Master thesis subjects

Unraveling the cellular and systemic effects of serum amyloids along the gut-brain axis in Alzheimer’s disease

Tutor: Aurélie Hofkens - Promoters: Peter Verstraelen, Winnok De Vos

Recent evidence suggests that gut inflammation and the enteric microbiome contribute to the progression of Alzheimer's Disease (AD), a devastating neurodegenerative disorder. We have identified curli, an amyloid produced by the gut microbiome, as a potent trigger of immunes response in the enteric nervous system, with serum amyloid A3 (SAA3) as a key regulator in this response. Since serum amyloids (SAAs) can penetrate the blood-brain barrier and are elevated in AD patients, they may represent a critical link in pathogenic gut-brain immune signaling. Therefore, we aim to elucidate the mode-of-action and systemic effects of the broader class of SAAs following peripheral inflammation. By exposing transgenic mouse models to intraperitoneal curli injections or dextran sulphate sodium (DSS)-induced colitis, we will explore inflammation-driven changes in the brain’s immune cell composition through multiplex immunofluorescence and spatial transcript mapping. In parallel, we will dissect amyloid-induced neuroimmune signaling pathways in human cell models using specific compounds that modulate SAA expression. By integrating in vivo and in vitro approaches, we intend to shed light on how peripheral inflammation shapes central neuropathology, with a specific focus on the role of SAAs in mediating gut-brain communication. Techniques such as cell culture, tissue prelevations from model mice, immunofluorescent stainings, microscopy and qPCR will be regularly used within this project.

Key words: Neurodegeneration, Enteric nervous system, Gut-brain axis, Inflammation, Microscopy

Uptake and immune sensing of bacterial amyloids in the intestinal mucosa

Tutor: Dizzy Deneve - Promoters: Peter Verstraelen, Winnok De Vos

There is an intricate crosstalk between the gut and the brain in both health and disease, with gut microbiota acting as key mediators. Among microbial factors, bacterial amyloids such as curli act as pathogen-associated molecular patterns (PAMPs) that trigger immune responses locally in the gut and systemically, where they have been linked to the progression of neurodegenerative disorders such as Alzheimer’s disease (AD). Yet, the exact pathogenic mechanisms of curli remain poorly understood. Therefore, we will investigate how bacterial amyloids are taken up and sensed by the gut’s nervous system and immune cells. To this end, we will use human intestinal organoids, derived from both healthy controls and AD patients, and transgenic mouse models for AD. In vitro, enteric neurospheres will be stimulated with curli to identify responsive cell types and characterize their activation states. In vivo, curli uptake will be studied under normal conditions and following intestinal barrier disruption (as occurs during colitis), allowing us to track how bacterial amyloids are internalized and processed. Immune cells that interact with curli will be identified and further characterized to understand their role in gut–brain axis communication.

Standard techniques used throughout the project include cell culture, tissue collection, qPCR, (immuno)fluorescent staining, and confocal microscopy, providing a combination of cellular, molecular, and imaging readouts. Through the integration of advanced organoid models and in vivo studies, this project will provide new insights into how microbial amyloids shape gut–brain communication and contribute to neuroinflammatory processes underlying neurodegenerative disease.

Key words: microbiome-gut-brain axis, bacterial amyloids, immune sensing, curli uptake

Blood platelets as biomarkers for Alzheimer’s Disease

Tutor: TBD – Promoter: Frederik Denorme

Alzheimer’s disease is often diagnosed only after significant neurodegeneration, limiting treatment opportunities. We propose that blood platelets, which share molecular features with neurons, can serve as minimally invasive biomarkers for pathological processes such as amyloid-beta and tau dysregulation. We hypothesize that platelet-based biomarkers allow earlier and more accurate detection of Alzheimer’s disease, improve distinction from other dementias, and provide a cost-effective tool to monitor disease progression and therapeutic response. Establishing platelets as a reliable biomarker source could transform diagnostic practice and support the development of new therapies. In this project, we will use murine models of Alzheimer’s disease which we will follow over time. Platelets will be isolated at defined stages of disease progression and subsequently profiled for Alzheimer’s disease–associated biomarkers using transcriptomic (qPCR) and proteomic (Western blotting) analyses. In parallel, in vitro and in vivo functional assays will be performed to investigate if Alzheimer’s disease progression affects the haemostatic and thrombotic potential of platelets.

Key words: platelets, neurodegeneration, thrombosis, bleeding, qPCR, western blotting, flow cytometry.

Characterization of the interplay between glioma-associated macrophages and tumor cells in human model systems

Tutor: Mariken Lathouwers – Promotor: Winnok De Vos

Glioblastoma (GBM) is a highly aggressive brain tumor characterized by infiltrative cancer cells that display significant phenotypic plasticity and an immunosuppressive microenvironment. Key players of this microenvironment are brain-resident microglia and bone marrow-derived macrophages, together known as the glioma-associated macrophages (GAMs), which are recruited and reprogrammed by the tumor to support its growth and survival. To better understand the interactions between cancer cells and GAMs, we are developing a model that recapitulates this interplay in a fully human 3D context. To this end, we generate both types of GAMs from human induced pluripotent stem cells and co-culture them with patient-derived glioblastoma stem-like cells (GSC) in 2D and 3D systems. We are currently mapping the molecular identity and cell state of the GAM and GSC populations, using cyclic immunofluorescence and molecular assays. Thereafter, we intend to document the impact of co-culture on their phenotype using single cell sequencing and to study their migration and functional behavior using live cell imaging.  Once optimized we will assess the impact of experimental aging paradigms on these interactions and evaluate the potential of pharmacological interventions aimed at re-educating GAM. This way we hope to unveil novel therapeutic entry points for a disease that still has no cure. Throughout the project, the main techniques consist of iPSC differentiation, organoids, immunofluorescence, high-resolution microcopy, live cell imaging, qPCR, western blotting, and sequencing.

Key words: Glioblastoma, glioma-associated macrophages, iPSC-derived organoids

Super-resolved analysis of DNA damage repair in glioblastoma

Tutor: Mirthe Vandenputte - Promoter: Winnok De Vos

Glioblastoma is among the most aggressive and lethal cancers worldwide. Despite intensive multimodal treatments, including fractionated radiotherapy, recurrence is almost inevitable primarily due to the persistence of glioma stem-like cells (GSCs), which exhibit strong intrinsic or acquired radioresistance. To address this challenge, more targeted and effective high-energy radiation regimens are currently under investigation. However, a major barrier to their clinical implementation is the limited insight into the complex DNA damage and repair mechanisms. Gaining a clearer understanding of how GSCs respond to irradiation requires precise quantification of DNA repair pathways and their interactions, ideally at the level of individual DNA lesions. To this end, we have optimized an expansion microscopy protocol that enables the quantification of repair factor recruitment and dissolution at DNA damage sites with near-nanoscale resolution. This powerful imaging approach will be complemented by a range of molecular biology techniques, including Western blotting and qPCR, to profile signaling and gene expression changes in response to various DNA-damaging agents and radiation. The established methodology will be applied to a panel of patient-derived GSCs with diverse genetic and phenotypic backgrounds. By correlating molecular responses to cellular phenotypes, tumor characteristics, and transcriptomic data, we aim to gain deeper mechanistic insight into the DNA damage response and molecular rewiring of glioblastoma that drives treatment resistance and tumor recurrence.

Key words: DNA damage, Radiation, Glioblastoma, Expansion microscopy

Nuclear envelope stress in glioblastoma

Tutor: Isa Decuypere - Promoter: Winnok De Vos

Glioblastoma (GBM) is the most common and aggressive primary brain tumor, with a median survival of only 15 months after diagnosis and no cure. A key challenge in treating GBM is its heterogeneity, as glioma stem-like cells (GSCs) can self-renew, change phenotype, and interact with the surrounding tumor microenvironment. While intensive GBM research has revealed a variety of molecular alterations, less attention has been given to the altered biophysical properties. Yet, GSCs experience vastly different mechanical forces than their non-transformed counterparts. We and others have found that these forces cause nuclear envelope (NE) stress disrupting cell homeostasis and triggering DNA damage and inflammation. To understand the impact of NE stress on GBM aggressiveness, we are now investigating how GSCs respond to mechanically induced NE stress and to what extent their vulnerability to NE stress correlates with cell state plasticity. Within the lab we have optimized an approach to mechanically compress cells and evoke NE stress. During this master thesis we will use this approach to shed light on the transcriptional and proteomics rewiring of GSCs and astrocytoma cell lines stably expressing NE stress reporters. To this end we will use a combination of live cell imaging, post-hoc immunostaining and RNA sequencing. In parallel we will investigate the relationship between NE stress and invasive potential using collagen and organoid invasion assays. This way, we intend to dissect the causal relationship between nuclear fragility/resilience and GBM aggressiveness and expose potential new therapeutic entry points.

Key words: glioblastoma, glioma stem-like cells, cell biology, nuclear envelope, live cell imaging, organoids

Single cell-based contextualized staging of glioblastoma cell plasticity

Tutor: Sarah De Beuckeleer/Ritik Mehta – Promoter: Winnok De Vos, Tim Van De Looverbosch

Glioblastoma (GBM) is a lethal brain cancer with no cure. Central to the pathology is a profound intra-tumoral heterogeneity and dramatic cell state plasticity driving therapy resistance and recurrence. Increasing evidence indicates that GBM cell fate critically depends on the local tumor microenvironment and intricate interactions with non-neoplastic cells. However, classical in vitro cultures fail to recapitulate this complexity, and patient-derived xenografts are limited by their non-human nature and scalability challenges, making it a daunting task to identify context-dependent GBM states and their vulnerabilities. To study GBM plasticity in a systematic and physiologically relevant manner, we are using a panel of patient-derived glioma stem-like cells, which we co-culture - either as suspension or as 3D tumoroids - with (sections of) cerebral organoids differentiated from induced pluripotent stem cells. To comprehensively map the spectrum of cell states within these complex matrices as a function of invasion and local microenvironment, we will deploy an optimized cyclic immunostaining approach based on validated marker panels and AI-enhanced image classification. To obtain in-depth understanding of the causal effect of invasive (such as mechanical) cues on cell state transitions, we will also develop transgenic cell lines that express sensitive fluorescent reporters. This way, we intend to shed light on the drivers of cell state plasticity and open avenues for the development of new targeted treatments.

Key words: glioblastoma, organoids, morphological phenotyping, image analysis, light-sheet microscopy, deep learning

Targeting Platelet-Immune Interactions in Stroke

Tutor: TBD – Promoter: Frederik Denorme

Ischemic stroke happens when a blood clot blocks blood flow to the brain, causing permanent brain damage and death. It affects about 15 million people each year, with 1 in 4 people at risk of having a stroke in their lifetime. Over the past decades, platelets have been firmly established as essential players in the pathophysiology of ischemic stroke. Despite this foundational understanding, current anti-platelet therapies remain suboptimal and are associated with a life-threatening bleeding risk. Work by our group identified platelet-immune cell cross talk as a key driver of brain injury in ischemic stroke. These platelet-immune cell interactions are unaffected by current anti-platelet agents. Our big picture goal is to find novel drugs that block these interactions and improve stroke outcomes. In this project, we will investigate how the immune receptor FCγRIIA worsens stroke outcomes by analysing brain inflammation and thrombosis as well as short and long-term stroke outcomes.

Key words: platelets, neutrophils, thrombosis, inflammation, confocal imaging, flow cytometry, neurological behaviour phenotyping.

 Pathogen-immune interactions in Pseudomonas aeruginosa and Staphylococcus spp pneumonia

Tutor: An Hotterbeekx – Promoter: Samir Kumar-Singh

Ventilator associated pneumonia (VAP) caused by Pseudomonas aeruginosa (PA) and Staphylococcus spp is a major contributor to high mortality and morbidity in the intensive care unit. In an immune-humanized mouse model we previously showed that both pathogens interact differently in the presence of a human or mouse immune system. Here the student will further investigate the pathogen-immune interactions in in vitro cell-culture based assays. To do so, the student will perform bacterial cultures, measure bacterial interspecies competition and host cell responses, perform RNA extraction and quantitative PCR and will investigate structural changes using microscopy.

Key words: Pneumonia, Pseudomonas aeruginosa, virulence factors, biofilm, antibiotic resistance