Digital Commons Network^™

Expression And Function Of Alpha3 And Beta2 Neuronal Nicotinic Acetylcholine Receptor Subunits In Hek-293 Cells, Nathan W. Steinhafel

Theses and Dissertations

Single-cell real-time quantitative RT-PCR was used to characterize the mRNA expression of rat neuronal nicotinic acetylcholine receptor (nAChR) subunits α3 and β2 in CA1 hippocampus stratum radiatum and stratum oriens interneurons. α3β2 co-expression was detected in 43% of interneurons analyzed. The nAChR subtype α3β2 was transiently expressed in cells derived from the human embryonic kidney cell line 293 at mRNA levels found in the CA1. The functional properties of α3β2 in HEK-293 cells were characterized by whole-cell patch clamping using acetylcholine (ACh) as an agonist. The kinetics of α3β2 channels were further analyzed by altering the level of α3 DNA …

The Influence Of Membrane Lipid Order On Cell Shape And Microvesiculation In Human Erythrocytes, Laurie Jackson Gonzalez

Theses and Dissertations

Exposure of human erythrocytes to elevated intracellular calcium causes alterations in cell shape and stimulates shedding of the cell membrane in the form of microvesicles. We hypothesized that both the shape transition and microvesiculation are influenced by microscopic membrane physical properties such as lipid order. To test this hypothesis, membrane properties were manipulated by varying the experimental temperature, membrane cholesterol content, and the internal ionic environment. Changes in membrane order were assessed using steady-state fluorescence spectroscopy with an environment-sensitive probe, laurdan. Our observations led us to the following conclusions: 1) the modest temperature dependence of membrane structure observed with laurdan …

Fgf4 And Wnt5a/Pcp Signaling Promote Limb Outgrowth By Polarizing Limb Mesenchyme, Keri Lynn Low

Theses and Dissertations

The focus of this study was to elucidate the molecular and cellular mechanisms whereby fibroblast growth factors (FGFs) mediate outgrowth of the limb. Specifically, we examined the epistatic relationship between FGF and Wnt/Planar cell polarity (PCP) signaling in establishing cell polarity as a mechanism for outgrowth. By implanting beads into embryonic limbs and lateral plate mesoderm, we established that FGF activates Wnt5a in a gradient fashion. Once it was established that Wnt5a was expressed at the right time and place to turn on PCP signaling, we investigated the ability of Wnt5a to influence cell migration and/or cell polarity. Our analysis …

Construction Of A Col11a1 Transgene Vector, Cameron Mckell Beck

Theses and Dissertations

Background: Cartilage disorders affect millions of people in the United States alone, with effects ranging from poor skeletal development and joint pain to shortened lifespan and perinatal lethality. Many of these disorders have their root in defects of collagen, type XI collagen being among the most important. A mouse model of such a type XI collagen defect is the chondrodysplasia (cho) mutant. Mice homozygous for this null mutation in the Col11a1 gene do not express the α1 chain of type XI collagen. This results in a functional knockout of type XI collagen, leading to insufficient skeletal development and perinatal lethality. …

Characterization Of Membrane Potentials In Vascular Smooth Muscle Of Hagfish, Lamprey And Trout, Prentiss Jones Jr.

Dissertations

The objective of this study was to characterize membrane potentials in systemic arteries of Pacific hagfish, Eptatretus stouti, Rainbow trout, Onchorhyncus mykiss, and Sea lamprey Petromyzon marinus. Previous studies have characterized membrane potentials in piscine tissue. However, these studies utilized non-vascular tissues such as cardiac muscle and skeletal muscle (69, 161). Characterization of membrane potentials in fish vasculature is without precedent in the literature. The hypothesis of this study was that membrane potentials in fish vascular smooth muscle differ little between species when comparable vasculature is evaluated.

Pka As An Upstream Kinase For Lkb1/Strad/Mo25, Seth Taylor Herway

Theses and Dissertations

The LKB1/STRAD/MO25 complex (LSMK) has been identified as the major upstream kinase for AMP-activated protein kinase (AMPK). PKA phosphorylates LKB1 at the Ser428 residue in humans and Ser431 residue in mice. We investigated PKA as an upstream kinase for LSMK. LKB1 that had been incubated with PKA prior to incubation with AMPK experienced up to a 51% increase in AMPK Kinase activity compared to LKB1 alone (p < 0.05). When blocked with a PKA Inhibitor, the kinase effect of PKA on LKB1 was eliminated. Rat epitrochlearis muscle tissue incubated with epinephrine experienced no increase in AMPK activity compared with controls indicating that epinephrine does not cause AMPK activity in this type of tissue. In conclusion, phosphorylation by PKA can increase the AMPKK activity of LKB1-STRAD-MO25 in vitro. Because LKB1 has been found to be constitutively active, it is postulated that phosphorylation by PKA may act to enhance LKB1-AMPK interaction and thus achieve its effect.

The Effects Of 3-Phosphoglycerate And Other Metabolites On The Activation Of Amp-Activated Protein Kinase By Lkb1/Strad/Mo25, William John Ellingson

Theses and Dissertations

Skeletal muscle contraction results in the phosphorylation and activation of the AMP-activated protein kinase (AMPK) by an upstream kinase, AMPKK. The LKB1-STRAD-MO25 complex is the major AMPKK in skeletal muscle; however, LKB1-STRAD-MO25 activity is not increased by muscle contraction. This relationship suggests that phosphorylation of AMPK by LKB1-STRAD-MO25 during skeletal muscle contraction may be regulated by allosteric mechanisms. In this study we tested an array of metabolites including glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose 1,6-bisphosphate (F1,6-P2), 3-phosphoglycerate (3PG), glucose-1-phosphate (G1P), glucose-1,6-bisphosphate (G1,6-P2), adenosine diphosphate (ADP), carnitine (Carn), acetyl-carnitine (Acarn), inosine monophosphate (IMP), inosine, and ammonia for allosteric regulation. We found that …

Development Of An In Vitro Protocol To Study The Effect Of Estrogen On Osteoblast Activity, Sourik Ganguly

Morehead State Theses and Dissertations

A thesis presented to the faculty of the College of Science & Technology at Morehead State University in partial fulfillment of the requirements for the Degree of Master of Science by Sourik Ganguly on July 7, 2006.

Molecular Analysis Of The Effects Of Atrazine On Xenopus Laevis Frogs, Ronald Edward Celestine

Dissertations

Atrazine, the most commonly used herbicide in the U.S., has been implicated as one of the factors that may be causing the worldwide decline of amphibians. First, I examined effects of atrazine on laboratory raised Xenopus laevis tadpoles, treated from 5 days post fertilization to NF stage 65/66 with a concentrationof 25 ppb of atrazine. This study showed no significant difference in body weight, length and metamorphic rate.

Second, I used DNA microarrays to examine mRNA expression levels of genes in atrazine treated and control Xenopus laevis tadpoles. I found significant changes in gene expression levels of 18 genes. These …

Exposure Of Nervous System Cells To Polychlorinated Biphenyls (Pcbs), Results In Alterations Of Neurotrophic Factor Expression, Gordon H. Gurley

Dissertations

Toxic insult by PCBs results in learning and memory deficits in humans. Alterations in expression of neurotrophic factors (NF) and/or their receptors have been linked to changes in learning and memory. How PCBs affect cognition is not known. We suggest that PCBs affect cognition by altering NF expression.

We exposed cultured C6 rat glioblastoma cells (an astrocyte cell-line) to medium containing PCB (Aroclor 1254 (10ppm)). Control cells were treated with dimethyl sulfoxide (DMSO) or regular medium. Enzyme-linked immunosorbent assays (ELISA) were used to determine glial cell-line derived neurotrophic factor(GDNF) and nerve growth factor (NGF) concentrations in all samples. We also …

Cellular Mechanism Of Arglabin-Dimethylaminohydrochloride Cytotoxicity, Xiaofei Qin

Theses and Dissertations in Biomedical Sciences

Cancer is the second leading killer in the United States. Anticancer drug development is always based on the understanding of molecular and cellular mechanisms of carcinogenesis, as well as comprehensive knowledge of potential anticancer drugs. Arglabin-dimethylaminohydrochloride (arglabin-DMA) represents one of the new classes of anti-cancer agents that have shown promise in suppressing the growth of various tumor cells. However, the cellular mechanism of arglabin-DMA cytotoxic effects on tumor cells is still unclear. The current study was to determine the farnesyltransferase (FTase) inhibitory activity of arglabin-DMA and to investigate the effects of arglabin-DMA on three proteins: Ras, Rho and cyclin kinase …

Properties Of Conductance And Inhibition Of Proton Channels: M2 From Influenza A Virus And Fo From Escherichia Coli Atp Synthase, Jeffrey C. Moffat

Theses and Dissertations

Proton channels are essential for many of the processes of life. The influenza A viral protein M2 is responsible for sensing the conditions necessary for viral RNA release. The proton-translocating FoF1 ATPase (ATP synthase) uses a proton gradient to drive adenosine triphosphate (ATP) synthesis. We have directly measured proton uptake in vesicles containing reconstituted M2 or FO by monitoring external pH after addition of valinomycin to vesicles with 100-fold diluted external [K+]. This proton flux assay was utilized to quantify proton flux through single M2 and Fo channels. Contrary to previous reports, proton uptake by M2 was not significantly altered …

Catalase Activity Mediates The Inhibitory Actions Of 24,25 Dihydroxyvitamin D3, Sven L. Peery

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The steroid hormone 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells, while the steroid 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃] inhibits the rapid stimulation by 1,25(OH)₂D₃. Earlier work in this laboratory has indicated that a cellular binding protein for the 24,25(OH)₂D₃ is the enzyme catalase. Since binding resulted in decreased catalase activity and increased H₂O₂ production, studies were undertaken to determine if pro-oxidant conditions mimicked the inhibitory actions of 24,25(OH)₂D₃, and anti-oxidant conditions …

Exogenous Expression Of Human Na, K-Atpase Isoforms In Insect Cells, Christal Renee' Carpenter

McCabe Thesis Collection

The Na-K-ATPase or Na pump is a membrane bound enzyme that maintains a high concentration of K+ and low concentration of Na+ in most animal cells. Three subunits constitute the Na pump, named alpha (ex), beta (~). and gamma (y). The ex subunit is responsible for the catalytic and transport of the enzyme, the ~ subunit is important for bringing the ex to the plasma membrane of the cell, and y is a regulatory subunit of the Na pump. In addition, there are several molecular variants or isoforms of the ex and ~ subunits. At present four different ex (ex …

Development Of Self-Assembled Monolayer-Based Cell Culture Platform Towards Fabrication Of A Three-Dimensional Bioreactor, Rajendra Kandoor Aithal

Doctoral Dissertations

The extracellular matrix (ECM) plays an important role in regulating a number of cellular properties and functions like cell differentiation, cell synthesis and degradation, cell viability and proliferation, cell function, and cell aging. Surface modification of planar substrates with self-assembled monolayers (SAMs) is a promising technique to achieve stable ECMs.

In this work, substrates such as silicon (Si), gallium arsenide (GaAs) and indium tin oxide (ITO) substrates were modified with SAMS containing amino (-NH2), methyl (-CH3), thiol (-SH) and carboxylic (-COOH) end groups and characterized using contact angle measurements, surface infrared (IR) spectroscopy and atomic force microscopy (AFM). Different cell …

Comparative Genomic Analyses And Expression Of A Set Of Overlapping Open Reading Frames Of Frog Virus 3, Type Species Of Ranavirus (Family Iridoviridae), Wendy Guat Hoon Tan

Dissertations

Frog virus 3 (FV3) is the type species member of the genus Ranavirus (family Iridoviridae ). To better understand the molecular mechanisms involved in the replication of FV3, including transcription of its highly methylated DNA genome, the complete nucleotide sequence of the FV3 genome has been determined. The FV3 genome is 105, 903 base pairs long excluding the terminal redundancy. The G+C content of FV3 genome is 55% and it encodes 98 non-overlappingpotential open reading frames (ORFs) containing 50 to 1293 amino acids. Eighty-four ORFs have significant homology to known proteins of other iridoviruses, whereas 14 are unique FV3 ORFs. …

Olfactory Bulb Neurons Of Adult Zebrafish Morphology, Distribution, Cellular Interactions And Structural Stability Following Deafferentation, Cynthia L. Fuller

Dissertations

The zebrafish is becoming an increasingly popular model for studies involving olfactory function, yet there is still much to be learned about the anatomy and circuitry of different cell types in the olfactory bulb. This study focuses on identifying the morphology and distribution of output neurons and interneurons in the olfactory bulb of adult zebrafish, Danio rerio . Furthermore, this investigation examines the cellular interactions of the primary output neuron, the mitral cell, and addresses the issue of neuronal plasticity by considering the structural stability of this cell type following loss of afferent innervation.

Using retrograde tract tracing with various …

Nanosecond Pulsed Electric Field Effects On Cell Cycle And Apoptosis, Emily H. Hall

Theses and Dissertations in Biomedical Sciences

Apoptosis, programmed cell death, is a highly regulated and complex pathway essential for embryonic development, immune-system function and maintenance of tissue homeostasis where cells induce their own cell death. Cells undergoing apoptosis exhibit a distinctive phenotype characterized by maintenance of membrane integrity, cell shrinkage, phosphatidylserine (PS) externalization at the plasma membrane, caspase protease activation, DNA fragmentation, release of cytochrome c from the mitochondrion, and membrane blebbing. An important regulatory protein in the apoptotic pathway is p53. The p53 protein functions to modulate the cell cycle by arresting cells in the G1 and G 2 phases to repair DNA damage, and/or …

The Study Of Nitric Oxide Synthase Expression, Function, And Regulation In The Renal Vasculature During Postnatal Renal Development, Brian Blake Ratliff

Theses and Dissertations in Biomedical Sciences

The newborn kidney is vulnerable to vasomotor acute renal failure (ARF) from adverse perinatal events or complications of prematurity. Nitric oxide (NO) vasodilation is vitally protective in this type of ARF, but its relationship with other vasoactive factors, such as angiotensin II (AII) has not been examined. In the immature kidney, nitric oxide synthase (NOS) isoforms, specifically eNOS and nNOS, are developmentally regulated, but their specific role and regulation are unknown.

The enhanced vasodilatory role of NO in the immature kidney was hypothesized to be attributed to regulatory, expressional, and functional differences in eNOS and nNOS isoforms from the adult. …

Modulation Of Tgfβ-Induced Pai -1 Expression By Changes In Actin Polymerization In Human Mesangial Cells, Keyur Patel

Theses and Dissertations in Biomedical Sciences

Chronic renal diseases show increased deposition of extracellular matrix (ECM) in the glomerulus (glomerulosclerosis). Glomerulosclerosis is associated with activation of normally quiescent glomerular mesangial cells into myofibroblast-like cells. The overall objective of this study is to delineate cellular mechanism/s of myofibroblast-differentiation in disease states. In cultured mesangial cells certain characteristics of myofibroblast differentiation (α-smooth muscle actin (α-SMA) and hypertrophy) are associated with an increase in polymeric actin microfilaments (stress fibers). It is likely that other genes are also regulated in an actin cytoskeleton-dependent manner during myofibroblast differentiation. In these studies, we therefore examined the hypothesis that changes in the actin …

The Role Of Fgfr4 In Trigeminal Placode Cell Development, Stephanie Beth Reynolds

Theses and Dissertations

In vertebrate embryos, the ophthalmic trigeminal (opV) placode contributes sensory neurons to the trigeminal ganglion during development of the peripheral nervous system. FGFR4 is expressed transiently in the trigeminal placode as cells undergo an epithelial-mesenchymal transition and begin to migrate toward the condensing ganglion. Because of the restricted spatiotemporal expression of FGFR4 in the opV placode, it was hypothesized that FGFR4 is necessary for the process of epithelial-mesenchymal transition in the opV placode. Using electroporation, an FGFR4 inhibitory gene construct was introduced into 6—10 somite stage chick embryos. This secreted inhibitory form of the FGFR4 gene blocked endogenous FGFR4 signaling, …

The Role Of Pax3 In Neuronal Differentiation Of The Ophthalmic (Opv) Trigeminal Placode And Neural Tube During Chicken Embryonic Development, James R. Bradshaw

Theses and Dissertations

Pax3 has been used as a valuable marker in research aimed at understanding tissue interactions involved in trigeminal ophthalmic (opV) placode development. While Pax3 expression coincides with opV neuron specification, the function of Pax3 in these cells has not previously been investigated. Splotch mutant mice (which lack Pax3) have a reduced trigeminal ganglion; however it is not clear whether this reduction is due to neural crest or placode cells. We have used electroporation in the chick model system to block or ectopically express Pax3 at key times in opV placode development. Using several markers of placode cell differentiation, we have …

Chmp1 Is Implicated In The Development Of Pancreatic Tumor Via The Retinoic Acid Signaling Pathway, Juliana Adedayo Akinsete

Theses, Dissertations and Capstones

In the present study, we investigated the involvement of Chmp1 (Chromatin ModifyingProtein 1/Charged Multivesicular body Protein 1) in the development of mousepancreatic acinar tumor cell line. CRL 2151 cell line was transfected with Chmp1/CS2vector to compare growth, morphology and expression of Chmp1, p53 and pp53 (ser37) with control-transfected cells. CRL 2151 cells were treated with all-trans retinoicacid (ATRA) to compare growth, morphology and expression of Chmp1 and p53 withcontrol-treated cells. Strabismus was used as control. Results showed inhibition ofgrowth but no morphological change in transfected cells. Western blot analysis showedthat Chmp1 transfection upregulated the expression of p53, pp53 and Stbm …

Characterization Of The Dna-Binding Properties Of Silent Information Regulator 3 Protein, Cotteka Nichisha Johnson

Theses, Dissertations and Capstones

The eukaryotic genome is organized into distinct domains with discrete regulatory potential, influenced by the variation in local chromatin structure. An example of chromatin-mediated consequences on gene expression is mating type determination of yeast Saccharomyces cerevisiae, which depends on silencing of specific gene cassettes. Silencing in yeast involves key proteins, amongst which the Silent Information Regulator (Sir) proteins are essential. Sir1p, Sir2p, and Sir4p are recruited to nucleate silencing events, while Sir3p serves as the pivotal factor in spreading the silent state. This investigation characterizes the DNA binding properties of Sir3p using DNA fragments with varying degrees of inherent flexibility, …

Population Genetic Structure Of Escolar (Lepidocybium Flavobrunneum) And A Molecular Phylogeny Of The Trichiuroidea, Kirsten Brendtro

Dissertations, Theses, and Masters Projects

No abstract provided.

Isolation And Functional Mapping Of Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein Dna-Damage Complexes, Sarah Saionz Durkin

Theses and Dissertations in Biomedical Sciences

Human T-cell Leukemia Virus Type 1 (HTLV-1) is a transforming retrovirus which causes Adult T-cell Leukemia (ATL) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). Cellular transformation can be caused by a single viral trans-activating protein, Tax. Tax may contribute to transformation through interaction with components of the DNA damage response pathway, promoting cellular genomic instability. We examined cellular Tax complexes in an effort to elucidate potential protein-protein interactions that can model the Tax-induced molecular events.

We also investigated the role of post-translational modification in regulating Tax function. We employed a direct physical analysis of Tax complexes isolated from mammalian …

Diverse Effects Of Nanosecond Pulsed Electric Fields On Mammalian Cells, Jody Anne White

Theses and Dissertations in Biomedical Sciences

The continuing effort to manipulate cell-signaling pathways for therapeutic benefit has lead to the exploration of electric field effects on cells. Current electric field applications include electroporation of the plasma membrane for introduction of drugs, genes, or other macromolecules into cells. Modeling of how these pulsed electric fields affect cells depicts the cell as an excitable circuit. In this model, the electric fields, administered in short pulses to a cell, charge the plasma and internal membranes, which act as dielectric layers, and between these the cytoplasm acts as a conductive medium. The pulse lengths of this treatment are traditionally in …

Development Of Proliferation Assays To Explore The Role Of Calcium Activated Potassium Channels In Cell Proliferation, Sean Foster

Honors Theses

Due to the large variety of K^† channel families implicated in cancer cell proliferation, it is desirable to develop techniques capable of quickly and reliably screening a variety of potential channels for a role in proliferation. While there are many established cell counting procedures, the time-consuming nature of such protocols limits their usefulness in broadly identifying particular channels in uncharacterized cell lines. For this reason, a primary goal of the present research was the development of techniques capable of implicating particular ion channels in proliferation with both qualitative and quantitative data. Once this goal was accomplished, the project sought …