Lipid signaling is an essential biological event/process in a plethora of pathophysiological conditions. The underlying idea of this thesis is that many of the roles and the complex interplay of... Show moreLipid signaling is an essential biological event/process in a plethora of pathophysiological conditions. The underlying idea of this thesis is that many of the roles and the complex interplay of the individual signaling lipids in inflammatory processes and related conditions in health and disease is not well known, and therefore has to be studied integrally as a complex network. In order to study this complex interplay, an improved broad analytical method is necessary to analyze a wide range of different signaling lipid classes such as oxylipins, (nitro) free fatty acids, endocannabinoids, bile acids and different subclasses of lysophospholipids. Therefore, the aim of this thesis is to develop a better method to study signaling lipids, and to apply it to study the role of these molecules in several relevant biological questions for a better understanding of inflammation related pathophysiology including autoimmune diseases, neurodegeneration and regulatory effect of exercise training. Show less
The work described in this thesis focuses on the development of linear or cyclized peptide probes against protein N-methyltransferases to characterize their specific binding behavior, providing... Show moreThe work described in this thesis focuses on the development of linear or cyclized peptide probes against protein N-methyltransferases to characterize their specific binding behavior, providing further binding details for inhibitory activity study. The thesis not only describes the extended application to produce peptide-based transition states mimicking PRMT inhibitors but builds an LC-MS/MS method to evaluate CARM1 inhibition and activity. Show less
Neurodegenerative diseases, including Parkinson’s disease (PD), are increasing in prevalence due to the aging population. Despite extensive study, these diseases are still not fully understood and... Show moreNeurodegenerative diseases, including Parkinson’s disease (PD), are increasing in prevalence due to the aging population. Despite extensive study, these diseases are still not fully understood and the lack of personalised treatment options that can target the cause of the diseases, rather than the symptoms, has led to a greater demand for improved disease understanding, therapies and diagnostic procedures. In this thesis, we use systems biology approaches to construct disease-specific models intended for biomarker discovery, therapeutic treatment strategy identification and drug repurposing in PD. Systems biology is a mathematical field of research that analyses biological systems via construction of a computational model using experimental data. This is achieved by integration of omics data, including genomics, proteomics, transcriptomics and metabolomics. A specific approach used to identify the physico- and biochemical bounds within a biological system is constraint-based modelling, which requires the input of absolute quantitative metabolomics data. To improve our absolute quantitative coverage of the metabolome, we developed and improved new quantitative metabolomics methods using a targeted mass spectrometry workflow to obtain data intended to be integrated into constraint-based metabolic models for the study of PD. Show less
Inflammation is a tightly regulated process. During the past decade it has become clear that the resolution of inflammation is an active process and its dysregulation can contribute to chronic... Show moreInflammation is a tightly regulated process. During the past decade it has become clear that the resolution of inflammation is an active process and its dysregulation can contribute to chronic inflammation. Several cells and soluble mediators, including lipid mediators, regulate the course of inflammation and its resolution. It is, however, unclear which signals and cells are involved in initiating the resolution process. Macrophages are tissue resident cells and key players in regulating tissue inflammation through secretion of soluble mediators, including lipids. We hypothesize that persistent inflammatory stimuli can initiate resolution pathways in macrophages.In this study, we detected 21 lipids in LPS-stimulated human monocyte-derived macrophages by liquid chromatography coupled to tandem mass spectrometry. Cyclooxygenase-derived Prostaglandins were observed in the first six hours of stimulation. Interestingly, a switch towards 15-lipoxygenase products, such as the proresolving lipid precursors 15-HEPE and 17-HDHA was observed after 24 h. The RNA and protein expression of cyclooxygenase and 15-lipoxygenase were in line with this trend. Treatment with 17-HDHA increased IL-10 production of monocyte-derived macrophages and decreased LTB4 production by neutrophils, indicating the anti-inflammatory property of this lipid.These data reveal that monocyte-derived macrophages contribute to the resolution of inflammation in time by the production of pro-resolving lipids after an initial inflammatory stimulus. Show less
Plants produce an astonishing variety of secondary metabolites (SMs) which are thought to play vital roles in the fitness of plants through ecological interactions. The most characteristic features... Show morePlants produce an astonishing variety of secondary metabolites (SMs) which are thought to play vital roles in the fitness of plants through ecological interactions. The most characteristic features of SMs are their striking chemical diversity and inter- or intraspecific variation. Due to the large number, high structural diversity and multifunctionality of SMs, it is still an ongoing challenge to understand how this SM diversity comes about, and why such a large diversity is maintained in nature. In this thesis this question was studied using the pyrrolizidine alkaloids (PAs) of Jacobaea species as the study system from an evolutionary and biosynthetic perspective. PA variations were studied among and within Jacobaea species, and species-specific PA profiles were observed. In order to understand how PA diversity is related to species phylogeny, the evolutionary histories and phylogenetic signals of individual PAs were investigated under the phylogenetic context of Jacobaea species and no strong phylogenetic signals were found. To shed light on the mechanisms underlying PA diversity, a gene-to-metabolite approach targeting cytochrome P450 monooxygenases which play an important role in the evolution of chemical diversity was applied to study their involvement in PA biosynthesis and PA diversity. Show less
The focus of this thesis is (oxy)lipid analysis. An introductory overview is given of lipids, lipidomics, and lipid mediators in inflammation; and in subsequent chapters the focus is on... Show moreThe focus of this thesis is (oxy)lipid analysis. An introductory overview is given of lipids, lipidomics, and lipid mediators in inflammation; and in subsequent chapters the focus is on development of lipidomic methods for the analysis of oxidized lipids. This touches on different extraction methods of the (target) analytes, sample handling/preparation and storage, separation techniques, and finally the MS detection/resolution/ specificity etc. These methods are then utilized, mainly on human plasma and synovial fluid (SF) samples. Special focus is on the targeted analysis of hydroxylated fatty acids (hFAs), specialized pro-resolving mediators (SPMs) and their intermediates, and other oxylipids present in inflammation and its resolution in rheumatic diseases. For example, the bioactivity of oxylipids and LMs is highly stereospecific, and some difficulties have arisen in the chromatographic separation and resolution of these stereoisomers using classical reversed phase liquid chromatography tandem MS (RPLC-MS/MS), perhaps sometimes resulting in wrong conclusions being drawn, one isomer being confused with another. The work in this thesis was therefore focused on further development and application of analysis platforms for oxidized lipids; their identification, separation and levels in different matrices. All of which, work that can be further used in inflammatory or rheumatoid research. Show less
The work described in this thesis is mainly focusing on setting up and application of a quantitative activity‐based proteasome profiling method. Chapter 1 provides a general introduction on the... Show moreThe work described in this thesis is mainly focusing on setting up and application of a quantitative activity‐based proteasome profiling method. Chapter 1 provides a general introduction on the ubiquitin proteasome system (UPS) and activity‐based proteasome profiling. Chapter 2 is a literature review of some new achievements in the activity‐based protein profiling field in the recent years, focusing on application in biochemistry, molecular and cellular biology, medicinal chemistry, pathology, physiology and pharmacology research. Chapter 3 is a protocol for performing quantitative activity‐based proteasome profiling experiments. In the protocol, both high throughput fluorescent ABPP and biotinylated probe plus LC/MS approaches are described. Chapter 4 is a brief technical report about bioorthogonal chemistry in ABPP. The commonly used secondary azide group is compared with a primary azide group in proteasome ABPs performing Cu(I) catalyzed azide‐alkyne cycloaddition and Staudinger‐Bertozzi reaction under native/denatured protein conditions Chapter 5 is focusing on the application of quantitative activity‐based proteasome profiling in the prognosis of cancer therapeutics. A combination of ABPP and global proteomics is performed to elucidate the bortezomib sensitivity and resistance mechanisms in leukemia and solid tumor cells. Chapter 6 describes the characterization of the newly discovered proteasome subunit β5t by ABPP and LC/MS proteomics. The subunit is proven to be catalytically active. A hydrophilic Thr residue on the P2 position of the proteasome inhibitor improves the inhibitory efficiency of β5t, which indicates it might prefer to cleave hydrophilic peptides. Chapter 7 describes the identification of O‐GlcNAcylation modifications on the ubiquitin receptor protein hHR23B and characterization of how the sugar moiety influences the conformation and functions of the protein. Show less
Krumpochova, P.; Bruyneel, B.; Molenaar, D.; Koukou, A.; Wuhrer, M.; Niessen, W.M.A.; Giera, M. 2015
Plants are attacked by a variety of (micro)organisms. In order to cope with potential attackers many plants synthesize a diversity of repellent, deterrent and/or toxic compounds. Pyrrolizidine... Show morePlants are attacked by a variety of (micro)organisms. In order to cope with potential attackers many plants synthesize a diversity of repellent, deterrent and/or toxic compounds. Pyrrolizidine alkaloids (PAs) are a well-known class of defense compounds, abundantly found in species of Senecio and Jacobaea. The objectives of the study were to acquire knowledge on the PA composition of plants and its interaction with soil-borne microorganisms. In order to do so, a more sensitive, PA-analysing method was applied, which allowed us to distinguish between PAs in tertiary amine and N-oxide form. The tertiary PA form is known to have a more negative effect on generalist insects. Our study clearly showed that high levels of tertiary PAs occur in Jacobaea vulgaris and not caused by an artefact as suggested in previous studies. Green-house experiments showed that the PA composition below- and aboveground was significantly aff ected by both soil-type and soil-inoculum. The inoculum-induced PA composition aboveground did affect thrips resistance for one J. vulgaris genotype. Experiments also showed that the fungal community did depend on the PA composition of the plant while this had less or no effect on bacterial and mycorrhizal communities in roots and rhizosphere soil. Show less
