Nuclear hormone receptors and fibroblast growth factor receptor signaling in Echinococcus multilocularis:
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| Main Author: | |
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2012
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| Subjects: | |
| Online Access: | Volltext Inhaltsverzeichnis |
| Item Description: | Zsfassung in dt. und engl. Sprache. - Online erschienen: 2014 |
| Physical Description: | IV, 144 Bl. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Nuclear hormone receptors and fibroblast growth factor receptor signaling in Echinococcus multilocularis |c von Sabine Förster |
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Record in the Search Index
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|---|---|
| adam_text | NUCLEAR HORMONE RECEPTORS AND FIBROBLAST GROWTH FACTOR RECEPTOR
SIGNALING IN ECHINOCOCCUS MULTILOCULARIS
SIGNALWEGE IN ECHINOCOCCUS MULTILOCULARIS AM BEISPIEL DER NUKLEAREN
HORMONREZEPTOREN UND
DES FIBROBLAST GROWTH FACTOR REZEPTORS
DOCTORAL THESIS FOR A DOCTORAL DEGREE
AT THE GRADUATE SCHOOL OF LIFE SCIENCES,
JULIUS-MAXIMILIANS-UNIVERSITAT WIIRZBURG,
SECTION INFECTION AND IMMUNITY
SUBMITTED BY
SABINE FORSTER
FROM
ZEITZ
WUERZBURG 2012
HTTP://D-NB.INFO/1054662444
LIST OF CONTENT
LIST OF CONTENT
1. ABSTRACT 1
2. ZUSAMMENFASSUNG 2
3. INTRODUCTION 3
3.1. THE SMALL FOX-TAPEWORM ECHINOCOCCUS MULTILOCULARIS 3
3.1.1. PHYLOGENY AND EPIDEMIOLOGY 3
3.1.2. BIOLOGY AND THE
LIFE CYCLE
OF E. MULTILOCULARIS 5
3.1.3. CLINICAL MANIFESTATION - ALVEOLAR ECHINOCOCCOSIS 6
3.2. HOST-PARASITE RELATIONSHIP 8
3.2.1. BIOCHEMICAL AND MOLECULAR BIOLOGICAL APPROACHES 8
3.2.2. CULTIVATION OF E. MULTILOCULARIS 8
3.2.3. EVOLUTIONARY CONSERVED SIGNALING CASCADES
IN E. MULTILOCULARIS 9
3.3. FIBROBLAST GROWTH FACTORS (FGF) AND FGF RECEPTORS
(FGFRS) 11
3.3.1. DIVERSITY AND FUNCTION OF FGF AND FGFRS 11
3.3.2. FGFRS IN PLATYHELMINTHES AND NEMATODES 12
3.4. NUCLEAR HORMONE RECEPTOR (NHR) SIGNALING 15
3.4.1. FUNCTION AND
STRUCTURE OF NHRS 15
3.4.2. NHRS IN PARASITICHELMINTHS 16
3.5. OBJECTIVES 17
4. MATERIALS AND METHODS 18
4.1. MATERIAL 18
4.1.1. EQUIPMENT 18
4.1.2. CONSUMABLES 19
4.1.3. CHEMICALS, ENZYMES AND
SOLUTIONS 19
4.1.4. COMMERCIAL KITS 21
4.2. OLIGONUCLEOTIDES 21
4.3. DETERMINATION OF NUCLEIC ACID CONCENTRATION AND PURITY 25
4.4. RNA APPLICATIONS 25
4.4.1. ISOLATION AND PURIFICATION OF TOTAL RNA FROM E. MULTILOCULARIS 25
4.4.2. FIRST STRAND CDNA SYNTHESIS 25
4.5. DNA PROCEDURES ^ 26
4.5.1. ISOLATION AND PURIFICATION OF CHROMOSOMAL DNA FROM THE
METACESTODES 26
4.5.2. ISOLATION OF PLASMID DNA FROM E. COLI 26
4.5.3. GEL ELECTROPHORESIS OF DNA 26
4.5.4. PURIFICATION AND DNA PRECIPITATION 26
4.5.5. SEQUENCING
OF DNA 27
4.5.6. AMPLIFICATION OF DNA VIA PCR 27
4.5.7. RAPID AMPLIFICATION OF CDNA ENDS (RACE) 27
4.5.8. SEMI-QUANTITATIVE RT PCR 28
4.5.9. PCR BASED MUTAGENESIS 28
4.5.10. TA CLONING 29
4.5.11. COLONY-PCR 29
4.5.12. RESTRICTION DIGEST OF DNA 29
I
LIST OF CONTENT
4.5.13. LIGATION OF DNA
FRAGMENTS 29
4.6. PROTEIN ANALYSIS 30
4.6.1. DETERMINATION OF PROTEIN CONCENTRATION 30
4.6.2. SDS-PAGE 30
4.6.3. COOMASSIE STAINING
OF PROTEIN GELS 31
4.6.4. SILVER STAINING OF PROTEIN GELS 31
4.6.5. WESTERN BLOTTING 31
4.6.6. MASS SPECTROMETRY 32
4.7. WORKING WITH BACTERIA 33
4.7.1. BACTERIA STRAINS AND
MEDIA 33
4.7.2. CHEMICALLY COMPETENT E. COLI 33
4.7.3. E. COLI TRANSFORMATION 34
4.7.4. HETEROLOGOUS EXPRESSION IN E. COLI AND PURIFICATION OF THE
RECOMBINANT PROTEINS 34
4.8. WORKING WITH YEAST 36
4.8.1. YEAST STRAINS AND MEDIA 36
YEAST-TWO HYBRID ANALYSIS 37
4.8.2. LIQUID CULTURE 0-GALACTOSIDASE ASSAY 38
4.9. WORKING WITH MAMMALIAN CELL LINES 39
4.9.1. CELL LINES AND MEDIA 39
4.9.2. CULTIVATION OF RH- CELLS UNDER
SERUM-FREE CONDITIONS: PANSERINTM 401 39
4.9.3. TRANSFECTION OF HEK293 CELLS 39
4.10. TRANSFECTION AND TREATMENT OF XENOPUS LAEVIS OOCYTES 40
4.11. WORKING WITH E. MULTILOCULARIS 41
4.11.1. E. MULTILOCULARIS ISOLATES 41
*
4.11.2. IN VIVO CULTIVATION OF METACESTODE LARVAE AND ISOLATION FROM
MONGOLIAN JIRDS 41
4.11.3. ISOLATION AND ACTIVATION OF PROTOSCOLECES 41
4.11.4. IN VITRO CULTIVATION OF METACESTODE LARVAE 42
4.11.5. IN VITRO CULTIVATION AND TREATEMENT OF ACTIVATED PROTOSCOLESES
43
4.11.6. TREATMENT OF THE METACESTODE VESICLES 43
4.11.7. ISOLATION AND.
CULTIVATION OF E. MULTILOCULARIS PRIMARY CELLS 43
4.11.8. TREATMENT OF E. MULTILOCULARIS PRIMARY CELLS 44
4.11.9. VERIFICATION OF PURITY OF ECHINOCOCCUS MATERIAL 44
4.11.10. BRDLL-PROLIFERATION ASSAY 44
4.11.11. SIRNA KNOCK-DOWN OF EMFR 45
4.12. COMPUTER ANALYSES AND
STATISTICS 46
4.12.1. ITERATIVE BLAST FOR THE
IDENTIFICATION OF PUTATIVE E. MULTILOCULARIS NHRS 46
4.13. ETHICS STATEMENT 47
5. RESULTS 48
5.1. FGFR SIGNALING -
EMFR 48
5.1.1. EMFR EXPRESSION ANALYSIS IN ECHINOCOCCUS LARVAL STAGES 48
5.1.2. PROTEIN-PROTEIN-INTERACTIONS STUDIES OF THE INTRACELLULAR DOMAIN
OF EMFR 49
5.1.3. HOST DERIVED FGFS STIMULATE
DE NOVO DNA
SYNTHESIS IN ECHINOCOCCUS LARVAE
IN VITRO- 50
5.1.4. GROWTH AND DEVELOPMENT OF E. MULTILOCULARIS IS STIMULATED BY HOST
FGFS
IN VITRO -
PRIMARY CELLS 51
II
LIST OF CONTENT
5.1.5. TARGETING THE FGF SIGNALING PATHWAY OF E. MULTILOCULARIS:
BIBF1120- EFFECTS ON
METACESTODE VESICLES AND
PRIMARY CELLS 52
5.1.6. THE EFFECT OF HOST FGFS AND BIBF1120 ON IN VITRO CULTIVATED
PROTOSCOLECES 55
5.1.7. TARGETING THE FGF SIGNALING PATHWAY OF E. MULTILOCULARIS: SU5042-
EFFECTS ON
METACESTODE VESICLES AND
PRIMARY CELLS 58
5.1.8. SIRNA KNOCK-DOWN OF EMFR
- PRELIMINARY DATA 61
5.1.9. EMFR IS ACTIVATED BY AFGF AND
BFGF, LEADING
TO MAP-KINASE SIGNALING
IN XENOPUS LAEVIS
OOCYTES 63
5.1.10. PHOSPHORYLATION STATUS OF EMMPKL IN METACESTODES AND
PRIMARY CELLS 65
5.1.11. PHOSPHORYLATION STATUS OF EMAKT IN METACESTODE VESICLES AND
PRIMARY CELLS 67
5.1.12. THE E. MULTILOCULARIS
GENOME DOES NOT ENCODE FOR OWN CANONICAL FGFS 68
5.2. NHR SIGNALING-EMNHRL 69
5.2.1. EXPRESSION ANALYSIS OF EMNHRL IN ECHINOCOCCUS LARVAL STAGES 69
5.2.2. RECOMBINANTLY EXPRESSED EMNHRL
- AN ATTEMPT TO RAISE A SPECIFIC ANTIBODY 70
5.2.3. PROTEIN-PROTEIN INTERACTION STUDIES OF EMNHRL AND ITS DOMAINS:
LBD AND DBD 71
5.2.4. EMNHRL-LBD DIMERIZATION STUDIES 72
5.2.5. EFFECT OF BILE SALTS AND CONJUGATED BILE SALTS ON AXENICALLY
CULTIVATED METACESTODE
VESICLES 74
5.3. NHR SIGNALING
- OTHER PUTATIVE EMNHRS 76
5.3.1. IN SILICO
ANALYSIS AND
IDENTIFICATION OF PUTATIVE E. MULTILOCULARIS NHRS 76
5.3.2. EXPRESSION OF OTHER EMNHRS IN ECHINOCOCCUS LARVAL STAGES 81
5.4. NHR SIGNALING-EMHNF4 82
5.4.1. ISOLATION AND CHARACTERIZATION OF THE EMHNF4 CDNA AND
THE GENOMIC LOCUS 82
5.4.2. EXPRESSION ANALYSIS EMHNF4A AND EMHNF4B
IN ECHINOCOCCUS LARVAL STAGES 86
5.4.3. PROTEIN-PROTEIN INTERACTION STUDIES OF EMHNF4 86
5.5. SERUM-FREE CULTIVATION OF E. MULTILOCULARIS 88
5.5.1. ADAPTATION OF RH- TO SERUM FREE CONDITIONS 88
5.5.2. CO-CULTIVATION OF E. MULTILOCULARIS UNDER SERUM-FREE CONDITIONS
AND IN VIVO PASSAGE- 90
5.5.3. AXENIC CULTIVATION OF METACESTODE VESICLES AND CULTIVATION OF
PRIMARY CELLS UNDER
SERUM-FREE CONDITIONS 92
5.5.4. INFLUENCE OF INSULIN AND SEROTONIN ON AXENIC METACESTODE VESICLES
CULTURED IN
PANSERIN*401 93
5.5.5. . CULTIVATION OF PRIMARY CELLS IN SERUM-FREE PANSERIN*401 97
5.5.6. MASS SPECTROMETRY AND WESTERN BLOT ANALYSES OF PRE-CONDITIONED
PANSERIN*401* 99
5.6. EMMPK3 - A SERUM-RESPONSIVE MAPK 101
5.6.1. PHOSPHORYLATION OF EMMPK3 IS NOT INDUCED BY SERUM IN METACESTODE
VESICLES,
DORMANT AND ACTIVATED PROTOSCOLECES 101
5.6.2. EXPRESSION ANALYSIS OF EMMPK3 102
6. DISCUSSION 104
6.1. FGFR SIGNALING
- EMFR 104
6.2. NHR SIGNALING
- EMNHRL, EMHNF4
AND OTHER PUTATIVE EMNHRS 109
6.3. SERUM-FREE CULTIVATION OF E.
MULTILOCULARIS 114
6.4. EMMPK3
- A SERUM-RESPONSIVE MAPK 115
7. SUPPLEMENT 117
III
LIST OF CONTENT
7.1. FULL LENGTH CDNA SEQUENCE OF EMNHRL (ISOLATE H95) 117
7.2. FULL LENGTH CDNA SEQUENCES
OF EMHNF4A AND EMHNF4B 118
7.3. LBD SEQUENCES OF E. MULTILOCULARIS NHRS FROM
IN SILICO ANALYSIS 120
7.4. PUTATIVE AMINO ACID SEQUENCE OF EMNDK 122
8. LIST OF ABBREVIATIONS 123
9. REFERENCES 127
10. ACKNOWLEDGEMENT - DANKSAGUNG 139
11. PUBLICATIONS 141
11.1. ORIGINAL PUBLICATIONS 141
11.2. CONFERENCE CONTRIBUTIONS 142
11.2.1. TALKS 142
11.2.2. POSTER 143
12. CURRICULUM VITAE 144
IV
|
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| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-027422342 |
| oclc_num | 884381170 |
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| physical | IV, 144 Bl. Ill., graph. Darst. |
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| spelling | Förster, Sabine 1982- Verfasser (DE-588)1053780117 aut Nuclear hormone receptors and fibroblast growth factor receptor signaling in Echinococcus multilocularis von Sabine Förster Signalwege in Echinococcus multilocularis am Beispiel der Nukleären Hormonrezeptoren und des Fibroblast Growth Factor Rezeptors 2012 IV, 144 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. und engl. Sprache. - Online erschienen: 2014 Würzburg, Univ., Diss., 2012 Fibroblastenwachstumsfaktor (DE-588)4310092-2 gnd rswk-swf Signaltransduktion (DE-588)4318717-1 gnd rswk-swf Fuchsbandwurm (DE-588)4290099-2 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Fuchsbandwurm (DE-588)4290099-2 s Signaltransduktion (DE-588)4318717-1 s Fibroblastenwachstumsfaktor (DE-588)4310092-2 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-85832 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-85832 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=027422342&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Förster, Sabine 1982- Nuclear hormone receptors and fibroblast growth factor receptor signaling in Echinococcus multilocularis Fibroblastenwachstumsfaktor (DE-588)4310092-2 gnd Signaltransduktion (DE-588)4318717-1 gnd Fuchsbandwurm (DE-588)4290099-2 gnd |
| subject_GND | (DE-588)4310092-2 (DE-588)4318717-1 (DE-588)4290099-2 (DE-588)4113937-9 |
| title | Nuclear hormone receptors and fibroblast growth factor receptor signaling in Echinococcus multilocularis |
| title_alt | Signalwege in Echinococcus multilocularis am Beispiel der Nukleären Hormonrezeptoren und des Fibroblast Growth Factor Rezeptors |
| title_auth | Nuclear hormone receptors and fibroblast growth factor receptor signaling in Echinococcus multilocularis |
| title_exact_search | Nuclear hormone receptors and fibroblast growth factor receptor signaling in Echinococcus multilocularis |
| title_full | Nuclear hormone receptors and fibroblast growth factor receptor signaling in Echinococcus multilocularis von Sabine Förster |
| title_fullStr | Nuclear hormone receptors and fibroblast growth factor receptor signaling in Echinococcus multilocularis von Sabine Förster |
| title_full_unstemmed | Nuclear hormone receptors and fibroblast growth factor receptor signaling in Echinococcus multilocularis von Sabine Förster |
| title_short | Nuclear hormone receptors and fibroblast growth factor receptor signaling in Echinococcus multilocularis |
| title_sort | nuclear hormone receptors and fibroblast growth factor receptor signaling in echinococcus multilocularis |
| topic | Fibroblastenwachstumsfaktor (DE-588)4310092-2 gnd Signaltransduktion (DE-588)4318717-1 gnd Fuchsbandwurm (DE-588)4290099-2 gnd |
| topic_facet | Fibroblastenwachstumsfaktor Signaltransduktion Fuchsbandwurm Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-85832 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027422342&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT forstersabine nuclearhormonereceptorsandfibroblastgrowthfactorreceptorsignalinginechinococcusmultilocularis AT forstersabine signalwegeinechinococcusmultilocularisambeispieldernuklearenhormonrezeptorenunddesfibroblastgrowthfactorrezeptors |