Nuclear pore complexes (NPCs) are giant macromolecular assemblies that are embedded into the double membrane of the nuclear envelope and control the transport processes between nucleus and cytoplasm. Vertebrate NPCs are composed of approximately 30 different nucleoporins, each present in multiple copies per pore. About one third of the nucleoporins contain characteristic Phe-Gly (FG) enriched domains on their N- or/and Ctermini. These FG domains are intrinsically unstructured protein stretches. In higher eukaryotes, they are typically modified at Ser/Thr residues by GlcNAc moieties. It is thought that nucleoporin FG repeat domains constitute the permeability barrier of NPCs. This barrier restricts passive diffusion of inert molecules larger than 20-40 kDa, but at the same time allows facilitated passage of selected substances, whose mass can reach several megadaltons. Nuclear transport receptors (NTRs) bind FG repeat domains and mediate facilitated translocation of cargo molecules through the permeability barrier of NPCs. The focus of my PhD work has been (i) to reconstitute the permeability barrier of vertebrate NPCs using Xenopus and human FG nucleoporins as a model system, (ii) to analyse the contribution of various FG nucleoporins to the total selectivity of the NPCs barrier, (iii) and to reveal the impact of O-glycosylation on the permeability properties of nuclear pores. Previously, such permeability barrier was reconstituted as hydrogels comprising yeast FG repeat domains. We now show that Xenopus and human FG repeat domains display the same behaviour. The hydrogel permeation assays showed interesting differences in passive exclusion limits of the different FG repeat domains that point to different sieving properties of individual barrier layers at the NPCs. Remarkably, the translocations of import and export complexes were put on hold in FG hydrogels derived from those nucleoporins that are located on the destination site of the pore and thus represent potential termination platforms for the respective complexes. A mutagenesis analysis revealed the importance of hydrophobic amino acids to initiate hydrogel formation as well as of Ala, Thr, Gln, Asn and Pro residues to maintain the selectivity of the barrier. Additional FG nucleoporin regions that are non-globular and lack FG clusters were considered as a part of permeability barrier as they bind NTRs and contribute to the selectivity. The permeability of FG hydrogels increased upon the addition of O-GlcNAc. This points to a so far unsuspected role of this modification in modulating NPCpermeability.