Bacteria release bacterial extracellular vesicles (BEV), which are nanometer-sized membrane particles transporting nucleic acids, metabolites, proteins and endotoxins. Recently, my host lab discovered that patients with a disrupted intestinal barrier (including cancer patients and HIV patients) have higher BEV counts in their blood circulation depicting them as valuable biomarker candidates. This discovery prompts the novel concept of exploring BEV traits in human body fluids, namely stool and blood samples, rather than in vitro bacterial cell cultures to gain unprecedented biological insights of BEV within their natural context. I aim to study their abundance, biophysical and biochemical traits and I will implement these insights to develop BEV-dedicated biomarker methodology. For this purpose, I will focus on blood plasma and stool samples from healthy donors, ovarian cancer patients, prostate cancer patients and non-cancer disease patients (HIV patients), and implement the in-house expertise for BEV separation and characterization. Furthermore, I will optimize and implement AF4 technology in combination with ICP-ToF-MS analysis for direct sensitive, accurate and multi-parametric BEV evaluation. This combined biological and technological approach holds the potential to provide a BEV-based multi-component diagnostic readout in body fluids.