Farmaceutische, Biomedische en Dier­geneeskundige Wetenschappen

Doctoraten 2022

Departement Farmaceutische Wetenschappen

Public defence Anna Twarogowska 30/06/2022 - Transformation of Belgian endive by-products into valuable products by applying a zero-waste biorefinery concept - Department Pharmaceutical Sciences

Public defence Anna Twarogowska 30/06/2022 - Transformation of Belgian endive by-products into valuable products by applying a zero-waste biorefinery concept - Department Pharmaceutical Sciences

Promotors: prof. dr. Luc Pieters - dr. ir. Bart Van Droogenbroeck

Public defence: Campus Drie Eiken - Q002 (Promotiezaal)

Abstract:

In recent years, the agrifood sector faced many global challenges, where climate issues are emerging, food insecurity and economic instability are increasing due to the ongoing pandemic (COVID-19), and geopolitical instability, including military conflicts. Furthermore, globalization of the food market caused the enormous growth of the food supply chain, where industrialized food processing generates significant amounts of residues and by-products. Thus, reducing the agrifood processing residues and by-products by valorizing them into value-added products are an emerging topic. The concepts such as circular economy, bioeconomy, biorefinery, and zero-waste are encouraged to improve the usage of resources and increase sustainability throughout the food system.
Belgian endive (Cichorium intybus var. foliosum) is an economically important vegetable in Europe, especially in Belgium, France, The Netherlands, Italy, and Germany. In 2020 in the EU, approximately 0.63 Mt of leafy crops (chicons) were produced, generating forced roots as by-products. Forced roots are a very interesting feedstock for the biorefinery concept thanks to their year-round availability and attractive chemical composition, being rich in sugars, dietary fibres (DF), and bioactive compounds such as phenolic compounds (PC) and sesquiterpenes lactones (SLs).
This PhD aimed to develop an innovative biorefinery processing method to transform underutilized forced roots into value-added products, which could be further used in multiple applications ranging from food and beverages to cosmetics, pharmaceuticals, and biomaterials.
Using the cost-efficient biorefinery process with water as solvent created zero-waste production process where two valuable fractions (solid and liquid) were obtained. A solid fraction was transformed into dietary fibre concentrate (DFC), a functional ingredient with low sugars, high dietary fibre content, and excellent functional properties such as water and oil holding capacities and swelling capacity, which can be used in various products. Incorporating the Belgian endive DFC into plant-based burger formulations resulted in lower caloric value and sugar content, higher dietary fibre content and improved baking properties.
The SL-rich aqueous fraction that is a by-product of the DFC production (liquid) was stabilized by spray-drying and used to extract and isolate sesquiterpene lactones. The simple, easy to upscale fractionation process by using EtOAc:H2O used in this work could be an alternative to expensive, labor-intensive, commonly used methods like flash chromatography and TLC. The spray-dried aqueous fraction showed potential to use as a promising starting material for isolation of SLs, which can be used to produce value-added applications, such as cosmetics and food, and novel drugs.

Public Defence Sofie De Moudt 09/06/2022 - The ageing aorta: pathophysiological investigation of aortic stiffness and its temporal relationship to peripheral hypertension and cardiac disease - Department Pharmaceutical Sciences

Public Defence Sofie De Moudt 09/06/2022 - The ageing aorta: pathophysiological investigation of aortic stiffness and its temporal relationship to peripheral hypertension and cardiac disease - Department Pharmaceutical Sciences

Promotors: dr. Paul Fransen - prof. dr. Wim Martinet

LIVE DEFENCE: Aula Q002 (Promotiezaal), building Q, Campus Drie Eiken

Abstract:

Arterial stiffness is defined as the impaired capacity of large arteries to cushion the pulsatile blood flow which is generated by the heart, and is recognized as an important and independent predictor of cardiovascular complications. In this doctoral thesis, we aimed to investigate the mechanisms of aortic stiffness in four well-known small animal models of cardiovascular ageing and disease, and we assessed the temporal relationship of aortic stiffness and associated cardiovascular disease parameters. We demonstrated that aortic stiffening precedes peripheral hypertension in ageing C57Bl/6 mice, chronic N-Ω-Nitro-L-arginine methyl ester (L-NAME) treated mice, and angiotensin II-treated mice. In endothelial nitric oxide synthase (eNOS) knockout mice, age-dependent arterial disease progression was attenuated versus C57Bl/6 controls. By means of in-depth biomechanical and physiological testing of isolated aortic rings, we can support the following 8 disease mechanisms of aortic stiffening as therapeutic target in arterial ageing: (1) contraction-independent aortic stiffening, (2) endothelial dysfunction, (3) heightened α1-adrenoreceptor mediated vascular smooth muscle cell (VSMC) contractility, (4) aberrant voltage-gated calcium channel (VGCC) signalling, (5) altered store operated calcium entry-mediated signalling, (6) VSMC intracellular calcium handling, (7) focal adhesions in mechanotransduction, and (8) VSMC phenotypic modulation. Therefore, we believe that the phenotypic evidence in support of these disease mechanisms will contribute to the development of an arterial stiffness-targeting treatment for arterial ageing and uncontrolled hypertension. Furthermore, from our comprehensive ex vivo biomechanical studies, we learned that the effect of vasoactive signalling pathways on aortic stiffening is a complex sum of VSMC contraction, VSMC adhesion, and extracellular matrix components, underlining the importance of studying aortic function using multiple methods. This work thus also contributes to the knowledge on how to study aorta biomechanical behaviour in an experimental ex vivo setting.

Public defence Laura Peeters 17/06/2022 - An integrated strategy to characterize active constituents and their metabolites in Herniaria hirsuta and Nauclea pobeguinii

Public defence Laura Peeters 17/06/2022 - An integrated strategy to characterize active constituents and their metabolites in Herniaria hirsuta and Nauclea pobeguinii

Promotors: Prof. Luc Pieters - Prof. Nina Hermans

Aula Q.002

Abstract:

During the past decades, researchers have shifted their focus towards natural products as a source of inspiration for potential new active compounds against diseases for which adequate treatment or prevention is lacking. Amongst them are urolithiasis and malaria. However, the fact that many natural products act as prodrugs which are biotransformed and activated after oral administration is usually overlooked when using classical approaches. Therefore, an integrated strategy to characterize new active compounds is developed. Firstly, comprehensive extraction followed by phytochemical profile elucidation is conducted. Then, gastrointestinal and hepatic biotransformation is simulated in vitro, avoiding extensive in vivo studies. Samples are taken at several timepoints during these biotransformation processes and analyzed using UHPLC-UV-HRMS for metabolic profiling. The longitudinal multiclass data are subjected to an automated data analysis workflow, allowing unbiased, rapid scoring of large amounts of data. Ultimately, the metabolites are tested in in vitro activity assays to assess antilithiatic and antiplasmodial activity of Herniaria hirsuta and Nauclea pobeguinii, respectively.
Comprehensive extraction of H. hirsuta resulted in the tentative identification of 63 compounds, mainly flavonoids and saponins, including 15 saponins that have not been reported before in H. hirsuta. In vitro gastrointestinal biotransformation followed by metabolomics profiling revealed a decrease in relative abundance over time for the majority of all identified compounds, especially during the colon phase. Additionally, the relative abundance of saponin aglycones increased, with medicagenic acid as the most abundant one. The antilithiatic effect of H. hirsuta may be attributed at least in part to metabolites of medicagenic acid, indicating that saponins with this aglycon may act as prodrugs. A dual effect was observed: a tentative effect on formation of crystals by inhibiting aggregation, together with significant inhibition of crystal binding to MDCK I cells.
Phytochemical profiling of N. pobeguinii led to the tentative identification of 54 compounds, mainly alkaloids and saponins, of which 32 are reported here for the first time in N. pobeguinii. In vitro gastrointestinal biotransformation showed that non-glycosylated alkaloids showed no biotransformation, while glycosylated alkaloids showed a decrease in intensity over time. The antiplasmodial activity of N. pobeguinii is suggested to be based upon an additive or synergistic effect of multiple minor alkaloids with a β-carboline moiety, present in the bark extract, and their metabolites.

Public defence Sofie Van Hees 20/04/2022 - Development of a PLGA-based nanoparticle drug delivery system for the targeting of macrophages- Department Pharmaceutical Sciences

Public defence Sofie Van Hees 20/04/2022 - Development of a PLGA-based nanoparticle drug delivery system for the targeting of macrophages- Department Pharmaceutical Sciences

Promotors: Prof. Filip Kiekens - Prof. Peter Delputte

LOCATION: Aula Q002 (Promotiezaal), building Q, Campus Drie Eiken, Universiteitsplein 1, Antwerpen (Wilrijk).

Abstract:

Macrophages are involved in many biological processes and are therefore, unfortunetaly, involved in several conditions. That’s why they may serve as therapeutic targets during infection, cancer or inflammatory diseases, but can also be addressed for antigen presentation. Due to their phagocytic nature, macrophages are attractive candidates for particle-based drug delivery systems. Nanoparticles gained a lot of interest lately as drug delivery platforms because of their unique properties.

This thesis focused on the evaluation of modified poly (lactic-co-glycolic acid) (PLGA) nanoparticles and their potential to target macrophages, as an alternative for free drugs enhancing the cellular uptake and reducing the toxicity.  Main points of interest were production process parameters and their influence on nanoparticle size, zeta potential and encapsulation efficiency. Equally important was the effect of these nanoparticle properties on in vitro nanoparticle uptake, cytotoxicity, and intracellular fate in macrophages.

Studies have shown benefits from chitosan coatings to target macrophages, however, cytotoxicity was considered an inconvenience. PLGA nanoparticles were altered with chitosan (glutamate) and the parameters incubation time, size class, PLGA derivative, and chitosan derivative were assessed for uptake kinetics and cell viability. It was found that chitosan coatings were the major determining factor for enhancing nanoparticle uptake, combined with the acid-terminated PLGA derivative and small size. Cytotoxicity was more favourable for small, chitosan glutamate-coated, acid-terminated PLGA nanoparticles compared to its larger-sized, uncoated and chitosan coated counterparts. Formulations based on ester-terminated PLGA nanoparticles showed the least toxic properties.

Furthermore, different sized nanoparticles consisting of ((polyethylene glycol) (PEG)) PLGA, loaded with fluorescein (dye) or auranofin (anti-rheumatic drug), were evaluated for nanoparticle uptake and intracellular fate; or cell viability in sialoadhesin-expressing macrophages, while being functionalized with anti-sialoadhesin antibody. The receptor sialoadhesin is expressed on certain populations of tissue macrophages, yet can be upregulated in inflammatory conditions. Small dye-loaded antibody-functionalized PEG PLGA nanoparticles showed the highest cellular uptake after 24 hours. No co-localization between small-sized (PEG) PLGA nanoparticles and (early/late) endosomes nor lysosomes could be observed. The functionalized auranofin-loaded PLGA nanocarriers showed a higher impact on cell viability in reference to the control groups in low dose, but often not in a higher dose. Surprisingly, auranofin-loaded PEG PLGA nanoparticles were found to be less effective.

To conclude, chitosan- and antibody-modified PLGA nanoparticles are promising tools for the targeting of macrophages. However, the production method, characteristics and surface properties need to be optimized according to the compound and purpose of the formulation.

Public defence Sedigheh Takhsha 30/03/2022 - Identification and validation of autophagy modulating small molecules - Department Pharmaceutical Sciences

Public defence Sedigheh Takhsha 30/03/2022 - Identification and validation of autophagy modulating small molecules - Department Pharmaceutical Sciences

Promotors: Prof. dr. Guido De Meyer - Prof. dr. Wim Martinet

ONLINE DEFENCE: 

https://eu.bbcollab.com/guest/a891ceadb0d540c39a3daaf2eb42e8e3

Abstract: 

Human health is strongly dependent of the accurate coordination and regulation of diverse cellular and metabolic processes, including autophagy. The latter is an evolutionarily conserved process which is active in all human cells. Accordingly, dysregulation of autophagic activity is involved in many pathologies. The main aim of this PhD thesis was the pharmacological modulation (inhibition or induction) of autophagy. The experimental work existed of two major parts. In part 1, we aimed to investigate the possible beneficial effects of UAMC-2526, a ATG4B inhibitor in a Panc02 mouse model of PDAC and in a HT-29 colorectal cancer model. In chapter 3 we used female c57/BL6 mice that were inoculated with Panc02 cancer cells. These mice were treated for 28 days with gemcitabine, autophagy inhibitor UAMC-2526, gemcitabine combined with UAMC-2526, or vehicle. We investigated the mechanism of the tumor growth inhibition to determine whether this inhibition is due to the autophagy inhibition or are other mechanisms involved such as inhibition of proliferation or cell death. The combination of oxaliplatin and bevacizumab is the backbone of therapy for advanced colorectal cancer. Based on the previous results of UAMC-2526 in chapter 4 we determined the influence of UAMC-2526 in combination with these two cancer therapies. We used CD1-/- xenograft mouse model inoculated with HT-29 colorectal cancer cells. These mice were treated for 28 days with oxaliplatin, bevacizumab, oxaliplatin combined with bevacizumab, oxaliplatin combined with bevacizumab and UAMC-2526, bevacizumab and vehicle. In part 2 of the thesis we focused on autophagy inducing molecules. In chapter 5, we used an image-based high-throughput method to screen 10,240 compounds. GFP-LC3 transfected L929 fibroblasts were used to identify novel autophagy inducing small molecules. Thirty compounds, for which their effects on autophagosome accumulation were confirmed in triplicate, were selected for further validation. These hits were re-evaluated in chapter 6, using HeLa cells transfected with mRFP-GFP-LC3 construct. The latter approach allowed us to distinguish compounds that can truly induce autophagic flux from those that induce autophagosome accumulation. mTOR independency of the compounds were investigated via western blot. Additionally, TEM was used as gold standard method to validate the inducers. In summary, we can conclude that modulation of autophagy is complicated and extremely condition dependent. In addition, it is crucial to note that monitoring autophagic flux is not straightforward and requires critical assessment. Combining different methods and comparing their results has to be a golden rule in monitoring autophagic activity.

Public Defence Borislav Lazarov 11/03/2022 - Characterisation of flame retardant emissions from treated consumer and building products in laboratory conditions and real life indoor environments - Department Pharmaceutical Sciences

Public Defence Borislav Lazarov 11/03/2022 - Characterisation of flame retardant emissions from treated consumer and building products in laboratory conditions and real life indoor environments - Department Pharmaceutical Sciences

Promotors: Prof. Adrian Covaci - Prof. Marianne Stranger

ONLINE DEFENCE: https://eu.bbcollab.com/guest/730bdae9912b45c7a42764a51ce7262e

Abstract:

The widespread use of flame retardants (FRs) in various consumer products and building materials has led to their ubiquitous distribution within indoor microenvironments with many studies reporting concentrations in indoor air and dust. Growing evidence that various FRs are associated with potential health risks and the exposure susceptibility of particularly sensitive groups, make many of these chemicals an issue of concern. Key factors towards a better understanding and quantification of the indoor levels of FRs, are the emission mechanisms and emission magnitude of FRs from treated products. The present work studies the emission processes of two widely used groups of organic FRs: polybrominated diphenylethers (PBDEs) and organophosphate FRs (OPFRs) in treated consumer products and building materials. Furthermore, their contribution to the overall indoor air quality was also assessed using standard emission test chambers and assessment studies in real-life indoor environments.

To perform a fast and reliable quantification of the FR concentrations during the experiments, an innovative method for air sampling and analysis of the target FR chemicals was developed. The method is based on low-volume active air sampling of gaseous and particulate air fractions onto mixed-bed (polydimethylsiloxane/Tenax) sorption tubes followed by thermal-desorption and gas chromatography mass-spectrometry analysis. The performance of the developed method was further compared with existing standardized methods for the determination of FRs. The study approach included establishing of a basic emission test protocol for evaluation of FR emission rates (ERs) from treated products. The test protocol was based on the existing harmonized method for evaluation of VOC emissions from construction products. An extensive exploratory study assessing the influence of various environmental parameters (e.g. temperature, humidity) and chamber design on the ERs was conducted. The established FR test protocol was further used for assessment of ERs for FRs from various products (furniture, toys, building materials). The ERs were determined at “standard” (23°C) as well at test conditions simulating a typical use scenario for the products. Furthermore, the relationship between the estimated FR emission rates and the resulted levels in indoor air were also investigated.

The outcomes of this work will further improve the general understanding of FR emissions from treated products and their overall contribution to the human exposure to FRs in indoor environments. Furthermore, the developed product emission test protocols could also contribute to ongoing standardization and product labelling initiatives of various products in term of SVOC emissions, leading to a healthy product policy.