The sequence-dependence of biomolecular interactions involving nucleic acids and proteins is essential for numerous processes inside the cell. Insights into the underlying molecular mechanisms have... Show moreThe sequence-dependence of biomolecular interactions involving nucleic acids and proteins is essential for numerous processes inside the cell. Insights into the underlying molecular mechanisms have been obtained using various biochemical and biophysical methods on two different levels — bulk and single-molecule. These have complemented each other as single-molecule studies excel in observing multi-state dynamic interactions, but perform only at low throughput; while bulk studies can probe many different sequences simultaneously, but providing limited kinetic information. To unite the strengths of both levels, we developed high-throughput Single-molecule Parallel Analysis for Rapid eXploration of Sequence space (SPARXS), that allows the study of molecular structure, kinetics and interactions for thousands of different sequences simultaneously at the single-molecule level. We, for the first time, combine single-molecule fluorescence with next-generation Illumina sequencing. As a proof of principle we apply SPARXS to study the sequence-dependent kinetics of the four-way DNA Holliday junction, occurring among others during homologous recombination. Using SPARXS we observe the dynamic behavior of 120,000 Holliday junction molecules covering 3750 distinct core sequences, a result unattainable with previous techniques. Overall, the mechanistic insights obtained using SPARXS will give an entirely new view on the relationship between sequence, structure and function. Show less
Protein glycosylation has profound implications in a wide range of molecular and biological processes occurring in cancer, where specific changes in the glycan structures have shown to be... Show moreProtein glycosylation has profound implications in a wide range of molecular and biological processes occurring in cancer, where specific changes in the glycan structures have shown to be associated with the development and progression of the disease paving the way for the development of new clinical biomarkers as well as providing specific targets for therapeutic intervention, patient stratification and personalized medicine. Protein glycosylation is also critical for the development of biopharmaceuticals, as even minor shifts in manufacturing procedures can substantially impact the bioactivity, safety, and efficacy of therapeutic proteins. Although a variety of mass spectrometric and chromatographic methods are available for the identification and characterization of glycans from complex sample mixtures, the lack of standardized protocols across platforms often results in inconsistent results, making data integration and comparison challenging. Furthermore, most of the current technology for the study of intact glycans would not be suitable for the rapid analysis of large sample sets, mainly due to limitations in sample throughput. The scope of this thesis is to establish standardized, high-throughput glycomics technologies for the quantitative analysis of protein N- and O-glycosylation and improve current methodologies in order to facilitate the characterization of intact oligosaccharides from in vitro established model systems. 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
The challenge of achieving fast quantification in metabolomics is the presence of severe matrix effects during the MS analysis of complex samples. Complex samples also result in challenges during... Show moreThe challenge of achieving fast quantification in metabolomics is the presence of severe matrix effects during the MS analysis of complex samples. Complex samples also result in challenges during metabolite identification as complex MS/MS spectra and peak overlap in 1H NMR complicate structure elucidation. The goal of this thesis is to tackle these challenges by the development and application of innovative fractionation approaches and state-of-the-art MS and NMR analyses. Show less
The human metabolome provides a direct physiological read-out of an individual's actual health state and includes biomarkers that may predict disease or response to a treatment. The discovery and... Show moreThe human metabolome provides a direct physiological read-out of an individual's actual health state and includes biomarkers that may predict disease or response to a treatment. The discovery and validation of these metabolomic biomarkers requires large-scale cohort studies, typically involving thousands of samples. This analytical challenge drives novel technological developments to enable faster, cheaper, and more comprehensive metabolomic analysis: more for less.This review summarises recent (2012–2018) developments towards this goal in all aspects of the analytical workflow, in relation to NMR but primarily to mass spectrometry (MS). Recent trends include miniaturisation and automation of extraction techniques, online coupling to fast analysis methods including direct infusion ion mobility MS, integrated microfluidic devices, and sharing and standardizing metabolomics software and data.The technological advances in metabolomics support its widespread application, integration with other -omics fields, and ultimately disease prediction and precision medicine. Show less
Ent, W. van der; Veneman, W.J.; Groenewoud, A.; Chen, L.P.; Tulotta, C.; Hogendoorn, P.C.W.; ... ; Langenau, D.M. 2016
Zebrafish embryos can be obtained for research purposes in large numbers at low cost and embryos develop externally in limited space, making them highly suitable for high-throughput cancer studies... Show moreZebrafish embryos can be obtained for research purposes in large numbers at low cost and embryos develop externally in limited space, making them highly suitable for high-throughput cancer studies and drug screens. Non-invasive live imaging of various processes within the larvae is possible due to their transparency during development, and a multitude of available fluorescent transgenic reporter lines. To perform high-throughput studies, handling large amounts of embryos and larvae is required. With such high number of individuals, even minute tasks may become time-consuming and arduous. In this chapter, an overview is given of the developments in the automation of various steps of large scale zebrafish cancer research for discovering important cancer pathways and drugs for the treatment of human disease. The focus lies on various tools developed for cancer cell implantation, embryo handling and sorting, microfluidic systems for imaging and drug treatment, and image acquisition and analysis. Examples will be given of employment of these technologies within the fields of toxicology research and cancer research. Show less
Ewing sarcoma (EWS) is a disease with a high need for novel therapeutic strategies. To aid in investigating such compounds in an in vivo setting, we have developed several zebrafish model systems... Show moreEwing sarcoma (EWS) is a disease with a high need for novel therapeutic strategies. To aid in investigating such compounds in an in vivo setting, we have developed several zebrafish model systems for EWS, which are presented in this thesis. The first is a manual xenograft model in 2-day-old zebrafish embryos, in which cell behaviour on the level of proliferation and migration has been described, as well as their interaction with the host innate immunesystem. With this model, we found that simultaneous induction of the p53 pathway and disruption of EWS-FLI1 transcriptional activity had an additive effect on the reduction of EWS malignancy in vivo. The same model was used to explore the mechanism of NOTCH-induced tumour suppression, via Sirtuin1 (SIRT1) inhibition. In addition to this manual xenograft model, we also established a high-throughput automated implantation model in blastula stage embryos. Furthermore, a flexible transgenic model has been developed, where human EWSR1-ERG expression has been placed under an UAS promoter, allowing the gene to be expressed in different tissues at different times. When this EWS-driving fusiongene was expressed neuronally, overlap in gene and protein expression was found with other EWS models, as well as a histologic similarity to EWS tumours. Show less
High-grade osteosarcoma is a primary bone tumor with complex genetic alterations, for which targeted therapy is lacking. The aim of this thesis was to use high-throughput molecular data analysis of... Show moreHigh-grade osteosarcoma is a primary bone tumor with complex genetic alterations, for which targeted therapy is lacking. The aim of this thesis was to use high-throughput molecular data analysis of high-grade osteosarcoma specimens and model systems, in order to learn more on osteosarcomagenesis and to find possible ways to inhibit this process. By analyzing different microarray data types using a systems biology approach, genomic instability was identified as an important driver of osteosarcomagenesis. A protective role of macrophages against metastasis of osteosarcoma was detected. In addition, the IR/IGF1R and PI3K/Akt signaling pathways were discovered as potential targets for treatment. This thesis provides the first steps in unraveling the genomic and transcriptomic landscape of high-grade osteosarcoma, and provides a biological rationale for certain new options for adjuvant treatment of this highly genomica lly unstable tumor. Show less
