Throughout this thesis, I have endeavored to apply an engineer’s mindset in my pursuit to better understand the marvelously convoluted immune system. In doing so, my colleagues and I have generated... Show moreThroughout this thesis, I have endeavored to apply an engineer’s mindset in my pursuit to better understand the marvelously convoluted immune system. In doing so, my colleagues and I have generated a number of new ‘hardware’(i.e., genetically engineered) tools and ‘software’ modules (i.e., custom analyses and models) that enable the investigation of several otherwise difficult-to- study concepts. Although we have used these modules here to study immune responses, I hope they may be utilized as tools and approaches to crack outstanding questions in other fields of research. Show less
This thesis pioneers diatom molecular identification and quantification through genome-scale methods, with four key aims: (i) reviewing DNA/RNA sequencing methods in aquatic biomonitoring to... Show moreThis thesis pioneers diatom molecular identification and quantification through genome-scale methods, with four key aims: (i) reviewing DNA/RNA sequencing methods in aquatic biomonitoring to highlight their strengths and limitations; (ii) unraveling the evolutionary history of Nitzschia palea and investigating species delimitation within the species complex; (iii) identifying silica genes in N. palea for insights into ecology and evolution; and (iv) assessing a genome-scale quantification method for diatom biomonitoring to improve accuracy and scalability in estimating abundances. The review (Chapter 2) emphasizes disparities between molecular and morphology-based approaches and introduces the challenges in accurately estimating species abundances. Chapter 3 explores N. palea's evolutionary history using transcriptome data and reveals reticulate evolutionary patterns resulting in a putative hybrid between populations with different morphological characteristics. Chapter 4 pinpoints silica genes in N. palea and reveals variations among different populations that may lead to differences in silica metabolism. Chapter 5 introduces a genome-scale quantification approach that provides a promising alternative for molecular diatom biomonitoring due to its improved taxonomic resolution and quantification accuracy. In summary, this thesis underscores that genome-scale methods' have a critical role in diatom identification and quantification, and in advancing our understanding of microalgal taxonomy, ecology, and evolution. Show less
The human ovary is responsible for producing eggs and steroid hormones necessary for reproduction. Ovarian factors, such as anovulation, polycystic ovarian syndrome, and decreased egg quality, can... Show moreThe human ovary is responsible for producing eggs and steroid hormones necessary for reproduction. Ovarian factors, such as anovulation, polycystic ovarian syndrome, and decreased egg quality, can lead to female infertility. Although advances have been made in assisted reproductive technologies (ART) and fertility preservation approaches, there is still a demand for new treatments and approaches for ovarian diseases and female infertility. The main obstacle to developing effective approaches is the lack of knowledge about the human ovary, especially the cellular development and molecular basis of oogenesis and folliculogenesis processes. The advances in single-cell RNA sequencing (scRNA-seq) techniques have opened up opportunities for studying the transcriptomes of human ovarian cells, decoding cell types and sub-populations, and identifying signature genes during oogenesis and folliculogenesis. In my research, we utilized the scRNA-seq technique to provide valuable transcriptomic datasets of human ovarian cells, contributing to the establishment of the molecular landscape of human oogenesis and folliculogenesis. Show less
The study of orchid flowers, fruits, and inflorescences is crucial due to the remarkable diversity of orchid species and their unique adaptations to pollinators and seed dispersers. However, our... Show moreThe study of orchid flowers, fruits, and inflorescences is crucial due to the remarkable diversity of orchid species and their unique adaptations to pollinators and seed dispersers. However, our understanding of the evolution and development of these organs within the orchid family remains limited. This research aims to fill this knowledge gap by investigating the genetic mechanisms underlying the evolution and development of floral structures, fruits and resupination in orchids, and the relationship between inflorescence stalk lignification and orientation. The research also includes a methodological chapter on the application of transcriptomics for plant species identification. Using advanced techniques such as microscopy imaging, 3D CT scanning, and anatomical analysis, the study provides detailed insights into the processes of root and fruit resupination and shows that inflorescence lignification is a heritable trait, with closely related orchid species displaying similar levels of lignification compared to distantly related species. The findings significantly advance our understanding of orchid biology by filling gaps in our knowledge of the evolutionary and developmental processes involved in flower and fruit development, resupination, and inflorescence lignification. By identifying specific genes and pathways associated with these traits, the study offers valuable insights into the genetic mechanisms that drive orchid diversity and adaptation. From a practical perspective, these findings hold great promise for the development of new orchid varieties with more robust and visually appealing varieties. The research also highlights the importance of conservation efforts to protect orchid diversity and their ecological relationships with pollinators and seed dispersal vectors. Show less
Major achievements in the field of immune oncology have demonstrated the ability of the immune system to induce a response against cancer. The prognostic impact of pre-existing immunity in several... Show moreMajor achievements in the field of immune oncology have demonstrated the ability of the immune system to induce a response against cancer. The prognostic impact of pre-existing immunity in several cancer types, including breast and colon cancer, demonstrates the influence of the immune system on disease progression. At the same time, immunotherapeutic approaches that aim to enhance antitumor immune reactions have significantly improved the clinical outcome for a subset of patients. However, a large proportion of patients (60-80%) do not respond to immunotherapeutic treatments. To extend the benefit of immunotherapeutic strategies to a larger number of patients, it is imperative to understand the mechanisms associated with immune responsiveness. Different variables have been described to influence the development of antitumor immunity in cancer patients, including the tumor’s genetic program, the genetic makeup of the patients, and environmental factors such as the microbiome. These factors likely act in concert to modulate antitumor immune responses. This thesis aimed to dissect the molecular determinants of cancer immune responsiveness in human tumors. A systems biology approach was used to define underlying factors that shape the tumor microenvironment and reveal potential mechanisms of immune escape. Show less
In this thesis, I study 1) metabolic alterations in tuberculosis related to wasting syndrome in human patients as well as in rodent and fish animal models. 2) effects of the mutation of the leptin... Show moreIn this thesis, I study 1) metabolic alterations in tuberculosis related to wasting syndrome in human patients as well as in rodent and fish animal models. 2) effects of the mutation of the leptin gene on cachexia and diabetes in rodent and zebrafish animal models. 3) how tuberculosis infection and resulting metabolic reprogramming are dependent on leptin signaling in mice and zebrafish larvae. Show less
Leprosy is a multifactorial chronic disease caused by Mycobacterium leprae or Mycobacterium lepromatosis that affects the skin and nerves. More than 200.000 new cases are diagnosed per year; thus,... Show moreLeprosy is a multifactorial chronic disease caused by Mycobacterium leprae or Mycobacterium lepromatosis that affects the skin and nerves. More than 200.000 new cases are diagnosed per year; thus, transmission is still ongoing. The most likely way of transmission is the respiratory route form human-to-human; however, transmission is still not clearly understood. Early diagnosis of leprosy is crucial to reduce and avoid transmission as well as leprosy-associated disabilities, which are also a cause of stigma. Currently, diagnosis is performed based on clinical signs and symptoms and late- or mis-diagnosis are not uncommon.In this thesis, we combined the study of pathogen transmission with host transcriptomic and genomic biomarkers. To explore M. leprae transmission a One Health approach was followed, where human, animal and environmental samples were studied.The combination of demographic characteristics, pathogen detection, genetic and/or transcriptomic biomarkers can be applied in a multifactorial leprosy signature applicable for early diagnosis of leprosy and/or to guide intervention strategies. Identification of predictive biomarkers will in due course lead to prompt treatment, preventing leprosy-associated irreversible disabilities as well as reducing M. leprae transmission. Show less
Autosomal Dominant Polycystic Kidney Disease (ADPKD) progression involves a complex interaction of different molecular pathways, ultimately leading to cyst growth and loss of kidney function. The... Show moreAutosomal Dominant Polycystic Kidney Disease (ADPKD) progression involves a complex interaction of different molecular pathways, ultimately leading to cyst growth and loss of kidney function. The exact mechanism behind cyst formation is still not clearly understood. Moreover, we know some of the molecular pathways involved in cyst initiation and progression, but we do not know at which stage of the disease they play a role. In this thesis, we investigated the molecular pathways involved in renal injury-repair mechanisms and ADPKD. According to the currently available literature, injury-repair and ADPKD are two extremely intertwined mechanisms, which not only are characterised by activation of similar molecular pathways but are also able to influence each other. In fact, injury is able to accelerate cyst formation and progression, and cyst growth can cause injury to the surrounding tissue. Thus, the introduction of injury in the context of ADPKD can help to characterize the steps of disease progression, particularly in the early phases of cyst initiation, and direct future research to new possible therapeutic targets. Show less
In this thesis, we aimed to better understand the underlying mechanisms involved in TNBC progression and metastasis formation and discover new targets to reduce breast cancer related deaths. We... Show moreIn this thesis, we aimed to better understand the underlying mechanisms involved in TNBC progression and metastasis formation and discover new targets to reduce breast cancer related deaths. We performed an imaging-based RNAi phenotypic cell migration screen in two highly motile TNBC cancer cell lines to provide a repository of signaling determinants that functionally drive TNBC cell motility. Interestingly, two modulators essential for cancer cell migration were known to be involved in RNA splicing, making us decide to focus on the role of RNA splicing in breast cancer progression. We next summarized the current knowledge about splicing factors in breast cancer development and progression and identified co-regulated splicing factors that were associated with aggressive breast cancer phenotypes and metastasis formation that was not only restricted to breast cancer, increasing the global understanding of the role of the spliceosome in cancer development and progression. Moreover, the role of splicing factors in two major processes in cancer progression, cell migration and proliferation, was examined. Finally, using RNA sequencing, we systematically compared the transcriptomes of 14 breast cancer cell lines cultured both in 2D and 3D conditions to unravel the reprogramming that is associated with the invasive phenotype of basal B TNBC. 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
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
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
