Title Promoter Affiliations Abstract "Nanodiamond labelling of neuronal cells for molecular resolution neuronal and network-level imaging" "Michele Giugliano" "Nanostructured and organic optical and electronic materials (NANOrOPT), Theoretical neurobiology" "This interdisciplinary project addresses the interactions of functionalised nanoparticles with neural tissues and their usage to probe the local environment and neural function. Fundamental progress must be made in two main research areas, the design of nanodiamond particles and the understanding of their chemical, electronic, and optical properties, as well as the biological and biophysics aspects, in particular insertion of the nanoprobes, their biocompatibility and non-contact detection. In parallel with the materials research, explored thanks to a recently granted FWO-project, and in a highly independent and complementary approach, the present request for a doctoral research project will focus on the bio-related aspects and the detection at the molecular and cellular level. The uptake and anchoring of functionalised nanodiamond labels will be investigated and monitored by electrophysiological techniques and confocal microscopy, keeping track of the influence on neural function. Neuronal cell culture with nanodiamond loading will be optimized as well as the use of drugs that affect neural interconnection. This work in the Neurobiology laboratory will be combined with the assessment and optimisation of the advanced detection techniques developed in the Physics research group. Foerster resonance energy transfer (FRET) and optically detected magnetic resonance (ODMR) will be applied and optimised for neural cell investigation in a confocal microscopy configuration and compared to the more classical patch-clamp methods for the high temporal-resolution detection of neural electrical activity." "Bead-enabled capturing and fluorescent labeling to quantify direct respiratory virus-microbiota interactions." "Irina Spacova" "Environmental Ecology & Applied Microbiology (ENdEMIC)" "Amidst the global pandemic, there is an acute urgency to understand which factors contribute to respiratory disease severity. Most research focuses on viral interactions with the host immune system. However, recent data shows that microbial communities inhabiting the airways (the airway microbiota) can influence viral infection through direct interactions with invading viral particles. This project aims to characterize the capacity of airway microbiota to bind the model respiratory syncytial virus (RSV) that puts more than 3 million children in the hospital every year. Specific methodology for targeted bacterial isolation from human nasopharyngeal samples will be implemented. Fluorescent labeling techniques will be used to visualize and quantify RSV binding to bacteria. Finally, the impact of this RSV binding on viral infectivity in host cells will be assessed using tripartite bacteria-virus-host cell assays. Implementation of the methodology developed in this project will provide insights into the ability of the resident airway microbiota to directly interact with invading viruses. This will help understand microbial interactions influencing respiratory disease severity, and can later be broadly applied to a wide range of bacteria and viruses from other body niches." "Dynamics in the Central Plant Cell Metabolism" "Bart Nicolai" "Laboratory of Protein Phosphorylation and Proteomics, Division of Mechatronics, Biostatistics and Sensors (MeBioS)" "As part of their development, plants have acquired adaptive mechanisms to cope with different stress situations they encounter in their environment. These adaptive mechanisms include the ability to alter their metabolism when faced with extreme environmental conditions such as low oxygen. In higher plants, oxygen (O2) availability is important for energy production through respiratory metabolism. Under conditions where O2 becomes limiting, respiratory metabolism can be impeded leading to impaired growth. Low O2 conditions in plants can be created by environmental and man-made factors such as soil flooding and control atmosphere (CA) storage, respectively. In addition, anatomical arrangements can create uneven gas distribution leading to low O2 conditions in cells located in the inner tissues of plants. The effect of low O2 stress on plants include stunted growth of field crops and development of storage disorders in fruits stored under CA. Taking into account that plants serve as an importantsource of food, it is important to study and understand how plant metabolism cope with low O2 stress.The main objective of this thesis is to study the effect of low O2 on plants through metabolome and fluxome analysis. Metabolome analysis involves the comprehensive quantitative analysis of all low molecular weight metabolites in an organism while fluxome analysis measures the rates at which metabolites are distributed through a reaction network. Together, these two techniques can be used to study and understand the response of plants to low O2 stress.Analysis of flux, especially using isotope labelling techniques, require feeding an organism with labelled substrate and measuring the incorporation of label in different metabolites. In whole plants, performing isotopic feeding experiments is limited by the long incubation times needed for metabolites to incorporate quantifiable amounts of label in their storage polymers like proteins and carbohydrates. To overcome this difficulty heterotrophic cell suspensions were used as a model system as they can be easily manipulated to grow on a defined medium, allowing a much faster incorporation of labelled substrate into their metabolome.In the first part of this thesis, techniques were developed to establish cellsuspension from tomato leaves. Subsequently, a gas chromatography-mass spectrometry (GC-MS) based protocol for separating, identifying and quantifying intracellular polar metabolites and their label accumulation during 13C-label feeding experiments was developed. Finally, 13C-label feeding experiments were carried out to determine the effect of low O2 stress on the polar metabolic profile of tomato cell suspension and to analyse the changes in fluxes of the central carbon metabolism under both metabolic dynamic and steady-state conditions.A cell suspension was established from dark grown friable callus of tomato leaves (Lycopersicum esculentum L. var cerasiforme). The growth of the cell suspension involved a lag period, a linear phase of growth and a stationary phase. Polar metabolites present in the cells were separated and detected on the GC-MS after methanol extraction and derivatisation using N,O-bis-(trimethylsilyl)trifluoroacetamide. A total of 70 polar metabolites could be identified and quantified with a GC-MS temperature programme of approximately 40 min duration. The polar metabolitespresent in the cells belonged to the functional groups of amino acids, organic acids, sugars and sugar alcohols. After performing feeding experiments, 13C-label accumulation could be detected in 47 of the 70 metabolites measured.The metabolic response of plant cells following the induction of low O2 stress was studied by analysing the changes in polar metabolite after incubating cell suspension at different O2 levels in a bioreactor. The cells were incubated at O2 levels of 21, 1 and 0 kPa and cell samples taken every hour for a period of 12 h with a final sampling after 24 h of incubation. 13C-glucose was added to the mediumof the cells four hours after the start of the incubation. The changes in metabolite levels as well as the incorporation of 13C-label was measured with GC-MS. Low O2 altered the polar metabolic profile of the cells.There was a general reduction in the levels of most amino acids, organic acids and sugars and an increase in the intermediates of glycolysis, lactate and some sugar alcohols. The 13C-label data showed reduced label accumulation in almost all metabolites except lactate and some sugar alcohols under low O2 stress. The results indicated that low O2 in plant cells activated fermentative metabolism and sugar alcohol synthesis while inhibiting the activity of the TCA cycle. Also, the levels of metabolites whose precursors are derived from the intermediates of the central carbon metabolism such as amino acids were reduced upon the induction of low O2 stress.To obtain a quantitative understanding ofthe response of the fluxome following the induction of low O2 stress inplant cells, the changing metabolite levels and 13C-label accumulation were used to construct a dynamic model of the central carbon metabolism.A compartmentalised metabolic network model containing glycolysis in the cytosol and plastid, the TCA cycle in the mitochondria and the syntheses of alanine, aspartate, lactate, glutamate, serine, sucrose and valinewas developed. The model contained differential equations describing both Michaelis-Menten and first-order kinetics and the model parameters were estimated using a non-linear least square optimisation approach. The dynamic modelling showed that incubating cell suspension under low O2 lead to a significant reduction in glucose uptake rate. Low O2 stress alsocaused a reduction in the activity of several enzymes involved in the TCA cycle resulting in the accumulation of intermediates of the glycolysis. An increase flux of lactate and ethanol synthesis was observed showing the enhanced role of fermentative metabolism in ensuring energy production under the low O2 stress. Analysis of energy production and utilisation showed similar amounts of ATP production at the different O2 levels even though the ATP produced under the low O2 conditions came at a cost of high substrate usage. Metabolic control analysis of glycolysis, fermentation and the TCA cycle showed that the uptake of external glucose controls most of the fluxes in the central carbon metabolism while thetransport of pyruvate into the mitochondria from the cytosol controls the activity of the TCA cycle. Also, enzymes which compete for a common substrate exerted negative control on each other.Steady-state metabolic flux analysis was carried out using the 13C-label incorporated into free intracellular metabolites instead of the conventional approach of utilising the label being incorporated in proteinogenic amino acids. This was done to avoid the long incubation times needed to achieve metabolic and isotopic steady-state in proteinogenic amino acids. For steady-state flux analyses, cell suspensions were incubated in a bioreactor at O2 levels of 21, 8, 5 and 0 kPa until metabolic and isotopic steady-state was reached (24 h after the start of the experiment). Free intracellular metabolites were extracted with methanol, derivatised with N-(tert-butyldimethylsilyl)-trifluoroacetamide and analysed using GC-MS. 13C-labelpresent in metabolites of the central carbon metabolism, amino acids and sugars were determined for steady-state fluxes analyses. Fluxes were estimated using the 13CFLUX2 software. The steady-state response to low O2 stress was similar to the observations made under dynamic conditions with a decrease in substrate uptake, an increase increased fermentative metabolism and a reduced TCA cycle activity and amino acid synthesis. Based on the similarity in fluxes through the central carbonmetabolism, the dynamic and steady-state modelling approaches were compared. Dynamic modelling offers several advantages including providing more detailed information on the structure and regulation of metabolic networks under different stress conditions and providing a time dependent response of an organism to stress. Steady-state flux analysis is, however, useful in obtaining a quick overview of the changes in metabolismupon stress induction especially in systems where metabolic and isotopic steady-state can be ascertained. " "Development of Imaging Methods towards Monitoring of Cell Therapy by In Vivo Magnetic Resonance Imaging" "Uwe Himmelreich" "Biomedical MRI, Stem Cell and Developmental Biology" "Despite recent advancements in medical research, many disorders still remain beyond the capabilities of current treatments. Cell based therapy is extensively being investigated to solve these unmet clinical needs. Several techniques are under development to assist the monitoring and validation of cell therapy. Of which, non-invasive imaging is one of the prime aides used to follow up on therapy efficacy and safety. By preloading therapeutic cells with MR contrast agents, it is possible to track thelocation of cells with magnetic resonance imaging (MRI). Current pre-labeling strategies are optimized mainly for non-phagocytic stem cells based on improved nanoparticle (NP) properties. With increasing knowledge about the cellular and molecular biology of tissue/ organ regeneration, extra-embryonic sources (like adult stem cells: MSCs, NSCs, HSCs or immune cells like dendritic cells, T-cells) have also been utilized as therapeutic vehicles. These cells exhibit different regenerative capacities but also different cell cytoskeleton organization and elasticity, cell shape, and adhesion strength and in vitro culture characteristics. Dueto these different parameters involved in the uptake mechanism, it is not always feasible to translate currently available labeling protocols from one cell type to other. Therefore, one aim of this PhD thesis was tocontribute to the development of cell labeling strategies with iron oxide based nanoparticles (NPs) to follow the fate of different therapeuticcells (adherent, in suspension and in differentiated mixed culture) in in vivo preclinical models. To achieve this goal, we utilize unspecific as well as specific (or targeted) labeling principles.In the contextof unspecific labeling, means of NPs uptake is normally achieved by a simple incubation step. Lot of attention has been paid to NP related characteristic where size, surface charge and surface coating of NPs shown to be responsible for differential uptake and toxic responses in different types of cells. Here, we determined that the cell based parameters (cell size, proliferation rate) are responsible for such variation with thehelp of electron microscopic investigation. This study also emphasizes the need for performing validation experiments by using, for example, transmission electron microscopy to determine the fate of NPs post endocytosis. Next, unspecific labeling approaches were employed for labeling ofpancreatic islets (PIs). Here we showed that magnetoliposomes (MLs), due to their positive surface charge and small size, were taken up by PIs in relatively shorter time (2-4 hours) when compared to commercially available agents like Endorem and Resovist (24-72 hours) without showing any apparent negative effects on the viability and functionality of PIs. These preliminary results indicate the potential of MLs as promising alternative for tracking transplanted PIs in diabetes model. Targeted labeling was studied in feasibility experiments to check if galactose functionalized MLs would be specific to hepatocytes in vitro and in vivo. Galactose MLs were tested in mixed cultures generated afterdifferentiation of embryonic stem cells. After particle labeling, cells were separated magnetically and the positive and negative magneticfractions were tested for the presence of hepatocyte specific marker. Due to the very small percentage of mature hepatocyte population, it was difficult to isolate hepatocytes from mixed cultures. Specificity of galactose MLs in vivo was evaluated by systemic administration of the particles in healthy mice. MR analysis, microscopic evaluation and immunostaining confirmed successful targeting of hepatocytes. The technology can further be implemented to determine the onset of liver diseases. This work contributes to the development of labeling strategies for monitoring cell based therapy in different disease models in vivo. This knowledge is currently being translated to other multiple applications including the development of cell therapy strategies and for determining theonset of diseases. Thus, this work will contribute to a solid basis forfuture applications and development in in vivo monitoring of cells withMR imaging." "Visualization of cell walls components in creal grains using fluorescently labeled enzymes." "Christophe Courtin" "Centre for Food and Microbial Technology" "Wheat bran is a by-product of wheat grain milling. Its use as a source of dietary fiber in human nutrition is increasing due to its nutritional profile in general and the presence of considerable levels of indigestible cereal cell wall polysaccharides in particular. In wheat bran, the most important polysaccharide is arabinoxylan (AX). For the degradation of wheat bran AX, both mechanical and enzymatic treatments can be used. In the enzymatic deconstruction of AXs, xylanases are the most important enzymes due to their endo-action. Accessory enzymes like arabinofuranosidases assist in the degradation process. The main objective of the project is to gain insight in the breakdown of cereal bran during processing, both enzymatically and mechanically, using fluorescently labeled enzyme probes and epifluorescence microscopy. This will provide insights complementary to the common quantification of hydrolysis products, since it will clarify which tissues are readily degraded or which remain intact given specific processes. Xylanases of different glycoside hydrolase families as well as arabinofuranosidases will be tested for their potential as probes and their staining specificity will be investigated. The generated probes will then be used to monitor breakdown of cereal bran by visualizing differences arising during processing." "Microwave sensing in microfluidics for cell and extracellular vesicle characterization (MESMERIC)" "Dominique Schreurs" "Waves: Core Research and Engineering (WaveCore), Mechatronics, Biostatistics and Sensors (MeBioS)" "A promising way to fight cancer in the early stages, among other diseases, is through information carried by single cells and extracellular vesicles (EVs). However, extracting this information often depends on the availability of biomarkers and their matching bioreceptors. To circumvent this necessity, we will explore in this interdisciplinary project microwave dielectric sensing combined with novel microfluidic solutions as a platform capable of differentiating single cells and EVs in a label-free manner. Thus, we will develop a unique microwave-microfluidic technology capable of single-cell seeding and retrieval, EV manipulation, micro- to pico-liter sample handling and label-free single cell and EV characterization through multi-frequency microwave sensing and actuation. By using microwaves, the novel enabling technology could alleviate biomarker discovery, drug screening at the single-cell level, EVs or single-cell analysis and as such have a significant impact on many fields, including cancer." "Towards understanding the extractability and functionality of fruit and vegetable derived cell wall polysaccharides using non-conventional extraction techniques" "Ann Van Loey" "Food and Microbial Technology (CLMT), Department of Microbial and Molecular Systems (M²S)" "Pectin, a cell wall polysaccharide in fruits and vegetables, is an important texturizing ingredient for the food industry. Since traditional pectin extraction results in degradation of pectic polymers and has a high environmental impact, novel extraction techniques have been proposed in recent decades. However, the potential of these non-conventional techniques is still unclear, mainly due to the fragmentary nature and ‘trial-and-error’ approach of the conducted studies, without mechanistic insight into these extraction techniques.In this context, an FWO-proposal was submitted in which novel in situ visualization techniques will be introduced to gain fundamental insights into several novel extraction techniques. An integrated approach will be applied, not only evaluating pectin extractability, but also the molecular properties and the functionality. The current PDM-proposal will be focused on (i) gaining insight into enzyme-assisted extraction of pectin from apple pomace using immunofluorescence co-labelling, and (ii) characterization of the extract in terms of molecular and functional properties." "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." "Studying global gene expression changes during hematopoietic development to identify novel regulators of hematopoietic stem cell function" "Catherine Verfaillie" "Stem Cell and Developmental Biology" "Self-renewal and differentiation are the two primary attributes of all kinds of stem cells. Hematopoietic stem cells (HSCs) are multi-potent stem cells that are responsible for producing all blood cells throughout the lifetime of an organism. Hematopoietic stem cell transplantation (HSCT) is an effective method for treatment of hematopoietic disorders including leukemia and other hematopoietic malignancies, as well as immunodeficiencies. The first HSCT was performed more than 50 years ago1. Today, more than 30,000 autologous and 15,000 allogeneic transplantations are performed annually2. Bone marrow (BM), mobilized peripheral blood (MPB) and umbilical cord blood (UCB) are three major sources for HSC. Although HSC transplantation can cure a significant proportion of patients, it still has certain limitations like availability of a matched donor, graft-versus-host diseases (GvHD) and delayed engraftment post-transplantation.The process of finding donors that have compatible histocompatibility antigens (HLA) is often a challenge. In this regard, UCB derived HSCs are considered an alternative source for HSCT, it requires less stringent HLA matching as compared with BM grafts3. One of the problems related to the use of UCB as cell source is the limited number of HSCs per graft, such that one UCB unit is insufficient to treat adult patients4-6. Some approaches could be useful to overcome the limitation such as ex vivo expansion of HSCs or improving the homing capacity of HSCs. Even though a lot of information is available on various regulators that control their function in vivo, maintenance and expansion of HSCs is still a major challenge and also the biggest hurdle in order to exploit their immense potential.During development, HSCs appear in various transient sites before they finally migrate to the bone marrow, which is a major site of hematopoiesis. In adult BM, long-term repopulating HSCs are mostly quiescent and are in G0 stage of cell cycle, while an expansion of HSC pools occurs in the fetal liver during development. Therefore, we aim at studying HSCs and their niche during hematopoietic development.Both intrinsic and extrinsic factors can influence the self-renewal capacity of HSCs. In order to uncover important regulators that govern self-renewal and proliferation of HSCs, we performed genome-wide transcriptome analysis of HSCs and their niche from various stagesof development using RNA-sequencing (RNA-Seq). The cell-intrinsic regulators of HSCs were shortlisted after comparing gene expression profile of HSCs isolated from fetal liver (FL) and adult bone marrow (BM) reflecting the proliferating and quiescence state, respectively. These intrinsic regulators were further validated for their functional role in hematopoiesis using a flk1:GFP/gata1:dsRed double transgenic Zebrafish line, in which the cells from blood and endothelial lineages are fluorescently labeled. Our study identified a previously unknown role for tdg, uhrf1, uchl5, and ncoa1 in the emergence of definitive hematopoiesis in zebrafish.Most of the studies on the hematopoietic niche have been done using adult BM as a model system where HSCs are mostly in a quiescent state. However, the niche in the FL has been less studied than the BM niche. Here, we performed transcriptome analysis of niche cells that directly surround HSCs by using laser capture microdissection from FL of E12.5, E14.5, and E16.5. The differentially regulated genes between FL 12.5 and FL16.5 relative E14.5 were shortlisted. From these lists of potential extrinsic factors, we identified a role of VEGF-C and S100A8 in increasing HSC number by an addition of recombinant proteins to the ex vivo culture of HSCs. Although the ex vivo expansion of HSC in the presence of these factors was achieved, These HSCs had reduced in long-term repopulation potential in vivo. The discrepancy between these findings will need further experimentation, including evaluation of homing or of possible exhaustion of HSCs in response to these factors.Lastly, we investigated the role of differentially regulated biological pathways between FL 14.5 and BM HSCs based on gene expression data obtained from RNA-seq. Interestingly, we observed that FL HSCs showed increased expression levels of genes involved in oxidative phosphorylation (OxPhos) and the tricarboxylic acid cycle  (TCA). On other hand, OxPhos pathways have been demonstrated to cause exhaustion of BM HSC; but this has not been extensively studied in fetal liver HSC. We here demonstrated that FL HSCs use the OxPhos pathway in addition to glycolysis, without apparent HSC exhaustion.The results presented in this thesis form a foundation for further functional validation of novel transcriptional regulators, extrinsic growth factors and metabolic regulators in mammalian models of hematopoiesis, to ultimately provide novel and clinically relevantmethods that will allow ex vivo expansion of HSCs."