The use of existing medications for diseases they were not originally developed for is called drug repositioning. A popular drug repositioning method to find new drugs against specific cancer types... Show moreThe use of existing medications for diseases they were not originally developed for is called drug repositioning. A popular drug repositioning method to find new drugs against specific cancer types is to search for drugs which are expected to bring back the gene expression activity of cancer cells to that of healthy cells (‘normalization’). One of the main research goals of this thesis was to investigate of this method could also be used on the gene expression profiles of individual tumors, enabling personalization of drug repositioning candidates for each patient. We initially had some success with this approach but this eventually lead to a systematic validation of the underlying principle using almost 10,000 tumor samples across 18 different tumor types. Unfortunately, the predictive power of the method was found to be much lower than previously reported and the part that remained could be nullified by correcting the gene expression profiles of the drugs for the downstream effects of reduced cell division. These results indicate that the current use of the method does not result in drug repositioning candidates specific for a tumor type but is only able to select generally cell-toxic drugs. Show less
This thesis sought to obtain a better understanding of the composition of the immune microenvironment in NSCLC and how to modulate this tumor immune microenvironment by RT to induce amplified... Show moreThis thesis sought to obtain a better understanding of the composition of the immune microenvironment in NSCLC and how to modulate this tumor immune microenvironment by RT to induce amplified antitumor immune responses to ICIs in advanced NSCLC patients. In the first part of this thesis, a multiangular approach of a combination of protein and mRNA expression with clinicopathological characteristics in a large cohort of early stage, resected NSCLC samples will be discussed. The second part focusses on the immune modulating effects of RT, in particular when combined with immunotherapy treatment in metastatic NSCLC. Show less
The work described in this thesis presents part of a framework that can be used to extract detailed disease biological information from peripheral tissue. This framework is based on the... Show moreThe work described in this thesis presents part of a framework that can be used to extract detailed disease biological information from peripheral tissue. This framework is based on the central dogma of biology “DNA to RNA to protein” and on a systems biology approach that aims to produce synergetic data whose disease pathological, prognostic and predictive value is greater than the sum of the individual experiment results. HD patients are often characterized by a multifaceted clinical profile, consisting of several symptoms and variable disease progression rates. Therefore, a systems approach such as the one described above is expected to be the most effective in identifying potential treatments and predictive biomarkers that will be most informative for the different patient subpopulations. 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 work described in this thesis focuses on the mechanisms that give rise to alternative mRNAs and their alternative translation into proteins. Each of the described studies has been based on a... Show moreThe work described in this thesis focuses on the mechanisms that give rise to alternative mRNAs and their alternative translation into proteins. Each of the described studies has been based on a specific set of high-throughput RNA sequencing technologies. An overview of the available RNA sequencing methods, together with an introduction to different regulatory layers which define the expression of a gene, are presented in Chapter 1. Our work in Chapter 2 and Chapter 3 investigates the process of alternative polyadenylation. Chapter 2 shows the role of alternative polyadenylation in the context of oculopharyngeal muscular dystrophy. Chapter 3 describes genetic variants associated with alternative polyadenylation. Chapter 4 focuses on mechanisms controlling protein synthesis (translation) during skeletal muscle differentiation, highlighting changes in the use of alternative translation initiation sites. In Chapter 5 we investigated the interdependence between alternative regulatory events in gene expression. In this study, based on single-molecule full-length RNA sequencing, we demonstrated coordination and interdependence between alternative transcription initiation, alternative splicing, and alternative polyadenylation. Finally, Chapter 6 connects fundamental research in the RNA field with clinical care, describing new diagnostic and therapeutic approaches. Show less
Recent studies indicate that there is a global rise in the prevalence of asthma and other allergic disorders. Several epidemiological studies conducted in countries endemic for parasitic worms ... Show moreRecent studies indicate that there is a global rise in the prevalence of asthma and other allergic disorders. Several epidemiological studies conducted in countries endemic for parasitic worms (helminths) have reported an inverse association between the presence of helminth infections and allergic disease. The main objectives of the study described in this thesis were to: i. To determine urban-rural differences in allergy outcomes in Ghana, West Africa ii. To examine the association between helminth infections and allergies iii. To characterize IgE responses associated with helminth infections and allergies The thesis describes cross-sectional studies among schoolchildren aged 5-16 years living in urban and rural areas of Southern Ghana. Study findings showed marked urban-rural differences in the prevalence of allergy outcomes with current infection with the waterborne helminth schistosoma being inversely associated with mite skin prick test allergic sensitization. In the study population, elevated levels of allergen specific IgE were observed that did not translate into skin reactivity or reported symptoms. Differences in gene expression profiles were also observed between urban and rural children. Overall, study findings indicate that factors associated with urbanization such as reduced exposure to parasitic worms are associated with the increased prevalence of allergy outcomes in Ghanaian children 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
Cellulose makes up one of the most abundant renewable materials, present in all kinds of plant biomass (Pauly and Keegstra 2010). However, to be able to utilize the cellulose as feedstock, it needs... Show moreCellulose makes up one of the most abundant renewable materials, present in all kinds of plant biomass (Pauly and Keegstra 2010). However, to be able to utilize the cellulose as feedstock, it needs to be separated from lignin which cements the cellulose and hemi-cellulose fibers. Lignolytic peroxidases can be produced by Aspergillus niger, and its production was found to be improved by heme supplementation, suggesting a limiting effect of this co-factor during heterologous expression. The research described in this thesis explores fungal heme biosynthesis and its regulation by means of heme deficient mutant strains and overexpression strains of heme biosynthesis genes or corroborated iron metabolism with the final aim to increase the available intracellular heme for peroxidase production. Using heme deficient strains, we demonstrated that A. niger is capable of heme uptake and utilzation and that siroheme synthesis derives from the first half of the heme biosynthesis pathway as well. The tight regulation on heme biosynthesis on transcription and (post)translational level prohibits large changes in heme content, and indicated a bottleneck on the level of ferrochelatase and possible uroporphyrinogen III decarboxylase and coproporphyrinogen III oxidase and questions whether A. niger would be the most suitable host for heterologous peroxidase production. Show less
DNA damage, mutations and genomic instability are established driving forces of cancer and other age-related diseases. Mutations in tumor suppressor genes and oncogenes are very frequently found in... Show moreDNA damage, mutations and genomic instability are established driving forces of cancer and other age-related diseases. Mutations in tumor suppressor genes and oncogenes are very frequently found in tumors and genomic instability is the most common enabling characteristic of cancer. Aging is also believed to be enabled, amongst others, by genomic instability. DNA repair pathways, like the nucleotide excision repair (NER) pathway and cell cycle control (e.g. p53-dependent) processes are therefore vital to organisms, since these processes counteract or prevent genomic instability, and are thought to underlie, when affected, aging and age-related diseases like cancer. To unravel the functions, mechanisms and pathways involved in the onset of aging and age-related diseases we have investigated several mouse models deficient in either DNA repair (NER) capacity (Chapter 3, 4), cell cycle control (p53) (Chapter 6) or both (Chapter 5), and compared this to a wild type situation (Chapter 2). The use of mouse models enabled us to investigate cancer and aging in a controlled environment, minimizing possible confounding factors. Additionally, the mouse models can be useful as an alternative tool to identify genotoxic and non-genotoxic carcinogens that can be harmful to the society and the environment (Chapter 5). 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 results obtained in this thesis suggest that the most explicit differences between normal and atypical melanocytes are subtle changes in pigment biosynthesis and the functioning of the... Show moreThe results obtained in this thesis suggest that the most explicit differences between normal and atypical melanocytes are subtle changes in pigment biosynthesis and the functioning of the antioxidant system. Impairment of the antioxidant system and increased levels of pheomelanin result in increased levels of oxidative stress. It is anticipated that these increased levels of oxidative stress contribute to early melanoma development by inducing DNA mutations, but additional studies are required to prove this hypothesis. Show less
Gene expression is a complicated process with multiple types of regulation, including binding of proteins termed transcription factors. This thesis looks at transcription factors and transcription... Show moreGene expression is a complicated process with multiple types of regulation, including binding of proteins termed transcription factors. This thesis looks at transcription factors and transcription factor binding site discovery through computational predictions and wet lab work to better elucidate their role in transcriptional regulation. This includes bioinformatics tools to extrapolate transcription factors common to a set of co-regulated sequences, such as genes differentially expressed in a microarray or next-generation sequencing experiment. It also includes a working pipeline to analyze next-generation sequencing data, used in the following projects: Next-generation sequencing of chromatin-immunoprecipitated CBP and p300 (two highly homologous transcription factors) bound DNA was performed to analyze their (different) roles in cell cycle regulation. Next-generation sequencing of RNA from differentiating muscle cells was also done to identify differential gene expression during myogenesis, as well as identify novel promoter regions (a common target of transcription factors). Taken together, computational and wet lab tools can enhance our knowledge of transcriptional regulation, as described by several applications to enhance our knowledge of myogenic and cell-cycle regulation in this thesis. Show less
Blood-flow-induced shear stress plays an important role in cardiovascular development and disease. How endothelial cells sense shear stress remains to be elucidated. We postulated that the primary... Show moreBlood-flow-induced shear stress plays an important role in cardiovascular development and disease. How endothelial cells sense shear stress remains to be elucidated. We postulated that the primary cilium is a component of the endothelial shear sensor. This luminal cell protrusion contains microtubules and is connected to the microtubular cytoskeleton. We identified cilia on endothelial cells of the embryonic heart in areas of low or oscillatory shear stress. This shear-related distribution is reminiscent of the distribution of atherosclerotic lesions in the adult arterial system, as lesions develop at sites of low or oscillating shear (athero-prone flow). Ciliated endothelial cells are exclusively present at these atherosclerotic predilection sites in adult mice. Athero-prone (oscillatory) but not athero-protective (steady or pulsatile) flow induces ciliation of cultured endothelial cells. Moreover, the endothelial shear response is dependent on the microtubular cytoskeleton and primary cilia sensitise the endothelium for shear. Taken together, these data demonstrate that primary cilia are induced by athero-prone flow and that ciliated cells are more sensitive to shear stress. We conclude that the endothelial biosensor for shear stress is the microtubular cytoskeleton and that the attached primary cilium functions as a signal amplifier in areas subjected to athero-prone flow. Show less
Total mesorectal excision (TME) is the standard treatment for rectal cancer, while transanal endoscopic microsurgery (TEM) is a recently introduced surgical approach for the treatment of rectal... Show moreTotal mesorectal excision (TME) is the standard treatment for rectal cancer, while transanal endoscopic microsurgery (TEM) is a recently introduced surgical approach for the treatment of rectal adenomas. Incorrect preoperative staging before TEM is a problem. Therefore the aim of this thesis was to identify molecular differences between rectal tumors of different stages, using gene expression profiling and genomic analysis. First protocols and data analysis algorithms for a new type of SNP array were developed. These studies showed that reliable LOH and copy number changes could be obtained from both frozen and paraffin embedded material. Consequently SNP arrays were used to type groups of TEM and TME treated samples. Five genomic events were found which could make a clear discrimination between adenomas and carcinomas. Early carcinomas treated by TEM, which were not recognized preoperatively as carcinomas, showed already carcinoma-associated aberrations. Analysis of tree core biopsies per patient showed a large degree of intra- tumor heterogeneity; although a good correlation was obtained between the biopsy with the largest number of aberrations and its corresponding tumor fraction. Gene expression array analysis was performed on the same samples as the SNP array series. A high concordance between chromosomal aberrations and changes in gene expression was observed. Finally a clinical application of these data is discussed in the preoperative staging of rectal tumours. Show less
Using newly developed single cell A3243G mutation load assays a novel mechanism of mtDNA segregation was identified in which the multi-copy mtDNA nucleoid takes a central position. Furthermore,... Show moreUsing newly developed single cell A3243G mutation load assays a novel mechanism of mtDNA segregation was identified in which the multi-copy mtDNA nucleoid takes a central position. Furthermore, likely due to low level changes in gene expression, no genes or gene sets could be identified with gene wide expression analysis that would hint to the molecular pathways that are altered upon loss of mitochondrial ATP production as a consequence of A3243G mtDNA mutation. Extensive post-transcriptional adaptation in the form of global translation repression, was however apparent. A comparison between two mtDNA haplotypes indicated, that these presumably neutral sequence variants can affect the nuclear expression program, which tentatively indicates that mtDNA haplotype can affect phenotype. Show less
Arsenic (As) is a notoriously poisonous metalloid with known hazardous effects to human health. The project described in this thesis was aimed at elucidating the probable mechanism of As-induced... Show moreArsenic (As) is a notoriously poisonous metalloid with known hazardous effects to human health. The project described in this thesis was aimed at elucidating the probable mechanism of As-induced neurotoxicity in vivo and in vitro. The animal studies in this thesis were designed to answer questions about the effect of As on the peripheral nervous system after sub-acute and chronic intoxication of laboratory rats. Protein composition analysis showed compositional changes in sciatic nerves proteins. Protein expression of neurofilament heavy (NF-H) and neurofilament medium (NF-M) remained unchanged. Neurofilament protein light (NF-L) expression was reduced, while _- and m-calpain protein expression was increased, both in a dose/time pattern. Furthermore, NF-H protein was hypophosphorylated; while NF-L and microtubule-associated protein tau (MAP-tau) proteins were phosphorylated. In the in vitro studies, effects of As species were tested in various cell culture models and the manner of their hyperphosphorylation was further studied for a better understanding of the disruption of neuroskeletal integrity by As. In vitro studies showed that the compositional changes were not caused by the changes on RNA expression levels, rather a post-translational activity. Cells treated with arsenite showed cleavage of p35 to p25 by calpain, which is mediated by an increase of Ca2+ in the cells. Over expression of calpain results in hyperphosphorylation of NF-L and activated calpain is also responsible for NF-L degradation. Show less
Through evolution the social amoebas have developed mechanisms to adapt to environmental changes and ensure survival. This thesis explores the evolutionary origins of cAMP signalling and regulation... Show moreThrough evolution the social amoebas have developed mechanisms to adapt to environmental changes and ensure survival. This thesis explores the evolutionary origins of cAMP signalling and regulation of developmental decisions in the model organism Dictyostelium discoideum. It also shows the first molecular-based phylogeny of the Dictyostelids. Development in Dictyostelium is characterized by the formation of a multicellular structure, the fruiting body, with a well-defined temporal and spatial pattern. cAMP, normally used as intracellular second messenger, in Dictyostelium is used also as an extracellular signal (chemoattractant) to mediate cell movement and cell differentiation. The study of the different components that control the formation of a multicellular fruiting body at a molecular level and from an evolutionary perspective shows that extracellular cAMP signalling was originally developed to control fruiting body morphogenesis. Furthermore it reinforces the idea that Dictyostelium is a simple but yet robust model to study the origins of multicellularity. Do to cAMP being so prevalent in Dictyostelium development I have studied the regulation of cAMP production during particular developmental stages showing in this thesis novel roles for the adenylyl cyclases that produce cAMP and their specific patters of expression during development. A thorough pharmacological analysis of these enzymes is also present in this work. Show less
Glomerulosclerosis is a general term describing the process of scarring of the glomeruli, the functional units in the kidney that filter urine from the blood. This severe, irreversible complication... Show moreGlomerulosclerosis is a general term describing the process of scarring of the glomeruli, the functional units in the kidney that filter urine from the blood. This severe, irreversible complication can occur secondary to various already established systemic or local diseases. However, not all patients with renal diseases show progression to end stage renal disease (ESRD). Thus renal patients can be subdivided into progressors and non-progressors based on clinical parameters1. Why patients with renal diseases become progressors or non-progressors is unclear, and better insight into the pathogenesis of glomerulosclerosis may improve our understanding of the process towards progression. However, the pathogenesis of glomerulosclerosis is complex and still poorly understood, although genetic factors probably play a role, given the considerable variation among individuals in both the risk of developing glomerulosclerosis and the rate of progression. Therefore, the first aim of the work described in this thesis was to identify genes involved in the progression and repair of glomerulosclerosis, using an animal model that allows a clear distinction between progression and repair after renal injury. The second aim was to gain better insight into the pathogenesis of glomerulosclerosis by investigating the expression and activity of fibrosis-related molecules in an animal model and in patients with renal diseases. Show less
