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Maren Depke Results Host Cell Gene Expression Pattern in an in vitro Infection Model SOCS1, and finally for the transcriptionally regulated genes PSMB8, TAP1, IRF1, IFI35, IFIT1, IFIT3, OAS1, and MX1. For the influence of staphylococcal infection on IFN signaling in in vivo kidney gene expression please refer to Results / Kidney Gene Expression Pattern in an in vivo Infection Model / Fig. R.2.9, page 86. Fig. R.4.8: Interferon Signaling (modified from IPA, www.ingenuity.com). Red color indicates increase of expression. More intense shade of color is used for higher absolute fold change values. The second result in significantly over-represented canonical pathways was “Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses” (p = 3.63E−08; ratio = 20/80). This pathway includes C3AR1 (fold change 3.6), receptor for complement component C3a, pentraxin PTX3 (3.7), a soluble pattern recognition receptor (PRR) and inflammation regulator involved in enhancing complement activation and clearance of apoptotic cells, toll-like receptor TLR3 (5.1) with its signal transduction molecule MYD88 (2.3), intracellular PRR RIG-1 (DDX58; 3.2) and MDA- 5 (IFIH1; 5.6), which are involved in viral double-stranded RNA recognition, and NOD1 (2.5), receptor for bacterial diaminopimelic acid with its signal transduction kinase RIPK2 (3.8). NOD1 is known to activate CASP1 (4.4), a protease cleaving the inactive IL-1β precursor. Three different 2'-5'-oligoadenylate synthetase genes OAS1/2/3 (2.5/2.4/1.9) were induced. They are part of the innate immune response, induced by interferon, and their product oligoadenylate activates RNASEL (1.6), which was additionally induced on mRNA level in this experiment. Cytokine genes IL6 (11.9), IFNB1 (3.6), IL12A (4.0), CCL5 (4.0; RANTES) are included in this pathway as end point of signal transduction starting with different TLRs via NFκB. Finally, three subunits of PI3K, phosphoinositide-3-kinase, were induced: PIK3R1 (1.7), regulatory subunit 1/alpha, PIK3R3 (2.4), regulatory subunit 3/gamma, and PIK3CA (1.7), catalytic subunit alpha polypeptide. Significant over-representation was also observed for members of the pathway “Death Receptor Signaling” (p = 1.21E−03; ratio = 12/64). The death receptor FAS (3.7) and cascade initiating caspases CASP8 (1.6) and CASP10 (2.2) were induced, but also CFLAR (2.6; FLIP) as inhibitor of CASP8/10. Ligands TNFSF10 (18.5; APO2L, TRAIL) and TNFSF15 (4.6; TL1) were strongly induced, but not their receptors (DR). Death receptor’s/TNF receptor’s signal transduction kinase RIPK1 (2.2) was induced together with MAP kinases MAP3K14 (2.0) and MAP4K4 (2.7) and IκB kinase (IKK) subunit IKBKE (2.3). Additionally, CASP3 was slightly induced 120
Maren Depke Results Host Cell Gene Expression Pattern in an in vitro Infection Model (1.6), but CASP9, the link to the intrinsic mitochondrial apoptosis pathway, was repressed (−1.6). Possibly, S9 cells were prepared to react to external apoptosis signals or give signals to infection relevant immune cells like neutrophils or macrophages. Of course, such situation does not occur in the S9 cell in vitro infection setting. Similarly, antigen presentation will not find any reaction partner in vitro. This canonical pathway was significantly overrepresented in the infection-dependent genes which were differentially expressed 6.5 h after start of infection (p = 1.21E−02; ratio = 8/39; Fig. R.4.9). Antigen presentation is divided into two parts, presentation of intracellular and extracellular antigens. Intracellular antigens are digested by cytosolic proteasome with sequential transport of peptides into the endoplasmatic reticulum (ER). These peptides are loaded to MHC-I molecules and transported via the Golgi to the cell surface for presentation. The immune-proteasome gene PSMB8 (LMP7), coding for a protein in the 20S-core of the proteasome, was induced (fold change 1.8). PSMB9 (LMP2) with a fold change of 1.499 was just marginally below the cutoff and could be regarded as induced, too. Both induced genes lead to an immune response-specific reorganization of the proteasomes’ catalytic center. Induced were the peptide transporters TAP1 (2.4) and TAP2 (2.2), the TAP-binding protein tapasin/TPN (TAPBP; 1.5), which attaches the TAP molecules to the MHC-I complex, and the ER aminopeptidases ERAP1 (1.6) and ERAP2 (4.4), which are responsible for N-terminal trimming of the peptide in the ER. At the end of this pathway, MHC-I molecules HLA-E (1.7) and HLA-F (1.8) and MHC class I-related molecule MR1 (2.0) were induced. MR1, a non-classical MHC-gene, features a strong homology to MHC-I molecules, is highly conserved in human and mouse, ubiquitously transcribed in different tissue or cell types, and speculated to present an unknown invariant ligand to certain “innate” mucosaassociated invariant T cells (MAIT cells) expressing an invariant T cell receptor (TCR) similar as innate natural killer T cells (iNKT) (Riegert et al. 1998, Hansen TH et al. 2007). Beta-2- microglobulin (B2M; MHC-I-β) as the second subunit of the MHC-I complex showed a trend of induction with a fold change of 1.3, but was not significant in statistical testing. Fig. R.4.9: Antigen Presentation (modified from IPA, www.ingenuity.com). Red color indicates increase of expression. 121
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G E N O M E W I D E C H A R A C T E
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Maren Depke C O N T E N T S GENOMEW
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Maren Depke Z U S A M M E N F A S S
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Maren Depke Zusammenfassung der Dis
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Maren Depke S U M M A R Y O F D I S
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Maren Depke Summary of Dissertation
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Maren Depke I N T R O D U C T I O N
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Maren Depke Introduction Besides op
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Maren Depke Introduction Extracellu
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Maren Depke Introduction with heigh
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Maren Depke Introduction 2005). SaP
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Maren Depke Introduction contains a
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Maren Depke Introduction via fibron
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Maren Depke Introduction cathelicid
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Maren Depke Introduction shock are
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Maren Depke Introduction STUDIES OF
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Maren Depke Introduction are effect
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Maren Depke Introduction cell stimu
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Maren Depke Introduction channel CF
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Maren Depke M A T E R I A L A N D M
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Maren Depke Material and Methods Li
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Maren Depke Material and Methods Ki
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Maren Depke Material and Methods GE
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corticosterone [pg/ml plasma] corti
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Maren Depke Results Liver Gene Expr
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lood glucose levels [nmol/l] resist
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Maren Depke D I S C U S S I O N A N
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Maren Depke Discussion and Conclusi
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Maren Depke References Athanasopoul
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Maren Depke References Byrne GI, Le
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Maren Depke References Demling R. T
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Maren Depke References Foss GS, Pry
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Maren Depke References Hagopian K,
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Maren Depke References Jin T, Bokar
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Maren Depke References Kwan T, Liu
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Maren Depke References Luster AD, U
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Maren Depke References Namiki S, Na
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Maren Depke References Puccetti P.
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Maren Depke References Siewert E, B
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Maren Depke References van den Akke
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Maren Depke References Yang XL, Sch
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Maren Depke A F F I D A V I T / E R
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Maren Depke Acknowledgments Kidney
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