Public defence Mélissa Lallier 16/12/2025 - Roadblock on the recycling route: development of novel autophagy inhibitors - Department Pharmaceutical Sciences

Promotors: Prof. dr. Pieter Van der Veken, prof. dr. Koen Augustyns and prof. dr. Wim Martinet

Location defence: Aula O1, gebouw O, Campus Drie Eiken

Abstract:

Autophagy is a conserved, lysosome-dependent process that maintains cellular homeostasis by degrading and recycling superfluous or damaged proteins and organelles. This catabolic pathway involves the formation of double-membraned vesicles, termed autophagosomes, which subsequently fuses with lysosomes, leading to degradation of their autophagic cargo by lysosomal enzymes. By limiting cellular damage and stress, basal autophagy exerts cytoprotective functions and prevents diverse pathologies, including cancer. However, the role of autophagy in cancer is highly complex and context-dependent. In established cancers, certain types of tumor cells can hijack autophagy to sustain their high metabolic demands and to resist therapy-induced stress. This dual role has made pharmacological modulation of autophagy an attractive, yet challenging, therapeutic target. Although preclinical studies have demonstrated that autophagy inhibition can sensitize tumors cells to conventional anticancer therapies, current clinical candidates, such as chloroquine and its derivatives, lack specificity and show limited efficacy. These limitations highlight the need for novel small molecule autophagy inhibitors with improved potency, selectivity, and translational potential.

To address this situation, we conducted a phenotypic high-throughput screening based on quantification of GFP-LC3-positive puncta to identify novel small molecule autophagy modulators. Among the top-ranked hits, the biarylacetamide family emerged as promising candidates and were selected for further development.

This thesis describes the optimization of the initial hits through three iterative rounds of structure-activity relationship. To explore the chemical space and improve potency we applied chemical modifications to biarylacetamide core in an explorative and diversity-oriented manner, generating a library of novel derivatives. The compounds were screened using the tandem fluorescent-tagged RFP-GFP-LC3 reporter in HeLa cells. The inhibitory activity of compounds that significantly increased RFP-GFP-LC3 puncta number (indicative of autophagosome accumulation) was validated by immunoblot. These compounds led to an accumulation of the autophagic markers LC3, GABARAP and p62/SQSTM1 in both HeLa and LNCaP cells. Moreover, autophagic cargo flux assays, including LDH sequestration and LDHB-mKeima, further confirmed that these compounds act at a post-sequestration step in the autophagy pathway.

To explore the biological effects induced by the biarylacetamides, we performed quantitative proteome profiling combined with untargeted metabolomic and lipidomic studies. These studies revealed that the biarylacetamides significantly alter lipid metabolism. These alterations included activation of the cholesterol biosynthesis pathway and changes in the distribution of key lipid classes. Further mechanistic studies indicated that the biarylacetamides triggered an ER stress response and may impair lysosomal function, as suggested by the accumulation of pro-cathepsin D.

This thesis underscores the biarylacetamides as a novel class of autophagy inhibitors. Ongoing target identification experiments will help define biarylacetamides mode of action and clarify how they disrupt autophagy. Further evaluation of these derivatives in combination with anticancer agents will be decisive to support their use as pharmacological tools and therapeutic candidates.

Public defence Rani Robeyns 11/12/2025 - The metabolic signature of autophagy modulators: an untargeted metabolomics approach for early mechanistic evaluation - Department Pharmaceutical Sciences

Promotors: Prof. Alexander van Nuijs and professor Sigrid Stroobants

Location: Aula O5, building O, Campus Drie Eiken

Abstract:

Autophagy is an evolutionary conserved cellular degradation process that maintains homeostasis by recycling cytoplasmic components such as damaged organelles and protein aggregates. Its tight regulation is essential for cellular health, and its dysfunction has been linked to a wide range of diseases, including age-related neurodegenerative diseases, inherited neuropathies, cardiomyopathies, and cancer. As pharmacological modulation of autophagy gains interest as a therapeutic strategy, there is a growing demand for robust tools to systematically assess how compounds influence this complex pathway. This work investigated untargeted metabolomics as a powerful approach to characterize autophagy modulation during early-stage mechanistic evaluation.

To that end, a robust and reproducible in vitro untargeted metabolomics and lipidomics workflow was developed, specifically optimized for autophagy research. This included tailored cell culture conditions, a biphasic extraction method suitable for both polar and apolar metabolites, LC-(DTIM)-HRMS analysis across multiple platforms, and a QA/QC framework. Special attention was given to normalization strategies, reproducibility, and the creation of a multidimensional in-house spectral library to improve metabolite annotation confidence. These methodological advances were guided by metabolomics best practices and designed to support both hypothesis generation and mechanistic interpretation.

The optimized workflow was applied to characterize the metabolic and lipidomic fingerprints of two widely used autophagy inducers, Torin1 (mTORC1/2 inhibitor) and Tat-Beclin1 (Beclin1 complex activator). Despite both increasing autophagic flux, they produced distinct metabolic profiles, revealing how upstream mechanisms of induction influence downstream biochemical pathways. Shared signatures were also identified which may represent conserved metabolic responses to autophagy.

The case study with compound 5j, a novel biarylacetamide derivate, further illustrated how metabolomics can be integrated with proteomics and autophagy validation assays to gain mechanistic insight into novel autophagy modulators during early mechanistic evaluation. In conclusion, this thesis provides both a conceptual and practical foundation for integrating untargeted metabolomics into autophagy research. The presented workflow and reference fingerprints lay the groundwork for future studies aimed at screening novel modulators, understanding compound mechanisms, and identifying translational metabolic biomarkers. Moving forward, combining untargeted metabolomics with complementary omics technologies, validation assays, and more pathophysiological relevant models will further enhance the clinical potential of this approach.

Public defence Maciej Rogacki 09/12/2025 - Phenotypic and target-based approach to antitubercular drug discovery: investigation of thienopyrimidine and hydantoin scaffolds as new hits against Mycobacterium tuberculosis - Department Pharmaceutical Sciences

Promotors: Prof. dr. Pieter Van der Veken, prof. dr. Koen Augustyns and prof. dr. Robert Bates

Location: Aula O5, building O, Campus Drie Eiken

Abstract:

Tuberculosis remains the leading cause of death amongst infectious diseases. Despite decades of drug discovery efforts, current treatment options still rely predominantly on suboptimal combinations of drugs developed more than half a century ago. With more then 10 million new cases annually and a growing threat from drug resistance, there remains an acute need for new antitubercular medicines. Under the framework of OpenMedChem - an academic-industrial partnership project, screening data generated by GlaxoSmithKline was shared with the University of Antwerp to fuel medicinal chemistry efforts aimed at identifying and advancing potential novel antitubercular chemotypes.
Research performed within this thesis was performed along two orthogonal strategies. The first approach focused on results of a phenotypic screening campaign which provided a number of hit compounds. Further analysis of active scaffolds, in comparison with chemotypes previously reported in literature, showed a common structural arrangement across otherwise not related chemical series, which suggested a possible shared, though not yet identified, biological target. Medicinal chemistry efforts led to the synthesis of a library of analogues with the main goal of identifying the minimal pharmacophoric features. Activity profiling showed a significant consensus between substitution patterns of the analyzed compound families, suggesting a potentially similar mechanism of action. At the same time correlation with cytotoxicity against a human cell line was also identified suggesting a potential overlap with a biological target relevant for eucaryotic cells. Although in view of these results further medicinal chemistry optimization was put on hold, the series yielded an active analogue with decreased toxicity, which may serve as a starting point for further research.
The second approach focused on a target-based approach against decaprenylphosphoryl-β-D-ribose 2-epimerase (DprE1) – a crucial enzyme involved in the synthesis of mycobacterial cell wall components. A high throughput screening campaign led to identification of a chemical scaffold with inhibitory activity against that enzyme. Subsequent medicinal chemistry efforts focused around hit expansion in order to understand the structure-activity relationship, improve the overall properties of the series and derisk the potential initial safety concerns. The data generated with the newly designed and synthesized compounds allowed to select most potent representatives for initial in vivo proof-of-concept study in mice, where reduction in bacterial burden was demonstrated.
Overall, research performed within this thesis allowed to generate valuable insights into chemical scaffolds with potent antitubercular properties, which may provide foundation for further development into novel antitubercular drugs.

Public defence Desirée Oliveira Alves 21/11/2025 - Structural analysis of the membrane-proximal venus flytrap domain (VFT2) of ESAG4 receptor-like adenylate cyclase from Trypanosoma brucei - Department Pharmaceutical Sciences

Promotors: 

Prof. Didier Salmon (Federal University of Rio de Janeiro): promotor

Prof. Yann Sterckx (UAntwerpen): promotor

Prof. Guilherme Augusto Piedade de Oliveira (Federal University of Rio de Janeiro): Co-promoter

Location: Federal University of Rio de Janeiro

Abstract:

The protozoan parasite Trypanosoma brucei possesses a large family of transmembrane receptor-like adenylate cyclases (TrypRACs), primarily located at the flagellar surface and involved in sensing of the extracellular environment. RACs exhibit a conserved topology characterized by a large N-terminal extracellular moiety harbouring two conserved Venus Fly Trap (VTF) bilobate structures separated from an intracellular catalytic domain by a single transmembrane helix. Activation of these enzymes requires the dimerization of the intracellular catalytic domain, which typically occurs under mild acid stress. While no ligand has been identified so far, the existence of VFT domains suggests their potential responsiveness to extracellular ligands in the absence of stress. Herein we report the biophysical characterization of the membrane-proximal VFT2 domain of the extracellular moiety of a bloodstream form-specific RAC, called ESAG4 (Expression Site-Associated Gene). We first attempted to produce the entire ectodomain of the RAC ESAG4, as well as the individual domains VFT1 and VFT2, as recombinant proteins in Escherichia coli. However, these were either unstable forming oligomeric structures or cleaved by proteases. By utilizing an AlphaFold2-based optimization of the construct’s domain boundaries, we successfully recombinantly produced and purified the VFT2 in its folded state as evidenced by analytical size exclusion chromatography (SEC), dynamic light scattering (DLS), circular dichroism (CD) spectroscopy, and one-dimensional nuclear magnetic resonance (1-D NMR) as quality controls. Furthermore, the protein’s in-solution behaviour was studied by small-angle X-ray scattering (SAXS), which fully supports a highly accurate AlphaFold2 model of a VFT2 monomer. Thermal shift assays (TSA) demonstrate that the protein has a marginal thermostability, with an apparent melting temperature (Tmapp) of around 42°C. The TSA curves display a highly cooperative unfolding event, with a steep transition region, making them suitable for screening of putative VFT2 ligands. In silico analyses of VFT2 genomic databases revealed its divergence among cyclase isoforms, suggesting a potential role in ligand specificity, with several groups displaying minor structural differences.

Attempts were made to recombinantly produce the full ectodomain in Leishmania tarentolae using the LESXY inducible expression system. Two different expression vectors were utilised for screening, but further optimization of the process will be required to express the full ectodomain. Taken together, these results demonstrate that we have successfully generated a stable monomeric domain, providing a valuable tool for further exploration of putative ligands using thermal shift assays (TSA) and other ligand screening methods. This, in turn, would enable a thorough structural characterization of its ligated structure through macromolecular X-ray crystallography. Hence, this study paves the way for the structural characterization of the only signalling receptor characterized to date in trypanosomatids.

Keywords: Trypanosoma brucei, Adenylate cyclase, Venus Flytrap domain, AlphaFold, ligands

Public defence Evelyn Rojas Vázquez 15/09/2025 - Integrating Psychosocial Perspectives in Medication Management Model - Department Pharmaceutical Sciences

Promotor: Prof. dr. Hans De Loof

Location defence: Universidad de Oriente (Cuba)

Abstract:

In Pharmaceutical Care (PhC), there remains a lack of focus on the SDOH and an insufficient understanding of medication use from the patient's perspective through their narratives. The Sociology of Health, also referred to as medical Sociology, examines the influence of social and cultural factors on individuals' health perceptions, healing processes, and their expressions in various societies. The primary aim of this research was to create a theoretical model that incorporates psychosocial elements into the process of identifying, preventing, and addressing drug related problems (DRPs) in the delivery of Comprehensive Medication Management services (CMM), to enhance patient-centered practice. The scoping review revealed that most studies still neglect to consider a broader perspective on patients' life circumstances and their relationship with pharmacotherapy, as well as how pharmacists integrate holistic elements to address or prevent DRPs. The qualitative study's findings indicated that PhC is not widely implemented and that pharmacists encounter similar challenges and barriers in its application. In Belgium, only a few pharmacists acknowledged the significance of understanding patients' psychosocial aspects and their experiences with medication. In Cuba, while patient interviews are infrequent, professionals emphasized the importance of grasping their life situations. Nevertheless, some social data and patient perspectives remain unrecorded. Based on the findings of the scoping review, the experiences of pharmacists from two distinct contexts, and McLeroy's methodology, a SEM was developed. This model encompasses several social levels: intrapersonal, interpersonal, institutional, and community, to illustrate the delivery of CMM service within the Cuban health system. The model features a theoretical framework and a proposal for pharmacists' actions through three indicators: 1) the incorporation of SDOH and their significance in PhC; 2) the exploration of patient experiences with medication and their connection to DRPs; and 3) the proposal of personalized pharmacist interventions. The sociological theories of Symbolic Interactionism, Social Constructionism, and Conflict were employed to elucidate and bolster the incorporation of a psychosocial perspective into the pharmaceutical care process. A case study aimed at implementing the CMM service, utilizing indicators from the SEM, facilitated the identification of DRPs and the execution of tailored pharmacist interventions. The use of indicators from the SEM has strengthened the patient-centered approach to CMM services, emphasizing the necessity for a more humanistic and efficient clinical pharmacy practice. The integration of the Sociology of Health enhances patient-centered practice in PhC, fostering a more holistic and effective approach that could benefit patients, caregivers, and the healthcare team.

Public defence Stef Lauwers 30/06/2025 - Investigations Into the Biotransformation, In Vitro, and Clinical Effects of Olive Polyphenols on Cardiovascular Health - Department Pharmaceutical Sciences

Promotors: Pof. Nina Hermans - Prof. Emmy Tuenter - Prof. Lynn Roth

Location defence: Aula Q002, building Q, Campus Drie Eiken

Abstract:

Cardiovascular diseases (CVDs), including coronary heart disease, cerebrovascular disease, and atherosclerosis, remain the leading cause of global mortality, accounting for nearly one-third of all deaths. Despite advancements in prevention strategies, the prevalence of CVDs has risen over the past decade, highlighting the urgent need for novel interventions. Among modifiable risk factors, diet plays a pivotal role in cardiovascular health. The Mediterranean diet, rich in polyphenol-containing foods such as olives, has been extensively associated with reduced CVD risk. In this thesis, the potential cardiovascular benefits of olive polyphenols are explored through a multidisciplinary approach.
The polyphenolic profiles of olive leaf and fruit extracts were characterised using an optimised UPLC-ESI-QToF-MS method. Olive leaf extract exhibited a broader spectrum of polyphenols, including oleuropein, hydroxytyrosol, verbascoside, flavonoids, and phenolic acids, compared to the fruit extract. The reduced diversity in the fruit extract is likely due to polyphenol degradation during maturation.
Biotransformation of oleuropein and olive leaf extract was studied using an in vitro gastrointestinal model simulating the stomach, small intestine, and colon phases, incorporating faecal samples from healthy young (20-30 years old) and elderly (≥ 65 years old) individuals. Despite age-related differences in gut microbiota composition, similar biotransformation reactions, primarily deglycosylation, demethylation, and hydrolysis, were observed, with hydroxytyrosol and 3-(4-hydroxyphenyl) propionic acid as key metabolites. Hydroxytyrosol was further evaluated for its vascular effects. In vitro and ex vivo studies demonstrated its ability to enhance antioxidant enzyme expression and autophagic activity in endothelial cells, and to reduce aortic stiffness in the Fbn1C1039G+/– mouse model.
A double-blind, placebo-controlled, randomised clinical trial assessed the effects of a commercial olive extract on blood pressure and cardiovascular biomarkers in 56 participants with elevated systolic blood pressure (≥130 mmHg). A significant decrease in systolic blood pressure was observed between the start and end of the trial in the intervention and placebo groups. However, the reported change was comparable with blood pressure reductions in placebo arms of hypertension trials, resulting in a non-significant difference between the groups. Both groups showed reductions in cholesterol markers, though no changes in oxidative stress biomarkers were detected.
In conclusion, olive polyphenols hold the potential to aid in CVD disease prevention. Further research into the bioavailability of olive polyphenols after biotransformation by the gut microbiome and investigations into liver biotransformation are necessary to clarify which metabolites are responsible for the reported beneficial effects and to expand the mechanistic insights into the cardiovascular health benefits of olive polyphenols.

Public defence Maria van de Lavoir 23/06/2025 - Building a human hair metabolome atlas for clinical diagnostics - Department Pharmaceutical Sciences

Promotors: Prof. Adrian Covaci - Prof. Alexander van Nuijs

Location: Aula O3, gebouw O, Campus Drie Eiken

Abstract:

Metabolites (small molecules <2000 Da) play an important role in metabolic processes and are influenced by lifestyle, diet, genetics, and disease states. Metabolomics, the study of these metabolites in clinical samples such as blood, urine, and hair, enables the detection of metabolic changes associated with disease. These metabolites act as signaling molecules, or biomarkers.
Hair offers unique advantages as a clinical sample due to its chemical stability and ability to preserve a long-term biochemical profile. Unlike blood or urine, which reflect short-term metabolic fluctuations, months. Segmental hair analysis is therefore a promising, non-invasive tool for monitoring chronic diseases and detecting disease at an early stage.
Despite its potential, hair metabolomics remains underexplored, particularly regarding the biochemical composition of hair and variations in metabolite concentrations along the hair strand.
This study presents the Human Hair Atlas, a comprehensive reference database of the chemical composition of human hair. To achieve this, advanced analytical techniques, such as liquid chromatography-high-resolution mass spectrometry and ion mobility spectrometry, were applied to precisely map the molecular structure of hair.

Key findings and contributions
A hair-specific, systematic 10-step analytical workflow was developed to optimize the extraction and identification of metabolites in hair with increased efficiency and accuracy. This improved method enabled the creation of a large-scale dataset: the Human Hair Atlas, which mapped over 1,200 metabolites in healthy individuals. This dataset serves as a reference profile for future comparisons, such as with patients with specific diseases.
Analysis of consecutive 1 cm hair segments revealed that metabolite concentrations varied by up to 50%, highlighting the need for correction of baseline variations in hair-based studies, particularly in chronic disease research.
Additionally, 122 metabolites were identified that are potentially exogenous, mainly originating from personal care products. By identifying and categorizing exogenous compounds, the Human Hair Atlas supports the interpretation of hair metabolomics data, preventing misattribution of these compounds as disease-related metabolic variations.
By detecting a wide range of metabolites, the Human Hair Atlas highlights both the potential of hair metabolomics and the value of hair as a clinical matrix. The Human Hair Atlas is publicly accessible (https://metabolomics.cloud/hair/) and serves as a reference for research in hair metabolomics, lipidomics, and exposomics.

Public defense Joni De Loose 11/03/2025 - On dipeptidyl peptidase 9 and the road towards understanding its role in human cells - Department Pharmaceutical Sciences

Promotors: prof. dr. Ingrid De Meester - prof. dr. ir. Yann Sterckx

Location defense: Aula Q002, gebouw Q, Campus Drie Eiken

Abstract:

This thesis focuses on human dipeptidyl peptidase 9 (DPP9), an intracellular serine protease that cleaves off the N-terminal dipeptide of its substrates. DPP9 is involved in cellular homeostasis and programmed cell death but remains less characterized than its family members DPP4 and fibroblast activation protein (FAP). Unlike these family members, DPP9 is essential for cell survival and neonatal development, highlighting the importance of this protease in the human body.
A significant challenge at the outset of this research was the absence of selective DPP9 compounds. To address this gap, we evaluated a small molecule-based toolset for studying DPP9. Novel inhibitors synthesized at the University of Antwerp’s Laboratory of Medicinal Chemistry (UAMC) were biochemically characterized. Among these, cpd-42 exhibited in vitro nanomolar affinity for DPP9 and high selectivity towards homologue DPP8. Cellular assays with cpd-42 and a second novel DPP8/9 inhibitor, cpd-6a, confirmed that DPP8/9 inhibition induces caspase-1-dependent lytic cell death in myeloid cells. Notably, gasdermin D was not essential for cell death in myeloid cells, suggesting the involvement of alternative cell death pathways beyond the expected gasdermin D-driven pyroptosis. The inhibitors also induced lytic cell death in primary human CD4+ T cells, monocytes, monocyte-derived macrophages and dendritic cells. However, further analyses will be necessary for a comprehensive interpretation of their intracellular effects.
We also investigated DPP9 expression and activity in peripheral blood mononuclear cells (PBMCs) and endothelial cells. DPP8/9 activity increased as monocytes differentiated into macrophages and dendritic cells. Intriguingly, DPP9 localized to the cytoplasm and the nucleus in PBMCs, displaying variable expression patterns across different cells and cell types, while in endothelial cells, DPP9 was primarily detected in the cytoplasm. The significance of DPP9’s nuclear localization and the related regulatory mechanisms remain unresolved.
We further characterized fluorescently labeled inhibitors that were synthesized at UAMC. Among these, an NBD-linked DPP8/9 inhibitor showed promise as the first intracellular DPP9 fluorescent probe. However, challenges such as photobleaching and compatibility issues with antibodies limited its applicability.
In the final experimental section, we evaluated proteolysis-targeting chimeras (PROTACs) for their ability to degrade DPP9 intracellularly. While these PROTACs inhibited DPP9 activity in vitro, intracellular DPP9 degradation was inconsistent in THP-1 cells, highlighting the need for further optimization of these molecules.
This thesis underscores the necessity for improved selective inhibitors, probes, and PROTACs to study DPP9’s functions. Future research should focus on fundamental research questions concerning DPP9’s homeostatic regulation, the nuclear functions, and the functional redundancy with DPP8. Advancing our understanding of DPP9’s role in cellular processes will be crucial in determining if and how DPP9 could ever become a druggable target.

Public defense Yentl Van Rymenant 18/02/2025 - Exploring Fibroblast Activation Protein (FAP) in the tumor microenvironment and Crohn’s disease using novel FAP-selective tools and methods - Department Pharmaceutical Sciences

Promotors: Prof. Ingrid De Meester - Prof. Pieter Van der Veken

Location defence: Aula Q002, gebouw Q, Campus Drie Eiken

Abstract:

Fibroblast activation protein (FAP) is a post-prolyl proteolytic enzyme that is prominently expressed in cancer-associated fibroblasts (CAFs) within the tumor microenvironment (TME) and in activated fibroblasts involved in tissue remodeling in chronic inflammatory diseases, such as Crohn’s disease (CD). Interestingly, FAP exhibits low expression and activity in healthy tissues, making it a promising diagnostic and therapeutic target for cancer and chronic inflammatory diseases. Unfortunately, FAP’s functional role in these pathological conditions remains poorly understood, and current tools and methods for studying FAP are often characterized by a lack of selectivity. Additionally, research has primarily focused on FAP mRNA and protein expression, often overlooking its enzymatic activity, which is critical for understanding its biological functions. These limitations highlight the urgent need for innovative, highly selective tools and methods to detect FAP and elucidate its role in disease mechanisms.
Hence, the goal of this doctoral research was to develop and validate novel FAP-selective tools and methods to enhance our understanding of FAP’s biological function in cancer and Crohn’s disease.
To achieve this, we established an in-house recombinant human FAP (rhFAP) production system to ensure a robust supply of high-quality active enzyme for downstream evaluation and validation of novel FAP-targeting tools and compounds. We produced and purified a highly specific antibody against FAP and developed fluorescent FAP-targeting activity-based probes (ABPs) that enable selective, high-affinity visualization of FAP’s enzymatic activity. Furthermore, we evaluated novel [18F]-labeled FAP inhibitors ([18F]-FAPIs) for PET/CT imaging as a non-invasive diagnostic approach for tumors and fibrotic diseases in which FAP plays a role.
With these tools in hand, we gained new insights into the expression and function of FAP in the TME, with a particular focus on its activity and role in natural killer (NK) cells. Our findings suggest that FAP is involved in NK cell activity and immune evasion. In addition, we used these FAP-specific tools to investigate its function in CD, revealing its involvement in chronic inflammation and intestinal fibrosis.
Overall, this research not only offers new insights into FAP’s role in the TME and CD, but also provides the scientific community with a comprehensive toolkit for the selective detection and functional analysis of FAP. By filling critical knowledge gaps in FAP’s biological function, our findings pave the way for novel diagnostic and therapeutic strategies targeting FAP in oncology, Crohn’s disease and beyond.

Public defence Agnese Compagnone 30/01/2025 - Implementation of in vivo models to identify potential candidates for DED treatment - Department Pharmaceutical Sciences

Promotors: Prof. Paul Cos - Prof. Peter Delputte

Location: Q002

Abstract:

This thesis explores the development of an evaporative dry eye (EDE) animal model based on Meibomian gland cauterisation, aiming to replicate the tear film alteration caused by Meibomian gland dysfunction (MGD), a major driver of EDE. Dry eye disease (DED), which causes ocular surface inflammation, pain, and discomfort, is a multifactorial condition with significant impact on quality of life. EDE is the most common subtype, linked to MGD, which impairs lipid secretion from Meibomian glands, increasing the tear evaporation rate. The cauterisation method, as suggested by existing literature, faced challenges: technique variability and unclear guidelines on how many glands to cauterise or whether both eyelids should be involved. These inconsistencies can compromise the model’s reliability, indicating the need for further optimisation to ensure reproducibility and consistency in experimental outcomes.
This thesis also presents a novel multiplex electrochemiluminescence immunoassay (ECLIA) for detecting key biomarkers in rat tear samples, critical for DED research. Biomarkers such as interleukin-17 (IL-17), matrix metalloproteinase-9 (MMP-9), and intercellular adhesion molecule-1 (ICAM-1) were targeted. The assay was validated with promising results, showing that the detection ranges for these biomarkers met the required parameters, though precision was identified as a limiting factor. Variations were mainly due to human error, such as pipetting inconsistencies and evaporation during incubation. Suggestions for improving precision include standardising operator expertise, minimising reagent variability, and controlling incubation conditions. These improvements would increase reliability and broaden the assay's detection range.
Additionally, the thesis investigates the role of oxidative stress and ferroptosis in DED. Ferroptosis, a form of iron-dependent cell death linked to lipid peroxidation, is thought to contribute to DED pathogenesis. In vitro studies with the ferroptosis inhibitor UAMC-3203 suggested potential in reducing oxidative stress. However, clear evidence for ferroptosis in DED models was not found. In vivo tests showed that UAMC-3203 could restore GPX4 gene expression closer to the control group, hinting at potential benefits of ferroptosis inhibition.
Finally, the thesis proposes optimising the EDE model with inflammation-triggering agents like LFA-1 and ICAM-1 inhibitors, or toll-like receptor agonists, to enhance chronicity and inflammation. The use of advanced 3D cell culture models for better simulation of the ocular surface is also discussed, highlighting their potential for testing new therapies. Further research is needed to address the multifactorial causes of DED and improve treatments for this widespread condition