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Abstract

Tumor heterogeneity is a term that refers to differences between tumors of the same type in distinct patients as well as to differences observed between cells within a tumor. The later is known as intratumoral heterogeneity (ITH) and is of high clinical relevance, since it directly affects the robustness of prognostic, diagnostics and prediction of biomarkers. Up to date ITH has been mainly investigated at the genomic level. Sequencing of multiple regions from the same cancer specimen have revealed that within a single tumor several clones of cells with distinct mutational landscapes exist, likely as a consequence of clonal evolution. However, ITH can also be driven by differences in the microenvironment that may rather be reflected in differential gene expression or protein turnover than in genomic changes. Nevertheless, to what extent the ITH is manifested on a proteome-wide scale remains largely unknown, mainly due to technical limitations. To overcome these limitations an efficient protocol that allows for proteomic analysis of limited amounts of formalin-fixed and paraffin-embedded (FFPE) material was developed and employed to characterize the proteomic changes in hepatocellular carcinoma (HCC). First, by comparing neoplastic to the adjacent, non-neoplastic tissues, I defined proteomic features that distinguish tumor from peritumoral tissues. The analysis revealed a decrease in abundances of various mitochondrial proteins including components of the NADH dehydrogenase complex I, possibly indicating the metabolic rearrangement in HCC. Subsequently, by analyzing different regions of HCC, I demonstrated the existence of a proteomic heterogeneity, beyond genetic variations, even in morphologically homogenous specimens, which affects various biological processes. Several clinically relevant proteins were identified as differentially expressed across the analyzed tumors or subject to ITH, thus underlying the importance of ITH studies for biomarker discovery and diagnostic applications. In the second part of my thesis, I focused on the functional characterization of gp210 – a transmembrane component of the nuclear pore complex (NPC). In eukaryotic cells the nuclear envelope constitutes a barrier separating the nucleoplasm and cytoplasm. The transport of macromolecules between these compartments occurs through NPCs which form channels across the inner and outer membrane of the nuclear envelope. Apart from regulating the nucleocytoplasmic transport, NPCs are also involved in the other cellular processes such as chromatin organization, regulation of gene expression or differentiation. The NPC is comprised 8 of multiple copies of around 30 proteins called nucleoporins (~1000 protein in total). While the stoichiometry of scaffold components is constant across cell lines, differences in the composition of peripheral sites have been observed. One example of a nucleoporin with a cell-type specific expression is gp210. It is a transmembrane nucleoporin that associates with the NPC via its short C-terminal domain. The remaining larger part of the protein is localized within the perinuclear space and it is not required for the interaction with the NPC. The luminal function of gp210 so far has been linked to muscle cell differentiation but apart from this, its role remains largely unknown. In order to investigate the luminal function of gp210, I attempted to draft a map of potential interacting proteins. This was achieved by in-situ proximity labeling combined with mass spectrometry-based proteomics using the so-called BioID approach. Data obtained in BioID experiments indicate a functional link between gp210 and endoplasmic reticulum (ER) related biological functions. I have identified multiple factors involved in the regulation of ER stress and several proteins involved in glycophosphatidylinositol anchor attachment.