In dit proefschrift worden de moleculaire mechanismen behandeld die onderliggend zijn aan artrose. Specifiek wordt genoomwijd onderzocht welke genen anders tot expressie komen in aangedaan... Show moreIn dit proefschrift worden de moleculaire mechanismen behandeld die onderliggend zijn aan artrose. Specifiek wordt genoomwijd onderzocht welke genen anders tot expressie komen in aangedaan vergeleken met gezond kraakbeen van artrose patienten. Dit in de context van epigenetische regulatie van gen expressie, specifiek door DNA methylatie in het licht van de lokale genetische context in de vorm van puntmutaties. Show less
This thesis provides insights into the mechanisms of renal I/R injury based on human kidney transplantation (i.e. the status of delayed graft function: DGF). A severe energetic crisis... Show moreThis thesis provides insights into the mechanisms of renal I/R injury based on human kidney transplantation (i.e. the status of delayed graft function: DGF). A severe energetic crisis differentiates DGF kidneys from adequately functioning controls. Although intact beta-oxidation, aerobic glycolysis and glutaminolysis provide Krebs Cycle intermediates, these intermediates are not able to enter the mitochondrial Krebs cycle. Hence, dysfunctional mitochondria disable efficient ATP production leading to the metabolic incompetence that causes DGF and underlies renal I/R injury. This finding sheds a whole new light on I/R injury and explains why ATP-dependent therapeutics are ineffective as treatment for I/R injury. A major difference in the vulnerability of mitochondria to ischemia and reperfusion between rodents and humans was found. This could explain the current differences in effectiveness of therapies in the experimental versus the clinical setting. Big cohort studies give insights in donor, recipient and transplant-procedure variables and challenge the reluctance towards the use of DCD donor kidneys. New preventive strategies could limit I/R injury by preserving mitochondria (hypothetically with peptide SS-31 or activation of mitochondrial aldehyde dehydrogenase). This will overcome the detrimental effects of I/R injury on graft function and survival - thereby increasing the success rate of kidney transplantation. Show less
Zanden, L.F.M. van der; Vermeulen, S.H.; Oskarsdottir, A.; Maurits, J.S.F.; Diekstra, M.H.M.; Ambert, V.; ... ; Oosterwijk, E. 2017
In chapter 2, the pancreas was used as a paradigm to study human organ development and assess the quality of our fetal material. In a descriptive, histochemical study, we investigated how blood... Show moreIn chapter 2, the pancreas was used as a paradigm to study human organ development and assess the quality of our fetal material. In a descriptive, histochemical study, we investigated how blood and lymphatic vascular networks develop and their association with basement membranes and smooth muscle cells between gestational weeks 9 and 22 (W9 and W22). In Chapter 3, 4 and 5, we analyzed a total of 21 fetal organs and maternal endometrium at three time points (W9, W18 and W22) at the transcriptional and epigenetic level, thereby, providing an atlas of human organ development. The rare fetal material also allowed us to investigate the presence of an epigenetic memory from the cell of origin in induced pluripotent stem cells (iPSCs). We generated isogenic iPSC lines from six fetal organs (brain, skin, kidney, muscle, lung and pancreas) in chapter 5. The six iPSC lines had very similar DNA methylation profiles, however, we showed that the two clones derived from the brain harbored 18 hypermethylated and 6 hypomethylated CpGs also found in the fetal brain and we demonstrated that the brain-iPSC clones appeared to have a differentiation bias towards neural derivatives when comparing neural differentiation of the brain- and skin-iPSC clones. Show less
The aim of the research described in this thesis entitled ‘The use of transcriptomics data in detecting non-genotoxic carcinogens’ was to develop in vitro tests to improve testing strategies for... Show moreThe aim of the research described in this thesis entitled ‘The use of transcriptomics data in detecting non-genotoxic carcinogens’ was to develop in vitro tests to improve testing strategies for cancer hazard assessment of chemicals, to reduce the use of in vivo experiments. The scope of this thesis was twofold. First, an improved in vitro approach to assess genotoxicity was developed, with the intention to reduce the number of misleading positive test results. The emphasis was on characterization of the cell system, primary hepatocytes derived from transgenic mice. Results showed that this cell system will be of added value in genotoxicity testing. In the second part of this thesis, the focus was on the development of a ‘trancriptomics’-based approach to detect modes of action of non-genotoxic carcinogens. It has been demonstrated that the described comparison approach is promising in recognizing gene expression patterns, which can be related to modes of action. In addition, the approach is also suitable to detect toxicity of chemicals in general. In conclusion, through the development of in vitro approaches, as described within this thesis, an important contribution in the improvement of testing strategies for cancer hazard assessment of chemicals has been delivered. Show less
The aim of this thesis was to identify in the human blood transcriptome, relevant pathways and potential biomarker profiles that associate with chronological age and discriminate between __healthy... Show moreThe aim of this thesis was to identify in the human blood transcriptome, relevant pathways and potential biomarker profiles that associate with chronological age and discriminate between __healthy agers__ from long-lived families and normative ageing controls. Such profiles may harbor determinants of the biological ageing rate. We studied genome-wide gene expression profiles in blood of members of the Leiden Longevity Study (LLS) and replicated our findings by extended sampling within the unique LLS cohort. The findings of the exploratory analysis prompted us to investigate multiple genes in the IL7R and MTOR pathways for association with familial longevity. The results obtained by examining mRNA from blood samples brought us to study mTOR protein levels and signalling in primary skin fibroblasts from the corresponding donors in the LLS. Finally, to discover robust, biologically relevant gene networks as markers of chronological ageing in larger sample sizes, we performed an explorative network-based meta-analysis on large publicly available transcriptomic datasets. We have identified several networks, pathways and candidate genes potentially marking the biological age and the rate of ageing Show less
In this thesis novel statistical methods that help scientists extract maximal information from high-dimensional data, in particular those derived by transcriptomics, are presented.
The work presented in this thesis has provided new insights into the mechanisms involved in the regulation of innate immune responses in zebrafish embryos. Furthermore, cell-specific transcriptome... Show moreThe work presented in this thesis has provided new insights into the mechanisms involved in the regulation of innate immune responses in zebrafish embryos. Furthermore, cell-specific transcriptome profiling studies identified novel marker genes for distinguishing immune cell types, which is highly useful information to fulfill the demand for new fluorescent reporter lines and lineage-specific antibodies in the zebrafish model. We have shown that Ptpn6, a protein tyrosine phosphatase homolog of human SHP1, functions as a critical negative regulator, required for a properly balanced innate immune response and for controlling infections with bacterial pathogens. In Salmonella typhimurium infection, ptpn6 deficiency caused a general hyperinduction of pro-inflammatory genes, which was contraproductive as it impaired the infection control. In Mycobacterium marinum infection, a more specific effect of ptpn6 deficiency on matrix metalloproteinase gene expression was found as a major underlying cause of increased bacterial burden. We further concluded that Ptpn6 functions as a much stronger negative regulator than infection-inducible miRNAs of the miR-146 family, which may be involved in more subtle fine-tuning of the innate immune response. Knowledge about the distinct roles of Ptpn6 and miR-146 miRNAs has practical applicability in regard to their potential as therapeutic targets for inflammatory diseases and cancer. Show less
This dissertation mainly focuses on interdisciplinary approaches for biomedical knowledge discovery. This required special efforts in developing systematic strategies to integrate various data... Show moreThis dissertation mainly focuses on interdisciplinary approaches for biomedical knowledge discovery. This required special efforts in developing systematic strategies to integrate various data sources and techniques, leading to improved discovery of mechanistic insights on human diseases. Chapter one looks at the possibility in which combining various bioinformatics-based strategies can significantly improve the characterization of the OPMD mouse model. We discuss that this approach in knowledge discovery, on the basis of our extensive analysis, helped us to shed some light on how this model system relates to OPMD pathophysiology in human. In Chapter two, we expand on this combinatory approach by conducting a cross-species data analysis. In this study, we have looked for common patterns that emerge by assessing the transcriptome data from three OPMD model systems and patients. This strategy led to unravelling the most prominent molecular pathway involved in OPMD pathology. The third chapter achieves a similar goal to identify similar molecular and pathophysiological features between OPMD and the common process of skeletal muscle ageing. Engaging in a study in which the focus was made on the universality of biological processes, in the light of evolutionary mechanisms and common functional features, led to novel discoveries. This work helped us uncover remarkable insights on molecular mechanisms of ageing muscles and protein aggregation. Chapters four and five take a different route by tackling the field of computational biology. These chapters aim to extend network inference by providing novel strategies for the exploitation and integration of multiple data sources. We show that these developments allow us to infer more robust regulatory mechanisms to be identified while translations and predictions are made across very different datasets, platforms, and organisms. Finally, the dissertation is concluded by providing an outlook on ways the field of systems biology can evolve in order to offer enhanced, diversified and robust strategies for knowledge discovery. Show less
The aim of the thesis was to develop metabolic analytical platforms for static and dynamic measurements that could answer biological questions for in vitro and in vivo animal models in the area of... Show moreThe aim of the thesis was to develop metabolic analytical platforms for static and dynamic measurements that could answer biological questions for in vitro and in vivo animal models in the area of lipid research. Gene profiling together with the transcriptome and metabolome data was used in combination with the LC/MS analytical platform. In terms of the analytical platforms developed, the focus was on high resolution LC/MS but not limited, as amalgamation with other platforms such as gradient gel electrophoresis (GGE) and fast protein liquid chromatography (FPLC) were explored in more detail to investigate the lipid composition of lipoprotein particles. These analytical strategies were applied to different lipid modulating biological targets as a mean to obtaining a more detailed and characteristic phenotype description directing decisions in drug search during the drug discovery process on the basis of the analytical results obtained. Additionally, the utilization of metabolic tracers was investigated further to probe dynamic changes in the biological target and animal models in question 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
As the zebrafish, Danio rerio, has been increasingly used as an animal model for biomedical research, we aimed to establish zebrafish cell line models for inflammation and cancer studies in this... Show moreAs the zebrafish, Danio rerio, has been increasingly used as an animal model for biomedical research, we aimed to establish zebrafish cell line models for inflammation and cancer studies in this thesis. Several zebrafish cell lines were characterized and their genetic and physiological properties were compared. We also developed a set of tool methods to investigate cellular signaling events in zebrafish cell lines. Our case studies illustrated that zebrafish cell lines are as reliable models as the widely used mammalian cell cultures. Taking advantage of the transparency of zebrafish embryos and cell implantation protocols, zebrafish cell lines can serve as a bridge platform between in vitro, in silico, ex vivo and in vivo studies in order to enhance our understanding of molecular mechanisms underlying disease progression. Show less
In the studies comprising this thesis we evaluated the potential usefulness of cDNA microarray based gene expression profiling and 1H-NMR based metabolomics platforms as tools for the evaluation of... Show moreIn the studies comprising this thesis we evaluated the potential usefulness of cDNA microarray based gene expression profiling and 1H-NMR based metabolomics platforms as tools for the evaluation of novel PPAR_ and -_ agonists in future clinical __proof of concept studies__. We investigated the effects of rosiglitazone, (prototype PPAR_ agonist ) and ciprofibrate (prototype PPAR_ agonist) on global (target) tissue gene expression profiles and endogenous urinary and plasma metabolites of type 2 Diabetes Mellitus (T2DM) patients and healthy volunteers (HVs).The results from the transcriptomic analyses indicated that none of the genes in any of the tissues in either study group displayed a significant treatment response with either rosiglitazone of ciprofibrate vs. placebo at Bonferroni adjusted values and _=0.05. The results of the metabolomic analyses revealed significant rosiglitazone and ciprofibrate induced changes in endogenous urinary and plasma metabolite profiles of T2DM patients but not in HVs. We conclude that from the two molecular profiling platforms evaluated in this thesis, metabolomics currently appears to be the most promising platform for future application in clinical __proof of concept__ studies with novel PPAR agonist compounds in T2DM patients.In the studies comprising this thesis we evaluated the potential usefulness of cDNA microarray based gene expression profiling and 1H-NMR based metabolomics platforms as tools for the evaluation of novel PPAR_ and -_ agonists in future clinical __proof of concept studies__. We investigated the effects of rosiglitazone, (prototype PPAR_ agonist ) and ciprofibrate (prototype PPAR_ agonist) on global (target) tissue gene expression profiles and endogenous urinary and plasma metabolites of type 2 Diabetes Mellitus (T2DM) patients and healthy volunteers (HVs).The results from the transcriptomic analyses indicated that none of the genes in any of the tissues in either study group displayed a significant treatment response with either rosiglitazone of ciprofibrate vs. placebo at Bonferroni adjusted values and _=0.05. The results of the metabolomic analyses revealed significant rosiglitazone and ciprofibrate induced changes in endogenous urinary and plasma metabolite profiles of T2DM patients but not in HVs. We conclude that from the two molecular profiling platforms evaluated in this thesis, metabolomics currently appears to be the most promising platform for future application in clinical __proof of concept__ studies with novel PPAR agonist compounds in T2DM patients. Show less
The introduction of the 'omics' techniques (transcriptomics, proteomics, and metabolomics) and systems biology, has caused fundamental changes in the drug discovery process and many other fields in... Show moreThe introduction of the 'omics' techniques (transcriptomics, proteomics, and metabolomics) and systems biology, has caused fundamental changes in the drug discovery process and many other fields in the life science area. In this thesis we explored the possibilities to apply these holistic technologies to investigate the effects of known and potential anti-inflammatory compounds on macrophages. For this purpose we made use of a monocyte-like human histocytic lymphoma cell line U937. U937 cells can be induced by phorbol 12-myristate 13-acetate (PMA) to undergo differentiation into a macrophage-like phenotype. The two differentiation stages, monocyte and macrophage, were compared by using oligonucleotide microarrays and 2-D gel electrophoresis in combination with principal component analysis (PCA). This differentiation study is described in Chapter 2. The differential expression of three protein biomarkers, gamma interferon inducible lysosomal thiol reductase (GILT), cathepsin D and adipocyte-fatty acid binding protein (A-FABP) were biologically validated by Western blot and real time polymerase chain reaction (real time PCR). GILT and A-FABP were also found to be differentially expressed at the mRNA level as indicated by the results of the microarray experiment. Moreover, the transcriptomics data revealed a large number of additional putative differentiation markers in U937 macrophages, many of which are known to be expressed in peripheral blood-derived macrophages. From the results presented in Chapter 2 can be concluded that the U937 cell line is an excellent model system for the blood-derived macrophage and that microarrays and 2-D gel electrophoresis are suitable methods to identify biomarkers for differentiation. Chapter 3 describes the use of a systems biology approach to categorize anti-inflammatory drugs based on their mRNA, protein and lipid expression pattern, as determined by oligonucleotide microarrays, 2-D gel electrophoresis and a LC-MS method for lipids, in combination with principal component discriminant analysis (PC-DA). The results described in this chapter demonstrate that different classes of anti-inflammatory compounds show distinct and characteristic mRNA, protein, and lipid expression patterns, which can be used to categorize known anti-inflammatory drugs, as well as to discover and classify new leads. The latter was exemplified by the categorization of zilpaterol, a poorly characterized ____-agonist. Exposure to zilpaterol gives rise to an almost identical expression pattern as that observed after exposure to the well-characterized __2-agonists clenbuterol and salbutamol, suggesting that zilpaterol is indeed a ____-agonist. In addition, this study revealed potential biomarkers for the different anti-inflammatory drugs under investigation. The categorization of the anti-inflammatory drugs on the basis of proteomics data alone was not successful. The most likely explanation for this is that by the analysis of whole cell lysates, only highly abundant proteins can be visualized, while the low abundant proteins, which are often involved in important metabolic pathways, are not. Therefore, a more focused approach was used to investigate the mechanism of action of zilpaterol, which is described in Chapter 4. In Chapter 4, U937 macrophages were stimulated with LPS to induce an inflammatory response. This response was inhibited by the addition of zilpaterol (LZ) and this inhibition was antagonized by the _2-adrenergic receptor antagonist propranolol (LZP). Two-dimensional difference gel electrophoresis (DIGE) in combination with Student__s t-test and two multivariate data analysis tools (PCA and partial least squares discriminant analysis PLS-DA) were used to examine the secreted proteome induced by the three treatments. This revealed 8 potential protein biomarkers. The protein spots were identified using nano LC-MS-MS. Only two of the identified proteins, namely macrophage inflammatory protein-1_ (MIP-1_) and macrophage inflammatory protein-1_ (MIP-1_) are known to be secreted proteins. The inhibition of MIP-1_ by zilpaterol and the involvement of the _2-AR and cyclic adenosine-3__,5__-cyclic monophosphate (cAMP) were confirmed using a specific immuno-assay. The experiments described in this chapter demonstrate the importance of pre-fractionation of complex protein samples before performing proteomics studies. The categorization of zilpaterol in Chapter 3 as a _2-adrenegic receptor agonist was further explored in Chapter 5. In this chapter we investigated the binding affinity of zilpaterol to the _1- and _2 receptor by using a receptor binding assay. Furthermore, we examined the role of the _1- and _2 adrenoceptor in the inhibition of the LPS induced tumor necrosis factor-alpha (TNF-_) production and the induction of cAMP by U937 macrophages. For this purpose we made use of a selective _1-receptor antagonist (atenolol), a selective _2-antagonist (ICI 118551) and a non-selective _-antagonist (propranolol). Finally, the inhibitory effect of zilpaterol on the TNF-_ production was investigated in LPS-treated male Wistar rats. The results obtained in this way clearly show that zilpaterol is a _2-adrenergic agonist and a inhibitor of the LPS-induced TNF-_ production by macrophages both in vivo and in vitro. The three _2-agonists specific biomarkers, Granulocyte Chemotactic Protein-2 (GCP-2/CXCL6), Oncostatin M (OSM), and Vascular Endothelial Growth Factor (VEGF) that were identified in Chapter 3, were further examined in Chapter 6. The three markers were significantly up-regulated both in U937 macrophages and in blood-derived macrophages exposed to a _2-agonist (clenbuterol and zilpaterol) in the absence or presence of LPS, as determined by a specific enzyme-linked immunosorbent assays (ELISA). Moreover, this up-regulation was also accomplished by other cyclic AMP elevating agents (forskolin, prostaglandins E2, and dibutyryl cAMP), suggesting a role of cAMP in the up-regulation of GCP-2/CXCL6, VEGF and OSM. We hypothesize that these proteins may be involved in some of the adverse effects in the treatment of asthma with _2-adrenergic receptor agonists. In the second part of this thesis we focussed on a multi-component drug, namely Cannabis sativa. In Chapter 7, the immuno-modulating effects of unheated and heated Cannabis extracts were investigated. This study revealed that unheated Cannabis extracts and its major non-psychoactive compound _9-tetrahydrocannabinolic acid (THCa) were able to inhibit the LPS induced TNF-_ production both in U937 macrophages and in blood-derived macrophages. The inhibitory effect on TNF-_ was not mediated by the cannabinoid receptors CB1 and CB2. Furthermore, this study showed that unheated Cannabis extracts and THCa exert their inhibitory effect on the TNF-_ production via a mechanism that is different from that of heated Cannabis extract and its main constituent the psychoactive compound _9-tetrahydrocannabinol (THC). The inhibition of TNF-_ release by unheated Cannabis extract and THCa was prolonged over a relatively long period of time. By contrast, although THC and heated extracts initially inhibit the release of TNF-_, after longer incubation times they seem to increase TNF-_ production to levels that are even higher than in the absence of THC or Cannabis extract. This difference in response of the U937 macrophages to THC and THCa was also observed in an experiment in which we examined the effects on phosphatidylcholine specific phospholipase C (PC-PLC) activity. Unheated Cannabis extract and THCa inhibited the PC-PLC activity in a dose-dependent manner, while THC induced PC-PLC activity at high concentrations. Finally, we studied the effect of THCa and unheated Cannabis extract in a pilot study using an Experimental Autoimmune Encephalomyelitis (EAE) mouse model. Unheated Cannabis extract and THCa had a favourable effect on the clinical and histological signs of EAE. However, these results are preliminary and not clearly significant, therefore further investigation is necessary. Chapter 8 describes the categorization of unheated and heated Cannabis extracts using the same model system as described in Chapter 3. The mRNA patterns obtained from U937 macrophages exposed to LPS in the absence or presence of different anti-inflammatory drugs and unheated and heated Cannabis extracts were analysed using PC-DA. The study revealed that heated and unheated Cannabis extracts give rise to different expression patterns, which is in agreement with the observations made in Chapter 7 that they exert their TNF-_ inhibitory effect via different pathways. Moreover, their expression patterns did not overlap with that of other classes of anti-inflammatory compounds known to inhibit the TNF-_ production. These results suggest that the Cannabis extracts can not be assigned to one of the above mentioned classes of inflammatory inhibitors. Further investigation is necessary to unravel the exact mechanism of action of unheated and heated Cannabis extracts. In conclusion, the studies in this thesis show that the application of systems biology approaches are very useful in the categorization of anti-inflammatory compounds based on their mRNA and lipid expression patterns and to find specific biomarkers for these compounds. The categorization based on the protein expression pattern was less successful. This is most probably due to the fraction of proteins that was analysed on the gel. With proteomics techniques only a small fraction of proteins can be analysed simultaneously. Pre-fractionation, enrichment techniques and different analytical methods are therefore necessary to analyse a wide range of proteins with diverse physiological properties and dynamic range. The datasets obtained by transcriptomics, proteomics and metabolomics were analysed using statistical and pattern recognition tools. The datasets often contained a limited number of samples with respect to the large number of variables. It is therefore important to use these techniques as an explorative tool only and to validate the potential biomarkers found by additional individual measurements. Taken together, the use of systems biology for the investigation of anti-inflammatory drugs yielded very promising results, even though only a small part of the systems biology circle was used. Show less
