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
Hiller, M.; Geissler, M.; Janssen, G.; Veelen, P. van; Aartsma-Rus, A.; Spitali, P. 2020
Muscle formation is a coordinated process driven by extensive gene expression changes where single cells fuse together to form multinucleated muscle fibers. Newly synthesized mRNAs are then... Show moreMuscle formation is a coordinated process driven by extensive gene expression changes where single cells fuse together to form multinucleated muscle fibers. Newly synthesized mRNAs are then regulated by RNA binding proteins (RBPs), affecting post-transcriptional transcript metabolism. Here, we determined how large-scale gene expression changes affect the catalog of RBPs by studying proliferating and differentiated muscle cells in healthy and dystrophic conditions. Transcriptomic analysis showed that the expression of more than 7000 genes was affected during myogenesis. We identified 769 RBPs, of which 294 were muscle-specific and 49 were uniquely shared with cardiomyocytes. A subset of 32 RBPs (half of which were muscle-specific) was found to be preferentially associated with target mRNAs in either myoblasts (MBs) or myotubes (MTs). A large proportion of catalytic proteins were bound to mRNAs even though they lack classical RNA binding domains. Finally, we showed how the identification of cell-specific RBPs enabled the identification of biomarkers that can separate healthy individuals from dystrophic patients. Our data show how interactome data can shed light on new basic RNA biology as well as provide cell-specific data that can be used for diagnostic purposes. Show less
Mendez, M.; Besse, A.; Besse, L.; Florea, B.I.; Christian, Z.; Overkleeft, H.S.; Driessen, C. 2019
Background Analysis of muscle biopsies allowed to characterize the pathophysiological changes of Duchenne and Becker muscular dystrophies (D/BMD) leading to the clinical phenotype. Muscle tissue is... Show moreBackground Analysis of muscle biopsies allowed to characterize the pathophysiological changes of Duchenne and Becker muscular dystrophies (D/BMD) leading to the clinical phenotype. Muscle tissue is often investigated during interventional dose finding studies to show in situ proof of concept and pharmacodynamics effect of the tested drug. Less invasive readouts are needed to objectively monitor patients' health status, muscle quality, and response to treatment. The identification of serum biomarkers correlating with clinical function and able to anticipate functional scales is particularly needed for personalized patient management and to support drug development programs.Methods A large-scale proteomic approach was used to identify serum biomarkers describing pathophysiological changes (e.g. loss of muscle mass), association with clinical function, prediction of disease milestones, association with in vivo(31)P magnetic resonance spectroscopy data and dystrophin levels in muscles. Cross-sectional comparisons were performed to compare DMD patients, BMD patients, and healthy controls. A group of DMD patients was followed up for a median of 4.4years to allow monitoring of individual disease trajectories based on yearly visits.Results Cross-sectional comparison enabled to identify 10 proteins discriminating between healthy controls, DMD and BMD patients. Several proteins (285) were able to separate DMD from healthy, while 121 proteins differentiated between BMD and DMD; only 13 proteins separated BMD and healthy individuals. The concentration of specific proteins in serum was significantly associated with patients' performance (e.g. BMP6 serum levels and elbow flexion) or dystrophin levels (e.g. TIMP2) in BMD patients. Analysis of longitudinal trajectories allowed to identify 427 proteins affected over time indicating loss of muscle mass, replacement of muscle by adipose tissue, and cardiac involvement. Over-representation analysis of longitudinal data allowed to highlight proteins that could be used as pharmacodynamic biomarkers for drugs currently in clinical development.Conclusions Serum proteomic analysis allowed to not only discriminate among DMD, BMD, and healthy subjects, but it enabled to detect significant associations with clinical function, dystrophin levels, and disease progression. 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