Understanding of the regulation mechanisms of CXCR4 signaling is essential for revealing its role in physiological and pathological processes. Though biochemical pathways following CXCR4 activation... Show moreUnderstanding of the regulation mechanisms of CXCR4 signaling is essential for revealing its role in physiological and pathological processes. Though biochemical pathways following CXCR4 activation by its ligand CXCL12 are well established, knowledge about the receptor dynamics on the plasma membrane remains limited. Here we used Ewing sarcoma-derived cells to unravel the processes that are involved in regulating CXCR4 dynamics on the plasma membrane during receptor signaling. Single-molecule epi-fluorescence microscopy showed that CXCR4 was present in monomeric state on the plasma membrane independent of receptor stimulation. However, upon activation freely diffusing receptors were immobilized in a ligand concentration-dependent manner. CXCR4 immobilization was strongly correlated with the ability for G-protein signaling and was a precursor of subsequent endocytotic events. Our data suggest that, a balanced regulation of G-protein dependent and independent pathways is required for controlling CXCR4 receptor mobility, and potentially subsequent controlled signal transduction. (C) 2015 Published by Elsevier B.V. Show less
Genetic alterations such as mutations, genomic rearrangements and aneuploidy, are commonly observed in tumors. To counteract this cells have multiple genome maintenance and surveillance systems to... Show moreGenetic alterations such as mutations, genomic rearrangements and aneuploidy, are commonly observed in tumors. To counteract this cells have multiple genome maintenance and surveillance systems to minimize the rate at which genomic alterations arise. The aim of the thesis is to gain understanding of processes and pathways that contribute to the maintenance of genome stability and to establish how defects in these processes and pathways abrogate the DNA damage response and consequently may promote genomic instability and development of cancer. The work described in this thesis addresses various aspects of the cellular response of mammalian cells to DNA damaging agents including changes in post translational modifications that occur after genotoxic stress. The role of poly-adenosyl ribose modification in nucleotide excision repair is investigated and found to be important for the recruitment of a chromatin remodeling protein and repair. Phosphoproteomic analysis identified changes in the global phosphorylation state of proteins following genotoxic stress. Key kinases responding to DNA damage are ATR and related kinases. A detailed study into the requirements for ATR activation after UV exposure indicates that at least two distinct modes of activation exist. Finally we describe the profound sensitivity of Cornelia de Lange Syndrome cells to DNA damage. Show less
Salicylic acid (SA) is involved in mediating defense against biotrophic pathogens. The current knowledge of the SA-mediated signaling pathway and its effect on the transcriptional regulation of... Show moreSalicylic acid (SA) is involved in mediating defense against biotrophic pathogens. The current knowledge of the SA-mediated signaling pathway and its effect on the transcriptional regulation of defense responses are reviewed in this thesis. PR-1 is a marker gene for systemic acquired resistance (SAR) and we have shown that the plant-specific transcription factor WRKY50 binds to the Arabidopsis PR-1 promoter at two positions close to binding sites of TGA proteins, bZIP class transcription factors previously shown to be important for PR-1 expression. We found that WRKY50 interacts with TGAs and together they synergistically activate PR-1 expression. In addition, a number of W-boxes in the PR-1 promoter have been shown to be also important for expression. We have shown that WRKY28 is able to bind to these W-boxes and to activate PR-1 gene expression in protoplasts. We also describe the effects of overexpression of the WRKY50 and WRKY28 proteins in transgenic plants. High constitutive expression of the WRKY50 gene resulted in accumulation of PR-1 mRNA when the plants were treated with SA. The overexpression of WRKY50 and WRKY28 had no clear effect on the plants__ resistance to the biotrophic pathogens, and neither was this the case with WRKY50 T-DNA knockout plants. To investigate the WRKYs__ influence on metabolite composition, a 1H NMR spectroscopy-based metabolomic approach was applied. The WRKY50 overexpression plants contained two- to three-fold more sinapic acid derivatives. This indicates a possible involvement of WRKY50 on metabolite production, in particular of hydroxycinnamates such as sinapic acid. As these compounds are components of lignin, this may point to a role of WRKY50 in stress-induced lignin modification. 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
Despite major advances in breast cancer diagnostics and treatment over the years, the disease is still a leading cause of death in women worldwide. Primary breast tumors can be treated relatively... Show moreDespite major advances in breast cancer diagnostics and treatment over the years, the disease is still a leading cause of death in women worldwide. Primary breast tumors can be treated relatively well with radiation, surgery, chemotherapy or a combination of these treatments. The occurrence of distant metastases derived from the primary tumor however, results in a considerable decrease in disease prognosis. Metastasis formation occurs through a series of distinct cell biological steps (outlined above). Understanding the molecular mechanisms that underlie each of these steps will help in the development of more successful anti-metastasis treatments. In this thesis, both in vitro and in vivo studies are described that aim at unraveling some of the processes involved in metastasis formation: signaling by components of the focal adhesions and cell migration. 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
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
In neonatal rat ventricular cardiomyocytes (NRVCs), we activated integrins by RGD to test whether integrin stimulation produced hypertrophy. Effect of RGD was compared with pro-hypertrophic effects... Show moreIn neonatal rat ventricular cardiomyocytes (NRVCs), we activated integrins by RGD to test whether integrin stimulation produced hypertrophy. Effect of RGD was compared with pro-hypertrophic effects of phenylephrine (chapter 2). Ventricular failure is associated with disturbed collagen turnover. Myocardial collagen turnover can be assessed by plasma PINP, PIIINP, and ICTP representing collagen synthesis (PINP, PIIINP) or degradation (ICTP). We investigated the effects of cardiac resynchronization therapy (CRT) on collagen turnover in patients at baseline and after 6 months of CRT (chapter 3). Monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) and RV failure are associated with MMP activation in RV, we investigated whether NO plays role in RV hypertrophy and failure (chapter 4). In chapter 5 we reviewed novel approaches to treat experimental PAH. We investigated whether MCT-induced PAH and RV failure can be treated with mesenchymal stem cells (MSCs) from donor rats with PAH caused by MCT. At day 14 after MCT, recipient rats were treated with MSCs. In chapters 6,7 the effects of MSCs on pulmonary pathology and RV function were examined. Isolated cardiomyocytes were investigated for PAH-related changes in excitability. In chapter 8 we reported on excitability properties dependent on Kv-channel expression, proposed to play a role in arrhythmias. Show less
The elucidation of the molecular and cellular mechanisms of ischemic ARF very important in finding new strategies to reduce or prevent renal injury. FAK is an important FA protein with tyrosine... Show moreThe elucidation of the molecular and cellular mechanisms of ischemic ARF very important in finding new strategies to reduce or prevent renal injury. FAK is an important FA protein with tyrosine kinase and scaffolding function. The general goal of this thesis was to investigate the role of FAK during I/R. Using a unilateral renal I/R rat model, we show the presence of tyrosine phosphorylated FAs in vivo and disruption of FAs and the F-actin network after ischemia and rebuild during reperfusion. FAK phosphorylation occured on different tyrosine residues during the reperfusion implicating a role of FAK. ERK is known to be involved in FA signaling. We studied the role of ERK signaling pathway during I/R in vivo using the inhibitor U0126. Inhibition prevented the changes in FA protein phosphorylation after ischemia and diminished injury. We used an inducible proximal tubule cell specific FAK knockout model to investigate the role of FAK in I/R. We show that FAK knockout mice are less susceptible to I/R injury compared to their wildtype littermates. Furthermore we studied FAK signaling under normal and ATP depletion in vitro. FAK deleted renal cells show no differences in morphology. However FAK knockout cells have increased FAs, aberrant stress fibers and impaired spreading. During recovery from ATP depletion, FAK deleted cells show impaired recovery of FAs and stress fibers. Show less
_-Catenin is an important protein for cancer research as it influences numerous events in the cell that lead to the development of cancer when gone awry. At the adherens junctions, _-catenin... Show more_-Catenin is an important protein for cancer research as it influences numerous events in the cell that lead to the development of cancer when gone awry. At the adherens junctions, _-catenin functions in cell-cell adhesion to maintain epithelial organisation. As an effector of Wnt signaling, _-catenin controls numerous developmental processes as well as homeostatic self-renewal. The effector function of _-catenin is to form a transcriptional complex in the nucleus with TCF/Lef transcription factors to regulate target gene expression. Due to the dual function of _-catenin in cell adhesion and signaling, there are different pools of the protein. The research described in this thesis focuses on the role of _-catenin in the Wnt signaling pathway. What is the pool of _-catenin that is active in signaling? Where is active _-catenin localized? Where and how is _-catenin activated and how is its nuclear export regulated to terminate Wnt signaling. Chapter 1 provides a general introduction about the different aspects of nuclear transport and the Wnt signaling cascade, putting it into the context of cancer development. Chapter 2 describes the identification of Ran-binding protein 3 (RanBP3) as a novel regulator of the active signaling form of _-catenin. We initiated this study to investigate the nuclear translocation of _-catenin and found that RanBP3 directly inhibits _-catenin signaling by stimulating nuclear export of the transcriptionally active form of _-catenin. The active form of _-catenin is unphosphorylated on its N-terminus, and covers only a small fraction of the total amount of _-catenin in the cell. We therefore continued to study the localization of this pool of _-catenin in Chapter 3. We describe that a relative large pool of unphosphorylated _-catenin resides at the adherens junctions, where it most likely functions in cell-cell adhesion. As Wnt treatment induces recruitment of unphosphorylated _-catenin to the plasma membrane, it is impossible to distinguish the resident junctional pool of unphosphorylated _-catenin from the signaling pool. We emphasize the importance of an E-cadherin null background in studying signaling competent unphosphorylated _-catenin. In Chapter 4, we study the unphosphorylated _-catenin pool at the plasma membrane upon Wnt signal induction in E-cadherin knock out cells. Plasma membrane recruitment of _-catenin in the early steps of the Wnt signaling cascade fits with recent new insights, which suggest recruitment of Axin and Dvl to the activated Wnt receptor LRP5/6. We expand these insights by showing that active _-catenin, Axin, APC and activated LRP6 receptor all localize to the plasma membrane upon Wnt stimulation. Moreover, we find that Wnt induced _-catenin is transcriptionally more active than overexpressed _-catenin. We suggest a model in which plasma membrane recruitment of _-catenin represents an important step in _-catenin processing and Wnt signal transduction. In Chapter 5, we determine the nuclear export kinetics of _-catenin in human cells and show that _-catenin exits the nucleus very fast, independently of the CRM1 export pathway and that _-catenin can enhance export of the small molecule GFP (green fluorescent protein). These observations fit into a model in which _-catenin can translocate quickly into and out of the nucleus independently of nuclear transport receptors. Therefore, the activity and localization of _-catenin are likely to be regulated by retention of the protein in the nucleus, cytoplasm and plasma membrane. Finally, in Chapter 6 we reconcile our findings with current knowledge of the Wnt signaling cascade. Show less
Multiple processes are influenced by a protein family named the function of Mitogen Activated Protein Kinases (MAPK), where among tissue morphogenesis, cell-proliferation, cell-differentiation,... Show moreMultiple processes are influenced by a protein family named the function of Mitogen Activated Protein Kinases (MAPK), where among tissue morphogenesis, cell-proliferation, cell-differentiation, cell-survival and immune reactions. A consequence of this broad role of these proteins is that disturbed MAPK signaling possibly leads to severe affects, and could even lead to the development of a disease or disturb embryogenesis. The human genome encodes for 14 mapk genes. One of the best characterized MAPK signaling transductions pathways is the Extracellular signal Regulated Kinase 1 and 2 (ERK1/ERK2). The zebrafish is a relatively new, genetic accessible model organism, which is because of their transparency of the embryos, very suitable to study the role of the MAPKs in embryonic development. The study described in this thesis shows that the zebrafish genome encodes for the same mapk genes, which makes it possible to study the functions of these proteins, using the zebrafish. Further investigations to the roles of ERK1 and ERK2 during embryonic development led to the new insight that ERK1 and ERK2 have distinct roles during crucial developmental cell migration processes and the differentiation of tissues and cell types. In addition, it was found that ERK1 and ERK2 affect the expression of common as well as distinct target genes. 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
