[ { "id": "thesis:4234", "collection": "thesis", "collection_id": "4234", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10242005-165226", "primary_object_url": { "basename": "HGdissertation.pdf", "content": "final", "filesize": 3024537, "license": "other", "mime_type": "application/pdf", "url": "/4234/1/HGdissertation.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Novel Methods for Studying Ras/Erk MAP Kinase Signaling in Developing T Cells", "author": [ { "family_name": "Green", "given_name": "Harry Miguel", "clpid": "Green-Harry-Miguel" } ], "thesis_advisor": [ { "family_name": "Alberola-Ila", "given_name": "Jose", "clpid": "Alberola-Ila-J" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "clpid": "Bjorkman-P-J" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Alberola-Ila", "given_name": "Jose", "clpid": "Alberola-Ila-J" }, { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The Ras/Erk MAPK pathway has been shown to be important in multiple developmental contexts. The development of T cells in the thymus is one such developmental system. Thymocytes undergo positive and negative selection, processes by which they are \"chosen\" for their ability to recognize MHC molecules loaded with peptide on the surface of cells, but only to react when the peptide is foreign. The Ras/Erk cascade has been shown to be indispensable during the onset of positive selection, but the mechanism of Erk signaling in this process is unknown. In addition, it is unclear if the Ras/Erk cascade is involved in the differentiation phase of positive selection called CD4/CD8 lineage determination, where thymocytes either become CD4+ or CD8+ T cells. Furthermore, Erk signaling has been shown to be activated during negative selection, but seems dispensable. In this thesis, we describe novel methods for analyzing Erk signaling by applying new technologies to gain a different perspective on Erk signaling in thymocytes during selection. To this end, we have utilized a technique of intracellular staining to obtain data for single-cell Erk activation in the context of a population of fixed thymocytes. We also pursued the development of FRET-based, genetically-encoded intracellular sensors of Erk activity that could be applied to the analysis of Erk signaling in live thymocytes in vivo. To examine the involvement of Ras/Erk signaling during CD4/CD8 lineage determination, we applied a recently described method of lentiviral transgenesis to examine dose-dependent effects of a dominant negative form of Mek, the Erk MAPK kinase, in a single mouse generation. These studies have yielded insights into Erk signaling events and advanced the development of novel techniques to examine signaling during thymocyte selection.", "doi": "10.7907/8wa9-t690", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:3939", "collection": "thesis", "collection_id": "3939", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10062004-073848", "primary_object_url": { "basename": "SDBarbeeThesis.pdf", "content": "final", "filesize": 12729386, "license": "other", "mime_type": "application/pdf", "url": "/3939/1/SDBarbeeThesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "The Functions of Phosphatidylinositol 3-Kinase in T Lymphocyte Development: Roles in Positive Selection and Thymic Exit", "author": [ { "family_name": "Barbee", "given_name": "Susannah Dale", "clpid": "Barbee-Susannah-Dale" } ], "thesis_advisor": [ { "family_name": "Alberola-Ila", "given_name": "Jose", "clpid": "Alberola-Ila-J" } ], "thesis_committee": [ { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Alberola-Ila", "given_name": "Jose", "clpid": "Alberola-Ila-J" }, { "family_name": "Baltimore", "given_name": "David L.", "clpid": "Baltimore-D-L" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "clpid": "Bjorkman-P-J" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Phosphatidylinositol 3-kinase (PI3K) is an important regulator of cell survival, proliferation, activation, and migration in multiple organisms and cell types. We have sought to determine how PI3K may regulate T lymphocyte development, a process that entails exquisitely coordinated phases of proliferation, differentiation, and intra-organ movement. We have generated transgenic mice that express a PI3K gain-of-function mutant specifically in thymocytes. The p110ABD transgene constitutes the adaptor binding domain of the PI3K catalytic subunit and this fragment associates with adaptor subunits in vivo. p110ABD expression induces constitutive PI3K function, as assessed by the activity of the downstream effector Akt. Furthermore, p110ABD-induced PI3K function potentiates Ca ++ influx induced by sub-optimal crosslinking of the antigen receptor (TCR) on immature thymocytes. Enhancing PI3K activity in developing T cells results in the specific accumulation of late-stage, mature HSA lo CD3hi thymocytes of both lineages. The increased numbers of mature thymocytes can be partly attributed to an improvement in positive selection. This is demonstrated by the ability of p110ABD to promote efficient positive selection of transgenic AND TCR thymocytes in a background that mediates sub-optimal differentiation. The improvement in selection is not biased to the CD4 lineage, since CD4 lineage development is not specifically improved in class I-restricted transgenic TCR animals expressing p110ABD. Furthermore, the effect is specific to positive selection since immature thymocyte survival and negative selection are unaffected by p110ABD expression. The increased mature populations are also partly the result of impaired thymocyte emigration. p110ABD T cells colonize the periphery of neonatal animals and irradiated recipients slower than do non-transgenic T cells. The ability of PI3K to regulate positive selection effect is probably due to enhancement of Itk-mediated Ca++ influx. By contrast, the role of PI3K in emigration appears to be independent of known chemotactic or adhesive factors and may instead reflect the importance of subcellular organization for chemokine receptor signaling.", "doi": "10.7907/3bbr-b515", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" } ]