The studies presented in this thesis focused on cutaneous CD30-positive lymphoproliferations, particularly on primary cutaneous anaplastic large cell lymphoma (C-ALCL), a distinct type of cutaneous... Show moreThe studies presented in this thesis focused on cutaneous CD30-positive lymphoproliferations, particularly on primary cutaneous anaplastic large cell lymphoma (C-ALCL), a distinct type of cutaneous T-cell lymphoma (CTCL). In the initial staging of patients with an anaplastic large cell lymphoma first presenting in the skin, bone marrow examination has limited value (chapter 4). C-ALCL patients usually have an excellent prognosis, although some patients follow an unfavorable course. These patients often have extensive disease with leg involvement (chapter 2). Several phenotypical markers have been reported to aid in the differentiation between different types of cutaneous CD30-positive lymphoproliferations, however, TRAF1, MUM1 and BCL2 are not useful for this purpose (chapter 3). The miRNA expression profile of C-ALCL does show differences with that of tumor stage mycosis fungoides (MF), another type of CTCL, suggesting a different contribution to the pathogenesis of these lymphomas (chapter 5). In patients with transformed MF, several parameters can be used as prognostic factors, including CD30 expression, folliculotropic MF, extent of skin lesions and extracutaneous transformations (chapter 6). Show less
The focus of the described research in this thesis is on the oxidative stress response (Nrf2 pathway). The aim of the research presented in this thesis is to obtain more information concerning... Show moreThe focus of the described research in this thesis is on the oxidative stress response (Nrf2 pathway). The aim of the research presented in this thesis is to obtain more information concerning microRNAs which are involved in the Nrf2 pathway, to determine and evaluate the application of microRNAs for the construction of novel mechanistic biomarkers. Furthermore, we aimed to obtain a better understandingwith respect to the dynamics of the Nrf2 pathway to repeated xenobiotic exposure.To investigate the effect of overexpression of microRNAs on the Nrf2 pathway response in general and in combination with chemical exposure, a microRNA mimic screen was performed. In this screen overexpression of microRNAs was induced by using synthetic microRNA mimics. Since repeated exposure may drive adaptation programs and may lead to different responses between single and repeated exposures. The effect of a second exposure on the dynamics of the Nrf2 pathway activation was conducted. Final, results of a study are shown where a panel of structurally different phenolic compounds were used to demonstrate the proof-of-concept that Nrf2 pathway reporters can successfully be applied as biomarkers to characterize the specific pro-oxidant responses of chemicals. Show less
Identification of the cellular mechanisms involved in the occurrence and persistence of autoreactive lymphocytes is key for understanding T1D etiology. Comparing autoreactive T lymphocytes from... Show moreIdentification of the cellular mechanisms involved in the occurrence and persistence of autoreactive lymphocytes is key for understanding T1D etiology. Comparing autoreactive T lymphocytes from healthy individuals and T1D patients can provide clues as to what the driving force is for the destruction of β-cells and might designate potential targets for (immune) intervention. Elucidating in what way T1D associated gene variants actually contribute to disease development, i.e. understanding the functional aspects of genetic risk, and how genetic control of autoantigens influences autoimmunity may provide crucial clues to the clarification of the enigma of T1D. This thesis aims to answer several of these issues by investigating the role of transcriptional and post-transcriptional gene control in T1D. Show less
To circumvent the dependency on prediction models, we developed a microRNA-screen-based assay to establish links between cellular phenotypes and microRNAs (miRNAs). To this end, a miRNA expression... Show moreTo circumvent the dependency on prediction models, we developed a microRNA-screen-based assay to establish links between cellular phenotypes and microRNAs (miRNAs). To this end, a miRNA expression library (miR-Lib) was built consisting of 300 annotated miRNAs and around 100 candidate miRNAs. These miRNA 'minigenes' were cloned from genomic DNA of human cells and inserted in a specially engineered retroviral expression vector. This vector, named miR-Vec, is a modified murine stem cell virus (pMSCV) vector, with the expression of the miRNA minigenes under the control of the CMV pol II promoter. The vast majority of naturally transcribed miRNA genes are known to be expressed by pol II promoters. In parallel with the miRNA library, a corresponding microarray was developed (miR-Array) containing DNA spots of the miRNA minigenes from the library. By combining the miRNA library and microarray tools, we designed and performed three distinct functional genetic approaches to identify cancerous microRNAs. Show less
We aimed at investigating the role of 14q32 microRNAs in intimal hyperplasia and accelerated atherosclerosis; two major contributors to restenosis. Restenosis occurs regularly in patients treated... Show moreWe aimed at investigating the role of 14q32 microRNAs in intimal hyperplasia and accelerated atherosclerosis; two major contributors to restenosis. Restenosis occurs regularly in patients treated for coronary artery disease and peripheral arterial disease. We have previously shown that inhibition of 14q32 microRNAs leads to increased post-ischemic neovascularization, and microRNA miR-494 also decreased atherosclerosis, while increasing plaque stability. We hypothesized that 14q32microRNA inhibition has beneficial effects on intimal hyperplasia, as well as accelerated atherosclerosis.Non-constrictive cuffs were placed around both femoral arteries of C57BL/6J mice to induce intimal hyperplasia. Accelerated atherosclerotic plaque formation was induced in hypercholesterolemic ApoE mice by placing semi-constrictive collars around both carotid arteries. 14q32 microRNAs miR-329, miR-494 and miR-495 were inhibited in vivo using Gene Silencing Oligonucleotides (GSOs). GSO-495 administration led to a 32% reduction of intimal hyperplasia. Moreover, the number of macrophages in the arterial wall of mice treated with GSO-495 was reduced by 55%. Inhibition of miR-329 and miR-494 had less profound effects on intimal hyperplasia. GSO-495 administration also decreased atherosclerotic plaque formation by 52% and plaques of GSO-495 treated animals showed a more stable phenotype. Finally, cholesterol levels were also decreased in GSO-495 treated animals, via reduction of the VLDL-fraction. GSO-495 administration decreased our primary outcomes, namely intimal hyperplasia, and accelerated atherosclerosis. GSO-495 administration also favourably affected multiple secondary outcomes, including macrophage in flux, plaque stability and total plasma cholesterol levels. We conclude that 14q32 microRNA miR-495 is a promising target for prevention of restenosis. Show less
We have previously shown that treatment with third-generation antisense oligonucleotides against miR-494-3p (3GA-494) reduces atherosclerotic plaque progression and stabilizes lesions, both in... Show moreWe have previously shown that treatment with third-generation antisense oligonucleotides against miR-494-3p (3GA-494) reduces atherosclerotic plaque progression and stabilizes lesions, both in early and established plaques, with reduced macrophage content in established plaques. Within the plaque, different subtypes of macrophages are present. Here, we aimed to investigate whether miR-494-3p directly influences macrophage polarization and activation. Human macrophages were polarized into either proinflammatory M1 or anti-inflammatory M2 macrophages and simultaneously treated with 3GA-494 or a control antisense (3GA-ctrl). We show that 3GA-494 treatment inhibited miR-494-3p in M1 macrophages and dampened M1 polarization, while in M2 macrophages miR-494-3p expression was induced and M2 polarization enhanced. The proinflammatory marker CCR2 was reduced in 3GA-494-treated atherosclerosis-prone mice. Pathway enrichment analysis predicted an overlap between miR-494-3p target genes in macrophage polarization and Wnt signaling. We demonstrate that miR-494-3p regulates expression levels of multiple Wnt signaling components, such as LRP6 and TBL1X. Wnt signaling appears activated upon treatment with 3GA-494, both in cultured M1 macrophages and in plaques of hypercholesterolemic mice. Taken together, 3GA-494 treatment dampened M1 polarization, at least in part via activated Wnt signaling, while M2 polarization was enhanced, which is both favorable in reducing atherosclerotic plaque formation and increasing plaque stability. Show less
In the past decade it became increasingly clear that tumor heterogeneity represents one of the major problems for cancer treatment, also in prostate cancer. The identification of the molecular... Show moreIn the past decade it became increasingly clear that tumor heterogeneity represents one of the major problems for cancer treatment, also in prostate cancer. The identification of the molecular properties of highly aggressive cells (Cancer Stem Cells, CSCs) dispersed within the tumor represents a challenge for the identification of new efficient therapies. In most of the cases, current treatments are indeed successful in eradicating the primary tumor. However, the clinical evidence of relapse and the occurrence of therapy resistance, suggest the presence of subpopulation of cells within the tumor, that can survive such treatments and can perpetuate the cancer. In this thesis we investigated the molecular properties of selected highly aggressive CSCs and indentified novel modulators responsible for the maintenance of their aggressive behavior. Collectively, the studies described in this thesis have increased our insights into the molecular properties of highly metastatic and tumorigenic prostate cancer stem-like cells and provided new targets for possible diagnostic and therapeutic applications. Show less
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
This thesis describes the role of 14q32 microRNAs in vascular remodelling. The 14q32 microRNA cluster contains 54 microRNAs in humans and is highly conserved in mammals. In part I of this thesis,... Show moreThis thesis describes the role of 14q32 microRNAs in vascular remodelling. The 14q32 microRNA cluster contains 54 microRNAs in humans and is highly conserved in mammals. In part I of this thesis, we describe the role of 14q32 microRNAs in several processes of vascular remodelling. We have shown that inhibition of several 14q32 microRNAs, miR-329, miR-494 and miR-495, results in increased neovascularisation after hindlimb ischemia in mice. In addition, inhibition of the same microRNAs reduced atherosclerotic plaque formation and restenosis in experimental mouse models under hypercholesterolemic conditions. In part II of this thesis, we zoom in to the post-transcriptional regulation of 14q32 microRNAs through RNA binding proteins. The third and last part of this thesis studies the expression of microRNAs in subcutaneous adipose tissue of critical limb ischemia patients and discusses the potential use of microRNAs as biomarker to predict the risk of amputation in these patients. In conclusion, this thesis provides novel insights in the role of 14q32 microRNAs in processes of vascular remodelling. Experimental studies have identified 14q32 microRNAs as potential therapeutic targets for treatment and prevention of atherosclerosis, restenosis and peripheral arterial disease. Show less
Cardiovascular disease (CVD) is the collective term for diseases that involve the heart or circulation and CVDs are a major cause of mortality and morbidity worldwide. The aim of thesis was to... Show moreCardiovascular disease (CVD) is the collective term for diseases that involve the heart or circulation and CVDs are a major cause of mortality and morbidity worldwide. The aim of thesis was to investigate the role of inflammation in CVD related cardiac and vascular remodelling, which may lead to potential therapeutic agents. We investigated the therapeutic potential of antibodies directed against phosphorylcholine (PC), an endogenous ligand capable of triggering the innate immune system, which is expressed by apoptotic cells and oxidized LDL, in mouse models for myocardial infarction (MI). We found that treatment with anti-PC antibodies reduces adverse cardiac remodelling after both permanent MI as myocardial ischemia reperfusion (MI-R) injury. Furthermore, we found that treatment with annexin A5 also reduces adverse cardiac remodelling after MI-R injury. Interestingly, both anti-PC as annexin A5 treatment reduced the post MI inflammatory response. Next, we investigated the role of PCAF, an inflammatory related epigenetic factor, in vascular remodelling. We found that PCAF deficiency and treatment with a PCAF inhibitor reduces adverse vascular remodelling. Finally, we investigated the role of microRNAs, small RNA molecules that can affect expression of many different gene simultaneously, in vascular remodelling. We show that inhibition of microRNA-495 reduces adverse vascular remodelling. Show less
This thesis details our studies assessing the role of the endothelial-enriched miRNA-126 in the regulation of vascular homeostasis. In Chapter 2 the current insight in the role of miRNA-126 in... Show moreThis thesis details our studies assessing the role of the endothelial-enriched miRNA-126 in the regulation of vascular homeostasis. In Chapter 2 the current insight in the role of miRNA-126 in vascular homeostasis is reviewed. Chapter 3 focuses on the role of miRNA-126 in ischemia induced angiogenesis, followed by Chapter 4 which describes the potential role of miRNA-126 the mobilization of vasculogenic progenitor cells upon ischemia. Both chapters utilize antagomir-technology to specifically silence miRNA-126 in vivo. This approach to silence miRNA-126 was also used in Chapter 5 to elucidate the regulatory role of miRNA-126 in vascular cell adhesion molecule-1 expression in the kidney vasculature. Chapter 6 details our findings that circulating miRNA-126 in the periphery is not exclusively derived from endothelial cells but can also originate from platelets. Consequently, the use of aspirin has to be taken into account when relating circulating miRNA-126 levels to the progression of cardiovascular disease. Chapter 7 demonstrates that the angiogenic potential of miRNA-126 as described in Chapter 3 might reach beyond the presence of this pro-angiogenic miRNA in endothelium, but that neovascularization can also be supported by miRNA-126 expressed in circulating cells. Finally, Chapter 8 provides a summary of research presented in this thesis, presents the major conclusions that could be drawn and further discusses the role of miRNA-126 in vascular homeostasis. Show less
Cardiovascular diseases (CVDs) remain the leading cause of death worldwide, and thus, novel therapies are required. CVDs generally result in local shortages in the blood supply, known as ischemia.... Show moreCardiovascular diseases (CVDs) remain the leading cause of death worldwide, and thus, novel therapies are required. CVDs generally result in local shortages in the blood supply, known as ischemia. Neovascularization is the body's innate response mechanism that stimulates the restoration of blood flow to ischemic tissues. During the last decade, microRNAs have emerged as critical regulators of both CVD and neovascularization. Recent studies demonstrated that microRNAs are altered in many ways; however, whether these microRNA modifications could be physiologically relevant remained unclear. We examined whether specific microRNAs with a known cardiovascular function are subject to particular microRNA-alterations and if they could be relevant in cardiovascular disease. Our experiments demonstrated that the level of specific microRNA alterations, including isomiR formation, adenosine-to-inosine editing, and N6-adenosine methylation, changed in response to cardiovascular pathology. Many of these alterations changed the microRNAs function, which had a direct effect on processes like neovascularization. For example, microRNA adenosine-to-inosine editing increased after ischemia in both mice and humans and promoted neovascularization. These findings suggest that microRNA modifications can potentially be harnessed as a biomarker for cardiovascular disease, or even a novel therapeutic target. Show less
Huntington Disease (HD) is a fatal neurodegenerative disease caused by a CAG repeat expansion in the exon 1 of the huntingtin (HTT) gene, which confers a toxic gain-of-function inducing protein... Show moreHuntington Disease (HD) is a fatal neurodegenerative disease caused by a CAG repeat expansion in the exon 1 of the huntingtin (HTT) gene, which confers a toxic gain-of-function inducing protein aggregation and cell death. Although HD has a well-defined monogenic cause and promising HTT-lowering therapies are being tested in clinical trials, mechanism of action studies can reveal relevant information about therapeutic targets and outcomes required for successful translation into patients.One of the most advanced HTT lowering therapies for HD is a micro(mi)RNA-based gene therapy which consists of an engineered miRNA targeting the exon 1 sequence of HTT (miHTT) and delivered by adeno-associated virus (AAV) into neuronal striatal cells (AAV-miHTT).The work in this thesis describes novel mechanistic features of AAV-miHTT treatment for HD, including the targeting of toxic HTT fragments, the therapeutic spread between neuronal cells and the development of translational biomarkers to monitor its efficacy in the affected brain regions. In the light of this work, we support the reduction of HTT exon 1 fragment, the persistent efficacy in most affected brain areas, and mechanisms that improve therapeutic spread to all affected cells, as processes that potentially contribute to the successful treatment of HD patients. Show less
Analysis of the transcriptome, the total of all expressed RNA transcripts in a cell or an organism, contributes to our understanding of gene regulation during development and disease processes and... Show moreAnalysis of the transcriptome, the total of all expressed RNA transcripts in a cell or an organism, contributes to our understanding of gene regulation during development and disease processes and is therefore of great importance in the field of genomic research. This thesis focuses on the analysis of transcriptome complexity during infectious disease and cancer. The zebrafish was applied as an immunological model organism, due to the remarkable similarities of its immune system to that of human. The studies took advantage of novel opportunities for transcriptome profiling provided by recent developments in microarray and next generation sequencing technology that have made an enormous impact on biology. In addition to studying expression of protein coding genes, the work addressed regulatory functions of microRNAs, small non-coding RNAs playing important roles in the function of the immune system and other processes. The transcriptome data provide a valuable reference set of infection-responsive genes and microRNAs in zebrafish models and have identified microRNAs conserved between human and zebrafish liver cancer. These genomic data sets provide a strong basis for future applications of zebrafish as an infection and cancer model and contribute to the understanding of pathogenesis and the development of novel strategies for disease treatment. Show less
Background Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disorder caused by genetic loss of dystrophin protein. Extracellular microRNAs (ex-miRNAs) are putative, minimally invasive... Show moreBackground Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disorder caused by genetic loss of dystrophin protein. Extracellular microRNAs (ex-miRNAs) are putative, minimally invasive biomarkers of DMD. Specific ex-miRNAs (e.g. miR-1, miR-133a, miR-206, and miR-483) are highly up-regulated in the serum of DMD patients and dystrophic animal models and are restored to wild-type levels following exon skipping-mediated dystrophin rescue in mdx mice. As such, ex-miRNAs are promising pharmacodynamic biomarkers of exon skipping efficacy. Here, we aimed to determine the degree to which ex-miRNA levels reflect the underlying level of dystrophin protein expression in dystrophic muscle. Methods Candidate ex-miRNA biomarker levels were investigated in mdx mice in which dystrophin was restored with peptide-PMO (PPMO) exon skipping conjugates and in mdx-Xist(Delta hs) mice that express variable amounts of dystrophin from birth as a consequence of skewed X-chromosome inactivation. miRNA profiling was performed in mdx-Xist(Delta hs) mice using the FirePlex methodology and key results validated by small RNA TaqMan RT-qPCR. The muscles from each animal model were further characterized by dystrophin Western blot and immunofluorescence staining. Results The restoration of ex-miRNA abundance observed following PPMO treatment was not recapitulated in the high dystrophin-expressing mdx-Xist(Delta hs) group, despite these animals expressing similar amounts of total dystrophin protein (similar to 37% of wild-type levels). Instead, ex-miRNAs were present at high levels in mdx-Xist(Delta hs) mice regardless of dystrophin expression. PPMO-treated muscles exhibited a uniform pattern of dystrophin localization and were devoid of regenerating fibres, whereas mdx-Xist(Delta hs) muscles showed non-homogeneous dystrophin staining and sporadic regenerating foci. Conclusions Uniform dystrophin expression is required to prevent ex-miRNA release, stabilize myofiber turnover, and attenuate pathology in dystrophic muscle. Show less
