In this thesis, nanochannels as well as nanofluidic phenomena are used to provide new and miniaturized bioanalytical tools for the life sciences. Isotachophoresis performed in nanochannels showed... Show moreIn this thesis, nanochannels as well as nanofluidic phenomena are used to provide new and miniaturized bioanalytical tools for the life sciences. Isotachophoresis performed in nanochannels showed the focusing and separation of analytes in a 0.4 picoliter volume, which is a volume in the order of a sample from a single cell. Depletion zone isotachophoresis (dzITP) is demonstrated which uses a nanofluidic phenomenon, concentration polarization, to enable isotachophoresis in a microchannel while using only a single electrolyte. A concept for surface enhanced Raman spectroscopy (SERS) sensor (SERSOR) is explored; a coating protecting the SERS surface from irreversible adsorption may enable dynamic measurements of biomolecules in solution in minute volumes. Working with nanochannels has led to the discovery of new unexpected fundamental phenomena: the very high surface to volume ratio in nanochannels causes acidification of solutions introduced in them, despite the presence of up to 1 mol/L of buffer. Also, extreme pressures of more than a 1000 bar may be induced in a nanochannel by what we named electrocavitation, an effects shown to impose a limit on further downscaling of ITP. 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
Dit proefschrift beschrijft de resultaten van aan aantal studies naar bewegingsstoornissen en pijn bij pati_nten met complex regionaal pijnsyndroom (CRPS). Ten eerste werd het effect onderzocht van... Show moreDit proefschrift beschrijft de resultaten van aan aantal studies naar bewegingsstoornissen en pijn bij pati_nten met complex regionaal pijnsyndroom (CRPS). Ten eerste werd het effect onderzocht van intrathecaal baclofen op verschillende pijnkwaliteiten bij CRPS-pati_nten, de invloed bestudeerd van verschillende infusiesnelheden op dystonie bij CRPS en het optreden van chorea na baclofen geanalyseerd bij enkele CRPS-pati_nten. Daarnaast werd het effect bestudeerd van intrathecaal glycine op CRPS-gerelateerde dystonie en pijn. Vervolgens werd aan de hand van een metabolomics benadering het metabole profiel in urine en hersenvocht van CRPS-pati_nten onderzocht. Tenslotte werd het effect bestudeerd van intramusculair magnesiumsulfaat op CRPS-gerelateerde pijn en dystonie Show less
Lignocellulosic biomass is the 2nd generation feedstock for biofuel production through fermentation processes. The material has a rigid structure, which needs to be broken down by a pretreatment... Show moreLignocellulosic biomass is the 2nd generation feedstock for biofuel production through fermentation processes. The material has a rigid structure, which needs to be broken down by a pretreatment procedure to expose cellulose for hydrolysis. The hydrolysis products, so called biomass hydrolysates, contain next to the sugar monomers, toxic compounds released and formed during the pretreatment process. These compounds inhibit the growth of the fermenting host(s). To improve the fermentability of biomass hydrolysates, identification of these inhibitory compounds is of great importance. Chapter 1 of this thesis reviews the approaches and techniques that have been used to study the inhibitors in various biomass hydrolysates, and introduces a non-targeted methodology to systematically identify biomass hydrolysate inhibitors: the exometabolomics approach. To identify hydrolysate inhibitors through an exometabolomics approach, a wide range of biomass hydrolysates needs to be prepared. Chapter 2 describes the detailed procedures of four pretreatment methods and the overall fermentability of the generated hydrolysates. The hydrolysis efficiency of the carbohydrate polymers in pretreated biomass was analyzed by using high-performance anion-exchange chromatography coupled with mass spectrometry (HPAEC-MS), and the results of this analysis are presented in Chapter 3. The last three chapters of the thesis focus on identifying inhibitory compounds in lignocellulosic biomass hydrolysates and studying their effects on fermenting yeasts during fermentation processes. Chapter 4 examines the fermentability of a series of biomass hydrolysates, in relation to the presence and dynamics of a target group of inhibitors in these hydrolysates. Chapter 5 reports the detailed experimental procedure and results of the actual exometabolomics approach introduced in Chapter 1. The research question, identification of inhibitors in biomass hydrolysates, was answered by statistically correlating the fermentability of different biomass hydrolysates with their biochemical compositions. Finally, in search for potential ethanologenic host organisms resistant to biomass hydrolysate inhibitors, a Pichia anomala strain was isolated. In Chapter 6, the properties and fermentation performance of this strain in biomass hydrolysates were tested. Through further research and possibly genetic modifications, the strain has the potential to become a suitable yeast for fermenting lignocellulosic biomass hydrolysates. Show less
Cellular responses to DNA damage are highly variable and strongly depend on the cellular and organismic context. Studying the DNA damage response is crucial for a better understanding of cancer... Show moreCellular responses to DNA damage are highly variable and strongly depend on the cellular and organismic context. Studying the DNA damage response is crucial for a better understanding of cancer formation and ageing as well as genotoxic stress-induced cancer therapy. To do justice to the multifaceted cellular changes, elicited by DNA damage, use of high-throughput techniques and integration with bioinformatics tools is of great value. This thesis summarizes recent advances in the field of systems biology studies of the DNA damage response and furthermore shows integrated approaches of the study of DNA damage response signaling networks in embryonic stem and cancer cells. By integration of transcriptional changes and the phosphorylation and metabolic response of cisplatin-treated embryonic stem cells, with RNAi-based knockdown screens we identify novel DNA damage response signaling networks, linking process such as Wnt signaling, translation arrest or altered metabolic pathways to the cellular response to DNA damage. Furthermore, genes, whose knockdown sensitizes embryonic stem cells to DNA damage-induced killing, are tested in cancer cells of varying genetic backgrounds identifying a small subset of genes, which represent potential drug targets for sensitization of cancer cells. Altogether, our systems approach for studying the DNA damage response identifies novel DNA damage-induced signaling networks and molecules, which modulate survival in the presence of DNA damage, potentially providing new targets for therapeutic intervention or biomarker discovery. 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
Cannabinoids are a group of terpenophenolic compounds and are naturally found in the cannabis plant (Cannabis sativa L). _9-Tetrahydrocannabinol (_9-THC) is the psychoactive cannabinoid. The high... Show moreCannabinoids are a group of terpenophenolic compounds and are naturally found in the cannabis plant (Cannabis sativa L). _9-Tetrahydrocannabinol (_9-THC) is the psychoactive cannabinoid. The high lipophilicity of _9-THC is a hindering factor in the further development of this compound into a large scale pharmaceutical product. In an effort to generate highly polar derivatives of _9-THC, we screened 206 alkane degrading bacterial strains. In total, eight derivatives were produced on milligram. All of these biotransformation products harbored modified alkyl chains, with hydroxy, carboxy and ester functionalities. We also investigated the potential of cell suspension culture of Catharanthus Roseus to transform _9-THC. Unlike bacterial strains, Catharanthus Roseus cells transformed _9-THC to its glycosylated and additionally hydroxylated derivatives. The behavioral effects of __9-THC are mediated by the centra l CB1 receptor in rats. CB1 receptors show high degree of sequence conservation in vertebrates. We used zebrafish embryos as a model to study the behavioral effects of cannabinoids. We conclude that cannabinoids have behavioural effects in zebrafish that are comparable to some of those reported in the literature for mammals. In particular, the acute exposure response resembles behavioural effects reported for adult. This suggests that some similarity in cannabinoid response pathways between zebrafish and mammals exists. We also performed the metabolomic profiling of zebrafish embryos treated with the concentrations of CB1 agonist and antagonist used in behavioural assays. The results of behavioural assays were successfully correlated with the metabolomic data of zebrafish embryos. Show less
Plants produce many diverse chemical compounds that are used in various industries. The aim of this thesis was to obtain an understanding of what is needed to convert an ornamental crop to a... Show morePlants produce many diverse chemical compounds that are used in various industries. The aim of this thesis was to obtain an understanding of what is needed to convert an ornamental crop to a pharmaceutical crop. This included knowledge about regulatory issues and expectations from the industry regarding quality and safety of such crops. Based on this knowledge, a further aim was to determine how the cultivation practices of the model ornamental crop Narcissus pseudonarcissus, which contains the medicinal compound galanthamine, needed to be adapted. Some key points in the cultivation process were identified that may need to be adapted in the production of Narcissus bulbs as raw material for the pharmaceutical industry. These points were investigated in field experiments using NMR-based metabolomics as the main analytical tool. This contributed to a validated production chain of Narcissus pseudonarcissus bulbs suita ble for use as raw material for the extraction of galanthamine. Results from exploratory investigations into other ornamental bulb material for novel industrial products are also presented. Show less
