Actinobacteria are Gram-positive bacteria that have a complex multicellular life cycle and are well known for their ability to produce a wide range of bioactive natural products (NPs). High... Show moreActinobacteria are Gram-positive bacteria that have a complex multicellular life cycle and are well known for their ability to produce a wide range of bioactive natural products (NPs). High throughput screening has failed to deliver the new antibiotics we so desperately need to combat multidrug-resistant pathogens. Therefore, new systematic approaches are needed to further explore the rich potential of Actinobacteria. The work described in this thesis entails systems biology approaches consisting of technologies such as proteomics, genomics, metabolomics and DNA binding studies. These were then applied to identify the biosynthetic gene clusters (BGCs) that are responsible for the production of novel antibiotics. Small molecules were thereby used as elicitors to activate the expression of cryptic BGCs in Streptomyces roseifaciens. Furthermore, S. coelicolor M1152 that was optimized for heterologous expression of antibiotics, was analysed for changes in protein expression, to understand which changes correlate to optimal antibiotic production. Finally, the role of the nucleoid associated protein SCO1839 in development and antibiotic production was studied. Chip-seq technology showed that it binds to thousands of DNA sequences on the S. coelicolor chromosome, which contain the motif GATC. I hope that this thesis contributes to utilizing multi-dimensional ‘omics approaches to answer major biological questions. Show less
Antibiotic resistance is an increasing problem in the battle against (bacterial) infectious diseases. The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) threatens to render... Show moreAntibiotic resistance is an increasing problem in the battle against (bacterial) infectious diseases. The emergence of drug-resistant Mycobacterium tuberculosis (Mtb) threatens to render tuberculosis (TB) untreatable. Efforts to develop novel antibiotics have so far been unsuccessful, calling for additional approaches for treatment of bacterial infections. Intracellular pathogens like Mtb and Salmonella can survive in the host by manipulating host cell signaling. This provides opportunities for novel therapeutic strategies by targeting the host, rather than the bacterium (host-directed therapy). In this thesis we report the development and application of novel (in vitro and in vivo) methods for identifying host genes and proteins involved in host control of intracellular bacteria, as well as chemical compounds that target host molecules as a basis for drug development for host-directed therapies. As a result, we report the identification of RTK inhibitors, the novel kinase inhibitor 97i, the human kinase family PCTAIRE and the host protein DRAM1 as promising leads for further drug development for host-directed therapeutic strategies for intracellular bacterial infections. Show less
Climate change is a challenge for both current and future generations. New biological resources have to be developed in order to meet the demand for energy as well as the demand for food. One way... Show moreClimate change is a challenge for both current and future generations. New biological resources have to be developed in order to meet the demand for energy as well as the demand for food. One way of doing this is to make use of so-called smart crops, as they have improved yields and a small environmental footprint. Model plant organisms, such as Arabidopsis thaliana, can contribute to the development of smart crops as they have been studied extensively by the research community. The purpose of this research is to unravel the pathways leading to the enhanced growth characteristics phenotype of two phenotypically engineered Arabidopsis mutants from a systems biology perspective, by using metabolomics. In metabolomics, metabolites are studied both qualitatively and quantitively under specific growth conditions. Since metabolites are the end products of cellular processes, the metabolome is most closely related to the phenotype of plants. To obtain the metabolic profile directly from the leaves of Arabidopsis, high-resolution magic angle spinning (HR-MAS) NMR is used. Combined with multivariate analysis, biomarkers, related to the mutant phenotype, are determined. The biological interpretation of the biomarkers will be performed in a systems biology approach to obtain a model to explain the mutant phenotype. Show less
The various models proposed for the mediation of auditory verbal hallucinations (AVH) implicate a considerable number of brain areas and mechanisms. To establish which of those mechanisms are... Show moreThe various models proposed for the mediation of auditory verbal hallucinations (AVH) implicate a considerable number of brain areas and mechanisms. To establish which of those mechanisms are actually involved in the mediation of AVH, we developed a novel method to analyze functional MRI data, which allows for the detection of the full network of mutually interacting brain states, and the identification of those states that are relevant to the mediation of AVH, while applying a minimum number of preconceived assumptions. This method is comparable to the draining of a pond to lay bare the full ecosystem that affects the presence of a particular fish species. We used this model to analyze the fMRI data of 85 psychotic patients experiencing AVH. The data were decomposed into 98 independent components (ICs) representing all major functions active in the brain during scanning. ICs involved in mediating AVH were identified by associating their time series with the hallucination time series as provided by subjects within the scanner. Using graph theory, a network of interacting ICs was created, which was clustered into IC modules. We used causal reasoning software to determine the direction of links in this network, and discover the chain of events that leads to the conscious experience of hallucinations. Hallucinatory activity was linked to three of the seven IC clusters and 11 of the 98 ICs. ICs with the most influential roles in producing AVH-related activity were those within the so-called salience network (comprising the anterior cingulate gyrus, right insula, Broca's homologue, premotor cortex, and supramarginal gyrus). Broca's area and the cerebellar regions were significantly, but more distantly involved in the mediation of AVH. These results support the notion that AVH are largely mediated by the salience network. We therefore propose that the mediation of AVH in the context of schizophrenia spectrum disorders involves the attribution of an excess of negative salience by anterior-cingulate areas to linguistic input from Broca's right homologue, followed by subsequent processing errors in areas further ‘downstream’ the causal chain of events. We provide a detailed account of the origin of AVH for this patient group, and make suggestions for selective interventions directed at the most relevant brain areas. Show less
Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for... Show moreAdverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field. Show less
Toledo, J.B.; Arnold, M.; Kastenmüller, G.; Chang, R.; Baillie, R.A.; Han, X.; ... ; Kaddurah-Daouk, R. 2017
IntroductionThe Alzheimer's Disease Research Summits of 2012 and 2015 incorporated experts from academia, industry, and nonprofit organizations to develop new research directions to transform our... Show moreIntroductionThe Alzheimer's Disease Research Summits of 2012 and 2015 incorporated experts from academia, industry, and nonprofit organizations to develop new research directions to transform our understanding of Alzheimer's disease (AD) and propel the development of critically needed therapies. In response to their recommendations, big data at multiple levels are being generated and integrated to study network failures in disease. We used metabolomics as a global biochemical approach to identify peripheral metabolic changes in AD patients and correlate them to cerebrospinal fluid pathology markers, imaging features, and cognitive performance.MethodsFasting serum samples from the Alzheimer's Disease Neuroimaging Initiative (199 control, 356 mild cognitive impairment, and 175 AD participants) were analyzed using the AbsoluteIDQ-p180 kit. Performance was validated in blinded replicates, and values were medication adjusted.Results Multivariable-adjusted analyses showed that sphingomyelins and ether-containing phosphatidylcholines were altered in preclinical biomarker-defined AD stages, whereas acylcarnitines and several amines, including the branched-chain amino acid valine and α-aminoadipic acid, changed in symptomatic stages. Several of the analytes showed consistent associations in the Rotterdam, Erasmus Rucphen Family, and Indiana Memory and Aging Studies. Partial correlation networks constructed for Aβ1–42, tau, imaging, and cognitive changes provided initial biochemical insights for disease-related processes. Coexpression networks interconnected key metabolic effectors of disease.DiscussionMetabolomics identified key disease-related metabolic changes and disease-progression-related changes. Defining metabolic changes during AD disease trajectory and its relationship to clinical phenotypes provides a powerful roadmap for drug and biomarker discovery. Show less
Treating chronic diseases such as rheumatoid arthritis and type 2 diabetes mellitus is a hot topic that has been discussed widely and investigated extensively, but never solved, due in part to... Show moreTreating chronic diseases such as rheumatoid arthritis and type 2 diabetes mellitus is a hot topic that has been discussed widely and investigated extensively, but never solved, due in part to their high complexity. Integrating disease-related information using a systems approach may help improve our knowledge of stages of the disease, thus improving the accuracy of diagnosing chronic disease. With respect to integrative thinking, traditional Chinese medicine (TCM)‒based concepts may provide a suitable holistic model, as TCM describes disease syndromes/phenotypes as an experience-based reference from the systems level. Systems-based metabolomics provides a comprehensive picture of small molecular metabolites as a readout and provides biological interpretations of the pathophysiology of disease. The rapid, highly sensitive, non-invasive measurement of ultra-weak photon emission (UPE) -- which measures spontaneously emitted photons at the surface of the skin--has been proposed for supporting TCM-based diagnostics and for reflecting the whole body’s physiological and pathological status. Combining metabolomics with TCM-based diagnostics will provide a robust model for investigating the biological processes that underlie UPE. This thesis aimed to investigate system-wide perturbations by using/combining metabolomics, UPE and TCM-based diagnostics, to provide i) a systems view of chronic disease, and ii) personalized phenotyping guided by TCM-based principles. Show less
On the basis of systems thinking, in this thesis metabolomics, Chinese medicine (CM), as well as Western medicine (WM) were combined to achieve a more comprehensive systems diagnosis of patients... Show moreOn the basis of systems thinking, in this thesis metabolomics, Chinese medicine (CM), as well as Western medicine (WM) were combined to achieve a more comprehensive systems diagnosis of patients with chronic diseases. Specifically, metabolomics has been applied to characterize patients by small molecule profiles, which can be applied in phenotyping patients and matching these with optimal therapies. Chinese and Western medicine have different perspectives on diagnosis and could be highly complementary to each other, so combining both could have advantages in personalized diagnosis. Therefore, in this thesis a combination of systems approaches is used, including metabolomics, CM-based diagnosis principles as well as WM to provide systems diagnosis of patients with chronic diseases, in particular rheumatoid arthritis (RA). With systems diagnosis, we could better identify potential biomarkers to predict the WM therapeutic response of patients with RA and monitoring disease progression. Show less
Ommen, B. van; Greef, J. van der; Ordovas, J.M.; Daniel, H. 2014
High-grade osteosarcoma is a primary bone tumor with complex genetic alterations, for which targeted therapy is lacking. The aim of this thesis was to use high-throughput molecular data analysis of... Show moreHigh-grade osteosarcoma is a primary bone tumor with complex genetic alterations, for which targeted therapy is lacking. The aim of this thesis was to use high-throughput molecular data analysis of high-grade osteosarcoma specimens and model systems, in order to learn more on osteosarcomagenesis and to find possible ways to inhibit this process. By analyzing different microarray data types using a systems biology approach, genomic instability was identified as an important driver of osteosarcomagenesis. A protective role of macrophages against metastasis of osteosarcoma was detected. In addition, the IR/IGF1R and PI3K/Akt signaling pathways were discovered as potential targets for treatment. This thesis provides the first steps in unraveling the genomic and transcriptomic landscape of high-grade osteosarcoma, and provides a biological rationale for certain new options for adjuvant treatment of this highly genomica lly unstable tumor. Show less
Cellular responses to DNA damage are highly variable and strongly depend on the cellular and organismic context. Studying the DNA damage response is crucial for a better understanding of cancer... Show moreCellular responses to DNA damage are highly variable and strongly depend on the cellular and organismic context. Studying the DNA damage response is crucial for a better understanding of cancer formation and ageing as well as genotoxic stress-induced cancer therapy. To do justice to the multifaceted cellular changes, elicited by DNA damage, use of high-throughput techniques and integration with bioinformatics tools is of great value. This thesis summarizes recent advances in the field of systems biology studies of the DNA damage response and furthermore shows integrated approaches of the study of DNA damage response signaling networks in embryonic stem and cancer cells. By integration of transcriptional changes and the phosphorylation and metabolic response of cisplatin-treated embryonic stem cells, with RNAi-based knockdown screens we identify novel DNA damage response signaling networks, linking process such as Wnt signaling, translation arrest or altered metabolic pathways to the cellular response to DNA damage. Furthermore, genes, whose knockdown sensitizes embryonic stem cells to DNA damage-induced killing, are tested in cancer cells of varying genetic backgrounds identifying a small subset of genes, which represent potential drug targets for sensitization of cancer cells. Altogether, our systems approach for studying the DNA damage response identifies novel DNA damage-induced signaling networks and molecules, which modulate survival in the presence of DNA damage, potentially providing new targets for therapeutic intervention or biomarker discovery. Show less
The current health care system is severely challenged by for instance rising costs, fewer new blockbuster drugs and increasing numbers of hospitalizations due to side effects. Especially in the... Show moreThe current health care system is severely challenged by for instance rising costs, fewer new blockbuster drugs and increasing numbers of hospitalizations due to side effects. Especially in the area of chronic diseases the current disease fighting strategy is failing and a more personalized medicine approach is needed. In this thesis new sub-types of rheumatoid arthritis are characterized with metabolomics analysis and symptoms patterns. The sub-types are based on diagnostic knowledge from Chinese medicine. The two sub-types of RA patients were found to have differences in apoptosis regulation of T-cells and differences in urine acylcarnitine levels. A questionnaire was designed to distinguish the two sub-types and to evaluate symptom patterns of arthritis patients. In the future the response to treatment of these sub-types of patients can be studied and specific treatment can be targeted to these sub-types. Show less
Metabolomics is the comprehensive analysis of small molecules involved in metabolism, on the basis of samples that have been obtained from organisms in a given physiological state. Data obtained... Show moreMetabolomics is the comprehensive analysis of small molecules involved in metabolism, on the basis of samples that have been obtained from organisms in a given physiological state. Data obtained from measurements of trait levels in twin families can be used to elucidate the importance of genetic and environmental variation for individual differences in trait levels. I describe the results of various analyses using metabolomics data from twin families. These data originated from analysis of blood plasma lipids by liquid chromatography-mass spectrometry, and from analysis of blood plasma and urine by proton nuclear magnetic resonance spectroscopy. Data analyses with a newly developed method, based on hierarchical clustering analysis of family members, suggested that shared genetic variation and shared environmental variation are important for similarities in blood plasma lipid profiles among individuals. Also, a method called __quantile equating__ was developed and applied that enables combination of semiquantitative metabolomics data sets originating from different measurement __blocks__. Univariate quantitative genetic analyses based on structural equation modeling revealed interesting differences in heritability among different metabolites. In multivariate analysis, relationships among genetic sources of phenotypic variation in different metabolites were investigated. These results bear relevance for the interpretation of the results from genome-wide association analyses. 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
