This doctoral thesis is an effort to understand how lipid phase-separation induced by diacylglycerol analogues in lipid-based nanoparticles affects their in vivo behavior, leading to specific... Show moreThis doctoral thesis is an effort to understand how lipid phase-separation induced by diacylglycerol analogues in lipid-based nanoparticles affects their in vivo behavior, leading to specific nanoparticle-protein communications and selective cell targeting. By studying how lipid composition affects morphology and this in turn affects the nano-bio interface, a comprehensive picture and prediction of nanoparticle behavior and cell selectivity is provided. More specifically, liposomes containing diacylglycerol analogues are found to phase separate and to be able to specifically target subsets of endothelial cells in zebrafish embryos. The mechanism behind this selective targeting is the result of a triglyceride lipase mediated mechanism due to phase separation and lipid composition, and is conserved in higher organisms (mice). Moreover, mRNA-based lipid nanoparticles that contain diacylglycerol analogues exhibit the same selectivity which leads to cell-specific mRNA delivery and transfection. Show less
β-Lactamases are enzymes that can break down β-lactam substrates, such as antibiotics, preventing the use of these antibiotics for the treatment of various infectious diseases. However, some... Show moreβ-Lactamases are enzymes that can break down β-lactam substrates, such as antibiotics, preventing the use of these antibiotics for the treatment of various infectious diseases. However, some compounds, β-lactamase inhibitors, can block these enzymes allowing for possible treatments using a combination of antibiotic and inhibitor. BlaC is the β-lactamase of Mycobacterium tuberculosis, the bacteria that cause tuberculosis, and is used as a model for protein evolution. To understand if and how BlaC can develop resistance against certain inhibitors we studied the evolutionary adaptability of this enzyme. We used laboratory evolution and various biochemical techniques to characterize several mutations in BlaC and subsequently tested the effect of combining mutations. One of the findings is that BlaC can easily become less sensitive to the inhibitor sulbactam by partially blocking the entrance to the active site. Interestingly, this was accompanied by increased sensitivity to another inhibitor, avibactam, that could not be compensated for by other mutations.Generally, Escherichia coli bacteria are used to test the effects of BlaC variants in cells, as they are easy and safe to use in the lab. We show that results obtained for E. coli can be extrapolated to conditions that resemble tuberculosis disease in humans: the M. marinum infection model of zebrafish. All these findings are of interest for the future development of combination therapies to treat tuberculosis. Show less
Neuraminidases are enzymes that cleave glycosidic linkages of sialic acid. These enzymes are involved in influenza infections as well as in many cellular processes in mammals and micro-organisms.... Show moreNeuraminidases are enzymes that cleave glycosidic linkages of sialic acid. These enzymes are involved in influenza infections as well as in many cellular processes in mammals and micro-organisms. Development of molecules that irreversibly inhibit these enzymes, as well as molecules that can covalently bind and also label these enzymes, is described in this thesis. These newly developed molecular tools can be used to study neuraminidsases. Better understanding of these enzymes could for example contribute to the timely invervention if new influenza strains emerge. Show less
Producing green energy has become the main goal in our society in the search of reducing or eliminating the carbon emission from fossil fuels. In this project, proteins were used as a tool to... Show moreProducing green energy has become the main goal in our society in the search of reducing or eliminating the carbon emission from fossil fuels. In this project, proteins were used as a tool to develop an artificial system capable of promoting the most demanding reaction in photosynthesis, water oxidation, as a new concept of producing energy from a green source. For this purpose, a screening study of the interaction between selected proteins and a library photocatalytic ruthenium and cobalt complexes was carried out. The results from such study allowed the production of two types of artificial metalloenzymes (ArM) which were proven to be able to perform water oxidation reaction via blue light irradiation. As well, an unusual interaction between proteins which promote the photocatalytic O2 evolution from water. These findings lead to develop ruthenium and cobalt metalloenzymes as promising candidates for artificial photosynthesis in bio-inspired systems Show less
This thesis describes the development and optimization of the first molecular tools to study the enzyme PLA2G4E. After remaining elusive for many years, in 2016, this enzyme was discovered to be... Show moreThis thesis describes the development and optimization of the first molecular tools to study the enzyme PLA2G4E. After remaining elusive for many years, in 2016, this enzyme was discovered to be responsible for the calcium-dependent formation of N-acylphosphatidylethanolamines (NAPEs) in cells. NAPEs are low-abundant lipid species that play roles in membrane stabilization, cell signaling and homeostasis. They are well-known as precursors to the signaling lipids of the N-acylethanolamine (NAE) class, but their own biological functions remain relatively poorly understood. To find inhibitors of PLA2G4E, a focused compound library was screened in a newly developed activity-based protein profiling (ABPP) assay. Hits were identified and optimized by building structure-activity relationships (SARs) through organic synthesis and activity assays. WEN091 was identified as potent inhibitor of PLA2G4E that was able to reduce cellular NAPE levels. Cellular target engagement was confirmed by use of a tailored activity-based probe. Using these molecular tools, the relevance of NAPEs and PLA2G4E in cellular processes and disease may be elucidated. Show less
Several single-stranded RNA viruses make use of Xrn1-resistant RNAs in their 3’ untranslated regions of their genome RNAs in order to increase their pathogenicity. This thesis focuses on two types... Show moreSeveral single-stranded RNA viruses make use of Xrn1-resistant RNAs in their 3’ untranslated regions of their genome RNAs in order to increase their pathogenicity. This thesis focuses on two types of Xrn1-resistant RNAs: those involving the “coremin” motif (xrRNAC) and those found in members of the Flaviviridae family (xrRNAF). While the structure for xrRNAFs has been solved, the xrRNAC structure is yet elusive. Therefore, we employed systematic mutational analysis in order to identify the features that are involved in halting the 5’-3’ exoribonuclease Xrn1 by xrRNAC. This led to the identification of novel variations of xrRNAC in viral families that were not yet known to employ an xrRNA. Regarding xrRNAF, we investigated their distribution and variability throughout the Flaviviridae family, and concluded that a universal xrRNAF structure is responsible for stalling Xrn1. Furthermore, the work in this thesis expands on the known, potential functions of xrRNAs by showing how xrRNAC is able to both inhibit scanning ribosomes and promote frameshifting. Show less
This thesis describes the design, synthesis, and immunological evaluation of varying (neo)antigenic peptide conjugates containing either a TLR2 or a TLR7 agonist. Chapter 2 discusses the synthesis... Show moreThis thesis describes the design, synthesis, and immunological evaluation of varying (neo)antigenic peptide conjugates containing either a TLR2 or a TLR7 agonist. Chapter 2 discusses the synthesis of a library of UPam functionalized (neo)antigenic peptides. Chapter 3 discusses the optimization of UPam and investigates the current paradigms surrounding UPam. Chapter 4 presents a new simplified agonist for TLR2, namely mini-UPam, which is then conjugated to some neoantigenic peptides and evaluated on its immunological properties. Chapter 5 discusses the design and synthesis of TLR2 and TLR7 dual functionalized antigenic peptides. Show less
This thesis has described novel synthetic methods to produce a variety of (glyco)peptides and their application in the study of various immunological processes. The first part of the thesis... Show moreThis thesis has described novel synthetic methods to produce a variety of (glyco)peptides and their application in the study of various immunological processes. The first part of the thesis describes novel insights into the pathogenesis of multiple sclerosis, in the form of new findings in the areas of lectin-driven immunotolerance and a biochemical comparison between human and animal model antigen. The next part describes novel multivalently glycosylated peptides, that can be used to study lectin binding and lectin mediated antigen uptake. The final part of the thesis describes a novel method to produce trans-cyclooctene protected peptides, an exciting new category of molecular tools within chemical biology that have recently been developed. Show less
This thesis describes the synthesis and biological evaluation of TLR2/6, TLR4, TLR7/8 and TLR9 ligands, of which the activity can be conditionally controlled. Trans-cyclooctenes are used to shield... Show moreThis thesis describes the synthesis and biological evaluation of TLR2/6, TLR4, TLR7/8 and TLR9 ligands, of which the activity can be conditionally controlled. Trans-cyclooctenes are used to shield the ligand's moiety critical for TLR activation, which can be removed fast and quantitatively by the addition of tetrazine derivatives. Photocages are used in parallel for the same purpose, which can be removed by exposure to low-energetic UV-light. A new photocage has been developed that can be decorated on two positions, i.e. it is bifunctional. This photocage has been used to synthesize a proteasome inhibitor-doxorubicin conjugate, in which the photocage bisects the two groups, which induces apoptosis in targeted multiple myeloma cells with an acquired resistance for proteasome inhibitors. The photocage was also used to synthesize a probe that enables targeting and isolation of CD8+ T cells in viable human melanoma and non-squamous cell lung cancer tissues. Show less
