This thesis introduces the concept of "physics-based inverse design", working on the notion that the physical driving forces governing functionality are inherently encoded in independently... Show moreThis thesis introduces the concept of "physics-based inverse design", working on the notion that the physical driving forces governing functionality are inherently encoded in independently parameterized energy functions, which can be resolved through the use of inverse design strategies.The thesis describes the development of EVO-MD, a Python-based implementation of the physics-based inverse design concept. EVO-MD is capable of automatically setting-up, performing, and analyzing molecular dynamics simulations, allowing for the evolutionary optimization of complex and dynamic features in peptides. Examples of such applications include the optimization of lipid composition and curvature sensors, and the development of peptides with antiviral properties. Show less
In the current global context, there is a pressing need to address sustainable energy supplies to safeguard our Planet and its ecosystems. The choices made by human society have a significant... Show moreIn the current global context, there is a pressing need to address sustainable energy supplies to safeguard our Planet and its ecosystems. The choices made by human society have a significant impact on genetic evolution and climate. To ensure a better future for all, it is crucial to exercise foresight, foster collaboration across various sectors, and reach agreements based on fair and ethical principles. Science plays a pivotal role in advancing energy conversion, offering the potential for significant scientific breakthroughs that contribute to the protection and respect of our World. Specifically, the development of solar-to-fuel devices holds promise for achieving this transition to green energy. This Ph.D. dissertation centers on the development and functionalization of 2D membranes and materials, which constitute integral components of these conversion devices. The optimization of functionalized 2D materials necessitates a comprehensive computational design approach. This involves the adoption of a multiscale computational framework for the thorough design of these materials and the precise prediction and understanding of molecular processes, encompassing molecular self-assembly, ion transport, and catalytic surface reactions. Show less
The research presented in this thesis explores the chemotherapeutic potential of metal-based compounds as chemotherapy agents, with an initial focus on the synthesis and DNA interaction studies of... Show moreThe research presented in this thesis explores the chemotherapeutic potential of metal-based compounds as chemotherapy agents, with an initial focus on the synthesis and DNA interaction studies of platinum and palladium compounds utilizing the [Pt(bapbpy)]2+ scaffold. The study identifies intercalation as the primary mechanism of action for these complexes. Furthermore, it provides a detailed structure-activity relationship analysis, highlighting the critical role of the complex's protonation state in influencing its biological activity and efficacy. Subsequently, the study delves into photoactivated chemotherapy (PACT) using ruthenium (II) complexes, where light activation of ruthenium complexes enables targeted drug delivery to tumor cells, thereby reducing adverse effects. This research emphasizes the development of ruthenium-based compounds that can photorelease a DNA repair inhibitor, specifically targeting the RAD51 protein, essential for Homologous Recombination (HR). By disrupting the DNA repair mechanisms in cancer cells, this approach seeks to enhance the cytotoxicity of the therapy and address drug resistance. Show less
The research aims to explore the evolutionary adaptability of enzymes and the impact of temperature on protein evolution pathways, using M. tuberculosis β-lactamase BlaC as the object of study.... Show moreThe research aims to explore the evolutionary adaptability of enzymes and the impact of temperature on protein evolution pathways, using M. tuberculosis β-lactamase BlaC as the object of study. Enzymes inherently embody a delicate balance between activity and stability, and the acquisition of new enzymatic functions is often accompanied by trade-offs, such as decreased stability or reduction of the original activity. Probing evolutionary adaptability of BlaC with laboratory evolution in combination with structural characterization can provide information about the mechanisms of rapid adaptations observed for β-lactamases in the clinic. The role of temperature as a conventional selection pressure in such evolutionary adaptation is unclear. The cooperative nature of enzyme unfolding over a narrow temperature trajectory raises the question whether evolution at temperatures well below the melting point is influenced by temperature. The approach used in this work to answer these questions is by simulating evolution under different selection pressures and characterize the variant enzymes in terms of activity, structure, dynamics and melting temperature. The research makes clear how enzyme kinetics and dynamics vary with different selection pressures and maps the evolutionary path that enzymes may take. The underlying structural mechanisms are established to provide a rationale for the observed effects. Show less
This work describes the use of click-to-release chemistry to get spatiotemporal control over immunocytokine activity. Until now, immunocytokines (cytokines coupled to a tumor-targeting-moiety)... Show moreThis work describes the use of click-to-release chemistry to get spatiotemporal control over immunocytokine activity. Until now, immunocytokines (cytokines coupled to a tumor-targeting-moiety) remained active throughout the body, being able to bind their respective receptors, causing mild to severe side-effects in cancer patients. Attempts have been made to improve the specific action of these immunocytokines, but these solutions remained very cytokine-specific and toxicity was not reduced significantly. Click-to-release chemistry allows us to inactivate a cytokine by blocking its free amines, present in lysines. This prevents the cytokine, IL-1β and TNF-α in particular, from binding its receptor. Removal of the blocking agent using a tetrazine restores the native amine and for IL-1β also its activity. By coupling the blocked cytokine to a targeting moiety allows for transport to the target, the tumor(-environment) upon which the unblocking or decaging can take place. This blocking-unblocking or caging-decaging was assessed using various cell-based assay. This technique can provide new opportunities in the immunocytokine field, as it is not cytokine-specific, and thereby opportunities in cancer therapy development. Show less
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
In this thesis, we consider various (electro)chemical phenomena at surfaces and nanoparticles and their underlying atomistic processes, which we studied using first-principles methods such as... Show moreIn this thesis, we consider various (electro)chemical phenomena at surfaces and nanoparticles and their underlying atomistic processes, which we studied using first-principles methods such as density functional theory. These phenomena range from CO2 reduction to C2 and C3 species, through solvation of adsorbates on various surface features of late transition metals, to the impact of graphene on hydrogen evolution reaction, cathodic corrosion and surface oxidation of Pt. With our thermodynamic and kinetic calculations, we provide explanations for experimental observations by unraveling underlying phenomena, support novel computational methods and techniques, and propose new atomic structures that explain prior findings and provide inroads into future electrocatalytic research. Show less
Global healthcare is on the verge of an antibiotic availability crisis as bacteria have evolved resistance to nearly all known antibacterials. Identifying new antibiotics that operate via novel... Show moreGlobal healthcare is on the verge of an antibiotic availability crisis as bacteria have evolved resistance to nearly all known antibacterials. Identifying new antibiotics that operate via novel modes-of-action is therefore of high priority.This thesis contains two drug discovery projects, originating from a antibacterial screen of a compound library. In both projects chemical hits are first structurally optimized, after which their mode-of-action is determined.The first project entails optimizing a hit with potency against MRSA into a submicromolar active antibiotic. By using a chemical proteomics approach, the targets of this compound were elucidated, along with the targets that are most important in its antibacterial activity.The second project concerns Gram-negative bacteria, where a hit molecule is optimized into the conformationally restricted LEI-800. The target of LEI-800 is found to be DNA gyrase, a common antibiotic target. However, it is that LEI-800 inhibits DNA gyrase differently, and more potently, than the status quo. Show less
This thesis focuses on the application of lipid-based nanomedicine in drug delivery, including small molecular antitumor drugs and biomacromolecules including mRNA, and evaluates their biological... Show moreThis thesis focuses on the application of lipid-based nanomedicine in drug delivery, including small molecular antitumor drugs and biomacromolecules including mRNA, and evaluates their biological performance. We have modified liposomes and LNPs with fusogenic coiled-coil peptides to enhance the drug/mRNA delivery efficiency (Chapter 2-4), and also investigated how the lipid composition of LNPs influences the immune response (Chapter 5). Show less
To design the ideal water oxidation catalyst, understanding of the catalytic mechanism and decomposition pathways is essential. The aim of the research described in this thesis is to provide... Show moreTo design the ideal water oxidation catalyst, understanding of the catalytic mechanism and decomposition pathways is essential. The aim of the research described in this thesis is to provide mechanistic insight on the water oxidation reaction catalyzed by homogeneous electrocatalysts and topostulate design criteria for the ideal water oxidation catalyst. Show less
Supramolecular polymers are class of materials that are formed by non-covalent interactions such as hydrogen bonding, π-π interactions, electrostatic interactions and the hydrophobic effect. The... Show moreSupramolecular polymers are class of materials that are formed by non-covalent interactions such as hydrogen bonding, π-π interactions, electrostatic interactions and the hydrophobic effect. The design and development of supramolecular polymers in aqueous solution gained a particular attention for the wide variety of applications in the biomedical field. In water, the self-assembly of well-defined nanostructures is mainly determined by the combination of hydrophobic effect with hydrogen bonding interactions in the monomer design. When squaramide-based monomer self-assemble, the formation of stable nanostructures in water is determined by the formation of directional hydrogen bonds which are strengthened by the partial aromatic character of the squaramide. In this thesis, the self-assembly properties of a panel of squaramide-based monomers is examined in aqueous solution through modulating the monomer chemical structure, co-assembly and introduction of light responsive chemistries. Show less
Ice, the solid state of water, plays an important role on our planet as well as the entire universe.Despite the fact that an individual water molecule has a very simple structure, its chemical... Show moreIce, the solid state of water, plays an important role on our planet as well as the entire universe.Despite the fact that an individual water molecule has a very simple structure, its chemical bonding in the solid phase can be surprisingly complex.Nowadays, atomistic computational models allow to describing and understanding these properties in a way that has not been possible for a long time.Chemical interactional potentials are at the heart of these atomistic models.In increasing order of complexity, these potentials range from simple pair potentials over polarizable force fields up to density functional theory (DFT).It is an ongoing scientific challenge is to improve and test these potentials.This thesis attempts to provide some answers to the following research questions:(i) How important is the contribution of zero-point energy to thermodynamical properties of ice phases?(ii) How accurately do available interaction potentials allow to model (small) differences between H2O and D2O ices related to nuclear quantum effects?(iii) Do interaction potentials need to be improved when nuclear quantum effects in ice are taken into account? Show less
