▼ Rapid interconversion between yeast, pseudohyphae and true hyphae is a hallmark of the growth of C. albicans, an opportunistic human fungal pathogen, in diverse host environments. Recently, the cAMP pathway has emerged as the central signaling pathway modulating morphogenic transitions and virulence of many pathogenic fungi, but the role of the pathway in virulence of C. albicans has not been well-studied. Therefore, the aim of this thesis is to characterize the role of the cAMP pathway in virulence of C. albicans by genetic and biochemical methods. To initiate studies on the relationship between cAMP signaling and virulence in C. albicans, we identified, cloned, characterized and disrupted the C. albicans CAP1 and PDE2 gene encoding adenylate cyclase-associated protein and high affinity phosphodiesterase, respectively. The cap1/cap1 mutant, where the cAMP pathway is partially inactivated, maintained basal levels of cAMP equivalent to the wild type but lacked pulse of cAMP during germ tube inducing conditions. The cap1/cap1 mutant is defective in filamentous growth and avirulent in a mouse model of systemic candidiasis. Conversely, the pde2/pde2 mutant, where the cAMP pathway is hyper-activated by lack of degradation of cAMP, showed increased basal levels of cAMP, which caused hyperfilamentous phenotypes. Importantly, the hyperfilamentous mutants were as avirulent as hypofilamentous mutants in a systemic model of candidiasis, indicating a need for yeast forms in candidiasis. Reduced budding of the pde2/pde2 mutants was accompanied by detrimental effects including sensitivity to nutritional starvation, defective entry into stationary phase, and reduced resistance to certain environmental stresses such as heat shock, high salt, and oxidative/nitrosative stresses. Given the importance of the cAMP pathway in virulence of C. albicans, we performed a genome-wide screening of cAMP dependent genes. The microarray data showed that during filamentous growth, the cAMP pathway stimulated a number of metabolic processes and induced genes encoding hypha-specific cell surface proteins. In contrast, various stress defense genes were found to be repressed by increased cAMP levels. The results suggest that morphological transitions and stress-responses should be coordinated by tight modulation of the cAMP signaling to adjust to various microniches in the host where C. albicans colonizes and establishes disease. Rapid interconversion between yeast, pseudohyphae and true hyphae is a hallmark of the growth of C. albicans, an opportunistic human fungal pathogen, in diverse host environments. Recently, the cAMP pathway has emerged as the central signaling pathway modulating morphogenic transitions and virulence of many pathogenic fungi, but the role of the pathway in virulence of C. albicans has not been well-studied. Therefore, the aim of this thesis is to characterize the role of the cAMP pathway in virulence of C. albicans by genetic and biochemical methods. To initiate studies on the relationship between cAMP signaling and virulence in C. albicans, we identified, cloned, characterized and disrupted the C. albicans CAP1 and PDE2 gene encoding adenylate cyclase-associated protein and high affinity phosphodiesterase, respectively. The cap1/cap1 mutant, where the cAMP pathway is partially inactivated, maintained basal levels of cAMP equivalent to the wild type but lacked pulse of cAMP during germ tube inducing conditions. The cap1/cap1 mutant is defective in filamentous growth and avirulent in a mouse model of systemic candidiasis. Conversely, the pde2/pde2 mutant, where the cAMP pathway is hyper-activated by lack of degradation of cAMP, showed increased basal levels of cAMP, which caused hyperfilamentous phenotypes. Importantly, the hyperfilamentous mutants were as avirulent as hypofilamentous mutants in a systemic model of candidiasis, indicating a need for yeast forms in candidiasis. Reduced budding of the pde2/pde2 mutants was accompanied by detrimental effects including sensitivity to nutritional starvation, defective entry into stationary phase, and reduced resistance to certain environmental stresses such as heat shock, high salt, and oxidative/nitrosative stresses. Given the importance of the cAMP pathway in virulence of C. albicans, we performed a genome-wide screening of cAMP dependent genes. The microarray data showed that during filamentous growth, the cAMP pathway stimulated a number of metabolic processes and induced genes encoding hypha-specific cell surface proteins. In contrast, various stress defense genes were found to be repressed by increased cAMP levels. The results suggest that morphological transitions and stress-responses should be coordinated by tight modulation of the cAMP signaling to adjust to various microniches in the host where C. albicans colonizes and establishes disease. Advisors/Committee Members: Sundstrom, Paula.

▼ The work presented in this dissertation aims to characterize molecular pathways involved in murine lung development, murine lung cancer development, and embryonic cancer development, in order to better understand the complex processes involved in the development and progression of lung cancer. Oligonucleotide microarray-based gene expression analysis was conducted across various stages of murine lung development to identify regulatory pathways at key developmental time points. Using Affymetrix U74Av2 Murine GeneChip® microarrays, 1,346 genes and ESTs were identified as having a significant change in expression. We also performed microarray analysis on a murine model of lung carcinogenesis, again using Affymetrix U74A GeneChips®. Epithelial lung cancers were induced in mice using NNK, a common carcinogen found in tobacco smoke, and gene expression analysis was performed on various stages of lung tumors. This analysis yielded 20 effectors involved in lung tumorigenesis and 50 effectors specific for tumor progression. We then compared the murine lung tumor results to the lung development study. This identified 24 developmentally regulated genes whose aberrant expression could contribute to tumor phenotype. To assess the relevance of murine lung cancer models in the study of human adenocarcinoma formation, we compared our murine lung tumor data to a study examining gene expression in human lung tumor samples and identified 39 genes with similar expression changes in human and murine lung tumors. This work links murine lung tumorogenesis to murine lung development and human lung tumor formation. Finally, we examined the formation of teratocarcinomas, embryonic cancers, to understand how dysregulation of the embryonic environment leads to tumor formation. We identified 3 gene methylation events by Restriction Landmark Genome Scanning that may play a role in teratocarcinoma development from embryonic stem cells. We performed microarray gene expression analysis on the tissues to determine expression changes taking place during transformation. Our study yielded 20 genes that have common expression pattern changes between ES cell cultures and teratocarcinoma cell lines, and between ES cell tumors and teratocarcinomas. The studies presented above have uncovered novel pathways involved in murine lung development, murine lung carcinogenesis, and embryonic tumor formation. These pathways have helped to further characterize lung carcinogenesis. Advisors/Committee Members: You, Ming.

▼ The cellular stress response is characterized by the expression of heat shock proteins (HSPs), and in particular hsp72. Elevated levels of hsp72 can mediate cellular stress conditioning, a state of heightened tolerance to noxious stimuli, where hsp72 persists into the post shock interval and allows cells to survive potential lethal injury. Paradoxically, increases in measles virus (MV) replication and gene expression are also seen in infected cells after induction of hsp72. The increase in MV transcription/replication has been attributed to a conserved motif on the MV nucleocapsid C-terminus, Box-3, where hsp72 can bind causing both structural and functional changes that increase transcription, replication, cytopathic effect and cell-free infectious progeny release. However, the outcome of MV-hsp72 interactions, in vivo, are completely unknown. The ability of hsp72 to increase viral gene expression may promote virulence. Conversely, HSPs have been shown to promote both innate and adaptive immune responses and may be host-protective. Using a Balb/c (H-2d) mouse model of MV-induced encephalitis we have demonstrated that heat shock (HS) increased viral clearance from brain where viral clearance is correlated to the onset of virus-specific cell mediated immune responses. Next, we ask if hsp72 expression alone can recapitulate the effects of HS. We generated a transgenic mouse (C57BL/6, H-2b) where hsp72 is overexpressed in neurons. Transgenic mice were highly susceptible to intracranial challenge with MV. In contrast, mice infected with Ed N-522D MV, a virus with an attenuated hsp72 responsiveness, showed no significant increase in mortality. The divergent outcomes are based on the mouse H-2 dependent ability to mount an effective antiviral immune response. These results suggest there would be selectional pressures on the virus to lose hsp72 responsiveness. N522D is a naturally occurring substitution frequently found in circulating wild type viruses and supports positive selection of this mutation. We establish fitness of a virus lacking hsp72 responsiveness and found that the N522D substitution diminishes fitness. Therefore, we hypothesize that immunological pressure is driving selection. Collectively, results presented here will be the first to show that hsp72 levels can directly influence the outcome of viral infection in an animal host. Advisors/Committee Members: Oglesbee, Michael J.

▼ Cyclic adenosine 5’ monophosphate (cAMP) response element binding protein-1 (CREB-1) belongs to the CREB/ATF leucine zipper family of transcription factors. CREB-1 is expressed in pro-B, pre-B, immature and mature B cells. Activation through the antigen receptor or CD40 molecule, but not with bacterial lipopolysaccharide, a B cell mitogen, results in time dependent phosphorylation of CREB-1 at Ser 119/133 . CREB-1 has been implicated in the regulation of antigen receptor induced B cell activation and cytokine signaling. Taken these evidences together, we hypothesized a potential role for CREB-1 in B cell development and functional maturation in vivo . To test this hypothesis, transgenic mice over expressing a dominant negative Ser 119-ala phosphomutant CREB-1 (mCREB-1) in B cells were generated. Analysis of these mice revealed reduced B220+IgM+ mature B cells associated with a block in pre-BI (CD43 + B220 + CD24 +(int) ) to pre-BII (CD43 + B220 + CD24 ++(high) ) transition resulting in the accumulation of pre-BI cells and decreased pre-BII, immature and mature B cells in the bone marrow. Over expression of either Bcl-2 or Bcl-xL transgenes in the mCREB-1 transgenic mice failed to rescue the B cell developmental defects. The decreased pre-BII B cell expansion in mCREB-1 transgenic mice is attributed to deregulated expression of c-Jun and JunB associated with decreased cell cycle entry from the G0/G1 phase to the S phase. More importantly, the transgenic pre-BII B cells failed to enter S phase in response to stimulation with IL-7 in vitro . In addition to the defects in the bone marrow, the transgenic mice exhibited decreased follicular B cells in the spleen and increased B1a and B1b B cell populations in the peritoneum. Further, while exhibiting normal antibody responses to T-independent antigens, defective secondary immune responses to T-dependent antigen was observed in the transgenic mice suggesting a role for CREB-1 in T-dependent immune responses. In vitro and in vivo CFSE labeling studies indicated that the increased B1 B cells were not due to altered proliferation of transgenic peritoneal B cells. Further, reconstitution analysis in recombination activation gene-2 (Rag-2) deficient recipients indicated that the increased B1 B cells in the peritoneum were attributed to neither the defective bone marrow cells nor fetal liver cells. Interestingly, the transgenic mice revealed decreased B1a B cell population in the spleen. Consistent with the increased B1 B cells, the mutant CREB-1 transgenic mice revealed increased serum IgM levels compared to wild type control littermates. These studies provide the first evidence of a role for CRE binding proteins in the differential regulation of B1 and B2 B cell development and functional maturation. Advisors/Committee Members: Muthusamy, Natarajan.

▼ Human populations are endowed with a sophisticated genetic diversity of complement C4 and its flanking genes RP, CYP21 and TNX in the RCCX modules of the major histocompatibility complex (MHC) class III region. Since the sequence polymorphisms and modular variations of the RCCX, and the polygenic and gene size variations of C4A and C4B are a potential root cause in the susceptibility to autoimmune and MHC-associated diseases, these areas are a topic of debate. Novel or improved techniques to accurately study the complex genetic patterns of the RCCX have been developed. Chromosomes with one- , two- , three- and four C4 genes were characterized in four informative families. Mutations that created polymorphic BglII and TaqI sites in RP and C4, respectively, were characterized. PshAI RFLP was developed to detect TNXB-XA recombinants, which causes congenital adrenal hyperplasia (CAH). TaqI and the novel BsaI RFLP’s revealed monomodular-L with CYP21A and bimodular structures with CYP21A-CYP21A at frequencies of 20.5% and 22.7% in 22 CAH patients, respectively. The transcript levels of C4A and C4B showed a direct correlation to the respective gene dosages in liver and kidney tissues. Primary cultures of human mesangial cells with equal numbers of C4A and C4B genes demonstrated a constitutively low expression of C4A, but not C4B. The relative level of C4B mRNA increased ~ 2- to 30-fold upon interferon-gamma induction. An analysis of 110 Caucasian SLE patients revealed increases of homozygous C4A deficiency (9.1% vs 0.7%, p=0.0008). Although homozygous C4A deficiency was inherently low in the southern Chinese population, partial C4A deficiency was increased in 291 SLE patients (15.5% vs 7.7%, p=0.007). Furthermore, the distribution of C4A gene dosages was lower in the Caucasian (p=0.0005) and Chinese (p=0.008) patient groups when compared to their respective normal controls. Therefore complete or partial deficiency of C4A is a common risk factor for SLE in two different ethnic populations. In conclusion, the genetic diversity of the RCCX modules and frequent variations in the numbers of C4A and C4B genes in humans may be related to different intrinsic strengths of the immune response and susceptibilities to autoimmune diseases. Advisors/Committee Members: Yu, Chack Yung.

▼ The immune system can be viewed as a diffuse sensory organ that is responsible for detecting and eliminating infiltrating pathogens. However, the immune system must strike a balance between limiting microbial replication and immune-induced pathology. The immune system has various regulatory mechanisms that can do this; one such immunoregulatory pathway is the bi-directional communication among the nervous, endocrine and immune system. Extrinsic factors, e.g. infection, and intrinsic factors, e.g. psychogenic stress, can activate the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous system (SNS). Activation of these systems by either stress or infection leads to the secretion of glucocorticoids (GCs), catecholamines and opioids. Chronic elevation of GCs by restraint (RST) stress modulated the immune response to an experimental influenza A/PR8 viral infection. As demonstrated by experiments contained herein, RST altered cytokine gene expression, suppressed NK cell activity, and attenuated lymphocyte trafficking. Finally, RST enhanced viral replication (probably as a consequence of the RST mediated immunomodulation). Thus, alteration of normal immunoregulatory mechanisms by RST modulated the inflammatory, innate and adaptive response to an influenza A/PR8 viral infection. Previous studies from our laboratory using a social stress paradigm, social disruption (SDR), showed that SDR induced a state of functional GC resistance in cultured splenocytes. SDR, depending on the timing of the infection relative to the SDR cycle, differentially affected immune response to an influenza viral infection. When SDR occurred concurrently with the influenza infection, cytokine gene expression in the lung was suppressed at early time points during the infection. However, cytokine gene expression returned to control levels after SDR was stopped. In contrast, SDR prior to the influenza challenge attenuated the infection-induced weight loss and corticosterone secretion. Furthermore, lung IFN-β cytokine gene expression was increased concomitantly with decreased viral replication. These data demonstrated that the timing of SDR relative to the infection significantly altered the impact on the anti-viral immune response. SDR prior to influenza A/PR8 viral infection also altered the generation of immunological memory. Assessment of the delayed-type hypersensitivity (DTH) to influenza viral antigen revealed that SDR prior to infection enhanced the generation of immunological memory. Additionally, the severity of the infection following re-challenge attenuated in the previously stressed mice compared to immune control mice. Attenuated weight loss and corticosterone secretion occurred concurrently with reduced Interleukin (IL)-6 and tumor necrosis factor (TNF)-α responses and decreased viral gene expression in the SDR mice. These data suggested that the experience of SDR prior to infection had a long term consequences on host susceptibility to a specific viral pathogen. Studies presented herein demonstrate that: 1) diverse psychological stressors differentially affect the immune system, 2) the timing of the stressor relative to the infection influences the impact on the immune response and 3) stress induced modulation of the primary response affects immunological memory. These findings revealed that neuroendocrine modulation of the immune system should be considered during disease treatment as well as during immunization protocols. Advisors/Committee Members: Sheridan, John F.

▼ Women with multiple sclerosis (MS) experience a decrease in relapse rate over the course of pregnancy, with the sharpest decline occurring during the third trimester. Abruptly following parturition, however, disease activity flares before returning to its baseline level three to six months later. As a result of these dramatic changes in disease, pregnancy and the post-partum period offer a unique opportunity to study both disease amelioration and disease exacerbation. We examined the effect of pregnancy on experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Our investigations focused on the effect of the different gestational stages on both the induction of disease as well as progression of pre-existing disease. We found that when EAE was induced in pregnant animals the clinical signs of disease were prevented while immunization with neuroantigen during the post-partum period led to increased relapse severity. These effects were not associated with alterations in lymphocyte activation or with changes in Th2 cytokine (IL-4 and IL-5) production. Instead, we observed a decrease in TNF-á and an increase in the immunoregulatory cytokine, IL-10, when animals were immunized during pregnancy, while a decrease in IL-10 occurred when the mice were immunized post-partum. Similar effects were observed when pregnancy was induced during pre-existing EAE. When pregnancy was induced prior to the onset of EAE clinical signs, disease was delayed until after parturition. When pregnancy was induced after the onset of clinical signs, the severity of disease was decreased. Thus, regardless of when pregnancy was induced, suppression of pre-existing EAE was observed. Similarly, this protection from disease was associated with an increase in IL-10. We extended these studies into a transgenic mouse model in order to determine the effect of the different gestational stages on autoreactive lymphocytes in an adjuvant free system. Each gestational stage was characterized by its own unique immunological environment, including a Th2 bias during mid pregnancy and lymphocyte hyperactivation in the post-partum period. The number of autoreactive cells present in the lymphoid organs, however, did not change. Together these investigations reveal that each gestational stage has its own distinctive effect on autoimmunity and demyelinating disease. Advisors/Committee Members: Whitacre, Caroline C.

▼ Cells from normal breast tissue have on their surface a specific protein known as HER2. Breast cancer cells over-express this protein around twenty thousand times that of normal tissue. Targeting of HER2 over-expressing tumor cells can be achieved using an agent called a monoclonal antibody (mAb) that specifically binds to over expressed HER2 on the surface of the breast cancer cells. Binding of this antibody to HER2 can potentially inhibit tumor cell growth or lead to tumor cell destruction by the immune system. Unfortunately, only 20-40% of patients with HER2 over-expressing breast cancers currently respond to this treatment. We show here that natural killer (NK) cells secrete a distinct profile of potent immune stimulatory cytokines and T cell-recruiting chemokines in response to dual stimulation with mAb-coated tumor cells and interleukin (IL)-12. Cytokine production was dependent upon synergistic signals mediated by the activating receptor for the Fc portion of IgG (Fc-gamma-RIIIa) and the IL-12 receptor expressed on NK cells. The importance of this NK cell cytokine profile was confirmed in a phase I trial of breast cancer patients receiving IL-12 in combination with the anti-HER2 mAb, trastuzumab. Upon examination of the intracellular signaling pathways within primary human NK cells responsible for the elevated cytokine production, we found that levels of activated Syk and extracellular signal-related kinase (Erk) were synergistically elevated in Fc-gamma-RIIIa and IL-12 co-stimuated NK cells as compared to either stimulation alone. Moreover, inhibitor studies suggested that these molecules were critical for synergistic produciton of IFN-gamma. Interestingly, inhibition of lipid rafts within NK cell membranes abroaged the enhanced Erk activation and subsequent IFN-gamma production, suggesting the importance of lipid raft signaling in mediating cytokine production. Confocal microscopy of NK cells confirmed co-localization of both Fc-gamma-RIIIa and the IL-12R within areas of lipid raft micro-domains. Our studies also reveal a role for the inositol phosphatase SHIP-1 in the down-regulation of Fc-gamma-RIIIa-mediated NK cell cytokine produciton. Taken together, these results enhance our overall understanding of the NK cell response to mAb-coated tumor cells and provide a rationale for our attempts to enhance NK cells function for therapeutic gain. Advisors/Committee Members: Carson, William E.

▼ Immunomodulatory therapies have been successful in MS and EAE. To determine a possible immunomodulatory target at the level of the T cell receptor/antigen/MHC interaction, the activation parameters of autoreactive T cells were assessed by differential stimulation of spleen cells from Va4/Vb8.2 TCR transgenic mice with MBP or the immunodominant epitope of MBP, NAc1-11. To determine the encephalitogenic potential of these cells, either MBP or NAc1-11 stimulated cells were adoptively transferred to syngeneic B10.PL recipients. Only recipients of NAc1-11 stimulated cells showed clinical signs of EAE. NAc1-11 stimulated cells secreted increased levels of IL-12, IFN-g and IL-2. MBP stimulated cells secreted increased IL-4 and IL-10. The MBP stimulated cells, as well as cells stimulated with an extended N-terminal peptide, NAc1-20, were able to suppress Th1 proliferation in vitro and protect from active EAE induction. In addition, a subset of CD4+ T cells, which are Vb8.2-, are being activated by MBP and NAc1-20, and this subset is protective against EAE induction. These findings support that MBP, and its N-terminal peptide NAc1-20 are capable of producing CD4+ T cells that can protect from EAE through activation of a regulatory CD4+ population and modulation of cytokine production. To determine potential immunomodulation at the level of cytokine production, the inflammatory cytokine macrophage migration inhibitory factor (MIF) was investigated. MIF has been implicated in the pathogenesis of inflammatory and autoimmune diseases. The role of MIF in the progression of EAE was explored using MIF-/- mice. Wild-type, C57BL6X129, mice showed a progressive disease course, while MIF-/- mice of the same strain exhibited acute signs but no further progression of clinical disease. MIF-/- mice displayed markedly elevated corticosterone (CORT) levels, significant decreases in the inflammatory cytokines TNF-a, IFN-g, IL-2, and IL-6, and significant increases in the anti-inflammatory cytokines IL-4 and IL-10 before, during and after EAE onset. These findings support that MIF is an important mediator of EAE progression through glucocorticoid antagonism and upregulation of the inflammatory response. Taken together, through the activation of a subset of MBP specific CD4+ Th2/regulatory cells or through targeting the cytokine MIF, new therapeutic targets for MS have been identified. Advisors/Committee Members: Whitacre, Caroline C.

▼ A dysregulated immune system that is either too weak or too strong may lead to disease states such as cancer or autoimmunity. Specifically, this dissertation presents data on a growth factor, interleukin-15 (IL-15), and its pre-clinical application for cancer treatment and its role in the pathophysiology of graft-versus-host disease (GVHD). Provision of tumor-specific T cells, or adoptive immunotherapy, has emerged as a valid approach for prevention or treatment of human cancers such as melanoma and Epstein Barr virus-positive lymphoma. To improve immunotherapy, we hypothesized that IL-15 could be applied as a single agent to enhance tumor immunity through the maintenance of tumor specific CD8+ memory T cells. Tumor-bearing mice were treated with an adoptive transfer of transgenic tumor specific T cells and then randomized to receive daily low dose IL-15 or PBS treatment. Mice receiving tumor-specific T cells and IL-15 experienced a significantly delayed tumor relapse or no relapse at all. Analysis of peripheral blood 21 days after adoptive transfer demonstrated the persistence of tumor specific T cells in mice treated with IL-15 as compared to PBS. These data support the notion that low dose exogenous IL-15 can sustain tumor-specific T cells in vivo and result in clinical benefit. IL-15 is a pleiotropic pro-inflammatory cytokine with inefficient post-transcriptional processing. We hypothesized that alteration of endogenous IL-15 gene regulation could adversely impact disease progression in the well described C57Bl/6 (B6) > (C57Bl/6 x DBA/2)F1 (B6D2F1) murine model of acute allogeneic graft versus host disease (GVHD). B6D2F1 allogeneic recipients were transplanted with B6 bone marrow cells expressing a murine IL-15 transgene (IL-15 tg) modified for efficient translation and secretion. These mice had a dramatically decreased median survival time (MST) compared to mice transplanted with wild type (wt) B6 bone marrow. Blinded analysis of histopathology revealed a significant increase in intestinal epithelial damage and inflammation of the liver. The new findings discussed here suggest that IL-15 may be a valuable component of cancer therapies that aim to expand and maintain tumor-specific T cells, but that it also may have adverse side effects when applied in the setting of allogeneic bone marrow transplantation. Advisors/Committee Members: Caligiuri, Michael A.

▼ The incidence of cancer is expected to be 1 in every 3 individuals. Onset of the disease in the population has been attributed to multiple genetic and environmental factors. Pathways leading to the development and progression of carcinomas, including head and neck squamous cell carcinomas (HNSCC) and non-small cell lung cancer (NSCLC), remain largely unknown. Common genetic alterations have been identified for many neoplasias, but many of the important genes of activation (oncogenes) or inactivation (tumor suppressor genes) have not yet been identified or characterized. This lack of identified cancer targets is in part due to limitations in detection techniques, as well as limited by the type of aberrations screened for. For example, researchers often search for mutations within a chromosomal region that is lost in order to identify candidate tumor suppressor genes. Epigenetic mechanisms, such as histone modifications and DNA methylation, have also become accepted modes of transcriptional inactivation in human malignancies, but are still in their beginning stages of evaluation in HNSCC, and have not been widely applied as an approach to identify tumor suppressor genes. The majority of DNA methylation studies in HNSCC have focused on genes previously identified as being inactivated in other cancer types. Efforts using genome-wide methylation scanning techniques, such as Restriction Landmark Genomic Scanning (RLGS) have identified novel methylation targets in HNSCC. Better understanding of the role of DNA methylation in human malignancies, as well as the targets of this epigenetic inactivation, may allow for more efficient and earlier detection screenings, as well as providing an additional mechanism for identifying important cancer-related genes. In the introductory chapter one, limited information from the literature regarding DNA methylation and HNSCC is reviewed, demonstrating the void that remains in the molecular etiology of the disease. Chapter two describes the difficulty and limitations of traditional experiments used to identify tumor suppressor genes from within regions of loss of heterozygosity (LOH). Often neglected in these studies that look for genetic mutations, DNA methylation has proven to be as important for gene silencing in cancer. In this chapter, a novel application of the genome-wide methylation technique Restriction Landmark Genomic Scanning (RLGS) is used to identify genes frequently hypermethylated in a localized region of the genome frequently lost in the progression of human neoplasia for which no tumor suppressor gene has been elucidated. TCF21 is shown to be targeted for hypermethylation in the majority of HNSCC and NSCLC samples. Chapter three describes further characterization of DNA methylation of TCF21, and determines the effect of such methylation on transcription. DNA methylation along the CpG island of TCF21 is tumor specific, and removal of methylation results in transcriptional upregulation. Chapters four and five summarize the antagonistic cancer properties elicited by TCF21 expression in cancer cell lines and how they translate in vivo. In chapter four the tumor suppressive function of TCF21 is investigated. The role of TCF21 in mesenchymal to epithelial transitions (EMT) had been described previously as important in embryogenesis and organ differentiation. The function of TCF21 in cancer had not been investigated. TCF21 expression results in a reduced tumor growth rate in vivo. In cell culture, TCF21 reduces the growth rate and ability of cancer cells to aggregate into colonies, oblivious to contact inhibition by neighboring cells. Epithelial expression patterns are restored to TCF21 positive cells. Chapter five experimentally determines a link to KiSS-1, a known metastasis suppressor gene from chromosome 1q32. TCF21 expression further results in a reduced ability to invade a collagen matrix, a protein found in the extracellular matrix that is degraded in cancer to allow for migration to distant sites. Together, these experiments substantiate the conclusions that TCF21 silencing is advantageous in cancer progression and that the dual function of TCF21 as a tumor and metastasis suppressor gene is important for human malignancies. Advisors/Committee Members: Plass, Christoph.

▼ Herpes simplex virus type 1 (HSV-1) DNA polymerase (pol) has an associated 3’ to 5’ exonuclease (exo) activity that plays an important role in maintaining the fidelity of DNA replication and viability of the virus. To assess the functional significance of the exo activity, the aspects of fidelity and translesion DNA synthesis affected by HSV-1 pol deficient in this exo activity (D368A) were examined and compared with those of wild-type pol. Because HSV-1 pol forms a stable heterodimer with UL42, a protein that stimulates the polymerizing activity and increases the processivity of pol, the impact of UL42 on fidelity was also evaluated. Pre-steady-state kinetic analysis revealed that the D368A mutation destroyed exo activity without significantly altering the inherent polymerization function. It was demonstrated that both wild-type and mutant pols could selectively incorporate correct vs. incorrect nucleotides, and exo activity enhanced nucleotide selectivity by roughly an order of magnitude under steady-state conditions. However, the primary effect of exo activity on fidelity was manifested during processive DNA synthesis, because it functioned to remove incorporated mismatches, the latter which impeded the extension ability of pol, particularly in the presence of UL42 processivity factor. UL42 did not alter the pre-steady-state rate constants for correct nucleotide incorporation, misincorporation, translocation, or nucleotide excision. However, UL42 decreased dissociation of pol from DNA, which not only made the enzyme more processive but also allowed enough time to excise replication errors. Lesions, such as abasic (AP) sites, also impeded extension beyond the sites, although both HSV-1 wild-type and exo-deficient pols could incorporate dATP opposite the AP site with reduced efficiencies compared to that for normal DNA synthesis. The exo activity facilitated sensing of the lesion through increased excision rate, thereby preventing extension through repeated incorporation and excision events. Taken together, the results of this study suggest that the associated exo activity of HSV-1 pol coordinates with the polymerizing activity by partitioning the primer terminus between the exo and polymerizing sites to proofread errors and prevent translesion synthesis, ultimately increasing the fidelity of HSV-1 DNA synthesis. Advisors/Committee Members: Parris, Deborah S.

▼ Uracil has the potential to be deleterious when introduced into DNA and because of this therapeutic approaches targeting viruses and cancer cells have been developed to increase the likelihood of uracil formation in DNA. However, organisms have evolved very efficient mechanisms to prevent uracil formation/retention in DNA. Two of the most well characterized mechanisms involve the enzymes uracil DNA glycosylase (UDG) and deoxyuridine triphosphate nucleotidohydrolase (dUTPase). In an attempt to further understand the roles of UDG and dUTPase in viruses and tumor cells, as well as determine whether these enzymes could possibly be targets of future chemotherapy, a protein transduction technique and a small interfering RNA (siRNA) approach were utilized. An inhibitor protein of UDG (UGI) coupled to the protein transduction domain of the human immunodeficiency virus type 1 Tat protein (TaT-UGI) was able to efficiently transduce multiple cell types and inhibit cellular UDG activity. UDG activity was decreased approximately 94.4 + 4.1% in U-937 cells, 29.6 + 5.6% in HeLa cells, 69.9 + 5.0% in HT29 cells, and 78.9 + 1.0% in SW620 cells. On the other hand, a double stranded siRNA synthesized against domain 3 (nucleotides 339 to 357) of human dUTPase (siRNA-dut3) was able to diminish cellular dUTPase activity in all cell types examined. dUTPase activity was decreased approximately 50.0 + 0.9% in HeLa and HT29 cells and approximately 27.0 + 11.0 in SW620 cells. While TaT-UGI decreased cellular protein concentration, dUTPase levels, and doubling times in cells treated for 72 hours with the highest concentration of TaT-UGI, siRNA-dut3 had no other statistically significant effects on the cells, other then decreasing dUTPase levels, when compared to non-treated controls. These results demonstrate that TaT-UGI and siRNA-dut3 can be used to decrease UDG and dUTPase activities, respectively, in human cells and that such an approach could be useful either alone or in combination with established therapies for anti-viral and/or cancer chemotherapy. Moreover, these reagents can be used to elucidate the functions/roles of UDG and dUTPase in normal cells and viral homeostasis so that rational therapeutic approaches can be developed targeting these enzymes. Advisors/Committee Members: Williams, Marshall V.

▼ There have been substantial advances in understanding the events that regulate gene expression at the cellular and molecular level, however, there has been limited progress integrating this information to understand how biological systems function in vivo. Complementary DNA and protein microarray technologies in combination with sophisticated bioinformatics may eventually provide important insight into how biologic systems work in vivo. We hypothesized that assessments of such events in vivo would provide new insights into the immune response that could not be predicted or discovered ex vivo. Here, we describe the use of quantitative real time RT-PCR to serially quantify expression of a variety of pro- and anti-inflammatory cytokine genes in a number of individual tissues before, during, and after challenge with lipopolysaccharide (LPS). The data provide new insight into the heterogeneity of cytokine gene expression from organ to organ following infectious insult in vivo, as well as a greater understanding of cytokine regulation. For example, the anti-inflammatory cytokine interleukin-10 (IL-10) is thought to down-regulate the effects of the pro-inflammatory cytokine interferon gamma (IFN-g) on monocyte activation following lipopolysaccharide (LPS) stimulation. However, the often-postulated reciprocal regulation of IL-10 gene expression by IFN-g has not been studied in vivo. Here we serially quantify the expression of IL-10 before, during, and after an in vivo challenge with LPS or a gram-negative organism. In our system, we demonstrate that the regulation of IL-10 gene expression has at least two phases. The early induction occurs independent of the signal transducer and activator of transcription 1 (STAT-1), while a delayed active repression of IL-10 gene expression is critically dependent on STAT-1, most or all of which is independent of IFN-g. Consistent with this, STAT-1 is absent from the IL-10 promoter during the early induction of the cytokine, but is bound to the IL-10 promoter in the delayed repression of the cytokine. Thus, STAT-1 binding to the IL-10 promoter is likely directly associated with STAT-1-mediated repression of IL-10 gene repression during infectious challenge with gram-negative organisms in vivo. This study provides new insights into the regulation of IL-10 following in vivo challenge with a gram-negative organism. Advisors/Committee Members: Caligiuri, Michael A.

▼ The two isotypes of immune effector proteins complement component C4, C4A and C4B, exhibit different biological functions. The number of C4 genes present in a diploid genome varies from 2 to 6 among different individuals. The variation of C4 gene dosage is always concurrent with three other flanking genes, including RP, CYP21 and TNX. The size of C4 genes can be long (21 kb) or short (14.6 kb), which differs in 6.4 kb due to the presence or absence of an endogenous retroviral sequence in intron 9. Complete or partial deficiency of C4A has been observed to be a risk factor for systemic lupus erythematosus (SLE). However, a deeply ingrained but inaccurate “two-locus” model of human C4 genes had impeded advances on epidemiological studies of C4 and diseases. The first objective of this dissertation was to determine the genetic complexity and protein polymorphism of C4. It was observed that there were good correlations between the number of long C4 genes with C4A protein levels, and the number of short C4 genes with C4B protein levels. The short C4B gene in the second locus not only increased the C4B protein level, but also slightly increased the C4A protein levels coded by the long C4A gene of the first locus. The second objective was to determine the molecular basis of C4 mutations from complete C4 deficiency subjects. Novel C4 mutations were discovered in seven complete deficient subjects. Sequence-specific polymerase chain reaction strategies were created for rapid screenings of known C4 mutations in human populations. The third objective was to study variations of C4 gene dosages and protein polymorphisms in Caucasian and Black SLE patients. When compared with case controls, both Caucasian and Black SLE patients had increased frequencies in deficiencies of total C4 and C4A genes. Protein levels of total C4, C4A and C4B decreased in SLE patients within the same gene dosage groups. The low protein levels of total C4 and C4A in SLE may result from low gene dosages in total C4 and/or C4A, C4A gene mutations, low C4 protein expression, and/ or high C4 protein consumption. Advisors/Committee Members: Yu, Chack-Yung.