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In Cytotoxic T Lymphocytes Hukelm University of Dundee DOCTOR OF PHILOSOPHY The Role of the Mammalian Target of Rapamycin (mTOR) in Cytotoxic T Lymphocytes Hukelmann, Jens Ludger Award date: 2014 Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. 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Oct. 2021 The Role of the Mammalian Target of Rapamycin (mTOR) in Cytotoxic T Lymphocytes Jens Ludger Hukelmann This thesis is submitted for the degree of Doctor of Philosophy (Ph.D.) to the University of Dundee September 2014 1 Contents Contents ............................................................................................................................ 1 List of figures .................................................................................................................... 7 Abbreviations .................................................................................................................. 12 Acknowledgements ......................................................................................................... 15 Declaration ...................................................................................................................... 16 Abstract ........................................................................................................................... 17 1. Introduction ................................................................................................................. 19 1.1. An introduction to the immune system ................................................................ 19 1.2. T cell development in the thymus ........................................................................ 20 1.3. T cell receptor signalling ...................................................................................... 22 1.4. T cell subpopulations ........................................................................................... 28 1.4.1. CD4+ T cells .................................................................................................. 29 1.4.2. CD8+ cytotoxic T lymphocytes ..................................................................... 30 1.4.3. CD8+ memory T cells .................................................................................... 34 1.5. Cytokine signalling .............................................................................................. 35 1.5.1. IL-2 (and other γc-cytokines) ........................................................................ 35 1.5.2. IL-12 .............................................................................................................. 41 1.6. mTOR and the integration of nutrient signalling ................................................. 44 1.6.1. Overview ....................................................................................................... 44 1.6.2. Signalling up-stream of mTOR ..................................................................... 46 1.6.3. Substrates down-stream of mTOR ................................................................ 50 2 1.6.4. mTOR control of CD8+ effector function and trafficking ............................ 54 1.7. Thesis aims ........................................................................................................... 56 1.8. Mass spectrometry ............................................................................................... 57 1.8.1. Introduction to mass spectrometry ................................................................ 57 1.8.2. Quantitative mass spectrometry based proteomics ....................................... 61 1.8.3. Computational analysis of MS-based proteomics ......................................... 64 2. Materials and Methods ................................................................................................ 67 2.1. Transgenic mice ................................................................................................... 67 2.1.1. P14 LCMV .................................................................................................... 67 2.1.2. PTENfl/fl LckCre+/-........................................................................................... 67 2.1.3. PDK1-K465E ................................................................................................ 67 2.2. Cell culture ........................................................................................................... 68 2.2.1. Reagents ........................................................................................................ 68 2.2.2. Cell culture media and solutions ................................................................... 68 2.2.3. In vitro cytotoxic T cell generation ............................................................... 69 2.2.4. Inhibitor treatments and stimulations ............................................................ 69 2.3. SDS-PAGE and Western Blotting ....................................................................... 70 2.3.1. Reagents ........................................................................................................ 70 2.3.2. Solutions ........................................................................................................ 70 2.3.3. Antibodies ..................................................................................................... 71 2.3.4. Sample preparation, SDS-PAGE and Western blotting ................................ 71 2.4. ELISA .................................................................................................................. 72 3 2.4.1. Reagents ........................................................................................................ 72 2.4.2. General ELISA protocol for CD62L and IFN-γ............................................ 72 2.5. Metabolic assays .................................................................................................. 73 2.5.1. Reagents ........................................................................................................ 73 2.5.2. O2 consumption and media acidification ...................................................... 73 2.5.3. Glutaminolysis assay ..................................................................................... 74 2.6. Quantitative Mass spectrometry ........................................................................... 75 2.6.1. Reagents ........................................................................................................ 75 2.6.2. Solutions ........................................................................................................ 75 2.6.3. Strong anion exchange .................................................................................. 75 2.6.4. Size exclusion chromatography .................................................................... 77 2.6.5. Desalting with tC18 Sep-Pak 96-well plate .................................................. 79 2.6.6. Liquid chromatography mass spectrometry analysis (LC-MS/MS) ............. 79 2.6.7. Mass Spectrometric Data Analysis by MaxQuant ........................................ 80 2.7. Flow Cytometry ................................................................................................... 81 2.7.1. Reagents ........................................................................................................ 81 2.7.2. Solutions ........................................................................................................ 81 2.7.3. Live cell staining ........................................................................................... 81 2.7.4. Fixed cell staining for intracellular IFN-γ staining ....................................... 81 2.8. Data analysis and statistical evaluation ................................................................ 82 3. Proteomic characterisation of Cytotoxic T Lymphocytes ........................................... 83 3.1. Introduction .......................................................................................................... 83 4 3.2. Results .................................................................................................................. 84 3.2.1. Label-free quantification (LFQ) based proteomics with strong anion exchange chromatography (SAX) fractionation is robust and highly reproducible 84 3.2.2. LFQ based proteomics with SAX fractionation is unbiased ......................... 86 3.2.3. Transcript and protein levels show a moderate correlation .......................... 87 3.2.4. Characterisation of CTL proteome ............................................................... 89 3.2.5. Relative quantification of key CTL molecules, nutrient transporters and protein isoforms .....................................................................................................
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