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
The research described in this thesis aims at the development of ubiquitin-based research tools to study the enzymes of the ubiquitination pathway, the ligase enzymes and the deubiquitinating... Show moreThe research described in this thesis aims at the development of ubiquitin-based research tools to study the enzymes of the ubiquitination pathway, the ligase enzymes and the deubiquitinating enzymes. These enzymes are responsible for the conjugation and the removal of the post-translational modifier ubiquitin. This small protein is involved in almost all cellular processes, and when conjugated onto a substrate protein it can signal for degradation and influence the localization, interaction, stability and activity of the protein. Therefore, the dysregulation of these processes can have detrimental effects of cell organization and survival which in turn has implications in numerous processes related to diseases. Hence, it is important to fully understand the ubiquitination pathway and how to interact with it. The ubiquitin-based research tools described in this thesis aim to shine light on parts of this pathway. Ranging from the selectivity and specificity of DUBs for specific Ub linkages in competition and the catalytic efficiency of these proteolytic cleavage processes to the selectivity and activity of ligases and the activity of DUBs in cells. All ubiquitin research tools are based on synthetic ubiquitin modified with unnatural amino acids, neutron-encoded amino acids, point mutations and/or fluorescent labels, in order to study the characteristics of the enzymes in vitro. Show less
Glycosylation is a widely occurring and complex modification found on lipids and proteins and is involved in the recognition, signaling and interaction events within the cell and between cells.... Show moreGlycosylation is a widely occurring and complex modification found on lipids and proteins and is involved in the recognition, signaling and interaction events within the cell and between cells. These events based on glycan structures result in adhesion, cell-matrix interaction and immune recognition. Alterations in the glycomic profile are considered a hallmark of various diseases, including cancer where it contributes to the development and progression of cancer, affecting cell-cell communication, cell-matrix interactions, tumor angiogenesis, invasion and metastasis. These functions are governed by different glycans and their terminal structures. In order to further explore these structures with regard to their potential as biomarkers and specific targets for diagnostic applications and therapeutical strategies for various diseases, in-depth glycomic analysis is needed. It is further noted that aberrant glycosylation not only results from the altered expression of glycosyltransferases (GTs) but also from the changed activity of GTs and glycosidases as well as the availability and abundance of sugar nucleotide donors. The aim of the research described in this thesis was to explore the glycomic signatures of colorectal cancer (CRC) in cell lines and tissues as well as of acute myeloid leukemia (AML) cell lines. Show less
This thesis describes very sensitive methods for peptide detection, obtained by the coupling of high efficiency capillary chromatographic techniques to mass spectrometry. The application of novel... Show moreThis thesis describes very sensitive methods for peptide detection, obtained by the coupling of high efficiency capillary chromatographic techniques to mass spectrometry. The application of novel data preprocessing methods to the analysis of such complex MS data generated is also described. The data analysis step was completed by the use of multivariate statistical analysis tools, such as principal component analysis (PCA). The developed platform could be applied to the study of complex peptide mixtures and of proteomic samples. Show less
