Life Sciences Commons^™

Scribble: A Potential Dual Kinase Inhibitor, Steven Christofakis

Theses and Dissertations

Extracellular signal-regulated kinases (ERKs) modulate cellular activities in response to extracellular stimuli and play important biological roles. Thus, perturbed kinase pathways induce pathological conditions, such as tumor development. Rit, a novel member of the Ras family GTPases, activase ERK6, and its over-expression confers tumorigenicity. We hypothesized the presence of scaffolding molecules specific to ERK6, similar to other known MAP kinases. We performed yeast two-hybrid assays using ERK6 as bait, and Scribble was identified as a binding partner. Scribble contains 16 LRR domains and four PDZ domains. We performed immunoprecipitation (IP) assays and discovered ERK2 as another binding partner. Surprisingly, no …

Neutrophil Products Control The Expression Of Progesterone Receptors And Matrix Metalloproteinase-1 In The Decidual And Myometrium And Are Possible Regulators Of Premature Labor, Anna Solotskaya

Theses and Dissertations

Neutrophils infiltrate myometrium and decidual tissue prior to parturition. Activated neutrophils release reactive oxygen species (ROS) and tumor necrosis factor α (TNFα), which might increase expression of pro-labor genes such as matrix metalloproteinase-1 (MMP-1), progesterone receptor (PR) A/B ratio, and cause demethylation of DNA. These changes might cause labor. Decidual tissue was obtained from consented, healthy women at term (37+ weeks of gestation) not in labor (no contractions, without cervical effacement), term labor and preterm labor (under 37 weeks of pregnancy). Decidual and myometrial cells in culture were treated with (1) ROS, (2) TNFα, or (3) 5-aza-2’-deoxycytidine. Total RNA was …

Mutation Of The Vesicular Monoamine Transporter-1 Gene Alters Its Protein Product, Abena Watson-Siriboe

Theses and Dissertations

The vesicular monoamine transporter 1 (VMAT1) is essential for storage of monoamines, such as epinephrine and serotonin, in secretory vesicles of neuroendocrine cells. Recently the VMAT1 protein was detected in human and mouse brain, and mutations of the VMAT1 gene at single DNA nucleotides (single nucleotide polymorphisms or SNPs) were associated with schizophrenia. In this study, Chinese hamster ovarian cells were stably transfected with either human VMAT1 DNA (GenBank: #NM_003053.1 or DNA with the Thr4Pro SNP, which results in a threonine to proline change in amino acid number 4 of the VMAT1 protein. Western blot analysis revealed that cells with …

Tied Together: A Molecular Role For Tie1 In Angiopoietin Tie2 Signaling, Tom Cm Seegar

Theses and Dissertations

The primary function of the vascular system is the maintenance of oxygen homeostasis for all metazoan tissue. Angiogenesis, the remodeling and maintenance of new blood vessels from an existing vessel, is primarily controlled through the endothelial specific receptor tyrosine kinase Tie2, and the orphan receptor tyrosine kinase, Tie1. Although these receptors share highly conserved, genetic and biochemical analysis has shown these receptors have distinct and essential roles in angiogenesis. Tie2 activation typically results in vessel stability and quiescences and has further been shown to interact with all four sub-types of the angiopoietin signaling factors, Ang1-4. Conversely, Tie1 is involved in …

Spatial And Temporal Distribution Of Larval Fishes In A Large Tidal River., Harold Seelig

Theses and Dissertations

There are few published studies of larval fish assemblages from unregulated, tidal freshwater rivers. Patterns in the spatial and temporal distribution of larval fishes in the Mattaponi River were examined. Sampling took place on a weekly basis from February through August, 2006 and 2007. Larval fishes were categorized by taxa, reproductive guild, and residency guild. Group comparisons using multi-response permutation procedures (MRPP) indicated significant spatial and temporal differences in assemblage composition on multiple scales. Differences in assemblage composition were analyzed using non-metric multidimensional scaling (NMS). Interannual differences were attributable to anadromous and semi-migratory species. Seasonal differences were attributable to herrings, …

Expression & Purification Of Recombinant Plasmodium Falciparum Erythrocyte-Binding Ligands, Seward Joann Cofie

Theses and Dissertations

Plasmodium falciparum, the most virulent malarial parasite, is capable of invading all known human blood types. Erythrocyte invasion depends on specific parasite ligand and erythrocyte receptor interactions. These interactions are mediated by Region II of the P. falciparum erythrocyte binding ligands. Although invasion does not seem dependent upon a singular ligand, their individual contributions to the invasion process are yet to be explained. In this study, Region II of P. falciparum binding ligands BAEBL and JESEBL were transiently expressed as hexahistidyl recombinant proteins in COS-1 cells. Purification by column chromatography yielded 0.52 mg of BAEBL and 0.433 mg of JESEBL. …

Seasonal Patterns Of Nutrient Retention In A Restored Tidal Freshwater Stream Of The Mid-Atlantic Coastal Plain, Joseph Wood

Theses and Dissertations

Nutrient retention is governed by the interplay between physical processes that control the throughput of water and materials (i.e., water residence time), and by biological processes that govern transformation and uptake (e.g., microbial denitrification). A partial breach of the dam located on Kimages Creek (VA) re-established the historical (pre-1920) connection to the James River and provided a well-defined channel to gauge tidal exchange. We quantified tidal exchange as well as non-tidal (watershed) inputs on a monthly basis to assess Nitrogen (N) retention. Water and N fluxes were dominated by tidal exchange which was typically three times greater than inputs from …

Small Molecule Inhibitors Of Mapk And Pi3k Pathways Enhance Mda-7 Lethality In Renal Cell Carcinoma, Patrick Eulitt

Theses and Dissertations

Renal cell carcinoma accounted for an estimated 57,760 new cases and estimated 12,980 deaths in the United States in 2009. Current treatment options for systemic renal cell carcinoma yield markedly low response percentages; however, recent cytokine therapy experiments have produced promising results. A novel adenovirus, Ad.5/3-mda-7, has been synthesized to efficiently infect renal cancer cells with the mda-7 gene. This gene encodes for the cytokine MDA-7/IL-24 that has the ability to specifically target transformed cells. Assays performed with this adenovirus resulted in an increased percentage of cell death in renal cancer cells when compared to infection with Ad.5/3-cmv empty vector. …

Size-Structured Competition And Predation In Red-Eyed Treefrog Tadpoles, Christopher Asquith

Theses and Dissertations

Body size is important in determining the outcome of competition and predator-prey interactions. Size structure of a population (i.e. relative proportion of large and small conspecifics) may be particularly important in organisms with prolonged breeding periods and rapid growth where populations may have multiple cohorts at different stages of development competing for one resource. Both the consumptive and nonconsumptive effects of predators can also be size-dependent and can alter competitive interactions. Here we study the importance of size structure in the Neotropical leaf-breeding tree frog, Agalychnis callidryas. This species is a prolonged breeder such that multiple overlapping cohorts of differing …

Role Of Jak/Stat Pathway In The Pathogenesis Of Breast Cancer, Qifang Zhang

Theses and Dissertations

The Jak/Stat signaling cascade mediates cell proliferation, differentiation, survival, apoptosis and immune responses. Aberrant activation of this pathway mediates neoplastic transformation and abnormal growth of many malignancies including breast cancer, the most common cancer among women, and the second leading cause of cancer deaths in women in United States. The mechanism by which the Jak/Stat pathway modulates the pathogenesis of breast cancer is unclear. This dissertation elucidates roles of Jak/Stat members that mediate the pathogenesis of breast cancer. For these studies, we used 4T1 mouse mammary tumor cells as a model which mimics human breast cancer. First, we investigated the …

Genetic Basis Of Growth Adaptation Of Escherichia Coli After Deletion Of Pgi, A Major Metabolic Gene, Pep Charusanti, Tom M. Conrad, Eric M. Knight, Karthik Venkataraman, Nicole L. Fong, Bin Xie, Yuan Gao, Bemhard O. Palsson

Study of Biological Complexity Publications

Bacterial survival requires adaptation to different environmental perturbations such as exposure to antibiotics, changes in temperature or oxygen levels, DNA damage, and alternative nutrient sources. During adaptation, bacteria often develop beneficial mutations that confer increased fitness in the new environment. Adaptation to the loss of a major non-essential gene product that cripples growth, however, has not been studied at the whole-genome level. We investigated the ability of Escherichia coli K-12 MG1655 to overcome the loss of phosphoglucose isomerase (pgi) by adaptively evolving ten replicates of E. coli lackingpgi for 50 days in glucose M9 minimal medium and …

Interactions Between Competition And Predation Shape Early Growth And Survival Of Two Neotropical Hylid Tadpoles [Poster], Sergio C. Gonzalez, Justin C. Touchon, James R. Vonesh

Biology Presentations

Background/Question/Methods

Recent literature reviews reveal that competition typically has stronger effects on growth than the presence of predators, while predation has larger effects on survival. Further, past studies show that predators typically lessen the negative effect of competition on growth and also make interspecific competition beneficial for the survival of focal species. We examine the independent and combined effects of competition and predation for survival and growth of the tadpoles of two co-occurring Neotropical hylid frogs (Agalychnis callidryas and Dendropsophus ebraccatus). Our experiment crossed tadpole species composition (single and mixed at single total density) with the presence or absence of …

Terrestrial Predators And Abiotic Conditions Affect Hatching Survival Of Arboreal Frog Eggs: Implications For Aquatic Food Web Dynamics [Poster], Jessica Hite, James R. Vonesh

Biology Presentations

Background/Question/Methods
Recent work in food web studies has highlighted the importance of terrestrial and aquatic linkages and the trait and density-mediated effects of predators and abiotic conditions. Many studies have focused on species with complex life histories (amphibians and aquatic insects) because they encompass a variety of trophic levels and form important linkages between terrestrial and aquatic food webs. For example, red-eyed treefrog, Agalychnis callidryas eggs are deposited on vegetation overhanging ponds and risk mortality from a variety of terrestrial predators and abiotic stressors. Red-eyed treefrog embryos, however, exhibit phenotypic plasticity in hatching time in response to these terrestrial stressors. …

Interplay Between Keratin And Vimentin Expression In Oral Cancer, Mary Catherine Mcginn

Theses and Dissertations

Previous research in our laboratory found that inhibiting expression of vimentin, a marker of epithelial-to mesenchymal transition, inhibited cell growth and motility in vitro and in vivo. Tumors derived from vimentin knockdown cells showed features of epithelial redifferentiation and increased expression of differentiation-specific keratins. It is unknown what causes re-expression of keratins when vimentin is inhibited. Although, canonical Wnt signaling may activate NF-κB and repress of keratin and/or induce vimentin expression through β-catenin. We hypothesize that downregulation of differentiation-specific keratins contributes to tumor progression, mediated directly or indirectly by expression of vimentin. Vimentin-negative HN4 cells were transfected with plasmids encoding …

The Role Of The Ca2+-Dependent Protein Kinase, Camk-Ii, In Heart And Kidney Development In The Zebrafish, Danio Rerio, Sarah Rothschild

Theses and Dissertations

Ca2+/calmodulin-dependent protein kinase type II (CaMK-II) is a multifunctional serine/threonine kinase that is ubiquitously expressed throughout the lifespan of metazoans. Mammals encode four genes (α, β, γ, δ) that generate over forty splice-variants. CaMK-II is important in a myriad of functions, including ion channel regulation, cell-cycle progression, and long term potentiation. In adults, alterations in activation of CaMK-II induce cardiac arrhythmias and heart failure. Developmental roles for CaMK-II are not as well understood since mouse knockouts are embryonic lethal. Therefore the identification of other vertebrate CaMK-II genes will add to our understanding of development. Zebrafish encode seven catalytically active CaMK-II …

Biochemical Characterization Of Suppressor Of Ikk-Ε And Nak-Associated Protein 1, Jonathan Forbes

Theses and Dissertations

Innate immunity provides the first line of defense against invading pathogen by recognizing and mounting a response to the pathogenic challenge. Among the cellular mechanisms of the innate immune response, Toll-like receptor 3 (TLR3) recognizes viral dsRNA and signals subsequent production of type-I interferon. The TLR3:interferon signaling cascades contains a kinase complex composed of two kinases and a scaffold protein, NAK-associated protein 1 (NAP1). The role of NAP1 in modulating kinase activation or regulation is unknown. A key inhibitory protein identified in the TLR3:interferon pathway, silencer of inhibitor of κBα kinase ε (SIKE), blocks the activity of this kinase complex …

Periodontal Bacterial-Dna Initiated Immuno-Inflammatory Responses In Human Osteoblastic Cells, Chebel Najib Bou

Theses and Dissertations

Periodontitis is a chronic inflammatory disease initiated by gram negative anaerobic bacteria. These bacteria possess pathogen-associated molecular patterns (PAMPs) that interact with various receptors including Toll like receptors (TLRs). Bacterial DNA (bDNA) is one of the PAMPs mainly recognized by TLR9. Interaction of bDNA and its receptors leads to activation of inflammatory signaling pathways potentially resulting in periodontal bone destruction. The aim of this study was to determine the production of IL- 6 and IL-8 in response to periodontal bDNA from human osteoblastic cells (MG-63). MG- 63 cells were stimulated in duplicate for 20 hours with 100ng/μl of bDNA from …

Influence Of Retinal States On The Development And Maintenance Of Retinofugal Projections, Duncan Morhardt

Theses and Dissertations

Vision provides a critical interface with the physical world. This work examines visual development and vision loss in mice to glean the influence of the retinal state on visual connections. I first assessed the impact of retinal activity on the eye-specific segregation of retinal afferents in the lateral geniculate nucleus (LGN) of young Gβ5 -/- mice. Gβ5 is the fifth member of the β subfamily of heterotrimeric G proteins. Gβ5 binds and stabilizes the R7 family of regulators of G-protein signaling (RGS), which accelerate Gi/o GTP hydrolysis. Gβ5 -/- mice, which lack R7RGS activity, have malformed synapses in the outer …

Cell-Cell Junction Signaling Regulating Dna Double-Strand Break Repair In Breast Cells, Sinduja Ethiraj

Theses and Dissertations

Genomic instability and acquisition of invasiveness through the basement membrane extracellular matrix (ECM) are two major processes for epithelial cell malignancy in breast cancer. DNA double-strand break repair (DSBR) is one of the processes that get misregulated during breast cancer progression. In addition, radiation induced breaks such as those induced during radiation therapy to treat breast cancer patients are repaired by DSBR, rendering this pathway relevant for therapy as well. DSBR can occur either by homologous recombination (HR) or non-homologous end-joining (NHEJ). HR is accepted as the more error-free pathway. HR is regulated by the cell cycle status such that …

Ipla2Β, Alternative Splicing And Apoptosis Of Pancreatic Islets, Bhargavi Emani

Theses and Dissertations

Ceramides are bioactive lipids that can promote splicing of apoptosis-related genes, including caspase 9 and BCL-x. A recent study demonstrated that expression of neutral sphingomyelinase (NSMase), an enzyme that hydrolyzes sphingomyelins to generate ceramide, is regulated by Group VIA phospholipase A2 (iPLA2β)-dependent mechanism during β-cell apoptosis. This prompted us to hypothesize that iPLA2 is upstream of ceramide generation in the process regulating splicing of apoptotic genes. To test this, Jurkat T cells were treated with the selective inhibitor of iPLA2β, bromoenol lactone (BEL), RNA was isolated and converted to cDNA, and caspase 9 and BCL-x mRNA viii species were amplified …

P53 Mediated Cell Motility In H1299 Lung Cancer Cells, Mi-Yon Choi

Theses and Dissertations

Studies have shown that gain-of- function mutant p53, AKT, and NFκB promote invasion and metastasis in tumor cells. Signals transduced by AKT and p53 are integrated via negative feedback between the two pathways. Tumor derived p53 was also indicated to induce NFκB gene expression. Due to the close relationship between p53/AKT and p53/NFκB, we hypothesized that AKT and NFκB can enhance motility in cells expressing mutant p53. Effects on cell motility were determined by scratch assays. CXCL5- chemokine is also known to induce cell motility. We hypothesized that enhanced cell motility by AKT and NFκB is mediated, in part, by …

Zooplankton Abundance, Community Composition And Grazing In The James River Estuary (Virginia, Usa), Laura E. Barry

Theses and Dissertations

This study examined the extent to which zooplankton in the James River Estuary (Virginia, USA) exploit the available algal-food resources. Zooplankton abundance, community composition and grazing rates were assessed at three locations which included a site with high algal production (near the VCU Rice Center) and two sites where algal production was lower. Grazing rates were measured by determining the rate of phytoplankton decline (as chlorophyll a) during 48-hour incubation experiments in the presence and absence of zooplankton. Significant differences in zooplankton abundance were observed among the three sites, with the greatest average zooplankton density (434 ± 69 ind/L) occurring …

Examining The Effects Of Penning On Juvenile Eastern Box Turtles (Terrapene Carolina Carolina), Nicolas Frederick

Theses and Dissertations

Box turtle (Terrapene carolina) populations have been declining over the last several decades, and one major cause is increasing urbanization. As a result of habitat fragmentation, wildlife managers are frequently turning to new and alternative management strategies. Traditional box turtle management has included relocation, which has been met with limited success. This study aims to combine these strategies with another less-studied one: forcing turtles to overwinter on site by penning them in an outdoor enclosure. Two sets of juvenile box turtles were released at the Virginia Commonwealth University Rice Center: one penned on site in a pen for one year, …

Prothonotary Warbler (Protonotaria Citrea) Plumage As An Indicator For Infection: The Relationship Between Haemosporidia Infection And Breast Feather Reflectance In A Neotropical Migrant Passerine, Robert Fithian

Theses and Dissertations

Yellow avian plumage is a direct result of carotenoid pigments obtained in a bird’s diet and may act as an indicator for individual health, parasite resistance, and status. This study describes breast feather reflectance of adult Prothonotary Warblers (Protonotaria citrea) (n=169), insectivorous Neotropical migrant passerines, throughout the Ultraviolet (UV) and human visible light spectra and examines the relationship between Haemosporidia (pathogen causing Avian Malaria) infection and feather reflectance (n=41). Reflectance was characterized using a Principle Component Analysis evaluating Intensity, Brightness, Hue, UV Intensity, UV Brightness, and UV Chroma. UV and visible light reflectance was higher in birds sampled earlier in …

Examination Of Platelet Adhesion By Streptococcus Sanguinis, Brian Mahoney

Theses and Dissertations

Streptococcus sanguinis is a leading cause of infective endocarditis. Bacterial adhesion to platelets is likely important in the pathogenesis of infective endocarditis. Bacterial cell wall-anchored (Cwa) proteins may mediate this adhesion. To begin to test this hypothesis, S. sanguinis adhesion to platelets was examined in vitro. The requirement of 12 Cwa proteins for S. sanguinis-platelet adhesion was individually assessed, measuring adhesion of purified platelets to polystyrene wells coated with S. sanguinis strain SK36 or 12 isogenic Cwa protein mutants. Significantly fewer platelets adhered to wells coated with one mutant strain, VT1614. However, results of a whole-cell enzyme-linked immunosorbent assay (ELISA) …

The Role Of Cannabinoids And Cannabinoid Receptors In Enteric Neuronal Survival, Yan Li

Theses and Dissertations

The Endocannabinoid system has been found in the gastrointestinal tract, where it plays an important role in gut under both physiological and pathological conditions. Although the major effects of cannabinoids in the gut are mediated through effects on enteric neurons, the role of cannabinoids in the enteric nervous system is poorly understood. In the present study, we have used the primary cultures of myenteric ganglia and a newly developed fetal enteric neuronal cell line to identify whether the endocannabinoid, anandamide, affects ganglionic and neuronal survival and the pathways involved. Anandamide had a biphasic effect on ganglionic survival, increasing survival at …

Systematics And Biogeography Of The Didymopanax Group Of Schefflera (Araliaceae), Pedro Fiaschi

Theses and Dissertations

Schefflera is the largest genus in the angiosperm family Araliaceae, with about 900 species, of which c. 300 belong to five subgeneric groups in the Neotropical region. Previous phylogenetic studies of Schefflera have been limited to a small number of species from this region, and very little is know about phylogenetic relationships in the Brazilian-centered Didymopanax group of this genus. Therefore, to gain a better understanding of the diversity and evolution of the Didymopanax group of Schefflera, I investigated the systematics of these plants in the broader context of the entire Neotropical clade. The main goals were (1) to investigate …

Sphingolipid-Induced Activation Of The Volume-Sensitive Cl− Current Is Mediated By Mitochondrial Reactive Oxygen Species, Frank Raucci

Theses and Dissertations

Swelling-activated Cl− current (ICl,swell) is an outwardly-rectifying current that plays an important role in cardiac electrical activity, cellular volume regulation, apoptosis, and acts as a potential effector of mechanoelectrical feedback. Persistent activation of ICl,swell has been observed in a number of models of cardiovascular disease. Previously we showed that angiotensin II (Ang II), endothelin-1 (ET-1), endothelial growth factor receptor (EGFR), and reactive oxygen species (ROS) produced by NADPH oxidase (NOX) and mitochondria are involved in the activation of ICl,swell by both osmotic swelling and Beta1 integrin stretch. Sphingolipid metabolism is modulated in several cardiopathologies and because sphingolipids are bioactive lipids …

The Regulation Of Alternative Splicing By Oncogenic Signaling Pathways., Jacqueline Shultz

Theses and Dissertations

In the presented study, we demonstrate that the alternative splicing of caspase 9 was dysregulated in a large percentage of NSCLC tumors and cell lines. These findings led to the hypothesis that survival pathways activated by oncogenic mutation regulated this mechanism. Indeed, the oncogenic PI3-Kinase/Akt pathway was demonstrated to regulate the alternative splicing of caspase 9. Further mechanistic studies demonstrate that multiple Akt isoforms can regulate the alternative splicing of caspase 9 in NSCLC. Akt was additionally shown to mediate the exclusion of the exon 3,4,5,6 cassette of caspase 9 via the phospho-state of the RNA trans-factor, SRp30a. Mutagenesis studies …

The Role Of Porphyromonas Gingivalis Lysine Specific Protease, Kgp In Hemin Transport And Pigment Accumulation, Anuj Phull

Theses and Dissertations

Porphyromonas gingivalis, a gram-negative anaerobic bacterium, is implicated as a major etiological agent in the initiation and progression of severe forms of periodontal disease. It has been reported that gingivitis and periodontal disease affect roughly 50.3% and 35% of the adult population, respectively. Therefore, approximately over 85% of the adult population may suffer from some form of gingival disease. Porphyromonas gingivalis, an established periodontopathogen, requires hemin for growth. Although multiple hemin uptake systems appear to be present in this organism, their specific role in hemin uptake and virulence remains unknown. Pigmentation is thought to result from the accumulation of iron …