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Expression kinetics from a synthetic model system for ERK signal duration
HEK293 with a stably transfected ∆RAF1:ER fusion protein were treated with ER antagonist 4‐hydroxytamoxifen (4OHT, ON scenario in B). To generate pulses, ERK signalling was turned off using the MEK inhibitor U0126 (ON/OFF scenario in B). To distinguish between primary and secondary response genes, translation was blocked with cycloheximide (CYHX) in parallel to 4OHT stimulation (ON/CYHX scenario in B). In addition, we used actinomycin D (ActD) to determine mRNA half‐lives via transcriptional shutdown and 4‐thiouridine (4SU) to determine mRNA half‐lives via metabolic labelling.

Log2 gene expression fold changes of significantly induced genes (FDR = 1%) across different treatment scenarios. Gene induction of immediate, delayed and late responding genes is sustained upon constant activation (ON scenario) and transient upon two‐hour pulse activation (ON/OFF scenario). Genes still significantly induced upon parallel CYHX treatment were considered primary response genes. Genes were ranked by their model‐derived response time.

Expression kinetics from a synthetic model system for ERK signal duration HEK293 with a stably transfected ∆RAF1:ER fusion protein were treated with ER antagonist 4‐hydroxytamoxifen (4OHT, ON scenario in B). To generate pulses, ERK signalling was turned off using the MEK inhibitor U0126 (ON/OFF scenario in B). To distinguish between primary and secondary response genes, translation was blocked with cycloheximide (CYHX) in parallel to 4OHT stimulation (ON/CYHX scenario in B). In addition, we used actinomycin D (ActD) to determine mRNA half‐lives via transcriptional shutdown and 4‐thiouridine (4SU) to determine mRNA half‐lives via metabolic labelling. Log2 gene expression fold changes of significantly induced genes (FDR = 1%) across different treatment scenarios. Gene induction of immediate, delayed and late responding genes is sustained upon constant activation (ON scenario) and transient upon two‐hour pulse activation (ON/OFF scenario). Genes still significantly induced upon parallel CYHX treatment were considered primary response genes. Genes were ranked by their model‐derived response time.

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Article
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The RAF‐MEK‐ERK signalling pathway controls fundamental, often opposing cellular processes such as proliferation and apoptosis. Signal duration has been identified to play a decisive role in these cell fate decisions. However, it remains unclear how the different early and late responding gene expression modules can discriminate short and long sign...

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Preprint
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Thesis
The genetic information of all life is encoded within DNA molecules that are translated into functional entities, so-called proteins. They are responsible for operating and controlling a vast array of molecular mechanisms in any biological system and ubiquitous in (patho)physiology as a result. Besides, proteins are the primary target of drugs and can have a central role as biomarkers for diagnostic, prognostic, or predictive purposes. Here, many regulatory mechanisms and spatiotemporal influences prevent an accurate prediction of a proteins’ abundance and its associated functionality based on the genome information alone. Nowadays, it has become possible to measure and quantify thousands of proteins simultaneously, however, involving comprehensive sample preparation procedures. Currently, no universally standardized method enables a routine application of proteome profiling in a clinical environment. In this thesis, an automated workflow for the efficient processing of the most common and quantity-limited specimens is described. In order to demonstrate the usefulness of the end-to- end pipeline, which was termed autoSP3, it was applied to the proteome profiling of histologically defined and WHO recognized growth patterns of pulmonary adenocarcinoma (ADC) that currently have a limited clinical implication. Secondly, we investigated the proteome composition of a molecularly well-defined cohort of Ependymoma (EPN) pediatric brain tumors. Despite the availability of substantial NGS data and their ability to differentiate nine distinct subgroups, the majority of tumors remained without a functional insight. Here, the proteome profiling could provide a missing link and emphasize several subgroup-specific protein targets. In summary, this thesis describes the optimization of SP3 and its automation into a robust and cost-efficient pipeline for quantity-limited sample preparation and biological insight into the proteome composition of ADC growth patterns and EPN tumor subgroups.
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