Inhalt

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   TABLE OF CONTENTS
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  ABBREVIATIONS
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   Escherichia coli
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   1 SUMMARY
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  2 INTRODUCTION
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  2.1 Cancer
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   2.1.1 Cancer Statistics
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   2.1.2 Cancer Cell Physiology
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   2.1.3 Cancer Cell Genetics
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   2.1.4 Tumor Suppressor Genes
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   2.1.5 Mechanisms of Inactivation of the Tumor Suppressor Gen
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   2.1.6 Cancer Therapeutics
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  2.2 Pdcd4 – A Novel Putative Tumor Suppressor
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   2.2.1 The Cloning of Pdcd4
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   2.2.2 The Sequence Motifs of Pdcd4
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   2.2.3 The Structure of Pdcd4
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   2.2.4 The Subcellular Localization of Pdcd4
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   2.2.5 The Regulation and Expression of Pdcd4
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   2.2.6 The Cellular Functions of Pdcd4
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   2.2.7 The Molecular Mechanisms of the Functions of Pdcd4
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  2.3 Objectives and Rationale of the Study
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   2.3.1 Pdcd4 Models
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   2.3.2 Pdcd4 Downregulation System
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   2.3.3 Pdcd4 Upregulation System
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  3 MATERIALS AND METHODS
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  3.1 Materials
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   3.1.1 Antibodies
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   3.1.2 Cell Culture Products
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   3.1.3 Chemicals and Reagents
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   3.1.4 Devices and Instruments
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   3.1.5 Enzymes
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   3.1.6 Genotype of Escherichia coli K12 strains
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   3.1.7 Kits
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   3.1.8 Plasmids and Constructs
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   3.1.9 Primers
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   3.1.10 Standard Buffers and Solutions
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  3.2 The Molecular Biology Techniques
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   3.2.1 Media and Agar plates
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   3.2.2 Preparation of Competent E.coli cells
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   3.2.3 Transformation of Competent Bacteria and Blue/White Sc
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   3.2.4 Plasmid DNA Isolation
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   3.2.5 Quantification of DNA
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   3.2.6 Modification of DNA by Enzymes
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   3.2.7 Agarose Gel Electrophoresis
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   3.2.8 Extraction of DNA Fragments from Agarose gels
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   3.2.9 Ligation
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   3.2.10 Polymerase Chain Reaction (PCR)
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   3.2.11 T/Acloning of PCR products with TOPO TA System
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   3.2.12 DNA Sequencing
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   3.2.13 Sequencing Gel
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   3.2.14 Isolation of Genomic DNA from the Eucaryotic cells
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   3.2.15 Isolation of PolyA RNA from the Eucaryotic cells
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   3.2.16 Genomic DNAAgarose gel Electrophoresis and Southern
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   3.2.17 RNAAgarose gel Electrophoresis and Northern Blotting
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   3.2.18 Radioactive Labeling of DNA with (32P-dCTP and with 3H-dUTP
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   3.2.19 Hybridization and Washing
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   3.2.20 Cell Cycle Analysis by FACS Method
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  3.3 The Cell Culture Techniques
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   3.3.1 Cell lines and Medium
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   3.3.2 Passage and Cultivation of cells
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   3.3.3 Cell Counting
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   3.3.4 Transfection and Harvesting of Adherent and Suspension
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  3.4 The Protein Biochemical Techniques
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   3.4.1 Reporter-gene Assays
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   3.4.2 2DimensionalSodium Dodecyl SulfatePolyacrylamide Ge
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   3.4.3 Coomassie Brilliant Blue Staining
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   3.4.4 Western Blotting and Immunodetection
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   3.4.5 35SMethionine Labeling
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   3.4.6 Immunoprecipitation
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   4 RESULTS
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  4.1 Downregulation of Pdcd4 – A KnockDown System
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   4.1.1 Targeted Silencing of the Human Pdcd4 gene
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   4.1.2 The effect of Hpdcd4 on CapDependent Translation
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   4.1.3 The effect of Hpdcd4 on IRESDependent Translation
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   4.1.4 The search for Novel Molecular Targets of Hpdcd4 – Tra
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   4.1.5 The effect of Hpdcd4 on Transcription Factor C/EBPβ
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   4.1.6 The search for Novel Molecular Targets of Hpdcd4 – A P
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   4.1.7 The Effect of Hpdcd4 on the Biochemical Modification o
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   4.1.8 Expression of Pdcd4 Targets at the RNA Level
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   4.1.9 The effect of Hpdcd4 on mRNA Stability
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   4.1.10 The effect of Hpdcd4 on Nonsense-Mediated mRNA Decay
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  4.2 Downregulation of Pdcd4 – Knock-Out-System
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   4.2.1 Targeted Disruption of the Chicken Pdcd4 gene
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   4.2.2 The effect of the Disruption of Cpdcd4 on the Growth C
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   4.2.3 The effect of Cpdcd4 on Translation
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   4.2.4 2D-SDS-PAGE Analysis of Cpdcd4 Knock-out Clones
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  4.3 Upregulation of Pdcd4 Overexpression System
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   4.3.1 Overexpression of the Human Pdcd4 gene
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   4.3.2 2D-SDS-PAGE Analysis of Cpdcd4 Overexpression Clones
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  5 DISCUSSION
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   5.1 Analysis of Pdcd4 in the HeLa Cells
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   5.1.1 Downregulation of Human Pdcd4 by siRNA Technology
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   5.1.2 Hpdcd4 inhibits Translation of Secondary Structured RN
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   5.1.3 Hpdcd4 inhibits IRES-Dependent Translation
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   5.1.4 Novel Molecular Targets of Hpdcd4
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   5.1.5 Expression of c-Myc is lower in the Absence of Hpdcd4
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   5.1.6 Phosphorylation of ATF-2 is decreased in the Absence o
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   5.1.7 Expression of C/EBPβ is increased in the absence of Hp
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   5.1.8 Biochemical Modification of Cytokeratin-8 in the absen
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   5.1.9 Expression of Cytokeratin-17 is higher in the absence
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   5.1.10 Expression of AKR1C2 and C3 are higher in the absence
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   5.1.11 Biochemical Modification of GluProRS in the absence o
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   5.1.12 Potential Role of Hpdcd4 in mRNA Stability
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   5.1.13 Potential role of Hpdcd4 in Nonsense-Mediated mRNA De
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  5.2. Analysis of Pdcd4 in the DT40 Cells
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   5.2.1 Downregulation of Chicken Pdcd4 by Homologous Recombin
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   5.2.2 Growth Characteristics are unaffected by Cpdcd4
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   5.2.3 Cpdcd4 has no Effect on Total Protein Synthesis
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   5.2.4 Cpdcd4 has no Effect on CapIndependent Translation
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   5.3 Analysis of Pdcd4 in A549 Cells
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   6 FUTURE PERSPECTIVES
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   7 REFERENCES
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  8 APPENDIX
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   8.1 Clone Charts
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   8.2 Sequence
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   8.3 Zusammenfassung
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   LEBENSLAUF
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   BIO-DATA
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   ACKNOWLEDGEMENT