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List of contents
Abstract ... 3 List of abbreviations ... 5 1. Introduction ... 7 1.1. Cancer ... 7 1.2. Biomarkers ... 9 1.3. Breast cancer ... 101.3.1. Breast cancer characteristics ... 11
1.3.2. Breast cancer biomarkers ... 14
1.4. Prostate cancer... 16
1.4.1. Prostate cancer characteristics ... 17
1.4.2. Biomarkers in prostate cancer ... 19
1.5. Aptamers ... 20
1.5.1. General information on aptamers ... 20
1.5.2. SELEX... 21
1.5.3. Aptamers as drugs ... 24
1.6. Cancer models ... 29
1.6.1. Orthotopic cancer models ... 29
1.6.2. Breast cancer cell lines – MCF7 ... 30
1.6.3. Prostate cancer cell line – PC3 ... 31
1.7. Proteomics as a branch of life sciences ... 32
2. Aims of the study ... 34
3. Materials and methods ... 35
3.1. Polyacrylamide gel separation - SDS-PAGE ... 37
3.2. Sample preparation protocol ... 38
3.3. nanoLC-MS/MS analysis ... 39
3.4. Bioinformatic tools ... 41
3.5. Proteome analysis of orthotopic breast cancer model ... 41
3.5.1. Qualitative analysis (orthotopic breast cancer model)... 42
3.5.2. Quantitative analysis (orthotopic breast cancer model) ... 43
3.5.3. Western blotting (orthotopic breast cancer model) ... 46
3.6. Comprehensive analysis of proteins targeted by aptamers possessing antitumor activity in cancer cell model PC3-luc. ... 47
3.6.1. Cell cultures ... 47
3.6.2. Aptamer binding assay ... 48
3.6.3. Optimization of aptamer's concentration ... 49
3.6.4. Homogenization method ... 50
3.6.5. Optimization of the aptamer-binding target enrichment process ... 52
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4. Results ... 58
4.1. Proteomic analysis of the orthotopic breast cancer MCF7 model ... 58
4.1.1. Qualitative analysis (orthotopic breast cancer model)... 58
4.1.2. The results of quantitative analysis (orthotopic breast cancer model) ... 61
4.1.3. Western blotting (orthotopic breast cancer model) ... 67
4.2. Results of the analysis of the proteins targeted by aptamers ... 68
4.2.1. Optimization of the aptamers' concentration ... 68
4.2.2. Optimization of the homogenization method ... 70
4.2.3. Enrichment process of the aptamer binding targets ... 74
4.2.4. Aptamers labeled with fluorescent dye Atto-488 ... 85
5. Discussion ... 88
6. Final conclusions ... 96
Streszczenie ... 96
Bibliography ... 99
