A structural investigation of coiled coil peptides used as membrane fusogens, mimicing naturally occuring coiled-coil fusion proteins. Synthetic modifications have been made to alter lipid... Show moreA structural investigation of coiled coil peptides used as membrane fusogens, mimicing naturally occuring coiled-coil fusion proteins. Synthetic modifications have been made to alter lipid attachment, secondary structure and to insert photoactive azobenzene moieties for active control over coiled coil structure. Finally, the underlying photocontrol mechanism investigated in coiled coil peptides is extended to beta-structured peptides, and was shown to be universally applicable. Show less
Dit proefschrift beschrijft een collectie aan alternatieve strategieën voor het begrijpen, ontwerpen en toepassen van lipide nanosystemen, waarin de rol van de bio-nano interacties centraal staan.... Show moreDit proefschrift beschrijft een collectie aan alternatieve strategieën voor het begrijpen, ontwerpen en toepassen van lipide nanosystemen, waarin de rol van de bio-nano interacties centraal staan. In het bijzonder wordt gekeken naar de interactie van RNA-lipide nanosystemen, bekend van de toepassing als vaccins en andere medicatie. Het onderzoeksgebied van de nanomedicatie kan gebruik maken van de specifieke voorbeelden die worden beschreven, maar er kan ook inspiratie worden opgedaan om de aanpak van onderzoek doen te verschuiven van een kostbare empirische aanpak naar rationeel gedreven ontwerpstrategie. Dit alles zal helpen in de ontwikkelen van nieuwe RNA therapieen in de toekomst. Show less
This dissertation contains four works during my PhD. Different biomaterials have been designed based on coiled-coil peptides. These biomaterials have a range of applications, inclusing drug... Show moreThis dissertation contains four works during my PhD. Different biomaterials have been designed based on coiled-coil peptides. These biomaterials have a range of applications, inclusing drug delivery, cell sorting to cell-cell fusion. By knowledge-guideddesign and modification, coiled-coil induced membrane fusion systems are expected to achieve drug delivery in vivo. Finally, L-forms obtained by coiled-coil induced fusion of different strains are expected to be an ideal model for studying questions related to the origin of life and novel antibiotics. Show less
In this thesis, bioorthogonal chemistry is combined with correlative light-electron microscopy to selectively label and study pathogenic intracellular bacteria within the host immune cell. This... Show moreIn this thesis, bioorthogonal chemistry is combined with correlative light-electron microscopy to selectively label and study pathogenic intracellular bacteria within the host immune cell. This technique combines the ultrastructural information of transmission electron microscopy with the functional information of fluorescence light microscopy in order to investigate the host-pathogen interactions that contribute to the diseases caused by pathogenic intracellular bacteria such as Salmonella Typhimurium and Mycobacterium tuberculosis. The technique is further expanded with super-resolution microscopy by combining stochastic optical reconstruction microscopy with transmission electron microscopy. Additionally, the bioorthogonal labeling method for the study of intracellular bacteria is validated through a bead-based stability assay, demonstrating the compatibility of alkyne and azide groups to label bacterial proteins within the degradative lysosomal environment. The technique developed in this thesis may contribute to a better understanding of the mechanisms behind bacterial diseases, as well as the development of novel antibiotics and other therapies to fight these important infectious diseases. Show less
In order to be able to develop effective medicine and treatments to prevent or cure autoimmune diseases or cancer we need to understand the mechanisms how they arise and what drives their course... Show moreIn order to be able to develop effective medicine and treatments to prevent or cure autoimmune diseases or cancer we need to understand the mechanisms how they arise and what drives their course.Unravelling the fundamental molecular mechanisms influencing the onset and course of diseases such as allergies, rheumatoid arthritis or cancer can be tackled using bioorthogonal antigens – chemically functionalized proteins.To tackle this challenge this thesis uses an inter-disciplinary approach. Combining standard immunological experimental methods with special, highly selective bioorthogonal chemical reactions. These reactions are bioorthogonal because, unlike normal organic chemistry, they are compatible with the physiological environment of a cell. This approach allows for following for example the location of the protein over time within a cell or alterations in the immune response due to disease related changes to the protein without disturbing the processes themself.This is a significant advantage because without changing the method used, new information can be retrieved from the same set of experiments, at any point in time during the process and a plethora of new readout options yielding additional data sets.This promises new insights into the causal relation of time, localisation and factors influencing effective anti-cancer vaccine-design and cancer immunotherapy or new biological drugs to prevent or delay onset and progression of autoimmune diseases. Show less
The work described in this Thesis is focused on the assembly of oligosaccharide fragments derived from a fungal polysaccharide, galactosaminogalactan (GAG) and fragments of the exopolysaccharide... Show moreThe work described in this Thesis is focused on the assembly of oligosaccharide fragments derived from a fungal polysaccharide, galactosaminogalactan (GAG) and fragments of the exopolysaccharide Pel, generated by Pseudomonas aeruginosa. To assemble the corresponding oligosaccharides, synthetic methodologies, enabling the stereoselective construction of the required cis-glycosidic linkages has been developed. These synthetic fragments will be valuable tools to elucidate the biosynthesis of GAG and Pel, and characterize the enzymes involved therein. Show less
The work described in this dissertation contributes to a better mechanistic understanding of nanoparticles in vivo. To achieve that goal, we used the zebrafish as a highly predictive pre-screening... Show moreThe work described in this dissertation contributes to a better mechanistic understanding of nanoparticles in vivo. To achieve that goal, we used the zebrafish as a highly predictive pre-screening model of nanoparticles. This approach enables the investigation of the fundamental behavior of nanoparticles, correlation of the physicochemical properties of the formulated nanoparticles with their biodistribution and identification of important nano-bio interactions. Zebrafish established transgenic lines were used to study specific interactions. In addition, genetically modified zebrafish applying CRISPR/Cas9 were generated. These strategies not only show key mechanistic features of nanoparticles in circulation, but also promote the rational design of more efficient nanoparticles systems.After understanding the fundamental behavior of nanoparticles, this thesis describes the identification of a key interaction between stabilins receptors (expressed in liver sinusoidal endothelial cells) and nanoparticles. Next, the scope is changed to design nano-systems that target specific cell types showing liposomes capable of switching the surface charge in situ and in vivo using light as an external trigger and a rationally designed lipid nanoparticle formulation containing mRNA able to preferentially target the hepatic reticuloendothelial system. In addition, a phase-separated liposomes hijacking a lipase mediated transport to selectively target endothelial lipase in vivo was studied. Show less
Synthetic hydrogels that mimic the natural extracellular matrix in the biophysical and biochemical cues it provides to cells are in high demand, however the cell phenotypes as they are observed in... Show moreSynthetic hydrogels that mimic the natural extracellular matrix in the biophysical and biochemical cues it provides to cells are in high demand, however the cell phenotypes as they are observed in vivo in numerous cases have yet to be attained. In this thesis, both chemically-defined supramolecular and covalent hydrogels are prepared that are encoded with bioactive peptides/ proteins, dynamics and porous structural features in order to explore how the modification of polymer materials with various biophysical cues affect cell behavior using various readouts. Through a chemical design, we have shown successful introduction of adhesive peptides that can be identified by cells, through an efficient supramolecular co-assembly approach to prepare biomaterials. We further examine this flexible strategy for coupling of specific peptides to supramolecular monomers to provide matrix interactions for the culture of spheroids based on liver (HepG2) and induced pluripotent stem cells. Also, a dynamic covalent hydrogel system based on thiosulfinate-chemistry to form disulfide bonds was designed and validated to play an important role in supporting the differentiation of induced pluripotent stem cell derived cardiomyocytes. Lastly, gas-forming chemistries that enable control over porosity of the hydrogel materials at the macroscale were examined through the Inverse electron-demand Diels-Alder (IEDDA) reaction with reaction pairs that react with distinct kinetics. Overall, we have engineered dynamic supramolecular and covalent biomaterials using different chemical tools to modulate their biophysical properties that the present to cells, providing guidelines for future biomaterials designs in the field of in vitro 3D cell culture Show less
This dissertation focused on computational methods based on first principles calculations using the Density Functional Theory (DFT) framework. Emphasis was laid on affordable methods that can... Show moreThis dissertation focused on computational methods based on first principles calculations using the Density Functional Theory (DFT) framework. Emphasis was laid on affordable methods that can provide a tradeoff between computational expense and accuracy. Specifically, we investigated solvation effects near the surface of the electrode, used thermodynamic cycles to compute solution-phase energies and also proposed a workflow to detect gas-phase errors on the free energies of target molecules. We used these simple methods to study complex adsorption processes at the PdMLPt(111) electrode surface. DFT and experimental studies (performed by Dr. Chen from our group) were crucial to guide the investigation forward. Show less
The inverse electron demand Diels-Alder (IEDDA) pyridazine elimination emerged in 2013 as a new bioorthogonal reaction and constitutes a prime example of what is now known as dissociative... Show moreThe inverse electron demand Diels-Alder (IEDDA) pyridazine elimination emerged in 2013 as a new bioorthogonal reaction and constitutes a prime example of what is now known as dissociative bioorthogonal chemistry. The research described in this Thesis aims to develop synthetic strategies which enable the IEDDA pyridazine elimination to be applied as a versatile toolbox in chemical biology studies. More specifically, it entails modification of antigenic (MHC-I) peptides and (CD1d) glycolipids with a trans-cyclooctene (TCO) moiety to allow chemical control over the recognition of these biomolecules by immune cells. Synthetic advances which encompass the entire scope of the IEDDA pyridazine elimination are additionally described. Show less
In surface science there is great effort to move from studying simple, flat model surfaces in vacuum to investigating more complex model catalysts in gas environments (in situ). This thesis gives... Show moreIn surface science there is great effort to move from studying simple, flat model surfaces in vacuum to investigating more complex model catalysts in gas environments (in situ). This thesis gives three examples of such studies using microscopy and spectroscopy.Exposure of ZnO(10-10) to moderate pressures of water in an in situ scanning tunneling microscope reveals that the surface roughens. The flat ZnO(10-10) is thus only conditionally suited as a model catalyst for reactions involving water.In the same microscope, surface gold oxide formation is observed on TiO2/Au(111) during CO oxidation at 1 bar pressure. Comparisons to the Au(111) surface suggest that the titania does not supply atomic oxygen to the Au(111) substrate as part of the reaction mechanism of the CO oxidation.Co(0001) is investigated as a model catalyst for Fischer-Tropsch synthesis, the reaction of CO and H2 to form hydrocarbons. In this thesis the oxidation behavior of the cobalt and the adsorption of carbon species during the reaction are investigated using near-ambient pressure X-ray photoelectron spectroscopy.Generally, this thesis exemplifies the significant influence that small concentrations of contaminants in gases and materials can have on the structure and behavior of surfaces in in situ studies. Show less
Metals surfaces form a group of effective catalysts for the reaction of small molecules such as hydrogen (H2). In order to improve the predictive power of theory with respect to the catalytic... Show moreMetals surfaces form a group of effective catalysts for the reaction of small molecules such as hydrogen (H2). In order to improve the predictive power of theory with respect to the catalytic activity of small molecules reacting at metal surfaces, the way in which metal surfaces modify the potential energy of molecules needs to be understood at a fundamental level. Currently density functional theory (DFT) is the only electronic structure method that is accurate enough to achieve chemical accuracy while being cheap enough to make large comparative studies feasible. The work in this thesis is concerned with the creation of highly accurate density functionals that can give a simultaneously good description of the metal surface, the molecule, and the molecule interacting with the metal surface, as well as the description and simulation of supersonic molecular beam experiments and associative desorption experiments needed to validate the obtained results. Show less
Cyclic peptides are investigated as a platform to induce different orientations between various ligands. Through click chemistry, a variety of Toll-like receptor ligands can be attached to the... Show moreCyclic peptides are investigated as a platform to induce different orientations between various ligands. Through click chemistry, a variety of Toll-like receptor ligands can be attached to the cyclic peptides, which are based on gramicidin S. Show less
Nucleoid associated proteins maintain the architecture of the bacterial chromosome and regulate gene expression, hinting that their role as transcription factors may involve local three-dimensional... Show moreNucleoid associated proteins maintain the architecture of the bacterial chromosome and regulate gene expression, hinting that their role as transcription factors may involve local three-dimensional chromosome re-modelling. This dissertation provides the first evidence to support this hypothesis. Using ensemble RT-qPCR and 3C-qPCR, in addition to in vivo and in vitro single molecule fluorescence microscopy techniques, the interplay between the expression of the H-NS-regulated, osmosensitive proVWX operon of Escherichia coli and structural re-modelling of the operon is shown. The formation of a loop anchored between the P2 promoter of proVWX and the terminus of the operon represses its expression. Destabilization of the loop activates transcription. The model presented in this thesis provides clues for how H-NS and H-NS-like proteins may regulate the expression of other operons and genes within their regulons. Show less
Multicopper oxidase, laccase, can efficiently reduce oxygen to water and are mostly used in the enzymatic biofuel cells. However, they suffer from low stability when functionalized over an... Show moreMulticopper oxidase, laccase, can efficiently reduce oxygen to water and are mostly used in the enzymatic biofuel cells. However, they suffer from low stability when functionalized over an electrode. This can be overcome by designing artificial catalysts for the oxygen reduction reaction based on the active site of laccase which requires a detailed understanding of the active site. The current research is aimed at characterizing the active site of small laccase from Streptomyces coelicolor using a combination of paramagnetic NMR spectroscopy, EPR spectroscopy, mutagenesis and quantum mechanical (QM) calculations. The presence of chemical exchange at the active site of laccase attributed to the coordinating histidines is reported. QM calculations showed the importance of the orientation of the coordinating water derived ligand. Mutagenesis study showed the importance of second shell residue in stabilizing intermediates during the oxygen reduction reaction. It is also reported that by changing the pH, a new intermediate could be experimentally observed however, further research is needed to characterize this. The resonance assignment shown in the current research can be used as spies to characterize the active site of laccase. This might in future provide insight into the catalytic mechanism of oxygen reduction reaction by laccase. Show less
Protein–protein complexes are formed via transient states called encounter complexes that greatly influence the formation of the stereospecific complex. Electrostatic charges on the protein... Show moreProtein–protein complexes are formed via transient states called encounter complexes that greatly influence the formation of the stereospecific complex. Electrostatic charges on the protein surfaces play a major role in encounter complexes of electron transfer proteins. The complex formed by cytochrome c (Cc) and cytochrome c peroxidase (CcP) has been studied intensively because it is an excellent model to explore the properties of transient protein-protein interactions. PRE experiments previously described the encounter complex formed by the two proteins in detail. In this thesis we tested to what degree the electrostatic patch on CcP is optimized to enhance the rate of the formation of the stereospecific complex. Using paramagnetic NMR in combination with Monte Carlo simulations and stopped flow spectrophotometry, we investigate several CcP mutants with reengineered charged patches to create new encounter complexes and measure their effects on electron transfer from Cc to CcP. The results indicate that the interactions with Cc are affected more by the total charge of CcP surface than the specific distribution of the charges, bringing into question the concept of electrostatic patches being highly optimized by evolution. Show less
Hydrodesulfurization (HDS) is an ubiquitous part of oil refining that ensures that fuels are cleaned of impurities and environment release of pollutants such as SOx and NOx gasses are minimized. In... Show moreHydrodesulfurization (HDS) is an ubiquitous part of oil refining that ensures that fuels are cleaned of impurities and environment release of pollutants such as SOx and NOx gasses are minimized. In this thesis, atomic level insights into the process of HDS are gained by exploring various methods of generating the catalytically active CoMoS phase as well as the effect of the reaction gasses like hydrogen and methylthiol on the atomic structure of the catalyst at industrially relevant conditions. For this purpose, a variety of techniques such as high-pressure scanning tunneling microscopy, X-ray photoelectron spectroscopy and electron diffraction are used. Furthermore, the studies presented in this thesis make several steps towards bridging the pressure and materials gap between the fundamental catalysis studies and industrial catalytic conditions. The results of this work pave way for more fundamental research with the help of theoretical methods such as DFT calculations which can help with designing more efficient catalysts to meet the future demands of clean fuels. Show less
In this thesis, the researcher developed a nanosystem based on the metallophilic Interaction between cyclometalated complexes. Using this strategy, the researcher achieved efficient photodynamic... Show moreIn this thesis, the researcher developed a nanosystem based on the metallophilic Interaction between cyclometalated complexes. Using this strategy, the researcher achieved efficient photodynamic therapy to several cancers, accompanied by the cell imaging property. Show less
The research described in this thesis combines the latest insights in lysosomal function with lysosome centred cell signalling. Novel imaging and labelling techniques are applied to provide in... Show moreThe research described in this thesis combines the latest insights in lysosomal function with lysosome centred cell signalling. Novel imaging and labelling techniques are applied to provide in depth characterization of lysosome function in health and disease. An integrative approach was used to study the physiological role of the lysosome, characterizing the function of lysosomal hydrolases and signalling on a cellular level as well as within the context of tissue. Show less
Tin electrodeposition applications have rapidly evolved in the past 25 years. Usage of tin coatings has advanced from being mainly used for corrosion protection and decorative purposes, to being... Show moreTin electrodeposition applications have rapidly evolved in the past 25 years. Usage of tin coatings has advanced from being mainly used for corrosion protection and decorative purposes, to being used in modern technology such in electronic devices, photovoltaic cells and Li-ion batteries. The new tin coating applications have also come with challenges that require the production of nanostructured deposits, multilayers coatings and composites. Furthermore, the need to reduce energy and source consumptions, and the implementation of more environment-friendly processes, require detailed and fundamental knowledge of the electrodeposition process.The emphasis throughout this thesis is therefore to obtain detailed mechanistic information of tin electrodeposition process.The experimental and theoretical work presented in this thesis attempts to understand the mechanism of tin electrodeposition, and the effect of electrolyte anions and naphthalene-based additives, during the early and subsequent stages of the process. Show less
