In recent decades, climate change has led to more frequent and severe drought events, causing serious consequences such as increased forest mortality and significant crop yield losses.... Show moreIn recent decades, climate change has led to more frequent and severe drought events, causing serious consequences such as increased forest mortality and significant crop yield losses. Understanding how drought affects plants, especially economically important herbaceous species, is crucial for predicting and developing drought-resistant crops. To address this issue, this study analyzed a comprehensive dataset of anatomical and hydraulic traits in different genotypes of Arabidopsis thaliana and tomato, including both wild-type and transgenic mutants. The study also investigated the expression of four well-known drought marker genes associated with ABA-dependent and ABA-independent pathways and the impact of overexpressing the JUNGBRUNNEN1 (JUB1) gene on drought response. The findings revealed that each genotype had a unique set of traits to cope with drought, which could be categorized into two response strategies. One group enhanced their drought resistance through traits like a more negative stem P50, thicker intervessel pit membranes, a more lignified inflorescence stem, and a gradual reduction of the low initial stomatal conductance during drought. This strategy enabled them to maintain a relatively high and stable leaf water potential (Ψl). The second group, represented by JUB1 overexpression genotypes, relied primarily on maintaining a high Ψl which is possibly due to osmoprotectant accumulation in leaves, while the other traits have not been recorded. Overall, this research demonstrated the adaptive capabilities of herbaceous plants to drought conditions, highlighting the intraspecific variation in drought responses that underscores the need for a detailed assessment of drought-responsive traits to improve crop yield in a warming world. Show less
Plant-microbe interaction resulted in different physio/chemical responses by host plant and interacting rhizobacteria. This thesis focuses on how different plants and rhizobacteria combinations... Show morePlant-microbe interaction resulted in different physio/chemical responses by host plant and interacting rhizobacteria. This thesis focuses on how different plants and rhizobacteria combinations modulate plant metabolism. Factorial combinations of different plant species, including Arabidopsis thaliana (model plant), Brassica oleracea var. italica (crop) and Artemisia annua (medicinal plant), and phylogenetically distinct rhizobacterial species, including Pseudomonas fluorescens SS101 (Pf SS101), Microbacterium and three Paraburkholderia species, were used as study model systems in this thesis. Untargeted metabolomics was used to assess the impact of these rhizobacteria on the shoot chemistry of the host plant species. This study revealed that root treatment of different plant species with rhizobacteria altered 18-78% of the detected plant secondary metabolites in the shoot. We also assessed the impact of a known bacterial trait on plant phenotype and chemistry. cysH mutation of Pf SS101 affected the chain elongation of aliphatic glucosinolate biosynthesis in Arabidopsis whereas it led to an accumulation of indolic glucosinolates and flavonoids in Broccoli. To further discover the bacterial traits affected during the interaction with Broccoli roots, genome wide transcriptome analysis was carried out, resulting in upregulation of genes involved in flagellar assembly, chemotaxis, and motility together with nutrient uptake and (an)ion transporter in Paraburkholderia species. 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
Image analysis of objects in the microscope scale requires accuracy so that measurements can be used to differentiate between groups of objects that are being studied. This thesis deals... Show more Image analysis of objects in the microscope scale requires accuracy so that measurements can be used to differentiate between groups of objects that are being studied. This thesis deals with measurements in yeast biology that are obtained through microscope images. We study the algorithms and workflow of image analysis of yeast cells in order to understand and improve the measurement accuracy. The Saccharomyces cerevisiae cell is widely used as a model organism in the life sciences. It is essential to study the gene and protein behaviour within these cells, and consequently making it possible to find treatment and solutions for genetic and hereditary diseases. This is possible since many processes that occurs at the molecular level in this organism are similar to those in human cells. In the research group Imaging and Bioinformatics, we have developed a framework for analysis of yeast cells. This framework is intended to serve as a support for research in yeast biology. The framework is integrated in one application and presented via a GUI. The application integrates modules and algorithms including segmentation, measurement, analysis and visualization. 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
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
Bio-informatica kan omschreven worden als het toepassen van algoritmen om meerwaarde te verkrijgen uit data afkomstig van biomedisch en/of biologisch onderzoek. In bio-informatica wordt onderzoek... Show moreBio-informatica kan omschreven worden als het toepassen van algoritmen om meerwaarde te verkrijgen uit data afkomstig van biomedisch en/of biologisch onderzoek. In bio-informatica wordt onderzoek gedaan met grote gegevens verzamelingen die afkomstig zijn uit biomedisch en/of biologisch experimenten. Het doel van dit onderzoek is komen tot nieuwe inzichten vanuit de gegevens verzameling. Deze inzichten komen tot stand door de goede organisatie van de data, het linken naar en integreren met complementaire gegevens verzamelingen en ontwikkelen en toepassen van analytische methodieken. Als bio-informatica groep onderzoeken wij het inrichten en ontwikkelen van een 3D spatio-temporele data omgeving voor ontwikkelingsstudies van het zebravis model organisme. De expressie van genen in spatio-temporale patronen vormt de basis van het ontwikkelingsproces. Voor onderzoekers is een begrip van deze patronen in sam enhang met de anatomische ontwikkeling belangrijk; hoe vormen de patronen de basis voor vorm verandering en welke genen kunnen bij dergelijke veranderende patronen betrokken zijn. In deze context hebben wij een omgeving ontwikkeld voor spatio-temporele gegevens uit embryonische studies van het zebravis modelsysteem. 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
In developmental biology, the expression of genes is studied to understand development, phenotypes and to construct models to understand disease. In this thesis, we explore and validate biological... Show moreIn developmental biology, the expression of genes is studied to understand development, phenotypes and to construct models to understand disease. In this thesis, we explore and validate biological as well as computerized tools, to address research questions in developmental biology. Based on these techniques, we developed a workflow to generate a large number of 3D spatio-temporal patterns of gene expression. Though several techniques for gene expression analysis are available, most spatial gene expression data are only in 2D. In order to study gene expression and differentiation of structures during development at the same time, both spatial 3D information, and temporal data are essential. These spatio-temporal patterns of gene expression have to be generated. To that end, we have developed a workflow based on fluorescent in situ hybridization (FISH) (ZebraFISH;), confocal laser scanning microscopy (CLSM) and subsequent three-dimensional modeling with, in our case, TDR-3Dbase software- resulting in a large amount of 3D spatio-temporal patterns of gene expression, obtained in a straightforward and non-destructive manner. In the work described in this thesis, we applied the workflow to 30 genes in 5 developmental processes. 3D modeling and data mining software are used to analyse gene expression patterns in zebrafish embryos and across species 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
