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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

IDENTIFICATION OF SUMOYLATED PROTEINS AND INVESTIGATION OF PROTEIN UBIQUITINATION IN THE NF-κB PATHWAY

Liu, Xiaoyan 01 January 2012 (has links)
SUMOylation and ubiquitination are important post-translational modifications. While ubiquitination is well known for targeting proteins for degradation, SUMOylation often regulates the intracellular localization of substrates. In the first project of this dissertation, we developed proteomic strategies to identify novel SUMOylated proteins in mammalian cells. In the second project, we investigated the regulation of protein ubiquitination in the NF-κB signaling pathway in the context of Paget’s disease of bone (PDB). Identification of SUMOylated proteins has been a challenge because of low abundance of SUMOylation substrates. Here, we utilized a mass spectrometry (MS)-based proteomic approach to identify novel SUMOylated proteins in mammalian cells. Seventy-four unique proteins were commonly identified in the collection of four SUMO-1 plasmids, thus considered candidate SUMOylated proteins. Many of these proteins are associated with the nucleus. The results were validated by confirming SUMOylation of a novel substrate Drebrin and a well known substrate Ran-GAP1. Furthermore, the potential SUMOylation sites in Drebrin have been identified and confirmed using site-directed mutagenesis. PDB is a disorder characterized by increased bone turnover containing hyperactive osteoclasts. Mutations in Sequestosome 1 (p62) are associated with 40% of familial PDB. P62 is a scaffold protein and plays a critical role in regulating ubiquitination of TRAF family signaling molecules and mediating the activation of NF-κB by RANK and TNFα ligands. P62 also plays a critical role in shuttling substrates for autophagic degradation. The objective of this project is to determine the effects of PDB-associated p62 mutants on NF-κB signaling and autophagy. We compared the effect of wild-type (WT) p62 and PDB mutations (A381V, M404V and P392L) on the TNFα-induced NF-κB signaling using an NF-κB luciferase assay. Our results show that these p62 mutations increased the NF-κB signaling. In addition, we found that the PDB mutations did not change the interaction between p62 and the autophagy marker protein LC3. In summary, the PDB mutations in p62 are likely gain-of-function mutations that can increase NF-κB signaling and potentially contribute to disease progression. Based on the results, we proposed a model to speculate the synergetic role of p62 PDB mutant on NF-κB signaling and autophagy.
2

Roles of Endothelial Cell Heat Shock Protein A12B and β-glucan, a reagent for trained Immunity in the Regulation of Inflammation in Sepsis

Tu, Fei 01 August 2020 (has links)
Sepsis is dysregulated host immune response to infection causing life-threatening organ dysfunction. Endothelial cell dysfunction and uncontrolled inflammatory responses are two contributors for sepsis-induced mortality. The crosstalk between endothelial and immune cells plays a critical role in the pathophysiology of sepsis. Therefore, understanding the mechanism of interaction between endothelial and immune cells will provide novel information to develop therapeutic strategies for sepsis. Pathogen associated moleculear patterns (PAMPs) and/or damage associated molecular patterns (DAMPs) produced during sepsis, activate endothelial cells to increase the expression of adhesion molecules, attracting immune cell infiltration into the tissues. Uncontrolled inflammatory responses during the early phase of sepsis contribute to organ failure and lethality. Over 100 clinical trials, targeting inflammatory responses in sepsis, have failed in the past three decades. Thereby, developing novel therapeutic strategies for sepsis are urgent. Heat shock protein A12B (HSPA12B), as one member of HSP70 family, predominately expressed in the endothelial cells, plays important roles in many pathophysiological processes. Currently, we observed endothelial cell specific HSPA12B deficiency (HSPA12B-/-) exacerbates mortality in sepsis induced by cecal ligation puncture (CLP). HSPA12B-/- septic mice exhibits increased expressions of adhesion molecule and infiltrated macrophages in the myocardium and activated macrophages in the peritoneal cavity. In vitro studies show that HSPA12B could be secreted from endothelial cells via exosome. HSPA12B carried by exosomes can be uptaken by macrophages to downregulate macrophage NF-kB activation and pro-inflammatory cytokine production. Trained immunity, induced by β-glucan, causes immune memory in innate immune cells, with an altered response towards another challenge. We have found that mice received β-glucan seven days before CLP sepsis exhibit attenuated mortality with decreased pro-inflammatory responses. We found that β-glucan significantly increased the levels of HSPA12B in endothelial cells and endothelial exosomes. β-glucan induced endothelial exosomes markedly suppress macrophage NF-kB activation and pro-inflammatory responses. The current data suggests that HSPA12B plays a novel role in the regulation of immune and inflammatory responses and that HSPA12B could be an important mediator for the crosstalk between endothelial cells and macrophages during sepsis. β-glucan regulates endothelial cell functions and immune/inflammatory responses, thus improving survival outcome in CLP sepsis.

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