Vaccine responses are in general measured by antibody titers. However, for many vaccines, antibody levels are not the correlate of protection and do not reflect the induction of immunological memory. A better parameter as indicator may be T-cells. Especially in elderly, a risk group in which waning of immunity is observed, T-cells are thought to be a more important correlate of protection. The T-cell repertoire is very unique per individual, and can present a personal signature of the immune status, indicating and may be indicative of disease susceptibility and vaccination response. In this project we aim to investigate whether we can monitor vaccination responses via T-cell receptor sequencing, whether we can detect differences between young and old individuals and if the immune repertoire may be a predictive of vaccine responsiveness. Therefore, we will make use of a unique clinical study in which we have blood samples at timepoints before and after Pneumococcal vaccination. This study will be the first step towards defining an individual’s health status and predict vaccine responses based on TCR sequencing, thereby identifying individuals at risk for infectious diseases, leading to more personal vaccination strategies to protect vulnerable older individuals.

An essential condition for organismal life is the ability to maintain an intact genome. The eukaryotic genome is under constant pressure from damaging insults that break or chemically modify the DNA. To protect the genome from this continuous damage, a plethora of DNA repair mechanisms have evolved. However, what is often overlooked is the fact that DNA in the eukaryotic nucleus is packaged into several distinct chromatin environments that, due to their different molecular and biophysical properties, each require unique responses to allow DNA damage repair to ensue safely. One such chromatin environment is heterochromatin, which forms a dense structure within the nucleus. Although originally named ‘junk DNA’, research in the past decades has revealed that heterochromatin in fact is essential to maintain nuclear integrity. Yet, surprisingly little is known about DNA damage repair in the context of heterochromatin. The aim of this proposal is to dissect the three-dimensional heterochromatin response to DNA damage, and to determine how this response promotes repair and maintains genome stability. To this end, we will employ my recently developed locus-specific DNA damage systems in Drosophila tissue, and will develop new in vitro DNA damage tools. Combining these unique in vivo and in vitro systems with proteomics, 3D-genome analyses and live imaging allows us to create a detailed picture of DNA damage-induced changes in heterochromatin-composition and -architecture, and their function in repair. Moreover, we will use these systems to define how cell cycle progression influences heterochromatic DNA damage repair. Deciphering the heterochromatin changes associated with DNA damage will have broader implications in understanding their role in other fundamental processes, such as transcription and replication. Finally, elucidating the heterochromatic-DNA damage response will be invaluable for understanding how defects in this response could lead to genomic instability and disease development in the long run.

Transformation of lysosomes is a long-known trait of cancer cells. Lysosomes are the main degradative compartments of the cell and central in regulating the delicate balance between anabolism and catabolism. How lysosome transformation occurs in cancer cells and how this translates into cancer progression is not known, but clearly of great importance. A major obstacle has been the lack of suitable imaging methods to follow single lysosomes in migrating cancer cells. Traditional ‘bulk’ microscopy yields averaged data, which hampers to distinguish ‘transformed’ lysosomes from ‘housekeeping’ lysosomes. Moreover, cancer cell migration should be studied in 3D, which poses another challenge for microscopy. Recently, I overcame these obstacles by developing a novel correlative 3D live-cell to electron microscopy (3D- CLEM) method which I applied to 3D cancer cell cultures. This resulted in 2 intriguing observations. First, I found a direct correlation between the invasive traits of cancer cells and accumulation of peripheral lysosomes at the cellular pseudopods. Second, in contrast to common theories, I found that most peripheral lysosomes are poor in lysosomal hydrolase activity and by Electron Microscopy (EM) show a drastically altered morphology. Based on these data I hypothesize that cancer cells form a special subpopulation of lysosomes that reposition to the cell periphery as a crucial step in cancer cell invasion. The aim of this proposal is to understand the role of these transformed lysosomes and establish the molecules and pathways that regulate their transformation. To this end I will advance the 3D-CLEM approach into a high throughput method to allow quantitative subcellular imaging in 3D cell cultures. I will complement this with mass spectroscopy to unravel the molecular composition of the transformed lysosomes. Moreover, I will investigate the cellular transport mechanisms that repositions lysosomes to the invading edge. These studies will lead to novel fundamental insights in lysosome biology and can reveal thus-far unexplored targets for cancer therapies.

NANOSPRESSO-NL specifically addresses the current mismatch between personalized therapeutic strategies and industrial centralized large-scale manufacture of medicines. The NANOSPRESSO-NL consortium is convinced that nucleic acid therapeutics are uniquely qualified for production in local hospital pharmacies in response to the needs of the individual patient. By switching towards a fully standardised platform formulation, quality control can be centred around the production process rather than the end product, quite similar to a popular method of decentralized high-quality espresso making.