All cells are delimited by membranes that protect the cell from the surrounding environment. In eukaryotic cells the same principle applies at subcellular level where membranes delimit functional cell organelles. The membrane structure, properties and function are defined in part by their lipid composition. Lipidomics is the large‐scale study of pathways and networks of cellular lipids in biological systems. It involves the identification and quantitation of cellular lipid molecular species and their interactions with other lipids, proteins, and other metabolites. Lipidomics has been greatly facilitated by recent advances in ionization technology and mass spectrometric capabilities which have simplified the sample processing prior to analysis, giving rise to shotgun lipidomics. Shotgun lipidomics is fast, highly sensitive, and can identify hundreds of lipids missed by other methods. However, Glycosphingolipids are an important lipid family that was out of the scope of shotgun lipidomics due to the lack of suitable analytical tools. The aim of my thesis was two‐fold. The first aim was the establishment of Glycosphingolipid identification and quantification by shotgun approach. This allowed us to perform lipidomic studies with unprecedented comprehensiveness (~300 lipid species from 15 different lipid classes) from low sample amounts and with minimal sample processing. The second was the application of this technology in studies of the role of lipids in several processes like vesicular carrier formation, cell polarization, protein delivery to the plasma membrane and viral budding. This work resulted in several findings. We found that there is sorting of sphingolipids and sterols into plasma membrane targeted vesicular carriers in budding yeast. When kidney cells change from a mesenchymal to an epithelial morphology there is a profound remodeling of their lipidome, with the synthesis of longer, more saturated, more hydroxylated, and more glycosylated sphingolipids. When these sphingolipids and sterols are depleted in epithelial cells, the apical transport in epithelial cells is impaired. These data strongly support the idea that lipid rafts play an important role in sorting and delivery of lipid and protein cargo to the plasma membrane. Finally, we found that the envelopes of vesicular stomatitis virus and Semliki forest virus assert little specificity in the incorporation of lipids from the plasma membrane. This weak specificity seems to be related to a combination of virus lipid bilayer asymmetry and curvature.

Covalent modification of histones is an important factor in the regulation of the chromatin structure implicated in DNA replication, repair, recombination, and transcription, as well as in RNA processing. In recent years, histone methylation has emerged as one of the key modifications regulating chromatin function. However, the mechanisms involved are complex and not well understood. Histone 3 lysine 4 (H3K4) methylation is deposited by a family of histone H3K4 methyltransferases (HMTs) that share a conserved SET domain. In mammalian cells, six family members have been characterized: Setd1a and Setd1b (the mammalian orthologs of yeast Set1) and four Mixed lineage leukemia (Mll) family HMTs, which share limited similarity with yeast Set1 beyond the SET domain. Several studies demonstrated that the H3K4 methyltransferases exist as multiprotein complexes. To functionally dissect H3K4 methyltransferase complexes, GFP tagging of the core subunit Ash2l and the complex-specific subunits Cxxc1 and Wdr82 (Setd1a/b complexes) Men1 (Mll1/2 complexes), and Ptip (Mll3/Mll4 complexes), was used. The fusion proteins were successfully expressed in mouse embryonic stem cells (ES cells), analyzed by confocal microscopy, Mass Spectrometry (MS) and ChIP-seq. Ptip was the only subunit able to bind mitotic chromatin. Additionally, both Ptip and Wdr82 were found to associate with cell cycle regulators, suggesting a possible role of the two proteins or respective complexes in cell cycle regulation. Mass Spectrometry revealed that Wdr82 and Ptip interact with members of he PAF complex, and ChIP-seq showed that Wdr82, Cxxc1 and Ptip positively modulate pluripotency genes. Thus, Setd1a/b and Mll3/4 complexes might act together in the regulation of embryonic stem cells identity. Protein pull downs identified at least one new Setd1a/b interactor, Bod1l that is orthologous to the yeast protein Sgh1, a component of the Set1C complex. Furthermore, our MS and ChIP-seq data suggested that only Mll2 complex binds to bivalent promoters, wheras Mll2 and Setd1a complexes might function together in a set of promoters.