Metabolomics has the potential to play a pivotal role in understanding disease onset and progression, and ultimately personalized treatments. One of its major challenges is its large-scale... Show moreMetabolomics has the potential to play a pivotal role in understanding disease onset and progression, and ultimately personalized treatments. One of its major challenges is its large-scale implementation, which is necessary to deal with the high variability of the metabolome. In this work we have developed tools for automated sample handling and preparation for metabolomics analysis, and bioanalysis in general. The tools are versatile, suitable for high-throughput, and able to deal with sensitive and biomass-limited samples. Sample transfer through segmented-flow can accommodate a wide range of samples and volumes, and can work seamlessly with many downstream processing or analysis. Two sample preparation tools based on droplets; one universal preconcentration tools using controlled evaporation, and one based on simultaneous extraction and enrichment, also provide a versatile interface and can be used to bridge gaps between processing steps. The working principles of these sample handling and preparation tools are universal and can be adapted for specific applications. Show less
Utilizing the polymeric platform of polypept(o)ides, this thesis describes synthesis and investigation of novel triblock copolymers to obtain carrier systems with multiple compartments for... Show moreUtilizing the polymeric platform of polypept(o)ides, this thesis describes synthesis and investigation of novel triblock copolymers to obtain carrier systems with multiple compartments for efficient siRNA delivery. Although the individual microstructure of nanoparticles differs depending on the polymeric building blocks, desired application and cargo, the final nanoparticles always combine a polysarcosine (pSar) shell with a polypeptide core, providing the ability of siRNA complexation by a polycationic segment. In addition, a third block enabled either covalent cross-linking, hydrophobic / π- π-stacking mediated stabilization or co-encapsulation of small hydrophobic drugs. Broadening the structural variety of such polypept(o)ides, a novel synthetic procedure was introduced to access AA'B- and ABC-type miktoarm star polymers.Investigations have been dedicated to the design of novel polymeric structures based on polypept(o)ides, to improve the delivery of siRNA by Polyion Complex Micelles (PICMs), provide access to different polymeric architectures, and to establish novel synthetic methods for the synthesis of these materials. Covering aspects from the synthesis of novel polymeric species up to advanced drug delivery strategies for siRNA in vivo, developments throughout this thesis extent the accessibility of the polypept(o)ide platform for nucleic acid delivery, highlight their potential in nanomedicine and further elaborate delivery strategies for next-generation nanomedical applications. Show less
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
Atherosclerosis is a progressive disease resulting in the formation of an arterial plaque. Despite lipid lowering, recurrent cardiovascular events remain a risk. While atherosclerosis is primarily... Show moreAtherosclerosis is a progressive disease resulting in the formation of an arterial plaque. Despite lipid lowering, recurrent cardiovascular events remain a risk. While atherosclerosis is primarily lipid-driven, the immune system plays a critical role in the pathophysiology. Additional treatment could be achieved via immunomodulation. We aimed to identify potential biomarkers for monitoring of immunomodulatory drugs in future clinical trials and investigated pharmacological modulation of atherogenic pathways. We identified smokers and elderly healthy people as suitable groups for future clinical trials. We investigated the impact of sample aging on LPS responses, and optimized methodology for evaluation of LPS-driven neutrophil responses, in vitro and in vivo. As potential anti-atherogenic strategy, we evaluated the effect of pneumococcal vaccination on circulating oxLDL-IgM levels in man. The immunomodulatory impact of hydroxychloroquine, a drug with potential anti-atherogenic effects, was evaluated in healthy volunteers. A novel OX40L inhibitor was tested in healthy volunteers, since the OX40-OX40L axis may play a role in atherogenesis. OX40L inhibition was safe and effectively reduced T cell activity. Lastly, we showed that PD-1 agonism reduced atherosclerosis in Ldlr-/- mice. This thesis adds to the future development of effective and specific immunomodulatory treatments for atherosclerosis. 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
Cardiovascular diseases are still a major concern for the global health. The main underlying pathology of this disease is atherosclerosis which is characterized by the accumulation of lipids and... Show moreCardiovascular diseases are still a major concern for the global health. The main underlying pathology of this disease is atherosclerosis which is characterized by the accumulation of lipids and immune cells in the arterial wall leading to a chronic local inflammation and lesion formation. In this thesis, we aimed to (1) validate the use of zebrafish in cholesterol metabolism and atherosclerosis research, (2) study the role of certain classes of scavenger receptors in lipoprotein uptake and cholesterol-based functions, and (3) validated two immune-based potential targets for atherosclerosis. Show less
In this thesis, we developed new ways to use a technique called electroextraction (EE) to extract charged molecules from one liquid into another using an electric field. We wanted to make EE more... Show moreIn this thesis, we developed new ways to use a technique called electroextraction (EE) to extract charged molecules from one liquid into another using an electric field. We wanted to make EE more practical and easy to use for extracting different types of molecules. We tackled three main challenges: making EE work seamlessly with other techniques, avoiding the need to dilute small samples, and making EE more robust and user-friendly. To do this, we used automation and made use of existing analysis systems. This allowed us to fully take advantage of EE without needing manual processing or dilution. As a result, we were able to expand the range of compounds and types of molecules that EE can extract, and even extract both positively and negatively charged molecules at the same time. Show less
Breast cancer has a high mortality in women worldwide. Tumor cells experience hypoxia, which is accompanied by alterations in cell metabolism and can drive metastasis by triggering an epithelial... Show moreBreast cancer has a high mortality in women worldwide. Tumor cells experience hypoxia, which is accompanied by alterations in cell metabolism and can drive metastasis by triggering an epithelial–mesenchymal transition (EMT) in the tumor cells. Yes-associated protein (YAP) and a transcriptional co-activator with PDZ-binding (TAZ) are two transcriptional co-activators involved in growth, metabolism, and metastasis in cancer. Breast cancer can be divided into different subtypes. One criterium underlying such subtypes is based on the levels of Human Epidermal growth factor Receptor 2 (HER-2), Estrogen Receptor (ER) and Progesterone Receptor (PR). The subtypes include luminal-like (luminal A and luminal B), HER-2 enriched and basal-like (often “triple negative”). Triple negative breast cancer (TNBC) has a lower survival rate due to the lack of therapeutic targets. Fundamental research exploring the molecular mechanisms at work in cancer cells and their response to a hypoxic environment may contribute to insights for future clinical treatment. This thesis focused on profiling breast cancer cells belonging to distinct subtypes under acute and chronic hypoxia, investigating the crosstalk between hypoxia regulated pathways and YAP/TAZ signaling in luminal breast cancer versus TNBC cells, and identification of the potential targets of TAZ in breast cancer cells. Show less
Drug-induced liver injury (DILI) is one of the main reasons for drug attrition during pre-clinical and clinical phases of drug development as well as for drug withdrawal post-marketing. The... Show moreDrug-induced liver injury (DILI) is one of the main reasons for drug attrition during pre-clinical and clinical phases of drug development as well as for drug withdrawal post-marketing. The development of DILI can result in severe outcomes giving high risk for liver failure. The activation of adaptive stress response pathways is a way for cells to cope with drug-induced stress and is one of the early key events in the development of DILI. In this thesis, the regulation of the activation of adaptive stress responses has been studied using high content imaging, gene silencing and high throughput transcriptomics approaches to improve the understanding and prediction of DILI. The tight regulation of the unfolded stress response was dissected by combining computational modelling and RNA interference screening. Improved insight in the crosstalk between oxidative stress and DNA damage response during chemical exposure was obtained. Different liver test systems were compared for their capability to identify stress response activation, which allows for well-considered selection of models fit-for-purpose for drug screening. Furthermore, the inter-individual variability in stress response activation was mapped to enable the usage of accurately defined uncertainty factors to account for human population variability for DILI liabilities during safety testing. 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
Heterozygous germ-line mutations in BRCA1 and BRCA2 predispose to several types of cancer. Owing to their roles in the error-free repair of DNA double-strand breaks (DSBs) via homologous... Show moreHeterozygous germ-line mutations in BRCA1 and BRCA2 predispose to several types of cancer. Owing to their roles in the error-free repair of DNA double-strand breaks (DSBs) via homologous recombination (HR), lack of BRCA1/2 in these tumors results in DNA damage defects that can be specifically targeted by the inhibition of Poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is a key sensor of DNA damage and its inhibition has been shown to be synthetically lethal with deficiencies in HR, resulting in the selective killing of BRCA1/2-deficient tumor cells, while sparing BRCA1/2-proficient non-tumor cells. The success of this approach has resulted in the approval of four PARP1 inhibitors (PARPi) for the treatment of ovarian, breast, prostate and pancreatic cancers. However, drug resistance poses a major obstacle as, despite initial responses, patients receiving PARPi often develop resistance to the treatment. Understanding the molecular mechanisms behind PARPi resistance is therefore crucial to identify key determinants of PARPi response and to find combination treatment strategies to overcome resistance to PARPi by preventing, delaying or targeting resistant clones. In this thesis, we expanded our insights into the molecular mechanisms underlying PARPi resistance by conducting functional genetic screens in PARPi-resistance cell lines. 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
Chondrosarcoma and giant cell tumour of bone (GCTB) are bone tumours characterized by recurrent mutations (IDH1/IDH2 and H3F3A, respectively) that induce remodelling of the epigenetic landscape.... Show moreChondrosarcoma and giant cell tumour of bone (GCTB) are bone tumours characterized by recurrent mutations (IDH1/IDH2 and H3F3A, respectively) that induce remodelling of the epigenetic landscape. The standard of care for both of these sarcoma subtypes is surgery and alternative treatment options for patients with inoperable disease are currently lacking (chondrosarcoma) or suboptimal (GCTB). Therefore, the aim of this thesis was to identify novel therapeutic targets for high-grade chondrosarcoma as well as GCTB, with a focus on potential therapies that could counteract the remodelling of the epigenome. PARP and HDAC inhibition, alone or in combination treatment strategies, were identified as promising therapeutic strategies for chondrosarcoma or both of these bone tumours, respectively. Additionally, this thesis describes the development and use of novel 3D cell culture models which can be used to improve the translation of preclinical findings to the clinic. Show less
Parkinson's disease is the second most common neurodegenerative disease in the world. One of its symptoms is the loss of dopaminergic neurons in the substantia nigra pars compacta. A number of... Show moreParkinson's disease is the second most common neurodegenerative disease in the world. One of its symptoms is the loss of dopaminergic neurons in the substantia nigra pars compacta. A number of phenotypes, including the aggregation of misfolded proteins, mitochondrial dysfunction, and neuroinflammatory chemicals released by microglia and activated astrocytes, may all play a role in its pathogenesis.Due to the multisystemic nature of Parkinson's disease, novel tools for developing mechanistic models that simulate its pathogenic processes have been proposed. Furthermore, as the amount of information in biological databases grows and the cost of omics experiments decreases, methods for integrating different types of biological data have become essential for increasing the level of detail in mechanistic models of biological systems.Constraint-based modelling is a valuable tool in bioengineering and biomedicine. It is used to estimate the reaction flux in a metabolic network. The constraints represent essential characteristics of a biological system, including connectivity between metabolites and reactions, thermodynamics, maximum and minimum flux rates, and the steady-state.This thesis presents studies and tools for integrating various types of specific information to genome-scale models used in constraint-based modelling. In addition, is presented the iDopaNeuro default models, genome-scale models of a culture of dopaminergic neurons derived from induced pluripotent stem cells. Show less
Acute cardiovascular clinical events such as myocardial infarction and cerebral stroke represent the major cause of death in Western societies. These pathologies are primarily resulting from... Show moreAcute cardiovascular clinical events such as myocardial infarction and cerebral stroke represent the major cause of death in Western societies. These pathologies are primarily resulting from atherosclerosis, a progressive condition characterized by the accumulation of lipids, immune cells, and fibrous elements in large arteries. The pathogenesis of atherosclerosis involves complex interactions between a wide variety of cells, including monocytes, macrophages, neutrophils, and lymphocytes. It is essential to identify novel targets for therapeutic application in order to reduce the residual atherosclerotic cardiovascular disease risk in current and future patients. Recent studies have suggested that members of the protein arginine methyltransferase (PRMT) family can potentially serve as novel therapeutic targets for atherosclerosis because of their regulatory role in inflammation and metabolism. To validate the contribution of PRMTs in the progression of atherosclerosis, in the studies presented in this thesis we have investigated the effect of inhibition of PRMT functionality on atherosclerosis susceptibility in established atherosclerotic mouse models.To address the role of PRMTs in atherosclerosis, we therefore made use of specific PRMT inhibitors, i.e. TC-E 5003 for PRMT1 inhibition, TP-064 for PRMT4 inhibition, and GSK3326595 for PRMT5 inhibition, that thus far have primarily been applied in vivo in the context of cancer treatment. Show less
Drug induced organ toxicity is the main problem of the drug development and drug usage in the clinic. The liver and kidneys are the most sensitive organs towards drug induced toxicity. The liver... Show moreDrug induced organ toxicity is the main problem of the drug development and drug usage in the clinic. The liver and kidneys are the most sensitive organs towards drug induced toxicity. The liver neutralizes xenobiotic to which human are exposed to while the kidneys remove waste products from the blood. Due to their detoxification function, these organs are continuously exposed to high amount of toxicants leading to potential injury. Investigating the molecular and cellular mechanisms contributing to drug-induced liver injury (DILI) and drug-induced kidney injury (DIKI) will open new avenues that can help in the prediction of induced organ injury caused by drugs as well as other xenobiotics.In this thesis, we mapped the dynamics of cellular stress responses in the liver and kidneys upon the exposure of a set of model compounds in order to gain a more holistic insight in DILI and DIKI. We conducted multiple extensive in vitro and in vivo studies to understand the dynamics of these cellular responses and determined the translation of our findings from in vitro to in vivo. Transcriptomics analysis was central in the research which was complemented with other methodologies, such as reporter cell assay, immunohistochemistry, to unravel the mechanisms of drug-induced organ toxicity in both liver and kidney. Show less
Antimicrobial drugs constitute a fundamental part of modern medicine. The global rise in antimicrobial resistance poses a major threat to global health. Optimising antimicrobial treatment... Show moreAntimicrobial drugs constitute a fundamental part of modern medicine. The global rise in antimicrobial resistance poses a major threat to global health. Optimising antimicrobial treatment strategies in patients offers an important direction to address this challenge. In this thesis, we describe how quantitative characterisation of the drug, the pathogen, and the patients, and how these three factors interact, can help to achieve this goal. To this end, we used a combination of state-of-the-art in silico model-based approaches to analyse and integrate experimental data from in vitro models, and clinical data from healthy volunteers and patients. We developed models describing infection site drug exposure, antimicrobial resistance evolution, and host response biomarker dynamics. We explored the impact of infection on pulmonary pharmacokinetics, evolutionary-based treatment strategies, and the utility host response biomarker for treatment monitoring. The work in this thesis builds towards developing novel strategies to optimise antimicrobial treatments and showcases the importance on interdisciplinary collaborations. Show less
Als gevolg van de grote technologische vooruitgang in de gezondheidszorg worden in toenemende mate gegevens verzameld tijdens de uitvoering van klinische onderzoeken. Het is evenwel essentieel om... Show moreAls gevolg van de grote technologische vooruitgang in de gezondheidszorg worden in toenemende mate gegevens verzameld tijdens de uitvoering van klinische onderzoeken. Het is evenwel essentieel om te beseffen dat gegevens op zich van weinig of geen waarde zijn. Ten behoeve van hun optimale bruikbaarheid dienen gegevens geanalyseerd, geïnterpreteerd en verwerkt te worden. Machine learning-strategieën kunnen hiertoe nuttige en adequate oplossingen bieden. Dit proefschrift bevat machine learning-benaderingen toegepast op verschillende klinische datasets. De klassieke gegevens bestaan uit elektrische signalen van het electrocardiogram (ecg) verkregen bijgezonde proefpersonen, de innovatieve gegevens zijn afkomstig vanmetingen in een rijsimulator, en de opkomende gegevens zijn afgeleid van dna-analyse van de micro-organismen die op de huid voorkomenvan patiënten met huidziekten. We toonden aan dat het aantal ECG’s van invloed was op de nauwkeurigheid van geschatte verlenging van het qt-interval voor alle ingezette qt-correctieformules. Met behulp van SHapley AdditiveexPlanations (shap)-waarden werd de impact van de individuele kenmerken op de voorspelling van fysiologische leeftijd van het hart bepaald. We maakten gebruik van machine learning voor een betere beoordeling van de rijprestaties van bestuurders die medicijnen gebruikten. Tot slot lieten we zien dat de belangrijkste micro-organismen voor discriminatie van seborrroische dermatitis – naast Cutibacterium en Staphylococcus – kwamen relatief weinig voor, waardoor men deze micro-organismen in standaardanalyses eenvoudig over het hoofd kan zien. Daarmee hebben we aangetoond dat machine learning kanworden toegepast op gegevens die zijn afgeleid van klinische onderzoeken om in een vroeg stadium het effect van medicijnen en andere interventies op te sporen en te evalueren. Show less
