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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Effect of Medicinal Herbal, Panax Notoginseng, on the Fate and Function of Professional Antigen Presenting Cells

Rhule, Ava-Gaye Tania 14 January 2008 (has links)
Antigen presenting cells (APCs) perform the essential task of integrating responses between the innate and adaptive immune system. Several approaches have been undertaken to manipulate the effects of APCs for therapeutic purposes. Panax notoginseng is a medicinal herb that is purported to possess a number of properties including modulation of the immune system. However, limited information exists on the effects and toxicities of this herbal on APCs. In this regard, we assessed the effects of Panax notoginseng on the fate and function of professional APCs in murine models using macrophages and dendritic cells (DCs). APCs were stimulated with the toll-like receptor ligands LPS, CpG and poly(I:C) and treated with notoginseng (0-200 ìg/ml). The LPS induced levels of the proinflammatory cytokine TNF-á, as well as the expression of accessory molecules MHC II, CD40 and CD86, were decreased dependent on notoginseng exposure time-points relative to LPS stimulation. LPS induced IL-1â, IL-6 and IL-12 production was also decreased with concurrent notoginseng treatment for 24 hours. Notoginseng decreased TNF-á and CD40 activation by CpG and poly(I:C), but had varied effects on the induction of IL-6 and CD86. Furthermore, treatment of APCs with ginsenosides Rb1 and Rg1 had differential effects on the production of TNF-á and IL-6. Phagocytosis of FITC-conjugated ovalbumin antigen by DCs was decreased by notoginseng. Furthermore, the uptake of FITC-conjugated modified LDL was reduced in notoginseng treated DCs. However, T cell proliferation in response to notoginseng treated-antigen-loaded DCs was not affected in vitro or in vivo. Mechanistically, notoginseng reduced nuclear levels of the transcription factor NFêB, but had no effect on glucocorticoid receptor activation. No immunotoxicities were observed with low dose notoginseng (660 ìg/kg) treatment of Balb/c mice in vivo. Collectively, our results indicate that notoginseng decreased inflammatory mediator production by APCs, without altering their ability to induce antigen specific CD 4+ T cell proliferation. Our research provides insight into the potential use of this herbal in the treatment of inflammatory diseases as a safe and effective complement to existing remedies.
2

PHYSICO-CHEMICAL STUDIES OF THE VESICULAR GLUTAMATE TRANSPORTER 1 (VGLUT1)

Guo, Jing 15 January 2009 (has links)
The vesicular glutamate transporter 1 (VGLUT1) is an important membrane protein located in glutamatergic synaptic vesicles. It is responsible for the storage and release of the excitatory neurotransmitter glutamate. VGLUT1 is a highly hydrophobic integral membrane protein with a molecular weight around 61 kD. The tertiary structure of VGLUT1 is still unknown. In our study, recombinant VGLUT1 was expressed in Pichia pastoris and purified using either a nickel chelating column or cobalt-coated Dynabeads. The HiTrapTM nickel chelating column proved to be more efficient in purification of recombinant VGLUT1 than Dynabeads. To study the physico-chemical properties and structure of VGLUT1 and advance our understanding of the membrane topology, FITC was used to modify VGLUT1 in solution. On average, 5.35 ¡À 1.10 lysines were labeled with FITC in each VGLUT1 molecule. Trypsin, endoproteinase Glu-C and Arg-C were used to digest FITC labeled VGLUT1 for mass spectrometry analysis. Mass spectrometry and other proteomics techniques were applied to identify labeled residues. Nine lysine residues were revealed to be labeled by FITC in total, among which 8 lysines (K10, K25, K140, K196, K272, K339, K378, and K507) are from native VGLUT1 and one is located at myc epitope (K569).
3

Design, Synthesis and Biological Evaluation of a Family of Excitatory Amino Acid Transporter 3 (EAAT3) Preferring Inhibitors.

Mavencamp, Terri Lynn 28 April 2009 (has links)
<p>This work describes the synthesis and initial characterization of the biological activity of a family of EAAT3 preferring inhibitors, L-&beta;-benzyl aspartate (L-&beta;-BA) and L-&beta;-BA derivatives. L-&beta;-BA and derivatives were initially synthesized in an approximate 2:1 ratio of diasteromers (threo:erythro), using base promoted enolate addition. Kinetic analysis of 3H-D-aspartate uptake into C17.2 cells expressing the hEAATs demonstrated that L-threo-&beta;-BA is the more potent diastereomer (Ki values of 9 µM for EAAT1, 10.0 µM for EAAT2 and 0.8 µM for EAAT3), acts competitively, and exhibits a 10-fold preference for EAAT3 compared to EAAT1 and EAAT2. Electrophysiological recordings of EAAT-mediated currents in Xenopus oocytes further identified L-&beta;-BA as a non-substrate inhibitor. Derivatives of L-&beta;-BA were prepared and characterized for the ability to inhibit 3H-D-aspartate uptake into hEAAT1-3 expressing C17.2 cells. Computational modeling and analysis of structure activity data suggest the area the aromatic moiety of L-&beta;-BA derivatives probe is 1) 3-dimentionally confined, 2) more tolerant of substitutions at the 3 and 5 positions than the 4 position, 3) at least partially distinct from the area probed by L-TBOA and 4) more accessible in the EAAT3 protein than EAAT1 and EAAT2. Computational modeling supports the pharmacological data and lends insight into the selectivity observed with L-&beta;-BA derivatives. Docking studies suggest that H-bonding interactions of L-&beta;-BA derivatives with key residues in the binding site position L-&beta;-BA analogues in a unique manner that is better tolerated in the EAAT3 protein than in the EAAT1 and EAAT2 proteins.</p>
4

STRUCTURE AND FUNCTION OF A NEURONAL GLUTAMATE TRANSPORTER

Leary, Gregory Patrick 28 April 2009 (has links)
<p>Glutamate transporters have a homotrimeric subunit structure with a large central water-filled cavity that extends partially into the plane of the lipid bilayer (Yernool et al., 2004). In addition to uptake of glutamate, the transporters also mediate a chloride conductance that is gated by Na+ and glutamate. Our data indicate that glutamate binding sites, transport pathways, and chloride channels reside in individual subunits in the trimer and function independently and that the anion channel is gated by alkali cations binding from either side of the membrane. We also investigated conformational changes during glutamate binding by incorporating a fluorescent probe into a site near the postulated external gate (HP2) of a mutant transporter that can bind but not transport L-Glu. Fluorescence changes were observed upon ligand binding that strongly depended on the number of subunits labeled; this suggests quenched fluorophore dimers form at the center of the trimer that are subject to HP2 loop closure upon substrate binding. This supports a model of gate motion that is also consistent with recent x-ray structural data (Boudker et al., 2007). Finally, we propose that the large aqueous central cavity in the trimeric complex (Yernool et al., 2004) may function to restrict the diffusion of molecules near the three ligand binding sites, resulting in an increase in the probability of rebinding.</p>
5

The Influence of SPARC on Collagen Deposition in Asbestos-Induced Pulmonary Fibrosis

Smartt, Aubrey Meghan 28 April 2009 (has links)
<p>Pulmonary fibrosis involves the invasion of lung tissue with fibrotic, scar tissue and affects roughly five million people total worldwide. Fibrotic development in the lung has several causes, including chronic inflammatory diseases, infections, medical compounds, and environmental agents. There is no known cure for the disease, but only therapies to improve quality of life. Scar tissue formation must be targeted in order to begin to provide any type of cure for fibrotic diseases. In this study, asbestos was used to induce pulmonary fibrosis in a mouse model and gene expression studies were then performed to identify potential candidate genes involved in asbestos response. One gene with the potential to regulate the fibrotic response is SPARC (secreted protein acidic and rich in cysteine), a matricellular protein involved in tissue repair, extracellular matrix (ECM) regulation, cellular proliferation, and cellular adhesion. The goal of this project was to determine the role of SPARC in fibrosis development after asbestos exposure, specifically targeting how lack of SPARC expression can influence collagen production. I hypothesize that SPARC is a necessary component involved in the fibrotic response to asbestos through an influence on collagen deposition in the lung. I have found that the expression of SPARC is increased in the lungs of C57Bl/6 wild-type mice exposed to asbestos. This increase in expression correlates to higher collagen deposition in the lung. The absence of SPARC in these treated mice resulted in a reduction of the level of collagen deposition back to baseline. To determine the therapeutic potential of these findings, SPARC expression was reduced by small interfering RNA (siRNA) in wild-type mice already suffering from fibrosis. Collagen deposition in the fibrotic mice that received the SPARC siRNA vector showed a significant decrease in collagen accumulation when compared to those that did not receive the vector. Overall, these results indicate that expression of SPARC is a significant step in the development of lung fibrosis through the modulation of collagen deposition and therefore, SPARC may be a potential therapeutic target.</p>
6

Novel roles of the endocannabinoid system in modulating synaptic plasticity

Angell, Alicia Ninet 07 August 2008 (has links)
Learning and memory formation are invaluable processes in human life; however, the cellular mechanisms that control these phenomena are largely unknown. Synaptic plasticity, which is the ability of the synapse between two neurons to change in strength based on activity, is believed to be a key process in the formation of memories and learning. Endocannabinoids (eCB) have recently emerged as important modulators of synaptic plasticity but their precise roles and mechanisms are not well understood and many contradictions exist in the current literature. We have investigated the roles of eCBs and their primary receptor, the CB1 receptor, in the central nervous system using electrophysiological recordings in rodent hippocampus. We find that a moderate frequency 10 Hz stimulation protocol produces long-term potentiation (LTP) that is modulated by eCBs in both mice and rats; but surprisingly, the roles played by eCBs differ greatly between species. In rats, 10 Hz LTP requires CB1 receptor activation, as it is completely abolished by the CB1 antagonists AM251 and SR141716. Unlike theta burst stimulation (TBS) induced LTP, 10 Hz LTP does not require NMDA receptor activation. However, it is prevented when both NMDA and group1 mGluR receptors are blocked. The 10 Hz LTP is also independent of GABAergic synaptic inhibition, suggesting it is a novel form of excitatory synaptic plasticity mediated by the eCB system in hippocampus. In mice, we find that CB1 has an inhibitory effect on 10 Hz induced LTP. When the receptor is genetically removed in CB1 (-/-) mice or pharmacologically blocked wild type mice, 10 Hz LTP is greatly facilitated. Similar to TBS LTP, 10 Hz LTP in mice is NMDA receptor mediated. Also, the ability to achieve successful long-term depression (LTD) is decreased in CB1 (-/-) mice; yet, the magnitude of successful LTD is not changed. Together, this data supports a role for the CB1 receptor in inhibiting the induction of LTP with moderate stimulation protocols in mice, while in rats CB1 activation is required for 10 Hz LTP. Overall, our data supports that eCBs are crucial modulators of synaptic plasticity, although the roles they play may differ among species.
7

Disruption of 8-hydroxy-2'-deoxyguanosine DNA Glycosylase (OGG1) Antioxidant Response Capacity by Sodium Arsenite

Cox, David Paul 07 August 2008 (has links)
8-hydroxy-2'-deoxyguanosine DNA glycosylase is the first step and rate-limiting enzyme involved in the removal of 8-hydroxy-2'-deoxyguanosine via the base excision repair pathway. Transcriptional regulation of human Ogg1 is sensitive to redox changes via modulation of intracellular glutathione. In response to changes in glutathione, changes in hOgg1 transcription occur similar to genes regulated by the transcription factor Nrf2. It was determined that positions - 47 to - 44 in the hOgg1 promoter are necessary for basal transcription of Ogg1 determined by site-directed deletion. This region is capable of interacting with nuclear protein determined by binding assays. Furthermore, transcription factor Nrf2 is identified as binding to this region determined by parallel, and competition EMSA binding assays. Exposure to arsenic has also been associated with oxidative stress and damage to DNA, specifically oxo8dG. This study identified significant increases in the cellular antioxidant glutathione, and alterations in superoxide dismutase activities subsequent to arsenite exposure in actively dividing and NGF treated PC12 cells. Assessment of Ogg1 activity and mRNA levels demonstrated a significant decrease for both measures subsequent to arsenite exposure. The effect seen was due in large part to alterations in gene transcription since direct testing revealed no effect by arsenite on Ogg1 activity. Levels of oxo8dG did not significantly change subsequent to arsenite exposure, however increased trends were evident. Characterization of Sp1 binding revealed that treatment with sodium arsenite could decrease Sp1 binding at two unique Sp1 sites in the human Ogg1 promoter. In summary, transcription factor Nrf2 is an important factor in the inducible regulation of Ogg1. Transcriptional changes in Ogg1 are further dependent on the redox status of the cell. Despite the role of Nrf2 in response to oxidative stress, sodium arsenite disrupted both the transcription and activity of Ogg1 in PC12 cells. This disruption occurred despite the induction of cellular stress response via increases in GSH and Mn SOD activity. This suggests that arsenite is acting through other mechanisms potentially through disruption of the Sp1 transcription factor.
8

The role of Zinc and Reactive Oxygen Species in the Regulation of Endothelial Nitric Oxide Synthase

Wilham, Jason Michael 12 July 2007 (has links)
Persistent pulmonary hypertension of the newborn (PPHN) is a very serious disease affecting nearly 5 in 1000 newborns each year. The development of PPHN has been linked to a decrease in the activity and expression of endothelial nitric oxide synthase (eNOS). Thus, it is critical to understand the mechanisms by which eNOS is regulated to identify new pathways and novel therapies for PPHN. eNOS is dynamically regulated at the transcriptional, post-transcriptional, post-translational, and developmental levels, however mechanisms of this regulation are unresolved. Our data indicates that increases in eNOS expression and activity correlate with increased cellular levels of labile zinc (Zn). In addition, our analysis of the eNOS promoter indicated the presence of a putative heavy metal response element (HRE) in the 5'-flanking sequence. We hypothesized that nitric oxide (NO) may be regulating eNOS expression and activity indirectly through its ability to regulate cellular levels of free Zn. Further, we hypothesized that Zn exerts an effect on eNOS at the transcriptional and post-translational levels. Our results suggested that both NO and hydrogen peroxide (H2O2) activate eNOS in a Zn-dependent manner by liberating cellular stores of Zn and activating the heavy metal-responsive transcription factor 1, MTF-1, which then would bind to the eNOS 5'-regulatory MRE. We found that basal zinc levels in endothelial cells (ECs) helped maintain eNOS promoter activity and total protein levels, but did not affect eNOS dimer levels. However, supplementing ECs with exogenous Zn increased eNOS total and dimer protein levels and activity, mostly through MTF-1. At higher, non-toxic doses of Zn, reactive oxygen species (ROS) production was much higher. eNOS was inhibited by high ROS levels through enzyme fragmentation. Lastly, an eNOS enzyme cofactor, tetrahydrobiopterin (BH4), was found to help maintain eNOS protein due to its high antioxidant potential. In conclusion, eNOS appears to be regulated by NO and ROS through an increase in intracellular Zn, thus activating MTF-1, which binds to the 5' flanking region of the eNOS promoter.
9

ACUTE CELLULAR UPTAKE OF ABNORMAL PRION PROTEIN IS CELL TYPE AND SCRAPIE STRAIN INDEPENDENT

Greil, Christopher Scott 01 October 2008 (has links)
<p>Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that include Creutzfeldt-Jakob disease, bovine spongiform encephalopathy and sheep scrapie. TSE disease pathology and mechanisms within the central nervous system (CNS) of an infected host largely remains unclear. At the cellular level, the uptake of protease resistant prion protein (PrP-res), which strongly correlates with infectivity and is a valid marker for TSE infection, is one of the earliest events that must occur during TSE infection. Given the difficulty of clearly distinguishing input PrP-res from either PrP-res or protease-sensitive PrP (PrP-sen) made by the cell, the uptake of PrP-res from an infectious inoculum into the host cell remains a poorly understood process. Through the development of a novel assay to exclusively detect input PrP-res we hypothesized that the acute infection of cells by PrP-res is mediated through general processes such as endocytosis, whereas internalization, retention, and propagation of PrP-res are dictated by specific characteristics of both the host cell and PrP-res. Using PrP-res tagged with a unique epitope to the mouse monoclonal antibody 3F4, we developed a detection system to specifically follow the acute cellular uptake of PrP-res. Mouse neural and fibroblast cells were exposed to three different mouse scrapie strains and PrP-res from the inoculum monitored. For all strains, PrP-res uptake was rapid and independent of both cellular prion protein expression and cell type. However, only 30%-40% of the cells were able to internalize PrP-res and PrP-res aggregate size influenced PrP-res uptake. Furthermore, infectious brain homogenate PrP-res was taken up more efficiently then PrP-res in either microsome or partially purified preparations. Our results suggest that PrP-res aggregate size, the PrP-res microenvironment, and/or host cell-specific factors can all influence whether or not a cell takes up PrP-res following exposure to TSE infectivity.</p>
10

NAD(P)H:QUINONE OXIDOREDUCTASE (NQO1)-DIRECTED LAVENDAMYCIN ANTITUMOR AGENTS: STRUCTURE-BASED DESIGN, MOLECULAR MODELING AND STRUCTURE-ACTIVITY STUDIES.

Hassani, Mary 14 September 2007 (has links)
NAD(P)H:quinone oxidoreductase 1 (NQO1) is a two-electron reductase that catalyzes an NAD(P)H-dependent activation of many quinone-based antitumor agents. NQO1, expressed at high levels in many human solid tumors, can be used as a target for enzyme-directed bioreductive antitumor drug development. We hypothesized that lavendamycins, quinolinedione antitumor antibiotics, can be activated by NQO1 in cancer cells that overexpress NQO1 to exhibit selective toxicity toward those cells. The effects of functional group changes on the metabolism of lavendamycins by recombinant human NQO1 were studied using a spectrophotometric assay. These structure-activity relationship (SAR) studies determined key structural features that were required for lavendamycin substrate specificity. Cytotoxicity toward human colon adenocarcinoma NQO1-deficient (BE) and NQO1-rich (BE-NQ) cells was also determined using colorimetric and clonogenic assays. The best lavendamycin substrates for NQO1 were also the most selectively toxic to the BE-NQ cells compared to BE cells. To facilitate structure-based design of more optimal lavendamycin substrates and NQO1-directed lavendamycin antitumor agent development, we developed a 1H69 crystal structure-based in silico model of the NQO1 active site and performed lavendamycin-docking studies. The docking was performed using the FlexX module of SYBYL software. Lavendamycin analogues were designed as NQO1 substrates utilizing our SAR and docking data as structure-based design criteria. Docking and biological studies on the analogues were performed and were consistent suggesting the in silico model of the enzyme possessed practical predictive power. Our results also suggested practicality of the design criteria resulting in the discovery of good NQO1 substrates with selective toxicity toward BE-NQ cells. The mechanisms of NQO1-mediated selective cytotoxicity of good lavendamycin substrates in BE and BE-NQ cells were also investigated including induction of oxidative stress and apoptosis. Biomarkers of oxidative stress including formation of 8-hydroxy-2'-deoxyguanosine (8-oxo-2dG), an indicator of oxidative DNA damage, and depletion of reduced glutathione (GSH) were examined using an HPLC-based method and a colorimetric assay, respectively. Induction of apoptosis was examined using a colorimetric assay. Our results revealed that oxidative stress and subsequent apoptosis induction by a good lavendamycin substrate was NQO1 dependent and that the poor substrate for NQO1 caused neither oxidative stress nor apoptosis.

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