Dissertation (Ph.D.)--University of South Florida, 2008. / Title from PDF of title page. Document formatted into pages; contains 181 pages. Includes bibliographical references.

Thesis (Ph.D. in Immunology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 103-118). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Dissertation (Ph.D.)--University of South Florida, 2009. / Title from PDF of title page. Document formatted into pages; contains 155 pages. Includes vita. Includes bibliographical references.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 97-109.

The insulin/IGF-1 signaling (IIS) was initially identified in C. elegansto control a developmental phenotype called dauer. Subsequently, it was realized that lifespan was extended by mutations in this pathway and became an intense focus of study. The IIS pathway regulates growth, metabolism and longevity across phylogeny and plays important roles in human disease such as cancer and diabetes. Given the large number of cellular processes that this pathway controls, understanding the regulatory mechanisms that modulate insulin/IGF-1 signaling is of paramount importance. IIS signaling is a very well-studied kinase cascade but few phosphatases in the pathway are known. Identification of these phosphatases, especially those that counteract the activity of the kinases, would provide a better insight into the regulation of this critical pathway. Study of serine/threonine phosphatases is hampered by the lack of appropriate reagents. In Chapter II, we discuss the design and results of an RNAi screen of serine/threonine phosphatases performed in C. elegans using dauer formation as a phenotypic output. We identified several strong regulators of dauer formation and in Chapter III, proceed to characterize one of the top candidates of our screen, pptr-1. We show that pptr-1 regulates the IIS and thereby affects lifespan, development and metabolism in C .elegans. pptr-1gene encodes a protein with high homology to the mammalian B56 family of PP2A regulatory subunits. PP2A is a ubiquitously expressed phosphatase that is involved in multiple cellular processes whose specificity determined by its association with distinct regulatory subunits. Our studies using C. elegans provides mechanistic insight into how the PP2A regulatory subunit PPTR-1 specifically modulates AKT-1 activity by regulating its phosphorylation status in the context of a whole organism. Furthermore, we show that this mechanism of regulation is conserved in mammals.

In a 4-cell C. elegans embryo the ventral blastomere EMS requires polarity signaling from its posterior sister cell, P2. This signaling event enables EMS to orient its division spindle along the anterior-posterior (A/P) axis and to specify the endoderm fate of its posterior daughter cell, E. Wnt pathway components have been implicated in mediating P2/EMS signaling. However, no single mutants or various mutant combinations of the Wnt pathway components disrupt EMS polarity completely. Here we describe the identification of a pathway that is defined by two tyrosine kinase related proteins, SRC-1 and MES-1, which function in parallel with Wnt signaling to specify endoderm and to orient the division axis of EMS. We show that SRC-1, a C. elegans homolog of c-Src, functions downstream of MES-1 to specifically enhance phosphotyrosine accumulation at the P2/EMS junction in order to control cell fate and mitotic spindle orientation in both the P2 and EMS cells. In the canonical Wnt pathway, GSK-3 is conserved across species and acts as a negative regulator. However, in C. elegans we find that GSK-3 functions in a positive manner and in parallel with other components in the Wnt pathway to specify endoderm during embryogenesis. In addition, we also show that GSK-3 regulates C. elegans germline development, a function of GSK-3 that is not associated with Wnt signaling. It is required for the differentiation of somatic gonadal cells as well as the regulation of meiotic cell cycle in germ cells. Our results indicate that GSK-3 modulates multiple signaling pathways to regulate both embryogenesis and germline development in C. elegans.

Activation of the basic helix-loop-helix (bHLH) gene TAL1 is the most common genetic event seen in both childhood and adult T cell acute lymphoblastic leukemia (T-ALL). Despite recent success in treating T-ALL patients, TAL1 patients do not respond well to current therapies. In hopes of leading the way to better therapies for these patients, we have sought to determine the mechanism(s) of Tal1 induced leukemia in mice. By generating a DNA-binding mutant Tal1 transgenic mouse we have determined that the DNA binding activity of Tal1 is not required to induce leukemia. We have also shown that Tal1 expression in the thymus affects thymocyte development and survival. We demonstrate that Tal1 heterodimerizes with the class I bHLH proteins E47 and HEB in our mouse models of TAL1 induced leukemia. Severe thymocyte differentiation arrest and disease acceleration in Tal1/E2A+/- and Tal1/HEB+/- mice provides genetic evidence that Tal1 causes leukemia by inhibiting the function of the transcriptional activators E47 and HEB which have been previously shown to be important in T cell development. In pre-leukemic Tal1 thymocytes, we find the co-repressor mSin3A/HDAC1 bound to the CD4 enhancer, whereas an E47/HEB/p300 complex is detected in wild type thymocytes. Furthermore, mouse Tal1 tumors are sensitive to pharmacologic inhibition of HDAC and undergo apoptosis. These data demonstrate that Tal1 induces T cell leukemia by repressing the transcriptional activity of E47/HEB and suggests that HDAC inhibitors may prove efficacious in T-ALL patients that express TAL1.

A subfamília p21 RAS de pequenas GTPases, incluindo KRAS, HRAS e NRAS, participa de muitas redes de sinalização, incluindo proliferação celular, organização do citoesqueleto e apoptose, e é o alvo mais freqüente de mutações ativadoras em câncer. Mutações germinativas em KRAS e HRAS causam graves anormalidades desenvolvimentais levando às síndromes de Noonan, cárdio-facial-cutânea e Costello, porem mutações ativadoras germinativas em NRAS não foram descritas até hoje. A síndrome autoimune linfoproliferativa (ALPS) é o mais comum defeito genético de apoptose linfocitária, cursando com autoimunidade e acúmulo excessivo de linfócitos, particularmente do tipo T + CD4- CD8-. As mutações causadoras de ALPS descritas até hoje afetam a apoptose mediada por Fas, uma das vias extrínsecas de apoptose. Nós demonstramos aqui que os principais achados clínicos de ALPS, bem como uma predisposição para tumores hematológicos, podem ser causados por uma mutação heterozigota ativadora G13D no oncogene NRAS, sem causar prejuízo na apoptose mediada por Fas. O aumento na quantidade intracelular de NRAS ativo, ligado a GTP, induziu a um aumento da sinalização na via RAF/MEK/ERK, o que suprimiu a expressão da proteína pró-apoptótica BIM, e atenuou a apoptose intrínseca mitocondrial. Desta forma, uma mutação germinativa ativadora em NRAS causou um fenótipo clinico diferente do visto em pacientes com mutações em outros membros da família p21 RAS, cursando com um defeito imunológico seletivo, sem distúrbios generalizados do desenvolvimento / The p21 RAS subfamily of small GTPases, including KRAS, HRAS, and NRAS, regulates cell proliferation, cytoskeletal organization and other signaling networks, and is the most frequent target of activating mutations in cancer. Activating germline mutations of KRAS and HRAS cause severe developmental abnormalities leading to Noonan, cardio-facial-cutaneous and Costello syndrome, but activating germline mutations of NRAS have not been reported. Autoimmune lymphoproliferative syndrome (ALPS) is the most common genetic disease of lymphocyte apoptosis and causes autoimmunity as well as excessive lymphocyte accumulation, particularly of CD4-, CD8- ab T cells. Mutations in ALPS typically affect Fas-mediated apoptosis, but certain ALPS individuals have no such mutations. We show here that the salient features of ALPS as well as a predisposition to hematological malignancies can be caused by a heterozygous germline Gly13Asp activating mutation of the NRAS oncogene that does not impair Fas-mediated apoptosis. The increase in active, GTP-bound NRAS augmented RAF/MEK/ERK signaling which markedly decreased the pro-apoptotic protein BIM and attenuated intrinsic, nonreceptor-mediated mitochondrial apoptosis. Thus, germline activating mutations in NRAS differ from other p21 Ras oncoproteins by causing selective immune abnormalities without general developmental defects

PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) by neuronal differentiation and partial growth arrest. Mouse c-myc and adenovirus E1A genes were introduced into PC12 cells to study the influence of these nuclear oncogenes on neuronal differentiation. Expression of myc and E1A blocked morphological differentiation and caused NGF to stimulate rather than inhibit cell proliferation. NGF binding to cell surface receptors, activation of ribosomal S6 kinase, and ornithine decarboxylase induction were similar in myc and E1A expressing clones compared with wild-type PC12 cells, suggesting that changes in the cellular response to NGF were at a post-receptor level. The ability of myc and E1A expression to block the transcription-dependent induction of microtubule associated proteins by NGF further suggested that these genes may inhibit differentiation by interfering with NGP's ability to regulate transcription. These results illustrate that NGF can promote either growth or differentiation of PC12 cells, and that myc or E1A alter the phenotypic responses to growth factors.

The p53 transcription factor responds to various cellular stressors by regulating the expression of numerous target genes involved in cellular processes such as cell cycle arrest, apoptosis, and senescence. As these downstream pathways are harmful to the growth and development of normal cells when prolonged or deregulated, p53 activity needs to be under tight regulatory control. The Mdm2 oncoprotein is the chief negative regulator of p53, and many mouse models have demonstrated that absence of Mdm2 expression leads to constitutive p53 activation in a variety of cell types. While unregulated p53 can be deleterious to cells, functional p53 is essential for tumor suppression, as many human cancers harbor p53 mutations and p53 knockout mice rapidly develop spontaneous tumors. Therefore, the mechanisms that control p53 regulation by Mdm2 are critical to ensure p53 activity in the appropriate cellular context. Many genetically engineered mouse models have been created to analyze p53 and Mdm2 functions and these studies have yielded valuable insights into their physiological roles. This dissertation will describe the generation and characterization of novel mutant Mdm2 mouse models and their use to interrogate the roles of p53-Mdm2 signaling in tissue homeostasis and cell stress responses. Deletion of Mdm2 in epidermal progenitor cells of the skin and hair follicles resulted in progressive hair loss and decreased skin integrity, phenotypes that are characteristic of premature aging. Furthermore, p53 protein levels, p53 target gene expression, and cellular senescence were all upregulated in the skins of these mice, and epidermal stem cell numbers and function were diminished. These results indicate that Mdm2 is necessary to limit p53 activity in adult tissues to ensure normal stem cell function. Additional mouse models used to determine the role of Mdm2 phosphorylation will also be presented. DNA damage triggers an extensive cellular response, including activation of the ATM kinase. ATM activity is necessary for p53 protein stabilization and, therefore, p53 activation, but in vivo evidence suggests that phosphorylation of p53 itself had little effect on p53 stability. ATM was previously shown to phosphorylate MDM2 at serine residue 395 (394 in mice), and we generated knock-in mutant mouse models to study the role of this posttranslational modification in vivo. Absence of this phosphorylation site led to greatly diminished p53 stability and function in response to γ-irradiation and increased spontaneous tumorigenesis in mice. Conversely, a phosphomimic model demonstrated prolonged p53 activation in cells treated with γ-irradiation, which revealed that phosphorylation of this Mdm2 residue controls the duration of the DNA damage response. Therefore, these mouse models have uncovered new roles for the p53-Mdm2 regulatory axis in vivo and will be useful reagents in future studies of posttranslational modifications in oncogene and DNA damage-induced tumorigenesis.