..................................................................................................................................................... 9 INTRODUCTION TO THE SUBJECT ...................................................................................................10 1. FEMALE MAMMARY GLAND...........................................................................................................10 2. BREAST CARCINOGENESIS ..............................................................................................................11 REVIEW OF THE LITERATURE ...........................................................................................................13 1. POSTTRANSCRIPTIONAL GENE REGULATION ......................................................................13 1.1 TURNOVER AND TRANSLATION REGULATORY RNA-BINDING PROTEINS (TTR-RBPS) ................ 13 2. HUMAN ANTIGEN R, HUR..................................................................................................................16 2.1 NUCLEOCYTOPLASMIC SHUTTLING OF HUR................................................................................................. 17 2.2 SIGNALING PATHWAYS RELATED TO HUR REGULATION ......................................................................... 18 2.3 MICRORNAS REGULATING HUR EXPRESSION ............................................................................................. 20 2.4 BINDING OF HUR TO ITS TARGET TRANSCRIPTS .......................................................................................... 21 3. HUR IN CARCINOGENESIS................................................................................................................22 3.1 HUR REGULATES MRNAS ENCODING PROTEINS INVOLVED IN CANCER DEVELOPMENT .............. 22 3.2 HUR EXPRESSION IN CANCER............................................................................................................................. 24 3.3 HUR IN MICE ............................................................................................................................................................ 27 3.4 HUR IN TREATMENT OF CANCER ...................................................................................................................... 27 AIMS OF THE STUDY................................................................................................................................29 MATERIALS AND METHODS ................................................................................................................30 1. PATIENT MATERIAL (I, II, III) ............................................................................................................................... 30 2. IMMUNOHISTOCHEMISTRY (I, II, III).................................................................................................................. 30 3. EVALUATION OF IMMUNOSTAINING (I, II, III) ................................................................................................ 31 4. TISSUE CULTURES (III) ........................................................................................................................................... 31 5. PRODUCTION OF HUR-SILENCING (III) AND -OVEREXPRESSING CONSTRUCTS (UNPUBLISHED DATA) ................................................................................................................................................................................ 32 6. VIRUS PARTICLE PRODUCTION (III).................................................................................................................... 33 7. CELL INFECTION WITH VIRUS PARTICLES (III)................................................................................................ 33 8. CELL TRANSFECTIONS (III) ................................................................................................................................... 34 9. HUR IMMUNOFLUORESCENCE (III) .................................................................................................................... 34 10. PROTEIN EXTRACTIONS AND WESTERN BLOT ANALYSIS (I, III) ............................................................ 34 11. RNA ISOLATION AND REAL-TIME REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION (RT-PCR, III) ................................................................................................................................................................. 35 12. MRNP IMMUNOPRECIPITATION FOR HUR (III) ............................................................................................ 36 13. CELL GROWTH MEASURED BY CELLTITERBLUE (III)................................................................................ 37 14. CELL GROWTH MEASURED BY CELL COUNTING (UNPUBLISHED DATA) .............................................. 37 15. ANCHORAGE-INDEPENDENT GROWTH IN SOFT AGAR (III) ....................................................................... 37 16. PROGRAMMED CELL DEATH (ANOIKIS) ASSAY IN METHYLCELLULOSE (III)...................................... 37