Arsenic trioxide exhibits potent antitumor effects in vitro and in vivo, and is widely used in the treatment of acute promyelocytic leukemia (APL) in humans. In addition to APL, arsenic is of potential therapeutic value for the treatment of other hematologic malignancies, including chronic myelogenous leukemia (CML). There is evidence...
Regulation of cell cycle and cell death are critical for normal cellular proliferation, tissue homeostasis, animal development, and the pathogenesis of human diseases such as cancer. Irregularities in cell cycle and cell death may result in tumorigenesis. In this dissertation, I describe my studies of a novel gene C53, which...
CD4-positive T cells express ligands for E- and P-selectins, carbohydrate-binding adhesion molecules that are induced on inflamed endothelium. The interaction between selectins and the selectin ligands facilitates the migration of T cells to sites of inflammation. The fucosyltransferase FucT-VII is essential for selectin ligand formation but the signaling and transcriptional...
The Gram-negative bacterium <em> Vibrio cholerae </em> elicits disease through the export of enterotoxins. The <em> V. cholerae </em> RTX toxin was identified due its ability to cause cell rounding. Characterization of the RTX toxin demonstrated that this cell rounding was due to the depolymerization of the actin cytoskeleton through...
Reactivation of latent human cytomegalovirus is of significant concern in immunocompromised transplant patients. However, the mechanisms controlling reactivation of latent CMV have not been understood. It is likely that reactivation is initiated by induction of IE gene expression. In this thesis, I studied molecular mechanisms leading to transcriptional activation of...
There is increasing evidence that links chronic activation of glial cells and the subsequent self-propagating cycle of neuroinflammation to the neurodegenerative progression of Alzheimer's disease (AD). Nevertheless, attempts to identify currently approved drugs capable of safely attenuating the neuroinflammatory processes in AD have proven disappointing to date, and the development...
><p><em>Vibrio cholerae</em>, the causative agent of the severe diarrheal disease cholera, secretes several "accessory" toxins including RTX toxin, which causes cross-linking of the actin cytoskeleton. The rtx locus of <em>V. cholerae</em> is located adjacent to the integrated CTX? prophage. The RTX toxin itself is encoded by the <em>rtxA</em> gene found...
Arsenic trioxide (As<sub>2</sub>0<sub>3</sub>) is an arsenic-derivative that exhibits potent growth inhibitory effects against malignant cells. The remarkable antitumor effects of As<sub>2</sub>0<sub>3</sub> in vitro and in vivo have prompted the development of various clinical trials that established its activity in acute promyelocytic leukemia (APL). As<sub>2</sub>0<sub>3</sub> is part of the standard treatment...
Most women diagnosed with epithelial ovarian carcinoma (EOC) initially present with metastases, but little is known about the regulation of ovarian tumor cell dissemination by the tumor microenvironment. Because ovarian cancer cell dissemination is mostly limited to the peritoneal cavity and often results in the accumulation of malignant ascites, the...
The primary function of the human urinary bladder is to store urine, while maintaining a permeability barrier that protects underlying tissues from noxious urinary components. Inflammatory diseases of the bladder, including urinary tract infection (UTI) and interstitial cystitis (IC), afflict millions of patients in the US annually and cause significant...
It is critical to genomic integrity that DNA is replicated completely and faithfully during each cell cycle. The essential replication factor Cdt1 is a critical protein in preventing reinitiation of replication. Overexpression of Cdt1 causes re-replication in p53 null cells and transforms NIH3T3 cells, causing them to form tumors in...
Epstein-Barr virus (EBV) is a human gamma-herpesvirus that primarily infects B cells and epithelial cells. While the glycoproteins required for entry into these two cell types differ, the gH/gL glycoprotein complex is essential for entry into both epithelial and B cells. Analysis of gH protein sequences from three gamma-herpesviruses: EBV,...
The ubiquitin-proteasome system degrades regulatory proteins and thereby controls a broad range of cellular processes such as the cell cycle, DNA-repair, gene transcription and signal transduction. The proteasome typically degrades its substrates completely into small peptides. However, biological exceptions do occur. For example, the activity of a handful of transcriptional...
Initial studies described the ability of CD4+CD25+ T regulatory (Treg) cells in suppressing autoimmune diseases in animal models. An emerging interest has focused on the potential role of Treg cells in cancer development and progression as they have been shown to suppress anti-tumor immunity. In the present study, CD4+CD25- T...
Breast cancer cells acquire many genetic alterations in apoptotic signaling pathways rendering them resistant to apoptosis. Human Epidermal growth factor Receptor-2 (HER-2/ErbB2/neu) is amplified or overexpressed in approximately 30% of breast and ovarian tumors and correlates with poor prognosis. Although HER-2 is an orphan receptor, it forms potent signaling heterodimers...
The response to reduced levels of oxygen (hypoxia) is essential for embryonic development by promoting the proper formation of vascular networks. Conversely, hypoxia also promotes the development of pathophysiological processes such as pulmonary hypertension and cancer. The main mediator of responses to hypoxia within the cell is the transcription factor...
Multiple Myeloma (MM) is an incurable plasma cell malignancy occurring within the bone marrow. Glucocorticoids (GCs) are widely used in the treatment of MM patients due to potent induction of apoptosis. Though known to occur via the glucocorticoid receptor (GR), the molecular details of GC-induced apoptosis in MM cells remain...
Prostate cancer (PCa) is the most commonly diagnosed cancer (1:6), and the second most common cause of cancer-related deaths in American men (>27,000/year). Metastasis is largely responsible for these deaths. During metastasis, tumor cells travel from the prostate to a distant organ where they establish a secondary tumor. Cell motility...
Barbed end regulation is critical to the formation of the actin-based protrusive structures, lamellipodia and filopodia. In this body of work we have chosen to focus on two main activities, filament termination by the heterodimeric Capping protein (CP) and continued filament elongation by the Ena/VASP family of proteins. We demonstrate...
Early growth response genes (Egr1-4) are induced as immediate early genes by a variety of extracellular stimuli that influence cellular growth and differentiation. Egr transcriptional regulators modulate gene expression by binding to Egr response elements (ERE) in target genes. In this study, the low affinity p75 neurotrophin receptor (p75NTR) was...
Although non-viral gene therapy has great potential for use in the lung, several problems including inefficient gene transfer and expression and the relative lack of cell-specific targeting have limited its applications. The two approaches that have been used to target genes to desired cell types are through specific ligand-receptor interactions...
HspB2 was discovered as a chaperone of muscular dystrophy protein kinase (DMPK) and originally named, DMPK binding protein (MKBP) and presently called HspB2. Functionally, MKBP binds to DMPK and stabilizes its kinase activity, thereby accelerating the progression of muscular dystrophy. Although other binding partners of HspB2 have been found, the...
Early-life seizures increase susceptibility to seizures in adulthood. However, mechanisms by which seizures in the developing brain lead to increased vulnerability to seizures and exacerbation of neurological injury in adulthood remain unknown. We test the hypothesis that upregulation of proinflammatory cytokine levels and chronic glial activation following early-life seizures result...
MHC class II-restricted T cell responses are a common driving force of autoimmune disease. Numerous therapeutic strategies target CD4+ T cells hoping to attenuate autoimmune responses and restore self-tolerance. We have previously reported that i.v. treatment with antigen-pulsed, ethylenecarbodiimide (ECDI)-fixed splenocytes (Ag-SP) is an efficient protocol to induce Ag-specific tolerance...
Cellular interactions are important for the development and maintenance of tissue structural integrity. In the developing neuroepithelium, the adherens junction proteins, N-cadherin and β-catenin, are highly enriched at the apical surface of the proliferative ventricular zone and have been suggested to have critical functions in ensuring the proper development of...
The perinucleolar compartment is a nuclear substructure that is associated with, but structurally distinct from the nucleolus. Previous studies have shown that the PNC preferentially forms in cancer cells and PNC prevalence (% of non-mitotic and non-apoptotic cells in a population with one or more PNC) is a prognostic in...
A primary limitation of non-viral gene therapy is the low level of expression achieved. One of the reasons that higher expression is not achieved is that there are many physical obstacles that prevent the DNA from reaching the nucleus to be expressed. A plasmid must cross the cell membrane, traverse...
Actin polymerization is responsible for the protrusion of filopodia and lamellipodia in immune, cancer, and other motile cells, as well as for propulsion of some intra cellular pathogens. Protrusion of a lamellipodium by the very filaments supporting the membrane load operates by the Brownian ratchet mechanism, with overall organization governed...
IL-6 is a pleiotropic cytokine involved in both immune and non-immune functions. In the immune system, IL-6 induces terminal differentiation of B cells into plasma cells, thereby promoting antibody production. IL-6 stimulates growth of T cells by enhancing the expression of the IL-2 receptor, and IL-6 also causes differentiation of...
Filopodia play a central role in proper cell navigation; they extend from the cell surface and scan the local environment to guide the cell to its proper destination. These dynamic events require precise mechanisms for the turnover of key molecules which determine the morphology of filopodia. As of yet, such...
The kinetoplastid protozoan <em>Trypanosoma brucei</em> is the causative agent of African trypanosomiasis, affecting humans, and Nagana disease, affecting cattle, prevalent in regions of sub-Saharan Africa. Our lab demonstrated that the flagellum of <em>T. brucei</em> is enriched in lipid rafts, membrane areas composed of sphingolipids, sterols, and proteins that serve as...
Tonia René Holverson The HPV genome contains a bipartite promoter system that regulates the expression of early and late gene transcripts. The early proteins include the replication proteins E1 and E2, the oncoproteins E6 and E7, and the late functioning proteins E4 and E5. The late proteins include the capsid...
Epstein Barr Virus (EBV) is associated with malignancies of lymphoid and epithelial origin. While the precise role of EBV in oncogenesis remains elusive, latent membrane protein 2A (LMP2A) is detected in all EBV-associated malignancies, implicating LMP2A in their pathogenesis. Interestingly, LMP2A is expressed in EBV-associated malignancies such as Burkitt's Lymphoma,...
The small heat shock protein αB-crystallin is expressed primarily in lens and muscle tissue, but it is also found in lung, kidney and many cancers. Regulators of αB-crystallin have been identified almost exclusively using mouse muscle and lens specific models. It has been well documented that αB-crystallin is expressed in...
Herpesviruses require axonal transport for the successful establishment of infection in peripheral ganglia (retrograde transport), and the subsequent spread to exposed body surfaces following reactivation from latency (anterograde transport). Viral progeny are assembled and spread to other tissues or hosts during egress. I focused on viral proteins which modulate directed...
Inactivation of tumor suppressors genes, which encode regulatory proteins critical for maintaining normal cellular function, is a common occurrence in cancer. Ikaros is a hematopoietic-specific zinc finger protein that functions as a differentiation regulator and has properties of a tumor suppressor. Ikaros functions to regulate gene expression as a component...
PilD is an enzyme that processes prepilins that are part of the type II protein secretion apparatus and the type IV piliation machinery. Using a <em>Legionella pneumophila pilDlacZ</em> fusion strain to measure transcription, we observed a 20% increase in beta-galactosidase levels at 30°C vs. 37°C. At 25°C and 17°C vs....
Embryonic stem cells have the potential to differentiate into ectodermal, mesodermal and endodermal derivatives. This property makes them a valuable source of tissue specific progenitor and differentiated cells that can be used for cell replacement therapy. The first goal of this thesis is to test if cardiogenesis from embryonic stem...
Plants, animals, and yeast have the capacity to produce and accumulate neutral lipids, sterol esters and triacylglycerol (TAG). However, their accumulation and the genes underlying their synthesis in intracellular microorganisms are poorly understood.
Plasmodium falciparum is a parasitic protozoan that causes the most virulent form of human malaria. During erythrocytic...
Senescence, permanent, irreversible replication arrest that occurs in all primary cells studied to date, is considered a cellular model of aging. Recently, senescence has gained attention as a potential tumor-suppressing mechanism. However, despite the obvious importance of senescence, its exact mechanisms remain unclear. One current hypothesis to explain senescence postulates...
The orderly process of hematopoietic differentiation is governed in large part by the coordinated transcription of specific genes. Deregulation of normal differentiation processes can lead to disease states including immunodeficiency, autoimmune disease, and leukemia. This research focuses on the family of transcription factors known as the early growth response (Egr)...
A tetramer model for HIV-1 Integrase (IN) with DNA representing the LTR termini was previously assembled which predicted amino acid residues on the enzyme surface that interact with the LTR termini (Chen et al, 2006). A separate structural alignment of HIV-1, SIV, and ASV INs predicted which of these residues...
Kinesin-1 is a motor protein that transports cargo along microtubules. Inside cells, the majority of kinesin-1 is regulated to conserve ATP and ensure its proper intracellular distribution and coordination with other motors. Regulated kinesin-1 is folded in half, and interactions between coiled-coil regions near the N-terminal enzymatically active heads and...
Gonadotropins LH and FSH are secreted by the pituitary in response to hypothalamic GnRH and act to stimulate ovarian follicle development, steroid production and ovulation. Gonadal steroids, including estrogen, enter the circulation and provide neuroendocrine feedback at the hypothalamus and/or pituitary. Estrogen provides negative feedback to suppress gonadotropin release throughout...
Phagocytosis is an essential mechanism for clearance of pathogens, dying cells, and other unwanted debris in order to maintain tissue health in the body. Macrophages execute this process in the peripheral immune system, but in the brain microglia act as resident macrophages to accomplish this function. In the peripheral immune...
Chagas heart disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is still regarded as a major public health problem in Central and South America. The finding of cardiac specific autoimmunity during infection in both humans and experimental animals has provided a basis for investigation of its potential role...
Herpes simplex virus (HSV) infection of host cells requires virus attachment to the cell surface and subsequent membrane fusion between the virus envelope and host cell membrane to deliver the nucleocapsid containing the viral genome into the host cell. A proposed mechanism for HSV glycoprotein-induced membrane fusion is gD binding...
Cardiovascular disease is a leading cause of morbidity and mortality worldwide. While many therapies have been used to treat vascular disease symptomatically, gene therapy offers a means of treating the molecular dysfunctions driving disease pathogenesis. Unfortunately, gene therapy has achieved only modest therapeutic success in clinical trials due to a...
Latent membrane protein 2A (LMP2A) and LMP2B are viral proteins expressed during Epstein-Barr virus (EBV) latency in EBV-infected B cells both in cell culture and in vivo. Although Lyn has been shown to be important in mediating LMP2A signaling, it is still unclear if Lyn is used preferentially or if...
The T-box transcription factor, Tbx5, is involved in heart development and congenital disease. For example mutations in human TBX5 lead to Holt-Oram syndrome, a disease characterized by a range of heart and arm malformations. Tbx5 gene activity has been perturbed in several animal models including the chicken, mouse, and zebrafish....
Epstein-Barr virus (EBV) is a human herpesvirus that is able to infect both epithelial cells as well as B lymphocytes, where the virus stablishes life-long latency in the host. Five glycoproteins are involved in infection of B cells: gp350/220 for initial tethering of the virus to the cell via CD21,...
Mechanisms of RNA Mediated Silencing in S. pombe Christina A. Lawrence Heterochromatin of eukaryotic genomes has classically been defined as condensed chromatin that is repressive to transcription and typically resides at highly repetitive regions of the genome. Genetic and molecular analyses have demonstrated that these regions are essential for genome...
The cause(s) of Aβ overproduction and accumulation in SAD are unknown; however, several lines of evidence indicate that impaired energy metabolism in the brain may be involved. Furthermore, the rate-limiting enzyme in Aβ production, BACE1, is elevated in SAD brains around amyloid plaques, indicating that BACE1 may also play a...
Desmosomes are adhesive complexes found at sites of intercellular contact that are essential for mediating cell-cell adhesion. These junctions undergo regulated assembly and reorganization during processes such as embryogenesis and wound healing. Plakophilins (PKPs) are armadillo family members related to the classic cadherin-associated protein p120ctn. PKPs localize to the cytoplasmic...
TGF-β has been named the molecular Jekyll and Hyde of cancer due to its ability to both suppress and promote tumor development. Components of the TGF-β signaling pathway are often mutated in cancer to inhibit the tumor suppressor roles of TGF-β. Our lab studies how a 3-alanine deletion in the...
Fusion of biological membranes is dictated by the interaction between specialized membrane proteins and the lipid bilayer. Parainfluenza virus 5 (PIV5) mediates fusion using two surface glycoproteins: the fusion protein (F) and the attachment protein hemagglutinin-neuraminidase (HN). Activation of membrane fusion of PIV5 typically occurs at neutral pH, and involves...
Early Growth Response genes (Egr1-4) are transcriptional mediators of signals governing growth and differentiation. In sympathetic neuron-derived cell lines, Egr1 is induced by NGF signaling, which is required for sympathetic neuron survival, differentiation and target organ innervation. In the absence of identifiable sympathetic defects in Egr1-deficient mice, we investigated the...
PRC is a PGC-1 coactivator family member responsive to serum growth factors and up regulated in proliferating cells. Unlike PGC-1α and PGC-1β, PRC has not been studied extensively and and its function or regulation remains largely unknown. Both PGC-1α and PGC-1β have been shown to be important regulators of mitochondrial...
The actions of luteinizing hormone (LH) to induce ovulation and luteinization of preovulatory follicles are mediated principally by activation of cAMP-dependent protein kinase (PKA) in granulosa cells. PKA activity is targeted to specific cellular locations by A-kinase anchoring proteins (AKAPs). I previously showed that follicle-stimulating hormone (FSH) induces expression of...
Embryonic stem cells have the potential to differentiate into ectodermal, mesodermal and endodermal derivatives. This property makes them a valuable source of tissue specific progenitor and differentiated cells that can be used for cell replacement therapy. The first goal of this thesis is to test if cardiogenesis from embryonic stem...