This thesis aims to increase the understanding of human osteoarthritis pathophysiology by developing reliable biomimetic ex vivo human osteochondral explant models and focussing on the role of... Show moreThis thesis aims to increase the understanding of human osteoarthritis pathophysiology by developing reliable biomimetic ex vivo human osteochondral explant models and focussing on the role of osteoarthritis-relevant triggers (mechanical stress) and interacting genetic factors for developing treatment targets. Human aged joint tissues were collected in the Research in Articular Osteoarthritis Cartilage (RAAK) biobank. To add knowledge of the osteoarthritis pathophysiological processes, aged human ex vivo osteochondral explants were subject to three osteoarthritis-relevant triggers, being inflammation, hypertrophy and injurious mechanical stress. Next, knowledge on early initiating processes occurring in mechano-pathology was investigated by applying RNA-sequencing to cartilage of aged human osteochondral explants subjected to mechanical stress. In addition, to show that the human osteochondral explant model could also be used for genetic interaction studies, we investigated expression of the osteoarthritis risk gene MGP in relation to rs1800801 genotypes. By combining information from RNA-sequencing datasets of cartilage and bone with osteoarthritis-relevant triggers in cartilage and bone explants we investigated the role of MGP and vitamin K in osteoarthritis. Lastly, the injurious mechanical explant model was exploited to determine the effectivity of inhibiting the osteoarthritis risk gene DIO2 by iopanoic acid treatment either by burst or prolonged release from PLGA-PEG nanoparticles. Show less
Background DNA methylation is a key epigenetic modification in human development and disease, yet there is limited understanding of its highly coordinated regulation. Here, we identify 818 genes... Show moreBackground DNA methylation is a key epigenetic modification in human development and disease, yet there is limited understanding of its highly coordinated regulation. Here, we identify 818 genes that affect DNA methylation patterns in blood using large-scale population genomics data. Results By employing genetic instruments as causal anchors, we establish directed associations between gene expression and distant DNA methylation levels, while ensuring specificity of the associations by correcting for linkage disequilibrium and pleiotropy among neighboring genes. The identified genes are enriched for transcription factors, of which many consistently increased or decreased DNA methylation levels at multiple CpG sites. In addition, we show that a substantial number of transcription factors affected DNA methylation at their experimentally determined binding sites. We also observe genes encoding proteins with heterogenous functions that have widespread effects on DNA methylation, e.g.,NFKBIE,CDCA7(L), andNLRC5, and for several examples, we suggest plausible mechanisms underlying their effect on DNA methylation. Conclusion We report hundreds of genes that affect DNA methylation and provide key insights in the principles underlying epigenetic regulation. Show less
In the field of Osteoarthritis (OA) research the step from genetics to biological functionality, also named ‘functional genomics’, is necessary to allow valorisation of genetic findings, thereby... Show moreIn the field of Osteoarthritis (OA) research the step from genetics to biological functionality, also named ‘functional genomics’, is necessary to allow valorisation of genetic findings, thereby augmenting the need for functional data of disease relevant tissues. Even so, it was estimated that pursuing druggable targets directed by genetic studies are twice as often successful as compared to those without it. In this thesis we apply the functional genomics methodology, to proceed from a genetic association to mechanistic understanding of the effect of genetic variation on gene expression and epigenetic regulation contributing to OA susceptibility. Particularly we set out to characterize and validate the pathophysiological processes that underlie the role of DIO2/thyroid hormone signalling in the onset of OA after identifying the DIO2 gene as a OA susceptibility locus. The results in this thesis show that intracellular T3 levels should be strictly regulated via DIO2 upon mechanical loading of the cartilage, to ensure cartilage tissue homeostasis. Future endeavours should be designed to demonstrate that local inhibition of DIO2 by intra-articular admission of a deiodinase-inhibitor (Iopanoic acid), could be an effective therapy to alleviate the burden of OA thereby increasing mobility, well-being and quality of life particularly among elderly. Show less
Osteoarthritis (OA) is an age related disorder of the joints characterized by pain, crepitus, and stiffness resulting in decreased mobility. Pathophysiology consists of cartilage degeneration and... Show moreOsteoarthritis (OA) is an age related disorder of the joints characterized by pain, crepitus, and stiffness resulting in decreased mobility. Pathophysiology consists of cartilage degeneration and bone remodeling, however, knowledge of OA etiology is still limited. Due to the growing population of elderly, OA prevalence rapidly increases. The fact that no reliable clinical markers are available for diagnosis, monitoring and progression is a major impediment in OA disease management and incurs high costs in drug development and clinical trials. Molecular markers were studied in OA affected cartilage compared to unaffected cartilage of the same joint (chapter 2) and in blood of OA patients (chapter 3). Perturbation of the application of traditional biochemical markers sCOMP and uCTX2 in the clinic due to genetic factors that, independent of OA, affect innate levels was investigated (chapter 4). Furthermore, we have tried to go beyond the results of molecular epidemiological studies to increase insights into underlying mechanisms (chapter 6 & 7). This shows how functional genomics can be achieved by combining genetic and functional data and will facilitate translation of knowledge of genetic variants to the needs of OA patients and thus to application in the clinic. Show less
Despite extensive studies to unravel molecular mechanisms underlying breast cancer metastasis, still 3500 women die of the results of this disease in the Netherlands each year. Improving our... Show moreDespite extensive studies to unravel molecular mechanisms underlying breast cancer metastasis, still 3500 women die of the results of this disease in the Netherlands each year. Improving our understanding of metastasis formation remains a challenge for further drug development. The scope of this thesis is the identification of novel candidate metastasis genes, with a main focus on candidate genes affecting tumor cell migration. For that purpose, a live cell imaging-based random cell migration assay that is suitable for screening has been developed. In addition, a mouse breast cancer model that allows to study tumor cell autonomous processes of metastasis formation is described. A RNA-interference tumor cell migration screen has been done and resulted in the identification of novel regulators of tumor cell migration that show clinical relevance in a breast cancer patient cohort. In addition, focused research has been conducted on two previously identified candidate metastasis genes to determine their role in breast cancer metastasis. Show less
The objective of the project described in this thesis was to study the complex induction of extracellular proteases in the filamentous fungus Aspergillus niger using information gathered with... Show moreThe objective of the project described in this thesis was to study the complex induction of extracellular proteases in the filamentous fungus Aspergillus niger using information gathered with functional genomics technologies. A special emphasis is given to the requirements for performing a successful systems biology study and addressing the challenges met in analyzing the large, information-rich data sets generated with functional genomics technologies. The role that protease activity plays in strain and process development of A. niger and other aspergilli is reviewed. The influence of several environmental factors on the production of extracellular proteases of A. niger in controlled batch cultivations was studied. Samples generated in this study were used for analysis with different functional genomics technologies. With a shotgun proteomics approach the A. niger secretome under different experimental conditions was determined. Furthermore, the effect of different quantitative phenotypes related to protease or glucoamylase activity on the information content of a metabolomics data set was investigated. Finally, the clustering of co-expressed genes is described. First, a set of conserved genes was used to construct gene co-expression networks. Subsequently, all protein-coding A. niger genes, including hypothetical and poorly conserved genes, were integrated into the co-expression analysis. Show less
