The outbreaks of AIDS and COVID-19 showed clearly how infectious viruses can influence people’s lives. Investigating the changes in the host metabolism may provide a paradigm shift to consider... Show moreThe outbreaks of AIDS and COVID-19 showed clearly how infectious viruses can influence people’s lives. Investigating the changes in the host metabolism may provide a paradigm shift to consider immune-metabolic interactions as therapeutic targets. The aim of this thesis is to examine the interplay between the immune system and metabolism during viral infections, such as HIV and coronavirus. These investigations will utilize metabolomic and lipidomic mass spectrometry techniques to gain a comprehensive understanding of the metabolic changes that occur during viral infections. To enhance the coverage of the lipidome, a new method will be developed. Show less
Viral hemorrhagic fever (VHF) is a group of acute diseases caused by highly infectious viruses including Ebola, Lassa, Dengue viruses. Its high mortality rate poses high risk to public health,... Show moreViral hemorrhagic fever (VHF) is a group of acute diseases caused by highly infectious viruses including Ebola, Lassa, Dengue viruses. Its high mortality rate poses high risk to public health, however, studies on VHF have been hampered due to the non-availability of proper models and incomplete knowledge on its mechanism. In order to fill this gap, this thesis presented new bioanalytical, lab-on-chip and single-cell assays to investigate changes in vascular biology and macrophage immunometabolism induced by VHF viruses. Firstly, an organ chip was developed to mimic the hemorrhagic shock syndrome caused by VHF viruses in vitro and test experimental drug candidates. In addition, acoustic force spectroscopy was applied to investigate the effect of Dengue on the cellular viscoelastic properties of endothelial cells at single-cell level. Then, metabolic profiling of endothelial cells and macrophages upon Ebola viral protein exposure was performed on bulk-level. Finally, the immunometabolism of human macrophages upon polarization was investigated by live single-cell metabolomics, setting the stage for future host-pathogen studies at single-cell level. Overall, this thesis will facilitate the understanding of VHF viruses and the development of treatment strategies. More importantly, the technologies developed here expectedly open up opportunities to combat the viruses that threaten global society. Show less
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
To increase clinical success rate of drugs, a better understanding of drug action mechanism and disease dynamics is required. Metabolomics, which studies small molecules involved in biochemical... Show moreTo increase clinical success rate of drugs, a better understanding of drug action mechanism and disease dynamics is required. Metabolomics, which studies small molecules involved in biochemical processes in organisms, has shown to be a useful tool for this better understanding. In this thesis, we focus on the endocannabinoid system (ECS) and profiling its related metabolic pathways using liquid chromatography - mass spectrometry (LC-MS) based metabolomics techniques. The endocannabinoid system (ECS) is a signaling system involved in multiple physiological and pathological processes. Due to its wide distribution and complex network of metabolic interactions, the development of drugs targeting the ECS has seen high failure rates. To get a better understanding of the behavior of the ECS and related pathways, LC-MS platforms with wide coverage of the major ECS-related metabolites, or with high sensitivity that reaches low levels of metabolites, were developed and optimized. Furthermore, these metabolomics platforms were applied in clinical studies looking into cardiometabolic health, and revealed correlations between endogenous metabolite signaling, cardiometabolic health and the benefits of exercise. Show less
This thesis focuses on the development of sample-preparation methods for small amounts of samples and applying the developed methods to muscle tissues to investigate the mechanisms involved in... Show moreThis thesis focuses on the development of sample-preparation methods for small amounts of samples and applying the developed methods to muscle tissues to investigate the mechanisms involved in sarcopenia. A fully automated, high-throughput, and high enrichment sample-preparation method, electroextraction, was developed. The metabolomics mechanism analysis of sarcopenia by using mouse models facilitate the understanding of metabolic processes underlying sarcopenia and can help to identify treatment options in the future. 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
The major and ultimate aim of metabolomics is to obtain an answer to a specific biological or clinical question. For that, many strategies have been applied in this field, including NMR and MS... Show moreThe major and ultimate aim of metabolomics is to obtain an answer to a specific biological or clinical question. For that, many strategies have been applied in this field, including NMR and MS-based approaches. CE-MS is one of the techniques in this field but remains underappreciated because of issues such as compromised sensitivity and poor reproducibility. In this thesis, we proposed standardized protocols for CE-MS studies using a sheathless interface and applied them in the metabolic analysis of ultra-small biological samples, such as low numbers of HepG2 cells. Another CE-MS technique used in this thesis is the traditional sheath liquid CE-MS, and it was successfully used in the metabolic profiling of plasma samples. We also introduced CE-MS with both interfaces in the field of nanosciences with our efforts in revealing the adsorption characteristics of polar metabolites to the surface of nanomaterials. This work clearly demonstrates that CE-MS can be reliably performed for metabolomics studies with acceptable repeatability. The use of sheathless CE-MS interface enables us to have in-depth profiling for ultra-small samples, and may become a powerful tool in the emerging field of single-cell analysis. Show less
The power of personalized nutrition lies in being able to conduct clinical research on healthy people while capturing metabolic markers sensitive to the impact of environmental and metabolic... Show moreThe power of personalized nutrition lies in being able to conduct clinical research on healthy people while capturing metabolic markers sensitive to the impact of environmental and metabolic stressors (e.g. diet, changing sex hormones and the menstrual cycle). Using clinical biomarkers, metabolomics, and diet interventions with intake analyses, we demonstrated the metabolic impact of vegan and animal diet interventions using fasting plasma analysis after 48 hours and using postprandial plasma analysis after meals and snacks. Sexually dimorphic responses were differentiated using proteomics and pathway analyses in two larger, sex-balanced cohorts. Finally, clinical biomarker and metabolomics analyses identified metabolic subtypes across menstrual cycle phases. Although challenges with integrating –omics technology and nutrition remain, the fundamental information generated from these research studies may provide a foundation for future novel personalized nutrition strategies. Show less
In recent decades, the use of a systems-based view of life has provided key insight into fundamental processes with respect to biology. In life sciences, important paradigm shifts are the way in... Show moreIn recent decades, the use of a systems-based view of life has provided key insight into fundamental processes with respect to biology. In life sciences, important paradigm shifts are the way in which we approach health and disease. Although modern medicine has traditionally emphasized pathology and acute conditions, our current understanding is that different interventions are needed for treating and preventing chronic disease. To design better interventions, new diagnostic tools are urgently needed in order to create new opportunities for achieving personalized health and medicine. The focus in diagnosis is, therefore, shifting from measuring single biomarkers such as glucose and cholesterol to creating complex maps of the dynamic patterns underlying regulatory processes. Moreover, the notion of "health" is viewed in a holistic context using biochemistry as a basis and then expanding this basis to include the psycho-social environment, including the individual's worldview. In the field of diagnostics, a highly promising new tool has recently emerged based on ultra-weak photon emission from biological systems, including all living cells. The aim of this thesis was to explore the applications of UPE and correlate it with biochemistry in order to obtain a deeper understanding of the processes that occur in living systems. Show less
Treating chronic diseases such as rheumatoid arthritis and type 2 diabetes mellitus is a hot topic that has been discussed widely and investigated extensively, but never solved, due in part to... Show moreTreating chronic diseases such as rheumatoid arthritis and type 2 diabetes mellitus is a hot topic that has been discussed widely and investigated extensively, but never solved, due in part to their high complexity. Integrating disease-related information using a systems approach may help improve our knowledge of stages of the disease, thus improving the accuracy of diagnosing chronic disease. With respect to integrative thinking, traditional Chinese medicine (TCM)‒based concepts may provide a suitable holistic model, as TCM describes disease syndromes/phenotypes as an experience-based reference from the systems level. Systems-based metabolomics provides a comprehensive picture of small molecular metabolites as a readout and provides biological interpretations of the pathophysiology of disease. The rapid, highly sensitive, non-invasive measurement of ultra-weak photon emission (UPE) -- which measures spontaneously emitted photons at the surface of the skin--has been proposed for supporting TCM-based diagnostics and for reflecting the whole body’s physiological and pathological status. Combining metabolomics with TCM-based diagnostics will provide a robust model for investigating the biological processes that underlie UPE. This thesis aimed to investigate system-wide perturbations by using/combining metabolomics, UPE and TCM-based diagnostics, to provide i) a systems view of chronic disease, and ii) personalized phenotyping guided by TCM-based principles. Show less
On the basis of systems thinking, in this thesis metabolomics, Chinese medicine (CM), as well as Western medicine (WM) were combined to achieve a more comprehensive systems diagnosis of patients... Show moreOn the basis of systems thinking, in this thesis metabolomics, Chinese medicine (CM), as well as Western medicine (WM) were combined to achieve a more comprehensive systems diagnosis of patients with chronic diseases. Specifically, metabolomics has been applied to characterize patients by small molecule profiles, which can be applied in phenotyping patients and matching these with optimal therapies. Chinese and Western medicine have different perspectives on diagnosis and could be highly complementary to each other, so combining both could have advantages in personalized diagnosis. Therefore, in this thesis a combination of systems approaches is used, including metabolomics, CM-based diagnosis principles as well as WM to provide systems diagnosis of patients with chronic diseases, in particular rheumatoid arthritis (RA). With systems diagnosis, we could better identify potential biomarkers to predict the WM therapeutic response of patients with RA and monitoring disease progression. Show less
In this thesis, metabolomics is used to study the role of the host-virus interaction on a metabolic level. A special emphasis is directed on the role of inflammation and oxidative stress on... Show more In this thesis, metabolomics is used to study the role of the host-virus interaction on a metabolic level. A special emphasis is directed on the role of inflammation and oxidative stress on the metabolic level, as part of the innate immune response against viral infection. We chose respiratory syncytial virus (RSV) and hepatitis B virus (HBV) as candidate viruses to metabolically study their role in acute respiratory infection and chronic hepatitis B infection. Secondly we also investigated infant metabolic and immunological consequences of in utero exposure to antiretroviral intervention and human immunodeficiency virus (HIV). Collectively, established targeted metabolomics approaches in conjunction with newly developed metabolomics methodologies and complemented with other “omics” techniques, were used to address pertinent questions related to host metabolic functioning and alterations during viral infection. In vitro RSV studies together with in vivo patient based studies relating to chronic HBV infection and in utero exposure too antiretroviral and HIV were used to address these questions. The work is divided into three research parts containing: i. the analytical methodology development work, ii. in vitro based metabolomics and iii. patient based metabolomics. Show less
The detailed description of the chemical compounds present in organisms, organs/tissues, biofluids and cells is the key to understand the complexity of biological systems. The small molecules ... Show moreThe detailed description of the chemical compounds present in organisms, organs/tissues, biofluids and cells is the key to understand the complexity of biological systems. The small molecules (metabolites) are known to be very diverse in structure and function. However, the identification of the chemical structure of metabolites is one of the major bottlenecks in metabolomics research. Hence, the annotation and the structure elucidation of the metabolites are essential to understand the biological system under study. Actually, no single analytical platform exists that can measure and identify all existing metabolites. Multistage mass spectrometry (MSn) is a powerful analytical technique that helps identifying all these metabolites. This technique provides detailed structural information of the unknown metabolite by fragmenting the metabolite and its fragments recursively. However, only computational tools can provide a fast and straightforward analysis of the large amount of complex data that is generated by using MSn spectrometry. The aim of this thesis was to develop a novel semi-automatic approach for the identification of metabolites using MS n data. Furthermore, these tools were to be integrated into a pipeline to assign identities to unknown metabolites present in databases but especially to unknown metabolites not present in a database Show less
The aim of this thesis was to develop concepts and methods to extract qualitative and quantitative information about metabolites from untargeted mass spectrometric data of biological samples.... Show moreThe aim of this thesis was to develop concepts and methods to extract qualitative and quantitative information about metabolites from untargeted mass spectrometric data of biological samples. Several typical challenges in data handling were addressed that prevent a straightforward interpretation (data analysis) of the data acquired with different types of mass spectrometric-based metabolomics methods (GC-MS, LC-MS, CE-MS or DI-MS) methods. The critical parameters causing variation in quantitative results were identified and studied at different stages in the metabolomics workflow such as data acquisition, data pre-processing and data analysis. Different methods and concepts were developed to address these and to improve the quantitation of metabolites and the comparison between metabolite data in different samples of the same study measured at different moments or between studies. The methods developed focused on improved normalization, data pre-processing of untargeted analysis and data pre-processing of high resolution direct infusion mass spectrometry data. Furthermore it was demonstrated that even for metabolomic studies with few samples cross-validation of multivariate models can be very time consuming and parallel implementation on a (large) cluster of computers is the way to make such computations feasible Show less
This dissertation describes the development and application of new mass spectrometry based tools which were used for the identification of various secondary metabolite pathways present in the... Show moreThis dissertation describes the development and application of new mass spectrometry based tools which were used for the identification of various secondary metabolite pathways present in the fungus Penicillium chrysogenum. Using these tools the biosynthesis of metabolites like roquefortine, meleagrin and fungisporin could be elucidated. Show less
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
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
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
Het is nu gangbaar in de kliniek om aan de hand van een enkele biochemische component (een gen, eiwit of metaboliet) vast te stellen of iemand ziek is. Echter, met geavanceerde meettechnieken zijn... Show moreHet is nu gangbaar in de kliniek om aan de hand van een enkele biochemische component (een gen, eiwit of metaboliet) vast te stellen of iemand ziek is. Echter, met geavanceerde meettechnieken zijn we nu in staat zijn om tientallen tot zelfs honderden van deze componenten tegelijk te meten. Hierdoor krijgen onderzoekers een veel breder overzicht van wat er allemaal verandert tijdens een ziekte en kunnen er betere diagnostische tests worden ontwikkeld. Voor dit proefschrift heb ik mij verdiept in deze technieken en hun toepasbaarheid in artrose. Artrose is een veel voorkomende rheumatische aandoening waarvan de exacte oorzaak nog onduidelijk is. Ik heb vastgesteld dat er met name nog weinig bekend is over het aandeel van de afbraakproducten van eiwitten en van vetten. Ik heb de benodige meettechnieken opgezet om honderden van deze afbraaktproducten en vetten te kunnen meten. Hieruit bleek dat een onstekingsmediator zeer verhoogd is in artrose en dat de samenstelling van vetten in het bloed van artrose patienten anders is dan bij gezonde mensen. De vindingen van dit proefschrift geven de kracht van deze meettechnieken aan om bij te dragen tot verbeterde diagnostische tests. Show less
