Analyzing the role of ciliary cAMP signaling in neurons:
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| Sprache: | English |
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2020
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| Beschreibung: | III, IX, 129 Seiten Illustrationen, Diagramme |
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| 003 | DE-604 | ||
| 005 | 20230118 | ||
| 007 | t | ||
| 008 | 220705s2020 gw a||| m||| 00||| eng d | ||
| 016 | 7 | |a 1255727055 |2 DE-101 | |
| 020 | |c Broschur | ||
| 035 | |a (OCoLC)1356803980 | ||
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| 044 | |a gw |c XA-DE | ||
| 049 | |a DE-355 |a DE-83 | ||
| 084 | |a 540 |2 sdnb | ||
| 100 | 1 | |a Klausen, Christina |e Verfasser |0 (DE-588)1267494212 |4 aut | |
| 245 | 1 | 0 | |a Analyzing the role of ciliary cAMP signaling in neurons |c vorgelegt von Christina Klausen aus Bonn |
| 264 | 1 | |a Köln |c 2020 | |
| 300 | |a III, IX, 129 Seiten |b Illustrationen, Diagramme | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Universität Köln |d 2020 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 856 | 4 | 2 | |m B:DE-101 |q application/pdf |u https://d-nb.info/1255727055/04 |3 Inhaltsverzeichnis |
| 856 | 4 | 2 | |m DNB Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=033693049&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
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| adam_text | TABLE
OF
CONTENTS
TABLE
OF
CONTENTS
................................................................................................................
I
ABSTRACT
.......................................................................................................................................................
I
LIST
OF
FIGURES
.....................................................................................................................
IL
LIST
OF
TABLES
......................................................................................................................
IV
ABBREVIATIONS
...........................................................................................................................................
V
1.
INTRODUCTION
...............................................................................................................
1
1.1
PRIMARY
CILIA
...............................................................................................................................
1
1.1.1
FUNCTION
OF
PRIMARY
CILIA
....................................................................................................................
1
1.1.2
STRUCTURE
OF
PRIMARY
CILIA
....................................................................................................................
2
1.1.3
INTRAFLAGELLAR
TRANSPORT
AND
THE
BBSOME
..........................................................................................
4
1.2
CILIOPATHIES
AND
THE
BARDET-BIEDL-SYNDROME
.........................................................................
5
1.3
OBESITY
AND
THE
REGULATION
OF
ENERGY
HOMEOSTASIS
...............................................................
6
1.4
THE
ROLE
OF
NEURONAL
PRIMARY
CILIA
IN
CONTROLLING
ENERGY
HOMEOSTASIS..................................8
1.5
THESIS
AIM
................................................................................................................................
10
2.
MATERIALS
AND
METHODS
.............................................................................................
11
2.1
CHEMICALS
................................................................................................................................
11
2.2
CELL
CULTURE
MATERIALS
..............................................................................................................
11
2.3
ANTIBODIES
...............................................................................................................................
11
2.3.1
PRIMARY
ANTIBODIES
............................................................................................................................
11
2.3.2
SECONDARY
ANTIBODIES
........................................................................................................................
13
2.4
PLASMIDS
...................................................................................................................................
13
2.5
ADENOASSOCIATED
VIRUSES
(AAVS)
...........................................................................................
14
2.6
MAMMALIAN
CELL
CULTURE
.................
......................................................................................
14
2.6.1
MAMTAMANCE
OF
STANDARD
CELL
LINES
.................................................................................................
14
2.6.2
ISOLATION
OF
MOUSE
EMBRYONIC
FIBROBLAST
.........................................................................................
16
2.6.2.1
IMMORTALIZATION
OF
MEFS
.........................................................................................................
16
2.6.3
TRANSIENT
TRANSFECTION
USING
PEI
.......................................................................................................
16
2.6.4
TRANSIENT
TRANSFECTION
USING
LIPOFECTAMINE
2000
...........................................................................
17
2.6.5
TRANSDUCTION
WITH
CADDIS
SENSOR
.....................................................................................................
17
2.6.6
PHARMACOLOGICAL
STIMULATION
............................................................................................................
17
2.6.7
LIGHT
STIMULATION
PROTOCOLS
..............................................................................................................
17
2.6.7.1
ACTIVATING
BPAC
IN
MLMCD-3
CELLS
..........................................................................................
17
2.6.7.2
ACTIVATING
LAPD
IN
MLMCD-3
CELLS
...........................................................................................
18
2.6.8
ISOLATION
OF
PRIMARY
HYPOTHALAMIC
NEURONS
.....................................................................................
19
2.6.9 FLUO-4
CALCIUM
IMAGING
.....................................................................................................................
19
2.6.10
PHARMACOLOGICAL
STIMULATION
WITH
PEPTIDE
YY
3-36
(PYY3-36)
..................................................
20
2.6.11
TRANSDUCTION
OF
AAVS
..................................................................................................................
20
2.6.12
MEASURING
CILIARY
CAMP
LEVELS
BY
THE
CADDIS
SENSOR
.................................................................
21
2.7
IMMUNOCYTOCHEMISTRY
.....................................
.
.........
21
2.7.1
FIXATION
AND
IMMUNOCYTOCHEMICAL
(ICC)
STAINING
OF
CELLS
...............................................................
21
2.8
MICE
..........................................................................................................................................22
2.8.1
LICENSES.............................................................................................................................................
22
2.8.2
MOUSE
LINES
.......................................................................................................................................
22
TABLE
OF
CONTENTS
2.8.2.1
AGRP/POMC-CRE
AC3-E4-FLOX
...................................................................................................
22
2.8.2.2
AGRP/POMC-CRE
R26-TDTOMATO
...............................................................................................
22
2.8.23
AGRP/POMC-CRE
IFT20-FLOX
......................................................................................................
23
2.8.2.4
BBS
KNOCKOUT
MICE
.....................................................................................................................
23
2.8.2.5
AGRP/POMC-CRE
R26-MT/MG
BBS6/8
.....................................................................................
23
2.8.3
ISOLATION
AND
GENOTYPING
OF
GENOMIC
DNA
FROM
MOUSE
TAILS
..........................................................
23
2.8.4
ISOLATION
OF
BRAIN
TISSUE
....................................................................................................................
25
2.8.4.1
DISSECTION
OF
MICE
......................................................................................................................
25
2.8.4.2
PERFUSION
OF
MICE
WITH
PARAFORMALDEHYDE
(
PFA)
...................................................................
25
2.8.43
CRYOPRESERVATION
TN
SUCROSE
GRADIENT
......................................................................................
26
2.8.4.4
CRYOSECTIONING
OF
MURINE
BRAIN
................................................................................................
26
2.9
HISTOCHEMICAL
STAININGS
...........................................................................................................26
2.9.1
NISSL
BODY
STAINING
.............................................................................................................................
26
2.10
IMMUNOFLUORESCENCE
...............................................................................................................26
2.11
MEASURING
ACUTE
FOOD
INTAKE
.................................................................................................
27
2.12
PROTEIN
BIOCHEMISTRY................................................................................................................
27
2.12.1
PREPARATION
OF
PROTEIN
LYSATES
AND
DETERMINATION
OF
PROTEIN
CONCENTRATIONS
...........................
27
2.12.2
SODIUM
DODECYL
SULFATE
POLYACRYLAMIDE
GEL
ELECTROPHORESIS
(SDS-PAGE)
..................................
28
2.123
WESTERN
BLOT
ANALYSIS
....................................................................................................................
29
2.12.4
IMMUNOSTAINING
OF
IMMOBILIZED
PROTEINS
...................................................................................
30
2.13
GENE
EXPRESSION
ANALYSIS
........................................................................................................30
2.13.1
RNA
EXTRACTION
...............................................................................................................................
30
2.13.2
CDNA
SYNTHESIS
..............................................................................................................................
31
2.133
QUANTITATIVE
REAL-TIME
PCR
(QPCR)
................................................................................................
32
2.14
SOFTWARE
APPLICATIONS
.......................................................................................................
33
2.15
STATISTICS
......................................
34
3.
RESULTS
......................................................................................................................
35
3.1
CHARACTERIZATION
OF
PRIMARY
CILIA
IN
VITRO
.....................................................................
35
3.1.1
PRIMARY
CILIA
IN
DIFFERENT
CELL
LINES
IN
VITRO
........................................................................................
35
3.1.2
ANALYZING
PRIMARY
CILIA
USING
CILIAQ
...................................................................................................
36
3.1.3
ESTABLISHING
CILIARY
MARKERS
...............................................................................................................
36
3.1.4
CHARACTERIZATION
OF
THE
ARLL3B-MCHERRY/CENTRIN-EGFP
MOUSE
LINE
...............................................
37
3.1.5
IMMORTALIZATION
OF
MEFS
...................................................................................................................
38
3.2
CHARACTERIZATION
OF
CAMP
SIGNALING
COMPONENTS
IN
THE
PRIMARY
CILIUM
.............................
39
3.2.1
ADENYLYL
CYCLASE
LOCALIZATION
IN
THE
PRIMARY
CILIUM
..........................................................................
39
3.2.2
PKA
SUBUNIT
LOCALIZATION
IN
PRIMARY
CILIA
..........................................................................................
41
3.3
CHARACTERIZATION
OF
MEFS
FROM
BBS6
AND
BBS8
WILD-TYPE
AND
KNOCKOUT
MICE
....................
44
33.1
VERIFICATION
OF
BBS6
AND
BBS8
ANTIBODIES
........................................................................................
44
3.3.2
PRIMARY CILIA
IN
BBS6
AND
BBS8
KNOCKOUT
MEFS................................................................................
46
3.3.3
ANALYZING
SHH
SIGNALING
AS
AN
INDIRECT
READOUT
FOR
CILIARY
CAMP
SIGNALING
IN
BBS
MEFS
.................
47
3.4
IMMUNOHISTOCHEMICAL
ANALYSIS
OF
THE
ARCUATE
NUCLEUS
.......................................................
52
3.4.1
DEFINING
THE
ARCUATE
NUCLEUS
IN
THE
BRAIN
........................................................................................
52
3.4.2
CHARACTERIZATION
OF
THE
ARC
IN
BBS6
AND
BBS8
WILD-TYPE
AND
KNOCKOUT
MICE
................................
53
3.4.3
NUMBER
OF
AGRP
AND
POMC
NEURONS
IN
THE
ARCUATE
NUCLEUS
OF
BBS6
AND
BBS8
WILD-TYPE
AND
KNOCKOUT
MICE
..................................................................................................................................................
55
3.4.4
ANALYZING
THE
CILIARY
LOCALIZATION
OF
CAMP-DEPENDENT
SIGNALING
COMPONENTS
IN
THE
ARCUATE
NUCLEUS
56
3.5
PRIMARY
HYPOTHALAMIC
NEURONS
AS
SYSTEM
TO
STUDY
NEURONAL
FUNCTION
.............................63
3.5.1
ARE
NEURONAL
CELL
LINES
INTERCHANGEABLE
WITH
PRIMARY
NEURONS
IN
AN
IN
VITRO
SYSTEM?
................
63
3.5.2
ESTABLISHING
THE
ISOLATION
OF
PRIMARY
HYPOTHALAMIC
NEURONS
..........................................................
66
II
TABLE
OF
CONTENTS
3.5.3
PRIMARY
CILIA
IN
PRIMARY
NEURONAL
HYPOTHALAMIC
CULTURES
...............................................................
70
3.5.4
CALCIUM
IMAGING
IN
BBS
HYPOTHALAMIC
NEURONS
...............................................................................
71
3.5.5
NPY2R
SIGNALING
IN
BBS8
HYPOTHALAMIC
PRIMARY
NEURONS
................................................................
74
3.6
NEURONAL
REGULATION
OF
FOOD
INTAKE
BY
NEURONAL
PRIMARY
CILIA
.........................................
75
3.7
OPTOGENETIC
TOOLS
TO
MEASURE
AND
MANIPULATE
CAMP
LEVELS
............................................
84
3.7.1
MEASURING
CILIARY
CAMP
LEVELS
..........................................................................................................
86
3.7.2
OPTOGENETIC
TOOLS
ENABLE
MANIPULATION
OF
CAMP
LEVELS
.................................................................
89
4.
DISCUSSION
.................................................................................................................
96
4.1
THE
BBSOME
-
A
COMPLEX
WITH
MULTIPLE
FUNCTIONS?
............................................................96
4.2
THE
ROLE
OF
CILIARY
CAMP
SIGNALING
IN
CONTROLLING
FOOD
INTAKE
..............................................98
4.3
CILIARY
CAMP
SIGNALING
AND
THE
ROLE
IN
BBS-DEVELOPED
OBESITY
........................................
101
5.
REFERENCES
....................................................................................................................................
106
6.
APPENDIX
.......................................................................................................................................
123
DANKSAGUNG
..........................................................................................................................................
127
III
|
| adam_txt |
TABLE
OF
CONTENTS
TABLE
OF
CONTENTS
.
I
ABSTRACT
.
I
LIST
OF
FIGURES
.
IL
LIST
OF
TABLES
.
IV
ABBREVIATIONS
.
V
1.
INTRODUCTION
.
1
1.1
PRIMARY
CILIA
.
1
1.1.1
FUNCTION
OF
PRIMARY
CILIA
.
1
1.1.2
STRUCTURE
OF
PRIMARY
CILIA
.
2
1.1.3
INTRAFLAGELLAR
TRANSPORT
AND
THE
BBSOME
.
4
1.2
CILIOPATHIES
AND
THE
BARDET-BIEDL-SYNDROME
.
5
1.3
OBESITY
AND
THE
REGULATION
OF
ENERGY
HOMEOSTASIS
.
6
1.4
THE
ROLE
OF
NEURONAL
PRIMARY
CILIA
IN
CONTROLLING
ENERGY
HOMEOSTASIS.8
1.5
THESIS
AIM
.
10
2.
MATERIALS
AND
METHODS
.
11
2.1
CHEMICALS
.
11
2.2
CELL
CULTURE
MATERIALS
.
11
2.3
ANTIBODIES
.
11
2.3.1
PRIMARY
ANTIBODIES
.
11
2.3.2
SECONDARY
ANTIBODIES
.
13
2.4
PLASMIDS
.
13
2.5
ADENOASSOCIATED
VIRUSES
(AAVS)
.
14
2.6
MAMMALIAN
CELL
CULTURE
.
.
14
2.6.1
MAMTAMANCE
OF
STANDARD
CELL
LINES
.
14
2.6.2
ISOLATION
OF
MOUSE
EMBRYONIC
FIBROBLAST
.
16
2.6.2.1
IMMORTALIZATION
OF
MEFS
.
16
2.6.3
TRANSIENT
TRANSFECTION
USING
PEI
.
16
2.6.4
TRANSIENT
TRANSFECTION
USING
LIPOFECTAMINE
2000
.
17
2.6.5
TRANSDUCTION
WITH
CADDIS
SENSOR
.
17
2.6.6
PHARMACOLOGICAL
STIMULATION
.
17
2.6.7
LIGHT
STIMULATION
PROTOCOLS
.
17
2.6.7.1
ACTIVATING
BPAC
IN
MLMCD-3
CELLS
.
17
2.6.7.2
ACTIVATING
LAPD
IN
MLMCD-3
CELLS
.
18
2.6.8
ISOLATION
OF
PRIMARY
HYPOTHALAMIC
NEURONS
.
19
2.6.9 FLUO-4
CALCIUM
IMAGING
.
19
2.6.10
PHARMACOLOGICAL
STIMULATION
WITH
PEPTIDE
YY
3-36
(PYY3-36)
.
20
2.6.11
TRANSDUCTION
OF
AAVS
.
20
2.6.12
MEASURING
CILIARY
CAMP
LEVELS
BY
THE
CADDIS
SENSOR
.
21
2.7
IMMUNOCYTOCHEMISTRY
.
.
.
21
2.7.1
FIXATION
AND
IMMUNOCYTOCHEMICAL
(ICC)
STAINING
OF
CELLS
.
21
2.8
MICE
.22
2.8.1
LICENSES.
22
2.8.2
MOUSE
LINES
.
22
TABLE
OF
CONTENTS
2.8.2.1
AGRP/POMC-CRE
AC3-E4-FLOX
.
22
2.8.2.2
AGRP/POMC-CRE
R26-TDTOMATO
.
22
2.8.23
AGRP/POMC-CRE
IFT20-FLOX
.
23
2.8.2.4
BBS
KNOCKOUT
MICE
.
23
2.8.2.5
AGRP/POMC-CRE
R26-MT/MG
BBS6/8
.
23
2.8.3
ISOLATION
AND
GENOTYPING
OF
GENOMIC
DNA
FROM
MOUSE
TAILS
.
23
2.8.4
ISOLATION
OF
BRAIN
TISSUE
.
25
2.8.4.1
DISSECTION
OF
MICE
.
25
2.8.4.2
PERFUSION
OF
MICE
WITH
PARAFORMALDEHYDE
(
PFA)
.
25
2.8.43
CRYOPRESERVATION
TN
SUCROSE
GRADIENT
.
26
2.8.4.4
CRYOSECTIONING
OF
MURINE
BRAIN
.
26
2.9
HISTOCHEMICAL
STAININGS
.26
2.9.1
NISSL
BODY
STAINING
.
26
2.10
IMMUNOFLUORESCENCE
.26
2.11
MEASURING
ACUTE
FOOD
INTAKE
.
27
2.12
PROTEIN
BIOCHEMISTRY.
27
2.12.1
PREPARATION
OF
PROTEIN
LYSATES
AND
DETERMINATION
OF
PROTEIN
CONCENTRATIONS
.
27
2.12.2
SODIUM
DODECYL
SULFATE
POLYACRYLAMIDE
GEL
ELECTROPHORESIS
(SDS-PAGE)
.
28
2.123
WESTERN
BLOT
ANALYSIS
.
29
2.12.4
IMMUNOSTAINING
OF
IMMOBILIZED
PROTEINS
.
30
2.13
GENE
EXPRESSION
ANALYSIS
.30
2.13.1
RNA
EXTRACTION
.
30
2.13.2
CDNA
SYNTHESIS
.
31
2.133
QUANTITATIVE
REAL-TIME
PCR
(QPCR)
.
32
2.14
SOFTWARE
APPLICATIONS
.
33
2.15
STATISTICS
.
34
3.
RESULTS
.
35
3.1
CHARACTERIZATION
OF
PRIMARY
CILIA
IN
VITRO
.
35
3.1.1
PRIMARY
CILIA
IN
DIFFERENT
CELL
LINES
IN
VITRO
.
35
3.1.2
ANALYZING
PRIMARY
CILIA
USING
CILIAQ
.
36
3.1.3
ESTABLISHING
CILIARY
MARKERS
.
36
3.1.4
CHARACTERIZATION
OF
THE
ARLL3B-MCHERRY/CENTRIN-EGFP
MOUSE
LINE
.
37
3.1.5
IMMORTALIZATION
OF
MEFS
.
38
3.2
CHARACTERIZATION
OF
CAMP
SIGNALING
COMPONENTS
IN
THE
PRIMARY
CILIUM
.
39
3.2.1
ADENYLYL
CYCLASE
LOCALIZATION
IN
THE
PRIMARY
CILIUM
.
39
3.2.2
PKA
SUBUNIT
LOCALIZATION
IN
PRIMARY
CILIA
.
41
3.3
CHARACTERIZATION
OF
MEFS
FROM
BBS6
AND
BBS8
WILD-TYPE
AND
KNOCKOUT
MICE
.
44
33.1
VERIFICATION
OF
BBS6
AND
BBS8
ANTIBODIES
.
44
3.3.2
PRIMARY CILIA
IN
BBS6
AND
BBS8
KNOCKOUT
MEFS.
46
3.3.3
ANALYZING
SHH
SIGNALING
AS
AN
INDIRECT
READOUT
FOR
CILIARY
CAMP
SIGNALING
IN
BBS
MEFS
.
47
3.4
IMMUNOHISTOCHEMICAL
ANALYSIS
OF
THE
ARCUATE
NUCLEUS
.
52
3.4.1
DEFINING
THE
ARCUATE
NUCLEUS
IN
THE
BRAIN
.
52
3.4.2
CHARACTERIZATION
OF
THE
ARC
IN
BBS6
AND
BBS8
WILD-TYPE
AND
KNOCKOUT
MICE
.
53
3.4.3
NUMBER
OF
AGRP
AND
POMC
NEURONS
IN
THE
ARCUATE
NUCLEUS
OF
BBS6
AND
BBS8
WILD-TYPE
AND
KNOCKOUT
MICE
.
55
3.4.4
ANALYZING
THE
CILIARY
LOCALIZATION
OF
CAMP-DEPENDENT
SIGNALING
COMPONENTS
IN
THE
ARCUATE
NUCLEUS
56
3.5
PRIMARY
HYPOTHALAMIC
NEURONS
AS
SYSTEM
TO
STUDY
NEURONAL
FUNCTION
.63
3.5.1
ARE
NEURONAL
CELL
LINES
INTERCHANGEABLE
WITH
PRIMARY
NEURONS
IN
AN
IN
VITRO
SYSTEM?
.
63
3.5.2
ESTABLISHING
THE
ISOLATION
OF
PRIMARY
HYPOTHALAMIC
NEURONS
.
66
II
TABLE
OF
CONTENTS
3.5.3
PRIMARY
CILIA
IN
PRIMARY
NEURONAL
HYPOTHALAMIC
CULTURES
.
70
3.5.4
CALCIUM
IMAGING
IN
BBS
HYPOTHALAMIC
NEURONS
.
71
3.5.5
NPY2R
SIGNALING
IN
BBS8
HYPOTHALAMIC
PRIMARY
NEURONS
.
74
3.6
NEURONAL
REGULATION
OF
FOOD
INTAKE
BY
NEURONAL
PRIMARY
CILIA
.
75
3.7
OPTOGENETIC
TOOLS
TO
MEASURE
AND
MANIPULATE
CAMP
LEVELS
.
84
3.7.1
MEASURING
CILIARY
CAMP
LEVELS
.
86
3.7.2
OPTOGENETIC
TOOLS
ENABLE
MANIPULATION
OF
CAMP
LEVELS
.
89
4.
DISCUSSION
.
96
4.1
THE
BBSOME
-
A
COMPLEX
WITH
MULTIPLE
FUNCTIONS?
.96
4.2
THE
ROLE
OF
CILIARY
CAMP
SIGNALING
IN
CONTROLLING
FOOD
INTAKE
.98
4.3
CILIARY
CAMP
SIGNALING
AND
THE
ROLE
IN
BBS-DEVELOPED
OBESITY
.
101
5.
REFERENCES
.
106
6.
APPENDIX
.
123
DANKSAGUNG
.
127
III |
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| spelling | Klausen, Christina Verfasser (DE-588)1267494212 aut Analyzing the role of ciliary cAMP signaling in neurons vorgelegt von Christina Klausen aus Bonn Köln 2020 III, IX, 129 Seiten Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier Dissertation Universität Köln 2020 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf https://d-nb.info/1255727055/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=033693049&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Klausen, Christina Analyzing the role of ciliary cAMP signaling in neurons |
| subject_GND | (DE-588)4113937-9 |
| title | Analyzing the role of ciliary cAMP signaling in neurons |
| title_auth | Analyzing the role of ciliary cAMP signaling in neurons |
| title_exact_search | Analyzing the role of ciliary cAMP signaling in neurons |
| title_exact_search_txtP | Analyzing the role of ciliary cAMP signaling in neurons |
| title_full | Analyzing the role of ciliary cAMP signaling in neurons vorgelegt von Christina Klausen aus Bonn |
| title_fullStr | Analyzing the role of ciliary cAMP signaling in neurons vorgelegt von Christina Klausen aus Bonn |
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| title_short | Analyzing the role of ciliary cAMP signaling in neurons |
| title_sort | analyzing the role of ciliary camp signaling in neurons |
| topic_facet | Hochschulschrift |
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