The polycystin signalling cascade in autosomal dominant polycystic kidney disease:
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| Format: | Abschlussarbeit Buch |
| Sprache: | German |
| Veröffentlicht: |
2014
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| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | XI, 135 S. Ill., graph. Darst. 30 cm |
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Datensatz im Suchindex
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| adam_text | U
OC
D
IT,
THE POLYCYSTIN SIGNALLING CASCADE IN
AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE
INAUGURAL DISSERTATION
ZUR
ERLANGUNG DER DOKTORWUERDE
DER FAKULTAET FUER BIOLOGIE
DER ALBERT-LUDWIGS-UNIVERSITAET NUR
LBSCHFAENKT FUES*
FREIBURG IM BREISGAU CAE@N AEUSTSUSCB
VORGELEGT VON
ALEXIS HOFHERR
FREIBURG IM BREISGAU
FEBRUAR 2014
HTTP://D-NB.INFO/1052563848
TABLE OF CONTENTS
LIST OF ILLUSTRATIONS IX
ABSTRACT XI
INTRODUCTION 1
AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE (ADPKD) 2
ADPKD IS A MULTI-SYSTEM DISORDER. 2
ADPKD IS CAUSED BY MUTATIONS IN EITHER PKD1 OR PKD2 2
A TWO-HIT MECHANISM EXPLAINS THE FOCAL NATURE OF ADPKD 3
VERY LIMITED INFORMATION IS AVAILABLE ON ADPKD GENOTYPE-PHENOTYPE
CORRELATION 3
LESSONS FROM ORTHOLOGOUS ADPKD ANIMAL MODEL SYSTEMS 3
CILIA ARE NEXUS IN CELLULAR SIGNALLING PATHWAYS 5
TRPP2 IS REQUIRED FOR LEFT-RIGHT PATTERNING 5
THE POLYCYSTIN PROTEINS FORM VERSATILE RECEPTOR-CHANNEL MODULES 6
TRPP2 IS
A
TRP ION
CHANNEL
7
TRPP2
IS
A
NON-SELECTIVE CATION CHANNEL WITH SIX TRANSMEMBRANE SEGMENTS
7
POST-TRANSLATIONAL GLYCOSYLATION OF TRPP2 8
PROJECT OUTLINE: THE TRPP2 SIGNALLING CASCADE IN ADPKD 9
MATERIAL 11
INSTRUMENTS & COMMERCIAL PRODUCTS 12
CHEMICALS 18
SOLUTIONS 21
ANTISERA & MONOCLONAL IMMUNGLOBULINS 24
RECOMBINANT DNA VECTORS 25
ENSEMBL REFERENCE SEQUENCES 26
PROTEIN TAGS 26
QUANTITATIVE
POLYMERASE
CHAIN REACTION
OLIGONUCLEOTIDES 26
MORPHOLINO ANTISENSE OLIGONUCLEOTIDES 27
DANIO RERIO LINES 27
DROSOPHILA MELANOGASTER
STOCKS
27
CELL CULTURE 28
COMMERCIAL SERVICES 28
SOFTWARE 29
METHODS 31
GENERAL CONSIDERATIONS 32
MOLECULAR CELL BIOLOGY 32
ISOLATION OF NUCLEIC ACIDS 32
SPECTROPHOTOMETRIC QUANTITATION OF NUCLEIC ACIDS 32
AGAROSE GEL ELECTROPHORESIS 33
POLYMERASE CHAIN REACTION (PCR) 33
REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION (RT-PCR) 33
REAL TIME QUANTITATIVE PCR (QPCR) 34
RESTRICTION ENDONUCLEASE-MEDIATED CLEAVAGE OF DNA. 34
TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASES (TALENS) 34
DNA LIGATION 34
DNA SEQUENCING 34
PROKARYOTIC CELL CULTURE & TRANSFORMATION 35
EUKARYOTIC CELL CULTURE 35
TRANSFECTION 35
TRANSDUCTION 36
DRUG-MEDIATED INTERFERENCE WITH CELLULAR PROTEIN METABOLISM 36
SGBM PHD THESIS - ALEXIS HOFHERR
TABLE OF CONTENTS
RESTRICTION OF PROTEIN BIOSYNTHESIS BY CYCLOHEXIMIDE 36
INHIBITION OF LYSOSOMAL ENZYMES BY CHLOROQUINE 36
PROTEASOME INHIBITION BY MG132 36
PROTEIN BIOCHEMISTRY 37
CELL DISRUPTION AND PROTEIN SOLUBILISATION 37
ACETONE PRECIPITATION OF PROTEINS 37
IMMUNOPRECIPITATION AND RECOVERY OF AFFINITY PURIFIED PROTEINS 37
N-IINKED GLYCOSYLATION 38
PROTEIN ANALYSIS BY GEL ELECTROPHORESIS 38
SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) 38
VISUALISATION OF PROTEINS BY COOMASSIE BRILLIANT BLUE G-250 38
BLUE NATIVE ELECTROPHORESIS (BNE) 39
WESTERN BLOT 39
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (HPLC-MS) 39
ADVANCED TECHNIQUES IN MICROSCOPY 40
BRIGHT FIELD MICROSCOPY 40
DIFFERENTIAL INTERFERENCE CONTRAST (DIC) 40
FLUORESCENCE MICROSCOPY 40
CONFOCAL MICROSCOPY 40
SCANNING ELECTRON MICROSCOPY (SEM) 41
DROSOPHILA MELANOGASTER 41
FLY HUSBANDRY 41
P ELEMENT MEDIATED TRANSFORMATION 41
FRUIT FLY FERTILITY ASSAY 41
PREPARATION OF TESTIS AND SPERM 41
LIVE IMAGING OF SPERM IN THE FEMALE REPRODUCTIVE TRACT 42
DANIO RERIO 42
STATISTICAL ANALYSIS 43
PROTOCOLS 47
MOLECULAR CELL BIOLOGY 48
DNACLEAN UP 48
GENOMIC DNA (CULTIVATED CELLS) 49
GENOMIC DNA (DROSOPHILA MELANOGASTER)... 51
RNA (DROSOPHILA MELANOGASTER/ DANIO RERIO) 54
QUIKCHANGE PCR * 56
PCR SEQUENCING 58
HIGH EFFICIENCY TRANSFORMATION 59
5 MIN TRANSFORMATION 60
TRANSFECTION CA
3
(P0
4
)
2
61
TRANSFECTION POLY(ETHYLENEIMINE) (PEI) 62
CELL COUNT DETERMINATION 63
PROTEIN BIOCHEMISTRY 64
SMALL MOLECULE INHIBITORS 64
CELL LYSATE 65
IMMUNOPRECIPITATION (IP) 67
ACETONE PRECIPITATION 69
SDS-PAGE 70
WESTERN BLOT (SEMI-DRY) 72
PROTEIN DETECTION 73
IMMUNOFLUORESCENCE 74
TABLE OF CONTENTS
CELLS 74
DROSOPHILA MELANOGASTER SPERMATOZOA 75
DROSOPHILA MELANOGASTER TESTIS 76
RESULTS 79
D. MELANOGASTER TRPP2 (ARNO) IS A MALE SPECIFIC PROTEIN 80
FLAGELLAR ARNO (TRPP2) IS REQUIRED FOR MALE FERTILITY AND FEMALE SPERM
STORAGE 81
D. MELANOGASTER SPERMATOZOA SWIM BACKWARDS IN THE FEMALE REPRODUCTIVE
TRACT 81
FRUIT
FLY
SPERMATOZOA ARE ACTIVATED VIA ARNO (TRPP2) ION
CHANNELS
84
AN UNBIASED FORWARD GENETIC SCREEN IN D. MELANOGASTER TO IDENTIFY AMO- -
PHENOCOPIES 87
Z3-2147 MUTANTS SHOW MALE INFERTILITY AND IMPAIRED FEMALE SPERM STORAGE
88
CG32103
R30AW
CAUSES THE Z3-2147 LOSS OF AMO (PKD2)-LIKE
PHENOTYPE IN D. MELANOGASTER. 88
AMO^ AND CG32103
R3OAW
SPERMATOZOA DISPLAY A CURLED ULTRA-STRUCTURE 88
THE PHENOCOPY OF PKD2^~ (AMO) AND SLC25A25+ (CG32103) IS CONSERVED IN
VERTEBRATES 91
A SCREEN FOR PHYSICAL INTERACTION PARTNERS OF TRPP2 BY MASS SPECTROMETRY
93
LIKE TRPP2, CINGULIN IS EXPRESSED IN PRIMARY CILIA 95
CGN DEPLETION PHENOCOPIES LOSS OF PKD2 BY CAUSING RANDOMISATION OF
UNILATERAL ORGAN PLACEMENT AND CONTRIBUTES TO RENAL CYSTOGENESIS 98
MAPPING POST-TRANSLATIONAL TRPP2 GLYCOSYLATION USING MASS SPECTROMETRY
98
N-GLYCOSYLATION OF TRPP2 IS REQUIRED FOR ADEQUATE TRPP2 PROTEIN LEVELS
101
TRPP2 IS SUBJECT TO LYSOSOMAL DEGRADATION 104
INACTIVATION OF GLUCOSIDASE II RESULTS IN DEFECTS IN N-GLYCAN TRIMMING
OF TRPP2 105
DISCUSSION 109
NOVEL
LEADS
INTO THE
TRPP2
SIGNALLING CASCADE
110
THE REFINEMENT OF THE D. MELANOGASTER AMO (PKD2) KNOCK-OUT PHENOTYPE
DEMONSTRATES
AMO
S FUNCTION AS AN ION CHANNEL CONTROLLING SPERM MOTILITY
110
A FORWARD UNBIASED GENETIC SCREEN FOR AMO (PKD2) KNOCK-OUT PHENOCOPIES
IN FLIES
HIGHLIGHTS CG32103 S (SLC25A25) ROLE IN THE ESTABLISHMENT OF
LEFT-RIGHT ASYMMETRY IN VERTEBRATES 111
A SCREEN FOR PHYSICAL INTERACTION PARTNERS OF TRPP2 BY MASS SPECTROMETRY
AND SUBSEQUENT
PHENOTYPIC EVALUATION IN ZEBRAFISH WAS FUNDAMENTAL IN ELUCIDATING THE
GENETIC
INTERACTION OF TRPP2 AND CINGULIN IN CYSTOGENESIS AND HETEROTAXY 113
MAPPING OF N-IINKED GLYCOSYLATION IN TRPP2 IDENTIFIED FIVE GLYCOSYLATED
ASPARAGINE RESIDUES
THAT ARE REQUIRED FOR ADEQUATE TRPP2 PROTEIN EXPRESSION 115
NEW STEPPING STONES 117
REFERENCES 119
ACKNOWLEDGMENTS 135
VIII
SGBM PHD THESIS - ALEXIS HOFHERR
|
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| dewey-raw | 610 570 |
| dewey-search | 610 570 |
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| dewey-tens | 610 - Medicine and health 570 - Biology |
| discipline | Biologie Medizin |
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| spelling | Hofherr, Alexis 1980- Verfasser (DE-588)136280781 aut The polycystin signalling cascade in autosomal dominant polycystic kidney disease vorgelegt von Alexis Hofherr 2014 XI, 135 S. Ill., graph. Darst. 30 cm txt rdacontent n rdamedia nc rdacarrier Freiburg (Breisgau), Univ., Diss., 2014 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027501635&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Hofherr, Alexis 1980- The polycystin signalling cascade in autosomal dominant polycystic kidney disease |
| subject_GND | (DE-588)4113937-9 |
| title | The polycystin signalling cascade in autosomal dominant polycystic kidney disease |
| title_auth | The polycystin signalling cascade in autosomal dominant polycystic kidney disease |
| title_exact_search | The polycystin signalling cascade in autosomal dominant polycystic kidney disease |
| title_full | The polycystin signalling cascade in autosomal dominant polycystic kidney disease vorgelegt von Alexis Hofherr |
| title_fullStr | The polycystin signalling cascade in autosomal dominant polycystic kidney disease vorgelegt von Alexis Hofherr |
| title_full_unstemmed | The polycystin signalling cascade in autosomal dominant polycystic kidney disease vorgelegt von Alexis Hofherr |
| title_short | The polycystin signalling cascade in autosomal dominant polycystic kidney disease |
| title_sort | the polycystin signalling cascade in autosomal dominant polycystic kidney disease |
| topic_facet | Hochschulschrift |
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