Receptors tyrosine kinases or RTKs are cell surface receptors that regulate numerous cellular processes, but also have a critical role in the development and progression of many types of cancer.... Show moreReceptors tyrosine kinases or RTKs are cell surface receptors that regulate numerous cellular processes, but also have a critical role in the development and progression of many types of cancer. The overexpression of EphA4, a member of the RTK family, has been observed in a variety of malignant carcinomas. The aim of the research project associated with this thesis was to develop high affinity inhibitors of the tyrosine kinase EphA4. Ligand discovery was based on two complementary approaches, a computational screen and an NMR based screen using Target Immobilized NMR Screening (TINS). In addition, orthogonal biophysical methods including Surface Plasmon Resonance (SPR) and protein observed NMR were employed to analyse fragment binding. The crystal structure of the EphA4 kinase domain was solved and the structure of the kinase domain in complex with dasatinib, a well-known kinase inhibitor, was also elucidated. The in silico approach discovered a potent inhibitor of EphA4 for which the binding mode was elucidated via X-ray crystallography. Moreover, the TINS approach identified two compounds that may constitute starting points for the generation of more potent EphA4 inhibitors. Show less
Cremers, R.; Asperen, C. van; Kil, P.; Vasen, H.; Wiersma, T.; Oort, I. van; Kiemeney, L. 2012
This thesis describes the PROMODE-study, which investigated in a pragmatic way whether a pro-active approach in primary care by screening for depressive symptoms, followed by an intervention offer... Show moreThis thesis describes the PROMODE-study, which investigated in a pragmatic way whether a pro-active approach in primary care by screening for depressive symptoms, followed by an intervention offer to persons of 75 years and over who screened positive, is (cost)effective to detect and relieve suffering from depressive symptoms at old age. We compared two screening methods regarding yield and costs. Furthermore, we found that scores of the 15-item Geriatric Depression Scale were higher when this screening questionnaire was self-administered than when interviewer-administered. In our intervention study, a cluster-randomised controlled trial, we found that the stepped-care intervention program was not (cost)effective compared with usual care in general practice, possibly due to a low uptake of the offered course being the main part of the intervention. In a qualitative study we explored the limiting and motivating factors for accepting course participation. This revealed that most persons were not (yet) prepared to accept the unsolicited intervention offer, although perceived needs to relieve depressive symptoms seemed to largely match the elements of the course. It is discussed that a more selective approach, aimed at high risk-groups and focussing on need for and readiness to accept help, might increase efficiency of a combined screening-intervention program. Show less
Membrane proteins are an interesting class due to the variety of cellular functions and their importance as pharmaceutical targets, but they pose significant challenges for fragment-based drug... Show moreMembrane proteins are an interesting class due to the variety of cellular functions and their importance as pharmaceutical targets, but they pose significant challenges for fragment-based drug discovery approaches. Here we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. Using the recently developed Target Immobilized NMR Screening (TINS) approach, we screened 1,200 fragments for binding to the enzyme Disulphide bond forming protein B. Biochemical and biophysical validation of the 8 most potent hits revealed an IC50 range of 7 to 200 uM, which could be categorized as cofactor binding inhibitors or mixed model inhibitors of both cofactor and substrate protein interaction. Our results establish the utility of fragment-based methods in the development of inhibitors of membrane proteins, making a wide variety3of important membrane bound pharmaceutical targets amenable to such an approach. We first tested the immobilization procedure on G protein coupled receptors and ion channels. Furthermore, we used Nanodiscs, an alternative solubilization strategy, to solubilize teh protein without detergents. This resulted in less broad NMR signals, less non-specific binding issues, and identification of the binders from the original screen, proving that the nanodisc solubilization technique is compatible with TINS. Show less
Growth impairment is considered a relatively early sign of poor health in children. Depending on its setting and the age of the child the impairment is expressed in several ways. In industrialized... Show moreGrowth impairment is considered a relatively early sign of poor health in children. Depending on its setting and the age of the child the impairment is expressed in several ways. In industrialized countries, where malnutrition is rare, the major purpose of growth monitoring, implying regular measurements of weight and length, is early detection of growth disorders, such as Turner__s syndrome (TS), Growth Hormone deficiency (GHD) and celiac disease (CD). For an early identification of children with abnormal growth it is important to have correct and well-defined referral criteria as well as a good growth monitoring system, with accurate growth charts and a well defined diagnostic work-up. In this thesis an effort is made to collect evidence on referral criteria and diagnostic procedures for the assessment of short stature in children, leading to a presentation of new guidelines. Show less
This thesis describes several topics on angiogenesis and screening in uveal melanoma, the most frequent eye tumour in adults. The expression of vascular endothelial growth factor and other... Show moreThis thesis describes several topics on angiogenesis and screening in uveal melanoma, the most frequent eye tumour in adults. The expression of vascular endothelial growth factor and other angiostimulating factors in this tumour are described. In a second part, the use of different screening tests in screening for metastases of uveal melanoma is investigated. Show less
