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Maren Depke Material and Methods Host Cell Gene Expression Pattern in an in vitro Infection Model Afterwards, the sequence sets were translated into EntrezGene records using the Rosetta Resolver annotation of December 2009, and an absolute fold change cutoff of 1.5 after combining the biological replicates was applied. Ingenuity Pathway Analysis (IPA) of gene expression data EntrezGene identifiers and fold change gene expression data of differentially expressed genes were imported to the Ingenuity Pathway Analysis tool version 8.6 (Ingenuity Systems Inc., www.ingenuity.com) and analyzed using all genes of the GeneChip Human Gene 1.0 ST array as reference set without further restriction. 54
Maren Depke Material and Methods PATHOGEN GENE EXPRESSION PROFILING Growth Media Comparison Study Bacterial cultivation, growth media, and sampling time points Staphylococcus aureus RN1HG was grown at 37°C with orbital shaking of 200 rpm in Erlenmeyer bacterial culture flasks. Culture volume did not exceed 1/5 of culture flask volume. Optical density (OD) was measured at 600 nm (Fig. M.5.1). Different media were included in this study in an international co-operation in the settings of the EU-IP-FP6-project BaSysBio (LSHG-CT2006-037469) consortium, e. g. the complete bacterial culture medium TSB, cell culture medium, minimal medium, and human serum. Here, only results from samples of the medium “pMEM” will be presented. The contents of the adapted cell culture medium pMEM (Schmidt et al. 2010) have already been listed above (see Material and Methods/Host Cell Gene Expression Pattern in an in vitro Infection Model/Bacterial growth medium, page 51). Bacterial samples were taken in the exponential growth phase and 2 h (t 2 ) and 4 h (t 4 ) after entry into stationary growth. OD600 10.00 1.00 0.10 TSB pMEM Fig. M.5.1: Example for bacterial growth in TSB and pMEM medium. 0.01 0 2 4 6 8 10 12 14 16 18 20 22 24 time / h Bacterial cell harvest and disruption At different growth phases, 5 to 15 optical density units of S. aureus RN1HG were harvested on ice with addition of at least one third volume of Killing Buffer (20 mM Tris pH 7.5, 5 mM MgCl 2 , 20 mM NaN 3 ). Pellets were flash-frozen in liquid nitrogen and stored at −70°C until cell disruption. For cell disruption, the pellet was resuspended on ice in 200 µl Killing Buffer, transferred to liquid nitrogen cooled Teflon vessels, and disintegrated mechanically at 2600 rpm for 2 min in a bead mill (Mikrodismembrator S, B. Braun Biotech International GmbH, Melsungen, Germany; now part of Sartorius AG, Göttingen, Germany). Frozen cell and buffer powder mix was resuspendend in 4 ml of 50°C pre-warmed lysis solution (4 M guanidine-thiocyanate, 25 mM sodium acetate pH 5.5, 0.5 % N-lauroylsarcosinate) until the solution appeared clear and homogeneous. Four aliquots with 1 ml each were intermittently frozen in liquid nitrogen and stored −70°C. 55
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G E N O M E W I D E C H A R A C T E
Page 3 and 4: Maren Depke C O N T E N T S GENOMEW
Page 5 and 6: Maren Depke Z U S A M M E N F A S S
Page 7 and 8: Maren Depke Zusammenfassung der Dis
Page 9 and 10: Maren Depke S U M M A R Y O F D I S
Page 11 and 12: Maren Depke Summary of Dissertation
Page 13 and 14: Maren Depke I N T R O D U C T I O N
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Page 39 and 40: Maren Depke M A T E R I A L A N D M
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Page 63 and 64: corticosterone [pg/ml plasma] corti
Page 65 and 66: Maren Depke Results Liver Gene Expr
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Page 71 and 72: Maren Depke Results Liver Gene Expr
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Page 81 and 82: Maren Depke BR1 sign DsigB vs wt BR
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Maren Depke Results Gene Expression
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Maren Depke Results Host Cell Gene
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Maren Depke Results Pathogen Gene E
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S. aureus RN1HG sample conditions a
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mean normalized intensity mean norm
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mean normalized intensity Maren Dep
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log 2 (ratio) log 2 (ratio) log 2 (
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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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