Recent advances in our understanding of immunotherapeutic strategies against cancer and the development of improved analysis tools and computing power have led to a highly anticipated progression... Show moreRecent advances in our understanding of immunotherapeutic strategies against cancer and the development of improved analysis tools and computing power have led to a highly anticipated progression of the cancer-vaccine field. The possibilities of personalized approaches to vaccinate patients against tumor-specific antigens by the prediction of MHC-binding peptides are already in clinical studies. Currently, the field is lacking behind in antigen identification tools and optimal delivery of vaccines. The studies in this thesis are divided in two major parts which address these topics. The first part reports a novel approach for the identification of relevant MHC class I and II binding epitopes. With the combined use of exome sequencing and mass-spectrometric analysis, relevant neoantigens were characterized from MHC class I and II in murine and human models. Meanwhile, significant genomic differences were observed in the MC38 murine colorectal cancer model of different sources. The second part shows the development of a molecular approach for the tracking of ligands and peptides to improve our understanding of vaccine delivery. Therefore, bioorthogonal ‘click’ chemistry was applied for in situ ligation of fluorophores to antigen, thus providing a quantification method of processed antigens. Subsequent optimizations were explored into bioorthogonal reactions independent of copper. Show less
This work involves the development of novel technologies to interrogate the complex interactions between eight differently linked diubiquitin chains and the ubiquitin binding domains (UBDs) or full... Show moreThis work involves the development of novel technologies to interrogate the complex interactions between eight differently linked diubiquitin chains and the ubiquitin binding domains (UBDs) or full-length proteins that are able to recognize them in a linkage-specific manner. Chapter 2 describes the development of a high-throughput (HT) fluorescence polarization assay used to discover alpha-helical ubiquitin binding domains. This leads to validation of known interacting binding domains and the discovery of a novel set of K6-linkage specific UBDs. In Chapter 3, all eight diubiquitin proteins are prepared using click chemistry that renders the diubiquitin proteins immune to proteolysis by deubiquitinases (DUBs). This allowed for pulldown efforts in different cell lysates and identification of novel linkage-specific interacting proteins using tandem mass spectrometry. The procedure of this effort is outlined in Chapter 4. The study in Chapter 5 details the interaction of one of the major hits found in Chapter 3: the linkage-specific interaction of K27 diubiquitin and UCHL3. The development of a panel of specific assay reagents combined with X-ray crystallography and kinetic modelling lead to a proposed model in which the consequences of the interaction between K27Ub2 and UCHL3 are explained. In Chapter 6 future directions are offered. Show less
In this thesis, bioorthogonal chemistry is combined with correlative light-electron microscopy to selectively label and study pathogenic intracellular bacteria within the host immune cell. This... Show moreIn this thesis, bioorthogonal chemistry is combined with correlative light-electron microscopy to selectively label and study pathogenic intracellular bacteria within the host immune cell. This technique combines the ultrastructural information of transmission electron microscopy with the functional information of fluorescence light microscopy in order to investigate the host-pathogen interactions that contribute to the diseases caused by pathogenic intracellular bacteria such as Salmonella Typhimurium and Mycobacterium tuberculosis. The technique is further expanded with super-resolution microscopy by combining stochastic optical reconstruction microscopy with transmission electron microscopy. Additionally, the bioorthogonal labeling method for the study of intracellular bacteria is validated through a bead-based stability assay, demonstrating the compatibility of alkyne and azide groups to label bacterial proteins within the degradative lysosomal environment. The technique developed in this thesis may contribute to a better understanding of the mechanisms behind bacterial diseases, as well as the development of novel antibiotics and other therapies to fight these important infectious diseases. Show less
This thesis describes the use of bioorthogonal proteins in immunological settings. It provides an introduction towards the field of protein modification, which was used throughout the thesis for... Show moreThis thesis describes the use of bioorthogonal proteins in immunological settings. It provides an introduction towards the field of protein modification, which was used throughout the thesis for the expression of proteins containing unnatural amino acids. It challenges this protein expression and the subsequent purification. With this successfully challenged, the use of the protein was assessed in antigen activation studies. It was analyzed whether the unnatural amino acids were tolerated by the T cell receptor and the ligation handles of the unnatural amino acids were subsequently used for visualization purposes. Furthermore, this thesis describes the possible use of azido HRP as a new tool for immunohistology. Show less
In photoactivated chemotherapy (PACT), a biologically active compound is caged by a light-cleavable protecting group. Light irradiation leads to the release of the active species which can then... Show moreIn photoactivated chemotherapy (PACT), a biologically active compound is caged by a light-cleavable protecting group. Light irradiation leads to the release of the active species which can then interact with the cell environment to induce cell death. In recent years, ruthenium polypyridyl complexes have proved to be promising candidates for PACT. To obtain more insight in the mode of action of these potential anticancer complexes, we explored their intracellular distribution. Post-treatment fluorophore labelling via click chemistry allowed for the visualization of the non-emissive ruthenium polypyridyl complexes in fixed cells. Show less
