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
The development of multicellular organisms involves an important balance between cell growth, cell division and cell death. In animals, programmed cell death (PCD) plays a key role by forming and... Show moreThe development of multicellular organisms involves an important balance between cell growth, cell division and cell death. In animals, programmed cell death (PCD) plays a key role by forming and deleting structures, controlling cell numbers and eliminating abnormal damaged cells. Caspases were found to be the key executioner of the cell suicide pathway. In plants, PCD plays an important role in development and in the responses to pathogens and abiotic stresses. Some common features of PCD were found to be conserved in both plants and animals (cytoplasm shrinkage, cytochrome c leakage out of mitochondria, chromatin condensation, altered nuclear morphology, DNA fragmentation in large fragments and DNA laddering). This indicates that a similar apoptotic machinery may be present in plants. After the elucidation of the complete sequences of Arabidopsis and rice, it has become clear that no genes for the caspases are present in plants. In this thesis, three model systems are described to determine the existence of caspase-like activities in plants during PCD. The first model used is a T-DNA integration arabidopsis mutant that shows necrotic spots on its leaves. The second model used is barley, during the transition from multicellular structures to globular embryos during androgenesis. The third model used was an artificial model of rice suspension cells with heat-shock as a PCD inducer. In these models, PCD was described and caspase-like activity was measured. In addition, the development and implementation of the protocol to purify plant caspase-3 like activity is described. Show less