Narcissus is an economically important crop with a large number of species, hybrids and/or varieties in cultivation. It is one of the most popular ornamental plants. The Netherlands is among the... Show moreNarcissus is an economically important crop with a large number of species, hybrids and/or varieties in cultivation. It is one of the most popular ornamental plants. The Netherlands is among the top producers of Narcissus in the world. The ornamental value of Narcissus is quite well known but they can also be a valuable resource of natural compounds. Among these compounds, galanthamine has been successfully commercialized as medicine for the treatment of early to mild Alzheimer’s Disease (AD). The characteristic symptoms of this disease are loss of memory and impairment of multiple cognitive and emotional functions. The objective was to find out how galanthamine levels could be enhanced to convert this floricultural crop into a medicinal crop. There are different factors that influence the production of galanthamine and other metabolites in the bulbs. Effect of various factors such as preanalytical methods, bulb location and age along with effect of biotic and abiotic factors on the growth and yield were studied. From the results, it is evident that factors have detrimental effect on the total bulb yield and galanthamine levels. Show less
BlaC is the β-lactamase of Mycobacterium tuberculosis. We show that it can recover from inhibition by clavulanic acid and that phosphate helps it do so. We also show that in solution, BlaC is a... Show moreBlaC is the β-lactamase of Mycobacterium tuberculosis. We show that it can recover from inhibition by clavulanic acid and that phosphate helps it do so. We also show that in solution, BlaC is a rigid protein on the pico-nanosecond timescale but shows dynamics around the active site on the catalytic timescale. These dynamics become more pronounced upon inhibitor binding. Lastly, we show that two mutations that both provide BlaC with inhibitor resistance have very different effects on the dynamic behaviour. Show less
The studies presented in the work show the potential of the integrative use of biophysical data in defining the structural basis of protein interactions. Even if the results obtained hold a... Show moreThe studies presented in the work show the potential of the integrative use of biophysical data in defining the structural basis of protein interactions. Even if the results obtained hold a degree of ambiguity, this approach allows to iteratively refine and validate the model and interpret its meaning for the molecular basis of protein function. Often all three points at the same time. This dynamic nature makes the use of structural models in the design of therapeutic compounds especially useful since the inhibition of a certain protein function might not require a structure to be accurate down to the last atom but rather highlight key interactions or structural features that can be addressed in context of small molecule or peptide inhibitors. Presented are the use of strucutral biochemistry techniques to investigate the mechanism of how the ubiquitine ligase PSIP1 obtains its target specificity. Furthermore, another epigenetic effector protein PSIP1 is investigated with the aim to develop a workflow for the design of potential peptide-based inhibitors. Show less
In the early nineties, Sidles (1991) came with a solution to combine the force microscopy techniques sensitive to atoms with that of magnetic resonance techniques: Magnetic Resonance Force... Show moreIn the early nineties, Sidles (1991) came with a solution to combine the force microscopy techniques sensitive to atoms with that of magnetic resonance techniques: Magnetic Resonance Force Microscopy (MRFM) was born. The technique was promising, big steps were taken, and the holy grail of atomic resolution imaging of biological tissues seemed within an arm’s reach. Unfortunately, the last steps are the most difficult. The technique is experimentally challenging and so far, the images of biological structures are no better than those obtained by other conventional techniques. In order to be an attractive technique, MRFM needs to be scientifically relevant while the technique is further improved towards the holy grail of imaging biological structures on the nanometer scale. In this thesis, we show how MRFM can usefully contribute to the field of condensed-matter. Show less
The explosive increase in infections by pathogens is a major problem in the clinic today. The theme of this thesis was to find novel antibiotics from actinomycetes. Next-generation... Show more The explosive increase in infections by pathogens is a major problem in the clinic today. The theme of this thesis was to find novel antibiotics from actinomycetes. Next-generation sequencing revealed that the biosynthetic potential of actinomycetes had been grossly underestimated. In this thesis, different antibiotics-eliciting strategies, including microbial cocultivation, streptomycin-resistant mutation, overexpression of pathway-specific activator, variation of culture conditions, were utilized to enforce fluctuations in the production of bioactive compounds in actinomycetes, after which, NMR-based metabolic profiling was used to facilitate uncovering those elicited molecules. This pipeline allowed the discovery of new antibiotics involving various chemical skeletons, such as 7-prenylisatin, methoxylated isocoumarins, endophenazines, and C-glycosylpyranonaphthoquinones. On the other hand, genome-mining methodology enabled the discovery of a group of endophenasides and leucanicidin in Kitasatospora sp. MBT66, whereby the rhamnosylation of both scaffold are executed by a same promiscuous glycosyltransferase. Last but not least, a novel antibiotic termed lugdunomycin with unprecedented chemical scaffold, as well as a number of new angucycline-type antibiotics, were characterized from Streptomyces sp. QL37. The biosynthetic pathway of lugdunomycin was deciphered by genetic knockout and OSMAC (One Strain MAny Compound) strategy. In summary, this thesis explores an interface of genomics and metabolomics to accelerate new antibiotics discovery. Show less
Nuclear magnetic resonance force microscopy (MRFM) is a technique which combines magnetic resonance imaging (MRI) with scanning probe microscopy (SPM). The final goal is to develop this technique... Show moreNuclear magnetic resonance force microscopy (MRFM) is a technique which combines magnetic resonance imaging (MRI) with scanning probe microscopy (SPM). The final goal is to develop this technique to such a level that the atomic structure of a virus or protein can be revealed by this microscope. This thesis shows nuclear magnetic resonance force measurements on copper in which the interaction of the magnetic moments of the nuclei of copper with a magnetic cantilever has delivered a detectable signal at a temperature of 50 millikelvin. Furthermore, we show measurements, which support a new theory where at low magnetic field and low temperature, non contact friction between the magnetic cantilever and paramagnetic electron spins is described. These measurements were enabled by technical improvements such as vibration reduction in a cryogen free dilution refrigerator. As a benchmark for the low vibration, we show atomic resolution scanning tunneling microscopy at 15 millikelvin temperature on graphite. We also show a method to create small magnets for MRFM from a thin magnet film. With these small magnets the field gradient and therefore the sensitivity may be significantly enhanced. Show less
The research presented in the thesis describes the development and implementation of solution based NMR methods that provide 3D structural information on the protein-small molecule complexes. These... Show moreThe research presented in the thesis describes the development and implementation of solution based NMR methods that provide 3D structural information on the protein-small molecule complexes. These methods can be critical for structure based drug design and can be readily applied in the early stages of preclinical drug discovery and fragment based drug discovery Show less
Urinary tract infection is a complex clinical entity a common infectious disease that encompasses a variety of clinical syndromes with a positive bacterial culture as common denominator. This... Show moreUrinary tract infection is a complex clinical entity a common infectious disease that encompasses a variety of clinical syndromes with a positive bacterial culture as common denominator. This thesis provides an exhaustive exploratory study of the metabolic pattern of patients affected by urinary tract infection and Here this complex clinical entity was investigated with a multiplatform approach. Each of the used platforms added a unique perspective to the further understanding of the infection process. The assessment of the bacterial growth (NMR), of the host response (LC__MS) and of the physiological status (GC-APCI-MS) could eventually be useful during the assessment of the disease severity and/or decision making Show less
The thesis aims at combining metabolomics with other methods to investigate the regulation of the TIA biosynthesis and how this is connected with other pathways and the plant__s physiology and... Show moreThe thesis aims at combining metabolomics with other methods to investigate the regulation of the TIA biosynthesis and how this is connected with other pathways and the plant__s physiology and development. It reviews the biosynthesis studies of Catharanthus roseus. An HPLC method is described for both precursors and alkaloids which was validated to analyze alkaloid contents in different organs, its relationship with leaf age and growth of C. roseus plants. A metabolomics investigation of C. roseus varieties with different flower colors by NMR profiling method is also reported. Besides, it explores the effect of JA/MeJA treatment on TIA biosynthesis in different organs and the influence of flowering on TIA levels in C. roseus plants. Overexpression of ORCA3 and G10H in C. roseus plants is reported and the effect on alkaloid biosynthesis and general metabolism is revealed by NMR-metabolomics. Finally, it reports on C. roseus plants fed with 13C-labeled glucose to study the flux into different metabolic pathways, and gives a summary and discussion about further perspectives. Show less
By utilizing paramagnetic NMR techniques, the structure and dynamics of the P450cam system were investigated. The analysis of PCS and RDC illuminated the stereo-specific final complex of Pdx and... Show moreBy utilizing paramagnetic NMR techniques, the structure and dynamics of the P450cam system were investigated. The analysis of PCS and RDC illuminated the stereo-specific final complex of Pdx and P450cam, while the results of PRE demonstrated the presence of a transient encounter complex. Furthermore, the significant insights of the interaction in the interface were uncovered by X-ray crystallography. Currently, the nature of Pdx effector activity is under debate. Since paramagnetic NMR experiments are applicable to solution studies at ambient temperature, PCS, RDC and PRE methods can further resolve the molecular mechanism of P450cam in the future. Show less
In this thesis, patterns of variation in plant metabolomes and insect communities were described in GM and non-GM potato plants in both laboratory and field experiments. Differences between plant... Show moreIn this thesis, patterns of variation in plant metabolomes and insect communities were described in GM and non-GM potato plants in both laboratory and field experiments. Differences between plant genotypes in insect abundances were small when compared to year-to-year differences, location effects and differences between developmental stages of plants. Standardized effect sizes are discussed as an alternative scale for measuring effects. Leaf age, aphid infestation and virus infection were found to cause significant alterations in leaf metabolism in a laboratory study as measured by 1H NMR. However, these changes were similar in quantity and quality in both GM and non-GM plants. Furthermore, 1H NMR metabolomic profiles of potato plants were measured in a 2-year field study. Large changes in metabolomic profiles occurred across years and locations, and throughout the growth period of plants within years. Only weak relationships were found between metabolomics data and insect abundances on the same experimental fields. GxE interactions imply that conclusions from field trials are not valid outside the range of tested environments. This emphasizes the need for a careful choice of representative geographical zones for risk assessment studies. However, it also shows that a residual level of uncertainty in NTO safety is unavoidable. Show less
This thesis describes the application of a variety of different classical and paramagnetic NMR to different protein system. To this end cytochrome P450cam was assigned using classical techniques... Show moreThis thesis describes the application of a variety of different classical and paramagnetic NMR to different protein system. To this end cytochrome P450cam was assigned using classical techniques and paramagnetic NMR was used to investigate the solution state of this enzyme in complex with its binding partner, putidaredoxin. The solution structure of Avr2 was also solved using classical NMR spectroscopy techniques. Additionally, an algorithm for the assignment of protein nuclei on the basis pseudocontact shifts, PARAssign, is presented. An investigation of the dynamic behaviour of a paramagnetic tag attached to T4 lysozyme using relaxation dispersion is described. Show less
Inflammation is an immune reaction of the body to the external stimuli such as toxins or pathogens, and is characterized by redness, swelling, pain, and heat. The process of inflammation is... Show moreInflammation is an immune reaction of the body to the external stimuli such as toxins or pathogens, and is characterized by redness, swelling, pain, and heat. The process of inflammation is regulated by several pro-inflammatory and antiinflammatory cytokines. Tumor necrosis factor-_ (TNF-_) is a major pro-inflammatory cytokine involved in the inflammatory response.Elevated TNF-_ expression has been found to be associated with the development of diabetes, atherosclerosis, septic shock, and tumorigenesis. Thus inhibition of TNF-_ at any step of inflammatory pathways provides an attractive treatment for inflammatory diseases as well as for series of other common diseases.Plants provide an alternative sources of medicines used traditionally by people worldwide since thousands of years ago. The aim of this thesis was to develop methods for the rapid identification of active compounds in plant extracts by correlating NMR metabolomics and bioassay results by means of multivariate data analysis. This work demonstrates the great potential of NMR spectroscopy in combination with chemometrics for the screening of large set of crude extracts, to study the effects of different variables on the activity, and identifying sets of active compounds in complex mixtures like plant extracts. Show less
Receptors tyrosine kinases or RTKs are cell surface receptors that regulate numerous cellular processes, but also have a critical role in the development and progression of many types of cancer.... Show moreReceptors tyrosine kinases or RTKs are cell surface receptors that regulate numerous cellular processes, but also have a critical role in the development and progression of many types of cancer. The overexpression of EphA4, a member of the RTK family, has been observed in a variety of malignant carcinomas. The aim of the research project associated with this thesis was to develop high affinity inhibitors of the tyrosine kinase EphA4. Ligand discovery was based on two complementary approaches, a computational screen and an NMR based screen using Target Immobilized NMR Screening (TINS). In addition, orthogonal biophysical methods including Surface Plasmon Resonance (SPR) and protein observed NMR were employed to analyse fragment binding. The crystal structure of the EphA4 kinase domain was solved and the structure of the kinase domain in complex with dasatinib, a well-known kinase inhibitor, was also elucidated. The in silico approach discovered a potent inhibitor of EphA4 for which the binding mode was elucidated via X-ray crystallography. Moreover, the TINS approach identified two compounds that may constitute starting points for the generation of more potent EphA4 inhibitors. Show less
Spin labeling and electron paramagnetic resonance (EPR) have been employed to study structure and dynamics of proteins. The surface polarity of four single cysteine mutants of the Zn-azurin in... Show moreSpin labeling and electron paramagnetic resonance (EPR) have been employed to study structure and dynamics of proteins. The surface polarity of four single cysteine mutants of the Zn-azurin in frozen solution were studied using 275 GHz EPR (J-band), with the advantage compared to 9 GHz (X-band) and 95 GHz (W-band) to allow differentiating between sites having small differences in polarity. The polarity/proticity profiles of the four mutants have been obtained and compared with data already reported in literature of spin labels in different solvents. In order to detect distances at the nm scale, azurin was used as a known model system for double electron-electron spin resonance. The conformation of the spin label linker was modelled. The model was found to be in good agreement with experimental results and can be extended to other proteins. The dynamics of the transient complex of Nostoc sp. PCC 7119 cytochrome f __ plastocyanin was investigated by NMR using the paramagnetic relaxation enhancements. The experimental distance restraints, used in docking calculations, are best interpreted by the presence of a dynamic ensemble of protein-protein orientations within the complex, rather than by a single, well-defined structure, and can be described with an encounter complex model. Show less
Despite the rich diversity in molecular structures and biological functionality, the complex matrix of the compounds with a broad dynamic range has limited the use of plants as an important source... Show moreDespite the rich diversity in molecular structures and biological functionality, the complex matrix of the compounds with a broad dynamic range has limited the use of plants as an important source for drug development. Adressing this issue, several papers have described the use of the more holistic approach targeting on a wide range of metabolite present in plant extract, i.e. metabolomics. This new approach involves the use of various analytical methods followed by appropriate multivariate data analysis. The aim is to shorten the bioassay guided isolation route, particularly in the identification and dereplication step This thesis describes a new strategy to improve dereplication and identification steps in drug discovery process from plants. The integration of comprehensive extraction coupled to NMR metabolomics and multivariate data analysis is found to be a potential new approach to uncovering bioactive compounds from crude plant extracts. Show less
Metabolomics is the comprehensive analysis of small molecules involved in metabolism, on the basis of samples that have been obtained from organisms in a given physiological state. Data obtained... Show moreMetabolomics is the comprehensive analysis of small molecules involved in metabolism, on the basis of samples that have been obtained from organisms in a given physiological state. Data obtained from measurements of trait levels in twin families can be used to elucidate the importance of genetic and environmental variation for individual differences in trait levels. I describe the results of various analyses using metabolomics data from twin families. These data originated from analysis of blood plasma lipids by liquid chromatography-mass spectrometry, and from analysis of blood plasma and urine by proton nuclear magnetic resonance spectroscopy. Data analyses with a newly developed method, based on hierarchical clustering analysis of family members, suggested that shared genetic variation and shared environmental variation are important for similarities in blood plasma lipid profiles among individuals. Also, a method called __quantile equating__ was developed and applied that enables combination of semiquantitative metabolomics data sets originating from different measurement __blocks__. Univariate quantitative genetic analyses based on structural equation modeling revealed interesting differences in heritability among different metabolites. In multivariate analysis, relationships among genetic sources of phenotypic variation in different metabolites were investigated. These results bear relevance for the interpretation of the results from genome-wide association analyses. Show less
This thesis concerns the symmetry, phase, and order parameter of the superfluid helium-3 in restricted geometries in combination with a magnetic field. Two cylindrical containers are constructed... Show moreThis thesis concerns the symmetry, phase, and order parameter of the superfluid helium-3 in restricted geometries in combination with a magnetic field. Two cylindrical containers are constructed for which the axis is aligned with the magnetic field. The first cell has a diameter (540 nm) of only a few times the size of the Cooper pairs, designed to find a new superfluid phase, namely the polar has. The second container has a diameter of 1 mm, which is the ideal size to create a potential (in the B-phase) for spin waves. To probe any superfluid phase, or spin waves, we use Nuclear Magnetic Resonance (NMR) Techniques. As the superfluids have an anisotropic susceptibility, it is an excellent tool to distinguish the different phases. However, as our samples are relatively small in volume, and the experiments needs to be performed in low magnetic field to prevent additional symmetry breaking, a very sensitive read-out magnetic resonance detection system needs to be developed, which is accomplish by creating an LC-circuit which maintains an ultra-high quality factor as it is combined with a weakly coupled transformer. Show less
The application of dedicated mass spectrometry (MS) and nuclear magnetic resonance (NMR) technologies for biomarker discovery and diagnostic purposes has increased substantially in the last decade.... Show moreThe application of dedicated mass spectrometry (MS) and nuclear magnetic resonance (NMR) technologies for biomarker discovery and diagnostic purposes has increased substantially in the last decade. In the studies presented in this thesis, we have used these technologies to identify parasite or host-derived products (biomarkers) related to infection and morbidity associated with schistosomiasis and to better understand the host-parasite interaction. The application of our peptidomics and metabonomics studies on schistosomiasis have provided some novel, valuable information but they are obviously only the first step. In addition to the potential biomarkers identified with the global biomarker discovery approaches, we showed in this thesis that a more targeted approach, looking at glycosylation, also resulted in novel information on S. mansoni infection. We have identified and characterized a set of human Apolipoprotein C-III peptides aberrantly glycosylated at the O-glycosylation site (Thr74), in urine of S. mansoni- infected individuals. The presented study is the first in which MS and NMR were used for the analysis of a cohort of human S. mansoni-infected individuals. This has resulted in the identification of a number of novel markers. Nevertheless, further studies are needed to evaluate the overall applicability of these putative biomarkers Show less
The effect of the incorporation of alpha- and beta-amino acids in model peptide gramicidin S is described. Several techniques were applied, such as NMR and X-ray crystallography. In addition the... Show moreThe effect of the incorporation of alpha- and beta-amino acids in model peptide gramicidin S is described. Several techniques were applied, such as NMR and X-ray crystallography. In addition the activity of the newly obtained peptides towards bacterial and mammalian cell membranes was investigated. Show less
