Antifibrotic therapy in the liver by adenovirus mediated expression of a TGF-beta1 antisense mRNA:
Saved in:
| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
Aachen
Shaker
2004
|
| Series: | Berichte aus der Biologie
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | VI, 96 S. Ill., graph. Darst. 21 cm, 162 gr. |
| ISBN: | 3832230548 |
Staff View
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| 245 | 1 | 0 | |a Antifibrotic therapy in the liver by adenovirus mediated expression of a TGF-beta1 antisense mRNA |c Mónica S. Arias-Kuhn |
| 246 | 1 | |a Antifibrotic therapy in the liver by adenovirus-mediated expression of a TGF-beta1 antisense mRNA | |
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Record in the Search Index
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| adam_text | A: Table of Contents
A Table of Contents I
B Index of Abbreviations v
1 Introduction 1
1.1 Liver Fibrosis 3
1.2 Transforming Growth Factor pi 4
1.3 Gene Therapy 5
1.4 Antisense Therapy 6
1.5 Gene Delivery 7
1.6 Adenoviral System 8
2 Material 9
2.1 Products 9
2.2 Chemicals 10
2.3 Enzymes 11
2.4 Antibodies 13
2.5 Primer 14
2.6 Reagent System and Radioactive Nucleotides 14
2.7 Other Biomolecular Reagents 15
2.8 Cell and Bacteria Culture Mediums 15
2.9 Biologic Materials 16
2.9.1 Bacteria 16
2.9.2 Cells 16
2.9.3 Plasmids 17
2.10 Solutions and Buffers 17
3 Methods 28
3.1 Isolation of Genomic DNA from Mammalian cells 28
3.2 Isolation of Plasmid ONA 28
3.2.1 Small Scale Preparation of Plasmid DNA 29
3.2.2 Large Scale Preparation of Plasmid DNA 29
A: Table of Contents
3.2.3 Preparation of Plasmid DNA by Ultracentrifugation 30
3.3 RNA Isolation 30
3.4 Measurement of Nucleic Acids Concentration 31
3.5 DNA Quantification (SYBR* Green I) 31
3.6 DNA Cutting by Restriction Endonucleases 31
3.7 Protein Isolation 32
3.8 Protein Quantification (BSA Protein Assay Reagent Kit) 32
3.9 Separation of Molecules by Gel Electrophoresis 32
3.10 Electrophoresis of DNA 33
3.11 Electrophoresis of RNA 33
3.12 Protein Electrophoresis 33
3.13 Cloning Strategies 34
3.14 Recovery of DNA from Agarose Gel by Electroelution 34
3.15 Filling up with Klenow Fragment 34
3.16 Dephosphorilation of Linearized DNA 35
3.17 Ligation of DNA Fragments 35
3.18 Sequence Analysis 35
3.19 Northern Blot Analysis 36
3.20 Southern Blot Analysis 36
3.21 Western Blot Analysis 36
3.22 Radioactive Labeling by Random Priming 37
3.23 Hybridization 37
3.24 Polymerase Chain Reaction (PCR) 37
3.25 Real Time PCR (RT PCR) 38
3.26 Preparation of Competent Bacteria (Hanahan Method 39
3.27 Transformation: Uptake of DNA by Competent Cells 39
3.28 Isolation and Culturing of Rat HSCs and MFBs 39
3.29 Cell Subculturing 40
3.30 Cell Freezing and Storage 41
3.31 Transfection and Infection in Cell Culture 41
3.32 Construction of a Replication defective Recombinant
Adenovirus 42
II
A: Table of Contents
3.33 Purification, Concentration and Titration of Adenoviruses 42
3.33.1 Purification by Cesium Chloride Gradient Centrifugation 42
3.33.2 Purification by Ion Exchange 43
3.34 Cell ELISA 43
3.35 TGF 01 ELISA 44
3.36 Metabolic Labeling, Immunoprecipitation and
Fluorography 45
3.37 Microarray Analysis 45
3.38 Proliferation Assay (Thymidine Incorporation) 46
3.39 Animal Models 46
3.40 Immunohistochemistry 47
4 Results 48
4.1 Construction of Antagonist Construct and Respective
Viruses 48
4.1.1 Construction of Chimaeric pCMV asTGF pi Antagonist
Construct 48
4.1.2 Construction of E1 Replacement Vectors 50
4.1.3 Examination of Functionality of Antagonist Construct 52
4.1.4 Testing of the Presence of E1 a E1 b Genes in
HEK293 Cells 53
4.1.5 Construction of Recombinant Adenoviruses 54
4.1.6 Proof of Functionality of Adenoviruses 55
4.1.7 High Level Expression of the Transgene 56
4.2 Testing of Functionality of the Antisense
Construct in vitro 56
4.2.1 Blockage of TGF 01 Synthesis 58
4.2.2 Effects of TGF pi in Cell Proliferation 58
4.2.3 Biological Activity 60
4.2.4 Expression of Matrix Related Proteins 61
4.2.5 Expression of TGF 01 Related Genes 62
4.3 Testing of Functionality of the Antisense
Construct in vivo 62
in
A: Table of Contents
4.3.1 Fibrosis Induced by Ligature of the Common Bile Duct 63
4.3.2 Analysis of Adenoviral Expression in Normal Tissue 65
4.3.3 Expression of Adenovirus in Experimentally Induced
Liver Fibrosis 67
4.3.4 Expression and Antifibrotic Capacity of the asTGF pi
Device 68
4.3.4.1 Histological Scoring of Liver Fibrosis in Rats 70
4.3.4.2 Suppression of Liver Fibrosis by Transfering the
Antisense Construct 70
4.4 Antifibrotic Potential by Selective Expresssion in HSC 73
4.4.1 Expression of the TIMP 1 promoter 73
4.4.2 Construction of TIMP 1 Recombinant Adenoviruses 74
4.4.3 Testing of Functionality of Adenoviral Expression 76
5 Discussion 78
6 Summary 82
7 References 84
8 Publications 93
9 Acknowledgements 95
10 Curriculum Vitae 96
IV
|
| any_adam_object | 1 |
| author | Arias-Kuhn, Mónica S. 1966- |
| author_GND | (DE-588)129424366 |
| author_facet | Arias-Kuhn, Mónica S. 1966- |
| author_role | aut |
| author_sort | Arias-Kuhn, Mónica S. 1966- |
| author_variant | m s a k msa msak |
| building | Verbundindex |
| bvnumber | BV019785199 |
| ctrlnum | (OCoLC)76502740 (DE-599)BVBBV019785199 |
| dewey-full | 610 |
| dewey-hundreds | 600 - Technology (Applied sciences) |
| dewey-ones | 610 - Medicine and health |
| dewey-raw | 610 |
| dewey-search | 610 |
| dewey-sort | 3610 |
| dewey-tens | 610 - Medicine and health |
| discipline | Medizin |
| format | Thesis Book |
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| id | DE-604.BV019785199 |
| illustrated | Illustrated |
| indexdate | 2024-07-09T20:06:03Z |
| institution | BVB |
| isbn | 3832230548 |
| language | English |
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| physical | VI, 96 S. Ill., graph. Darst. 21 cm, 162 gr. |
| publishDate | 2004 |
| publishDateSearch | 2004 |
| publishDateSort | 2004 |
| publisher | Shaker |
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| series2 | Berichte aus der Biologie |
| spelling | Arias-Kuhn, Mónica S. 1966- Verfasser (DE-588)129424366 aut Antifibrotic therapy in the liver by adenovirus mediated expression of a TGF-beta1 antisense mRNA Mónica S. Arias-Kuhn Antifibrotic therapy in the liver by adenovirus-mediated expression of a TGF-beta1 antisense mRNA Aachen Shaker 2004 VI, 96 S. Ill., graph. Darst. 21 cm, 162 gr. txt rdacontent n rdamedia nc rdacarrier Berichte aus der Biologie Zugl.: Aachen, Techn. Hochsch., Diss., 2003 Leberfibrose (DE-588)4167063-2 gnd rswk-swf Transforming Growth Factor beta 1 (DE-588)4404907-9 gnd rswk-swf Adenoviren (DE-588)4141412-3 gnd rswk-swf Antisense-RNS (DE-588)4324981-4 gnd rswk-swf Gentherapie (DE-588)4296957-8 gnd rswk-swf Heterologe Genexpression (DE-588)4342015-1 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Leberfibrose (DE-588)4167063-2 s Gentherapie (DE-588)4296957-8 s Transforming Growth Factor beta 1 (DE-588)4404907-9 s Antisense-RNS (DE-588)4324981-4 s Heterologe Genexpression (DE-588)4342015-1 s Adenoviren (DE-588)4141412-3 s DE-604 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=013111065&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Arias-Kuhn, Mónica S. 1966- Antifibrotic therapy in the liver by adenovirus mediated expression of a TGF-beta1 antisense mRNA Leberfibrose (DE-588)4167063-2 gnd Transforming Growth Factor beta 1 (DE-588)4404907-9 gnd Adenoviren (DE-588)4141412-3 gnd Antisense-RNS (DE-588)4324981-4 gnd Gentherapie (DE-588)4296957-8 gnd Heterologe Genexpression (DE-588)4342015-1 gnd |
| subject_GND | (DE-588)4167063-2 (DE-588)4404907-9 (DE-588)4141412-3 (DE-588)4324981-4 (DE-588)4296957-8 (DE-588)4342015-1 (DE-588)4113937-9 |
| title | Antifibrotic therapy in the liver by adenovirus mediated expression of a TGF-beta1 antisense mRNA |
| title_alt | Antifibrotic therapy in the liver by adenovirus-mediated expression of a TGF-beta1 antisense mRNA |
| title_auth | Antifibrotic therapy in the liver by adenovirus mediated expression of a TGF-beta1 antisense mRNA |
| title_exact_search | Antifibrotic therapy in the liver by adenovirus mediated expression of a TGF-beta1 antisense mRNA |
| title_full | Antifibrotic therapy in the liver by adenovirus mediated expression of a TGF-beta1 antisense mRNA Mónica S. Arias-Kuhn |
| title_fullStr | Antifibrotic therapy in the liver by adenovirus mediated expression of a TGF-beta1 antisense mRNA Mónica S. Arias-Kuhn |
| title_full_unstemmed | Antifibrotic therapy in the liver by adenovirus mediated expression of a TGF-beta1 antisense mRNA Mónica S. Arias-Kuhn |
| title_short | Antifibrotic therapy in the liver by adenovirus mediated expression of a TGF-beta1 antisense mRNA |
| title_sort | antifibrotic therapy in the liver by adenovirus mediated expression of a tgf beta1 antisense mrna |
| topic | Leberfibrose (DE-588)4167063-2 gnd Transforming Growth Factor beta 1 (DE-588)4404907-9 gnd Adenoviren (DE-588)4141412-3 gnd Antisense-RNS (DE-588)4324981-4 gnd Gentherapie (DE-588)4296957-8 gnd Heterologe Genexpression (DE-588)4342015-1 gnd |
| topic_facet | Leberfibrose Transforming Growth Factor beta 1 Adenoviren Antisense-RNS Gentherapie Heterologe Genexpression Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=013111065&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT ariaskuhnmonicas antifibrotictherapyintheliverbyadenovirusmediatedexpressionofatgfbeta1antisensemrna |