Impact of hypophosphatasia on the biology of the nervous system: the molecular role of tissue-nonspecific alkaline phosphatase beyond bone mineralization
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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Würzburg
2015
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| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | IV, 234 Seiten Illustrationen, Diagramme |
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| 245 | 1 | 0 | |a Impact of hypophosphatasia on the biology of the nervous system |b the molecular role of tissue-nonspecific alkaline phosphatase beyond bone mineralization |c vorgelegt von Stephanie Graser, geboren in Schweinfurt |
| 264 | 1 | |a Würzburg |c 2015 | |
| 300 | |a IV, 234 Seiten |b Illustrationen, Diagramme | ||
| 336 | |b txt |2 rdacontent | ||
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| 502 | |b Dissertation |c Julius-Maximilians-Universität Würzburg |d 2015 | ||
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Datensatz im Suchindex
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| adam_text | IMPACT OF HYPOPHOSPHATASIA ON THE BIOLOGY OF THE NERVOUS SYSTEM
- THE MOLECULAR ROLE OF TISSUE-NONSPECIFIC ALKALINE PHOSPHATASE
BEYOND BONE MINERALIZATION
DISSERTATION ZUR ERLANGUNG DES
NATURWISSENSCHAFTLICHEN DOKTORGRADES
DER JULIUS-MAXIMILIANS-UNIVERSITAET WUERZBURG
VORGELEGT VON STEPHANIE GRASER
GEBOREN IN SCHWEINFURT
WUERZBURG, 2015
TABLE OF CONTENTS
1.
SUMMARY...............................................................................................................................
1
1.1
SUMMARY...........................................................................................................................1
1.2
ZUSAMMENFASSUNG............................................................................................................2
2.
INTRODUCTION.........................................................................................................................
5
2.1 MOLECULAR CHARACTERISTICS OF ALKALINE PHOSPHATASE WITH SPECIAL
REGARD TO THE TISSUE-
NONSPECIFIC
ISOFORM..........................................................................................................5
2.1.1 GENES ENCODING THE FOUR HUMAN ISOFORMS OF ALKALINE PHOSPHATASE
.
....................................................5
2.1.2 PROTEIN STRUCTURE OF ALKALINE
PHOSPHATASE.............................................................................................7
2.1.3 TNAP S SUBSTRATES AND THE ENZYME*S ROLE DURING
MINERALIZATION........................................................8
2.1.4 TNAP-DEFICIENT MOUSE
MODELS..............................................................................................................11
2.2 HYPOPHOSPHATASIA - IMPLICATIONS OF HEREDITARY TNAP
MALFUNCTION...............................12
2.2.1 CLINICAL CLASSIFICATION AND SYMPTOMS OF
HPP.......................................................................................
13
2.2.2 INHERITANCE OF
HPP...............................................................................................................................
14
2.2.3 THERAPEUTIC
APPROACHES.......................................................................................................................
14
2.3 TNAP*S ROLE IN THE NERVOUS
SYSTEM.................................................................................16
2.3.1 NERVOUS SYSTEM RELATED ISSUES IN
HPP.................................................................................................16
2.3.2 TNAP*S LOCALIZATION IN THE
BRAIN..........................................................................................................
16
2.3.3 TNAP*S INVOLVEMENT IN NEUROTRANSMITTER
METABOLISM.......................................................................17
2.3.4 TNAP*S PARTICIPATION IN PURINERGIC
SIGNALING......................................................................................19
2.3.5 INFLUENCE OF TNAP ON ADDITIONAL PROCESSES IN THE NERVOUS SYSTEM
..................................................
20
2.3.6 TNAP*S ROLE IN NEURODEGENERATIVE
DISORDERS......................................................................................21
2.4 AIM OF THE
PROJECT..........................................................................................................
24
3. MATERIALS AND
METHODS.....................................................................................................
25
3.1
MATERIALS......................................................................................................................25
3.1.1 TECHNICAL
EQUIPMENT............................................................................................................................25
3.1.2 CHEMICALS AND
REAGENTS........................................................................................................................26
3.1.3 BUFFERS AND
SOLUTIONS............................................................................................................................27
3.1.4
CONSUMABLES.........................................................................................................................................
31
3.1.5 KITS AND PURCHASED
SOLUTIONS...............................................................................................................
32
3.1.6
PRIMERS..................................................................................................................................................33
3.1.7
MARKERS.................................................................................................................................................35
3.1.8
ANTIBODIES.............................................................................................................................................35
3.1.9 CELL
CULTURE...........................................................................................................................................
36
3.1.10
VECTORS.................................................................................................................................................38
3.1.11
ENZYMES..............................................................................................................................................
38
3.1.12 BACTERIA
CULTIVATION.............................................................................................................................
39
3.1.13 MICE
STRAINS........................................................................................................................................
39
3.1.14 SOFTWARE AND ONLINE
SOURCES..............................................................................................................
39
3.1.15
STATISTICS..............................................................................................................................................
40
3.2
METHODS.......................................................................................................................
41
3.2.1 ISOLATION OF RNA AND REVERSE TRANSCRIPTION (CDNA SYNTHESIS)
...........................................................
41
3.2.2 ISOLATION OF GENOMIC DNA
(GDNA)......................................................................................................42
3.2.3 SEMI-QUANTITATIVE PCR (RT-PCR) AND AGAROSE GEL ELECTROPHORESIS
..................................................
42
3.2.4 QUANTITATIVE REAL-TIME PCR
(QPCR)............................................................. 44
3.2.5
DNA-SEQUENCING..................................................................................................................................
45
3.2.6 PREPARATION OF PROTEIN LYSATES FROM CELLS AND DETERMINATION OF
TOTAL PROTEIN CONTENT
....................
46
3.2.7 ANALYSIS OF MOUSE
BRAINS.....................................................................................................................46
3.2.8 SDS-PAGE AND WESTERN
BLOTTING..........................................................................................................49
3.2.9
IMMUNOCYTOCHEMISTRY........................................................................................................................
52
3.2.10 MICROARRAY AND GOSTAT
ANALYSIS........................................................................................................
52
3.2.11 BASIC CELL
CULTURE.................................................................................................................................
53
3.2.12 NEUROGENIC
DIFFERENTIATION................................................................................................................
55
3.2.13
TRANSFECTION........................................................................................................................................57
3.2.14 MACS (MAGNETIC CELL SEPARATION) SORTING OF HMSCS AND SUBSEQUENT
NEUROGENIC DIFFERENTIATION
............................................................................................................................................................
58
3.2.15
CSPD-ASSAY.........................................................................................................................................
59
3.2.16 CELL TITER GLO ASSAY FOR DETERMINATION OF ATP
CONVERSION.............................................................
61
3.2.17 ALKALINE PHOSPHATASE STAINING OF
HMSCS...........................................................................................62
3.2.18 DETERMINATION OF VITAMIN B6 CONTENT IN MOUSE BLOOD
SAMPLES.......................................................62
4.
RESULTS.............................................................................................................................
63
4.1 COMPARISON OF NEUROBLASTOMA CELL LINES WITH DIFFERENT TNAP
EXPRESSION LEVELS
.....
63
4.1.1 BASIC CHARACTERIZATION OF SH-SY5YTNAPIOW, SH-SYSY^0^ 1, AND
SH-SY5YTNAPHIGH CELL LINES
.............
63
4.1.2 DIFFERENTIAL GENE EXPRESSION IN SH-SY5YTNAPHIGH AND SH-SYSYTNAPIOW
ON RNA LEVELS
.......................
65
4.1.3 NEUROGENIC DIFFERENTIATION OF SH-SY5YTNAPIOW, SH-SYSY**0*3 1, AND
SH-SYSYTNAPHIGH
......................
70
4.1.4 EFFECTS OF HIGH DOSE TREATMENT WITH ATP, ADP, AND AMP IN
SH-SY5YTNAPLOW, SH-SYSY^0^ 31, AND
SH-SY5YTNAPHI6H
CELLS.........................................................................................................................
90
4.2 ANALYSIS OF TNAP*S ROLE DURING NEUROGENIC DIFFERENTIATION OF
HMSCS..........................94
4.2.1 BASAL TNAP EXPRESSION IN
HMSCS.......................................................................................................
94
4.2.2 MORPHOLOGY AFTER TREATMENT WITH NEUROGENIC DIFFERENTIATION MEDIUM
...........................................
96
4.2.3 TNAP LOCALIZATION IN DIFFERENTIATING
HMSCS.......................................................................................
98
4.2.4 MAP2 STAINING OF CELLS AFTER 6 DAYS NEUROGENIC DIFFERENTIATION
........................................................
99
4.2.5 EXPRESSION OF TNAP, NESTIN, TAU, AND MAP2 AFTER TREATMENT WITH
NEUROGENIC DIFFERENTIATION
MEDIUM.............................................................................................................................................101
4.2.6 TNAP SPECIFIC SORTING OF
HMSCS........................................................................................................102
4.3 ANALYSIS OF THE MOUSE MODELS BAP023 AND SAP007 CHARACTERIZED BY A
LOW SERUM
ALKALINE PHOSPHATASE
ACTIVITY.......................................................................................
108
4.3.1 DETERMINATION OF ALKALINE PHOSPHATASE ACTIVITY IN BRAIN SAMPLES
...................................................
109
4.3.2 COMPARISON OF GENE EXPRESSION IN BRAIN SAMPLES OF YOUNG MICE
.....................................................
113
4.3.3 DETERMINATION OF DOPAMINE, SEROTONIN, AND THEIR METABOLITES
........................................................
115
5.
DISCUSSION.........................................................................................................................
123
5.1 SH-SY5Y NEUROBLASTOMA CELL
LINE..................................................................................123
5.1.1 CONFIRMATION OF TNAP OVEREXPRESSION ON RNA, PROTEIN, AND ACTIVITY
LEVELS
..................................
123
5.1.2 TNAP OVEREXPRESSION CHANGES THE TRANSCRIPTOME OF THE SH-SY5Y CELL
LINE
...................................
124
5.1.3 TNAP PROMOTES NEUROGENIC DIFFERENTIATION IN THE SH-SY5Y CELL LINE
.............................................
126
5.1.4 EFFECTS OF TREATMENT WITH ATP, ADP, AND AMP AND STUDY OF ATP
TURNOVER IN THE COURSE OF TIME .. 139
5.2 PRIMARY
HMSCS.............................................................................................................
142
5.2.1 VARIABLE RATIO OF TNAP POSITIVE TO NEGATIVE CELLS IN UNTREATED
HMSC POPULATIONS
..........................
142
5.2.2 NEUROGENIC DIFFERENTIATION OF UNTREATED HMSCS AND AFTER TNAP
SPECIFIC MACS SORTING
............
143
5.3 WT, BAP023 AND SAP007
MICE....................................................................................
150
5.3.1 CHARACTERIZATION OF TNAP-DEFICIENT MOUSE
MODELS...........................................................................150
5.3.2 COMPARISON OF AP-LEVELS IN THE BRAIN OF THE MOUSE MODELS REVEALED
CORRELATION WITH AGE, GENOTYPE,
AND
GENDER........................................................................................................................................151
5.3.3 DIFFERENT AP-ACTIVITY LEVELS IN VARIOUS MOUSE TISSUE
SAMPLES...........................................................152
5.3.4 ANALYSIS OF BRAINS FROM WT, BAP023, AND SAP007 MICE NEITHER SHOWED
GENOTYPE SPECIFIC PATTERNS
OF TNAP EXPRESSION NOR RELIABLE EFFECTS OF TNAP ON THE EXPRESSION OF
GENES OF INTEREST
.............
153
5.3.5 NEUROTRANSMITTER ANALYSIS OF BRAIN SAMPLES GAINED FROM WT, BAP023,
AND SAP007 ANIMALS
......
154
6. CONCLUSION AND
OUTLOOK...................................................................................................159
7.
LITERATURE.........................................................................................................................
163
8.
APPENDIX..........................................................................................................................
177
8.1
ABBREVIATIONS................................................................................................................
177
8.2 LIST OF
FIGURES................................................................................................................
181
8.3 LIST OF
TABLES..................................................................................................................185
8.4 SUPPLEMENTARY
DATA.....................................................................................................
187
8.5
PUBLICATIONS...................................................................................................................231
8.6
ACKNOWLEDGEMENT........................................................................................................233
|
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| spelling | Graser, Stephanie Verfasser (DE-588)1099990238 aut Impact of hypophosphatasia on the biology of the nervous system the molecular role of tissue-nonspecific alkaline phosphatase beyond bone mineralization vorgelegt von Stephanie Graser, geboren in Schweinfurt Würzburg 2015 IV, 234 Seiten Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier Dissertation Julius-Maximilians-Universität Würzburg 2015 Zusammenfassung in deutscher und englischer Sprache Nervensystem (DE-588)4041643-4 gnd rswk-swf Alkalische Phosphatase (DE-588)4141894-3 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Alkalische Phosphatase (DE-588)4141894-3 s Nervensystem (DE-588)4041643-4 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028964072&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Graser, Stephanie Impact of hypophosphatasia on the biology of the nervous system the molecular role of tissue-nonspecific alkaline phosphatase beyond bone mineralization Nervensystem (DE-588)4041643-4 gnd Alkalische Phosphatase (DE-588)4141894-3 gnd |
| subject_GND | (DE-588)4041643-4 (DE-588)4141894-3 (DE-588)4113937-9 |
| title | Impact of hypophosphatasia on the biology of the nervous system the molecular role of tissue-nonspecific alkaline phosphatase beyond bone mineralization |
| title_auth | Impact of hypophosphatasia on the biology of the nervous system the molecular role of tissue-nonspecific alkaline phosphatase beyond bone mineralization |
| title_exact_search | Impact of hypophosphatasia on the biology of the nervous system the molecular role of tissue-nonspecific alkaline phosphatase beyond bone mineralization |
| title_full | Impact of hypophosphatasia on the biology of the nervous system the molecular role of tissue-nonspecific alkaline phosphatase beyond bone mineralization vorgelegt von Stephanie Graser, geboren in Schweinfurt |
| title_fullStr | Impact of hypophosphatasia on the biology of the nervous system the molecular role of tissue-nonspecific alkaline phosphatase beyond bone mineralization vorgelegt von Stephanie Graser, geboren in Schweinfurt |
| title_full_unstemmed | Impact of hypophosphatasia on the biology of the nervous system the molecular role of tissue-nonspecific alkaline phosphatase beyond bone mineralization vorgelegt von Stephanie Graser, geboren in Schweinfurt |
| title_short | Impact of hypophosphatasia on the biology of the nervous system |
| title_sort | impact of hypophosphatasia on the biology of the nervous system the molecular role of tissue nonspecific alkaline phosphatase beyond bone mineralization |
| title_sub | the molecular role of tissue-nonspecific alkaline phosphatase beyond bone mineralization |
| topic | Nervensystem (DE-588)4041643-4 gnd Alkalische Phosphatase (DE-588)4141894-3 gnd |
| topic_facet | Nervensystem Alkalische Phosphatase Hochschulschrift |
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