C TO R S
.................................................42
3.2.5. COMBINED TREATMENT OF CELLS WITH DSRNA AND PROTEIN EXPRESSION
.... 43
3.2.6. ESTABLISHMENT OF STABLE CELL LIN E S
..................................................................
43
3.3. PROTEIN B IO C H E M ISTRY
................................................................................................
44
3.3.1. CELL LYSIS AND PROTEIN P U RIFIC A TIO N
...............................................................
44
3.3.2. PROTEIN QUANTIFICATION BY BCA ASSAY
............................................................
44
3.3.3. PRESERVATION OF REDOX STATE OF PROTEIN
THIOLS.................................................45
3.3.4. STREPTAVIDIN PULLDOW N
....................................................................................
45
3.3.5. SD S-PA G E
......................................................................................................
45
3.3.6. IM M U N O B LO TTIN G
.............................................................................................
46
3.4. ROGFP2-BASED A S S A Y S
................................................................................................
47
3.5. CELL CHARACTERIZATION
TECHNIQUES..............................................................................
47
3.5.1. ADP/ATP Q U A N TIFIC A TIO N
..............................................................................
47
3.5.2. OXYGEN CONSUMPTION RATE M EASUREM
ENTS....................................................48
3.5.3. QUANTITATIVE RT P C R
....................................................................................
48
3.5.4. CELL ABUNDANCE Q U A N TIFIC A TIO N
.....................................................................
48
4 RESULTS 51
4.1. DEPLETION OF ND-B8 AND ND-B17 INDUCES MITOCHONDRIAL DYSFUNCTION AND
M TH 20 2
EMISSIONS......................................................................................................
51
4.1.1. RNAI-SCREEN TO IDENTIFY MITOCHONDRIAL PROTEIN-DEPLETIONS LEADING
TO
M TH 20 2 EM
ISSIONS..........................................................................................
51
4.1.2. DEPLETION OF ND-B8 AND ND-B17 GENERATES MITOCHONDRIAL DYSFUNCTION
55
4.1.3. INCREASED M TH 20 2 EMISSIONS ARE COMPATIBLE WITH CELLULAR
VIABILITY. . . .57
4.2. EMISSION OF MTH 20 2 CORRELATES WITH THE INDUCTION OF P38 AND NRF2
SIGNALLING
P ATH W AY
S.....................................................................................................................58
4.3. JAFRACL IS REQUIRED FOR M TH 20 2 DEPENDENT P38 SIGNALLING
ACTIVATION..................59
4.4. OXIDISED JAFRACL INTERACTS WITH THE MAP3K MEKKL TO INDUCE P38
PHOSPHORYLA
TION
.............................................................................................................................
61
5 DISCUSSION 67
5.1. DEVELOPING A MITOCHONDRIAL DYSFUNCTION M O D E L
..................................................
68
5.1.1. RNAI-BASED SCREEN TO IDENTIFY MITOCHONDRIAL PROTEINS INVOLVED IN
M TH 20 2
REGULATION..........................................................................................68
5.1.2. MITOCHONDRIAL PROTEINS WHOSE DEPLETION LED TO INCREASED M TH 20 2
EMIS
SIONS
.................................................................................................................
70
5.1.3. MITOCHONDRIAL DYSFUNCTION A N ALY SIS
............................................................
72
5.2. M TH 20 2-DEPENDENT ACTIVATION OF MITOCHONDRIAL RETROGRADE SIG N A
LLIN G
.............
74
5.2.1. MITOCHONDRIAL RETROGRADE SIGNALLING P ATH W AY
S...........................................74
5.2.2. JAFRACL MEDIATION OF P38 MARK PATHWAY ACTIVATION
.................................
75
5.3. JAFRACL MEDIATED REDOX RELAY BETWEEN M TH 20 2 AND THE MAP3K M EKKL
.............
76
5.3.1. MAP3K MEKKL MEDIATES P38 A C TIV A TIO N
...................................................
76
5.3.2. REDOX SENSITIVITY OF M E K K L
................................
78
5.3.3. H 20 2-DEPENDENT INTERACTION BETWEEN JAFRACL AND MEKKL
......................
80
5.4. FUTURE
DIRECTIONS........................................................................................................
82
5.5.
CONCLUSIONS.................................................................................................................
83
REFERENCES 84
LIST OF FIGURES
LIST OF TABLES.
|
| any_adam_object | 1 |
| author | Gomes Barata, Ana |
| author_facet | Gomes Barata, Ana |
| author_role | aut |
| author_sort | Gomes Barata, Ana |
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| building | Verbundindex |
| bvnumber | BV045277933 |
| ctrlnum | (OCoLC)1035526351 (DE-599)GBV1022178814 |
| discipline | Biologie |
| format | Thesis Book |
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| institution | BVB |
| language | English |
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| physical | IX, 105 Seiten Illustrationen, Diagramme 30 cm |
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| spelling | Gomes Barata, Ana Verfasser aut Peroxiredoxin-mediated mitochondrial H2O2 signalling presented by Ana Gomes Barata, M.Sc. Heidelberg 2018 IX, 105 Seiten Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier Im Titel ist "2" tiefgestellt Dissertation Ruperto-Carola University of Heidelberg 2018 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1159997519/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030665571&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Gomes Barata, Ana Peroxiredoxin-mediated mitochondrial H2O2 signalling |
| subject_GND | (DE-588)4113937-9 |
| title | Peroxiredoxin-mediated mitochondrial H2O2 signalling |
| title_auth | Peroxiredoxin-mediated mitochondrial H2O2 signalling |
| title_exact_search | Peroxiredoxin-mediated mitochondrial H2O2 signalling |
| title_full | Peroxiredoxin-mediated mitochondrial H2O2 signalling presented by Ana Gomes Barata, M.Sc. |
| title_fullStr | Peroxiredoxin-mediated mitochondrial H2O2 signalling presented by Ana Gomes Barata, M.Sc. |
| title_full_unstemmed | Peroxiredoxin-mediated mitochondrial H2O2 signalling presented by Ana Gomes Barata, M.Sc. |
| title_short | Peroxiredoxin-mediated mitochondrial H2O2 signalling |
| title_sort | peroxiredoxin mediated mitochondrial h2o2 signalling |
| topic_facet | Hochschulschrift |
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