Schistosomiasis is an acute and chronic disease caused by blood dwelling parasitic trematodes of the genus Schistosoma, and it is classified as the second most socioeconomically devastating... Show moreSchistosomiasis is an acute and chronic disease caused by blood dwelling parasitic trematodes of the genus Schistosoma, and it is classified as the second most socioeconomically devastating parasitic disease, second only to malaria. Currently the wormload is determined by the Kato-Katz method, which is not always reliable. In order to prepare diagnostic tools able to capture specific anti-carbohydrate antibodies or develop conjugate vaccines targeting these carbohydrate structures, sufficient amounts of well-defined fragments are needed. This thesis describes the synthesis of several glycans of these glycans present on the S. mansoni parasite, focusing mainly on the Circulating Anodic Antigen (CAA) and glycans bearing the unique α-(1-2)-L-Fucose-α-(1-2)-L-Fucose motifs. These glycans have been attached to gold nanoparticles and these particles were screened against several monoclonal antibodies and sera of individuals suffering from schistosomiasis. Show less
This thesis describes the use of a combined approach of computational and experimental techniques to gain novel insights to understand the glycosylation reaction and its reactive intermediates. The... Show moreThis thesis describes the use of a combined approach of computational and experimental techniques to gain novel insights to understand the glycosylation reaction and its reactive intermediates. The research in this thesis shows that glycosyl cations can act as reactive intermediates in glycosylation reactions for the introduction of glycosidic linkages. Furthermore, computational and experimental evidence has been provided showing that dioxolenium ions, formed by participation of remote acyl groups, are relevant reactive intermediates and can effectively steer the stereochemical course of glycosylation reactions. Ultimately, the techniques developed and insights gained in these studies were used in the synthesis of a complex mycobacterial glycolipid. The fundamental knowledge presented in this thesis can be further exploited in future synthetic endeavors, delivering more and more complex glycans to fuel glycobiological and glycomedical research. Show less
In this thesis an activity-based probe was discovered that could visualize the activity of PLAATs. With an optimized gel-based ABPP assay in hand, screening of a compound library led to the... Show moreIn this thesis an activity-based probe was discovered that could visualize the activity of PLAATs. With an optimized gel-based ABPP assay in hand, screening of a compound library led to the discovery of alpha-ketoamides as a hit for PLAAT3. Through extensive structural modifications of the hit, LEI110 was identified as the most potent inhibitor (Ki = 20nM) for PLAAT3. LEI110 reduced cellular arachidonic acid levels in PLAAT3 overexpressing U2OS cells and oleic acid-induced steatosis in human HepG2 cells. Gel-based ABPP and chemical proteomics showed that LEI110 is a selective pan-inhibitor of the Hrasls-family of thiol hydrolases (i.e. PLAAT2, PLAAT3 and PLAAT5). LEI110 could be an excellent starting point for the structure-based drug development of novel molecular therapies for obesity and/or common cold. In addition, a competitive, gel-based ABPP method for PLA2G4E using TAMRA-FP was successfully developed and applied to screen a focused library of lipase inhibitors. This resulted in the discovery of two clusters of inhibitors with different scaffolds. Optimization of the potency and selectivity of the inhibitors is required to the study of the biological role of PLA2G4E in an acute and dynamic setting with these novel tools. Together these novel chemical tools and methods will allow for a better understanding of the biosynthesis of the NAPEs and to study their biological role. Show less
Glycoconjugate vaccines are composed of microbial poly- or oligosaccharides covalently linked to a carrier protein. In the absence of structural information, long poly- or oligosaccharides... Show moreGlycoconjugate vaccines are composed of microbial poly- or oligosaccharides covalently linked to a carrier protein. In the absence of structural information, long poly- or oligosaccharides extracted from bacteria are employed to cover all relevant glyco epitopes and elicit an effective immune response. The comprehension of the structural basis for the immune recognition of carbohydrate antigens and the elucidation of minimal epitopes is key to understand their mechanism of action and support the rational design of modern glycoconjugate vaccines.Group B streptococcus (GBS) is a Gram-positive bacterium, cause of infections in pregnant women and newborns, whose capsular polysaccharide (CPS) is a major virulence factor.A small library of GBS glycans from serotypes Ia, Ib and III CPSs was synthesized. These oligosaccharides were used to model the conformational preferences of the corresponding CPS and to characterize the interactions to protective anti PS monoclonal antibodies. Conjugation to carrier proteins of the most promising fragments and evaluation in vivo of the obtained glycoconjugates showed that a hexasaccharide corresponding to the minimal structural epitope of GBS III was able to elicit anti PSIII functional IgGs. Overall, the findings described in this Thesis open the path to the design of GBS vaccines based on synthetic oligosaccharides. Show less
Lysosomal storage disorders (LSDs) are a group of orphan diseases characterized by lysosomal dysfunction or impaired lysosomal catabolism and affect collectively about 1 in 5000 live births. A... Show moreLysosomal storage disorders (LSDs) are a group of orphan diseases characterized by lysosomal dysfunction or impaired lysosomal catabolism and affect collectively about 1 in 5000 live births. A common LSD is Gaucher disease, which is characterized by a defect in glucocerebrosidase (GCase) degrading glucosylceramide (GlcCer) in lysosomes. In this thesis, the zebrafish is evaluated as vertebrate animal model for the investigation of lysosomal storage disorders, in particular Gaucher disease. Zebrafish are an appealing model organism to study genetic disorders with a high evolutionary conservation of genes and proteins compared to humans, easy maintenance and simple genetic and pharmacological manipulation. Zebrafish larvae are of particular use as zebrafish can generate hundreds of off-spring which have a rapid embryonal development, are transparent and fit in a 96-wells plate. In this thesis several biochemical and genetic techniques have been developed in order to 1) compare the catalytic features of zebrafish GCase with human GCase, 2) investigate the consequences of its defect in zebrafish larvae and adults as well as a concomitant defect in non-lysosomal GBA2 and 3) study the potential toxicity of excessive glucosylsphingosine during GCase deficiency as consequence of a defect in lysosomal acid ceramidase. GCase-deficient zebrafish showed similar symptoms and affected molecular mechanisms as patients and mouse models. Therefore the zebrafish offers exciting new possibilities to study molecular mechanisms underlying pathological processes during lysosomal hydrolase deficiencies. Show less
Cytotoxic T-cells (CTLs) are involved in the clearance of viruses and killing of tumor cells. The capacity of these killer cells to clear viruses and kill tumor cells can be harbored by the... Show moreCytotoxic T-cells (CTLs) are involved in the clearance of viruses and killing of tumor cells. The capacity of these killer cells to clear viruses and kill tumor cells can be harbored by the creation of vaccines. Improving understanding of T-cell activation and the possibility to understand, influence or even have control over this process might lead to better anti-cancer vaccines. Chemical tools can be a good addition to fill in the gaps of knowledge about T-cell activation. Show less
This thesis describes the design and synthesis of fragments of various cell wall carbohydrates of the Streptococcus species, including the branched Group B-specific antigen (GBC) of Group B... Show moreThis thesis describes the design and synthesis of fragments of various cell wall carbohydrates of the Streptococcus species, including the branched Group B-specific antigen (GBC) of Group B Streptococcus, glycerol phosphate (GroP) modified group A carbohydrate (GAC), and the O-acetylated type 1 capsular polysaccharide of Streptococcus pneumonia (Sp1). All the synthesized fragments were equipped with a spacer at the reducing end for further conjugation with proteins or active small molecules to explore the mechanisms of carbohydrate-based vaccines in immune responses and to develop novel vaccines. To investigate the structure-activity relationship, several fragments of each polysaccharide were assembled varying in length. Show less
Bacteria often experience external challenges, such as changes in environmental conditions or attacks by bacteriophages. To cope with these challenges, bacteria need to be able to adapt quickly to... Show moreBacteria often experience external challenges, such as changes in environmental conditions or attacks by bacteriophages. To cope with these challenges, bacteria need to be able to adapt quickly to the challenges. Key to the survival of bacteria is to be able to adapt to environmental stresses, to acquire new genetic characteristics through horizontal gene transfer to remain competitive and to silence these foreign genes as long as they do not provide any benefits. The Histone-like nucleoid structuring (H-NS) protein is a key regulator of the dynamic bacterial genome. The protein is conserved among enterobacteria and plays a determinant role in the architecture of their nucleoid acting as a global genome organizer and gene regulator. We used a homolog of H-NS, MvaT from P. aeruginosa, in which we scrutinized its structural/function relationship in response to changes in the surrounding ionic strength. We have combined integrative structural biology methods and biochemical assays to decipher the structural changes in MvaT that drive the switch between its DNA stiffening and bridging activities under different salt conditions. These structural changes appear to be conserved within the H-NS family of proteins: analysis of primary sequences of H-NS family members revealed conserved positions of charged residues. Show less
Vitamin A or retinol is essential in embryonic development, the visual cycle and the immune system. Vitamin A is converted to retinoic acid (RA) by aldehyde dehydrogenases (ALDHs). The family of... Show moreVitamin A or retinol is essential in embryonic development, the visual cycle and the immune system. Vitamin A is converted to retinoic acid (RA) by aldehyde dehydrogenases (ALDHs). The family of ALDHs consists of 19 members, three of which (ALDH1A1, ALDH1A2 andALDH1A3) have retinal as their preferred substrate. Due to a lack of selective and potent inhibitors for these enzymes, it is difficult to study their individual contribution to Vitamin A metabolism in biological systems.Therefore an activity-based probe based on the chemical structure of retinal has been synthesized to enable activity-based protein profiling (ABPP) of ALDHs. The probe covalently binds to the catalytic cysteine of ALDH enzymes which can then be visualized on gel or analyzed by proteomics using ligation chemistry.After biological evaluation of the probe this chemical tool has been used to study the influence of individual ALDH enzymes on the mucosal immune system and to determine the ALDH profile of several breast cancer cell lines. Thus showcasing its use to study Vitamin A metabolism in a wide variety of biological settings including but not limited to: immunology, cancer and (cancer) stem cells. Show less
In this dissertation iron-based homogeneous catalysts were synthesized, characterized and investigated for water oxidation activity. The catalysts were studied under electrochemical conditions in... Show moreIn this dissertation iron-based homogeneous catalysts were synthesized, characterized and investigated for water oxidation activity. The catalysts were studied under electrochemical conditions in order to compare the electrochemical approach to the catalysis based on the use of sacrificial oxidants. The mechanisms under which these catalysts operate have been explored with particular attention to the O−O bond formation step. The combination of electrochemical techniques and in situ characterization techniques allowed for the identification of the active intermediates responsible for catalysis. The influence of the presence of water oxidation catalysts in solution on the evolution of carbon dioxide from the surface of a pyrolytic graphite working electrode was also investigated. Overall, the results of this work demonstrate that the combination of in operando and in situ (spectro)electrochemical techniques allows for a complete investigation of the catalytic mechanism of the water oxidation reaction. Show less
Nature uses a special class of histone proteins, histone variants, to modulate the properties of chromatin at defined genomic locations. H2A.B is one of the most divergent H2A variants and is... Show moreNature uses a special class of histone proteins, histone variants, to modulate the properties of chromatin at defined genomic locations. H2A.B is one of the most divergent H2A variants and is involved in important cellular functions, such as transcription and mRNA splicing. Incorporation of H2A.B in nucleosomes causes unwrapping of ~15 bp entry/ exit nucleosomal DNA from the histone octamer core. Yet, the molecular basis of such peculiar nucleosome conformation is unclear. The work described in this thesis aimed to determine the impact of H2A.B incorporation on the structural and dynamical properties of the nucleosome, primarily using nuclear magnetic resonance (NMR) spectroscopy. Show less
Large parts of the research described in this Thesis aims at the development of oligopeptide-masked toxins and their in situ immunoproteasome-mediated activation. Chapter 2 focuses on the... Show moreLarge parts of the research described in this Thesis aims at the development of oligopeptide-masked toxins and their in situ immunoproteasome-mediated activation. Chapter 2 focuses on the development of selective fluorogenic substrates for each of the iCP and cCP proteasome subunits. Chapter 3 describes the utilization of proteasome cleavable linkers in order to release a toxic entity in immunoproteasome expressing cells. By selecting oligopeptide sequences of previously described immunoproteasome-selective fluorogenic substrates and inhibitors, and linking these to doxorubicin via a cleavable linker, new constructs were obtained. Chapter 4 describes an immunoproteasome inhibitor-doxorubicin conjugate that targets multiple myeloma derived cells and releases doxorubicin upon low-dose photon irradiation. Chapter 5 describes how a trans-cyclooctene (TCO) is utilized for the release of a toxic payload via a click to release reaction in conjunction with selective proteasome targeting. TCOs were used in order to facilitate toxin activation via an inverse electron demanding Diels-Alder reaction with tetrazine as a dienophile. Chapter 6 gives insight in the development of pan-immuno subunit selective proteasome inhibitors based on P3 and P4 scaffold exchanges. Finally, the research described in this thesis is summarized in Chapter 7, and future prospects based the presented results are discussed. Show less
The studies described in this thesis deal with glycosidases, in particular alpha-galactosidases. Activity-based probes are a versatile research tool in many of the investigations on glycosidases... Show moreThe studies described in this thesis deal with glycosidases, in particular alpha-galactosidases. Activity-based probes are a versatile research tool in many of the investigations on glycosidases and are examined regarding diagnostic application. A specific element of the thesis investigations is the focus on plants, either as source of endogenous glycosidases as well as production platform for therapeutic human enzymes. Show less
The aim of this thesis is to study cell-cell interactions and the development of an assay to explore and quantify the exchange of membrane compounds. In the first part the rate of lipid exchange... Show moreThe aim of this thesis is to study cell-cell interactions and the development of an assay to explore and quantify the exchange of membrane compounds. In the first part the rate of lipid exchange was studied, during both natural cell-cell contacts and prolonged enforced contacts. In the second part lipidated coiled-coil peptides were used to study the behaviour and potential fusogenic behaviour of these compounds on cell-wall-deficient bacteria. Show less
The long-held desire - to link structure directly to function and to explain molecular mechanisms based on basic chemical or physical principles - is finally coming closer, satisfying not only our... Show moreThe long-held desire - to link structure directly to function and to explain molecular mechanisms based on basic chemical or physical principles - is finally coming closer, satisfying not only our scientific curiosity but also offering new solutions to the many challenges in the field of health, energy and sustainability. This thesis aims to understand the mechanisms of the largest and most efficient antenna complex in photosynthetic bacteria, the chlorosome, at the molecular level. By linking classical methods for the description of molecular motions to quantum models for electronic movements, and comparison with experimental data, I was able to unambiguously determine the molecular structure of chlorosomes. Moreover, this hybrid approach provided new and unique insight into how these antennae encode robust and efficient functions in structure and structural dynamics. This elucidates the origin of their photosynthetic efficiency, which can be useful for the design of new generations of solar cells. Furthermore, we gain unique insight into the versatile ways in which nature encodes its functions: not only in DNA, but also in the way molecules arrange themselves in a larger aggregate, and the dynamics that results from it. Show less
This thesis investigates the relations between metabolism and anatomy through the use of Magnetic Resonance Imaging (MRI). Two model systems are studied: Botryococcus braunii, a green oleaginous... Show moreThis thesis investigates the relations between metabolism and anatomy through the use of Magnetic Resonance Imaging (MRI). Two model systems are studied: Botryococcus braunii, a green oleaginous algae and Medicago truncatula, a small leguminous plant in symbiosis with Sinorhizobium meliloti bacteria. In order to map the variation of metabolic profiles across tissues, MRI is used extensively, including Chemical Shift Imaging, Magnetic Resonance Spectroscopy and Diffusion-Weigthed Imaging. Home-built microcoils for ultra-high magnetic field strength are developed for studying the model organisms. Show less
Glycosidases are important enzymes in the turnover of polysaccharides and glycoconjugates, and are involved in a range of human pathologies including genetic disorders such as Gaucher and Pompe... Show moreGlycosidases are important enzymes in the turnover of polysaccharides and glycoconjugates, and are involved in a range of human pathologies including genetic disorders such as Gaucher and Pompe disease, but also in various cancers. The discovery of potent and selective glycosidase inhibitors for fundamental glycobiology studies and as leads for drug discovery requires access to suitable (glycomimetic) compound libraries, as well as easily applicable assays and screening formats. The research described in this Thesis was aimed to explore how covalent and irreversible glycosidase inhibitors can be applied in the screening of focused compound libraries on various glycosidases using fluorescence polarization activity-based protein profiling (FluoPol-ABPP). Show less
This thesis describes the development of a variety of mannosylated conjugates. Antigen presenting cells bear mannoside recognizing receptors that actively transport antigen into the cell. This... Show moreThis thesis describes the development of a variety of mannosylated conjugates. Antigen presenting cells bear mannoside recognizing receptors that actively transport antigen into the cell. This thesis exploits this feature for the development of improved vaccines. Show less
Advanced sensing techniques require graphene with high quality and well-controlled surface chemistry. The intrinsic high mobility, low electrical noises and uniform graphitic crystallinity are the... Show moreAdvanced sensing techniques require graphene with high quality and well-controlled surface chemistry. The intrinsic high mobility, low electrical noises and uniform graphitic crystallinity are the prerequisites for high-performance graphene electronics. More importantly, chemical functionalization contributes to unlock the sensing potential of the graphene basal plane. This thesis focuses on manipulating the surface chemistry of a graphene monolayer and explores the impacts on the electrical and electrochemical properties for sensing applications. Heteroatoms like hydrogen, nitrogen and oxygen were systematically introduced into the graphene lattice as defect sites to modify the surface chemistry, and consequently the electronic properties and sensing performance. In summary, a correlation between the in-plane electron transport and the electrochemical activity of hydrogenated graphene was studied by modulating the density of H-sp3 defects. Moreover, cleaning effect on the graphene surface caused by hydrogenation process and the corresponding mechanism were discussed. The electrocatalysis of oxygen reduction reaction on nitrogen doped monolayer graphene was conducted to pinpoint the catalytic active sites. The mechanics of a centimeter-scale graphene floating on water was characterized by biaxial compression. Finally, the chemically modified graphene was tested for field-effect sensing of gas molecules. Show less
