Titel "Korte inhoud" "Dynamics in the Central Plant Cell Metabolism" "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." "The effect of dissolved organic matter on the stability and activity of a pesticide-degrading bacterial consortium" "Benjamin Horemans" "The uncontrolled use of pesticides to secure food production has resulted into the contamination of surface water and groundwater bodies. Microbial biodegradation is considered as a major process that contributes to the removal of pesticides from the environment. Bacteria have been isolated which display specialized catabolic pathways that enable them to use pesticides as sole source of energy and carbon. In natural ecosystems, pesticides are considered micropollutants which are pollutants present in water at trace concentrations in the pg L-1 to µg L-1 range and are often only temporarily available. It has been questioned whether such concentrations can sustain pesticide degrading populations and whether degradation of pesticides at micropollutant concentrations still occurs. Natural environments contain dissolved organic matter (DOM) at concentrations of 1-20 mg L-1 and is considered as the major source of carbon and energy for heterotrophic communities. The DOM is a supplementary source of nutrients and energy for residing pesticide degrading populations. However, DOM can also have negative effects on pollutant biodegradation, for instance by exerting catabolic repression. Up till now, the net effects of natural DOM on pesticide biodegradation are scarcely explored especially in case of the biodegradation of pesticides occurring at relevant micropollutant concentrations of pesticides The overall aim of this research was to explore the effects of the quantity and quality of DOM on pesticide degradation at macro- and micropollutant concentrations. The DOM quantity in European surface waters is measured at a median concentration of 5 mg L-1 dissolved organic carbon (DOC). DOM quality is determined by the molecular properties of the occurring organic compounds (e.g. spectroscopic properties) and is related to its biodegradability. As an experimental system, the degradation of the phenylurea herbicide linuron was examined by members of the genus Variovorax and/or by a triple-species bacterial consortium. This consortium consists of three strains which synergistically mineralize linuron. Variovorax sp. strain WDL1 initiates linuron mineralization by transforming linuron to 3,4-dichloroaniline (3,4-DCA) and N,O-dimethylhydroxylamine (N,O-DMHA). Comamonas testosteroni WDL7 lives on excreted 3,4-DCA while Hyphomicrobium sulfonivorans WDL6 feeds on N,O-DMHA.In a first part of the research, the metabolic capabilities of the linuron degrading bacteria were explored. By determining C-source utilization profiles in Biolog GN2 microtiter plate assays, it was found that the consortium displays an extended metabolic capability compared to individual linuron degrading strains and its members. This was due to the respiration of C-sources by the consortium for which respiration was unobtainable by the individual members. The data suggest that the consortium members cooperate in the utilization of C-sources other than linuron. This feature can contribute in consolidating community composition in the presence of micropollutant concentrations of linuron or in its absence. By examining the biodegradable DOC in environmental DOM (eDOM) by the consortium and its members and concomitant growth, we observed that this cooperation extends towards the utilization of eDOM as carbon source. The data suggest that for both DOM of low and high recalcitrance, cooperation existed within the consortium. All consortium members benefited from the cooperation, however the balance of benefits for each strain shifted according to DOM recalcitrance. All together, the results show that compared to individual heterotrophic pesticide degrading bacteria, consortia can benefit to a greater extent of dissolved carbon resources available in an environment due to metabolic cooperation and that consortia have to be included to evaluate the effect of DOM on pesticide degradation.In a second part of the research, the activity of bacterial cultures towards the degradation of linuron at concentrations ranging from 10 mg L-1 to 1 µg L-1 was explored in batch experiments. Several linuron degrading pure Variovorax strains and the consortium were tested. All cultures tested were able to degrade linuron at initial concentrations as low as 1 µg L-1 and that degradation proceeded beyond the detection limit of 1 ng L-1. Degradation rates however varied up to a factor 70 between the different strains and were concentration dependent. Inclusion of strain WDL1 in the consortium did not dramatically improve the degradation of linuron at concentrations lower than 100 µg L-1. In addition, the effect of supplementary C-substrates including eDOM of varying quality and quantity on linuron degradation was assessed. Effects of supplementary C-sources on linuron degradation were especially noted at a concentration of 10 mg L-1 linuron. In case of pure cultures, citrate as a supplementary C-source repressed linuron degradation and increased accumulation of 3,4-DCA. eDOM, even when only 20% biodegradable, increased the linuron degradation rate 2-fold. At micropollutant concentrations a 2-fold increase in linuron degradation rates was observed with strain WDL1 for eDOM, suggesting that eDOM can stimulate linuron degradation by pure cultures at micropollutant concentrations. However, with the consortium, the magnitude of both negative and positive effects of eDOM obtained with pure cultures diminished greatly.Bacteria grow in the environment as a community on surfaces and form so-called biofilms. That is why in the last part of the research we explored the effects of eDOM on linuron degradation in continuously irrigated biofilm setups with focus on the consortium. It was first shown that the consortium was able to form biofilms in flow chambers irrigated with eDOM of both low and high quality at environmentally relevant concentrations of 5 mg L-1 DOC. The structure and composition of the biofilms clearly depended on the DOM formulation applied. The biofilms were afterwards irrigated with linuron at a concentration of either 10 mg L-1 or 100 µg L-1. Clearly, the linuron degrading activity of the biofilm depended on the DOM used for growing the biofilm and the linuron concentration. In case of a linuron feed of 10 mg L-1, for some DOM complete failure of linuron degradation was observed. The ability of these biofilms to degrade linuron correlated with the relative abundance and co-localization of WDL1 and WDL7, while the linuron degrading activity correlated with biomass. Moreover, degradation of 100 µg L-1 linuron was maximally 30% for all biofilms. Second, the effect of eDOM on linuron degradation at concentrations of 10 mg L-1 was explored. Easily degradable DOM like citrate inhibited linuron degradation and was accompanied with the accumulation of 3,4-DCA. More recalcitrant DOM stimulated linuron degradation as the effluent concentrations of linuron decreased. After changing the feed to only linuron, both positive and negative effects of the DOMs were rapidly lost and degradation occurred with an efficiency similar to this recorded for control biofilms which had never been in contact with DOM. In a final experimental setup, the effect of DOM on linuron degradation of concentrations as low as 10 µg L-1 in biofilms was explored. While the consortium was shown to form biofilms on linuron concentrations of 1000 µg L-1, 100 µg L-1 and 10 µg L-1 with removal efficiencies going from 80% to 30%, no significant effect of DOM of both low and high quality was recorded, despite changes in biofilm structure and composition. It can be concluded that at high linuron concentrations, the presence of DOM as supplementary C-source affects linuron degradation by pure bacterial cultures positively at high linuron concentrations. In microbial communities, smaller effects of DOM on linuron degradation were found than with isolates, likely related to the robustness of communities. Microbial communities are the rule rather than the exception in natural ecosystems. All previously isolated linuron degrading cultures showed the ability to degrade linuron at micropollutant concentrations. The effect of DOM did however no longer apply at these concentrations. On the other hand, biofilm experiments suggest that growth on DOM in periods of absence of linuron can significantly affect subsequent linuron degradation." "Age differences in wealth accumulation and retirement saving behavior of wageworkers" "Chavis Ketkaew" "Thailand has now become an aging society. However, the fact that the majority of Thai wageworkers do not effectively save for their retirement may result in several elderly living below the poverty threshold during retirement. The objectives of this research project were 1) to identify the relationships among variables that explain the wealth accumulation behavior and retirement contribution of Thai wageworkers, 2) to explore demographic characteristics influencing retirement preparedness and perform behavioral segmentation of Thai wageworkers based on their traits, 3) to explore in-depth retirement planning obstacles of Thai wageworkers based on the segments. This thesis involves five chapters: 1) Research Background, 2) The Effect of Expected Income on Wealth Accumulation of Thai Wageworkers, 3) Demographic Analysis, Segmentation, and Perceived Retirement Preparedness of Thai Wageworkers, 4) Identifying Retirement Planning Obstacles of Thai Wageworkers, and 5) Conclusion and Policy Recommendations. The three objectives, as mentioned earlier, are translated primarily into chapters 2), 3), and 4). A total of 794 wageworkers from the Northeastern Region of Thailand were recruited to participate in 3 episodes of this research project [chapter 2) n=350, 3) n= 398, and 4) n=46]. The theoretical framework of this research is based on the theory of Life-cycle Hypothesis. This thesis employed several methods: a statistical analysis using the Structural Equation Modeling approach (SEM) using age as a moderator, cross-tabulation and cluster analyses, and qualitative analysis using in-depth interviews, thematic analysis, and Lean Entrepreneur approaches. We found that expected income, wealth accumulation, career status, and health status were the main constructs influencing an individual’s ability to contribute to his or her retirement. We also found that the most common obstacles for young workers are urgent health and emergency issues that cost money. However, the absence of financial knowledge is the most crucial barrier for young and low-income workers. Nevertheless, low financial literacy is a big problem for old and low-income workers. In contrast, high consumption expense is the main problem for old and high-income workers. This paper suggested that a wage worker should first provide his/her income through wealth accumulation schemes such as investment in financial assets, e.g., stocks, bonds, mutual funds, and properties, investment in other businesses as a second job, and cash deposit. The results suggested that wealth accumulation was the essential mediator allowing a wage worker to contribute to retirement effectively in the long term. This paper recommended that the government and authorized bodies (e.g., the Bank of Thailand and the Stock Exchange of Thailand) should provide more investment alternatives and improve the investment knowledge of the citizens. Most importantly, the First Pillar (public mandated, publicly managed, defined benefit system) should be more supportive." "Discrete modeling of strain accumulation in granular soils under low amplitude cyclic loading" "Stijn François, Geert Degrande" "An advanced understanding of the strain accumulation phenomenon in granular soils subjected to low amplitude cyclic loading with relatively high frequency is needed to enhance the ability to predict the settlement of granular soils induced by vibrations. In the current study, the discrete element method is used to study this phenomenon. A loose and a medium dense sample composed of a relatively large number of spheres are considered. A series of stress controlled cyclic triaxial tests with different excitation amplitudes and frequencies is performed on these samples at different static stress states. The response of these samples at the macroscopic and microscopic scales is analyzed. The sample density, the cyclic stress amplitude and the static stress state importantly affect strain accumulation. However, the cyclic excitation frequency has a small effect on strain accumulation. At the microscopic scale, frictional sliding occurring at a few contacts continuously dissipates energy and the fraction of these contacts varies periodically during cyclic loading. The coordination number of these samples increases slightly as strain accumulates. However, the anisotropy remains almost constant during low amplitude cyclic excitation. A qualitatively good agreement between numerical and experimental results is found. © 2014 Elsevier Ltd." "Application of Lemna minor in site remediation strategies" "Isabelle VAN DYCK" "Pollutants such as radionuclides and heavy metals are present in the environment due to the exploitation of nuclear installations, mining and milling processes, industrial activities (e.g. involving naturally occurring radionuclides), nuclear accidents and the dumping of radioactive waste. Polluted surface waters can become an environmental problem for both aquatic and terrestrial organisms and therefore remediation is needed. Phytoremediation uses the biological processes of living plants (and their associated microbiome) to remove, degrade or stabilise pollutants from their surrounding environment. Phytoremediation is recognised as an efficient site remediation technology for various types of pollutants and can be used as a complementary waste water treatment technique for specific scenarios. Within this PhD project, phytoremediation of water, polluted with radionuclides and metals, using the macrophyte Lemna minor has been systematically studied. L. minor is one of the 36 Lemnaceae species that can absorb and accumulate various pollutants in its biomass, making it a candidate plant species for phytoremediation purposes. L. minor plants are used and studied for wastewater evaluation and treatment due to their fast growth, ability to form extended surface mats on the water, high removal capacity and sensitivity towards different kinds of stress (e.g. heavy metals, radionuclides). Due to their easy growth in a wide variety of laboratory conditions, L. minor is often used as a model organism for environmental studies and fundamental plant research. Therefore, L. minor is suitable for studying toxicity of wastewater and for investigating in detail its applicability for phytoremediation purposes. Commercial phytoremediation technologies appear to be globally underutilised notwithstanding their effectiveness in field applications and the academic research studies available in support of this methodology. More in-depth research is required to demonstrate that phytoremediation can be a valuable and eco-friendly solution for the treatment of polluted environmental or industrial water. L. minor can be used to remove radioactive or heavy metal pollutants from water but can also be affected by the pollutants that are present in the water, hence this study covers also pollutant interactions with L. minor. We started this study by providing a brief introduction on environmental pollution, the concept of phytoremediation and the test organism L. minor (Chapter 1). Subsequently, the scope and objectives are described in Chapter 2. L. minor growth was studied under different optimal and sub-optimal environmental conditions (temperature, light irradiation and variable nutrient concentrations), while varying one environmental parameter per experiment. By combining experimental and mathematical modelling approaches, it was possible to develop a L. minor growth model as a function of biomass, temperature, light irradiation and variable nutrient concentrations, suitable for application in phytoremediation of radionuclides and other water pollutants (Chapter 3). A previously described L. minor growth model was used as a starting point for developing an extended and more process-based growth model. Important changes were made to this model, such as modification of the crowding term to a logistic biomass function and addition of a light irradiance function with variable photoperiod. We made growth curves for each experimental condition and fitted the model parameters to the experimental data using ModelMaker 3. Equations for the associated environmental conditions were tested and adapted where necessary (e.g. new equation for temperature and light irradiance function, changes to the nutrients equations). A sensitivity analysis indicated that parameters related to growth (ropt, rmax,L, rmax,N, rmax,P and r), optimum and experimental temperature (Topt and Texp) and maximum biomass allowed by the system on the used surface area (hB) are the most sensitive parameters of the model. Finally, after an overall parameter optimisation of the model, it was clear that our improved L. minor growth model can predict L. minor growth under different environmental conditions. It is an improvement on the way to establish a full remediation model capable of evaluating different remediation options in real case scenarios. Not only growth of L. minor is affected by changing environmental conditions, but also starch and soluble sugars contents are affected (Chapter 4). Accumulation of starch can either be connected with typical stress related responses (e.g. salinity, nutrient deprivation, low or high temperatures, pollutants) or related to increased growth rates when more substrates are needed for metabolism and growth. By performing experiments under different environmental conditions (temperature, light irradiance and nutrients) it was possible to distinguish between these processes. Non-optimal temperatures and low nitrogen concentrations resulted in decreased growth rates and higher starch and soluble sugars accumulation, which are typical stress associated responses. Results suggest that lower temperatures are more stressful to L. minor compared to higher temperatures. We also demonstrated that higher light intensities and longer photoperiods led to increased growth rates and more starch formation. These results contribute to a better understanding of L. minor growth, starch and soluble sugar content under different environmental conditions and form a basis for optimising plant cultivation conditions to improve the suitability of L. minor biomass for practical applications (e.g. biofuels, biomaterials, animal feed and even human nutrition). Next, we studied the removal and effects of pollutants (Co, 60Co, Cs, 137Cs, Mn, Ni and Zn) by and on L. minor. Information related to the uptake mechanisms of some radionuclides and metals by L. minor (biosorption versus bioaccumulation) and possible effects on L. minor’s physiological and biochemical functions is not readily available. However, this knowledge is needed to generally better understand uptake and effects on L. minor when exposed to multiple pollutants, to assess the possibility of using L. minor in phytoremediation applications and to improve environmental impact assessments. We studied the uptake and effects of stable Co and Cs, together with radiation effects of 60Co and 137Cs on L. minor’s physiological and biochemical functions, in order to differentiate between chemo- and radiotoxicity (Chapter 5). Regarding effects of 60Co and 137Cs, some studies have investigated the effects of external gamma radiation on L. minor, but studies that investigated the internal radiation exposure effects or chemical effects are scarce. Since the usage of high concentrations of 60Co and 137Cs in solution was not practically feasible in our laboratory (due to safety restrictions related to working with open radioactive sources), we used an innovative approach that combined the external irradiation (from a 60Co and 137Cs source) together with the direct uptake of a corresponding amount of stable Co or Cs. By doing this, the radiological and chemical components of 60Co and 137Cs are combined and the uptake by and effects on L. minor when applying higher activity concentrations of 60Co and 137Cs can be imitated. Experiments were performed by exposing L. minor plants to different concentrations of stable Co and Cs, different external dose rates of 60Co and 137Cs and the combined exposure experiments. Dose-response curves, based on percentages of growth inhibition, were established for each exposure condition and (radio)element. The derived EDR50 values indicated that the combined exposure caused a higher toxicity compared to (a) external irradiation applied as a single stressor or (b) exposure of L. minor to the stable isotope. The highest toxicity was observed for 137Cs. In addition, exposure 60Co or 137Cs caused a dose rate-dependent increase of the starch content. Since we considered gamma radiation and chemical pollutants as stressors that probably affect different physiological systems of an organism, a combined effect assessment was performed. We used the two reference models/approaches originally developed for assessing the toxicity of mixtures of chemicals, namely independent action and concentration addition. Both approaches resulted in a relatively good fit between our predicted growth curve and the experimental data points from the 60Co/Co and 137Cs/Cs combined exposure experiments, but independent action led to a slight overestimation. Therefore, this is the most conservative approach regarding assessing the combination of metal exposure and gamma radiation. Our method is a practical tool for predicting the effects on L. minor growth caused by a mixture of stable elements and gamma radiation. Besides stable Co and Cs, also the uptake of Mn, Ni and Zn on L. minor was extensively studied as function of pollutant concentration and time (Chapter 6). L. minor plants were exposed to different concentrations of Mn, Ni and Zn, in order to produce dose-response curves. From the derived EC50 values, an ordering was made based on the toxicity of the elements on L. minor: Ni > Co > Zn > Cs = Mn. Pollutant removal parameters were also determined; a similar but reversed order was obtained as for the toxicity (Mn = Cs > Zn > Ni > Co). Generally, the higher the initial exposure concentration, the lower the specific growth rate, removal percentage and bioconcentration factor of L. minor for the elements. Elements with a high toxicity (low EC50 values) demonstrated lower removal per L. minor dry mass. For essential elements, such as Mn, L. minor showed a high removal capacity. The removal capacity for Cs was also high, most likely resulting from the high similarity with the essential element K. Our detailed study on the influence of K on Cs uptake by L. minor confirms that the presence of high K concentrations results in more Cs-K competition, less Cs uptake and eventually less toxicity. Our previously developed L. minor growth model was further extended by adding all uptake and release time series data of the five elements (Co, Cs, Mn, Ni and Zn) leading to an experimentally-based mathematical single pollutant uptake model for L. minor, based on the concepts of biokinetic modelling (first order kinetics) and population modelling. All key uptake and release parameters were numerically optimised and it can be concluded that they depend on the applied pollutant. Our L. minor uptake and release model makes it possible to predict plant growth and pollutant removal for single elements present in water, opening the door to predicting the concentrations of pollutants in L. minor in real-life remediation applications. Lastly, we focussed on a broad study about the possible effects of pollutants (Co, Cs, Mn, Ni and Zn) on L. minor’s physiological and biochemical functions (Chapter 7). We also compared uptake mechanisms (biosorption and bioaccumulation), but mainly focused on effects of single pollutants on growth, photosynthetic pigments, photosynthesis, starch and soluble sugars content. When applying increased concentrations of Co, Cs, Mn, Ni and Zn, toxic effects got visible as decreases in photosynthetic pigments (Chl a, Chl b and carotenoids) and an increase in starch content in a concentration-dependent manner. Soluble sugars contents were also higher for all exposure conditions. Photosynthesis only displayed pronounced effects during exposure to toxic concentrations of Mn and Zn. Too high Zn concentrations caused damage to the light harvesting capacity of PSII and by consequence a smaller fraction of the incoming energy could be used for photosynthesis trough PSII. In contrast, the light harvesting capacity of PSII remained intact during exposure to Mn and we even observed an enhanced capability to perform photosynthesis. Meanwhile, Mn exposure also negatively affected the photosynthetic pigment concentrations which led to a reduced light absorption. By performing this study, a broad overview of the effect of metals and radionuclides on L. minor’s uptake, physiological and biochemical functions was obtained. Together with the L. minor growth study under different environmental conditions, the possibility is there to use L. minor for remediation purposes due to our quantification of the relevant processes. This work provides also essential information about how the remediation ability of L. minor is influenced by the physico-chemical and biological characteristics together with environmental factors such as cations, anions, radionuclide concentration and speciation, metal concentration, growing conditions (e.g. light, nutrients, temperature) and set-up (e.g. biomass/water ratio, contact time)." "A pin-based pyro-electrohydrodynamic jet sensor for tuning the accumulation of biomolecules down to sub-picogram level detection" "Simona Itri, Danila del Giudice, Martina Mugnano, Volodymyr Tkachenko, Sanna Uusitalo, Annukka Kokkonen, Inka Pakkila, Prof. Dr. Ir. Heidi Ottevaere, Yunfeng Nie, Emanuela Mazzon, Agnese Gugliandolo, Pietro Ferraro, Simonetta Grilli" "The detection of low abundant biomarkers is of great interest in different clinical cases, such as the early diagnosis of Alzheimer's disease. Here we present what we call pin-based pyro-electrohydrodynamic jet (p-jet) sensor that can linearly tune the accumulation rate of biomolecules in tiny droplets. In this newly revealed pin -based configuration we demonstrate the ability to detect biomolecules down to sub-picogram concentration. Two different fluorescent probes at a varying number of accumulated droplets, up to 50, have been used to investigate the p-jet sensor. Moreover, a comparative study with a commercial piezo-driven bio-spotter has been performed. The spots of biomolecules exhibit high reproducibility by achieving a coefficient of variation" "[99mTc]duramycin for cell death imaging: Impact of kit formulation, purification and species difference" "Sigrid Stroobants" "INTRODUCTION: [99mTc]duramycin is a SPECT tracer for cell death imaging. We evaluated the impact of kit formulation, purification and species difference on the pharmacokinetic profile and cell death targeting properties of [99mTc]duramycin in order to define the optimal conditions for (pre-)clinical use. METHODS: Three kits were prepared (A: traditional formulation, B: containing 1/3 of ingredients, C: containing HYNIC-PEG12-duramycin). Following labeling, the kits were used without purification, or with SPE or HPLC purification. The pharmacokinetic profile was evaluated in mice and rats at 24 h post tracer injection (p.i.). Non-specific accumulation of [99mTc]duramcyin was studied by μSPECT imaging in chemotherapy treated COLO205 tumor bearing mice pre-treated with cold duramycin (0.01-50 μg). Cell death targeting ability of the kits displaying the best pharmacokinetic profile was compared in a treatment response study in COLO205 tumor bearing mice treated with conatumumab (anti-DR5 antibody). RESULTS: HPLC purification of kit prepared [99mTc]duramycin and reducing the amount of kit ingredients resulted in the best pharmacokinetic profile with low accumulation in liver, spleen and kidneys. The use of PEGylated [99mTc]duramycin required longer circulation times (> 4 h pi) to obtain good imaging characteristics. Pre-treatment with duramycin significantly decreased tracer uptake in chemotherapy treated tumors in a dose-dependent manner. A blocking dose of 50 μg significantly increased non-specific accumulation in liver and spleen. Non-specific accumulation of [99mTc]duramycin was however demonstrated to be species dependent. HPLC purified kit A (5.21±1.71 %ID/cc) and non-purified kit B (1.68±0.46 %ID/cc) demonstrated a significant increase in tumor uptake compared to baseline following conatumumab treatment. CONCLUSIONS: To obtain [99mTc]duramycin with favorable imaging characteristics for cell death imaging in mice [99mTc]duramycin needs to be prepared with high specific activity by applying HPLC purification. The need for HPLC purification appears to be a species dependent phenomenon and might therefore not be required for clinical translation." "Identification and characterization of Sul1 and Sul2 in Saccharomyces cerevisiae as first sulfate sensors in cell biology" "Saccharomyces cerevisiae is being used for many years by humans in the production of bread, beer and wine. It is now also used for the production of renewable energy sources such as bioethanol. Apart from being vital for the economy of the beverage sector, it is also the simplest eukaryotic model organism which has been most extensively studied to understand the basics of life. Many cellular processes in S. cerevisiae are well conserved in higher organisms, providing an advantage for researchers to easily study these cellular processes. In nature, cells are exposed to fluctuating environmental conditions to which they have the ability to adjust because of a myriad of adaptive mechanisms obtained over millions of years of evolution. In particular, microorganisms such as S. cerevisiae, are continuously exposed to rapid environmental changes from mere physiochemical properties such as temperature to nutrient availability. It is therefore very crucial for these organisms to coordinate their life cycle with these changes for their survival and perpetuation of their species.Nutrients play a pivotal role in the growth and proliferation of cells. They are used as energy sources and building blocks to scaffold all cellular components. Many studies have shown that nutrients also act as signaling molecules which trigger various signaling pathways inside the cells which lead to adaptations needed for the survival. Nutrient limitation conditions trigger cells to enter a low proliferative state characterized by phenotypes indicative of low protein kinase A (PKA). During this state, cells substitute growth and proliferation mechanisms by cell survival mechanisms such as increased carbohydrate storage, reduced ribosomal synthesis and increased expression of stress tolerant proteins which provide resistance in the unfavorable conditions. These phenotypic responses are reversed when the missing nutrient is restored in the medium, suggesting a strong signaling role for the nutrients in activating/deactivating growth and proliferation versus survival pathways.Recently, it has been shown that nutrient-specific receptor-like proteins called transceptors (because of their dual transporter-receptor function) scan for specific nutrients and trigger pathways leading to growth and proliferation upon their sensing of nutrient availability. As explained above, the absence of a single nutrient can trigger cells to enter a quiescent state in which among other processes, they induce the expression of specific nutrient transceptors at the plasma membrane. Reappearance of the missing nutrient in the medium is sensed by the nutrient-specific transceptor/s which then rapidly activates the PKA signaling cascade. In S. cerevisiae, transceptor sensing of nutrients and subsequent PKA activation is always dependent on the presence of a fermentable sugar. In contrast to classical PKA activation, this activation occurs independently of intracellular cAMP increase. Transceptor-dependent PKA activating pathways have been collectively designated as Fermentative Growth Medium (FGM) pathway to differentiate them from the classical cAMP-dependent activation of PKA pathway. Nutrient transceptors such as Gap1 and Pho84 have, for example, been shown to activate FGM signaling pathway upon sensing amino acids and phosphate, respectively.In this work, we have studied the effect of re-addition of sulfur to sulfur-starved cells. We have investigated the mechanisms of sulfate sensing and identified two sulfate transceptors that trigger PKA activation leading to cell growth and proliferation upon sulfate availability. We first optimized the sulfur starvation conditions by studying accumulation of trehalose and entry of cells in quiescent state. We then confirmed activation of the PKA signaling pathway upon re-addition of sulfate to sulfur starved cells. We verified this activation by analyzing the upregulation and downregulation of determined PKA downstream targets. Furthermore, we confirmed that sulfate signaling is mediated by Sul1 and Sul2, two previously known high affinity sulfate transporters. This uncovered novel signaling functions for Sul1,2 in addition to their previously known transporting function.Using two different approaches, we further demonstrated that the signaling function of Sul1 and Sul2 can occur independently from their transporting function. We first identified a non-transporting agonist, D-glucosamine 2-sulfate, which triggers PKA signaling in spite of not being transported into the cell. Then we identified residues in Sul1 and Sul2, whose mutation blocks the transporting ability of these two proteins without eliminating their sulfate-sensing and PKA signaling capacities. Moreover, these approaches confirmed that transport of sulfate into the cell is not necessary to trigger transceptor-mediated signaling and that metabolism of sulfate is not involved in this mode of PKA activation. After characterizing sulfate transporters Sul1 and Sul2 as sulfate transceptors, we have shown that these plasma membrane proteins undergo substrate-mediated endocytosis and are downregulated in the same way as classical receptors. Re-addition of sulfate to sulfate-starved cells downregulated sulfate transceptors both at the transcriptional and post transcriptional levels. We also identified that the conserved C-terminus STAS domain of the sulfate transceptors is important for both transport and sensor functions of Sul1 and Sul2.Finally, in this study we have additionally discovered and annotated ORF YIL166c as a low affinity sulfate transporter which we referred as Siu1. In this work we show that this transporter can compensate for the lack of Sul1 and Sul2 and allow growth of a sul1Δ sul2Δ mutant in the presence of high sulfate concentrations." "Ecological niche, genetic variation in natural populations, and harvest maturity of Senegalia macrostachya (Rchb. ex DC) Kayl. & Boatwr., a promising wild and perennial edible-seeded crop" "Moustapha Drabo, Habtu Shumoy Abraha, Jonas Koala, Aly Savadogo, Katleen Raes" "Senegalia macrostachya is a wild, perennial, and promising legume that can help alleviate food insecurity in sub-Saharan Africa's drought-prone regions. However, it has been for a long time overlooked, underutilized, and scarcely studied. This study shed light on its ecological niche, genetic variation in natural populations, and seeds' harvest maturity. Three populations of S. macrostachya were selected along an environmental gradient, and their structures, based on their plants' pod characteristics (i.e., unique morphological differentiation trait amongst the plants), were assessed. Then, the seeds' development was investigated following their morphological transformation, mass accumulation, and the mobilization of their proteins and secondary metabolites. S. macrostachya has revealed low genetic variation and fitness to poor soils, warm and semi-arid climates in sub-Saharan Africa, prone to droughts and low to medium agricultural productivity. The maturation of the seeds involved the accumulation of specific late embryogenesis abundant proteins (60, 48, and 14 kDa), identified as chilling-responsive dehydrins, and the degradation of the chlorophylls and carotenoids, which could be used as biochemical landmarks of the harvest maturity. Besides, an aging symptom (high seed abortion rate (14-20%)) was observed in the study populations. This work provides unprecedented information that will assist in predicting the eco-climate suitability and climate resilience, identifying the conservation strategies, and planning the harvesting, reforestation programs, and domestication of S. macrostachya. In parallel, the dehydrins identified from S. macrostachya can be tapped into for drought-resistant crop development. Further research is needed to evaluate the impacts of the mother plants' aging and larger climate gradient on the performance (i.e., seed productivity and regeneration ability) of S. macrostachya." "Evaluation of [18F]CP18 as a Substrate-Based Apoptosis Imaging Agent for the Assessment of Early Treatment Response in Oncology" "Sigrid Stroobants" "PURPOSE: The substrate-based positron emission tomography (PET) tracer [18F]CP18 is capable of detecting the activity of caspase-3/7, two key executioner proteases in the apoptosis pathway, through selective cleavage of the ligand by the activated proteases and subsequent accumulation in apoptotic cells. Using an in vitro and in vivo model of colorectal cancer (CRC), we investigated whether [18F]CP18 tracer accumulation provides a measure for apoptosis and reliably reflects early treatment response to chemotherapeutics. PROCEDURES: [18F]CP18 cell uptake was assessed in treated Colo205 cells (saline, 5-fluorouracil (5-FU), irinotecan or their combination) and correlated with caspase-3/7 activity. [18F]CP18 imaging was performed in Colo205 xenografts, starting with a baseline μPET/micro X-ray computed tomography (μCT) scan, followed by a 3-day treatment with saline (n = 5), 5-FU (low sensitivity, n = 4), irinotecan (high sensitivity, n = 5), or a combination of both (n = 7). The study was concluded with a second [18F]CP18 scan, 24 h after final treatment administration, followed by tumor removal for gamma counting (%ID/g) and for cleaved caspase-3 immunohistochemistry (apoptotic index/necrosis). Tumors were delineated on μCT images and, using the obtained volumes of interest, average percentage injected dose per cubic centimeter (%ID/cm3) was calculated from every μPET image. RESULTS: In vitro, [18F]CP18 cell uptake was positively correlated with caspase-3/7 activity (r = 0.59, p = 0.003). A drug-dependent increase in [18F]CP18 tumor uptake compared to baseline was observed in animals treated with 5-FU (+14 ± 25 %), irinotecan (+56 ± 54 %), and their combination (+158 ± 69 %, p = 0.002). %ID/cm3 showed a positive relationship with both %ID/g (r = 0.83, p "