Expression and functional analysis of the RasGAPs Rasal1, Rasal2 and Rasal3:
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| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
Marburg
2018
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Physical Description: | iii, 126 Seiten Illustrationen, Diagramme 21 cm |
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| 245 | 1 | 0 | |a Expression and functional analysis of the RasGAPs Rasal1, Rasal2 and Rasal3 |c vorgelegt von Rui Xu |
| 264 | 1 | |a Marburg |c 2018 | |
| 300 | |a iii, 126 Seiten |b Illustrationen, Diagramme |c 21 cm | ||
| 336 | |b txt |2 rdacontent | ||
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| 502 | |b Dissertation |c Philipps-Universität Marburg |d 2018 | ||
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|---|---|
| adam_text | CONTENTS
ABBREVIATIONS.................................................................................................................................1
SUMMARY........................................................................................................................................3
ZUSAMMENFASSUNG......................................................................................................................
4
1
INTRODUCTION................................................................................................................................
5
1.1 RAS
PROTEINS.......................................................................................................................
5
1.2 RASGTPASE-ACTIVATING PROTEINS
(RASGAPS).................................................................6
1.2.1 RASAM
.........................................................................................................................
9
1.2.2 RASAL2 AND
RASAL3..................................................................................................
10
1.3 GASTRIC
EPITHELIUM...........................................................................................................
11
1.4 ENTEROENDOCRINE
SYSTEM................................................................................................
13
1.4.1 OVERVIEW OF THE ENTEROENDOCRINE
SYSTEM............................................................13
1.4.2 GASTRIC ENTEROENDOCRINE CELLS AND ACID
SECRETION...............................................14
2 AIM OF THE
STUDY......................................................................................................................
17
3
MATERIALS...................................................................................................................................18
3.1 CHEMICALS AND
REAGENTS................................................................................................
18
3.2 BUFFERS AND
SOLUTIONS......................................................................................................
20
3.3
KITS....................................................................................................................................
24
3.4
MEDIUM.............................................................................................................................
24
3.5
INSTRUMENTS......................................................................................................................
25
3.6
ANTIBODIES........................................................................................................................
25
3.7 BACTERIAL
STRAINS...............................................................................................................
26
3.8
ENZYMES...........................................................................................................................
26
3.9
PRIMERS.............................................................................................................................
26
3.10
PLASMIDS.........................................................................................................................
30
3.11 M
ICE................................................................................................................................
31
4
METHODS...................................................................................................................................
32
4.1 MOLECULAR CLONING AND
PCR...........................................................................................32
4.2 RNA EXTRACTION AND QUANTITATIVE REAL-TIME P C R
.......................................................
34
4.3 ORGANOID
CULTURE..............................................................................................................34
4.3.1 INTESTINAL ORGANOID
CULTURE......................................................................................34
4.3.2 PYLORIC ORGANOID
CULTURE..........................................................................................36
4.4 FLUORESCENCE-ACTIVATED CELL SORTING (FA
CS)...............................................................38
4.4.1 SINGLE CELL
PREPARATION............................................................................................
38
4.4.2 IMMUNO-STAINING AND
SORTING.................................................................................40
4.5 PROTEIN PURIFICATION AND
PULL-DOWN................................................................................40
4.6 MASS
SPECTROMETRY.........................................................................................................
42
4.7 SOUTHERN
BLOT...................................................................................................................
43
4 .8 GENERATION OF TRANSGENIC REPORTER MICE AND GENE-DEFICIENT M ICE
.........................
43
4.8.1 GENERATION OF TRANSGENIC REPORTER
MICE................................................................43
4.8.1 GENERATION OF GENE DEFICIENT
MICE........................................................................45
4.9 IMMUNOFLUORESCENCE
STAINING.......................................................................................46
4.9.1 IMMUNOFLUORESCENCE STAINING FOR
TISSUE...............................................................46
4.9.1 IMMUNOFLUORESCENCE STAINING FOR
ORGANOID..........................................................47
4.10 HISTOLOGICAL
ANALYSIS.....................................................................................................
47
4.10.1 X-GAL
STAINING.........................................................................................................
47
4.10.2 H&E
STAINING...........................................................................................................
48
4.11 GLUCOSE TOLERANCE
TEST.................................................................................................
49
4.12 BLOOD PLASMA
PREPARATION...........................................................................................
50
4.13 GASTRIN
ELISA................................................................................................................
50
4.14 MAG FIX
ASSAY...............................................................................................................
50
4.15 GASTRIC ACID COLLECTION AND
TITRATION............................................................................51
4.16 PHENOMASTER
ASSAY......................................................................................................
51
4.17
STATISTICS.........................................................................................................................
52
5
RESULTS.................................................................................................................................
53
5.1 GENERATION OF GENETICALLY-MODIFIED MOUSE LINES FOR RASAM, RASAL2 AND
RASAL3 53
5.1.1 GENERATION OF TRANSGENIC REPORTER MOUSE LINES FOR RASAM. RASAL2
AND
RASA!3.................................................................................................................................
53
5.1.2 GENERATION OF RASAM, RASAL2 AND RASAL3 KNOCKOUT MOUSE LINES
.................
59
5.2 DETERMINATION OF EXPRESSION PATTERNS FOR RASAM. RASAL2, AND RASAL3
.................
64
5.2.1 EXPRESSION ANALYSIS OF
RASAM...............................................................................65
5.2.4 EXPRESSION OF RASAL2 AND
RASAL3.....................................................................74
5.3 ANALYSIS OF THE FUNCTION OF RASAM IN ENTEROENDOCRINE CELLS
....................................
78
5.3.1 EXPRESSION ANALYSIS OF DIFFERENT CELL TYPE-SPECIFIC MARKERS IN THE
GASTRIC AND
INTESTINAL EPITHELIUM OF RASAM-DEFICIENT
MICE...............................................................78
5.3.2 GASTRIC ENTEROENDOCRINE CELL NUMBERS ARE INCREASED IN
RASAM-DEFICIENT MICE
5.3.3 RASAM KNOCKOUT MICE HAVE NORMAL BODY WEIGHT AND BLOOD GLUCOSE
LEVELS.. 82
5.3.4 RASALL-DEFICIENT MICE DO NOT DISPLAY ABNORMALITIES IN GLUCOSE
TOLERANCE
.....
83
5.3.5 MICE LACKING RASALL EXHIBIT NORMAL METABOLIC FUNCTIONS
..................................
84
5.3 .6 PLASMA GASTRIN LEVELS IN RASALL-DEFICIENT MICE ARE ELEVATED
......................
85
5.3.7 RASALL-DEFICIENT MICE SHOW AN INCREASE IN GASTRIC ACID SECRETION
..............
87
5.4 IDENTIFICATION OF CRE LINES FOR EFFICIENT RECOMBINATION IN
ENTEROENDOCRINCE CELLS.. 88
5.4.1
ATOH1-CRE..................................................................................................................
89
5.4.2
VILLIN-CRE....................................................................................................................
90
5.4.3
NGN3-CRE..................................................................................................................
91
5.5 DETERMINATION OF RASALL EXPRESSION IN ORGANOIDS OF THE STOMACH AND
SMALL
INTESTINE...................................................................................................................................
92
5.5.1 RASALL IS EXPRESSED IN GASTRIC AND INTESTINAL ORGANOIDS
...................................
92
5.5.2 RASALL IS EXPRESSED IN ENTEROENDOCRINE CELLS OF GASTRIC ORGANOIDS
...............
93
5.6 ANALYSIS OF GASTRIC ORGANOIDS FROM RASALL-DEFICIENT MICE
......................................
94
5.6.1 NORMAL MORPHOLOGY OF RASALL-DEFICIENT GASTRIC ORGANOIDS
.............................
94
5.6.2 RASALL-DEFICIENT PYLORIC ORGANOIDS DISPLAY INCREASED MRNA
EXPRESSION OF
ENTEROENDOCRINE MARKER
GENES.......................................................................................95
5.7 IDENTIFICATION OF INTERACTION PARTNERS OF RASAL PROTEINS
.............................................
96
5.7.1 EXPRESSION OF RASAL PROTEINS AND DOMAINS
........................................................
97
5.7.1 MASS SPECTROMETRY FOR IDENTIFICATION OF INTERACTION PARTNERS OF R
A S A LL
.......
100
6
DISCUSSION.............................................................................................................................
102
6.1 EXPRESSION ANALYSIS FOR RASAL
GENES.........................................................................102
6.2 THE ROLE OF RASALL IN EPITHELIAL PROLIFERATION AND
DIFFERENTIATION............................105
6.3 CELL TYPE-SPECIFIC KNOCKOUT OF R A S A
LL........................................................................107
6.4 ANALYSIS OF GASTRO-INTESTINAL
ORGANOIDS......................................................................109
6.5 RASALL AND GASTRIC ACID
SECRETION..............................................................................I
L L
6.6 PERSPECTIVE ON THE INVESTIGATION OF MOLECULAR MECHANISMS BY THE USE
OF GASTRIC
ORGANOIDS..............................................................................................................................
112
6.7 ROLE OF RASALL IN THE
INTESTINE....................................................................................112
6.8 INTERACTION PARTNERS OF RASALL, RASAL2, AND RASAL3
.................................................
114
7
REFERENCES............................................................................................................................
115
LIST OF ACADEMIC
TEACHERS......................................................................................................
123
ACKNOWLEDGEMENTS.................................................................................................................
125
EHRENWOERTLICHE
ERKLAERUNG.......................................................................................................
126
|
| any_adam_object | 1 |
| author | Xu, Rui 1987- |
| author_GND | (DE-588)1172677204 |
| author_facet | Xu, Rui 1987- |
| author_role | aut |
| author_sort | Xu, Rui 1987- |
| author_variant | r x rx |
| building | Verbundindex |
| bvnumber | BV045893983 |
| ctrlnum | (OCoLC)1136357678 (DE-599)HEB43738621X |
| discipline | Biologie Medizin |
| format | Thesis Book |
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| indexdate | 2024-07-10T08:29:38Z |
| institution | BVB |
| language | English |
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| owner | DE-355 DE-BY-UBR |
| owner_facet | DE-355 DE-BY-UBR |
| physical | iii, 126 Seiten Illustrationen, Diagramme 21 cm |
| publishDate | 2018 |
| publishDateSearch | 2018 |
| publishDateSort | 2018 |
| record_format | marc |
| spelling | Xu, Rui 1987- Verfasser (DE-588)1172677204 aut Expression and functional analysis of the RasGAPs Rasal1, Rasal2 and Rasal3 vorgelegt von Rui Xu Marburg 2018 iii, 126 Seiten Illustrationen, Diagramme 21 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Philipps-Universität Marburg 2018 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1172051402/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=031276941&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Xu, Rui 1987- Expression and functional analysis of the RasGAPs Rasal1, Rasal2 and Rasal3 |
| subject_GND | (DE-588)4113937-9 |
| title | Expression and functional analysis of the RasGAPs Rasal1, Rasal2 and Rasal3 |
| title_auth | Expression and functional analysis of the RasGAPs Rasal1, Rasal2 and Rasal3 |
| title_exact_search | Expression and functional analysis of the RasGAPs Rasal1, Rasal2 and Rasal3 |
| title_full | Expression and functional analysis of the RasGAPs Rasal1, Rasal2 and Rasal3 vorgelegt von Rui Xu |
| title_fullStr | Expression and functional analysis of the RasGAPs Rasal1, Rasal2 and Rasal3 vorgelegt von Rui Xu |
| title_full_unstemmed | Expression and functional analysis of the RasGAPs Rasal1, Rasal2 and Rasal3 vorgelegt von Rui Xu |
| title_short | Expression and functional analysis of the RasGAPs Rasal1, Rasal2 and Rasal3 |
| title_sort | expression and functional analysis of the rasgaps rasal1 rasal2 and rasal3 |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/1172051402/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=031276941&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT xurui expressionandfunctionalanalysisoftherasgapsrasal1rasal2andrasal3 |
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