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​Celine Gys​

• 30 November 2020
• Supervisors: Adrian Covaci and Hugo Neels
• ​​
• Time: 5:00 PM - 7:00 PM

Abstract

The toxicity and occurrence of bisphenol A (BPA) have been studied extensively, but despite the amount of available research, there is no consensus on its impact on human health and the environment. BPA is used in a variety of applications, such as electronic equipment, construction materials, food packaging and thermal paper. Because of growing concern regarding BPA, restrictive regulations are implemented worldwide, resulting in the suspectedly toxic but well-documented BPA being replaced by less studied bisphenols. We have addressed some of the knowledge gaps that endure regarding BPA and the most applied alternative bisphenols.

Using high-resolution mass spectrometry combined with suspect and non-target screening workflows, we identified the biotransformation products generated by human metabolism and environmental degradation of BPA-analogues and the new alternatives to BPA in thermal paper. Employing a human in vitro metabolization assay, a full screening and structural elucidation of in vitro BPS metabolites was performed. The transformation of bisphenols in an aqueous environment was investigated through photochemical and biological degradation processes and the generated transformation products were identified. As dermal contact with thermal paper (e.g. receipts and tickets) is regarded a substantial source of exposure to BPA and its alternatives, we have screened > 300 thermal paper samples from 14 different countries. While BPA was still the main developer, eight alternatives including BPS were detected. Different trends were observed between countries, which will likely affect human exposure.

To assess the total internal exposure to bisphenols via biomonitoring, an analytical method based on gas chromatography coupled to tandem mass spectrometry was developed and validated. Levels of several BPA-analogues were measured for the first time in multiple samples collected from ten individuals over five consecutive days, showing high within-individual variability. Recommendations for future sampling strategies were formulated based on these results.

Determinants of urinary bisphenol levels were investigated in study populations of Japanese school children and Flemish adolescents, using questionnaire data. Although measured urinary levels of bisphenols are subject to regional differences, it was clear that exposure to BPA decreased significantly over the past decade, likely due to restrictions and replacement. As a risk assessment, estimated daily intakes and measured urinary levels were compared to scarcely available health-based guidance values. Even in high-exposure scenarios, no health concerns were expected for the studied populations. Further studies are needed to characterize the exposure to bisphenols in the general population and their potential adverse health effects.

​Michiel Bastiaensen​

• 24 November 2020
• Supervisors: Adrian Covaci and Greet Schoeters
• ​​
• Time: 5:00 PM - 7:00 PM

Abstract

Organophosphate flame retardants (PFRs) and alternative plasticizers (APs) are two classes of emerging contaminants widely used in commercial products, such as textiles, plastics, electronic devices, food contact materials, paints and furniture. Exposure to these environmental chemicals is of concern for humans due to widespread indoor contamination and potential adverse health effects but has not yet been thoroughly investigated.

In the first part of this work, two multi-target analytical methods were developed and validated for the quantification of a broad range of candidate biomarkers for PFRs and Aps in biological matrices. Solid phase extraction and liquid chromatography coupled to tandem mass spectrometry provided the required sensitivity to detect the metabolites at trace levels in urine.

Secondly, the distribution of PFR and AP biomarkers was studied in Japanese children and Flemish adolescents. Several hydroxylated and diester PFR metabolites could be frequently detected in urine. Increased levels of all three TBOEP metabolites were observed for children from Japan, likely as a result of dust ingestion because significant correlations were found with TBOEP in house dust. Flemish adolescents had comparably higher levels of both EHDPHP metabolites possibly due to its migration from food packaging. Metabolites of APs, such as DINCH and DEHTP were also frequently detected in Flemish adolescents indicating widespread exposure. Multiple regression analysis of questionnaire data revealed determinants of exposure to PFRs and APs: product use such as decoration and building materials, socio-economic status and seasonality.

The third part of the thesis investigated the short-term temporal variability of PFR and AP biomarker concentrations. AP metabolites showed weak reproducibility with high within-day variance in samples collected from ten individuals during five consecutive days. Similar observations were made for DPHP and EHPHP, while PFR metabolites with more continuous sources had fair-to-good reproducibility. Recommendations on sampling strategies for future studies were formulated based on these results.

Finally, estimated daily intakes calculated based on urinary PFR metabolite levels were all well below the current reference doses. None of the Flemish adolescents had concentrations higher than the health-based guidance values for the sum of DINCH metabolites, indicating low risk of individual chemical exposures. However, higher levels of TDCIPP in dust and of TDCIPP, TBOEP, and TCIPP metabolites in urine were associated with allergy symptoms of Japanese children. Moreover, metabolite concentrations of DPHP, EHPHP and BBOEP were associated with increased levels of oxidative stress biomarkers. Further experimental and epidemiological studies are needed to elucidate the association between continuous PFR and AP exposure and the potential adverse health effects thereof.

​Eline Byl​

• 27 October 2020
• Supervisors: Filip Kiekens and Sarah Lebeer

Abstract

The morbidity rate of respiratory infections is high. Viral infections are cured with over-the-counter products that treat the symptoms. However, a virus is a disruptive occupant of the respiratory tract that can cause opportunistic infections. Bacterial infections are treated with antibiotics. Although, an extensive and incorrect use, can lead to antibiotic resistance, which is one of the biggest threats to global health. Therefore an alternative strategy for the prevention and treatment of infectious diseases is highly necessary. Recent years, the knowledge in the human microbiome and its functionalities has been increasing continuously, resulting in a growing awareness of the potential application of probiotic bacteria for acute respiratory prophylaxis and treatment.

Tablets are easy to use, have a good patient acceptance and are suitable for large-scale production. Therefore, tablets are an interesting dosage form for oral probiotics. However, due to the complex nature of the probiotic bacteria, the manufacturing of a stable and high-quality probiotic tablet remains a challenge. Specifically, the probiotic cells are exposed to high mechanical stresses during tablet manufacturing, which may affect probiotic survival, efficacy and safety. Therefore, extensive research is needed to develop a successful probiotic tablet. This PhD-project aimed to investigate which process and formulation parameters have a major impact on the survival of the prototype probiotic strain Lacticaseibacillus rhamnosus GG during tablet manufacturing.

The results demonstrated that the survival rate is highly affected by the applied pressure and the tableting properties of the probiotic powder blend. Specifically, the survival rate was significantly better when L. rhamnosus GG was compacted with a powder blend that needs time and pressure to deform and which, after some deformation, fractures. Additionally, it may be important that the powder particles show elastic recovery during decompression. Furthermore, the results suggest that the large galactose-rich cell wall polysaccharides act as shielding molecules. This implies that bacterial cells that lack these molecules, may experience more stress and may be therefore more sensitive to the tableting process. The results also suggest that larger bacterial cells are better protected within plastically deforming powders.

These results are promising and show that the probiotic cells can be protected during tablet production by selecting the appropriate manufacturing determinants, resulting in a better survival and therefore in a probiotic tablet with a sufficient amount of viable cells.

​Dorien De Munck​

• 1 October 2020
• Supervisors: Guido De Meyer and Wim Martinet

Abstract

Recent evidence showed that autophagy, a catabolic cellular mechanism responsible for nutrient recycling, plays a major role in the physiology of vascular cells such as endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). Moreover, dysregulation of this process is associated with the development of age-related cardiovascular disorders including heart failure, atherosclerosis, hypertension and arterial stiffness. To increase the understanding about the role of autophagy on the vasculature, with implications for the development of age-related pathologies we investigated in this project the role of VSMC and EC autophagy on the vasculature using mouse models with a knockout of the essential autophagy gene Atg7 in VSMCs or ECs. 

First we focused on the role of VSMC autophagy on the vascular structure and its, reactivity and biomechanics using a traditional organ bath setup, wire myograph and an in-house developed Rodent Oscillatory Tension Set-up to study Arterial Compliance (ROTSAC). Vascular reactivity measurements at 2 months of age showed enhanced voltage gated calcium channel (VGCC)-mediated contraction in the aorta and femoral artery segments of mice with an VSMC autophagy defect, which resulted in increased sensitivity to depolarization induced contractions. In addition, femoral artery segments showed a large increase in IP₃-mediated contraction at the age of 2 months while this was absent in the aorta. Surprisingly, deletion of Atg7 in VSMCs also affected the relaxing capacities of the aorta and the femoral artery. In aortic segments basal unstimulated nitric oxide (NO) release as well as stimulated NO release was enhanced, while femoral artery segments display increased VSMC sensitivity to exogenous NO.

Aortic segments of mice with a VSMC autophagy defect also displayed attenuated compliance and higher arterial stiffness, which was more evident at higher distention pressures. At the age of 3.5 months, passive aortic wall remodeling, rather than differences in VSMC tone, was responsible for these phenomena, since differences in compliance and stiffness were more pronounced when VSMCs were completely relaxed by the addition of exogenous NO. These observations are supported by histological data showing extracellular matrix remodeling. Short-term adaptations in the aorta, measured at 2 months, also included changes in the active modulation of arterial stiffness since depolarization induced contraction significantly increased vascular stiffness more in aortic segments of mice with an VSMC autophagy defect. As an increase of the focal adhesion protein vinculin was observed, we speculate that the enhanced active component is possibly due to an increase in focal adhesion sites.

Similar to VSMC autophagy, we also investigated the effect of an EC selective Atg7 deficiency on the aortic reactivity and biomechanical properties using a ROTSAC setup and a traditional isometric organ bath setup. Short-term as well as long-term consequences were evaluated by measurements at the age of 2 and 3 months and 1 year. Surprisingly, no differences were seen between the EC autophagy deficient mice and control mice at the age of 1 year. This was in contrast to younger mice where isometric tension measurements of EC-specific autophagy deficient aorta segments showed features of endothelial dysfunction such as decreased acetylcholine sensitivity. Moreover, isobaric measurements however, showed a decline in basal NO bioavailability, which was mostly pronounced at the age of 3 months. In addition, a higher stiffness was present in aortic segments of mice with an EC autophagy defect as compared to control mice in unstimulated conditions. Blocking of basal NO with the eNOS inhibitor L-NAME eliminated this difference indicating this effect of EC autophagy on arterial stiffness were solely dependent on effects on basal NO bioavailability. This was in contrast with stimulated conditions where aortic segments of mice with an EC autophagy defect showed increased stiffness, independent on the presence of L-NAME.

Overall we can conclude that autophagy in VSMCs and ECs plays a major role in normal vascular function since both the contractile and relaxing capacities of large elastic arteries as well as the smaller muscular arteries in mice are affected by a selective Atg7 knockout. Moreover, disruption of this homeostatic process results in an increased development of arterial stiffness as measured by an ex-vivo experimental setup.

​Stefaniya Velichkova​

• 25 September 2020
• Supervisors: Luc Pieters and Kenn Foubert

Abstract

Since centuries, medicinal plants have been providing human civilisation with remedies for health maintenance and disease control. Nowadays, natural sources are still a starting point for drug discovery investigations. They attract attention for their potential application as novel therapeutic agents in the treatment of current diseases with big social and economic impact (e.g. type 2 diabetes, cardiovascular diseases). Various mechanisms have been proposed to explain the causes of chronic conditions, and on biochemical level, protein glycation (formation of advanced glycation endproducts (AGEs)) correlates with many pathological complications. Similar to AGEs, autophagy has been associated with a plethora of different pathologies including heart diseases, cancer, neurodegeneration, infectious diseases, diabetes and autoimmune diseases. Therefore, AGEs and autophagy represent novel therapeutic targets in natural product research. The purpose of this PhD project was to isolate and identify several selected classes of natural products (polymethoxy­flavonoids (PMFs), biflavonoids, quinazoline alkaloids) from four different plant species: Citrus sinensis, Citrus depressa, Ginkgo biloba and Adhatoda vasica; and to evaluate their properties as AGEs inhibitors and autophagy modulators through a set of experimental procedures.

The phytochemical investigation was performed through various chromatographic techniques: open column, flash and semi-preparative liquid chromatography. The structure of the compounds was elucidated by 1D- and 2D- NMR spectroscopy and mass spectrometry. The preliminary testing for AGEs inhibitory properties of the obtained pure compounds was achieved by means of the bovine serum albumin (BSA) / glucose, fructosamine adducts formation and alfa-dicarbonyl compounds formation assays. Due to certain limitations of the standard approaches, a method was developed to evolve from non-selective colorimetric / fluorimetric techniques to a more reliable chromatography-based method. An HILIC UPLC/MS method was aimed to be developed and validated, and consequently, used to investigate the AGEs inhibiting properties of pure compounds and selected commercial standards. To evaluate the autophagy modulation by some isolated pure compounds and commercial standards, different assays were applied: LC3 detection and quantification by western blot analysis, and the Cyto-ID autophagy detection kit. As part of the analytical work, a method for quantifying vasicine – the main quinazoline alkaloid in Adhatoda vasica leaves, was developed and validated. Additionally, the established method was applied for quality control of commercially available herbal products containing Adhatoda powder or extract. Overall, advanced glycation and modulation of autophagy are leading causes for the progression and pathogenesis of many chronic diseases, therefore, the use of validated methods and proved techniques can contribute to the unambiguous discovery of new potent anti-AGEs and autophagy modulating agents.

​Lindsey Dugaucquier​

• 22 September 2020
• Supervisors: Gilles De Keulenaer and Vincent Segers

Abstract

In this thesis, we aimed to study ERBB4 signaling in non-myocytes, specifically in ECs and inflammatory cells, and challenge the current concept that the cardiac effects of NRG1 are mediated by paracrine activation of ERBB4/ERBB2 receptors on cardiomyocytes. We studied how NRG1/ERBB4 signaling in ECs or inflammatory cells could contribute to the beneficial effects of NRG1 during cardiac remodeling. The central hypothesis of this thesis is that NRG1/ERBB4 receptor signaling in non-myocytes contributes to the beneficial effects of NRG1 during cardiac remodeling.​Therefore, we first generated mice with myeloid-specific deletion of Erbb4 and tested the effects of myeloid-specific Erbb4 deletion in 2 models of cardiac remodeling—MI and pressure overload. We observed that myeloid-specific Erbb4 deletion accentuated the early increase of myocardial macrophage density in the viable myocardium after MI, but that subsequent ventricular dilation and dysfunction, CM hypertrophy, or interstitial myocardial fibrosis remained unaffected. Interestingly, myeloid-specific Erbb4 deletion reduced infarct scar. In the TAC model, pressure overload–induced myocardial inflammation remained absent, and LV remodeling progressed independently of myeloid ERBB4. Overall, this study shows a modulatory role of NRG1/ERBB4 signaling in myeloid cells during the early inflammatory phase of MI.​Secondly, we generated EC–specific Erbb4 KO mice to specifically eliminate endothelial autocrine ERBB4 signaling without affecting paracrine NRG1/ERBB4 signaling in the heart. First, we observed no basal cardiac phenotype in these mice up to 32 weeks. We next studied these mice following TAC, exposure to Ang II or MI in terms of cardiac performance, myocardial hypertrophy, myocardial fibrosis and capillary density. In general, no major differences between EC–specific Erbb4 KO mice and control littermates were observed. However, 8 weeks following TAC both myocardial hypertrophy and fibrosis were attenuated by EC–specific Erbb4 deletion, albeit these responses were normalized after 20 weeks. Similarly, 4 weeks after Ang II treatment myocardial fibrosis was less pronounced compared to control littermates. These observations were supported by RNA-sequencing experiments on cultured ECs showing that NRG1 controls the expression of various hypertrophic and fibrotic pathways. Overall, these data contribute to our understanding of myocardial cell-cell communication during cardiac remodeling, and to the role of autocrine NRG1/ERBB4 signaling specifically. NRG1 has direct effects on human ECs in vitro, and endothelial autocrine NRG1/ERBB4 signaling plays a role in the modulation of hypertrophic and fibrotic responses during early cardiac remodeling.​

​Joachim Mertens​

• 7 September 2020
• Supervisors: Dirk Hendriks and Anne-Marie Lambeir

Abstract

Currently, only 20% of acute ischemic stroke (AIS) patients can be successfully treated. New treatment strategies are therefore urgently needed. The antifibrinolytic enzyme carboxypeptidase U (CPU) is an appealing target to improve stroke therapy. CPU circulates in plasma as an inactive zymogen, proCPU, that can be activated by thrombin, the thrombin-thrombomodulin complex, or plasmin. We aimed to assess the CPU system as a potential target to improve ischemic stroke treatment.

Activity-based, immunologic and functional assays were optimized and further characterize  for application in observational and clinical studies as well as in preclinical animal models of ischemic stroke.  Special attention was paid to the pre-analytical phase which is key to accurate CPU measurement. Especially the impact of in vitro hemolysis on the measurement of the CPU system was assessed. Assay-specific cut-off values reflecting maximal allowable oxyhemoglobin levels were determined as significant inhibition of CPU activity was observed.

In the preclinical part, a CPU inhibition strategy for the treatment of AIS was tested in a rat model of transient middle cerebral artery occlusion. Clear activation of the CPU system was observed in both saline- and rtPA-treated animals. Administration of the CPU inhibitor AZD9884 resulted in complete inhibition and reduced fibrinogen levels in the brain, which is a parameter of microvascular thrombosis (MT).

Two observational studies were performed on AIS patients. CPU activity and CPU+CPUi antigen increased in patients upon arrival in the hospital compared to controls. In AIS patients receiving either rtPA or rtPA with endovascular thrombectomy (EVT), the CPU system was clearly activated. Maximum CPU and CPU+CPUi levels tended to be higher in patients undergoing additional EVT compared to those that received rtPA alone. Some findings point towards a potential role of MT in these patient populations but, large inter-individual variation was observed in CPU and CPU+CPUi kinetics.

ProCPU levels were also quantified in the cerebrospinal fluid (CSF) of stroke patients that did not receive thrombolytic treatment and were increased compared to controls Patients with progressive stroke or poor outcome had higher proCPU CSF levels. The proCPU levels were also associated with blood-brain barrier dysfunction, which was found to be the likely cause of the increase.

In the last part of this project, we performed the pharmacodynamic assessment of a novel CPU inhibitor in a first-in-man trial. The combined use of the CPU activity assay and the two in vitro clot lysis assays proved to be valuable to confirm the target engagement of the inhibitor.

​Gwendolyn Vliegen​

• 22 June 2020
• Supervisors: I. De Meester and A.M. Lambeir

Abstract

During this PhD, several proline-specific enzymes of the dipeptidyl peptidase (DPP)-family were studied, being DPP4, fibroblast activation protein α (FAP), DPP8 and DPP9. These enzymes process substrates preferentially after a proline when it is in the penultimate position at the N-terminus. The goals of this PhD were twofold. On the one hand, it was aimed to produce human DPP8 and DPP9 recombinantly, to have sufficient stock for further research. On the other hand, some functional aspects of these enzymes were examined. To this end, DPP4 and FAP were examined in blood samples from patients with septic shock (defined by sepsis-2) on days 1, 3, 5 and 7 to see if they can serve as diagnostic or prognostic biomarkers and whether there are associations with sepsis-related parameters. In addition, an anti-inflammatory effect of the DPP8/9 inhibitor 1G244 on human and murine macrophages has been reported. Furthermore, this inhibitor induced cell death in the J774 cell line. However, it is not known how this inhibitor effects these effects. Attempts were made to decipher these underlying mechanisms.

DPP8 and DPP9 were successfully expressed and purified from insect cells. The identity of the enzymes was confirmed by PCR, western blot and enzymatic activity measurements. In addition, their characteristics were evaluated by determining their pH profile, the K_m- and k_cat-values of Gly-Pro-pNA and Gly-Pro-AMC and the IC₅₀ values for 1G244 and Val-boroPro. These experiments yielded results within the expectations.

Both DPP4 and FAP were significantly lower in the septic shock patients compared to an ICU control group for all days. ROC curves gave AUC values for FAP of 0.94 (CI: 0.89-0.99) and for DPP4 of 0.86 (CI: 0.77-0.95). A limited association was seen between DPP4 and survival at day 90. Higher DPP4-activity was associated with an increase in survival. These results demonstrate that FAP and DPP4 should be further investigated as possible diagnostic (and for DPP4 also as prognostic) biomarkers.

To unravel the mechanisms behind the effects of the DPP8/9 inhibitor, the potential involvement of kinases was investigated using the PamGene kinomics platform. In experiments with primary murine macrophages, protein kinase C (PKC) and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) were identified as being less active after inhibition. In the J774 mouse macrophage cell line, CaMKII and src family kinases (SFKs) showed less activity in the inhibited samples. These results need to be further validated with specific methods.

​Eduardo Figueiredo Pires Ruivo​

• 30 March 2020
• Supervisors: Koen Augustyns and Pieter Van der Veken

Abstract

Pretargeted in vivo imaging using the bioorthogonal IEDDA reaction between TCO and Tz holds promise to allow imaging of mAbs with shorter living isotopes, to reduce radiation burden to the patient. This strategy can allow the development of a single radiolabeled probe that can be used with many mAbs, facilitating its use in R&D and clinical practice. For application of the TCO-Tz ligation, the radiolabeled probe should be stable and have fast reaction kinetics allowing reaction with the tag within minutes at low μM concentration. The two-step in vivo labeling approach also facilitates the use of shorter living radioisotopes like 18F (T1/2 = 110 min) that would otherwise not be compatible with the long circulation times of high molecular weight molecules (mAb).

Considerable research has been devoted to the development of mAb-TCO conjugates, and their application for pretargeted tumor imaging has been reported using diverse 18F-labelled tetrazines. However, it has been shown that TCO has the tendency to isomerize to its isomer, cis-cyclooctene (CCO), that is several orders of magnitude less reactive with tetrazine after prolonged exposure to physiological conditions.

Therefore, in this thesis we explored the inverse approach where a mAb is modified with tetrazine instead of TCO. A range of tetrazines was developed and evaluated regarding their stability and reactivity, and the most promising tetrazine was conjugated to mAb trastuzumab. In a proof of concept study we demonstrated the usefulness of the tetrazine-mAb conjugate for bioorthogonal pretargeted imaging using a live-cell fluorescent imaging experiment, using a TCO-fluorescent probe. Furthermore, we envisaged the translation of this strategy for in vivo pretargeted PET imaging applications. For this, a novel Al18F-NOTA labeled TCO radioligand was developed as a potential counterpart for IEDDA reaction with a tetrazine-tagged antibody. The radiotracer showed improved in vivo metabolic stability (51.9 ± 5.16% after 1 h) compared to previous reported 18F-labeled TCOs, allowing a clear visualization of tumor tissue in a small-animal pretargeted PET imaging experiment.

In an effort to increased absolute tumor uptake, we further developed next-generation TCO derivatives. The novel 18F-labeled dTCO-amide probe showed an extremely fast kinetic rate (10,553 M-1s-1 in 50:50 methanol:water), good stability in saline and plasma up to 4h at 37°C and a favorable biodistribution in healthy mice. Pretargeted µPET imaging experiments in mice bearing LS174T colorectal tumors, previously treated with a tetrazine-modified anti-TAG-72 monoclonal antibody (CC49), showed clear visualisation of tumor tissue with a significant higher uptake when compared to the control.

​Fuchao Xu​

• 16 December 2019
• Supervisors: Adrian Covaci and Hugo Neels

Abstract

Used as alternative flame retardants (FRs) for the banned polybrominated diphenyl ethers (PBDEs), organophosphate flame retardants (PFRs) have shown negative effects to human health and eco-systems. Widely used in commercial products, such as electronic and electrical products, textiles, paints, and plastics to reduce the fire risk, PFRs and related FRs can be released into the ambient environment throughout the lifetime of the product. Humans are possibly exposed to PFRs via several pathways, including inhalation, dermal absorption, diet, and dust ingestion. This thesis aims to investigate different human exposure pathways to PFRs and other FRs and to discuss the appropriate sampling strategies for exposure assessment. The thesis also explores the potential link between the internal and external exposure of PFRs via human biomonitoring data (urine, serum and hair) and exposure matrices (air, dust, handwipes and food). As a result, the thesis provides a comprehensive picture of the human exposure to PFRs.

As first, methods for analysing external exposure matrices and human biological samples were developed and validated. Besides optimizing available analytical methods for air, dust and handwipes, a new method for determining FRs in various types of foods was developed. The method allowed the simultaneous analysis of PFRs, PBDEs, and emerging halogenated FRs (EHFRs), with a cost-efficient, fast and easy procedure, while significantly improving the analytical sensitivity compared to existing methods. A biomonitoring method to determine PFR metabolites in urine and serum has also been developed to determine di-alkyl/aryl and hydroxyl metabolites of PFRs. These methods have been applied to the assessment of human exposure to PFRs and related compounds in latter studies.

Most studies claim that dust ingestion is the major pathway of human exposure to FRs. This thesis thoroughly discussed the key factors in dust sampling that might influence the accuracy of FR exposure assessment. Results showed that the FR profile in dust could be significantly influenced by local/national regulation, geographical location, microenvironment, sampling season, and even particle size. These factors demonstrate the necessity of an appropriate dust sampling strategy for human exposure assessment.

Human exposure to PFRs in Chinese e-waste recycling areas has been discussed as the worst-case exposure scenario. PFRs in house dust collected from these areas had higher levels than dust from other countries. Although PBDE concentrations in dust from e-waste recycling area were lower than for PFRs, they were still significantly higher than in normal indoor dust. Low levels and detection frequencies of PFRs were found in local free-range eggs, which was negligible comparing to PBDE levels in these eggs. These results suggested that PFRs behave differently from PBDEs and do not bioaccumulate in eggs, even in a highly contaminated environment. However, considering the larger intake of eggs compared to dust, the dietary intake of PFRs via egg consumption for local people was still substantial. Health and environmental concerns regarding e-waste recycling business is in these situations of concern.

This thesis also presents a comprehensive human exposure survey to PFRs, which was conducted in Oslo, Norway. Sixty-one participants were recruited in this campaign to provide floor dust, surface dust, personal air, stationary air, handwipe, duplicate food, urine, serum, hair, and detailed questionnaires for exposure assessment. PFR profiles in surface dust were similar to floor dust, but had significantly higher levels. However, PFR profiles in personal air were completely different from those in stationary air. PFR levels in handwipes could not be associated with levels in air and dust. Results indicated that the selection of sampling strategy might significantly affect the accuracy of exposure assessment. Ethylhexyl diphenyl phosphate (EHDPHP) was the major PFR in Norwegian duplicate diets. This exposure pathway accounted for nearly 75% of total PFR exposure. Inhalation and dust ingestion were the following exposure pathways for the participants. Results showed that each PFR has its individual major exposure pathway and that dust ingestion is not the major pathway for all PFRs. PFR metabolites were measured in urine, serum and hair of the participants. A higher number of metabolites were detected in urine. Some metabolites were associated with external exposure matrices, among which floor dust is shown to be a better matrix to predict internal exposure of PFRs. In summary, this exposure survey shows how sampling strategies affect the human exposure assessment. A comprehensive picture of human exposure to PFRs via different pathways was also profiled for the Norwegian cohort.

​Eveline Torfs​

• 12 December 2019
• Supervisors: Paul Cos and Louis Maes

Abstract

Despite advances in modern medicine and healthcare, tuberculosis (TB) still poses a major threat worldwide. With approximately 10.0 million new cases and the attributable cause of death of an estimated 1.3 million people, TB is part of the top ten leading causes of death worldwide and has been declared the world’s deadliest infectious disease for several years. In addition, the ever-increasing emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains places an enormous strain on current anti-TB chemotherapy. Accordingly, the World Health Organization (WHO) has set up several strategies to improve TB management, in which the discovery and development of novel anti-TB drugs is key. Although the pipeline for novel anti-TB drugs is steadily filling up with novel candidates, the current arsenal is still limted and likely not sufficient due to high attrition rates. Therefore, there is still an urgent need for new anti-TB compounds.

To support and broaden the early discovery phase with knowledge on novel compound classes, the present thesis primarily reports on the in vitro biological evaluation of a series of triazenes, quinones and thiazolidinedione-hydroxamates. The studied compounds were synthesized by chemist of the University of Antwerp (Belgium), University of Ghent (Belgium), University of Ljubljana (Slovenia) and Palacký University (Czech Republik). To evaluate the small compound libraries for antimycobacterial hits and provide a primary evaluation of their in vitro anti-TB properties at the Laboratory for Microbiology, Parasitology and Hygiene (LMPH), an anti-TB drug screening platform was set up. Using this cascade of state-of-the-art assays, several triazene and quinone hits were described in terms of extra- and intracellular antimycobacterial activity, acute cytotoxicity, early genotoxicity and metabolic stability. In addition to the phenotypically identified hits, a small library of target-based synthesized thiazolidinedione-hydroxamates was evaluated. In vitro anti-TB properties of the discovered hits were described and compared to their activity results in an enzymatic assay, which was performed by the Palacký University (Czech Republik).

Secondly, the present thesis reports on the improvement of compound assessment methodologies and development of new research tools as these are equally important as the discovery and development of novel anti-TB drugs. In an effort to improve the standard biosafety level two (BSL2) TB infection model, calcium-alginate encapsulation of Mtb H37Ra was envisaged. Although micro-bead encapsulation was successful, the in vivo course of infection was not enhanced during both the acute and chronic phase of infection. Moreover, the generation of inflammation during infection was not influenced as well. Though, experiments confirmed the disputed statement that Mtb H37Ra is attenuated in mice, rather than avirulent. Further, a next-generation bioluminescent Mtb H37Ra reporter model was developed to facilitate future in vitro and in vivo anti-TB drug screening at the LMPH. Upon optimization of the bioluminescent reaction, the selected reporter was determined to be a sensitive and robust alternative for the prokaryotic luciferase reporter strain currently used in the LMPH’s in vitro anti-TB screening platform. Finally, proof-of-concept studies indicated the presence of a bioluminescent signal in living BALB/c mice, illustrating the novel bioluminescent reporter strain might be used to develop a bioluminescent imaging model (BLI). This would in turn facilitate in vivo anti-TB drug screening at the LMPH.

​Andrés Rivera Mondragón​

• 21 November 2019
• Supervisors: Kenn Foubert and Luc Pieters

Abstract

Herbal medicines are described as any form of plant or plant product used in the maintenance of health as well as in the prevention, improvement, diagnosis or treatment of diseases. Due to the growing relevance of these products, we aimed to contribute to the development of scientifically based and quality controlled herbal substances. Some species of the genus Cecropia (Urticaceae) and Crescentia cujete (Bignonaceae) collected in Panama were selected as case study. Although food supplement-derived products from these plant species are commercially available, a comprehensive description of their phytochemical composition and appropriate analytical methods for guaranteeing their quality and safety are still lacking.

A detailed phytochemical investigation on 4 Cecropia species led to the full identification or tentative characterization of 47 compounds, including 2 phenolic acids, 33 flavonoids, 3 flavonolignans and 9 triterpenoid saponins. Among these, two new antiplasmodial flavonolignans were isolated from C. obtusifolia and identified as ent-mururin A and ent-vaccinin A.

The conditions for the extraction of chlorogenic acid, total flavonoids and flavonolignans from leaves of Cecropia species were optimized by using a design of experiment (DOE). A HPLC-DAD method for the quantification of these chemical markers was validated according to the ICH guidelines. The multivariate data analysis of the Cecropia species under investigation revealed the implications for phytochemical analysis in further taxonomic studies.

The gastrointestinal and colonic biotransformation of the crude extract of the leaves of C. obtusifolia, was investigated under in vitro conditions, and the processing and interpretation of results were facilitated by using an automated machine-learning model. This investigation revealed that flavone C-glycosides and flavonolignans were stable throughout their passage in the simulated gastrointestinal tract including the colon phase. On the other hand, the colon bacteria extensively metabolized chlorogenic acid, flavonol and triterpenoid O-glycosides.

An untargeted metabolomics approach combining UPLC-MS/MS-based molecular networking with conventional isolation and NMR methods was carried out for the phytochemical profiling of the fruit pulp of Crescentia cujete. Sixty-six products, including 9 n-alkyl glycosides, 23 phenolic acid derivatives (such as cinnamoyl and benzoyl derivatives), 15 flavonoids, 4 phenylethanoid derivatives and 15 iridoid glycosides were fully or tentatively identified. Among these, 8-epi-eranthemoside, crescentiol A and crescentiol B were reported as three new iridoid glucosides.

In view of future work, the implementation of the validated analytical methods for the quantification of markers in Cecropia spp. and Crescentia cujete will be a great help for chemical standardization and authentication of their commercial derived products.

​Adrienn Baán​

• 19 November 2019
• Supervisors: Koen Augustyns and Filip Kiekens

Abstract

Formulation development of two poorly water-soluble compounds, UAMC 00523 (developed by the Medicinal Chemistry Laboratory of the University of Antwerp) and mangiferin, has been performed using the following techniques: cyclodextrin complexation, PLGA nanoparticle formulations, amorphous solid dispersions and dry amorphization with mesoporous silica. UAMC 00523 is a non-nucleoside transcriptase derivate with potential activity against Human African Trypanosomiasis. Mangiferin is a natural compound with a broad range of potential health-improving activities including anti-inflammatory, anticancer and antidiabetic effects.

UAMC 00523 was first formulated with different β-cyclodextrin derivatives, with a maximum solubility enhancement factor of 80. This formulation was tested in vivo (performed by the Laboratory of Microbiology, Hygiene and Parasitology of the University of Antwerp) with no detectable effect. Subsequently, PLGA nanoparticle formulations were prepared with different PLGA’s and different preparation conditions. Formulations were tested in a 50 mg/kg concentration in vivo after oral administration with no detectable effect. Next, a metabolic stability study was performed, confirming the lack of in vivo activity due to the extensive metabolic clearance of the compound.

Mangiferin was formulated by spray-drying of PLGA nanoparticles with an entrapment efficiency above 70% but with a low yield (less than 40%) due to the adhesion of particles on the drying chamber and the cyclone. Considering the low solubility of mangiferin in a common solvent with PLGA and the technical limitations, no further development was performed with this technique. Amorphous solid dispersions were also prepared using different HPMC grades as carrier. Partial amorphization of the compound was achieved with increased solubility. The poor solubility of mangiferin limited the further formulation development with solvent-based techniques, therefore a solvent-free method, the dry amorphization with mesoporous silica using a planetary mono mill was further applied. Binary and ternary systems have been prepared using HPMC and Soluplus^® as third component in the formulations. By adjusting the milling settings, amorphous samples were prepared in a significantly shorter time than already reported with other carriers. The stability of the samples was evaluated at accelerated stability conditions in open and closed containers. Increased solubility and amorphous stability of 6 months were achieved for the high energy milled sample. The addition of polymer enhanced the amorphization rate of the samples and improved the stability in open and closed conditions.

​Carlos Moreno Cinos​

• 9 October 2019
• Supervisors: Koen Augustyns and Hans De Winter

Abstract

The serine protease family is the most widely studied group of proteins in biology. These enzymes are of special interest due to their notoriously diverse and well-characterized role in physiological and pathological processes. Under normal circumstances, complex and accurate systems regulate the proteolytic activity of serine proteases. Dysregulation of their function results in pathological disorders.

Diaryl esters of a-amino phosphonates are a group of irreversible inhibitors of serine proteases. Their potent inhibition of serine proteases added to their absolute lack of activity against cysteine or threonine proteases confer them advantage over alternative serine protease inhibitors lacking this selectivity such as chloromethyl ketones, ketoesters or ketoamides. These attributes open for them a broad range of applications in the Medicinal Chemistry field, from the design of small molecules for target inhibition, to the synthesis of chemical tools such as ABPs. The focus of this PhD research lies on these two options.

Design, synthesis and characterization of small inhibitors was undertaken for the antimicrobial drug discovery field. Increased Gram-negative bacteria resistance to antibiotics is becoming a global problem and new classes of antibiotics with novel mechanisms of action are required. The caseinolytic protease subunit P (ClpP) is a serine protease conserved among bacteria that is considered as an interesting drug target. ClpP function is involved in protein turnover and homeostasis, stress-response and virulence among other processes. The focus of this study was to identify new inhibitors of Escherichia coli ClpP and to understand their mode of action. A focused library of serine protease inhibitors based on diaryl phosphonate warheads was tested for ClpP inhibition and a chemical exploration around the hit compounds was conducted. Altogether 14 new potent inhibitors of E. coli ClpP were identified. Compounds 4.85 and 4.92 emerged as most interesting compounds from this study due to their potency and, respectively, to its moderate but consistent antibacterial properties as well as the favorable cytotoxicity profile.

Utilization of a-amino diaryl phosphonates as chemical tools for the target identification was the second goal of this PhD research, focusing on the design and synthesis of activity-based probes for serine proteases involved in the patho-physiological mechanism of visceral hypersensitivity.

Establishment of an efficient synthetic route with the potential of combining a variety of reporter tags in a last step was achieved. A small library of 12 ABPs with an acceptable inhibitory activity for different subclasses of serine proteases was synthesized.

​Matthias Cuykx​

• 2 July 2019
• Supervisors: Adrian Covaci, Tamara Vanhaecke and Kris Laukens

Abstract

The liver is a multifunctional organ that has a central role in human physiology. The importance of the liver and its metabolic capacity make it vulnerable to stress, and liver toxicity is often observed as a side effect of xenobiotic exposure.

Effective strategies in risk assessment and toxicology are shifting towards mechanistic interpretations and in vitro techniques to offer an alternative to descriptive in vivo toxicology. Metabolomics, the holistic study of small endogenous molecules, is the closest representation of the actual phenotype of the organism and includes downstream effects of the genes and proteins as well as their response to stimuli outside the cell.

This thesis is the result of first experiments within the Toxicological Centre at the University of Antwerp in close collaboration with the research groups In Vitro Toxicology and Dermato-Cosmetology at the Vrije Universiteit Brussels and Adrem/Biomina at the University of Antwerp. The scope of the thesis is towards high quality-based acquisition of samples for metabolomics purposes in order to increase both metabolic coverage and data confidence.

The development of a reliable acquisition platform based on liquid chromatography and accurate mass spectrometry included an initial screening of the separation performance for different stationary and mobile phases using a known mix of standards to accurately estimate retention characteristics. Additional optimisation performed with metabolite extracts further improved intra-class separation. In comparison with a generic platform, the optimised, complementary platforms for polar and non-polar extracts practically doubled metabolic coverage and reduced intra-batch variability.

Improvements concerning precision and stability for sample preparation procedures included cell culture in chamber slides, the introduction of buffers, anti-oxidants and chelating agents in the liquid/liquid extraction and adjustments in reconstitution procedures.

Metabolic alterations observed during exposure to sodium valproate describe the hallmark metabolic profile of NAFLD, including the initiating carnitine depletion and the downstream diacyl- and triacylglycerol accumulation. Metabolic alterations during a low-dose exposure to bosentan include a carnitine upregulation, polyamine alterations and activation through phosphorylation. High-dose exposure resulted in a moderate triacylglycerol accumulation through a carnitine depletion, potentially related to mitotoxicity.

Next to common metabolic alterations upon exposure to both reference toxicants, differences between their metabolic fingerprints provided the opportunity to describe the different modes of action. The obtained mechanistic information can provide valuable evidence to construct and consolidate adverse outcome pathways.

​Annelies Verlaet​

• 24 May 2019
• Supervisors: Nina Hermans and Huub Savelkoul

​Kelly Schats​

• 24 April 2019
• Supervisors: Ingrid De Meester and Marc Kockx

​Géraldine Broeckx​

• 21 March 2019
• Supervisors: Filip Kiekens and Sarah Lebeer

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At present time, every hour 80 people die due to the consequences of antimicrobial resistance. A ‘post-antibiotic’ era threatens the global population, as warned by the World Health Organization (WHO). This has prompted the scientific world to search for alternative strategies to manage and prevent diseases. With the increasing awareness of the role that our human microbiome plays in health and disease, one of the strategies explored is the application of probiotics as a preventive and/or therapeutic agent for infectious diseases. Probiotics are defined as ‘live microorganisms that, when administered in adequate amounts, confer a health benefit to the host’ (Hill et al., 2014).

The road to develop successful pharmabiotics, i.e. probiotics in pharmaceutical formulations, is very challenging. One of the key hurdles for formulators, is to keep the bacteria alive and active during a longer period of time (preferably up to several years). To enhance shelf-life stability, bacteria are dried, which decreases the available free water content, and this slows down the bacterial metabolism. Freeze drying is the commonly used method in the pharmaceutical industry to dry biopharmaceuticals, such as probiotics. However, in recent years, spray drying has been brought forward as a promising alternative to freeze drying. It is a cheap, fast, and continuous process, where the end products consist of a particulate powder. However, due to the high temperatures used during spray drying and the lack of sufficient long-term storage data (to date), the pharmaceutical industry is skeptical to implement this drying technology to process probiotics.

Therefore, the goal of this research is to investigate if spray drying can be used to produce viable probiotics, which can be stored during a sufficient amount of time. Three protection strategies were investigated, namely, addition of protective agents, controlling the process parameters and pretreating the bacterial cells prior to spray drying. Noteworthy, promising results regarding long-term stability (for up to more than two years) were obtained with the addition of small disaccharides (like lactose and trehalose) and the presence of phosphates in the drying medium, even when storing at room temperature.   Although, the road to develop successful pharmabiotics is still full of hurdles to overcome and problems to be solved, it can be concluded that an exciting future lies ahead for us and our friendly microbes.