Year: 2012
Type: dissertation
Abstract: Proteomics is defined as large-scale study of proteins, and with current proteomic methods thousands of proteins can be characterized in a single experiment. Mass spectrometry (MS) has an important role in the characterization of complex protein samples. In addition, various bioinformatics tools have become increasingly important in the interpretation of complex proteomic data. The combination of proteomics and bioinformatics is nowadays an important tool to study cellular signaling mechanisms. When host cell recognizes the invading virus, multiple cell signaling cascades are activated resulting in antiviral immune responses, inflammation and programmed cell death of the infected cell. The detailed mechanisms of host cell defense responses activated upon viral infection are still partially unknown. The aim of this project was to develop and utilize proteomic and bioinformatic methods to characterize host responses to viral infection. Three different proteomic approaches were used in this project to study virus-induced changes in human epithelial cells and macrophages. Viral RNA-induced responses in HaCaT keratinocytes were studied using two-dimensional gel electrophoresis and MS. Influenza A virus-induced changes in the intracellular compartments and secretomes of human primary macrophages were characterized using iTRAQ labeling-based quantitative proteomics. Finally, viral RNA-triggered protein secretion from human primary macrophages was studied using SDS-PAGE, liquid chromatography and MS. In addition, two computational tools, Compid and Pripper, were developed to simplify the analysis of our proteomic data. Our studies showed that both influenza A virus and viral RNA trigger significant changes in the proteomes of macrophages and HaCaT keratinocytes. Virus-induced changes in the expression of 14-3-3 signaling proteins as well as rearrangement of host cell cytoskeleton were detected in HaCaT keratinocytes. Caspase-3-dependent apoptosis was detected in HaCaT keratinocytes and macrophages after viral stimulation. Our studies with macrophages also showed that several inflammatory pathways, and especially the NLRP3 inflammasome, are activated as a result of viral RNA and influenza A virus infection. Additionally, we showed that cathepsins, src tyrosine kinase and P2X7 receptor were involved in the inflammasome activation. Finally, we showed that viral stimulation triggered extensive protein secretion from macrophages. In conclusion, our proteomic experiments have given an in-depth view of cellular events activated in human primary macrophages and HaCaT keratinocytes after viral infection.
Source: Työväentutkimus Vuosikirja
Author: Niina Lietzén
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
Funders: Helsingin Yliopisto, Turun Yliopisto
OpenAlex ID: W1534212731