Title Promoter Affiliations Abstract "HIV nuclear import and chromatin reading as novel drug targets" "Zeger Debyser" "Molecular Virology and Gene Therapy" "The replication cycle of HIV is a complicated interplay between viral and cellular proteins. Until now  antiretroviral therapy mainly targets the viral proteins although the interaction between the viral and cellular proteins could be interesting new drug targets. This strategy is mainly of interest because cellular proteins are much less prone to mutations which gives the virus less opportunity to develop drug resistance.  My laboratory has a track record in research on cellular cofactors of the viral enzyme integrase.  In 2003 Cherepanov et al. discovered LEDGF/p75 as an interaction partner of integrase and in 2008 Christ et al. found transportin-SR2 in a yeast-two-hybrid screen as an interaction partner of  integrase. Transportin-SR2 plays an important role in the nuclear import of the virus and LEDGF/p75 tethers the viral DNA to actively transcribed chromatin where it can integrate. Both proteins form interesting new targets for the treatment of HIV. Next to its role in HIV replication, LEDGF/p75 plays a role in mixed lineage leukemia. This type of cancer is driven by translocations involving the MLL1-gene and mainly affects children. Targeting the chromatin reading function of LEDGF/p75 could therefore be an interesting drug target in the treatment of both HIV and mixed lineage leukemia.In the first chapter of this PhD thesis I focus on the TRN-IN interaction and try to gain more insight into their binding mode. Since nuclear import is generally considered as a bottleneck in the viral replication cycle this could be an interesting new target for antiretroviral therapy. Previous studies have already revealed that mainly the catalytic core domain (CCD) and the C-terminal domain (CTD) of integrase interact with transportin-SR2. The aim of this work was to gain more insight into this protein complex and to pinpoint the important regions in TRN-SR2 that are necessary for integrase binding. In collaboration with the bio-crystallography lab I revealed that one TRN-SR2 molecule can bind a CCD-CTD dimer. Next, I divided transportin-SR2 into small peptides, each corresponding to a single HEAT repeat and used AlphaScreen binding assays to determine if these peptides could still bind integrase. This study revealed that it is mainly the N-terminal region of TRN-SR2 that interacts with integrase, principally through HEAT repeats 4, 10 and 11. Based on these results in combination with small-angle X-ray scattering data for the complex of TRN-SR2 with a truncated integrase, I propose a model of the complex in which the nuclear import of the pre-integration complex can proceed in parallel with nuclear transport of its endogenous cargoes.In the second part of this thesis the aim was to identify novel antivirals that do not suffer from cross-resistance with existing drugs. This chapter describes the development and use of an AlphaScreen-based high-throughput screening cascade for inhibitors of the TRN-IN interaction. In total, 25608 small molecules were tested and after eliminating false positives and nonspecific protein-protein interaction inhibitors two active compound series were discovered. Although the inhibitory effect of these compounds in a multiple and single round antiviral activity assay was only moderate, they significantly reduced the nuclear import of fluorescently labelled HIV particles. This study shows that it is possible to use the AlphaScreen technology as a high throughput platform to screen for a novel class of inhibitors. These results again confirm the important role of the TRN-IN interaction in nuclear import. Our hit compounds represent the first small molecule inhibitors of this step in the viral replication cycle and hold promise for future drug development.The last part of this work focuses on the protein-chromatin interaction between LEDGF/p75 and the trimethylated lysine 36 on histone 3 (H3K36me3). The PWWP domain of LEDGF/p75 plays an important role in tethering the HIV pre-integration complex to the host chromatin. Also in the case of mixed lineage leukemia, LEDGF/p75 is responsible for targeting the leukemogenic MLL-fusion/MENIN complex to actively transcribed genes. Targeting the LEDGF/p75-H3K36me3 interaction could therefore have beneficial therapeutic effects in the treatment of both HIV and mixed lineage leukemia. This part of the thesis describes the development and use of a fragment-based drug discovery campaign. Molecular modelling was used to virtually screen over 4 million compounds for binding to the PWWP domain and the top 525 molecules were selected. An AlphaScreen-based screen was next used to pick initial hit compounds from this small compound library. All compounds were also tested in a nano-DSF set-up in which the intrinsic tryptophan fluorescence of the PWWP domain is monitored upon thermal denaturation in the presence and absence of compounds. Initial results indicate that some of our compounds can indeed inhibit the LEDGF/p75-H3K36me3 interaction, although the effect is only moderate. In parallel we have therefore worked on resolving the crystal structure of the PWWP domain. This structural information could guide further chemical optimization of our compounds.In brief, this work aims to discover and validate novel targets for drug discovery targeting HIV and/or mixed lineage leukemia. Protein-protein and protein-chromatin interactions can be interesting new targets since the risk for cross-resistance with already existing drugs is rather low. However, the main challenge in targeting these interactions is to develop selectivity against the specific interaction while keeping interference with other proteins to a minimum." "Chromatin structure analysis of rare likely pathogenic inherited Copy Number Variants" "Catia Attanasio" "Laboratory of Gene Regulation and Disease, Department of Human Genetics" "Chromatin structure has been shown to play important roles in the orchestration of gene expression programs during development. Spatio-temporal specific cis-regulatory sequences often lie at a long distance from the gene(s) they regulate, requiring spatial chromatin folding to move them in close proximity of their target promoters and fine-tune the time, place and level of gene expression. Interestingly, several whole-genome chromatin interaction analyses have also recently revealed the existence of topologically associated domains (TADs) which seem to act as fundamental regulatory units of the genome by delimiting the scope of action of regulatory elements in the 3D nuclear space. In other words, TADs organize the genome into regulatory islands by defining regions that interact more frequently with themselves than with the rest of the genome. Few studies have reported that the alteration of TADs by structural genomic rearrangements could disrupt the normal activity of TADs and play significant roles in human diseases. Copy-Number Variants (CNVs) are DNA sequence deletions or insertions of ≥50bp which have been shown to play important roles in human biology. The overall contribution of CNVs to human phenotypic variability and diseases is, however, still largely unclear. Few studies have now reported that deletion or duplication of DNA segments could alter gene expression regulation by disrupting, duplicating or shifting TADs boundaries causing gene(s) misexpression and diseases. Here, we propose to investigate the contribution of rare likely pathogenic CNVs into human disorders by analyzing theirs effect on chromatin architecture, and by extension on gene regulatory programs, in patients’ and controls’ derived cells. This project is part of a larger research effort that will combine our analyses with deep phenotyping, whole genome sequencing and evolutionary analyses in selected families to assess the contribution of rare likely pathogenic CNVs to phenotypic variability of developmental disorders." "Exploration of the therapeutic potential of protein phosphatases involved in chromatin signaling." "Mathieu Bollen" "Biochemistry, Molecular and Structural Biology, Laboratory of Biosignaling & Therapeutics" "Our research group has a long-standing expertise in protein phosphatase 1 (PP1) and chromatin signaling and we want to develop chromatin-associated PP1 as a successful therapeutic target. Specific research objectives of this project are: 1. To delineate the role of PP1 in the dynamics of histone phosphorylation. 2. To identify the protein that target PP1 to histones. 3. To explore how PP1 cross-talks with other histone-modifying enzymes. 4. To select PP1 holoenzymes with a high therapeutic potential. 5. To determine the 3D-structues of selected PP1 holoenzymes. 6. To screen for small-molecule effectors of selected PP1 holoenzymes." "Optimizing an experimental approach to silence disease-related genes by interfering with their chromatin structure. Epigenetic silencing of the human androgen receptor." "Annemie Haelens" "Laboratory of Molecular Endocrinology" "In this CREA-project, we aim to develop methods to induce alterations in the chromatin structure at a specific locus in the genome, to shut down one singe gene. Three approaches will be testen: (1) expression of shRNAs, (2) expression of non-coding RNAs and (3) transfection of methylated oligonucleotides. Epigenetic silencing by these mechanisms has been described for other test organisms, but has not been testen thoroughly in human/mammalian cells. Such ""epigenetic therapy"" has the advantage that it acts before the gene is actually expressed, preventing the protein from appearing. The use of mRNA targeting siRNAs to experimentally control protein levels has only short term effects, since they target the mRNA which is continuously synthesized as long as the gene is active. In contrast, a transient exposure to an epigenetic modifying agent is expected to suffice for the stable shut down of the gene. The changed epigenome will even be inherited by the daughter cells. During the two years of this CREA project, we will test the possibility to shut down the expression of the androgen receptor gene in prostate cancer cells. Prostate cancer is the most common cancer excluding skin cancer, and the second leading cause of cancer-related dead. It is likely to become a more promiment and pressing problem as the percentage of elderly men increases. Current treatments have severe side effects and a high chance for recurrence. Therefore, the need for development of new highly effective therapies with a small impact on the quality-of-life and minimal side effects is obvious. The fact that the AR is crucial for both the initiation and maintenance of prostate cancer makes it the ideal target for therapy. It has been shown that the knock down of androgen receptor (AR) expression at the mRNA level, leads to cell death of prostate cancer cells. Our approach is innovative because silencing the AR promoter depletes the cell of AR protein. This may enhance the benefits of androgen ablation by reducing of preventing AR cross activation by other ligands and other signaling pathways. Our new epigenetic silencing protocol will be a major step forward in the treatment of prostate cancer. When our approach is successful, we plan to target other genes with a proven role in prostate and/or other cancers, such as oncogenes or tumor survival genes. Of course the same protocols will be useful in fundamental research in which shutting down specific genes (cfr. knock out) has provided many insights in protein functions." "Unraveling the structural/topological aspects and the energetics related to complex DNA or chromatin interactions of high order nucleoprotein complexes involved in HIV/AIDS and/or leukemia." "Steven De Feyter" "Molecular Virology and Gene Therapy, Molecular Imaging and Photonics, Soft Matter and Biophysics" "The aim of the project is to develop and combine different single-molecule techniques in a parallel fashion as well as in integrated setups, together with in silico approaches and tools from molecular biology and virology, to dissect the molecular interactions that determine the structure and energetics of higher-order nucleoprotein assemblies in vitro and in vivo. The project addresses several questions in contemporary biomedical research related to the development and treatment of the acquired immune deficiency syndrome (AIDS) and leukemia, and the design of safer vectors for gene therapy." "Effects of Screotal Insulation on Sperm Kinetics, Chromatin Protamination and Gene Expression Pattern in Belgian Bleu and Holstein-Friesian AI Bulls." "Ann Van Soom" "Department of Internal Medicine, Reproduction and Population Medicine, Department of Veterinary and Biosciences, Bangladesh Agricultural University" "In this research we will look into transient rises of temperature effects on sperm kinetics, DNA chromatin protamination and gene(s) expression pattern within and among BB and HF bulls and effects of heat- stressed spermatozoa on fertilization and subsequent embryo development in vitro." "The Chromatic Illusion within the Photographic Universe" "Steven Humblet" "Thinking Tools" "The Chromatic Illusion within the Photographic Universe is a practice-based research project that aims to examine the hidden impact technology has on color rendering within the photographic. To carry out this work, I will refer to Vilém Flusser's notions of black box and program, and I will re-enact the scientific experiments carried out in the 1660s by Isaac Newton with sunlight and prisms. By stripping photography down to its very essence, my work will seek to disclose how light-sensitive supports and output devices record and render the colors of the visible spectrum. I will examine photographic films, digital sensors, tv/computer monitors, and chemical papers. The research will deepen the notion of ready-made colors and explore the elementary properties of photographic matter. The experiments will address the following research questions: - Given that color in photography is always manufactured and never merely registered, what influence does the chosen technology have on the produced color charts? Would it be possible to link different worldviews to these different color charts? - What is the influence of a given culture on the development of color technology? Do the cultural stratifications of a geographic area determine how visual technologies decode colors? - What remains of the chromatic heritage left by past technologies? For instance: when a certain photographic film is discontinued, is it possible to talk about extinct colors?" "Regulatory mechanisms of PEAR1 expression in myeloid cell-lineage specification." "Marc Hoylaerts" "Centre for Molecular and Vascular Biology" "Platelet Endothelial Aggregation Receptor 1 (PEAR1) is a membrane protein on blood platelets, first described in 2005. Platelets are small circulating cells playing a crucial role in the arrest of bleeding after vascular trauma. Our laboratory revealed that PEAR1 contributes to platelet function by stabilizing the platelet plug, formed upon platelet adhesion to the injured vessel wall and their aggregation to each other. PEAR1 is present on other cells, including endothelial cells, which line the blood vessel surface, but is also expressed in those cells that are the precursors of platelets and in other cells, present in the bone marrow, including granulocyte precursors. The present work aims at understanding the role of PEAR1 in myeloid lineage maturation, i.e. proliferation and differentiation, under normal and pathological conditions, with special focus on its interplay with the transcription factor GATA1. To do so, the project will apply very new methodologies, based on recent insights in gene regulation. Such insights have taught us that control of gene expression is highly complex, involving the structure (DNA sequence), physical organization (chromatin conformation) and interacting proteins (the so-called transcription factors) of a certain genomic region. Some of those features are fixed, such as the genetic component, while others undergo dynamic changes over time, dependent on environmental factors, i.e. the epigenetic component. This work will determine how the reversible expression of PEAR1 participates in bone marrow myeloid differentiation and will clarify whether defective PEAR1 regulation participates in the development of well-defined types of blood cell cancers." "Variable tandem repeats in regulatory genes as a source of ""evolvability""." "Kevin Verstrepen" "Centre of Microbial and Plant Genetics" "Tandem repeats are extremely unstable DNA sequences. Such variable repeats also occur within coding regions. In this project, we investigate whether variable repeats located within genes encoding regulatory proteins (transcription factors, chromatin modifiers) confer phenotypic variability and evolvability. First, we will identify all S. cerevisiae genes containing and internal repeat. The variability of some selected repeats will be investigated in a set of different S. cerevisiae strains. Subsequently, we will use genetic transformation to generate a set of yeast strains that only differ in the number of repeat units they carry in a specific regulatory gene. For these yeast, we will investigate the phenotypic consequences of repeat variation in the regulatory gene will be measured (e.g. expression patterns of target genes)." "Investigation of nutritional and/or environmental epigenetic modulators in health and disease." "Wim Vanden Berghe" "Proteinchemistry, proteomics and epigenetic signalling(PPES)" "Short exposure to nutrients, toxines, endocrine disruptors, hormones, famine or stress, can have longlasting consequences by epigenetic mechanisms (chromatin code, DNA methylation, noncoding RNAs) which affect the interpretation and expression of the genetic DNA blueprint. As such chronic inflammation is epigenetically recorded in the human genome and increase incidence and progression of various diseases, including metabolic disease, autoimmune disease, cancer, neurodegeneration, cardiovascular disease and ageing. This research will explore how diet and environmental factors interfere with epigenetic regulation of immune homeostasis in health and disease."