Several studies have highlighted the uniqueness of the human immune system in early life. Due to the scarceness of human fetal tissues and technical limitations, a system-wide and detailed... Show moreSeveral studies have highlighted the uniqueness of the human immune system in early life. Due to the scarceness of human fetal tissues and technical limitations, a system-wide and detailed phenotypical characterization of the composition and development of the human fetal immune system was lacking. Here, I delineate the composition and development of the human fetal immune system using an array of advanced high-throughput technologies. First, mass cytometry analysis of the innate lymphoid cells revealed a previously unrecognized subset named int-ILC in the fetal intestine, which can give rise to NK cells and ILC3s. Moreover, by combining the acquired datasets from (imaging-) mass cytometry, single-cell RNA-sequencing and TCR sequencing with advanced computational analysis tools and functional analysis this revealed that memory-like CD4+ T cells were already generated in the developing human fetal intestine, indicative of in utero exposure to foreign antigens. Additionally, (imaging-) mass cytometry analysis of the immune cells in the fetal intestine, spleen and liver revealed an early-life immune compartmentalization in these different fetal tissues. Overall, our results deepens the understanding of prenatal immunity and may ultimately be useful for the development of “early” intervention strategies to prevent the development of immune mediated diseases later in life. Show less
The work described in this thesis is mainly focusing on setting up and application of a quantitative activity‐based proteasome profiling method. Chapter 1 provides a general introduction on the... Show moreThe work described in this thesis is mainly focusing on setting up and application of a quantitative activity‐based proteasome profiling method. Chapter 1 provides a general introduction on the ubiquitin proteasome system (UPS) and activity‐based proteasome profiling. Chapter 2 is a literature review of some new achievements in the activity‐based protein profiling field in the recent years, focusing on application in biochemistry, molecular and cellular biology, medicinal chemistry, pathology, physiology and pharmacology research. Chapter 3 is a protocol for performing quantitative activity‐based proteasome profiling experiments. In the protocol, both high throughput fluorescent ABPP and biotinylated probe plus LC/MS approaches are described. Chapter 4 is a brief technical report about bioorthogonal chemistry in ABPP. The commonly used secondary azide group is compared with a primary azide group in proteasome ABPs performing Cu(I) catalyzed azide‐alkyne cycloaddition and Staudinger‐Bertozzi reaction under native/denatured protein conditions Chapter 5 is focusing on the application of quantitative activity‐based proteasome profiling in the prognosis of cancer therapeutics. A combination of ABPP and global proteomics is performed to elucidate the bortezomib sensitivity and resistance mechanisms in leukemia and solid tumor cells. Chapter 6 describes the characterization of the newly discovered proteasome subunit β5t by ABPP and LC/MS proteomics. The subunit is proven to be catalytically active. A hydrophilic Thr residue on the P2 position of the proteasome inhibitor improves the inhibitory efficiency of β5t, which indicates it might prefer to cleave hydrophilic peptides. Chapter 7 describes the identification of O‐GlcNAcylation modifications on the ubiquitin receptor protein hHR23B and characterization of how the sugar moiety influences the conformation and functions of the protein. Show less
