Title Promoter Affiliations Abstract "Development and evaluation of specific strategies for the targeted labeling of intracellular structures with quantum dots in live cells" "Kevin Braeckmans" "Department of Pharmaceutics" "The current project focuses on the intracellulair delivery of fluorescent nanomaterials (quantum dots), where several technologies will be evaluated to obtain cytoplasmic delivery. Both heat-generating particles and the combination of ultrasound and quantum dots will be tested in generating membrane pores, where cytoplasmic delivery will be compared to the level obtained by microinjection" "Kit-based labeling of anti-CEA nanobodies for in vivo imaging and therapy of colorectal cancer" "Guy Bormans" "Radiopharmaceutical Research" "In Belgium, colorectal cancer is the third most diagnosed cancer in men and second most diagnosed cancer in women. For efficient treatment and follow-up of patients, it is important to know if the cancer has spread to distant sites (metastases). Positron emission tomography (PET) uses a scanner to track radioactive molecules in the body. Fluorine-18 (18F, a radioactive form of fluorine) is the radionuclide of choice for PET. However, the most used fluorine-18 labeled molecule, [18F]FDG, has limited specificity for the detection of colorectal cancer. Therefore, the development of novel, more specific fluorine-18 labeled tracers is highly needed. Nanobodies (Nbs) are small proteins that can specifically target colorectal cancer cells (anti-CEA Nb). They are sometimes called “magic bullets” for imaging and therapy. Nevertheless, the incorporation of fluorine-18 and therapeutic radionuclides (e.g. astatine-211) in these proteins is challenging. In this project, the aim is to develop a mild labeling method that allows rapid and efficient labeling of Nbs, while reducing kidney retention. The same labeling method will also be used for the incorporation of therapeutic radionuclides (astatine-211), which will destroy cancer cells when the Nb binds to them. The labeled Nbs will be fully evaluated and ready for clinical translation by the end of this project. In conclusion, this project will result in a more accurate staging, treatment and follow-up of colorectal tumor patients." "Terbium labeling of anti-CEA nanobodies for in vivo imaging and therapy of colorectal cancer" "Guy Bormans" "Radiopharmaceutical Research" "In Belgium, colorectal cancer is the third most diagnosed cancer in men and second most diagnosed cancer in women. Nuclear medicine plays an important role in the diagnosis, follow-up and treatment of cancer. Ideally, the diagnostic radiopharmaceutical has an identical chemical structure than the therapeutic radiopharmaceutical, so that the pharmacokinetic properties of both compounds are equal, and that accurate dosimetry is possible. This can only be achieved if radionuclides for both diagnostic and therapeutic use of the same element are available. This is the case for the radiometal Terbium which has four medically interesting isotopes both for diagnostic and therapeutic use. The aim of this project is to develop and evaluate new terbium-based radiopharmaceuticals for diagnosis and therapy of colorectal cancer. For this purpose, nanobodies (Nbs) that specifically target colorectal cancer cells (anti-CEA Nb) will be used as vector molecule and radiolabeled with diagnostic and therapeutic terbium isotopes. The labeled Nbs will be fully evaluated and ready for clinical translation by the end of this project. In conclusion, these innovative terbium-based radiopharmaceuticals will allow a more accurate diagnosis, treatment and follow-up of colorectal tumor patients, which is highly needed." "Programmable proximal protein labeling of long non-coding RNAs in cancer" "Department of Biomolecular Medicine, Department of Pediatrics and medical genetics" "The human genome is estimated to contain approximately 20.000 to 25.000 genes, which accounts for about 2% of the genomic DNA. Since the 1960s, the remaining 98% or non-coding DNA has been referred to as “junk” DNA and was thought to have no molecular function in the cell. Recently, however, it has been shown that this “junk” DNA has a prominent role in normal cellular functioning. The bulk of “junk” DNA is being actively transcribed into long non-coding RNAs (lncRNA). These lncRNAs exert their function through interaction with other molecules, and an important mechanism underlying lncRNA function involves the interaction with proteins. Over the last decennium, an increasing number of studies report the involvement of lncRNAs in human diseases such as cancer. Due to their specific attributes, lncRNA-protein interactions are highly suitable candidates as therapeutic targets. However, the study of lncRNA-protein interactions is still in its infancy and robust methods to identify these proteins are still lacking. In this project outline, we propose the development of a new and efficient method to identify lncRNA-interacting proteins. In a second part, we will apply these methods on a set of cancer-specific lncRNAs. Finally, we will elucidate and validate the molecular mechanisms behind the lncRNA in its relevant cancer setting. At the end of this project, we expect to have a universal protein identification platform that readily can be applied to any lncRNA." "The BD FACS Symphony FACS cell sorter: the most advanced multiparameter fluorescence activated cell sorter that allows the identification and purification of rare cells for further functional characterization" "Department of Pharmaceutics, Department of Applied Mathematics, Computer Science and Statistics, Department of Biomedical molecular biology, Department of Internal Medicine and Pediatrics, Department of Biomolecular Medicine" "To be in a position to better understand the roles played by different immune cells in healthy and disease settings we must first be able to purify these cells. Only once we can obtain these cells as a pure fraction can we truly assess their individual contributions in any given setting. In recent years, our ability to discriminate between different cells types that often look similar has improved dramatically. By marking the cells with many different fluorescently-labelled antibodies recognising proteins on the surface of specific cell types, we can now accurately identify the cells.However, this often requires combinations of more than 20-25 antibodies or ‘markers’ each labelled with a different colour. While we have the infrastructure to allow us to design such panels of markers and begin to analyse the cells, the current technology available in Belgium, does not allow us to isolate these cells for downstream functional studies to enable us to assess exactly what roles these cells might play in health and disease. However, the recent development of the BD FACSymphony 30-parameter cell sorter allows just this. Thus, here in this application we are applying for funding to purchase this machine and install it within the institute’s core facility to enable many researchers within Ghent University to take their research to the next level, allowing them for the first time to purify and hence specifically assess the functions of these, often, rare and important cells." "Synthesis of novel fluorescently quenched activity-based probes for serine proteases and the application in live cell imaging using various detection techniques" "Steven Verhelst" "Laboratory of Chemical Biology" "For its immune system to function properly, the human body relies on a multitude of signaling processes executed by many different types cells. The most common one of these cell types is the neutrophil. Neutrophil cells patrol the blood and once encountering pathogens, they react to kill it making use of their armory contained in different types of granules. The neutrophil serine proteases are an important part of this armory and they can, for instance, be released in the phagosome containing a phagocytosed pathogen to be killed. Proteases, like the neutrophil serine proteases, are enzymes that hydrolyze peptide bonds and as such cut their substrate peptides. They can have a variety of functions but need to be tightly controlled as aberrant activity of a protease in question may lead to disease. For example, aberrant control of the neutrophil serine proteases can play a part in diseases such as chronic obstructive pulmonary disease, cancer and Alzheimer dementia. This regulation, however, means that protease presence does not necessarily equal protease activity. Therefore, activity-based probes have been developed as a special tool enabling distinguishing of active from inactive protease. To achieve this activity-direction, these small molecule probes contain an electrophilic group that reacts with the active site residue of an active protease. The probes eventually stay connected to the protease and usually also contain a tag, either a dye or a group allowing for the attachment of a dye, to make the protease-probe complex visible. Furthermore, quenched probes exist which turn fluorescent only after having reacted with a protease. In this work we design, develop and apply new activity-based probes directed against the neutrophil serine proteases. In chapter 2, we develop amino phosphinates as irreversible inhibitors for serine proteases. These are similar to amino phosphonates which find wide application as serine protease inhibitors. We establish a facile synthetic path towards the phosphinates and incorporate different carbon substituents at the phosphorus and evaluate their influence on activity, thereby assessing their reactivity and fit into the protease active site. We show that phosphinates incorporating a phenyl substituent are better inhibitors than analogous phosphonates and perform docking experiments giving evidence on a preferred configuration at the phosphorus. We then go on to use these novel phosphinate inhibitors as reactive group for serine protease activity-based probes in chapter 3. The phosphinates are fused to a linker and a tag and then used on neutrophil serine proteases neutrophil elastase, proteinase 3 and cathepsin G as well as the digestive protease chymotrypsin. We are able to separate different diastereomers of some of the probes and show that these possess distinct labeling capacities providing indirect proof for the docking results of parent inhibitors. Finally, we use some of these probes for imaging of neutrophil serine proteases in primary human neutrophils. Chapter 4 is directly related to the one before and follows a similar path. Here we apply the same phosphinate warhead but use it to develop quenched activity-based probes. These contain a quencher furnishing a dark molecule. The quencher gets expelled from the molecule upon reaction with protease restoring fluorescence. We make quenched probes for neutrophil elastase and cathepsin G, show that they can be used for purified protease labeling and labeling of protease in neutrophil lysates. Furthermore, we show unquenching of one of the probes by neutrophil elastase. This chapter establishes the phosphinate scaffold as a new and only the second group that allows synthesis of quenched activity-based probes for serine proteases. Lastly, in chapter 5 we develop quenched activity-based probes for the three most abundant neutrophil serine proteases, this time using the already established mixed phosphonate group as the reactive group. We make a set of probes, demonstrate labeling of purified protease and in neutrophil lysates as well as in the supernatant of neutrophils stimulated for degranulation. We then go on to use the probe designed to target neutrophil elastase in a live-cell imaging experiment of neutrophils undergoing NETosis. As we can see background fluorescence in this experiment, we go on to improve the probe, fitting a different quencher and dye and end with establishing its labeling capacity. Finally, we show using this new probe in a similar live-cell experiment of neutrophils undergoing NETosis. However, this experiment needs to be improved and is thus placed in chapter 6, the general discussion, as an outlook to future experiments we plan to perform." "Treatment of erectile dysfunction following cavernous nerve injury with adipose-tissue derived stem cells: efficacy and insights into the mechanisms of action of stem cell therapy." "Dirk De Ridder" "Woman and Child" "Radical prostatectomy is for most urologists the preferred treatment option for localized prostate carcinoma in young patients. During this surgery, irrespective of the extent of neurovascular budle preservation, neuropraxia of the cavernous nerves occurs, and Wallerian degeneration of these nerves ensues, resulting in fibrosis in the corpus cavernosum, anda loss in smooth muscle mass. These changes in turn result in changed compliance of the erectile tissue and thus a defective veno-occlusive mechanism. As a result of this denervation and the secondary changes to theerectile tissue, often severe and difficult-to-treat erectile dysfunction develops. This erectile dysfunction can be temporary, and resolve when reinnervation of the corpus cavernosum occurs, or, in a large proportion of men, definitive.Clinical attempts to preserve erectile function following radical prostatectomy with the use of so-called penile rehabilitation schemes, has resulted in disappointing outcomes. Therefore, researchers have been searching for alternative treatment options aimed at preservation of smooth muscle in the penis, protection of the cavernous nerve from degenerating after injury, or enhancing the endogenousneuroregenerative process. With the initial presumption that stem cellscould engraft in the erectile tissue and replace apoptotic smooth muscle cells, preclinical experiments directed at improving erectile functionwith stem cells have been set up. In this PhD, our primaryobjective was to test the feasibility of the intracavernous injection of adipose tissue-derived stem cells (ADSC) in an animal model of cavervous nerve crush injury. Crushing the nerve resembles a state of neuropraxia such as occurs during radical prostatectomy. In an initial study, we observed a beneficial effect of injection of ADSC on recovery of erectile function, as illustrated by intracorporal pressure increase upon electrostimulation of the cavernous nerve 4 weeks after injury and injection of cells. This functional observation was accompanied by a restoration of neuronal nitric oxide synthase (nNOS; the enzyme that produces NO, themain pro-erectile neurotransmitter in the corpus cavernosum) expressionin the penile nerves. Furthermore, smooth muscle content in the penis was preserved, and fibrosis was diminished. Four weeks following injection of labeled ADSC in the corpus cavernosum, scarce ADSC were found in the erectile tissue, in numbers too low to possibly explain treatment effects. Furthermore, the injection of lysate of an equivalent number of ADSC into the corpus could partially replicate the functional and structural effects of stem cell treatment. With this approach, we exposed the injured tissues to soluble factors contained in ADSC, without allowing live cells to directly act on the host tissue. Therefore we suggested that ADSC may not act by engraftment in the erectile tissue and transdifferentiation into smooth muscle cells, but rather may exert their effects by a paracrine interaction with the injured host tissue. As ADSC did not engraft in the penis, we were intrigued by the fate of these cells after injection. ADSC were therefore labeled withEdU, a DNA marker, and injected in both injured and non-injured animals. When traced, we observed that cells dissapear from the injection site rather quickly, to appear in the major pelvic ganglion, from which the cavernous nerve arises. At 28 days, few ADSC were found in the penis as well as in the ganglia. In injured rats treated with ADSC, we further observed a time-dependent increase in nNOS indicating neuroregeneration, rather than neuroprotection as a treatment effect of stem cells. As it is known that chemokines, a class of small signaling molecules, are involved in chemo-attraction, the migration of cells towards a gradient of a certain molecule, we tested which chemokines may play a role in recruitment of ADSC towards the major pelvic ganglion following cavernous nerve injury. We identified the ligand/chemokine receptor pairs CCL2/CCR4, CX3CL1/CX3CR1 and XCL1/XCR1 as potentially responsible for ADSC recruitment in this nerve injury model by a thorough multistep analysis of chemokiine receptor expression in ADSC. Surprisingly in the light of what was previously proposed, CXCR4-CXCL12 (SDF-1) interaction is not likely a major homing factor for ADSC. Conversely, we wondered what the effect would be of early blocking of the inflammatory cascade that leads to chemokine release in injured tissues, as this inflammatory reaction draws macrophages into the neural tissue, which are much needed for clearing debris, but often cause more damage than desired by the release of reactive oxygen species. Therefore, we blocked the inflammatory response at the level of TNFA  and TGF-β signalling with orally administered Pentoxifylline. Rats that underwent cavernous nerve crush injury and were treated daily with Pentoxifylline for four weeks had better recovery of erectile function than untreated rats, and this effect was dose dependent. Furthermore there was enhancement of nervegrowth in vitro, implying that besides anti-inflammatory effects, Pentoxifylline may induce neurotrophic signalling via other pathways. In conclusion, this work shows that ADSC have the capacity to induce neuroregeneration in rats with cavernous nerve injury. These effects are mediated by a neuro-inflammation induced chemoattraction of ADSC towards the major pelvic ganglion where they induce nerve regeneration. These insights may help translation of this autologous cellular therapy into clinical practice, but can aso be helpful in future experiments aimed at potentiating the effects of ADSC therapy. " "Development of drug-injection system using plasmonic endoscopy toward single cell study." "Hiroshi Ujii" "Molecular Imaging and Photonics" "In biology and biomedical research, where the microscopic organization of proteins and other molecules play a key role in the functioning of cells and organisms, it is imperative to zoom into the single cell and look at the underlying processes. Fluorescence-based microscopy techniques have been developed to get superresolution images of the structural organization inside living cells with resolution down to 10-50 nm. But these techniques rely on labeling of the bio-system with suitable fluorophores, and so label-free techniques like Raman imaging etc are important although yet to be implemented. Mechanical insertion of a needle into single-cells, know as single cell endoscopy, has been recognized as a useful technique for gene/drug delivery, bio-sensing, and single-cell electrophysiology etc. While large conical pipettes or tapered optical fibers have been widely used for such purpose, their insertion through the plasma membrane often causes cell damage. Recently, researchers have found thin 1D nanowires to be useful alternative to avoid such damage. In this project, I propose to use nanometer-scaled 1D metal nanowire for single-cell endoscopy. Metal nanowires offer two main advantages: (1) possibility to reduce its diameter to < 100 nm with enough strength, (2) subwavelegth plasmonic waveguiding that allows for optical access to the inside a single cell. In this project, the applicants will establish molecular injection system using plasmonic waveguide-based endoscopy." "Quantum dot nanoparticles for live cell imaging and intracellular targeting." "Kevin Braeckmans" "Department of Pharmaceutics" "This project is aimed at enabling fluorescent labeling of organelles in living cells by quantum dots. At the one hand, silica encapped quantum dots will be targeted to the cytosol by making use of a membrane-disruptive liposomal carrier. At the other hand, a new concept will be tested using thermoresponsive nanoparticles to enable endosomal escape to the cytosol." "Data driven modeling of cell shapes and movements in multicellular systems." "Rob Jelier" "Mechatronics, Biostatistics and Sensors (MeBioS), Microbial and Plant Genetics (CMPG)" "Unraveling how cells move and self organize in a multicellular settingis crucial for understanding a variety of biological processes likeembryogenesis, tissue formation and many diseases. This is a highlydynamic process, where cells move and change shape guided bybiochemical as well as physical cues. A precise reconstruction of cellshape over time provides detailed information on force generation ina cell and force transmission between neighboring cells. Howeverextracting 3D cell shapes from microscopy remains a challenge,especially when cells are irregularly shaped. In this project a novelapproach is proposed that leverages a biophysical model of cellshape to automatically reconstruct cell shapes from microscopyimages of stained cell membranes. The developing C. elegansembryo will be used as a model. The reconstructed cell shapes aresubsequently used to quantitatively assess the effect of a geneticperturbation on cell geometry. Next, cell shapes will be analyzed toinfer biomechanical parameters like cortical tension and cell-celladhesion, together with the active forces that drive shape changeand cell movement. Finally, the approach will be extended to offer anovel framework for data driven mechanical modeling of multi-cellularmovement. As a proof of concept, an explanatory model will be madeof early gastrulation in the C. elegans embryo to quantitativelyevaluate competing hypotheses on force generation."