This thesis describes the use of zebrafish to study Noonan-(NS) and LEOPARD syndromes (LS), two autosomal dominant disorders with overlapping symptoms, caused by mutations in protein-tyrosine... Show moreThis thesis describes the use of zebrafish to study Noonan-(NS) and LEOPARD syndromes (LS), two autosomal dominant disorders with overlapping symptoms, caused by mutations in protein-tyrosine phosphatase, non-receptor type 11 (PTPN11). Intriguingly, while NS mutations result in a more __active__ state of Shp2, LS mutations give rise to a PTP defective protein. First, we studied the role of ptpn11 in zebrafish embryonic development. Whereas ptpn11a is crucial for development ptpn11b seems dispensible. Moreover, using phoshoproteomics we show hypo- and hypertyrosyl phosphorylation of Fer kinase and PZR (Protein zero related), respectively and study their roles as (potential) interacting proteins in the etiology of both NS and LS. Defective heart development is a prominent symptom of NS and LS. Embryos expressing NS and LS Shp2 showed impaired expression of laterality markers, impaired cilia function in Kupffer__s vesicle and at later stages, heart function defects. Finally we identified several mutations in Alpha-2-Macroglobulin-Like-1 (A2ML1) in patients diagnosed with NS. Functional characterization of these mutations in zebrafish showed NS-like developmental defects. This is the first example of an extracellular factor in a disorder clinically related to NS and LS, providing potential new leads for better understanding of the molecular basis of these developmental syndromes. Show less
With the persistent efforts to improve healthcare while the costs are ever-increasing, there is a growing demand for healthcare innovations including faster and cheaper analytical methods to... Show moreWith the persistent efforts to improve healthcare while the costs are ever-increasing, there is a growing demand for healthcare innovations including faster and cheaper analytical methods to support clinical decisions. The in-depth phenotyping of a biological system, such as comprehensive small-molecule profiling, will be key for breakthroughs in biomedical and pharmacological research towards the development of personalized medicine. In this thesis the development of new methods towards high-throughput, mass spectrometry-based profiling of small molecules in complex biological samples is presented. One option for high-throughput mass spectrometric profiling is direct infusion in which no separation techniques are employed prior to MS detection. With the pre-analytical part being a major bottleneck in a typical direct-infusion MS-based analytical workflow, the focus was set on the development of new sample pretreatment and sampling procedures, including miniaturized and automated concepts with the potential for high-throughput application. Show less
Cells communicate in multicellular organisms; by secreting and sensing signals, in order to adjust their behavior to the environment. Extracellular signals such as cytokines and growth factors bind... Show moreCells communicate in multicellular organisms; by secreting and sensing signals, in order to adjust their behavior to the environment. Extracellular signals such as cytokines and growth factors bind to cell surface receptors and trigger the activation of multiple protein signal transduction cascades that mediate cellular responses such as proliferation, differentiation, apoptosis and motility. The Mitogen-activated protein kinase (MAPK) family is a group of homologous proteins forming several linear signal transduction pathways. The MAPK family is conserved among eukaryotes and most vertebrates contain at least 14 MAPKs. We are interested in the molecular mechanisms of MAPK signalling that facilitate proper development of the zebrafish embryo. The zebrafish is an excellent model to delineate MAPK associated embryonic processes. The bodyplan is completed within 24hours and within a week most organs are formed. With the current available zebrafish tools molecular mechanisms could be identified and linked to cellular processes and morphological observations. We constructed constitutive active zebrafish Erk2 mutants for identification of new signalling events. We completed expression analysis of all P38 isoforms by expression analysis of the P38_ and P38_ isoforms. We also performed a comparative study between P38_ and Erk2 MAPK in gastrulation. Finally, We addressed the role of P38_ in zebrafish angiogenesis. Show less
