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Cartilage is an important tissue in the skeleton. During skeletal development transient cartilage forms a template for the skeletal elements before it is replaced by bone. In the joints a non-transient form of cartilage, the articular cartilage, caps the ends of the bones and ensures very low friction between the bones to support mobility. Damage to this articular cartilage is a main feature of joint diseases. Osteoarthritis is the most common chronic joint disease, affecting millions of people world-wide and is characterized by progressive structural damage to the articular cartilage and the underlying subchondral bone, leading to pain and disability. Currently, no disease modifying treatments are available for patients with osteoarthritis, thereby defining an important unmet medical need.The Wnt signaling pathway has been identified as a key player in cartilage development, homeostasis and disease. Hyperactivation of Wnt signaling inhibits early cartilage differentiation but stimulates the last stages of the developmental differentiation process. In the articular cartilage, low levels of active Wnt signaling appear important for cell survival, but hyperactivation of the pathway is associated with abnormal differentiation of the cells and an increase in the production of tissue-destructive enzymes thereby contributing to disease processes active in osteoarthritis. Hence, Wnt signaling is considered to be a therapeutic target for this disease. Therefore, understanding of the mechanisms that regulate this pathway in cartilage is important.In this thesis, two regulatory mechanisms are investigated and demonstrated. First, activation of Wnt signaling is demonstrated to be dependent on the presence of heparin sulfate proteoglycans. Exostosin-1 (Ext1) encodes a glycosyltransferase that is required for heparan sulfate (HS) chain elongation in proteoglycan biosynthesis. HS chains serve as binding partners for signaling proteins, affecting their distribution and activity. In knockdown experiments in a chondrocyte development model, HS levels were reduced and this positively impacted on chondrogenic differentiation and proteoglycan accumulation. Ext1 knock-down reduced active Wnt signaling. Conversely, Ext1 overexpressing cells, with higher HS content, showed decreased chondrogenic differentiation and enhanced Wnt. Wnt signaling activation led to a down-regulation of Ext1 expression in chondrocytes.EXT1 therefore affects chondrogenic differentiation of precursor cells, in part via changes in the activity of Wnt signaling. As Wnt signaling also controls Ext1 expression, a regulatory loop between EXT1 and Wnt signaling during chondrogenesis is proposed.Second, interactions between Wnt signaling and intracellular multifunctional molecule ANP32A were demonstrated. ANP32A was previously shown to protect against cartilage damage by limiting oxidative stress through regulation of key anti-oxidant regulator molecule ATM expression. However, anti-oxidant treatment only partially rescued joint damage in Anp32a KO mice with osteoarthritis. Analysis of global gene expression levels by microarray in the articular cartilage of Anp32a KO mouse cartilage suggested that ANP32A also can regulate Wnt signaling. Lack of Anp32a in a cartilage differentiation process resulted in inhibition of differentiation and lack of proteoglycan accumulation, associated with Wnt hyper-activation. In human and mouse articular cartilage, Anp32a deficiency was also linked to hyper-activation of Wnt signaling.Therefore, this work reveals that EXT1 and ANP32A regulate the activity of Wnt signaling and can be considered potential targets to modulate this pathway in the treatment of osteoarthritis.

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Environmental monitoring (EM) is of utmost importance to demonstrate contamination control in a sterile production environment. Active air sampling (AAS), which is the traditional method for monitoring viable airborne particles, requires an incubation process. Results can only be read out at least five days after sampling. At Organon Heist, a new instrument, the BioTrak®, has been purchased, which can be used for monitoring airborne particles. The BioTrak® makes use of biofluorescent particle counting (BFPC) technology and is classified under the alternative and rapid microbiological methods (ARMM). The instrument uses laser induced fluorescence (LIF) and can therefore distinguish viable and non-viable particles. Results are generated in real-time. This brings great benefits as well as cost savings over time. For implementation of the BioTrak® as an EM instrument, an equivalence study between the BFPC technology and the traditional method is expected from regulatory agencies. Therefore, alert and action limits must be set, which define whether a process is in control or not. This master thesis focuses on setting reliable limits for the BFPC technology per classified area. Afterwards, a comparative study should be done with the air sampler to show non-inferiority of the alternative method. AAS requires incubation, after which the number of colony forming units (CFU) can be read out. The BioTrak® uses cellular detection to detect microorganisms, expressed as autofluorescence units (AFU). As test method, the BioTrak® will be installed at several locations of different classified areas, performing three continuous 24-hour measurements per location. Multiple air samplings will also be taken in parallel to compare the behavior of CFU versus AFU counts. The obtained BioTrak® data will be statistically analyzed, in an attempt to set up limits for the BFPC technology. Once conclusive limits have been determined based on thorough research, it is possible to determine whether non-inferiority can be demonstrated versus AAS. The results of the parallel measurements showed that the BFPC technology is clearly more sensitive compared to AAS. The BioTrak® data indicated that activities in proximity of the instrument have a major impact on the viable particle counts measured. Based on this study, preliminary limits were set for the different classified areas. The gathered data were too limited to conclude reliable and final limits for the BFPC technology. The BFPC technology already proves to be promising as EM tool, but future research is needed to determine conclusive limits for a sterile production environment.