Background Hypertrophic cardiomyopathy (HCM) is the most common genetic disease of the cardiac muscle, frequently caused by mutations in MYBPC3. However, little is known about the upstream pathways... Show moreBackground Hypertrophic cardiomyopathy (HCM) is the most common genetic disease of the cardiac muscle, frequently caused by mutations in MYBPC3. However, little is known about the upstream pathways and key regulators causing the disease. Therefore, we employed a multi-omics approach to study the pathomechanisms underlying HCM comparing patient hearts harboring MYBPC3 mutations to control hearts. Results Using H3K27ac ChIP-seq and RNA-seq we obtained 9310 differentially acetylated regions and 2033 differentially expressed genes, respectively, between 13 HCM and 10 control hearts. We obtained 441 differentially expressed proteins between 11 HCM and 8 control hearts using proteomics. By integrating multi-omics datasets, we identified a set of DNA regions and genes that differentiate HCM from control hearts and 53 protein-coding genes as the major contributors. This comprehensive analysis consistently points toward altered extracellular matrix formation, muscle contraction, and metabolism. Therefore, we studied enriched transcription factor (TF) binding motifs and identified 9 motif-encoded TFs, including KLF15, ETV4, AR, CLOCK, ETS2, GATA5, MEIS1, RXRA, and ZFX. Selected candidates were examined in stem cell-derived cardiomyocytes with and without mutated MYBPC3. Furthermore, we observed an abundance of acetylation signals and transcripts derived from cardiomyocytes compared to non-myocyte populations. Conclusions By integrating histone acetylome, transcriptome, and proteome profiles, we identified major effector genes and protein networks that drive the pathological changes in HCM with mutated MYBPC3. Our work identifies 38 highly affected protein-coding genes as potential plasma HCM biomarkers and 9 TFs as potential upstream regulators of these pathomechanisms that may serve as possible therapeutic targets. Show less
The research described in this thesis combines the latest insights in lysosomal function with lysosome centred cell signalling. Novel imaging and labelling techniques are applied to provide in... Show moreThe research described in this thesis combines the latest insights in lysosomal function with lysosome centred cell signalling. Novel imaging and labelling techniques are applied to provide in depth characterization of lysosome function in health and disease. An integrative approach was used to study the physiological role of the lysosome, characterizing the function of lysosomal hydrolases and signalling on a cellular level as well as within the context of tissue. Show less
Photosynthesis is the process that harvests energy from light, and fixes it as chemical energy. It is performed by cyanobacteria, algae, and plants. The overall solar energy to biomass conversion... Show morePhotosynthesis is the process that harvests energy from light, and fixes it as chemical energy. It is performed by cyanobacteria, algae, and plants. The overall solar energy to biomass conversion efficiency of plant photosynthesis is widely considered to be very low. Recent models have indicated that the human demand for plant biomass will exceed the current production capacity in the near future. Improving the efficiency of photosynthesis has since been designated as one of the primary targets for improving crop yield. In order to acquire novel insights into the process of engineering more efficient photosynthesis I have investigated the use of zinc finger artificial transcription factor (ZF-ATF) mediated genome interrogation as a novel tool for the enhancement of photosynthesis-related traits, the results of which are described in this thesis. Show less
Chapter 1 presents a general introduction to the factors involved in crop domestication and the specific role of transcription factors in this process. Chapter 2 describes the functional analysis... Show moreChapter 1 presents a general introduction to the factors involved in crop domestication and the specific role of transcription factors in this process. Chapter 2 describes the functional analysis of a Dof-type transcription factor, named OsDof24, from rice.chapter 3 describes the role of OsDof25 in the regulation of OsC4PPDK. Interactions between OsDof25 and OsC4PPDK were confirmed in experiments involving EMSA, yeast one-hybrid analysis and transient expression in rice protoplasts. The regulatory role of OsDof25 in OsC4PPDK expression was confirmed using OsDof25 misexpression plants. The results suggested that OsDof25 is a trans-activator of the OsC4PPDK gene in rice. Chapter 4 describes the effects of OsDof24 and OsDof25 on the content of rice seeds storage proteins. First the interaction between both OsDof24 and OsDof25 with the GluB-1 promoter was investigated by a set of in vitro and in vivo experiments. Results showed that both OsDof24 and OsDof25 can affect seed storage protein content probably by activating the transcription of the GluB-1 promoter. Finally, in Chapter 5, a general discussion of the results from Chapters 2, 3 and 4 is presented. Show less
Overload of nutrients can lead to diet-induced inflammation, also called metabolic inflammation, which is thought to play an important role in many metabolic diseases, including the development of... Show moreOverload of nutrients can lead to diet-induced inflammation, also called metabolic inflammation, which is thought to play an important role in many metabolic diseases, including the development of nonalcoholic fatty liver disease (NAFLD). NAFLD encompasses a spectrum of pathologies that range from simple hepatic steatosis to nonalcoholic steatohepatitis (NASH) and fibrosis. The pathogenesis of NAFLD, including the sequence of events in time and the underlying mechanisms that initiate the transition from a fatty liver to NASH and fibrosis, remain poorly understood. Effective and reliable therapeutic approaches that are based on the understanding of the pathogenesis of NASH are therefore still lacking. In order to gain more insight into the mechanisms of NASH pathogenesis, we started with comparison of human NASH and experimental NASH. Subsequently, we provided evidence that activation of AP-1 and associated neutrophil infiltration is important for NAFL progression towards NASH and this can be induced experimentally by __metabolic__ dietary triggers of inflammation.Furthermore, we explored novel nutritional and pharmacological agents as potential strategies to combat NASH. Finally, we investigated the effects of high fat diet-induced metabolic overload on the liver in relation to inflammation in white adipose tissue and kidney, and the dysfunction of these tissues. Show less
The salicylic acid (SA) signaling pathway triggered by attack of biotrophic pathogens leads to broad spectrum resistance against a plethora of pathogenic fungi, bacteria and viruses and is known as... Show moreThe salicylic acid (SA) signaling pathway triggered by attack of biotrophic pathogens leads to broad spectrum resistance against a plethora of pathogenic fungi, bacteria and viruses and is known as systemic acquired resistance (SAR). One of the hallmarks of SAR is the accumulation of PR proteins and the induced expression of the PR-1 gene is often used as a marker for SAR. In this thesis we describe the characterization of a WRKY-class transcription factor (NtWRKY12) that specifically binds to the sequence TTTTCCAC. This sequence differs significantly from the consensus WRKY protein binding site TTGAC[C/T] (W-box), and was designated as the __WK-box__. The activation of PR-1a has always been proposed to occur mainly via TGA-factors. We show that NtWRKY12 can bind to TGA2.2 and has a more essential role in PR-1a activation. Using bioinformatic analysis various candidate genes have been prospected to have a role in regulation of the SA, jasmonate (JA) and ethylene (ET) biosynthesis or signaling pathways. The connection between AtWRKY28 and ICS1 was further studied. In protoplasts AtWRKY28 can activate ICS1 promoter::GUS reporter constructs and endogenous ICS1 mRNA levels. Using EMSA assays and ChIP experiments the bindingsite has been determined in vitro and confirmed in vivo. Show less
Plants defend themselves against stress, including pathogen or herbivore attack, via biosynthesis of defense proteins and of protective compounds called secondary metabolites. Stress induces these... Show morePlants defend themselves against stress, including pathogen or herbivore attack, via biosynthesis of defense proteins and of protective compounds called secondary metabolites. Stress induces these responses via a complex signal transduction network with jasmonic acid (JA) and related compounds as major players. In some defense responses JA acts synergistically with the stress hormone ethylene. It is largely unknown how JA induces expression of defense genes. It is also not well understood how the synergistic interaction between JA and ethylene is brought about. Previous research has identified several genes encoding AP2/ERF-domain transcription factors, called ORAs, whose expression is rapidly induced by JA in Arabidopsis. In this thesis, the function of several ORAs is addressed and shows that certain ORAs, by regulating the expression of defense-related genes, play major roles in JA signaling and defense. Show less
