Structural characterization of the TFIIH subunits p34 and p44 from C. thermophilum: = Strukturelle Charakterisierung der TFIIH Untereinheiten p34 und p44 aus C. thermophilum
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| Main Author: | |
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| Format: | Thesis Book |
| Language: | English |
| Published: |
Würzburg
2014
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis kostenfrei Inhaltsverzeichnis |
| Item Description: | veröffentlicht 2017 |
| Physical Description: | Seite A-F, 142 Seiten Illustrationen, Diagramme 30 cm |
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MARC
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| 100 | 1 | |a Schmitt, Dominik |d ca. 21. Jh. |e Verfasser |0 (DE-588)1149192704 |4 aut | |
| 245 | 1 | 0 | |a Structural characterization of the TFIIH subunits p34 and p44 from C. thermophilum |b = Strukturelle Charakterisierung der TFIIH Untereinheiten p34 und p44 aus C. thermophilum |c submitted by Dominik Schmitt from Heidelberg |
| 246 | 1 | 1 | |a Strukturelle Charakterisierung der TFIIH Untereinheiten p34 und p44 aus C. thermophilum |
| 264 | 1 | |a Würzburg |c 2014 | |
| 300 | |a Seite A-F, 142 Seiten |b Illustrationen, Diagramme |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a veröffentlicht 2017 | ||
| 502 | |b Dissertation |c Julius-Maximilians-Universität Würzburg |d 2014 | ||
| 546 | |a Zusammenfassung in deutscher und englischer Sprache | ||
| 650 | 0 | 7 | |a Chaetomium |0 (DE-588)4122997-6 |2 gnd |9 rswk-swf |
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| 650 | 0 | 7 | |a DNS-Reparatur |0 (DE-588)4150347-8 |2 gnd |9 rswk-swf |
| 653 | |a TFIIH | ||
| 653 | |a General Transcription Factor II H | ||
| 653 | |a p34 | ||
| 653 | |a p44 | ||
| 653 | |a Tfb4 | ||
| 653 | |a Ssl1 | ||
| 653 | |a Chaetomium thermophilum | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
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| 776 | 0 | 8 | |i Erscheint auch als |n Online-Ausgabe |o urn:nbn:de:bvb:20-opus-104851 |a Schmitt, Dominik |t Structural Characterization of the TFIIH Subunits p34 and p44 from C. thermophilum |d Würzburg : Universität Würzburg, 2017 |h Online-Ressource |
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| _version_ | 1804178393907855360 |
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| adam_text | TABLE OF CONTENTS
ABSTRACT.................................................................................................................................................................A
ZUSAMMENFASSUNG.................................................................................................................................................B
TABLE OF
CONTENTS...................................................................................................................................................
C
I.
INTRODUCTION......................................................................................................................................................1
1.1 DNA DAMAGE AND
REPAIR..............................................................................................................................
1
1.1.1 THE IMPORTANCE OF DNA
REPAIR.............................................................................................................
1
1.1.2 DNA REPAIR
MECHANISMS........................................................................................................................
2
1.1.3 EUKARYOTIC NUCLEOTIDE EXCISION REPAIR
(NER)......................................................................................3
1.2 BASIC CONCEPTS OF DNA TRANSCRIPTION
..................................................................
.......................................
4
1.3 THE GENERAL TRANSCRIPTION FACTOR II H
(TFIIH).....................................
.
......................................................
5
1.3.1 MOLECULAR COMPOSITION OF THE TFIIH
COMPLEX......................................................................................5
1.3.2 STRUCTURAL KNOWLEDGE ON TFIIH
SUBUNITS..............................................................................................
6
1.4 THE TFIIH COMPLEX IN DNA REPAIR AND TRANSCRIPTION
................................................................................
7
1.4.1 TFIIH IN NUCLEOTIDE EXCISION
REPAIR......................................................................................................
7
1.4.2 THE ROLE OF TFIIH IN TRANSCRIPTION AND CELL CYCLE
CONTROL...................................................................
8
1.4.3 CROSSTALK BETWEEN DNA REPAIR AND
TRANSCRIPTION................................................................................8
1.5 DISEASES CAUSED BY MUTATIONS IN TFIIH
SUBUNITS........................................................................................
9
1.6 THE FUNGUS C.
THERMOPHILUM.......................................................................................................................10
II. MATERIAL
...........................................................
11
II. 1
EQUIPMENT..............................................................................................................................................11
II. 1.1
INSTRUMENTS.....................................................................................................................................
11
II.1.2
CONSUMABLES....................................................................................................................................
13
11.2 CHEMICALS AND BIOCHEMICAL
SUBSTANCES....................................................................................................
14
11.2.1
CHEMICALS............................................................................................................................................
14
11.2.2 READY-TO-USE
KITS..............................................................................................................................
15
11.2.3 CRYSTALLIZATION SCREENS
.................
16
11.2.4 PRIMERS AND DNA
SUBSTRATES..............................................................................................................
16
11.2.5
ENZYMES..............................................................................................................................................17
11.3 CELL
CULTURE..............................................................................................
18
11.3.1 BACTERIAL AND INSECT CELL
STRAINS.........................................................................................................
18
11.3.2 VECTORS FOR PROTEIN
EXPRESSION...........................................................................................................
18
11.3.3 BACTERIAL CULTURE MEDIA AND MEDIA
SUPPLEMENTS..............................................................................
19
11.3.4 INSECT CELL CULTURE MEDIA AND MEDIA
SUPPLEMENTS............................................................................19
11.4 BUFFERS AND
SOLUTIONS................................................................................................................
20
11.4.1 BUFFERS AND SOLUTIONS FOR PURIFICATION OF
PROTEINS.............................................................................
20
11.4.2 BUFFERS AND SOLUTIONS FOR BIOCHEMICAL ASSAYS
...................................................................................
20
11.4.3 BUFFERS AND SOLUTIONS FOR GEL ELECTROPHORESIS AND SAMPLE STAINING
.................................................
21
II. 5 SOFTWARE AND
DATABASES.........................................................................................................................
22
11.5.1 SOFTWARE ON LOCAL
HARDWARE...............................................................................................................22
11.5.2 SOFTWARE ON THE WORLD WIDE
WEB......................................................................................................23
11.5.3
DATABASES.............................................................................................................................................23
III.
METHODS...................................................................................................................
.
..................................25
III. 1 MOLECULAR
BIOLOGY..................................................................................................................................
25
111.1.1 POLYMERASE CHAIN REACTION
(PCR).....................................................................................................
25
111.1.2 DNA ISOLATION
.....................................
26
111.1.3 DNA DOUBLE
DIGESTION........................................................................................................................
26
111.1.4 DNA GEL
ELECTROPHORESIS....................................................................................................................
26
111.1.5 DNA CLONING AND LIGATION
......
.
..........................................................................................................
27
111.1.6
COLONY-PCR.........................................................................................................................................
28
111.1.7 SITE-DIRECTED
MUTAGENESIS.................................................................................................................
29
111.1.8 DNA
SEQUENCING..................................................................................................................................29
111.1.9 DNA HYBRIDIZATION......................................... 29
111.1.10 BACMID DNA
GENERATION...................................................................................................................30
111.1.11 BACMID DNA
ISOLATION.......................................................................................................................30
111.2
MICROBIOLOGY.............................................................................................................................................
31
111.2.1 TRANSFORMATION OF
BACTERIA................................................................................................................
31
111.2.2 GROWING OF BACTERIA FOR PROTEIN EXPRESSION
.................................................
.
...................................31
111.2.3 GROWING AND MAINTENANCE OF INSECT CELL
CULTURES.............................................................................
32
111.2.4 TRANSFECTION OF INSECT
CELLS................................................................................................................
32
111.2.5 PLAQUE
ASSAY.......................................................................................................................................
32
111.2.6 VIRUS
AMPLIFICATION.............................................................................................................................33
111.2.7 GROWING OF INSECT CELLS FOR PROTEIN
EXPRESSION.................................................................................33
111.3 PROTEIN
BIOCHEMISTRY................................................................................................................................34
111.3.1 CELL LYSIS.............................................. 34
111.3.2 PURIFICATION OF PROTEINS AND CHROMATOGRAPHY
TECHNIQUES...............................................................34
111.3.3 ANALYSIS OF PROTEINS VIA
SDS-PAGE.....................................................................................................36
111.3.4 ULTRA-FILTRATION AND STORAGE OF PROTEIN
SAMPLES...............................................................................
36
111.3.5 ANALYTICAL SIZE EXCLUSION CHROMATOGRAPHY
(A-SEC).........................................................................
36
111.3.6 ELECTROPHORETIC MOBILITY SHIFT ASSAY
(EMSA)....................................................................................
37
111.3.7 ISOTHERMAL TITRATION CALORIMETRY
(ITC).............................................................................................38
111.4
SPECTROSCOPY.............................................................................................................................................
39
111.4.1 DETERMINATION OF DNA AND PROTEIN
CONCENTRATION...........................................................................
39
111.4.2 THERMOFLUOR
(TF)...............................................................................................................................
39
111.4.3 CIRCULAR DICHROISM
(CD).....................................................................................................................40
111.4.4 MULTI ANGLE LIGHT SCATTERING
(MALS).................................................................................................
40
111.4.5 MASS SPECTROMETRY
(MS)....................................................................................................................41
------------------------------------------------------------- D
-------------------------------------------------------------
III. 5 X-RAY
CRYSTALLOGRAPHY.............................................................................................................................
42
111.5.1 GENERAL
INTRODUCTION.........................................................................................................................
42
111.5.2 PRINCIPLES OF X-RAY CRYSTALLOGRAPHY
...................................
.
............................................................
42
111.5.3 PHASE
DETERMINATION.........................................................................................................................
43
111.5.4 PROTEIN
CRYSTALLIZATION.......................................................................................................................44
111.5.5 DATA
COLLECTION...................................................................................................................................
45
111.5.6 DATA PROCESSING AND STRUCTURE DETERMINATION
................................................................................
46
111.5.7 MODEL BUILDING AND
REFINEMENT........................................................................................................46
IV. RESULTS AND
DISCUSSION............................................................................................................................
47
IV. 1 TOWARDS CRYSTALLIZATION OF TFIIH SUBUNITS FROM H.
SAPIENS...................................................................
47
IV. 1.1 INITIAL ATTEMPTS WITH HUMAN XPD, XPB, P44 AND
P52....................................................................47
IV. 1.2 THE HUMAN P44
PROTEIN..................................................................................................................48
IV.1.3 THE P34H P44H MINIMAL COMPLEX
(HMC)...........................................................................................49
IV.2 CHARACTERIZATION OF TFIIH SUBUNITS FROM C.
THERMOPHILUM.....................................................................52
IV.2.1 PURIFICATION OF P34CT
VARIANTS............................................................................................................52
IV.2.2 PURIFICATION OF P44CT
VARIANTS............................................................................................................54
IV.2.3 PREPARATION OF THE P34CT P44CT MINIMAL COMPLEX (MC)
...................................................................
56
IV.2.4 DNA BINDING STUDIES WITH P34CT AND
P44CT......................................................................................
58
IV.2.5 THERMOSTABILITY OF P34CT AND P44CT IN
SOLUTION................................................................................60
IV.2.6 OLIGOMERIC STATE OF P34CT AND P44CT IN
SOLUTION...............................................................................
63
IV.3 STRUCTURE DETERMINATION OF DPS - A SUCCESSFUL
FAILURE..........................................................................
65
IV.4 CRYSTALLIZATION OF P44CT VARIANTS - GETTING
CLOSER...................................................................................67
IV.5 CRYSTAL STRUCTURE OF P34CT - A NOVEL VWA LIKE DOMAIN
....................
.
..........
.
..........................................
68
IV.5.1 CRYSTALLIZATION OF
P34CT......................................................................................................................68
IV.5.2 STRUCTURE DETERMINATION OF
P34CT.....................................................................................................
69
IV.5.3 CRYSTALLIZATION OF P34CT
1-277...........................................................................................................70
IV.5.4 STRUCTURE DETERMINATION OF P34CT 1-277 - THE P34CT VWA LIKE
DOMAIN
..........................................
71
IV.5.5 ANALYSIS OF P34CT CRYSTAL PACKING AND ELUCIDATION OF BINDING
SITES FOR P44CT..................................75
IV.5.6 COMPARISON OF P34CT VWA TO OTHER VWA
DOMAINS...........................................................................
77
IV.5.7 THE MULTIFACETED ROLES OFVWA
DOMAINS...........................................................................................79
IV.5.8 IMPLICATIONS FOR PROTEIN-PROTEIN INTERACTIONS WITHIN
TFIIH..................................................... 81
IV.6 CRYSTAL STRUCTURE OF P34CT P44CT MC - A NOVEL BINDING MODE FOR RING
DOMAINS..................................82
IV.6.1 CRYSTALLIZATION OF P34CT P44CT
MC....................................................................................................
82
IV.6.2 STRUCTURE DETERMINATION OF P34CT P44CT
MC....................................................................................
83
IV.6.3 COMPARISON OF THE P44CT C4C4 DOMAIN TO OTHER RING
DOMAINS..................................................... 85
IV.6.4 ANALYSIS OF P34CT P44CT MC CRYSTAL
PACKING....................................................................................
67
IV.7 ANALYSIS OF THE P34CT P44CT COMPLEX INTERFACE
......................................................................................
88
IV.7.1 DECIPHERING OF INTERACTION SITES WITHIN P34CT P44CT MC
.................................................................
88
IV.7.2 PURIFICATION OF P34CT AND P44CT
VARIANTS..........................................................................................90
IV.7.3 CHARACTERIZATION OF P34CT AND P44CT
VARIANTS..................................................................................91
E
IV.7.4 DETERMINATION OF CRITICAL
RESIDUES....................................................................................................93
IV.7.5 DETERMINATION OF BINDING
AFFINITIES..................................................................................................
96
V. CONCLUSION AND
OUTLOOK................................................................................................................................
99
V.L THE TFIIH SUBUNITS P34 AND P44 FROM C.
THERMOPHILUM..........................................................................99
V.2 THE P34CT VWA
DOMAIN............................................................................................................................100
V.3 THE P34CT P44CT MINIMAL
COMPLEX..........................................................................................................101
V.4 IMPLICATIONS FROM THE P34CT P44CT MINIMAL
COMPLEX.............................................................................102
V.5 THE ROLE OF THE P34CT AND P44CT ZINC FINGER
DOMAINS...........................................................................
103
V.6 THE IMPORTANCE OF TFIIH
RESEARCH..........................................................................................................
104
VI. LITERATURE
REFERENCES................................................................................................................................105
VII. PROTEIN DATA BANK
REFERENCES................................................................................................................121
VIII. FIGURES AND
TABLES..................................................................................................................................
122
VIII.L LIST OF
FIGURES.......................................................................................................................................
122
VIII.2 LIST OF
TABLES.........................................................................................................................................
123
IX.
ABBREVIATIONS..................................................................................
124
X.
APPENDIX........................................................................
127
X.1 TABLE OF AMINO
ACIDS................................................................................................................................
127
X.2 DETAILED MAPS OF THE VECTORS USED FOR PROTEIN
EXPRESSION.....................................................................128
X.2.1
PBADM-11..........................................................................................................................................128
X.2.2
PETM-11.............................................................................................................................................128
X.2.3 PFASTBAC
HTA.....................................................................................................................................
129
X.2.4 PFASTBAC
DUAL....................................................................................................................................
129
X.3 DETAILED INFORMATION ON P34CT AND P44CT
CONSTRUCTS.............................................................................131
X.3.1 CONSTRUCTS OF
P34CT............................................................................................................................131
X.3.2 CONSTRUCTS OF
P44CT............................................................................................................................131
X.4 SECONDARY STRUCTURE PREDICTION OF P34CT USING THE PHYRE2
ALGORITHM...................................................132
X.5 SECONDARY STRUCTURE PREDICTION OF P44CT USING THE PHYRE2
ALGORITHM...................................................133
X.6 DATA COLLECTION AND REFINEMENT STATISTICS FOR
P34CT................................................................................134
X.7 DATA COLLECTION AND REFINEMENT STATISTICS FOR P34CT P44CT MC
...............................................................
135
ACKNOWLEDGMENT
........................................
137
AFFIDAVIT................................................. 139
CURRICULUM
VITAE.................................................................................................................................................141
F
|
| any_adam_object | 1 |
| author | Schmitt, Dominik ca. 21. Jh |
| author_GND | (DE-588)1149192704 |
| author_facet | Schmitt, Dominik ca. 21. Jh |
| author_role | aut |
| author_sort | Schmitt, Dominik ca. 21. Jh |
| author_variant | d s ds |
| building | Verbundindex |
| bvnumber | BV044866320 |
| collection | ebook |
| ctrlnum | (OCoLC)1135401992 (DE-599)DNB1149192453 |
| discipline | Biologie |
| format | Thesis Book |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV044866320 |
| illustrated | Illustrated |
| indexdate | 2024-07-10T08:03:16Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-030260857 |
| oclc_num | 1135401992 |
| open_access_boolean | 1 |
| owner | DE-384 DE-473 DE-BY-UBG DE-703 DE-1051 DE-824 DE-29 DE-12 DE-91 DE-BY-TUM DE-19 DE-BY-UBM DE-1049 DE-92 DE-739 DE-898 DE-BY-UBR DE-355 DE-BY-UBR DE-706 DE-20 DE-1102 DE-860 DE-2174 |
| owner_facet | DE-384 DE-473 DE-BY-UBG DE-703 DE-1051 DE-824 DE-29 DE-12 DE-91 DE-BY-TUM DE-19 DE-BY-UBM DE-1049 DE-92 DE-739 DE-898 DE-BY-UBR DE-355 DE-BY-UBR DE-706 DE-20 DE-1102 DE-860 DE-2174 |
| physical | Seite A-F, 142 Seiten Illustrationen, Diagramme 30 cm |
| psigel | ebook |
| publishDate | 2014 |
| publishDateSearch | 2014 |
| publishDateSort | 2014 |
| record_format | marc |
| spelling | Schmitt, Dominik ca. 21. Jh. Verfasser (DE-588)1149192704 aut Structural characterization of the TFIIH subunits p34 and p44 from C. thermophilum = Strukturelle Charakterisierung der TFIIH Untereinheiten p34 und p44 aus C. thermophilum submitted by Dominik Schmitt from Heidelberg Strukturelle Charakterisierung der TFIIH Untereinheiten p34 und p44 aus C. thermophilum Würzburg 2014 Seite A-F, 142 Seiten Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier veröffentlicht 2017 Dissertation Julius-Maximilians-Universität Würzburg 2014 Zusammenfassung in deutscher und englischer Sprache Chaetomium (DE-588)4122997-6 gnd rswk-swf Transkriptionsfaktor (DE-588)4303350-7 gnd rswk-swf DNS-Reparatur (DE-588)4150347-8 gnd rswk-swf TFIIH General Transcription Factor II H p34 p44 Tfb4 Ssl1 Chaetomium thermophilum (DE-588)4113937-9 Hochschulschrift gnd-content Chaetomium (DE-588)4122997-6 s Transkriptionsfaktor (DE-588)4303350-7 s DNS-Reparatur (DE-588)4150347-8 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-104851 Schmitt, Dominik Structural Characterization of the TFIIH Subunits p34 and p44 from C. thermophilum Würzburg : Universität Würzburg, 2017 Online-Ressource B:DE-101 application/pdf http://d-nb.info/1149192453/04 Inhaltsverzeichnis https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-104851 Resolving-System kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030260857&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Schmitt, Dominik ca. 21. Jh Structural characterization of the TFIIH subunits p34 and p44 from C. thermophilum = Strukturelle Charakterisierung der TFIIH Untereinheiten p34 und p44 aus C. thermophilum Chaetomium (DE-588)4122997-6 gnd Transkriptionsfaktor (DE-588)4303350-7 gnd DNS-Reparatur (DE-588)4150347-8 gnd |
| subject_GND | (DE-588)4122997-6 (DE-588)4303350-7 (DE-588)4150347-8 (DE-588)4113937-9 |
| title | Structural characterization of the TFIIH subunits p34 and p44 from C. thermophilum = Strukturelle Charakterisierung der TFIIH Untereinheiten p34 und p44 aus C. thermophilum |
| title_alt | Strukturelle Charakterisierung der TFIIH Untereinheiten p34 und p44 aus C. thermophilum |
| title_auth | Structural characterization of the TFIIH subunits p34 and p44 from C. thermophilum = Strukturelle Charakterisierung der TFIIH Untereinheiten p34 und p44 aus C. thermophilum |
| title_exact_search | Structural characterization of the TFIIH subunits p34 and p44 from C. thermophilum = Strukturelle Charakterisierung der TFIIH Untereinheiten p34 und p44 aus C. thermophilum |
| title_full | Structural characterization of the TFIIH subunits p34 and p44 from C. thermophilum = Strukturelle Charakterisierung der TFIIH Untereinheiten p34 und p44 aus C. thermophilum submitted by Dominik Schmitt from Heidelberg |
| title_fullStr | Structural characterization of the TFIIH subunits p34 and p44 from C. thermophilum = Strukturelle Charakterisierung der TFIIH Untereinheiten p34 und p44 aus C. thermophilum submitted by Dominik Schmitt from Heidelberg |
| title_full_unstemmed | Structural characterization of the TFIIH subunits p34 and p44 from C. thermophilum = Strukturelle Charakterisierung der TFIIH Untereinheiten p34 und p44 aus C. thermophilum submitted by Dominik Schmitt from Heidelberg |
| title_short | Structural characterization of the TFIIH subunits p34 and p44 from C. thermophilum |
| title_sort | structural characterization of the tfiih subunits p34 and p44 from c thermophilum strukturelle charakterisierung der tfiih untereinheiten p34 und p44 aus c thermophilum |
| title_sub | = Strukturelle Charakterisierung der TFIIH Untereinheiten p34 und p44 aus C. thermophilum |
| topic | Chaetomium (DE-588)4122997-6 gnd Transkriptionsfaktor (DE-588)4303350-7 gnd DNS-Reparatur (DE-588)4150347-8 gnd |
| topic_facet | Chaetomium Transkriptionsfaktor DNS-Reparatur Hochschulschrift |
| url | http://d-nb.info/1149192453/04 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-104851 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030260857&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT schmittdominik structuralcharacterizationofthetfiihsubunitsp34andp44fromcthermophilumstrukturellecharakterisierungdertfiihuntereinheitenp34undp44auscthermophilum AT schmittdominik strukturellecharakterisierungdertfiihuntereinheitenp34undp44auscthermophilum |
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