Doctor of Philosophy, The Ohio State University, 2004, Molecular, Cellular, and Developmental Biology

Ets transcription factors have been implicated in the development, regulation, and survival of numerous hematopoietic derived cells, including B and T-lymphocytes. E26 avian leukemia oncogene-2 (Ets-2) is an Ets family member transcription factor. Strong expression of Ets-2 is observed in developing thymocytes from the DN1 stage and in B-cells from the pro-B stage. A conserved sequence within the Ets-2 pointed domain amino acids 69 to 73 PLLTP is able to interact with the Map kinase ERK. Threonine seventy-two is phosphorylated by ERK1, and permits transactivation by Ets-2. Mutation of this residue from a threonine to an alanine, Ets-2 T72A, abrogates Ras mediated transactivation of Ets-2. Based on this evidence, we hypothesized that Ets-2 was important in T- and B-cell development, activation, and function. To test this hypothesis we analyzed Ets-2 activation in T-cell lines, and created two transgenic mice lines that express Ets-2 T72A in either developing T-cells or B-cells. Transactivation of Ets-2 can be induced by both Ras activation and by phorbol 12-myristate 13-acetate and ionomycin stimulation in the Jurkat T-cell line. In-vitro analysis of Ets-2 activation by ionomycin results in a rapid, but transient Ets-2 band shift as evidenced by western blot analysis of protein extracts from thymocytes. Transgenic mice that over-express Ets-2 T72A in the thymus displayed a dramatic reduction in thymus size associated with hypocellularity. This reduction was associated with a partial developmental block at the double negative 2 and double negative 3 stage of development, a twenty-fold increase in c-Kit+ expression, and a five-fold increase the CD5low population. Further, thymocytes from the transgenic mice have increased apoptosis both in-vitro and in-vivo compared to non-transgenic littermates. Transgenic mice that over-express Ets-2 T72A in B-lymphocytes revealed a loss of the B220Hi population of B-cells in the bone marrow. This population consists of mature B-cell (open full item for complete abstract)

Committee: Natarajan Muthusamy (Advisor) Subjects: Biology, Cell

Master of Science, Miami University, 2005, Microbiology

Previous research in this laboratory demonstrated that protein malnourishment causes increased levels of serum corticosterone and thymic atrophy in mice. The purpose of this study was to determine the effects of up-regulated serum corticosterone on thymocyte apoptosis, and the effects of a stress response on corticosterone-induced apoptosis. This study confirmed that protein malnourishment induces thymic atrophy. When mouse thymocytes were treated in vitro with corticosterone and assayed for apoptosis by quantifying phosphatidylserine externalization, mitochondrial permeabilization, andDNA fragmentation, corticosterone was shown to induce thymocyte apoptosis. When thymocytes from protein deficient mice were assayed for apoptosis, phosphatidylserine externalization and mitochondrial permeability were significantly altered, but DNA fragmentation was not. To determine how a stress response could alter thymocyteapoptosis, protein sufficient thymocytes were heat shocked and then treated with corticosterone in vitro. Heat shock decreased corticosterone-induced apoptosis and increased heat shock protein (hsp) 70 and hsp90 levels in these normal mouse thymocytes. However, when hsp70 and hsp90 were quantified in thymocytes from protein deficient mice, hsp70 or hsp90 were not significantly increased. In conclusion this study describes the significance of corticosterone-induced thymocyte apoptosis during protein malnourishment.

Committee: John Stevenson (Advisor) Subjects: Biology, Molecular