Contakt

Ondra Martin M.Sc.

Phone: +420605356858
Groups: Doctoral Student, Staff, Master Student, LEM
Curriculum Vitae: martin_ondra_cv.pdf

Master training

Cellular model of ribosomal stress.

The theoretical part of the diploma thesis summarizes information about nucleolus, ribosomal biogenesis, Mdm2 and p53 dependent and independent ribosomal stress. In the experimental part, vectors expressing nucleolin (NCL) and fibrillarin (FBL) fused with green fluorescent protein at its N-terminus were created. Transfection of human U2OS cells with plasmids expressing GFP-NCL and subsequent selection resulted in a stable fluorescent- labeled nucleoline (U2OS-pEGFP-NCL) cell line. By transfecting the U2OS cell line with plasmids expressing GFP-FBL and subsequent selection, fluorescent-labeled fibrillarin cell line (U2OS-pEGFP-FBL) was created. For the validation of FBL and NCL as possible markers of ribosomal stress, selected cytostatics known to disturb ribosomal biogenesis have been applied to U2OS-pEGFP-FBL and U2OS-pEGFP-NCL cell lines. Double RNA fluorescent in situ hybridization on specific rRNA locus was performed after application of cytostatic. Cisplatin, oxaliplatin and actinomycin-D caused the breakdown of the nucleus integrity and the release of FBL and NCL from the nucleolus to the nucleus, whereas 5-fluorouracil did not affect the integrity of the nucleus and the localization of FBL and NCL. From the results of the double rRNA FISH was evident that cisplatin, oxaliplatin and actinomycin-D blocked transcription of 47S rRNA, but did not block processing of pre-rRNA. Application of 5-fluorouracil caused stopping pre-rRNA processing, but transcription of 47S rRNA was not affected. The U2OS-pEGFP-NCL cell line was transfected with siRNA-RPL11 to silence expression of RPL11. The RPL11 depletion did not cause the loss of integrity of the nucleus and did not trigger p53 dependent ribosomal stress. Cells transfected with siRNA-RPL11 have been identified to accumulate pre-rRNA precursors. The combination of localization of these fluorescently labeled proteins with the double rRNA FISH method for pre-rRNA specific regions has proven to be a suitable system for identifying substances that may cause ribosomal stress.

Status: Graduated from 2015 to 2017.
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