Doctor of Philosophy, Case Western Reserve University, 2023, Pathology

T-cells are a central component of the adaptive immune response that promote the clearance of pathogens, immune tolerance, and tissue homeostasis. T-cell development, survival, and function are coordinated by NOTCH1, T-cell receptor (TCR), and cytokine signaling in a stage and location specific manner. The strength/persistence of these signals directly influences thymocyte development and peripheral T-cell maintenance. However, mechanisms that precisely interpret these signals to transcriptional and proteostasis machinery are poorly understood. In this dissertation, I investigate the hypothesis that developmental and homeostatic T-cell signals are translated by “adaptor proteins”. Adaptors modulate expression and activity of this machinery by facilitating protein-protein interactions. Specifically, I describe how two recently discovered adaptor proteins, SEL1L and CHMP5, play critical roles in T-cell development, homeostasis, and malignancy. During thymocyte development, signals from the pre-TCR and NOTCH1 coordinately instruct β-selection to generate mature T-cells. I discovered that SEL1L, an adaptor for the ER-associated degradation complex is induced by NOTCH1 to mitigate ER stress and apoptosis during β-selection. Consequently, deletion of SEL1L abrogated early thymocyte development. This research supports that SEL1L couples developmental signals to proteostasis machinery to enable thymocyte differentiation. In the thymus, CHMP5's adaptor function was discovered to be induced by positive selection TCR signals to ensure post-selection thymocyte survival. Subsequently, I found that CHMP5 is also required for the maintenance of peripheral T-cells. CHMP5 is stabilized downstream of TCR and IL-7 signaling, and loss of CHMP5 dramatically impairs the survival of CD4 and CD8 T-cells. Because dysregulation of developmental signals in thymocytes contributes to T-cell leukemogenesis, I hypothesized that CHMP5 could facilitate thymocyte survival downstream of onco (open full item for complete abstract)

Committee: Stanley Adoro (Advisor) Subjects: Biochemistry; Biology; Cellular Biology; Immunology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 1983, Graduate School

Committee: Not Provided (Other) Subjects: Chemistry

Doctor of Philosophy, The Ohio State University, 1981, Graduate School

Committee: Not Provided (Other) Subjects: Health Sciences

Doctor of Philosophy, The Ohio State University, 1960, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 1953, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 2017, Biomedical Sciences

In medicine, successful patient treatment relies on early and accurate diagnosis. Following diagnosis disease specific and effective treatments are necessary, targeting affected cells while sparing normal tissue. While past studies have focused on genomics, the importance of transcriptomics and proteomics is increasingly understood. Proteomics, the study of proteins, will be the focus of this dissertation. Proteomics provide insight in the post transcriptional and translational regulation of proteins, information not available through the study of DNA and RNA alone. These effects play an important role in protein quantity and physiological function. It is well established that changes in protein homeostasis are associated with disease conditions, hence providing the grounds for biomarker discovery. It has been shown that if homeostasis can be restored, disease conditions can be reversed, further emphasizing the role of proteomics in therapeutic target discovery. Chapter 1 highlights the importance of proteomics in the field of biomedical research with an emphasis on clinical translational sciences in moving discoveries from bench to bedside. Chapters 2 of this dissertation describe the development of methodology for the study of archived clinical biopsy samples. Following biopsy, patient tissue is preserved with formalin fixation and paraffin embedding (FFPE) and archived. Such tissue is stable for research for decades. This document will describe a method to prepare this tissue in for proteomic studies using LC-MS/MS with a novel on-slide digestion technique to be used with manual microdissection. Then using this technique, possibility to distinguish the different thymoma subtypes with a proteomics approach was investigated to provide an objective diagnosis tool to complement the current histological diagnosis. In Chapter 3, desmoyokin, a protein found to be unique to the medulla of the thymus and not present in the cortex will be described. This protein holds g (open full item for complete abstract)

Committee: Michael Freitas PhD (Advisor); Mark Parthun PhD (Committee Member); Kun Huang PhD (Committee Member); Charles Hitchcock MD, PhD (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy, The Ohio State University, 2008, Integrated Biomedical Science

CD24 is a glycosylphosphatidylinositol (GPI) anchored cell surface glycoprotein that is expressed in hematopoietic cells and cells of the central nervous system (CNS). Although CD24 is commonly used as a maturation marker for T lymphocytes, its role in thymocyte development is not clear. CD24 has been reported to function as a co-stimulatory molecule independent of CD28 and is required for the induction of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Bone marrow chimera experiments suggest that CD24 expression on both bone marrow-derived cells and CNS cells are required for EAE development. Studies of single nucleotide polymorphisms (SNPs) suggest that CD24 is related to the risk and progression of MS. We have used a series of genetic models to delineate the role of CD24 in the pathogenesis of EAE. It has previously shown that CD24 is required for the induction of EAE. In CD24-/- mice, normal levels of myelin oligodendrocyte glycoprotein (MOG) specific T cells were primed, however these T cells were non-pathogenic. To understand this issue, we bred CD24-/- mice with 2D2 TCR transgenic mice, which bear TCR specific to MOG, and generated 2D2 TCR transgenic mice with or without CD24. Here I show that 2D2 TCR transgenic mice with CD24-deficiency (2D2+CD24-/-) have remarkably withered thymi. In peripheral lymphoid organs, transgenic T cells from 2D2+CD24-/- mice have an immature phenotype (CD4-CD8-), do not respond to MOG peptide stimulation, and fail to cause autoimmune inflammation in the CNS and optical nerves. In contrast, OTII TCR transgenic mice with CD24 deficiency (OTII+CD24-/-), which bear TCR specific to chicken ovalbumin (OVA), have normal thymi and their peripheral T cells have a normal response to OVA peptide. These data suggest that CD24 inhibits thymic deletion of myelin antigen, but not foreign antigen-reactive T cells. To understand the role of CD24 on the resident cells in the CNS during EAE development, (open full item for complete abstract)

Committee: Bai Xue-Feng PhD (Advisor); Caroline Whitacre PhD (Committee Member); Phillip Popovich PhD (Committee Member); James Waldman PhD (Committee Member) Subjects: Immunology

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