Title Affiliations Abstract "Optimization and elucidation of ultrasound-induced transfection of cardiac endothelial cells with SDF-1 for the purpose of stem cell migration" "Department of Pharmaceutics" "geen abstract" "Combining photoporation with electrophoresis for improved transfection of hard-to-transfect cells" "Kevin Braeckmans, Kristiaan Neyts" "Department of Diagnostic Sciences, Department of Pharmaceutics, Department of Electronics and information systems" "Intracellular delivery of high molecular weight biological molecules is a prerequisite both for fundamental cell biological research as well as for the production of engineered therapeutic cells. This project aims at substantially improving this process for hard-to-transfect cells by combining the strengths of photoporation and electrophoresis, two unrelated techniques that belong to the respective expertises of both promoters." "Replic-ON project – Pioneering the investigation of self-amplifying messenger RNA electroporation for long-lasting ex vivo transfection of immune cells." "Diana Campillo Davó" "Laboratory for Experimental Hematology (LEH)" "Recently, there has been growing interest in the use of self-amplifying mRNA (saRNA) in therapeutic vaccines for infectious diseases and cancer. SaRNA is a type of messenger RNA (mRNA) that contains the non-structural proteins (nsP1-4) of an alphavirus replicase that copies the original strand of mRNA upon delivery into the cell. The nsP1-4 replicon is followed by a subgenomic promoter and the sequence of a gene of interest, allowing the expression of proteins of interest in the host cell. The self-replicating property means that proteins of interest encoded in the transfected saRNA will be expressed for a longer period of time compared to conventional mRNA. However, since there is no integration into the genome of the host cell, insertional mutagenesis is prevented. Thus, saRNA-based strategies combine the best of stable viral- or non-viral-based and transient mRNA-based engineering strategies. SaRNA is usually delivered in vivo as ""naked"" saRNA with or without intradermal electroporation or formulated into nanoparticle vaccines, with which expression of the protein of interest may last for 28 days. However, the exploitation of this technology for ex vivo modification of T cells in a therapeutic product has never been explored thus far. The primary objective of the Replic-ON project is to explore saRNA transfection as an innovative technology for genetically engineering immune cells in the context of the development of cell-based therapies. If successful, this project will provide groundbreaking data for the further development of ex vivo saRNA transfection technology as an amenable approach for T-cell genetic engineering in larger fundamental research project applications. We expect that this project will be the cornerstone for the much-needed development of more efficient and long-lasting non-integrating cellular immunotherapies while straddling the boundary between short-lived conventional mRNA technologies and integrating technologies such as viral transduction. Finally, this pioneering research would consolidate our leadership on ex vivo saRNA-based cellular therapies within the research community." "Generation of autologous T cells against acute myeloid leukemia by transduction of hematopoietic stem cells with T cell receptor genes and subsequent differentiation to T cells" "Department of Clinical chemistry, microbiology and immunology" "Hematopoietic stem cells will be transduced to express a T Cell Receptor. This receptor will be optained by immunizing against WT1 tumor antigen. The T cell receptor genes will be cloned from WT1 speciific T cells and subsequently used for transduction of the stem cells. Stem cells will be differentiated in vitro to T cells and functionality will be assayed in an in vivo animal model" "Generation of autologous T cells against acute myeloid leukemia by transduction of hematopoietic stem cells with T cell receptor genes and subsequent differentiation to T cells" "Hematopoietic stem cells will be transduced to express a T Cell Receptor. This receptor will be optained by immunizing against WT1 tumor antigen. The T cell receptor genes will be cloned from WT1 speciific T cells and subsequently used for transduction of the stem cells. Stem cells will be differentiated in vitro to T cells and functionality will be assayed in an in vivo animal model" "Electrical impedance spectroscopy and 3D cell cultures as emerging technologies for studying signal transduction pathways: the curious case of cathelicidins" "Liliane Schoofs" "Animal Physiology and Neurobiology" "This doctoral dissertation has two important parts. The first part covers experiments on elucidating the signal transduction pathways of recently discovered candidate bioactive peptides. The second part introduces 3D cell culture systems and expands on differential gene expression between 2D and 3D cell cultures using RNA-sequencing.Bioactive peptides perform countless functions in multicellular organisms as key players in cell-cell communication. They are involved in the regulation of various biochemical and biophysical processes such as cell growth, cell metabolism, reproduction, and homeostasis. Endogenous peptides can either signal over a short distance (auto- or paracrine signaling) or a long distance (endocrine signaling). Whatever their range of action, peptide signaling is usually accomplished through binding to a receptor protein expressed on target cells. Both the peptide as its cognate receptor(s) are potential targets for therapeutic interference, in case the peptide is implicated in a certain physiological or pathological process.Continuous improvement of peptide-extraction methods as well as mass spectrometry-based techniques enabled researchers to identify endogenous peptides in complex mixtures such as tissue extracts. By adapting peptidomics technologies previously used for invertebrates, the laboratory of prof. Schoofs systematically discovered more than 1000 new candidate bioactive peptides in various mouse and human tissues, from which approximately 700 were selected for synthesis and functional assessment.In this thesis project, candidate peptides were tested for activity on a collection of cultured cell lines representative for almost all cell types present in humans or mice. In a first stage, screening for activity was done using electrical impedance spectroscopy, a technique capable of detecting a cellular response regardless of the intracellular signaling cascade that is triggered. Once a response was detected upon exposure to a candidate peptide, further experiments using pharmacological agents were performed to identify its cognate receptor(s) and unravel its mechanism of action.  Screening experiments indicated that the purity level of candidate peptides is important for adequate interpretation of results, as several effects evoked by crude (> 70% purity) peptides could not be reproduced with purified (> 95%) peptides. However, a significant number of pure peptides were shown to be biologically active, which opened ample perspectives to study their mode of action.We primarily focused on one peptide, codenamed P318. P318 is a novel fragment of the murine cathelicidin-related antimicrobial peptide (CRAMP). Cathelicidins play a crucial role in the immune system as they exhibit both immunomodulatory and direct antimicrobial activities. Probably the most widely studied member of the cathelidicin family is LL-37, the sole cathelicidin peptide found in humans. Various reports suggest that LL-37 exerts pleiotropic effects through interacting with various putative cell surface receptors and/or intracellular targets in a wide variety of cell lines. However, the molecular details by which LL-37 exerts its effect on host cells are often incompletely understood.Impedance measurements showed that LL-37 and P318 were able to activate a wide variety of cultured cell lines. Various cell lines exhibited unique impedance responses to LL-37 or P318, which may reflect different signaling events induced by the peptides. Subsequent experiments with pharmacological agents further indicated that the action of LL-37 or P318 is cell type-dependent. For example, LL-37 dose-dependently evoked calcium mobilization in HEK293T cells via the PLC-IP3R pathway. Strong indications were also found that LL-37 binds to the cell membrane rather than to specific ligand-binding sites of a receptor. However, in RAW264.7 macrophages as well as in A549 lung carcinoma cells (and not in HEK293T cells), the activation of a currently unknown Gai-coupled receptor(s) is involved in the signaling cascade induced by LL-37. Intriguingly, endocytic pathways also seem to play a role in A549 cells.Even though several parallel effects of P318 and LL-37 were discovered (e.g. in HEK293T cells), P318 exerted an effect on B16 melanoma cells through a different molecular mechanism than LL-37. In this cell line, P318 increased levels op cAMP and caused hyperpolarization across the cell membrane, after which the signal was transduced through an Akt-dependent pathway.In the final part of this doctoral dissertation, 3D cell culture systems were covered as they mimic in vivo cell and tissue biology more closely than 2D monolayer cultures. Multiple studies report differences in gene and protein expression between 3D spheroids and 2D cell cultures. These divergent gene expression profiles can lead to differences in receptor expression which in turn drive an altered response to candidate drugs. However, an easily accessible overview of differential receptor expression among cells cultured in 2D and 3D is currently not available. Therefore, we used RNA-sequencing to examine differentially expressed receptor genes between 2D and 3D cell cultures of HT-29 colorectal adenocarcinoma cells and identified 17 differentially expressed genes encoding GPCRs, as well as several clinically relevant RTKs and ion channels. In addition, we performed pathway enrichment analysis and showed that a number of pathways were altered among HT-29 spheroids and 2D cell cultures, including pathways associated with cell cycle progression and glucose metabolism." "Influence of therapy on T cell signal transduction in Th1 and Th2 type diseases." "Translational Pathophysiological Research (TPR)" "The aim of the project is to study the role of intracellular signalling molecules in T helper cell differentiation in patients with rheumatoid arthritis (as a model of T helper 1-mediated disease) and wasp venom allergy (Th2). Relevant signalling molecules include STAT4, STAT5, STAT6 and p38 MAPK. Using a cell-based technique, the effect of stimuli from dendritic cells and regulatory T cells on the activation of cell signalling is studied, as well as the effect of therapy that is known to induce a clinically ""tolerant"" state." "Innovation mandate with spin-off: Photoporation for in vitro / ex vivo transfecting of cells" "Kevin Braeckmans" "Department of Pharmaceutics" "Optimizing the laser-induced photoporation technology (expanding the transfection portfolio, including larger nucleic acids and plant cells) and translating it into a disruptive, market-worthy product portfolio (device and reagents) and demonstrating the added value compared to the current state-of-the-art transfection methods adhv some user cases.- increasing the transfection efficiency of large, (negatively charged) molecules. Here it is the objective to overcome the current limitation in molecular size that one can bring. The aim here is to create conditions (composition of a 'booster' buffer, optimization of nanoparticles and laser parameters) that allow functional transfections of cells to an efficiency of more than 50% for mRNA and more than 15% for pDNA.- extrapolating the photoporation technology to hard transferable cells such as plant cells. The aim is to create conditions that allow plant cells to be photoporated (possibly in the form of protoplasts) so that external material (eg sgRNA-CAS9 RNA-protein complex) can be introduced.- gain insight into the effects of photoporation on the homeostasis of cells with benchmarking on current commercial technologies including electroporation and lipofection.- elaboration of user cases in which the unique possibilities and added value compared to the state-of-the-art of photoporation technology (in the form of a demonstrator device) are used by researchers where the current methods prove insufficiently adequate: - transfection of fluorescently labeled nano- and antibodies for 'life cell imaging'; - screening study of locked nucleic acides (LNA) in T cells; - CRISPR-CAS9 mediated genetic modification of cells.The obtained results must allow us to put together a product portfolio (appliance + reagents) and launch this via a spin-off in the market. These products will allow the user to efficiently transfect cells (including difficult transfectable cells) with a wide range of components." "cGMP signal transduction in microvascular endothelial cells of the pressure overloaded and dysfunctional heart." "Stefan Janssens" Cardiology "High blood pressure is a global problem. Untreated, it leads to decreased function of the heart muscle or heart failure with serious consequences for human health and survival. This is one of the reasons why cardiovascular disease remains the leading cause of death worldwide. Failure of our current therapies emphasizes the need for a better understanding of the mechanisms in the evolution of the disease. There are several factors that play a role. Heart failure is not only affected by decreased contraction of muscle cells, also the vessels in the cardiac wall play an important regulating role. However the underlying mechanisms of this phenomenon are insufficiently known to date. The proposed project is designed to investigate the control mechanisms that keep the weakened heart in balance. This balance sometimes derails in patients with valvular heart disease, high blood pressure or diseases of the heart muscle. We will investigate this mechanisms by isolating cells and studying them in several mouse models. This will allow us to find out what exactly goes wrong and how we can affect this problem. In the same philosophy, we will take human tissue during heart valve surgery to investigate whether the same mechanisms are found in humans. Our findings would lead to better therapies, a decrease in burden on health care expenses and healthier people." "Improved analysis of microRNA mediated regulation of signal transduction in lymphatic endothelial cells." "Filip Claes" "Switch Laboratory (VIB-KU Leuven)" "This research project tries to effectively optimize the analysis of microRNA-mediated regulation of signal transduction in lymphatic endothelial cells."