Pharmaceutical, Biomedical and Veterinary Sciences

Defences 2020

Attented a phd of find the archive of concluded doctoral research

Human exposure to bisphenol A and its alternatives: sources, levels, patterns and determinants - Celine Gys (30/11/2020)

​Celine Gys

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


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.

Human biomonitoring of phosphate flame retardants and alternative plasticizers: from method development to exposure assessment - Michiel Bastiaensen (24/11/2020)

​Michiel Bastiaensen

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


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.

Formulation and process research to safeguard Lacticaseibacillus rhamnosus GG during tablet manufacturing - Eline Byl (27/10/2020)

​Eline Byl

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


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.

Role of autophagy in the cardiovascular system and implications for the development of age-related vascular pathologies - Dorien De Munck (01/10/2020)

​Dorien De Munck

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


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.

Natural products as potential inhibitors of Advanced Glycation Endproducts (AGEs) and modulators of autophagy - Stefaniya Velichkova (25/09/2020)

​Stefaniya Velichkova

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


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.

Role of non-myocyte NRG1/ERBB4 signaling in cardiac remodeling - Lindsey Dugaucquier (22/09/2020)

​Lindsey Dugaucquier

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


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.​

The carboxypeptidase U system in acute ischemic stroke: translation from bench to bedside - Joachim Mertens (07/09/2020)

​Joachim Mertens

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


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.

Proline-specific enzymes in infection and inflammation - Gwendolyn Vliegen (22/06/2020)

​Gwendolyn Vliegen

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


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 Km- and kcat-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.

Development of novel chemical tools for bioorthogonal pretargeted PET imaging - Eduardo Figueiredo Pires Ruivo (30/03/2020)

​Eduardo Figueiredo Pires Ruivo

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


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.