To advance our understanding of the histone acylation regulators and the response to DNA replication stress, we employ a variety of genetic screening techniques together with follow-up experiments... Show moreTo advance our understanding of the histone acylation regulators and the response to DNA replication stress, we employ a variety of genetic screening techniques together with follow-up experiments in yeast. By employing Epi-ID, we interrogated a common chromatin locus in the yeast deletion and DaMP collection. This was successful in identifying new histone acetylation and acylation regulators and we describe the crotonyltransferase activities of the ADA and NuA4 complexes. Increased crotonylation levels stimulated gene expression dependent on the activity of the HATs Gcn5 and Esa1, suggesting a role for these enzymes in the crotonylation-dependent transcriptional regulation. Additionally, extensive antibody validation studies revealed that non-acetyl pan-lysine specific acylation antibodies had a severe lack of specificity.To identify new regulators of replication stress, we developed a novel approach called Replication-Identifier (Repli-ID), which we used to investigate the accumulation of replicative polymerase ε near a barcoded origin of replication in mutant yeast collections. We demonstrate that this method is effective for studying DNA polymerase occupancy directly on chromatin and we identify new regulators of DNA replication fork progression/stability.Finally, we identify and characterize the Mediator complex in the replication stress response. We show how Mediator reduces replication stress by preventing R-loop formation. Show less
Melanoma is the most aggressive and lethal type of skin cancer since it has the ability to spread to other organs in the body making it harder to control the disease.In this thesis, we aim to... Show moreMelanoma is the most aggressive and lethal type of skin cancer since it has the ability to spread to other organs in the body making it harder to control the disease.In this thesis, we aim to explore the degree to which epigenetics play a role in melanoma, namely, inherited and acquired epigenetic alterations in melanoma susceptibility and development. Show less
The studies presented in the work show the potential of the integrative use of biophysical data in defining the structural basis of protein interactions. Even if the results obtained hold a... Show moreThe studies presented in the work show the potential of the integrative use of biophysical data in defining the structural basis of protein interactions. Even if the results obtained hold a degree of ambiguity, this approach allows to iteratively refine and validate the model and interpret its meaning for the molecular basis of protein function. Often all three points at the same time. This dynamic nature makes the use of structural models in the design of therapeutic compounds especially useful since the inhibition of a certain protein function might not require a structure to be accurate down to the last atom but rather highlight key interactions or structural features that can be addressed in context of small molecule or peptide inhibitors. Presented are the use of strucutral biochemistry techniques to investigate the mechanism of how the ubiquitine ligase PSIP1 obtains its target specificity. Furthermore, another epigenetic effector protein PSIP1 is investigated with the aim to develop a workflow for the design of potential peptide-based inhibitors. Show less
In human cells, a meter-long DNA is condensed inside a micrometer-sized cell nucleus. Simultaneously, the genetic code must remain accessible for its replication and transcription to functional... Show moreIn human cells, a meter-long DNA is condensed inside a micrometer-sized cell nucleus. Simultaneously, the genetic code must remain accessible for its replication and transcription to functional proteins. Such plasticity of the genome is maintained by dynamic folding and unfolding of DNA-protein spools called nucleosomes. It is unclear, however, how this process is controlled when multiple nucleosomes stack on top of each other and form compact chromatin fibers. This is particularly important since nucleosomes are rarely present in isolation inside a densely packed cell nucleus. Therefore, the aim of this thesis was to increase the understanding of the chromatin fiber structure and its dynamics. Knowing these details would provide many new insights into the mechanisms of gene expression (epigenetic regulation) which, upon malfunction, may cause severe diseases. The presented work consists of an experimental approach involving the application of single-molecule force spectroscopy, and makes use of theoretical modelling based on statistical mechanics. By using magnetic tweezers, we stretched and twisted individual chromatin fibers reconstituted in vitro in order to unfold its nucleosomes. These studies show that folding of nucleosomes into chromatin fibers opens up a plethora of regulatory pathways for controlling the level of DNA organization in cells. Show less
Cardiovascular disease (CVD) is the collective term for diseases that involve the heart or circulation and CVDs are a major cause of mortality and morbidity worldwide. The aim of thesis was to... Show moreCardiovascular disease (CVD) is the collective term for diseases that involve the heart or circulation and CVDs are a major cause of mortality and morbidity worldwide. The aim of thesis was to investigate the role of inflammation in CVD related cardiac and vascular remodelling, which may lead to potential therapeutic agents. We investigated the therapeutic potential of antibodies directed against phosphorylcholine (PC), an endogenous ligand capable of triggering the innate immune system, which is expressed by apoptotic cells and oxidized LDL, in mouse models for myocardial infarction (MI). We found that treatment with anti-PC antibodies reduces adverse cardiac remodelling after both permanent MI as myocardial ischemia reperfusion (MI-R) injury. Furthermore, we found that treatment with annexin A5 also reduces adverse cardiac remodelling after MI-R injury. Interestingly, both anti-PC as annexin A5 treatment reduced the post MI inflammatory response. Next, we investigated the role of PCAF, an inflammatory related epigenetic factor, in vascular remodelling. We found that PCAF deficiency and treatment with a PCAF inhibitor reduces adverse vascular remodelling. Finally, we investigated the role of microRNAs, small RNA molecules that can affect expression of many different gene simultaneously, in vascular remodelling. We show that inhibition of microRNA-495 reduces adverse vascular remodelling. Show less
Numerous studies have contributed to our current understanding of autoimmune diseases (AIDs), however, pathogenesis of many AIDs can still not be fully explained. Both genetic factors and... Show moreNumerous studies have contributed to our current understanding of autoimmune diseases (AIDs), however, pathogenesis of many AIDs can still not be fully explained. Both genetic factors and environmental factors are involved in the onset of autoimmunity. Which mechanisms explain the contribution of these genetic and environmental factors to disease pathogenesis, and how the different factors interplay remain unanswered key questions. The studies presented in this thesis aimed at identifying and unravelling some of the enigmatic mechanisms in rheumatoid arthritis (RA) and systemic sclerosis (SSc). Show less
The work presented in this thesis provides important new clues on the neurobiology of juvenile psychopathic traits in clinically antisocial juveniles. The data specifically shows that these... Show moreThe work presented in this thesis provides important new clues on the neurobiology of juvenile psychopathic traits in clinically antisocial juveniles. The data specifically shows that these traits are ostensibly underpinned by highly specific corticolimbic network dysfunctions, in which amygdala subregional networks seem particularly relevant. That data additionally suggests that some of these network dysfunctions and their associated neurocognitive deficits are possibly driven by alterations in the oxytocinergic system. Interestingly, the data also provides preliminary neurobiological support for the scientific utility of using juvenile psychopathic traits to subtype the highly heterogeneous group of clinically antisocial teens. While these data may represent important new steps towards a deeper understanding of clinical youth antisociality, their significance has to be evaluated by replication studies that further explore and validate the findings presented here. Show less
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 ultimate goal of translational colon and rectal cancer research is to turn these types of cancer into curable or manageable chronic diseases. The approach to achieve this is to enable... Show moreThe ultimate goal of translational colon and rectal cancer research is to turn these types of cancer into curable or manageable chronic diseases. The approach to achieve this is to enable clinicians to make (adjuvant) treatment decisions, based on the individual patient characteristics and individual characteristics of a patient__s tumor. Identification of new prognostic and predictive biomarkers, based on the biology of individual tumor characteristics, is therefore warranted to further refine the current TNM classification. This thesis describes the use of molecular techniques for the identification of prognostic biomarkers for clinical outcome in (sporadic) colon and rectal cancer. We here present compelling candidate biomarker combinations for validation in further studies. Retrospective and prospective validation of these prognostic biomarker combinations in international and independent patient series is therefore the crucial next step. Additionally, the presented studies stress the importance of -1- combining biomarkers based on tumor biology, -2- integrative analysis of cancer hallmark related processes at all different cellular regulatory levels (genetics, epigenetics and protein level), -3- assessment of tissue specificity between colon and rectal tumors, and -4- studying age-related effects in future colorectal cancer research. Show less
Colorectal cancer is one of the most common diagnosed cancers worldwide, and is the second most important cause of cancer mortality in Europe. The current TNM staging system used at the time of... Show moreColorectal cancer is one of the most common diagnosed cancers worldwide, and is the second most important cause of cancer mortality in Europe. The current TNM staging system used at the time of diagnosis is insufficient, as patients with the same tumor stage show wide variations in survival and tumor recurrence. Therefore, there is a need for identification of new biomarkers in colorectal cancer in order to identify high-risk patients and to guide treatment decision-making. In this thesis, epigenetic markers, including DNA methylation and histone modifications were studied in colorectal cancer patients. Several epigenetic clinically prognostic biomarkers were identified in colorectal cancer in this thesis, including both genome-wide and gene-specific patterns of DNA methylation and histone modifications. Knowledge of tumor biology is of key importance in the development of new therapies and the making of informed treatment decisions. Pathway-focused approaches, as presented in this thesis, provide information regarding possible synergistic interactions of biomarkers. Epigenetic mechanisms are unquestionably tied to the tumorigenic process and should be considered as a grand new source of information not only for identification of prognostic and predictive biomarkers, but also for the development of new, possibly tumor- and therefore patient-specific, anti-cancer therapies. Show less
This thesis is a study on the link between early development and adult health. Studies in animal models indicate that so-called epigenetic marks may be influenced by nutrition during development,... Show moreThis thesis is a study on the link between early development and adult health. Studies in animal models indicate that so-called epigenetic marks may be influenced by nutrition during development, changing the expression of genes implicated in disease. Epigenetics may therefore link development and disease. To investigate this hypothesis in humans we studied DNA methylation, a key epigenetic mark, in individuals exposed during early gestation to the Dutch Famine and individuals born growth restricted, which is also alleged to relate to malnutrition. DNA methylation at metabolic and developmental genes was associated with early gestational famine exposure to the Dutch Famine and the patterns of the associations mirrored the epidemiological findings. The associations found with prenatal famine exposure did not relate to prenatal growth restriction, adding evidence that prenatal growth restriction is not linked with m alnutrition in Western cohorts. Further characterization showed that DNA methylation differences associated with prenatal famine exposure are independent of genetic variation, cluster along biological pathways and within regulatory regions and may relate to the phenotypic consequences of prenatal malnutrition. The work described in this thesis gives credence to the hypothesis that epigenetic marks may be the molecular link between development and later disease in humans Show less
Cardiovascular diseases remain the major cause of death throughout the world and can be primarily attributed to atherosclerotic vascular disease leading to stroke and coronary heart disease (CHD).... Show moreCardiovascular diseases remain the major cause of death throughout the world and can be primarily attributed to atherosclerotic vascular disease leading to stroke and coronary heart disease (CHD). Improved primary prevention and the introduction and subsequent optimization of percutaneous coronary interventions (PCI) for myocardial ischemia due to obstructive CHD have significantly improved patient outcome and reduced morbidity and mortality. The insight into disease pathology has however expanded tremendously over the past decade and continuing research has shifted the focus of interest towards post-interventional accelerated atherosclerosis development due to a dysfunctional (auto) immune inflammatory response, responsible for vascular remodeling, re-occlusion and recurrence of symptoms. The aim of this thesis therefore was to investigate the role of the immune system in this pathophysiological process that ultimately results in post-interventional atherosclerotic vascular remodeling and apply this insight for the development of new immune-modulatory therapies in a preclinical setting. Show less
Uveal melanoma (UM) is the most common malignancy of the eye in adults and it is the second most common form of melanoma after cutaneous melanoma (CM). The identification of patients who have a... Show moreUveal melanoma (UM) is the most common malignancy of the eye in adults and it is the second most common form of melanoma after cutaneous melanoma (CM). The identification of patients who have a high risk of developing metastases would allow the possibility of providing adjuvant therapies to prevent metastases. The application of FISH on transvitreal fine-needle aspiration biopsies is thought to be a reliable method for assaying genetic parameters such as chromosome 3 loss. However, this is based on the assumption that this chromosomal abnormality is distributed homogeneously throughout the tumor. We show that UM can be heterogeneous for the number of copies of chromosome 3 and investigated whether any evidence can be found for heterogeneity in the regulation of tumor-suppressor genes (TSG). Recently, a segregation study identified a potential locus harboring a TSG. One of the genes in this area, RASEF, was analyzed whether the RASEF gene was affected by mutations or gene silencing due to promoter methylation. The MAPK pathway is involved in the balance between melanocyte proliferation and differentiation. Whereas mutant B-RAF and N-RAS are responsible for the activation of the MAPK pathway in most CM, mutations in these genes are usually absent in UM. Nowadays, an assay with increased potential to identify mutations is available and we set out to reanalyze UM cell lines and primary UM for B-RAF mutations. We set out to explore the MAPK pathway by using MAPK profiling and tyrosine kinase arrays. Finally, conclusions drawn from above mentioned studies are summarized and put into perspective. Show less
The first study described the colon tumor-specific methylation of a low CG-dense CpG island that is located in the first intron of the PTPRG gene (PTPRGint1). High levels of specificity and... Show moreThe first study described the colon tumor-specific methylation of a low CG-dense CpG island that is located in the first intron of the PTPRG gene (PTPRGint1). High levels of specificity and sensitivity of this region were observed in sporadic and familial colon cancer which makes this region interesting for application in epigenetic screening panels. In the second study the relation between mutations and DNA methylation was investigated in an attempt to identify initiating factors for DNA methylation in colon cancer. In this study, we were unable to identify an intrinsic tendency towards CpG island hypermethylation other than aberrant accumulation of CpG island methylation via a somatic mutation of BRAF. The relationship between BRAF mutations and DNA methylation was explored further in Chapter 4, which describes an improved study of DNA methylation in BRAF mutationassociated colon cancer. We describe BRAF mutation-specific promoter methylation of the FOX transcription factor genes FOXB1, FOXB2 and FOXD3 and speculate that this methylation might help these tumors escape BRAF-induced senescence. In the final study the DMH technique was combined with oligonucleotide microarrays that contained high CpG island coverage to determine the methylation patterns of infant B-ALL patients. The majority of MLL-rearranged infant ALL cases (i.e., those who are characterized by a t(4;11) or t(11;19) translocation) represent hypermethylated leukemias. In contrast, infant ALL patients with a t(9;11) translocation and those without any MLL translocation (wild-type MLL) displayed DNA methylation patterns that closely resembled the pattern seen in normal bone marrow. This study indicates that patients with a t(4;11) or t(11;19) translocation who have high levels of DNA methylation might be promising candidates for therapies that inhibit DNA methylation. Show less
This thesis examines the impact of genetic and epigenetic factors on several aspects of vascular disease. Part 1 addresses the influence of genetic variation in genes involved in the different... Show moreThis thesis examines the impact of genetic and epigenetic factors on several aspects of vascular disease. Part 1 addresses the influence of genetic variation in genes involved in the different processes that lead to the occurrence of adverse events after percutaneous coronary intervention, mainly restenosis after bare metal stent placement, but also late acquired stent malapposition after implantation of a drug-eluting stent. Part 2 discusses the role of a relatively new area of research, which we refer to as 'epigenetic epidemiology', in restenosis and other aspects of coronary heart disease. In this part we show that polymorphisms in genes encoding lysine acetyltransferases, which are able to modify chromatin structure to allow gene transcription, can influence restenosis and mortality from coronary heart disease in several large prospective follow-up studies. Show less
The general objective of this thesis was to investigate associations between genetic variants involved in inflammation and epigenetics and age-related diseases in an elderly cohort to get more... Show moreThe general objective of this thesis was to investigate associations between genetic variants involved in inflammation and epigenetics and age-related diseases in an elderly cohort to get more insights in the patho-physiological mechanisms involved in age-related diseases, like cardiovascular disease, cognitive decline and cancer. For all analyses we used data of the participants of the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER). We have shown that subjects carrying genetic variants coding for a high pro-inflammatory profile or a low anti-inflammatory profile have an increased risk to develop cardiovascular disease and cognitive decline. Moreover, they tend to have an increased risk of dying as a consequence of cancer. Furthermore we have provided first evidence that the process of epigenetics can play an important role in the patho-physiology of age-related diseases. Future research is necessary to investigate how we can corporate these results into clinical practice. For example, Anti-inflammatory and immunosuppressive mechanisms may be attractive targets for disease prevention and/or treatment. Show less
Survival rates after surgical treatment of gastric, colon and rectal cancer can improve with preoperative and/or postoperative adjuvant treatment with chemo- and/or radiotherapy. The role of... Show moreSurvival rates after surgical treatment of gastric, colon and rectal cancer can improve with preoperative and/or postoperative adjuvant treatment with chemo- and/or radiotherapy. The role of epigenetic aberrancies such as DNA methylation is established to play a pivotal role in gastrointestinal carcinogenesis and malignant progression. This thesis describes novel epigenetic biomarkers that could tailor (neo)adjuvant treatment regimens to the patients who would benefit from them. The same markers could further be used to exclude those patients who would not gain more survival-years and would be unnecessarily exposed to the morbidity/mortality of additional chemo- and/or radiation therapy. Currently, nodal status is the single most important prognostic indicator in gastrointestinal cancers however this can only be reliably assessed by the pathologist after surgery. Currently, neoadjuvant regimens are more and more being studied and shown to be of benefit. This development necessitates biomarkers that are available before the operation. An important derived conclusion from this thesis is that the preoperative availability of primary tumor DNA methylation biomarkers holds great potential for tailoring neoadjuvant therapy. The thesis also describes technical advancements that will enable primary tumor DNA methylation assessment in small amounts of tissue as preoperative diagnostic tumor biopsies harvested during gastroscopy or colonoscopy Show less
