Endosomal lysosomal proteases in epithelial to mesenchymal transition (EMT) and invasion of normal and malignant mammary epithelium:
Saved in:
| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
2013
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Item Description: | Enth. Sonderabdr. |
| Physical Description: | 121, [39] S. Ill., graph. Darst. 30 cm |
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Record in the Search Index
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| adam_text | ZUSAMMENFASSUNG
TABLE OF CONTENTS
1 ZUSAMMENFASSUNG 6
2 ABSTRACT 8
3 INTRODUCTION 10
3.1 BREAST CANCER METASTASIS: A WORLDWIDE
HEALTH PROBLEM 10
3.2 PROTEASES PROMOTE
HALLMARKS OF CANCER DEVELOPMENT 10
3.3 SPECIFIC PROTEASES FACILITATE
CANCER CELL
INVASION AND
METASTASIS 11
3.4 NORMAL AND
ONCOGENIC EMT 12
3.5 CYSTEINE CATHEPSINS: A GROUP OF MULTIFUNCTIONAL LYSOSOMAL
PROTEASES 14
3.6 SPECIFIC CYSTEINE CATHEPSINS PROMOTE
BREAST CANCER PROGRESSION
AND METASTASIS 17
4 OBJECTIVES OF THIS DOCTORAL THESIS 19
5 MATERIALS 20
5.1 TECHNICAL EQUIPMENT 20
5.2 CONSUMABLES 21
5.3 REAGENTS 21
5.3.1 CHEMICALS 21
5.3.2 REAGENTS, MEDIA AND SUPPLEMENTS 21
5.4 COMMERCIAL KITS 22
5.5 ANTIBODIES 23
5.6 PCR PRIMERS 23
5.7 SOFTWARE 25
6 METHODS 26
6.1 CELL CULTURE
METHODS 26
6.1.1 GENERATION AND
CULTURE OF CELL
LINES 26
6.1.2 GROWTH FACTOR TREATMENT OF CELLS 26
6.1.3 PHARMACOLOGICAL CYSTEINE CATHEPSIN
INHIBITION IN CELLS 27
6.1.4 INDUCTION OF CTSB AND
CTSL OVEREXPRESSION 27
6.1.5 GENERATION OF CELL
CONDITIONED MEDIUM 27
6.2 CELL-BASED ASSAYS 28
6.2.1 LDH RELEASE CELL
VIABILITY ASSAY 28
6.2.2 DETERMINATION OF CELL
NUMBERS 28
6.2.3 REAL TIME
CELL MIGRATION
AND INVASION ASSAY 28
6.2.4 FLOW CYTROMETRY 28
6.3 MOLECULAR BIOLOGY METHODS 29
6.3.1 DNA GEL ELECTROPHORESIS 29
6.3.2 PREPARATION OF RNA FROM
CELLS OR TISSUES 29
6.3.3 CDNA SYNTHESIS 30
6.3.4 QRT PCR 30
3
HTTP://D-NB.INFO/1049563999
ZUSAMMENFASSUNG
6.3.5 GENE TARGETING
WITH CTSL
ZINK FINGER NUCLEASES 30
6.3.6 DETERMINATION OF MOUSE
GENOTYPES 32
6.4 BIOCHEMICAL METHODS 33
6.4.1 PREPARATION OF PROTEIN
LYSATES 33
6.4.2 PREPARATION OF ORGANELLE
ENRICHED FRACTIONS 33
6.4.3 DETERMINATION OF PROTEIN CONCENTRATION 34
6.4.4 WESTERN BLOT 34
6.4.5 CYSTEINE CATHEPSIN ACTIVITY ASSAY 35
6.4.6 PROTEASOME ACTIVITY ASSAY 36
6.5 QUANTITATIVE PROTEOME
COMPARISON 36
6.5.1 STABLE ISOTOPIC LABELING WITH AMINO
ACIDS OF CELLS
(SILAC) 36
6.5.2 PROTEIN PREPARATION
FOR MS ANALYSIS 37
6.5.3 TRYPTIC DIGEST AND
PEPTIDE PREPARATION 37
6.5.4 LC MS/MS MEASUREMENT 38
6.5.5 DATA ANALYSIS 38
6.6 MICROSCOPY 39
6.6.1 HISTOLOGY 39
6.6.2 FLUORESCENT IMMUNSTAIRRING OF CELLS 40
6.7 ANIMAL KEEPING 40
6.8 STATISTICAL DATA
ANALYSIS 40
7 RESULTS 41
7.1 FUNCTIONAL ANALYSIS OF CYSTEINE
CATHEPSIN CONTRIBUTION
TO INVASION 41
7.1.1 EXPERIMENTAL INDUCTION OF CLASSICAL
EMT IN NORMAL
AND PEMT IN MALIGNANT MAMMARY
EPITHELIAL CELLS 41
7.1.2 EFFECT OF PHARMACOLOGICAL
CYSTEINE CATHEPSINS INHIBITION
ON INVASION AND
CELL MOTILITY.
43
7.1.3 CYSTEINE CATHEPSIN
EXPRESSION IN RESPONSE TO TGFP-1 50
7.1.4 INFLUENCE OF CYSTEINE
CATHEPSINS ON EMT 52
7.1.5 CELL VIABILITY AND
PROLIFERATION UPON
CYSTEINE CATHEPSIN
INHIBITION 57
7.2 PROTEOME WIDE ANALYSIS
OF THE ROLE
OF CYSTEINE CATHEPSINS DURING
TGFP-1 INDUCED EMT....
59
7.2.1 EFFECTS OF CYSTEINE CATHEPSIN
INHIBITION ON THE
INTRACELLULAR PROTEOLYTIC COMPARTMENT
OF
MALIGNANT MAMMARY EPITHELIAL
CELLS 65
7.2.2 EFFECT OF CYSTEINE CATHEPSIN
INHIBITION ON CELL-CELL ADHESION 68
7.3 TARGETED CTSL
KNOCK-OUT 72
7.4 EFFECT OF CATHEPSIN
H DEFICIENCY ON LUNG SURFACTANT GENERATION 74
8 DISCUSSION 77
8.1 CYSTEINE CATHEPSIN
ACTIVITY PROMOTES TGFP-1
INDUCED CELL INVASION 77
8.2 TGFP-1 INDUCES SPECIFIC CYSTEINE CATHEPSINS 78
8.3 REGULATION OF EMT,
PROLIFERATION AND
CELL VIABILITY
ARE NOT AFFECTED
BY CYSTEINE CATHEPSIN
INHIBITION 80
4
ZUSAMMENFASSUNG
8.4 IDENTIFICATION OF TGF|3-1 INDUCED
CHANGES IN THE
PROTEOMES OF NORMAL AND
MALIGNANT
MAMMARY EPITHELIAL CELLS 81
8.5 IDENTIFICATION OF PROTEOME
ALTERATIONS UPON CYSTEINE
CATHEPSIN INHIBITION 82
8.6 CYSTEINE CATHEPSIN
INHIBITION DISTURBS
THE HOMEOSTASIS OF INTRACELLULAR DEGRADATION 83
8.7 ALTERED CELL-CELL
JUNCTION DYNAMICS AS POTENTIAL CAUSE
FOR REDUCED INVASION 85
8.8 SYNOPSIS AND PERSPECTIVES 88
9 REFERENCES 89
10 LIST OF ABBREVIATIONS 101
11 LIST OF PUBLICATIONS AND PRESENTATIONS 103
12 ACKNOWLEDGEMENTS 105
13 APPENDIX 106
5
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| author | Kern, Ursula |
| author_GND | (DE-588)1047631741 |
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| building | Verbundindex |
| bvnumber | BV042055729 |
| ctrlnum | (OCoLC)891167067 (DE-599)DNB1049563999 |
| discipline | Biologie Medizin |
| format | Thesis Book |
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| indexdate | 2024-07-10T01:11:33Z |
| institution | BVB |
| language | English |
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| physical | 121, [39] S. Ill., graph. Darst. 30 cm |
| publishDate | 2013 |
| publishDateSearch | 2013 |
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| spelling | Kern, Ursula Verfasser (DE-588)1047631741 aut Endosomal lysosomal proteases in epithelial to mesenchymal transition (EMT) and invasion of normal and malignant mammary epithelium vorgelegt von Ursula Kern 2013 121, [39] S. Ill., graph. Darst. 30 cm txt rdacontent n rdamedia nc rdacarrier Enth. Sonderabdr. Freiburg (Breisgau), Univ., Diss., 2014 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027496720&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Kern, Ursula Endosomal lysosomal proteases in epithelial to mesenchymal transition (EMT) and invasion of normal and malignant mammary epithelium |
| subject_GND | (DE-588)4113937-9 |
| title | Endosomal lysosomal proteases in epithelial to mesenchymal transition (EMT) and invasion of normal and malignant mammary epithelium |
| title_auth | Endosomal lysosomal proteases in epithelial to mesenchymal transition (EMT) and invasion of normal and malignant mammary epithelium |
| title_exact_search | Endosomal lysosomal proteases in epithelial to mesenchymal transition (EMT) and invasion of normal and malignant mammary epithelium |
| title_full | Endosomal lysosomal proteases in epithelial to mesenchymal transition (EMT) and invasion of normal and malignant mammary epithelium vorgelegt von Ursula Kern |
| title_fullStr | Endosomal lysosomal proteases in epithelial to mesenchymal transition (EMT) and invasion of normal and malignant mammary epithelium vorgelegt von Ursula Kern |
| title_full_unstemmed | Endosomal lysosomal proteases in epithelial to mesenchymal transition (EMT) and invasion of normal and malignant mammary epithelium vorgelegt von Ursula Kern |
| title_short | Endosomal lysosomal proteases in epithelial to mesenchymal transition (EMT) and invasion of normal and malignant mammary epithelium |
| title_sort | endosomal lysosomal proteases in epithelial to mesenchymal transition emt and invasion of normal and malignant mammary epithelium |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027496720&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT kernursula endosomallysosomalproteasesinepithelialtomesenchymaltransitionemtandinvasionofnormalandmalignantmammaryepithelium |