The role of the E3-ubiquitin ligase HUWE1 in podocyte signaling and homeostasis:
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| Format: | Abschlussarbeit Mikrofilm Buch |
| Sprache: | English |
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Köln
2017
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| Beschreibung: | 2 Mikrofiches (X, 94 Seiten) Illustrationen |
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| 100 | 1 | |a Bertsch, Sabine |d 1987- |e Verfasser |0 (DE-588)1153516403 |4 aut | |
| 245 | 1 | 0 | |a The role of the E3-ubiquitin ligase HUWE1 in podocyte signaling and homeostasis |c vorgelegt von Sabine Bertsch |
| 264 | 1 | |a Köln |c 2017 | |
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| 502 | |b Dissertation |c Universität zu Köln |d 2017 | ||
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Datensatz im Suchindex
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| adam_text | CONTENT
LIST OF
FIGURES........................................................................................................................
VI
LIST OF
TABLES.........................................................................................................................VII
ABBREVIATIONS......................................................................................................................
VIII
1.
ABSTRACT..............................................................................................................................1
2.
ZUSAMMENFASSUNG.............................................................................................................2
3.
INTRODUCTION......................................................................................................................
4
3.1 RENAL AND PODOCYTE
PHYSIOLOGY..................................................................................
4
3.2 PODOCYTE RESPONSES TO CELLULAR
STRESS.........................................................................5
3.3 PODOCYTE HOMEOSTASIS AND PROTEIN TURNOVER
.............................................................
6
3.4 THE UBIQUITIN PROTEASOME
SYSTEM..............................................................................6
3.5 THE E3-UBQUITIN LIGASE
HUWE1................................................................................8
3.6 THE LYSOSOMAL PROTEIN DEGRADATION SYSTEM
............................................................
11
3.7 BI-DIRECTIONAL CROSSTALK BETWEEN THE PROTEASOMAL AND LYSOSOMAL
DEGRADATION
PATHWAYS...................................................................................................................14
4.
AIMS................................................................................................................................16
5. MATERIALS AND
METHODS...................................................................................................
17
5.1
MATERIALS..................................................................................................................
17
5.1.1 CHEMICALS AND
REAGENTS....................................................................................
17
5.1.2 BUFFERS AND
SOLUTIONS........................................................................................
19
5.1.3 ASSAYS AND
KITS.................................................................................................22
5.1.4 CELL
LINES...........................................................................................................
22
5.1.5
ANTIBODIES........................................................................................................22
5.1.6
OLIGONUCLEOTIDES...............................................................................................23
5.1.7
CONSUMABLES....................................................................................................24
5.1.8
EQUIPMENT........................................................................................................
25
5.1.9
SOFTWARE............................................................................................................26
5.2
METHODS...................................................................................................................
27
5.2.1 WORKING WITH NUCLEIC
ACIDS..............................................................................
27
5.2.1.1 RNA ISOLATION AND CDNA SYNTHESIS
.........................................................
27
5.2.1.2 QUANTITATIVE POLYMERASE-CHAIN-REACTION
(QPCR).....................................27
5.2.2 CELL
CULTURE.......................................................................................................
27
5.2.2.1 CULTURE OF IMMORTALIZED CELL
LINES............................................................ 27
5.2.2.2 FREEZING AND THAWING CELLS
.......................................................................
28
5.2.2.3 IMMUNOFLUORESCENCE STAINING OF CELLS
......................................................
28
5.2.3 PROTEIN
BIOCHEMISTRY.........................................................................................29
5.2.3.1 PREPARATION OF PROTEIN
LYSATES....................................................................
29
5.2.3.2 PROTEIN
QUANTIFICATION................................................................................
29
5.2.3.3 SDS-POLYACRYLAMIDE GEL PREPARATION AND ELECTROPHORESIS
......................
29
5.2.3.4 WESTERN
BLOTTING........................................................................................30
5.2.3.5 PROTEASOME ACTIVITY
ASSAY.........................................................................
30
5.2.3.6 IMMUNOPRECIPITATION FOR
MS/MS.............................................................. 31
5.2.3.7 N-LC-MS/MS ANALYSIS OF CELLULAR
LYSATES.................................................32
5.2.3.8 PULSED SILAC (STABLE ISOTOPE LABELING WITH AMINO ACIDS IN CELL
CULTURE)32
5.2.4 MOUSE
EXPERIMENTS............................................................................................
34
5.2.4.1 MOUSE
LINES................................................................................................34
5.2.4.2 DNA EXTRACTION FROM MOUSE TISSUE
..........................................................
34
5.2.4J POLYMERASE-CHAIN-REACTION FOR MOUSE GENOTYPING
...................................
35
5.2.4.4 AGAROSE GEL ELECTROPHORESIS FOR GENOTYPING
.............................................
36
5.2.4.5 BODYWEIGHT ANALYSIS AND URINE COLLECTION
...............................................
36
5.2.4.6 TAMOXIFEN
TREATMENT.................................................................................
36
5.2.4.7 IMMUNOHISTOCHEMICAL STAINING ON RENAL TISSUE
........................................
36
5.2.4.S PERIODIC ACID SCHIFF (PAS)
STAINING..........................................................37
5.2.4.9 METHYLENBLUE STAINING AND ELECTRON MICROSCOPY
.....................................
37
5.2.4.10 COLLOIDAL COOMASSIE
STAINING....................................................................38
5.2.4.11 ALBUMIN
ELISA........................................................................................38
5.2.4.12 CREATININE AND UREA
MEASUREMENTS...........................................................38
6.
RESULTS..........................................................................................................................
39
6.1 GENERATION AND CHARACTERIZATION OF A NOVEL PODOCYTE-SPECIFIC HUWEL
KNOCKOUT
MOUSE
MODEL...............................................................................................................
39
6.1.1 GENERATION AND VALIDATION OF THE MOUSE
MODEL............................................... 39
6.1.2 HUWEL PKO MICE DISPLAY SIGNS OF GLOMERULAR DAMAGE
....................................
39
6.1.3 HUWEL PKO MICE DIE OF RENAL
FAILURE.................................................................43
6.1.4 HUWEL DEFICIENCY CAUSES PODOCYTE DAMAGE, FOOT PROCESSES EFFACEMENT
AND,
FORMATION OF CYTOPLASMIC VACUOLES
..................................................................
43
6.2 SEARCH FOR HU WEI INTERACTORS BY MS/MS
...........................................................
46
6.2.1 IMMUNOPRECIPITATION WITH POLYCLONAL HU WEI ANTIBODY REVEALS FIVE
SIGNIFICANT
INTERACTORS.......................................................................................46
6.2.2 IMMUNOPRECIPITATION WITH MONOCLONAL HU WEI ANTIBODY
..............................
49
6.2.3 PRE-TREATMENT WITH THE PROTEASOME INHIBITOR MG 132 REVEALS NO HU
WEI
SPECIFIC
INTERACTORS............................................................................................
51
6.3 GENERATION AND EVALUATION OF HUWE1 KNOCKDOWN HUMAN PODOCYTES
................
52
6.3.1 PROTEOMIC SCREEN IDENTIFIES SIGNIFICANTLY CHANGED PROTEINS
.............................
52
6.3.1.1 KNOWN HUWE1 INTERACTORS ARE DIFFERENTIALLY EXPRESSED IN
KNOCKDOWN CELLS.
...................................................................................................................
55
6.3.1.2 INCREASED EXPRESSION OF LYSOSOMAL PROTEINS IN HUWE1 KNOCKDOWN
CELLS.
...................................................................................................................
56
6.3.1.3 HUWEJ KNOCKDOWN RESULTS IN DECREASED PROTEASOMAL ACTIVITY
..............
58
6.3.1.4 DECREASED EXPRESSION OF DNA REPAIR AND -DAMAGE ASSOCIATED
PROTEINS IN
HUWE1 KNOCKDOWN
CELLS.........................................................................58
6.3.2 HU WEI KNOCKDOWN CELLS DISPLAY AN INCREASED NUMBER OF
LYSOSOMES............59
6.3.3 HUWE1 KNOCKDOWN LEADS TO A REDUCTION OF PHOSPHORYLATED 86 PROTEIN
......
60
6.3.4 HU WEI LOSS GLOBALLY INCREASES PROTEIN HALF-LIFE AND IMPACTS ON
THE
ABUNDANCE OF SPECIFIC PROTEIN NETWORKS
..........................................................
61
7.
DISCUSSION......................................................................................................................
65
7.1 PODOCYTE-SPECIFIC LOSS OF HUWE1 CAUSES GLOMERULAR DISEASE ASSOCIATED
WITH FOOT
PROCESS EFFACEMENT AND ACCUMULATION OF LYSOSOMES IN MICE
.................................
65
7.2 THE IDENTITY OF SPECIFIC INTERACTORS OF HU WEI IN PODOCYTES REMAINS
ELUSIVE
......
66
7.3 PROTEOMIC APPROACH IDENTIFIES PROTEINS DIFFERENTIALLY REGULATED UPON
HU WEI
KNOCKDOWN IN
PODOCYTES.........................................................................................68
7.4 HU WEI LOSS IMPACTS ON PODOCYTE LYSOSOMAL COMPOSITION/CONTENT AND
PROTEASOMAL
ACTIVITY.................................................................................................
69
7.5 MTOR SIGNALING IS REDUCED UPON LOSS OF HU WEI IN
PODOCYTES...........................72
7.6 HU WEI -DEFICIENT PODOCYTES EXHIBIT A GLOBAL DECREASE IN DNA DAMAGE-
AND DNA
REPAIR-ASSOCIATED
PROTEINS........................................................................................73
7.7 HU WEI LOSS GLOBALLY INCREASES PODOCYTE PROTEIN HALF-LIFE AND
IMPACTS ON THE
ABUNDANCE OF SPECIFIC PROTEIN NETWORKS
.................................................................
74
8.
CONCLUSION......................................................................................................................
75
9.
PUBLICATIONS....................................................................................................................
76
9.1 PUBLICATIONS IN ACADEMIC
JOURNALS............................................................................76
9.2 PUBLICATIONS ON
CONFERENCES....................................................................................76
10.
REFERENCES..................................................................................................................
77
11.
ACKNOWLEDGEMENT.......................................................................................................
92
12.
ERKLAERUNG....................................................................................................................
93
13. CURRICULUM
VITAE.........................................................................................................
94
|
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| bvnumber | BV045054567 |
| ctrlnum | (OCoLC)1136267366 (DE-599)DNB1155027159 |
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| spelling | Bertsch, Sabine 1987- Verfasser (DE-588)1153516403 aut The role of the E3-ubiquitin ligase HUWE1 in podocyte signaling and homeostasis vorgelegt von Sabine Bertsch Köln 2017 2 Mikrofiches (X, 94 Seiten) Illustrationen txt rdacontent h rdamedia he rdacarrier 24x Dissertation Universität zu Köln 2017 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1155027159/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030446238&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Bertsch, Sabine 1987- The role of the E3-ubiquitin ligase HUWE1 in podocyte signaling and homeostasis |
| subject_GND | (DE-588)4113937-9 |
| title | The role of the E3-ubiquitin ligase HUWE1 in podocyte signaling and homeostasis |
| title_auth | The role of the E3-ubiquitin ligase HUWE1 in podocyte signaling and homeostasis |
| title_exact_search | The role of the E3-ubiquitin ligase HUWE1 in podocyte signaling and homeostasis |
| title_full | The role of the E3-ubiquitin ligase HUWE1 in podocyte signaling and homeostasis vorgelegt von Sabine Bertsch |
| title_fullStr | The role of the E3-ubiquitin ligase HUWE1 in podocyte signaling and homeostasis vorgelegt von Sabine Bertsch |
| title_full_unstemmed | The role of the E3-ubiquitin ligase HUWE1 in podocyte signaling and homeostasis vorgelegt von Sabine Bertsch |
| title_short | The role of the E3-ubiquitin ligase HUWE1 in podocyte signaling and homeostasis |
| title_sort | the role of the e3 ubiquitin ligase huwe1 in podocyte signaling and homeostasis |
| topic_facet | Hochschulschrift |
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