The role of the histone demethylase PHF8 in acute myeloi leukemia:
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[2021]
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| 100 | 1 | |a Felipe-Fumero, Eloísa |d 1989- |e Verfasser |0 (DE-588)1252700725 |4 aut | |
| 245 | 1 | 0 | |a The role of the histone demethylase PHF8 in acute myeloi leukemia |c vorgelegt von Eloísa Felipe-Fumero |
| 264 | 1 | |a Münster |c [2021] | |
| 300 | |a 114 Blätter |b Illustrationen, Diagramme |c 29 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Westfälische Wilhlems-Universität Münster |d 2022 |g Beschränkt für den Austausch | ||
| 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/1253039070/04 |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=034097931&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 999 | |a oai:aleph.bib-bvb.de:BVB01-034097931 | ||
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| 883 | 2 | |8 2\p |a dnb |d 20220714 |q DE-101 |u https://d-nb.info/provenance/plan#dnb | |
Datensatz im Suchindex
| _version_ | 1804184934830571520 |
|---|---|
| adam_text | TABLE
OF
CONTENTS
I.
LIST
OF
FIGURES
7
II.
LIST
OF
TABLES
9
III.
LIST
OF
ABBREVIATIONS
10
1
ABSTRACT
............................................................................................................................................
I
2
INTRODUCTION
3
2.
1
HEMATOPOIESIS
3
2.2
ACUTE MYELOID
LEUKEMIA
5
2.3
EPIGENETICS
IN
AML
5
2.3.1
POST-TRANSLATIONAL
HISTONE
MODIFICATIONS
6
2.4
THE
METHYLTRANSFERASE
MIXED
LINEAGE
LEUKEMIA
(MLL)
9
2.4.1
MLL-REARRANGED
LEUKEMIAS
11
2.5
KDM7
HISTONE
LYSINE
DEMETHYLASE
SUBFAMILY
14
2.5.1
THE
KDM7
FAMILY
MEMBER
PHF8
18
2.6
AML
TREATMENT
20
2.7
SPECIFIC
AIMS
23
3
MATERIAL
24
3.
1
CHEMICALS
24
3.2
EQUIPMENT
27
3.3
SUPPLIES
AND
CONSUMABLES
29
3.4
KITS
30
3.5
CELLS
30
3.6
ANTIBODIES
31
3.7
PRIMERS
..
.................................................
32
3.8
PLASMIDS
32
3.9
SOFTWARE
34
3.10
BUFFERS
AND
SOLUTIONS
34
4
METHODS
42
4.1
CELL
CULTURE
42
4.2
RETROVIRAL/LENTIVIRAL
TRANSDUCTION
OF
MURINE
HEMATOPOIETIC
PROGENITOR
CELLS
42
4.2.1
ISOLATION
OF
MURINE
C-KIT
POSITIVE
HEMATOPOIETIC
PROGENITOR
CELLS
42
4.2.2
VIRUS
PRODUCTION
43
4.2.3 RETROVIRAL/LENTIVIRAL
TRANSDUCTION
44
4.2.4
SERIAL
COLONY
FORMING
ASSAY
45
4.3
RETROVIRAL/LENTIVIRAL
TRANSDUCTION
OF
HUMAN
AML
CELL
LINES
45
4.3.1
INDUCTION
OF
GENE
EXPRESSION
IN
HUMAN
AML
CELL
LINES
WITH
4-HYDROXYTAMOXIFEN
47
4.4
IN
VIVO
ASSAYS
........................................................
47
4
4.5
TREATMENT
OF
HUMAN
AML
CELL
LINES
WITH
OKA
AND/OR
A
TRA
....................................................
48
4.6
INT
STAINING
..............................................................................................................................
48
4.
7
CELL
VIABILITY
ASSAYS..................................................................................................................
49
4.8
GENERATION
OF
GROWTH
CURVES
....................................................................................................
49
4.9
CYTOMORPHOLOGY
ANALYSIS
.........................................................................................................
49
4.10
FLOW
CYTOMETRY
.......................................................................................................................
49
4.10.1
APOPTOSIS
ANALYSIS
..........................................................................................................
49
4.10.2
CELL
CYCLE
ANALYSIS
............................................................................................................
50
4.10.3
DETECTION
OF
HUMAN
CD33*
CELLS
......................................................................................
50
4.11
GENE
EXPRESSION
ANALYSIS........................................................................................................
50
4.11.1
RNA
ISOLATION
...................................................................................................................
50
4.11.1.1
RNA
LIBRARY
PREPARATION
...........................................................................................
50
4.11.1.2
SEQUENCING
OF
THE
RNA
SAMPLES
............................................................................
51
4.11.2 CDNA
SYNTHESIS
...............................................................................................................
51
4.11.3
RELATIVE
QUANTITATIVE
REAL-TIME
PCR
(QPCR)
................................................................
51
4.12
PROTEIN
DETECTION
.....................................................................................................................
52
4.12.1
ISOLATION
OF
PROTEINS
.........................................................................................................
52
4.12.1.1
PIERCE
BCA
PROTEIN
ASSAY
......................................................................................
53
4.12.1.2
ISOLATION
OF
NUCLEAR
AND
CYTOPLASMIC
PROTEINS
.........................................................
53
4.12.2
SDS-PAGE
.......................................................................................................................
53
4.12.3
WESTERN
BLOT
....................................................................................................................
54
4.12.4
IMMUNODETECTION
.............................................................................................................
54
4.13
CHROMATIN
IMMUNOPRECIPITATION
(CHIP)
..................................................................................
54
4.13.1
CHLP-QPCR
......................................................................................................................
56
4.13.2
DNA
LIBRARY
PREPARATION
....................................................................................................
56
4.13.3
SEQUENCING
OF
THE
CHIP
SAMPLES
.....................................................................................
57
4.14
CLONING
OF
LENTICRISPR
V2
CONSTRUCTS
....................................................................................
57
4.14.1
SANGER
SEQUENCING
.........................................................................................................
60
4.14.2
PLASMID
PREPARATION
..........................................................................................................
61
4.14.3
CONTROL
DIGESTION
.............................................................................................................
61
4.15
STATISTICAL
AND
BIOINFORMATIC
ANALYSIS
.......................................................
.
..............................
62
5
RESULTS
............................................................................................................................................
63
5.1
PROGNOSTIC
IMPACT
OF
THE
KDM7
FAMILY
MEMBER,
PHF8,
IN
AML
.............................................
63
5.1.1
PHF8
DEMETHYLASE ACTIVITY
COMPROMISES
THE
TRANSFORMATION
CAPACITY
OF
AML
ASSOCIATED
ONCOGENES
IN
MURINE
HEMATOPOIETIC
PROGENITOR
CELLS
...................................................
63
5.1.2
PHF8
IMPAIRS
CLONAL
GROWTH
OF
HUMAN
AML
CELLS
AND
COMPROMISES
IN
VIVO
LEUKEMOGENESIS
.............................................................................................................................
68
5.1.2.1
CHARACTERIZATION
OF
THE
ROLE
OF
PHF8
DEMETHYLASE
ACTIVITY
IN
CELL
LINES
OF
HUMAN
MLL
R
AML
IN
VITRO
..............................................................................................................................
68
5.1.2.2
INFLUENCE
OF
PHF8
DEMETHYLASE
ACTIVITY
IN
HUMAN
MLL-R
AML
CELLS
IN
VIVO
..............
71
5.1.3
PHF8
DEMETHYLASE
ACTIVITY
INDUCES
DIFFERENTIAL
TRANSCRIPTOME
PROFILE
IN
AML
CELLS
FAVORING
DIFFERENTIATION
PROCESSES
...................................................................................................
72
5.1.4
KNOCK
OUT
OF
PHF8
DOES
NOT
IMPACT
LEUKEMIC
TRANSFORMATION
OF
PRIMARY
MURINE
HSPCS
NOR
CLONOGENIC
GROWTH
OF
HUMAN
AML
CELLS
....................................................................................
73
5.2
THE
ROLE
OF
PHF8
PHOSPHORYLATION
IN
AML
..............................................................................
75
5.2.1
PHF8-EFFECT
ON
CLONOGENIC
GROWTH
OF
HUMAN
AML
CELLS
IS
REGULATED
BY
SERINE
PHOSPHORYLATION
AS
WELL
AS
ITS
CATALYTIC
ACTIVITY
..............................................................................
76
5
5.2.1.1
SERINE
PHOSPHORYLATION
OF
PHF8
SIGNIFICANTLY
DELAYS
LEUKEMOGENESIS
OF
HUMAN
AML
IN
VIVO
.....................................................................................................................................
78
5.2.2
DEMETHYLASE
ACTIVITY
OF
PHOSPHORYLATED
PHF8
MODULATES
PROMOTER
OCCUPANCY
AND
GENE
EXPRESSION
IN
HUMAN
AML
.....................................................................................................
79
5.2.2.1
DEMETHYLASE
ACTIVITY
OF
PHOSPHORYLATED
PHF8
TRIGGERS
SMAD6
AND
ID2
EXPRESSION
AS
WELL
AS
NUCLEAR
TRANSLOCATION
OF
SMAD6
IN
HUMAN
AML
CELLS
...............................................
81
5.2.2.1
.1
PHF8-MEDIATED
RESPONSE
TO
ATRA
IS
INDUCED
BY
INCREASED
EXPRESSION
LEVELS
OF
ID2
AND
SMAD6
IN
AML
CELLS
..................................................................................................
85
6
DISCUSSION
..................................................................................................................................
...90
7
CONCLUSIONS
...................................................................................................................................
98
8
REFERENCES
.....................................................................................................................................
99
9
CURRICULUM
VITAE
..........................................................................................................................
112
10
ACKNOWLEDGEMENTS
.................................................................................................................
114
6
|
| adam_txt |
TABLE
OF
CONTENTS
I.
LIST
OF
FIGURES
7
II.
LIST
OF
TABLES
9
III.
LIST
OF
ABBREVIATIONS
10
1
ABSTRACT
.
I
2
INTRODUCTION
3
2.
1
HEMATOPOIESIS
3
2.2
ACUTE MYELOID
LEUKEMIA
5
2.3
EPIGENETICS
IN
AML
5
2.3.1
POST-TRANSLATIONAL
HISTONE
MODIFICATIONS
6
2.4
THE
METHYLTRANSFERASE
MIXED
LINEAGE
LEUKEMIA
(MLL)
9
2.4.1
MLL-REARRANGED
LEUKEMIAS
11
2.5
KDM7
HISTONE
LYSINE
DEMETHYLASE
SUBFAMILY
14
2.5.1
THE
KDM7
FAMILY
MEMBER
PHF8
18
2.6
AML
TREATMENT
20
2.7
SPECIFIC
AIMS
23
3
MATERIAL
24
3.
1
CHEMICALS
24
3.2
EQUIPMENT
27
3.3
SUPPLIES
AND
CONSUMABLES
29
3.4
KITS
30
3.5
CELLS
30
3.6
ANTIBODIES
31
3.7
PRIMERS
.
.
32
3.8
PLASMIDS
32
3.9
SOFTWARE
34
3.10
BUFFERS
AND
SOLUTIONS
34
4
METHODS
42
4.1
CELL
CULTURE
42
4.2
RETROVIRAL/LENTIVIRAL
TRANSDUCTION
OF
MURINE
HEMATOPOIETIC
PROGENITOR
CELLS
42
4.2.1
ISOLATION
OF
MURINE
C-KIT
POSITIVE
HEMATOPOIETIC
PROGENITOR
CELLS
42
4.2.2
VIRUS
PRODUCTION
43
4.2.3 RETROVIRAL/LENTIVIRAL
TRANSDUCTION
44
4.2.4
SERIAL
COLONY
FORMING
ASSAY
45
4.3
RETROVIRAL/LENTIVIRAL
TRANSDUCTION
OF
HUMAN
AML
CELL
LINES
45
4.3.1
INDUCTION
OF
GENE
EXPRESSION
IN
HUMAN
AML
CELL
LINES
WITH
4-HYDROXYTAMOXIFEN
47
4.4
IN
VIVO
ASSAYS
.
47
4
4.5
TREATMENT
OF
HUMAN
AML
CELL
LINES
WITH
OKA
AND/OR
A
TRA
.
48
4.6
INT
STAINING
.
48
4.
7
CELL
VIABILITY
ASSAYS.
49
4.8
GENERATION
OF
GROWTH
CURVES
.
49
4.9
CYTOMORPHOLOGY
ANALYSIS
.
49
4.10
FLOW
CYTOMETRY
.
49
4.10.1
APOPTOSIS
ANALYSIS
.
49
4.10.2
CELL
CYCLE
ANALYSIS
.
50
4.10.3
DETECTION
OF
HUMAN
CD33*
CELLS
.
50
4.11
GENE
EXPRESSION
ANALYSIS.
50
4.11.1
RNA
ISOLATION
.
50
4.11.1.1
RNA
LIBRARY
PREPARATION
.
50
4.11.1.2
SEQUENCING
OF
THE
RNA
SAMPLES
.
51
4.11.2 CDNA
SYNTHESIS
.
51
4.11.3
RELATIVE
QUANTITATIVE
REAL-TIME
PCR
(QPCR)
.
51
4.12
PROTEIN
DETECTION
.
52
4.12.1
ISOLATION
OF
PROTEINS
.
52
4.12.1.1
PIERCE
BCA
PROTEIN
ASSAY
.
53
4.12.1.2
ISOLATION
OF
NUCLEAR
AND
CYTOPLASMIC
PROTEINS
.
53
4.12.2
SDS-PAGE
.
53
4.12.3
WESTERN
BLOT
.
54
4.12.4
IMMUNODETECTION
.
54
4.13
CHROMATIN
IMMUNOPRECIPITATION
(CHIP)
.
54
4.13.1
CHLP-QPCR
.
56
4.13.2
DNA
LIBRARY
PREPARATION
.
56
4.13.3
SEQUENCING
OF
THE
CHIP
SAMPLES
.
57
4.14
CLONING
OF
LENTICRISPR
V2
CONSTRUCTS
.
57
4.14.1
SANGER
SEQUENCING
.
60
4.14.2
PLASMID
PREPARATION
.
61
4.14.3
CONTROL
DIGESTION
.
61
4.15
STATISTICAL
AND
BIOINFORMATIC
ANALYSIS
.
'.
.
62
5
RESULTS
.
63
5.1
PROGNOSTIC
IMPACT
OF
THE
KDM7
FAMILY
MEMBER,
PHF8,
IN
AML
.
63
5.1.1
PHF8
DEMETHYLASE ACTIVITY
COMPROMISES
THE
TRANSFORMATION
CAPACITY
OF
AML
ASSOCIATED
ONCOGENES
IN
MURINE
HEMATOPOIETIC
PROGENITOR
CELLS
.
63
5.1.2
PHF8
IMPAIRS
CLONAL
GROWTH
OF
HUMAN
AML
CELLS
AND
COMPROMISES
IN
VIVO
LEUKEMOGENESIS
.
68
5.1.2.1
CHARACTERIZATION
OF
THE
ROLE
OF
PHF8
DEMETHYLASE
ACTIVITY
IN
CELL
LINES
OF
HUMAN
MLL
R
AML
IN
VITRO
.
68
5.1.2.2
INFLUENCE
OF
PHF8
DEMETHYLASE
ACTIVITY
IN
HUMAN
MLL-R
AML
CELLS
IN
VIVO
.
71
5.1.3
PHF8
DEMETHYLASE
ACTIVITY
INDUCES
DIFFERENTIAL
TRANSCRIPTOME
PROFILE
IN
AML
CELLS
FAVORING
DIFFERENTIATION
PROCESSES
.
72
5.1.4
KNOCK
OUT
OF
PHF8
DOES
NOT
IMPACT
LEUKEMIC
TRANSFORMATION
OF
PRIMARY
MURINE
HSPCS
NOR
CLONOGENIC
GROWTH
OF
HUMAN
AML
CELLS
.
73
5.2
THE
ROLE
OF
PHF8
PHOSPHORYLATION
IN
AML
.
75
5.2.1
PHF8-EFFECT
ON
CLONOGENIC
GROWTH
OF
HUMAN
AML
CELLS
IS
REGULATED
BY
SERINE
PHOSPHORYLATION
AS
WELL
AS
ITS
CATALYTIC
ACTIVITY
.
76
5
5.2.1.1
SERINE
PHOSPHORYLATION
OF
PHF8
SIGNIFICANTLY
DELAYS
LEUKEMOGENESIS
OF
HUMAN
AML
IN
VIVO
.
78
5.2.2
DEMETHYLASE
ACTIVITY
OF
PHOSPHORYLATED
PHF8
MODULATES
PROMOTER
OCCUPANCY
AND
GENE
EXPRESSION
IN
HUMAN
AML
.
79
5.2.2.1
DEMETHYLASE
ACTIVITY
OF
PHOSPHORYLATED
PHF8
TRIGGERS
SMAD6
AND
ID2
EXPRESSION
AS
WELL
AS
NUCLEAR
TRANSLOCATION
OF
SMAD6
IN
HUMAN
AML
CELLS
.
81
5.2.2.1
.1
PHF8-MEDIATED
RESPONSE
TO
ATRA
IS
INDUCED
BY
INCREASED
EXPRESSION
LEVELS
OF
ID2
AND
SMAD6
IN
AML
CELLS
.
85
6
DISCUSSION
.
.90
7
CONCLUSIONS
.
98
8
REFERENCES
.
99
9
CURRICULUM
VITAE
.
112
10
ACKNOWLEDGEMENTS
.
114
6 |
| any_adam_object | 1 |
| any_adam_object_boolean | 1 |
| author | Felipe-Fumero, Eloísa 1989- |
| author_GND | (DE-588)1252700725 |
| author_facet | Felipe-Fumero, Eloísa 1989- |
| author_role | aut |
| author_sort | Felipe-Fumero, Eloísa 1989- |
| author_variant | e f f eff |
| building | Verbundindex |
| bvnumber | BV048832360 |
| ctrlnum | (OCoLC)1338229920 (DE-599)DNB1253039070 |
| format | Thesis Book |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV048832360 |
| illustrated | Illustrated |
| index_date | 2024-07-03T21:35:34Z |
| indexdate | 2024-07-10T09:47:14Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-034097931 |
| oclc_num | 1338229920 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR |
| owner_facet | DE-355 DE-BY-UBR |
| physical | 114 Blätter Illustrationen, Diagramme 29 cm |
| publishDate | 2021 |
| publishDateSearch | 2021 |
| publishDateSort | 2021 |
| record_format | marc |
| spelling | Felipe-Fumero, Eloísa 1989- Verfasser (DE-588)1252700725 aut The role of the histone demethylase PHF8 in acute myeloi leukemia vorgelegt von Eloísa Felipe-Fumero Münster [2021] 114 Blätter Illustrationen, Diagramme 29 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Westfälische Wilhlems-Universität Münster 2022 Beschränkt für den Austausch (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf https://d-nb.info/1253039070/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=034097931&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p emakn 0,38079 20220714 DE-101 https://d-nb.info/provenance/plan#emakn 2\p dnb 20220714 DE-101 https://d-nb.info/provenance/plan#dnb |
| spellingShingle | Felipe-Fumero, Eloísa 1989- The role of the histone demethylase PHF8 in acute myeloi leukemia |
| subject_GND | (DE-588)4113937-9 |
| title | The role of the histone demethylase PHF8 in acute myeloi leukemia |
| title_auth | The role of the histone demethylase PHF8 in acute myeloi leukemia |
| title_exact_search | The role of the histone demethylase PHF8 in acute myeloi leukemia |
| title_exact_search_txtP | The role of the histone demethylase PHF8 in acute myeloi leukemia |
| title_full | The role of the histone demethylase PHF8 in acute myeloi leukemia vorgelegt von Eloísa Felipe-Fumero |
| title_fullStr | The role of the histone demethylase PHF8 in acute myeloi leukemia vorgelegt von Eloísa Felipe-Fumero |
| title_full_unstemmed | The role of the histone demethylase PHF8 in acute myeloi leukemia vorgelegt von Eloísa Felipe-Fumero |
| title_short | The role of the histone demethylase PHF8 in acute myeloi leukemia |
| title_sort | the role of the histone demethylase phf8 in acute myeloi leukemia |
| topic_facet | Hochschulschrift |
| url | https://d-nb.info/1253039070/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=034097931&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT felipefumeroeloisa theroleofthehistonedemethylasephf8inacutemyeloileukemia |
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