RNA modifications

Selection and characterization of synthetic riboswitches by RNA-Capture-SELEX and fluorescence-based Screening in S. cerevisiaeDuring my Master's thesis I aimed to provide a better understanding of how we can use RNA aptamers to control gene expression. I focused on employing them as a synthetic riboswitch to modulate translation initiation in eukaryotes.Following methods and approaches were mainly used:- RNA-Capture-SELEX to generate RNA aptamers that bind to a target with high affinity and specificity (using magnetic beads, radioactive labeling of RNA, RT-PCR, in vitro transcription)- fluorescence-based cellular screening in S. cerevisiae using flow cytometry- characterization of a riboswitch by randomization and rational design- yeast growth curve

Interaction of S. aureus with human B cellsThe main focus was to investigate the interaction of Staphylococcus aureus, a major human pathogen, and human B cells to gain a better understanding of how this pathogen circumvents the human immune response. Acquired techniques and methods:- working with primary human leukocytes: cultivation, isolation of leukocyte subsets by magnetic separation and stimulation with microbial compounds or bacteria - determination and analysis of cell proliferation, viability and surface marker expression by flow cytometry - investigation of cytokine-production and characterization of antibody-response by ELISA and ELISpot - bacterial cell culture and working with S2 genetically modified S. aureus strains - characterization of the used strains by SDS-PAGE and Western Blot, and growth curve- scientific writing (written protocol in a thesis-like manner) and presentation

Chromosome analyses to investigate one-ended double-strand breaks by oxidative stressI aimed to investigate the induction of DNA double-strand breaks by oxidative stress (H2O2). Acquired techniques and methods:- established chromosome analysis to detect structural chromosome aberrations based on immunofluorescence microscopy (internship)- mammalian cell culture and working with chemical inhibitors for distinct repair pathways - preparation of chromosome spreads: premature chromosome condensation and fixation of cells - chromosome staining and identifying the cell cycle stage by DAPI staining and labeling S phase cells with EdU - analysis of chromosome fusions to confirm a DNA double-strand generation by oxidative stress in a replication-dependent manner

Establishment of chromosome analyses to investigate one-ended double-strand breaks by oxidative stress