Glycosylation is a widely occurring and complex modification found on lipids and proteins and is involved in the recognition, signaling and interaction events within the cell and between cells.... Show moreGlycosylation is a widely occurring and complex modification found on lipids and proteins and is involved in the recognition, signaling and interaction events within the cell and between cells. These events based on glycan structures result in adhesion, cell-matrix interaction and immune recognition. Alterations in the glycomic profile are considered a hallmark of various diseases, including cancer where it contributes to the development and progression of cancer, affecting cell-cell communication, cell-matrix interactions, tumor angiogenesis, invasion and metastasis. These functions are governed by different glycans and their terminal structures. In order to further explore these structures with regard to their potential as biomarkers and specific targets for diagnostic applications and therapeutical strategies for various diseases, in-depth glycomic analysis is needed. It is further noted that aberrant glycosylation not only results from the altered expression of glycosyltransferases (GTs) but also from the changed activity of GTs and glycosidases as well as the availability and abundance of sugar nucleotide donors. The aim of the research described in this thesis was to explore the glycomic signatures of colorectal cancer (CRC) in cell lines and tissues as well as of acute myeloid leukemia (AML) cell lines. Show less
The experiments described in this thesis employ local lentiviral knockdowns in brain areas of female zebra finches followed by behavioural assays consisting of preference and Go/Nogo tasks.... Show moreThe experiments described in this thesis employ local lentiviral knockdowns in brain areas of female zebra finches followed by behavioural assays consisting of preference and Go/Nogo tasks. Ultimately, the targeted brain areas are extracted for gene expression analyses.The findings suggest that localised reduction of FoxP1 expression in HVC or CMM of female zebra finches does not impair the establishment or maintenance of auditory memories of conspecific song nor the females’ ability to discriminate or categorise auditory stimuli based on spectral or sequential features. Females which received a knockdown of FoxP1 in HVC as adults requested fewer familiar and unfamiliar playbacks and had a lower preference for familiar song than their matched controls. This might suggest that FoxP1 contributes to motivational behaviours in female zebra finches.Gene expression analyses links FoxP1 to pathways that have previously also been associated with FOXP2 in mammals including retinoic acid signalling and the SLIT-ROBO signalling cascade. Altered energy metabolism in different brain areas might also contribute to the observed phenotypes.Ultimately, the results presented in this thesis suggest implications of the transcription factor FoxP1 beyond vocal motor learning which need to be investigated in future studies. Show less
Background DNA methylation is a key epigenetic modification in human development and disease, yet there is limited understanding of its highly coordinated regulation. Here, we identify 818 genes... Show moreBackground DNA methylation is a key epigenetic modification in human development and disease, yet there is limited understanding of its highly coordinated regulation. Here, we identify 818 genes that affect DNA methylation patterns in blood using large-scale population genomics data. Results By employing genetic instruments as causal anchors, we establish directed associations between gene expression and distant DNA methylation levels, while ensuring specificity of the associations by correcting for linkage disequilibrium and pleiotropy among neighboring genes. The identified genes are enriched for transcription factors, of which many consistently increased or decreased DNA methylation levels at multiple CpG sites. In addition, we show that a substantial number of transcription factors affected DNA methylation at their experimentally determined binding sites. We also observe genes encoding proteins with heterogenous functions that have widespread effects on DNA methylation, e.g.,NFKBIE,CDCA7(L), andNLRC5, and for several examples, we suggest plausible mechanisms underlying their effect on DNA methylation. Conclusion We report hundreds of genes that affect DNA methylation and provide key insights in the principles underlying epigenetic regulation. Show less
Mollazadeh, S.; Bazzaz, B.S.F.; Neshati, V.; Vries, A.A.F. de; Naderi-Meshkin, H.; Mojarad, M.; ... ; Kerachian, M.A. 2019
Background Skeletal development and its cellular function are regulated by various transcription factors. The T-box (Tbx) family of transcription factors have critical roles in cellular... Show moreBackground Skeletal development and its cellular function are regulated by various transcription factors. The T-box (Tbx) family of transcription factors have critical roles in cellular differentiation as well as heart and limbs organogenesis. These factors possess activator and/or repressor domains to modify the expression of target genes. Despite the obvious effects of Tbx20 on heart development, its impact on bone development is still unknown. Methods To investigate the consequence by forced Tbx20 expression in the osteogenic differentiation of human mesenchymal stem cells derived from adipose tissue (Ad-MSCs), these cells were transduced with a bicistronic lentiviral vector encoding Tbx20 and an enhanced green fluorescent protein. Results Tbx20 gene delivery system suppressed the osteogenic differentiation of Ad-MSCs, as indicated by reduction in alkaline phosphatase activity and Alizarin Red S staining. Consistently, reverse transcription-polymerase chain reaction analyses showed that Tbx20 gain-of-function reduced the expression levels of osteoblast marker genes in osteo-inductive Ad-MSCs cultures. Accordingly, Tbx20 negatively affected osteogenesis through modulating expression of key factors involved in this process. Conclusion The present study suggests that Tbx20 could inhibit osteogenic differentiation in adipose-derived human mesenchymal stem cells. Show less
Aspergillus niger is an important industrial enzyme producer. Highly efficient production of enzymes mediated by modulating transcriptional regulation is meaningful. This thesis focuses on the... Show moreAspergillus niger is an important industrial enzyme producer. Highly efficient production of enzymes mediated by modulating transcriptional regulation is meaningful. This thesis focuses on the complex regulation of the expression of pectinolytic genes in A. niger. Chapter 1 gives an introduction of filamentous fungi with a focus on A. niger, and their industrial application. Chapter 2 describes a split marker approach in combination with NHEJ mutants for efficient targeted gene deletion. In Chapter 3, we constructed a set of isogenic auxotrophic strains by recycling pyrG marker. In Chapter 4, we selected five galacturonic acid (GA) induced genes to analyze the regulation of promoter activity of these genes in vivo by constructing promoter_amdS reporter strains. In Chapter 5, we identified the GA responsive transcription activator GaaR of A. niger by homology to BcGaaR of Botrytis cinerea. In Chapter 6 full genome sequencing of five mutants showing constitutive pectinolytic activity revealed allelic mutations in one particular gene gaaX. GaaX is likely to act as a transcription repressor which inhibits GaaR activity under non-inducing conditions. Chapter 7 summarizes and discusses the major conclusions of this thesis and proposes some future directions to study the regulatory mechanism of pectin degradation in A. niger. Show less
Jasmonates (JAs) are plant signaling molecules that play important roles in defense against insects and necrotrophic pathogens. The receptor-repressor-transcription factor module COI1-JAZ-MYC is a... Show moreJasmonates (JAs) are plant signaling molecules that play important roles in defense against insects and necrotrophic pathogens. The receptor-repressor-transcription factor module COI1-JAZ-MYC is a key component in JAs signaling in Arabidopsis thaliana. JAs stimulate their own biosynthesis by inducing the expression of genes encoding the biosynthetic enzymes. The research aim was to study the molecular mechanisms controlling this positive feedback loop. The results show that the AP2/ERF-domain transcription factor ORA47 is the key regulator. Overexpression of ORA47 resulted in increased expression of all JAs biosynthesis genes tested and in elevated levels of several JAs, including jasmonic acid (JA) and the main bioactive compound JA-Isoleucine. The expression of the ORA47 gene and the activity of the ORA47 protein are stimulated by JA. The JA-responsive expression of the ORA47 gene is controlled by the transcript ion factors MYC2, MYC3 and MYC4 which interact with a G-box in the ORA47 promoter. The expression of ORA47 target genes is negatively affected by members of the JAZ repressor family, suggesting that they regulate ORA47 activity. However ORA47 does not directly interact with JAZ repressors in yeast two-hybrid assays. Therefore it is hypothesized that ORA47 is regulated via interaction with an adaptor protein that recruits certain members of the JAZ family. Members of the BTB-TAZ (BT) protein family were found to interact with ORA47 and with certain JAZ proteins. However analysis of quadruple bt mutants failed to detect an effect on the expression of JAs biosynthesis genes, indicating that BT proteins are not the hypothetical adaptor proteins. 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
Jasmonic acid (JA) is a plant hormone that plays an important role in defense against wounding, insects and microbial pathogens. In defense responses against microbial pathogens JA acts... Show moreJasmonic acid (JA) is a plant hormone that plays an important role in defense against wounding, insects and microbial pathogens. In defense responses against microbial pathogens JA acts synergistically with the stress hormone ethylene. ORA59, belonging to the plant-specific class of AP2-domain transcription factors, is the main regulator of JA/ethylene-responsive defense gene expression in the model plant species Arabidopsis thaliana. The aim of the research described in this thesis was to study how the activity of ORA59 is regulated by JA. Studies on the promoter of the defense gene PDF1.2 showed that ORA59 regulates gene expression by interaction with GCCGCC motifs. Studies of ORA59 at the protein level indicated that JA controls ORA59 activity by promoting the nuclear localization and stabilization of the protein. Based on the findings it was postulated that there is an F-box protein that targets ORA59 for degradation, and a repressor protein that sequesters ORA59 in the cytoplasm. A protein interacting with ORA59 was identified by yeast two-hybrid screening. Further studies showed that this protein acts as a repressor of ORA59 by retaining it in the cytoplasm, which leads to fine-tuning of basal resistance against pathogens. Show less
