Browsing by Author "Gregg Dean, Committee Member"

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Biochemical and Functional Analysis of Homeoprotein Nkx3.1
(2006-06-08) Simmons, Steven O'Neal; Jonathan Horowitz, Committee Chair; Robert Smart, Committee Member; James Mahaffey, Committee Member; Gregg Dean, Committee Member
Nkx3.1 is a homeodomain-containing transcription factor that is expressed early in the development of the prostate gland and is likely to play an important role in the differentiation of prostatic epithelia. Loss of Nkx3.1 protein expression is often an early event in prostate tumorigenesis, and the abundance of Nkx3.1-negative epithelial cells increases with disease progression. In a number of systems, homeodomain proteins collaborate with zinc-'finger'-containing transcription factors to bind and regulate target genes. Herein I report that Nkx3.1 collaborates with Sp-family members in the regulation of prostate specific antigen (PSA) in prostate-derived cells. Nkx3.1 forms protein complexes with Sp proteins dependent on their respective DNA-binding domains and an amino-terminal segment of Nkx3.1 and Nkx3.1 negatively regulates Sp-mediated transcription via Trichostatin A-sensitive and —insensitive mechanisms through a distal portion of the PSA promoter. Nkx3.1 DNA-binding activity is not required for trans-repression of Sp-driven PSA activity. I conclude that Nkx3.1 negatively regulates Sp-mediated transcription via the tethering of histone deacetylases and other co-repressors and/or inhibiting the association of Sp proteins with co-activators. Additionally, I outline my efforts to characterize the subcellular localization of Nkx3.1. I report that Nkx3.1 is a nuclear protein and contains at least one nuclear localization signal (NLS), likely within the carboxy-terminal 20 amino acids of the homeodomain. I also show that Nkx3.1 associates with the nuclear matrix likely via a nuclear matrix targeting sequence (NMTS) that may be coincident with the NLS within Nkx3.1 homeodomain. I show further that a functionally intact Nkx3.1 homeodomain is not required for nuclear localization but is required for association with the nuclear matrix. In addition to these results I show that Nkx3.1 is associated with mitotic chromatin throughout most, if not all, of mitosis and that a functionally intact Nkx3.1 homeodomain is sufficient for inclusion within mitotic chromosomes. Finally, I report the expression of Nkx3.1 in insect and mammalian cells, including LNCaP prostate epithelial cells does not lead to the detection of Nkx3.1 DNA-binding activity in cell extracts. My inability to detect Nkx3.1 protein/DNA binding activity does not appear to be due to an inhibitory phosphorylation event or to inhibitory protein-protein interactions. I also report my efforts to identify Nkx3.1 target genes using a genome-wide approach. This genome-wide screen for putative Nkx3.1 target genes yielded 42 clones containing novel, human genomic DNA. Ten of these clones harbored unique genomic fragments, while the remaining 32 clones carried sequences that were isolated repeatedly and could be subdivided into three sequence classes. Many of the recovered sequences mapped to locations that are within or near known genes and most carried one or more consensus Nkx3.1 DNA-binding sites. Further work must be performed to corroborate that the results from this genome-wide screen represent in vivo Nkx3.1 targets.
Identification and Investigation of Conserved Innate Immune Response Genes Between Zebrafish and Humans.
(2010-12-09) Heffelfinger, Amy; Jeffrey Yoder, Committee Chair; Susan Clarke, Committee Member; Gregg Dean, Committee Member; John Rawls, Committee Member
Role of Epicutaneous Exposure to Dermatophagoides farinae in the Development of IgE-dependent and Independent Allergic Dermatitis in the Dog.
(2006-11-15) Pucheu-Haston, Cherie Marie; Thierry Olivry, Committee Member; Bruce Hammerberg, Committee Chair; Gregg Dean, Committee Member; Keith Linder, Committee Member
Atopic dermatitis (AD) is an inflammatory and pruritic allergic skin disease of humans and several species of domestic animals. It presents as a clinical syndrome with characteristic features, including a young age of onset, genetic predisposition, dermatitis of flexural skin and predisposition to secondary infections. Clinical disease is most frequently observed with IgE antibodies to environmental allergens, especially those associated with several species of housedust mite (HDM), including Dermatophagoides pteronyssinus and Dermatophagoides farinae. Although the immune response in established AD has been well studied, many questions remain regarding the development and perpetuation of clinical disease. For example, it remains unclear as to whether naturally occurring sensitization and the development of clinical disease can be induced by cutaneous exposure to HDM allergens. Most research on the role of epicutaneous allergen exposure during the sensitization phase of AD has been performed in mouse models. However, extrapolation of the results of murine studies to spontaneous human disease is limited by the existence of subtle but important differences in the cellular distribution and function of several receptors and mediators critical to the development and maintenance of cutaneous hypersensitivity. A complementary alternative model is the dog, a species in which spontaneous allergic diseases are common and artificial sensitization is both possible and commonly performed. Historically, research in this species has been limited due to the lack of canine-specific reagents, but recent advances in the development of monoclonal and polyclonal antibodies and primer sequences for use in the dog now allows research to be performed at a high level of sophistication. The current work describes the results of three experiments designed to further understanding of the role of epicutaneous allergen exposure in the development and perpetuation of AD. First, the gross, microscopic and inflammatory mediator responses of normal dog skin following IgE-mediated challenge were evaluated by intradermal injection of cross-linking anti-IgE antibodies. This study provided important baseline data and established optimized sample processing protocols for the following two studies. Second, the cutaneous and systemic response to cutaneous allergen exposure was evaluated by repeated epicutaneous application of a sonicated slurry of Dermatophagoides farinae house dust mites to the intact skin of allergy-predisposed Maltese-Beagle cross-bred dogs. This study demonstrated that both cutaneous and systemic inflammation similar to naturally-occuring AD can be induced in the dog by cutaneous exposure to a mix of mite allergens. Finally, the role of allergen proteolytic activity in facilitating epicutaneous sensitization was evaluated by repeated epicutaneous application of either proteolytically-active or —inactive Der f 1 house dust mite allergen. This study demonstrated demonstrate that cutaneous exposure to Der f 1 allergen through intact canine skin may be sufficient to induce sensitization, and suggest that this response may be facilitated by the proteolytic activity of the allergen.
T regulatory Cell Suppression of CD8+ Lymphocyte Responses During FIV Infection.
(2009-01-07) Fogle, Jonathan Edward; Fred Fuller, Committee Member; Gregg Dean, Committee Member; Ed Breitschwerdt, Committee Member; Mary B. Tompkins, Committee Chair
BSTRACT FOGLE, JONATHAN EDWARD. T regulatory Cell Suppression of CD8+ Lymphocyte Responses During FIV Infection. (under the direction of Mary Tompkins.) The action of activated CD8+ lymphocytes is critical to the control and elimination of viral pathogens. Impaired CD8+ immune responses are well recognized in lentiviral infections; however, the mechanisms underlying CD8+ impairment are incompletely understood. Using the FIV model for human AIDS, we reported previously that CD4+CD25+ Treg cells in both the acute phase and long-term, asymptomatic phase of infection are constitutively activated and suppress CD4+CD25- T cell immune responses. Building upon these observations, we tested the hypothesis that CD4+CD25+ Treg cells suppress CD8+ responses to immune stimulation during both the acute and chronic, asymptomatic stages of FIV infection. SPF cats were infected with NCSU1 FIV. During the acute stage of infection, plasma viremia as well as PBMC and LN lymphocyte phenotype was assessed at regular intervals. Unfractionated lymph node, CD4+CD25+ depleted lymph node, and CD8+ / CD4+CD25+ co-cultures were assayed for IFNï § production via a feline specific ELISpot. During the chronic, asymptomatic phase of infection, IFNï § mRNA in CD8+ lymphocytes was assessed using real time RT-PCR following CD8+ co-culture with CD4+CD25+ lymphocytes. Our results demonstrated that the CD8+ nadir at 14 days corresponds to peak plasma viremia and is followed by an increase in CD8+ number to greater than pre-infection values. Ex-vivo depletion of CD4+CD25+ lymphocytes from lymph node suspensions significantly enhanced the production of IFNï § during the acute phase of infection. Furthermore, co-culture of CD8+ lymphocytes with CD4+CD25+ lymphocytes results in suppression of CD8+ IFNï § production during both the acute and chronic stages of infection. The same observations were not evident in uninfected cats evaluated in an identical manner. These results demonstrate the profound suppressive effect of CD4+CD25+ T regulatory cells on the CD8+ immune response during the acute and chronic stages of FIV infection. Although the mechanism of CD4+CD25+ T cell-mediated suppression is controversial, there is strong evidence to suggest that, at least in some models, it occurs via a TGFb / TGFbRII signaling pathway. We hypothesize that during the early acute stage of FIV lentiviral infection, TGFï ¢ is up-regulated on the plasma membrane of Treg cells (mTGFï ¢), which engages TGFï ¢RII on the surface of antigen activated CD8+ cells thus transducing a signal through the Smad pathway for G1 cell cycle arrest (anergy) and effectively aborting CD8+ T cell expansion and a sustained CD8+ immune response. The experiments that follow demonstrate up-regulation of mTGFï ¢ in the CD4+CD25+ subset and up-regulation of TGFï ¢RII in the CD8+ subset of FIV+ cats as assessed by FACS analysis. Furthermore, we demonstrate Smad 2 phosphorylation in CD8+ targets following CD4+CD25+ / CD8+ co-culture.