Public Defence Pauline Puylaert 09/12/2022 - Prevention of atherosclerotic plaque destabilization: focus on necroptosis, pyroptosis and ferroptosis - Department Pharmaceutical Sciences

Promotors: prof. Guido De Meyer - prof. Wim Martinet

Aula Q002 (Promotiezaal) building Q, Campus Drie Eiken,

Abstract:

The stability of an atherosclerotic plaque determines its fate, either toward a low-risk lesion with a thick fibrous cap, or toward a high-risk lesion with an inflammatory necrotic core and a thin fibrous cap prone to rupture. Because a large necrotic core, induced by necrosis, is a prominent feature of advanced plaques with a major impact on atherogenesis and plaque destabilization, pharmacological modulation of necrosis represents a promising therapeutic approach to prevent plaque destabilization. Markers of regulated necrosis pathways, in particular necroptosis, pyroptosis and ferroptosis are present in advanced atherosclerotic plaques. Therefore, we targeted necroptosis, pyroptosis and ferroptosis in atherosclerosis and evaluated their potential as plaque stabilizing strategies.

To limit intraplaque necroptosis, we targeted RIPK1 (receptor-interacting serine/threonine protein kinase 1) enzymatic activity using a genetic and a pharmacological approach. First, we repressed RIPK1 kinase activity using a RIPK1^S25D/S25D mutation in atherosclerotic ApoE^-/- mice. Interestingly, ApoE^-/- RIPK1^S25D/S25D mice developed larger plaques with increased deposition of extracellular matrix components. Next, we administered the pharmacological RIPK1 kinase inhibitor GSK’547 in ApoE^-/- Fbn1^C1039G+/- mice, a model of advanced atherosclerosis. Importantly, GSK’547 did not affect plaque size and necrosis but increased apoptosis. Thus, clearly targeting RIPK1 kinase activity is not an ideal approach to limit plaque progression and necrosis.

Gasdermin D (GSDMD) is the executor-protein of pyroptosis and we were the first to report its expression in human plaques. Next, we targeted GSDMD in atherosclerosis using ApoE^-/- Gsdmd^-/- mice. While stable plaque formation in the aorta was not altered, ApoE^-/- Gsdmd^-/- mice developed smaller plaques with smaller necrotic cores and a less inflammatory plaque phenotype in the brachiocephalic artery. Therefore, inhibiting GSDMD appears as a promising plaque stabilizing approach.

Intraplaque (IP) hemorrhage contributes significantly to plaque instability, but exact mechanisms are not clear-cut. We showed that phagocytosis of free erythrocytes by macrophages (erythrophagocytosis) induces ferroptosis and that markers of erythrophagocytosis-induced ferroptosis are expressed in advanced plaques. The ferroptosis inhibitor UAMC-3203 blocked erythrophagocytosis-induced ferroptosis in vitro and limited plaque progression in ApoE^-/- Fbn1^C1039G+/- mice with more advanced stages of atherosclerosis, especially in plaques with confirmed IP hemorrhage, but not in earlier stages of the disease or in hemorrhage-free plaques. Thus, erythrophagocytosis-induced ferroptosis provides a link between IP hemorrhage and plaque destabilization, and can be efficiently blocked with UAMC-3203.

In conclusion, targeting RIPK1 kinase activity did not limit plaque necrosis and progression while inhibition of pyroptosis and ferroptosis are promising strategies to prevent plaque destabilization.

Public defence Daniel Méndez Rodríguez 05/11/2022: Antimicrobial activity of Coccoloba cowellii Britton leaves, an endemic endangered plant from Cuba - Department Pharmaceutical Sciences

Promotors: prof. dr. Paul Cos - prof. dr. Luc Pieters

Place of defence: University of Oriente, Santiago de Cuba, Cuba

Abstract:

Coccoloba cowellii Britton (Polygonaceae) is an endemic and critically endangered plant species that only grow in Camagüey, a province of Cuba. This research aimed to determine the chemical composition and antimicrobial activity of Coccoloba cowellii Britton leaves, to encourage the implementation of ex situ conservation strategies for this species. The phytochemical screening of C. cowellii showed a high production of phenolic compounds, mainly flavonoids, and tannins. The combination of multiple isolation and chemical characterization techniques (UHPLC-HRMS and semipreparative HPLC-DAD-MS/NMR) allowed identifying 31 new compounds (all of them first report for the species, while 24 for the genus), with the prevalence of quercetin and myricetin glycosides, methoxyflavonoids, proanthocyanidins, and lignin oligomers. C. cowellii total extract showed a strong direct antifungal activity against Cryptococcus neoformans (IC₅₀ 2.7 µg·mL^-1) and various Candida spp. (Candida albicans with the lowest value, IC₅₀ 1.7 µg·mL^-1), with no cytotoxicity (CC₅₀ > 64.0 µg·mL^-1) on MRC-5 SV2 cells. In addition, demonstrated an indirect antimicrobial activity acting as an inhibitor of the quorum sensing in a Vibrio sp. model, and the enzymes COX-1 (IC₅₀ 4.9 µg·mL^-1) and COX-2 (IC₅₀ 10.4 µg·mL^-1), while showing a good to medium effect against C. albicans biofilm formation (IC₅₀ 49.73 µg·mL^-1). The bio-guided fractionation protocol implemented allows us to suggest a probable synergistic effect as a result of the combination of different molecular mechanisms conditioned by the diverse kind of polyphenols produced by the species. Those findings might contribute to the development of a future natural medicine to treat fungal diseases, alone or mixed with commercial antifungals. The results presented constitute the first phytochemical and pharmacological report for C. cowellii leaves, contributing to a better understanding of the phytochemistry and biological activities of the genus Coccoloba.

Public defence Eline Feyen 13/09/2022 - The identification of small-molecule ERBB4 agonists - Department Pharmaceutical Sciences

Promotors: prof. Gilles De Keulenaer - Prof. Vincent Segers

Auditorium Kinsbergen
UZA
Drie Eikenstraat 655
2650 Edegem

Abstract:

ERBB4 is a member of the epidermal growth factor subfamily of receptor tyrosine kinase receptors and regulates signaling pathways involved in cardiac development, biology and disease. The endogenous ligand NRG1 is of particular importance in the heart, signaling through the ERBB4 and ERBB2 receptor. Activation of ERBB4 by NRG1 plays well-defined roles in heart failure and elicits antifibrotic effects in the heart by attenuating cell death, inflammation and extracellular matrix production. In tumor cells that overexpress ERBB2, NRG1 may promote formation of ERBB2/ERBB3 heterodimers which accelerates tumor growth and is a feared side effect when NRG1 would be used as a drug. Furthermore, NRG1 is a protein and has to be delivered intravenously, limiting applicability in chronic diseases. In this thesis, we aimed to discover a selective small-molecule ERBB4 agonist with protective cardiac properties, biasing signaling away from ERBB2/ERBB3 interactions and allowing oral administration. In chapter 4, we performed a high-throughput screening of 10,240 compounds to characterize small-molecule ERBB4 agonists, using an ERBB4/ERBB4 homodimerization assay. The screen resulted in 62 confirmed hits, in which we were able to identify a highly enriched pharmacophore. We selected eight compounds, all containing the pharmacophore or an analogue. In chapter 5, we demonstrated that the compounds behaved as allosteric agonists, providing agonistic stimulation of ERBB4 on a dimerization level and post-receptor level, without phosphorylation of ERBB1/ERBB2/ERBB3. Moreover, some compounds potentiated NRG1-induced ERBB4/ERBB4 homodimerization but attenuated NRG1-induced ERBB2/ERBB3 dimerization. Cytotoxicity was absent and compounds did not induce proliferation of tumor cells. In chapter 6, we examined biological effects of the EF-compounds. We showed that the most effective compounds induced concentration-dependent survival effects in cardiomyocytes during H₂O₂-induced oxidative stress and collagen-decreasing effects in dermal and atrial fibroblasts during TGFβ-stimulation. Importantly, negative control compounds NA1 and NA2 had no effect, suggesting that the effects of EF-compounds are ERBB4-mediated. Silencing ERBB4 in cardiac fibroblasts attenuated the effects, further endorsing the role of ERBB4. After evaluation of microsomal and plasma stability of EF-1 in mice and human, we showed that in vivo administration of EF-1 attenuated AngII-induced effects on cardiac collagen mRNA levels and myocardial interstitial fibrosis, whereas administration of negative control compound NA1 had no effect. Taken together, our discoveries provide basic information for a structure-activity relationship needed for further development of a cardioprotective drug. Moreover, further analyzing effects of selective ERBB4 agonists will contribute to a better understanding of the translational potential of the ERBB signaling system.

Public defence Karen Claesen 25/08/2022 - The carboxypeptidase U system in atherosclerosis: Insights in carboxypeptidase U mediated pleiotropic effects of statins - Department Pharmaceutical Sciences

Promotors: Prof. Ingrid De Meester - Prof. Dirk Hendriks

LIVE: Aula Q002 (Promotiezaal) building Q, Campus Drie Eiken, Universiteitsplein 1, 2610 Antwerpen (Wilrijk)

Abstract:

The carboxypeptidase U system in atherosclerosis: Insights in carboxypeptidase U mediated pleiotropic effects of statins

Carboxypeptidase U (CPU) provides a molecular link between coagulation and fibrinolysis. By cleaving C-terminal lysines from partially degraded fibrin it counteracts efficient plasminogen activation, thereby attenuating the fibrinolytic rate. CPU circulates in plasma as the zymogen procarboxypeptidase U (proCPU). With this doctoral research we aimed to contribute to a better understanding of the clinical context and expression of CPU, mainly focusing on hyperlipidemia & atherosclerosis.

In a first part, the influence of statin therapy on proCPU biology in statin-eligible patients with hyperlipidemia was explored in a proof-of-concept observational study. Treatment of hyperlipidemic individuals with statin therapy normalized their proCPU levels and on a functional level a resulting improvement of the fibrinolytic rate was observed. Moreover, this pilot study revealed that the statin-dependent decline in plasma proCPU concentrations is highly different interindividually with the largest improvement in fibrinolysis seen in patients with the highest pre-treatment proCPU levels. Additionally, evidence that the proCPU downregulation in response to statins is a pleiotropic effect of this therapy was also provided in a murine model of atherosclerosis. In a larger clinical study, we confirmed that plasma proCPU concentration and its expected effect on the fibrinolytic rate are increased in hyperlipidemic patients and these effects can be normalized by treatment with atorvastatin. High interindividual variation in proCPU levels was again observed in hyperlipidemic individuals, with proCPU levels up to 80% higher compared to normolipidemic controls. Interestingly, patients with the highest proCPU levels were not necessarily the individuals with the highest cholesterol levels. Lastly, we observed that the proCPU downregulation and the improvement of the plasma fibrinolysis by atorvastatin were dose-dependent.

In the second part of this research, the expression of proCPU in (primary) human monocytes and macrophage-subsets was studied. CPB2 mRNA and proCPU protein was detected in all investigated cell types and it was found that CPB2 mRNA expression and proCPU secretion in monocytes and (activated) macrophages are related to the degree to which these cells are differentiated. Moreover, it was demonstrated that proCPU can be activated into functionally active CPU in the in vitro cell culture environment.

During the conduct of this PhD research, some very interesting, CPU-related research questions came into the spotlight that we explored further. The first one was the functional characterization of a new mutation in the CPB2 gene of proCPU. This mutation resulted in a proCPU deficiency that was found to be clinically asymptomatic, but accelerated fibrinolysis in vitro.

Finally, CPU-related parameters were evaluated in hospitalized COVID-19 patients, showing an initial significant generation of CPU with concomitant proCPU consumption during the early phase of SARS-CoV-2 infection, with a subsequent progressive increase in both proCPU concentration and CPU+CPUi antigen levels.

Het carboxypeptidase U systeem in atherosclerose: Inzichten in carboxypeptidase U gemedieerde pleiotrope effecten van statines

Abstract:

Carboxypeptidase U (CPU) zorgt voor een moleculaire link tussen coagulatie en fibrinolyse. Door C-terminale lysines te splitsen van gedeeltelijk afgebroken fibrine, gaat het efficiënte activatie van plasminogeen tegen, waardoor de fibrinolytische snelheid wordt verminderd. CPU circuleert in plasma als het zymogeen procarboxypeptidase U (proCPU). Met dit doctoraatsonderzoek wilden we de kennis omtrent de klinische context en expressie van CPU uitbreiden, met de focus op hyperlipidemie en atherosclerose.

In een eerste deel werd de invloed van statine therapie op de proCPU-biologie onderzocht in een proof-of-concept observationele studie bij patiënten met hyperlipidemie die in aanmerking kwamen voor opstart van statine therapie. Behandeling van hyperlipidemische personen met statine therapie normaliseerde hun proCPU concentratie en op functioneel niveau werd een resulterende verbetering van de fibrinolytische snelheid waargenomen. Bovendien onthulde deze pilotstudie dat de statine-afhankelijke afname van de plasma proCPU concentratie sterk verschilt van persoon tot persoon, waarbij met de grootste verbetering in fibrinolyse werd gezien bij de patiënten met de hoogste proCPU concentratie vóór de behandeling. Bovendien werd in een muismodel van atherosclerose bewijs geleverd dat de proCPU downregulatie door statines een pleiotroop effect is van deze therapie. In een grotere klinische studie hebben we bevestigd dat plasma proCPU concentratie en het verwachte effect op de fibrinolytische snelheid verhoogd zijn bij hyperlipidemische patiënten en dat deze effecten genormaliseerd kunnen worden door behandeling met atorvastatine. Hoge interindividuele variatie in proCPU concentratie werd opnieuw waargenomen bij hyperlipidemische individuen, met proCPU concentraties die tot 80% hoger waren in vergelijking met normolipidemische controles. Interessant is dat de hoogste proCPU waarden niet noodzakelijk gezien werden bij de personen met de hoogste cholesterol waarden. Ten slotte zagen we dat de proCPU downregulatie en de verbetering van de plasmafibrinolyse door atorvastatine dosisafhankelijk waren.

In een tweede deel van dit onderzoek werd de expressie van proCPU bestudeerd in (primaire) humane monocyten en macrofaag-subsets. CPB2 mRNA en proCPU eiwit werden gedetecteerd in alle onderzochte celtypen en er werd waargenomen dat de CPB2 mRNA expressie en proCPU secretie in monocyten en (geactiveerde) macrofagen gerelateerd zijn aan de mate waarin deze cellen gedifferentieerd zijn. Bovendien werd aangetoond dat proCPU kan worden geactiveerd tot functioneel actief CPU in de in vitro celcultuuromgeving.

Tijdens het uitvoeren van dit onderzoek kwamen enkele zeer interessante, CPU-gerelateerde onderzoeksvragen op ons pad die we verder onderzocht hebben. Een eerste was de functionele karakterisering van een nieuwe mutatie in het CPB2 gen van proCPU. Deze mutatie resulteert in een proCPU deficiëntie die klinisch asymptomatisch bleek te zijn, maar in vitro de fibrinolyse versnelde.

Ten slotte werden CPU-gerelateerde parameters geëvalueerd bij gehospitaliseerde COVID-19-patiënten. Hierbij werd een significante generatie van CPU waargenomen met gelijktijdig proCPU verbruik tijdens de initiële fase van de SARS-CoV-2-infectie, met een daaropvolgende progressieve toename van zowel de proCPU concentratie als CPU+ CPUi-antigeenniveaus.

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

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

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

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

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.

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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.