​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.

​Arthur Leloup​

• 27 May 2019
  
• Supervisors: Gilles De Keulenaer and Paul Fransen

Abstract

Arteriële stijfheid is een toestand waarbij de grote arteriën een verminderde capaciteit hebben om de pulsatiele stroom – die gegenereerd wordt door het linker ventrikel van het hart – te dempen. Het is een belangrijke indicator en initiator van cardiovasculaire, neurovasculaire en renovasculaire aandoeningen. Een significant deel van de arteriële wand bestaat uit gladde spiercellen waarvan de contractiele tonus onder andere gereguleerd wordt door circulerende en lokale factoren. De endotheelcellen aan de binnenkant van de elastische arteriën produceren grote hoeveelheden van het vasodilaterende stikstof monoxide en een verminderde beschikbaarheid hiervan wordt gelinkt aan verhoogde arteriële stijfheid. Hoewel dit suggereert dat de contractiele tonus van de gladde spiercellen een rol speelt in de fysiologische en pathofysiologische regulatie van arteriële stijfheid is de precieze rol van deze ‘actieve’ vaatwandcomponenten nog onduidelijk.

Het primair doel van dit project was om een beter begrip te krijgen van de rol van gladde spiercel contractie en relaxatie als regulatoren van arteriële stijfheid in gezonde condities. Daarnaast werd ook onderzocht of dysfunctie van deze actieve vaatwandcomponenten een rol speelt in de ontwikkeling van acute of chronische vasculaire pathologieën in diermodellen. Onze resultaten bevestigen dat de contractiele tonus van vasculaire gladde spiercellen inderdaad het biomechanische gedrag van gezonde bloedvaten kunnen moduleren, grotendeels bepaald door de basale productie van stikstof monoxide in de endotheelcellen. Bovendien toonden we aan dat arteriële verstijving die optreedt na angiotensine-II infusie grotendeels het gevolg is van veranderingen in de contractiele tonus van gladde spiercellen in de aorta. Dit bleek niet het geval in een genetisch muismodel met chronische endotheelfysfunctie, wat suggereert dat de ‘actieve’ componenten van de elastische bloedvatwand vooral een belangrijke rol spelen in de vroege stadia van arteriële dysfunctie. Het verbeterde begrip over de bijdrage van de gladde spiercellen en de endotheelcellen tot het bewaren van  een centraal hemodynamisch evenwicht is een belangrijke stap naar de ontwikkeling van nieuwe therapeutische strategieën die zich specifiek richten op de stijfheid van de centrale arteriën.

​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

Abstract​

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.