This thesis describes the development and evaluation of a new laboratory tool to improve early kidney injury diagnosis. In the first part, we define the unmet clinical needs in the clinical care... Show moreThis thesis describes the development and evaluation of a new laboratory tool to improve early kidney injury diagnosis. In the first part, we define the unmet clinical needs in the clinical care pathways in kidney injury, by a questionnaire and s literature review to summarize current state-of-the-art diagnostics. The second part of this thesis comprises the analytical phase of test development. Liquid-chromatography (LC) coupled to tandem MS was chosen as analytical platform for protein-based kidney injury biomarker quantitation from human urine specimens. The third part of this thesis covers aspects of the postanalytical phase of medical test development, which included the establishment of reference intervals and clinical observational cohort studies. Elevated levels of the studied kidney injury biomarkers, and their combined signatures, were associated with acute kidney injury induced by ischemia-reperfusion injury after kidney transplantation, but not with stable chronic kidney disease. In a general perspective, this thesis highlights the role of quantitative protein mass spectrometry for biomarker translation from research towards the clinical laboratory. Show less
Metabolomics, proteomics, and genomics analyses provide profound insight into human biology and disease pathophysiology. In this thesis, we explored the methodological challenges facing these OMICs... Show moreMetabolomics, proteomics, and genomics analyses provide profound insight into human biology and disease pathophysiology. In this thesis, we explored the methodological challenges facing these OMICs technologies and illustrated their applications in epidemiological studies. In part one, we focused on some of the methodological challenges facing OMICs research. These challenges included handling missing data in metabolomics, measurement agreement between high throughput proteomic measurements with standard clinical measurements, and challenges in developing prediction models using metabolomic data. The second part of this thesis addressed various epidemiological research questions by utilizing genomic data and metabolomics measurements (Metabolon and Nightingale platforms) and using advanced data analysis methods. These studies provided important insights into the associations between metabolites and hepatic triglyceride content, the associations of between the size of cytosine-adenine-guanine nucleotide repeats in the huntingtin gene with metabolomic profile, and the associations of the man-made per- and polyfluoroalkyl substances (PFAS) with metabolite levels. Show less
Filamentous Actinobacteria, such as Streptomyces, produce a plethora of chemically diverse bioactive metabolites that have found applications across medicine, agriculture and biotechnology. Yet,... Show moreFilamentous Actinobacteria, such as Streptomyces, produce a plethora of chemically diverse bioactive metabolites that have found applications across medicine, agriculture and biotechnology. Yet, the vast majority of the biosynthetic potential of Actinobacteria remains uncharacterised, largely because their biosynthetic gene clusters (BGCs) are poorly expressed in the laboratory, preventing the discovery of the cognate natural products. Additionally, only a narrow band of environments and a few taxonomic groups have been explored for gifted Actinobacteria. In this thesis different approaches are described, wherein we combined drug discovery with ecology, aimed at accessing the full potential of Actinobacteria. Bioactive Actinobacteria were isolated from a faecal sample of a 28,000-year-old-mammoth and their taxonomic and metabolic diversity was analysed. Furthermore, the effect of human stress hormones on the production of antibiotics by Streptomyces was investigated, resulting in the discovery of adrenaline as elicitor of siderophore production. This was later shown to be caused by the adrenaline analog catechol, which is ubiquitous in nature. Catechol also elicited the production of angucycline glycosides, well known for their therapeutic potential as anticancer and antibiotic compounds. Lastly, zebrafish were used as an in vivo model to explore the bioactive and functional potential of Actinobacteria within the animal microbiome. Show less
This thesis highlights, firstly, the importance of early CRC detection by presenting results of a CRC diagnostic proteomic biomarker signature with high discriminative power. Secondly, a strong... Show moreThis thesis highlights, firstly, the importance of early CRC detection by presenting results of a CRC diagnostic proteomic biomarker signature with high discriminative power. Secondly, a strong robust, independent prognostic tumor stroma ratio (TSR) biomarker, which confirms to be of important clinical value. The TSR has the ability to stratify colon cancer patients according to their prognostic outcome in a highly reproducible and low-cost manner. It has shown to link patients with a high intra tumor stromal content and a worse prognosis. Literature shows a wealth of evidence that supports this prognostic value in CRC as well as in other cancers. This PhD research therefore concludes that it should be implemented in the official guidelines of the TNM classification to improve stratification for CRC patients in daily routine pathological evaluation. The prospective, international, multicentre UNITED study will hopefully overcome the last hurdle for this clinical implementation. Lastly, this thesis offers more insight in the elusiveness of the tumor microenvironment and stromatogenesis that contributes to the aggressiveness of some CRC tumors. The biological differences, interconnections and changes in the microenvironment presented give multiple leads for further research and new personalized treatment possibilities. Show less
Genotoxic stressors may induce various types of DNA damage triggering the activation of the DNA damage response. However, the dose-dependency and the drug compound-specificity of the activated... Show moreGenotoxic stressors may induce various types of DNA damage triggering the activation of the DNA damage response. However, the dose-dependency and the drug compound-specificity of the activated responses are unclear. Here, I first investigated the phospho-signalling responses induced by four chemical stressors with distinct modes of action and uncovered that, at equitoxic doses, even two ‘similar’ DNA damaging agents induced discrete and complex phosphorylation signalling cascades. Next, I investigated the ionising radiation (IR) dose-dependent responses using multi-omics and systems analyses to investigate the molecular and cellular responses to low and high doses of IR. I uncovered that the IR induced molecular responses have different dose-effect relationships. A minor part of the phosphorylation signalling and transcription involved in the DSB-related responses displayed a linear dose-effect relationship. Contrastingly, the reactive oxygen species (ROS) production-related molecular and cellular responses and DNA replication stress revealed complex dose-response relationships. LD uniquely activates ROS-related responses at multiple molecular levels, including proteome, phosphoproteome, nascent transcriptome and metabolome. Further work will be required to determine how these differences in molecular and cellular responses observed over hours after low and high doses impact the risk for cancer development that occurs over months and years. Show less
The human body consists of hundreds, perhaps thousands of different types of cells, each with different morphologies and functions, despite having the same genome. This diversity is created by gene... Show moreThe human body consists of hundreds, perhaps thousands of different types of cells, each with different morphologies and functions, despite having the same genome. This diversity is created by gene regulation, a set of mechanisms that determine, which genes are used to make proteins and which genes are kept silent. During embryonic development, gene are turned on and off in a tightly orchestrated manner, to make sure that the right cell type is created at the right time and place.In this thesis we report several studies pertaining to gene regulation in embryonic development. Each of the four chapters will cover a different layer of the gene regulation toolbox: gene inactivation by DNA methylation, transcriptional regulation in the developing kidney, regulation of protein turnover and translational regulation through micro-RNAs. Together, these studies provide a refined understanding of the crucial role of gene regulation for embryonic development. Show less
Actinobacteria are Gram-positive bacteria that have a complex multicellular life cycle and are well known for their ability to produce a wide range of bioactive natural products (NPs). High... Show moreActinobacteria are Gram-positive bacteria that have a complex multicellular life cycle and are well known for their ability to produce a wide range of bioactive natural products (NPs). High throughput screening has failed to deliver the new antibiotics we so desperately need to combat multidrug-resistant pathogens. Therefore, new systematic approaches are needed to further explore the rich potential of Actinobacteria. The work described in this thesis entails systems biology approaches consisting of technologies such as proteomics, genomics, metabolomics and DNA binding studies. These were then applied to identify the biosynthetic gene clusters (BGCs) that are responsible for the production of novel antibiotics. Small molecules were thereby used as elicitors to activate the expression of cryptic BGCs in Streptomyces roseifaciens. Furthermore, S. coelicolor M1152 that was optimized for heterologous expression of antibiotics, was analysed for changes in protein expression, to understand which changes correlate to optimal antibiotic production. Finally, the role of the nucleoid associated protein SCO1839 in development and antibiotic production was studied. Chip-seq technology showed that it binds to thousands of DNA sequences on the S. coelicolor chromosome, which contain the motif GATC. I hope that this thesis contributes to utilizing multi-dimensional ‘omics approaches to answer major biological questions. Show less
The aim of this thesis is to identify emerging risk factors for VTE. To achieve this goal, we describe the supposedly causal role of statin and glucocorticoid use (i.e. two drugs that can influence... Show moreThe aim of this thesis is to identify emerging risk factors for VTE. To achieve this goal, we describe the supposedly causal role of statin and glucocorticoid use (i.e. two drugs that can influence inflammation) with changes in hemostasis and VTE risk. Systemic glucocorticoid use increases the relative risk of first VTE by more than three-fold and confers an 5% absolute risk of recurrent VTE per year. On the other hand, rosuvastatin use may reduce the risk of first VTE by 40%. Although the mechanisms behind this association are not fully elucidated, this thesis shows that rosuvastatin is capable of decreasing the thrombin generation potential by 10% in patients with a prior VTE. This thesis has shown that both statins and systemic glucocorticoids can affect the risk of VTE, improving the knowledge on the influence of these two commonly prescribed drugs on VTE pathophysiology. These findings have the potential to further refine the assessment of VTE risk since they highlight that the use of these drugs should be considered when evaluating the risk of VTE. Finally, this thesis provides insight into new therapeutic approaches since the results underscore that treatment strategies on VTE prevention in patients already taken statins, which may be sufficient for VTE prevention, are lacking. Treatment strategies to prevent glucocorticoid-associated VTE are also needed. 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
This thesis describes the introduction of the zebrafish animal model to the field of proteomcis. The work presented provided the research community with the first zebrafish spectral library, which... Show moreThis thesis describes the introduction of the zebrafish animal model to the field of proteomcis. The work presented provided the research community with the first zebrafish spectral library, which is an important first step to introduce zebrafish to proteomics research. This thesis also provides proteomics resourses to automate and simplify the creation of spectral libraries. Furthermore, it describes the use of the software programme CompareMS2 to provide information on similarities across proteomics databases. In addition, the programme has shown to be applicable as a quick first analysis step on large proteomics datasets. Besides, the thesis demonstrates the advantages and applicability of a multidisciplinary approach, addressing multiple omics, exemplified by using two closeley related model species (zebrafish and common carp). Finally, an overview is given on the advantages and disadvantages of the zebrafish animal model within the field of proteomics. The practical approach to zebrafish models in proteomics described in this thesis resulted in experimental protocols, a standard spectral library and data analysis tools, and significantly contributed to maturing of the zebrafish animal model system for proteomics research. Show less
The thesis describes the development of a number of novel mass spectrometric methods for the protein analysis of Gram-negative bacteria. These applications are developed with the aim of finding new... Show moreThe thesis describes the development of a number of novel mass spectrometric methods for the protein analysis of Gram-negative bacteria. These applications are developed with the aim of finding new and improved diagnostic routes for the typing of bacteria and their antibiotic resistance. The research is application driven and the focus is on utilizing high-end mass spectrometric instrumentation in diagnostic clinical microbiology, in a complimentary nature to already established techniques. Show less
The work presented in this thesis describes the improvement and application of on-tissue chemistry for in-situ biomolecular analysis using matrix assisted-laser desorption/ionization mass... Show moreThe work presented in this thesis describes the improvement and application of on-tissue chemistry for in-situ biomolecular analysis using matrix assisted-laser desorption/ionization mass spectrometry imaging (MALDI-MSI). We have proposed new methodologies, applying on-tissue (enzymatic) chemistry, to increase the molecular information obtained in a MALDI-MSI analysis. We have also developed an automated histology-guided MSI platform, based on state-of-the-art image processing tools, to facilitate high mass and spatial resolution MALDI-MSI while maintaining reasonable data loads and acquisition times. We have shown the importance of these methods in a clinical biomarker discovery study on myxoid liposarcoma tissues. Show less
Parasitic helminths are important organisms to study because their infections have both adverse and beneficial effects on the human host. Helminth infections are considered a burden, as these... Show moreParasitic helminths are important organisms to study because their infections have both adverse and beneficial effects on the human host. Helminth infections are considered a burden, as these infections cause significant morbidity in a large proportion worldwide. However, helminth infections, by means of their ability to modify host immune responses can also provide protection against inflammatory diseases (inflammatory bowel disease, diabetes, and asthma). It is important to better understand the underlying mechanisms of these Yin (positive) and Yang (negative) consequences of helminth infections. The general objective of this thesis is to track helminths at different levels. On the one hand to improve the detection of helminth infections, essential for the studying helminths and the interaction with their human host. Moreover, a more sensitive diagnostics is instrumental for monitoring the distribution of helminth infections and to evaluate the helminth infections elimination program. On the other hand, to understand the mechanistic insights of the interplay between helminths and the host immune system results in priming of Th2 and regulatory T cell responses. This could contribute to the identification of targeted pathways to manipulate immune responses, as part of developing therapeutics to treat inflammatory disorders characterized by deregulated Th2 and/or Treg responses. Show less
The Middle to Upper Palaeolithic transition in Europe witnesses the local replacement of Neanderthals by Anatomically Modern Humans. During this period several “transitional” technocomplexes are... Show moreThe Middle to Upper Palaeolithic transition in Europe witnesses the local replacement of Neanderthals by Anatomically Modern Humans. During this period several “transitional” technocomplexes are present that seem to combine behavioural characteristics from both the preceding Middle and following Upper Palaeolithic. These technocomplexes therefore play a critical role in our understanding of Neanderthal behavior, cognition and extinction, the dispersal of AMHs into Europe, and the interactions between Neanderthals and AMHs. Unfortunately, biological remains of the hominins responsible for the transitional technocomplexes are notoriously scarce. This dissertation proposes to utilize recent advances in mass spectrometry to identify additional hominin specimens through ZooMS screening and shotgun proteomics. Novel approaches towards de novo/error-tolerant proteomic analysis in Pleistocene contexts were developed and tested, allowing phylogenetic analysis of ancient protein sequences from now-extinct Mammalian species and hominin populations. ZooMS screening is shown to be highly successful for all included Châtelperronian sites. The discovery of additional hominin specimens at one archaeological site allowed demonstrating that ancient protein sequences can be used to differentiate between hominin populations, in this case indicating a Neanderthal affiliation. As a result, palaeoproteomics now allows studying the evolutionary relationships between hominin individuals based on ancient protein sequences. Show less
In this thesis mass spectrometry based protein profiling was applied as a new biomarker screening modality and it was evaluated whether or not this could be translated into early detection... Show more In this thesis mass spectrometry based protein profiling was applied as a new biomarker screening modality and it was evaluated whether or not this could be translated into early detection of breast cancer and pancreatic cancer. The status of breast cancer screening by proteomic profiling is discussed. Which steps have already been made? What is essential to implement this techniques in a clinical setting? Furthermore, the new protein profiling screening methods for pancreatic cancers are evaluated. Future studies will be suggested that are needed to translate this promising biomarker into a clinical application. Show less
Recent developments in the coupling of liquid phase separations and mass spectrometry have yielded some new source designs for electrospray ionization. The research in this thesis covers the... Show moreRecent developments in the coupling of liquid phase separations and mass spectrometry have yielded some new source designs for electrospray ionization. The research in this thesis covers the application of a newly developed electrospray ionization source design and it's application for the coupling of capillary electrophoresis to mass spectrometry. The initial chapters cover improvements in sensitivity and separation power in capillary electrophoresis mass spectrometry (CE-MS) by allowing electrospray at ultra low flow rates and therefore optimal ionization and separation conditions. We showed strong complementarity of this CE-MS system with reversed phase liquid chromatography in the identification of peptides and proteins from e.coli digests. Finally a sample preparation method for peptide and protein identification from laser micro-dissected tissue samples from human kidneys was developed. It was shown that a few hundred proteins could be identified from amounts of material representative of one full human glomerulus. On the whole great strides were made in development of strategies employing CE-MS for proteomics analysis. Show less
In part 1 of the thesis quantitative proteomics was used to determine changes in abundance and phosphorylation status of host proteins during infection with the human pathogen chikungunya virus ... Show moreIn part 1 of the thesis quantitative proteomics was used to determine changes in abundance and phosphorylation status of host proteins during infection with the human pathogen chikungunya virus (CHIKV). Several proteins were identified that may be specifically downregulated during CHIKV infection to create a suitable environment for viral replication. eEF2 was identified as a factor that becomes strongly phosphorylated during infection with several viruses which may be a mechanism to stall translation in response to viral infection. In part 2 of the thesis the discovery of a novel and unusual -2/-1 programmed ribosomal frameshifting (PRF) mechanism is described that is used during translation of the nonstructural open reading frame of the economically important porcine reproductive and respiratory syndrome virus (PRRSV). This mechanism relies on a slippery site and stimulatory RNA signal in the PRRSV genome and is stimulated by the viral protein nsp1beta and host poly (C) binding proteins 1 and 2. Frameshifting results in the synthesis of two previously unidentified proteins, nsp2 TF (-2 PRF) and nsp2N (-1 PRF). Virus mutants that can no longer make the frameshift products are attenuated and may be used for vaccine development. Show less
SUMOylation is a reversible lysine post-translational modification critical for eukaryotic life, and involved in many diseases such as cancer. This thesis describes five years of research into... Show moreSUMOylation is a reversible lysine post-translational modification critical for eukaryotic life, and involved in many diseases such as cancer. This thesis describes five years of research into SUMOylation using cutting edge high-resolution mass spectrometry, at the system-wide level and in an unbiased manner. Various novel and highly efficient methods for purifying SUMOylation from complex samples are described, and validated through mass spectrometry. Overall, we report an unprecedented number of SUMOylation sites, shed light on the dynamic nature of SUMOylation in response to various cellular treatments and stress conditions, and provide an unsurpassed level of depth through extensive bio-informatics analysis. Show less
A large part of modern biology is dedicated to the functional annotation and interpretation of genetic information and its influence on the subject__s phenotype. Proteomics describes the state of... Show moreA large part of modern biology is dedicated to the functional annotation and interpretation of genetic information and its influence on the subject__s phenotype. Proteomics describes the state of the system from the perspective of expression, structure, localization, interaction and function of the proteins. It is a complex field which requires a combination of various disciplines such as cellular biology and biochemistry for sample preparation, analytical chemistry for sample measurement and bioinformatics for the processing of data. Different chapters of this thesis illustrate various aspects of the proteomics pipeline and emphasize the importance and connection between them. Therefore the information in this dissertation can be divided into three major parts depicting these aspects of a proteomics experiment. First, sample preparation for proteomics has been addressed through two studies aimed at decreasing sample complexity and increasing proteome coverage. The second part is dedicated mostly to the technical step of the mass spectrometry measurement and the analysis of obtained spectra. The final part describes example- and proof-of-principle applications. Show less
Myxoid tumours of soft tissues are a heterogeneous group of mesenchymal origin characterized by their so-called myxoid extracellular matrix. although their histological overlap, their clinical... Show moreMyxoid tumours of soft tissues are a heterogeneous group of mesenchymal origin characterized by their so-called myxoid extracellular matrix. although their histological overlap, their clinical behaviour ranges form benign to malignant. In this thesis we studied the exact nature and possible function of the components of the myxoid matrix using a broad range of techniques (including (immuno)histochemistry and (imaging) mass spectrometry. Furthermore, we studied the molecular (cyto)genetic aberrations in different myxoid soft tissue tumours, using direct sequence analysis and COBRA FISH. Show less
