Theses and Dissertations
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21598
Browse
Browsing Theses and Dissertations by Author "Agarwal, Neeraj"
Now showing 1 - 9 of 9
- Results Per Page
- Sort Options
Item Alterations in mRNA Levels of Selected Gene Products During Hypoglycemia, Hypoxia, and Ischemia Induced Apoptosis of Cultured Rat Retinal Ganglion Cells(2001-08-01) Vopat, Kelly S.; Agarwal, Neeraj; Wordinger, Robert J.; Pang, Iok-HouVopat, K., Alterations in mRNA Levels of Selected Gene Products during Hypoglycemia, Hypoxia, and Ischemia Induced Apoptosis of Cultured Rat Retinal Ganglion Cells. Master of Science (Biomedical Science), August 2001. 54 pp., 2 tables, 10 illustrations, bibliography, 105 titles. In order to explore the mechanisms involved in the signal transduction pathways of ischemia-induced apoptosis of RGCs in glaucoma, an in vitro ischmia model of transformed rat retinal ganglion cells (RGC-5) was utilized. RGC-5 cells were exposed to hypoglycemia, hypoxia, and ischemia for six hours. Hypoxia and ischemia resulted in apoptosis of RGC-5 cells as determined by TUNEL assay. The bax mRNA levels increased significantly in cells exposed to hypoxia. The mRNA levels of hemoxygenase, c-fos HSP 70, and BDNF showed a trend of increase in both the hypoxic and ischemic conditions. These results demonstrate that retinal ganglion cells undergo apoptosis in hypoxic conditions likely via an increase in bax/bcl-2. The up-regulation of BDNF and some stress proteins may be part of a cellular rescue effort trying to overcome the damage created by hypoxic and ischemic stresses.Item Brain Derived Neurotrophic Factor Regulates Müller Cell Survival via MAPK and PI3K Pathways(2003-05-01) Taylor, Sara A.; Agarwal, Neeraj; Wordinger, Robert J.; Pang, Iok-HouTaylor, Sara A., Brain Derived Neurotrophic Factor Regulates Müller Cell Survival via MAPK and PI3K Pathways. Master of Science (Biomedical Sciences), January, 2003, 112 pp., 4 tables, 39 illustrations, bibliography, 68 titles. Purpose: Glutamate has been implicated in many pathologies affecting the Central Nervous System including those in the retina, but the exact nature of the role of glutamate in neuronal degeneration remains unclear. In the retina. Müller cells are resistant to glutamate insults that are normally toxic to other cells of the retina, however the molecular and biochemical mechanisms that control their death or survival are not well understood. We used a series of pharmacological inhibitors and molecular biology agents on cultured Müller cells to dissect two key signaling pathways normally involved in cell survival, the Mitogen Activated Protein Kinase – Extracellularly Regulated Kinase (MAPK(ERK) pathway and the Phosphatidylinositide 3 Kinase (PI3K) pathway. Since preliminary data in our laboratory showed that Müller cells upregulate their secretion of neurotrophins including Brain Derived Growth Factor (BDNF) in response to glutamate treatment, we also examined the effect of BDNF on the activation of these two signaling pathways. Methods: Early passaged Müller cells were treated with various concentrations (5 nM -50 μM) of inhibitions of the MAPK(ERK) pathway (GW5074, U0126, and PD98059) or with various concentrations (1-50 μM) of inhibitors of the PI3K pathway (LY294002 or Akt inhibitor) in the presence and absence of 50 ng/ml of BDNF for 24 hours. These experiments were repeated in Müller cells transfected with either NFκB or Bc12 DNA. Cell cultures were then analyzed for surviving cells with an MTS/PMS assay, a colorametric method for determining the number of viable cells in a proliferation assay. Results: The MAPK (ERK) inhibitors PD98059 and GW5074 both resulted in decrease in Müller cell survival. PD98059 did not decrease Müller cell survival until concentrations were high enough to suppress ERK2 phosphorylation. Müller cells transfected with NFκB or Bc12 DNA were able to resist treatment with concentrations of PD98059 that reduced cell number in untransfected cells. The PI3K inhibitor LY294002 also resulted in significant decreases in Müller cell survival in both untransfected cells and cells transfected with NFκB or Bc12 DNA. Treatment with an inhibitor farther down in the PI3K pathway, Akt inhibitor, did not significantly decrease Müller cell survival. Finally, BDNF was not able to increase cell survival in Müller cells treated with PD98059 or U0126, although it did increase the survival of cells treated wit GW5074. BDNF was also able to reverse the decrease in cell survival caused by LY294002 in both untransfected Müller cells or Müller cells transfected with NFκB or Bc12 DNA. Conclusions: Our data shows that Mitogen Activated Protein Kinase – Extracellularly Regulated Kinase (MAPK(ERK) and Phosphatidylinositide 3 Kinase (PI3K) are both essential for Müller cell survival. There is modulation between the pathways and they may interconnected far upstream at a protein previously associated with only the MAPK(ERK) pathway. These results are consistent with a role for both pathways in Müller cell survival.Item Cellular and Molecular Mechanisms that Distinguish the Effects of Progestorone and Medroxyprogesterone Acetate on Neuroprotection(2006-07-28) Kaur, Paramjit; Goldfarb, Ronald; Singh, Meharvan; Agarwal, NeerajKaur, Paramjit. Cellular and Molecular Mechanisms That Distinguish the Effects of Progesterone and Medroxyprogesterone Acetate on Neuroprotection., Doctor of Philosophy, (Pharmacology and Neuroscience), July, 2006, 203 pp., 5 illustrations, 20 figures and bibliography. Women have a higher prevalence for Alzheimer’s disease (AD) than men, suggesting that the precipitous decline in gonadal hormone levels following the menopause may contribute to the risk of developing AD. However, principal results from the Women’s Health Initiative concluded that women taking conjugated equine estrogens combined with medroxyprogesterone acetate (MPA, tradename: Prempro) incurred more harmful than beneficial outcomes versus the placebo group (Rossouw et al., 2002). This dissertation was aimed at determining if the discrepancy between basic science reports and these clinical studies could have been due to the synthetic progestin, MPA. I hypothesized that P4 and MPA differed in their ability to protect against the excitotoxic/oxidative insult, glutamate. Further, I proposed that this difference in neuroprotective potential would be reflected in the difference in the ability of these hormones to elicit key effectors of two neuroprotection-associated signaling pathways, the ERK/MAPK and P13-Kinase pathways. Finally, studies were initiated to evaluate the potential importance of BDNF (brain-derived neurotrophic factor) in mediating the protective effects of P4. I used organotypic explants of the cerebral cortex, and found that both P4 and MPA elicit the phosphorylation of ERK and Akt, two signaling pathways implicated in neuroprotection, with maximal phosphorylation occurring at a concentration of 100 nM. Interestingly, P4 protected against glutamate- induced toxicity however, while an equimolar concentration of MPA (100nM) did not. Further, P4 resulted in an increase in BDNF, while MPA did not. Our data bring into question the relevance of using MPA as a component of hormone therapies in postmenopausal women, and instead, argue that the relevant progestin for use in treating brain-related disorders is progesterone. Collectively, the data presented here suggest that P4 is protective via multiple, and potentially related mechanism, and importantly, its neurobiology is different from the clinically used progestin, MPA.Item Combined Chemo/Anti-Angiogenic Cancer Therapy in Lewis Lung Metastases(2002-05-01) Sinha-Datta, Anjuli; Goldfarb, Ronald H.; Agarwal, Neeraj; Mathew, Porunelloor A.Datta, Anjuli. Combined Chemo/Anti-Angiogenic Cancer Therapy in Lewis Lung Metastases. Master of Science (Microbiology and Immunology), May 2002. 41 pp., 17 illustrations, bibliography. The focus of my dissertation studies is an eight amino acid peptide (Å6) derived from the non-receptor binding region of urokinase plasminogen activator (uPA), which partially inhibits the binding of uPA to its receptor (uPAR). Å6 has been synthesized as a potential novel anti-cancer agent and kindly provided by Ångstrom Pharmaceuticals, Inc. (San Diego, CA). We further examined potential therapeutic properties of Å6 in vivo and in vitro. Å6 appeared to directly inhibit the invasion of Lewis lung carcinoma cells through Matrigel by approximately 40-45% compared to control. In addition, Å6 had a morphological effect resulting in thicker tubes on small vessel endothelial tube formation compared to no treatment. Interestingly, doxorubicin had similar effects when added to growing endothelial cells. Moreover, Å6 was administered alone and in combination with a standard clinically used chemotherapeutic agent, doxorubicin, in a Lewis lung carcinoma mouse model to test possible synergy between an anti-angiogenic compound (Å6) and a chemotherapeutic agent. This is the first observation that Å6 has the potential to display a direct anti-metastatic therapeutic effect for established pulmonary metastases in this model. Therefore, we believe that Å6 in combination with doxorubicin has the potential to provide better therapy to cancer patients with tumor metastases than potent chemotherapeutics agents alone, by increasing the dose of non-toxic Å6 and reducing the recommended dose of doxorubicin.Item In Vitro Effect of CNTF, FGF-9, IL-1α on Human Optic Nerve Head Astrocytes(2004-08-01) Tovar-Vidales, Tara; Wordinger, Robert J.; Alvarez-Gonzales, Rafael; Agarwal, NeerajTovar, Tara., In Vitro Effect of CNTF, FGF-9, and IL-1α on Human Optic Nerve Head Astrocytes. Master of Science (Biomedical Sciences), August 2004, 100 pp., 4 tables, 35 illustrations, bibliography, 163 titles. Glaucoma is a leading cause of blindness worldwide. A major risk factor for glaucoma is increased intraocular pressure that leads to pathological changes in the optic nerve head (ONH). Astrocytes within the ONH become activated in glaucoma and may create an environment detrimental to retinal ganglion cell axons. The factors that cause activation of the ONH astrocytes (ONA) are unknown, although there is evidence that CNTF, FGF-9, and IL-1α activate glial cells within the CNS. The purpose of this research was to determine if exogenous CNTF, FGF-9, and/or IL-1α activate human ONH astrocytes.Item Obesity Genetics: The Prevalence of DRD2, DAT1 and DBH Genes in the Obese Individual(1998-08-01) Davis, Karla R.; Eisenberg, Arthur; Agarwal, Neeraj; Sherman, MarkDavis, Karla R., Obesity Genetics: The prevalence of DRD2, DAT1 and DBH Genes in the obese individual. Master of Science (Biomedical Sciences), August, 1998, 106 pp., 3 tables, 14 illustrations, reference, 44 titles. Obesity has been presented in research literature as a polygenic or multiple gene disorder. Currently, 3 genes have been associated with obesity, dopamine receptor D2 (DRD2), dopamine transporter (DAT1), and dopamine beta hydroxylase (DBH). The primary objective of this study is to analyze the DRD2, DAT1 and DBH genes to determine if a correlation exists between certain allelic variations of these 3 genes and the body mass index of obese individuals. We have developed an assay for the DRD2, DAT1 and DBH genes, utilizing polymerase chain reaction (PCR) technology. Within the DRD2 gene, 2 allelic variants have been identified, the A1 and A2 alleles. The A1 allele consists of a 310 bp fragment in which the Taq 1 restriction site has been deleted. The A2 allele consists of 180 bp fragment and a 130 bp fragment. The presence of the A1 allele after enzyme digestion has shown a strong correlation to obesity in prior studies. With respect to the DAT1 gene, a VNTR of 40 bp’s has been correlated to other disorders within the ‘reward deficiency syndrome’. The fragment length identified most often is 440 or 480 bp, with 480 as the primary fragment in obesity. The DBH gene is similar to the DRD2 in that it also contains a Taq I restriction. Two allelic variants are also identified, B1 and B2. The B1 allele contains no Taq I site and produces a 316 bp fragment while the B2 does cleave, exhibiting an 86 bp and a 230 bp fragment after enzyme digestion. The presence of one or more of the aberrant alleles could be associated with and a predisposing factor to obesity.Item Role of Nonfeminizing Estrogen Analogues in Neuroprotection of Rat Retinal Ganglion Cells Against Glutamate-Induced Cytotoxicity(2007-05-01) Kumar, Domalapalli Maneesh; Agarwal, Neeraj; Gracy, Robert; Garner, MargaretKumar, Domalapalli Maneesh, Role of Nonfeminizing Estrogen Analogues in Neuroprotection of Rat Retinal Ganglion Cells against Glutamate-Induced Cytotoxicity, Doctor of Philosophy (Cell Biology and Genetics), May, 2007, 210 pp., 3 tables, 23 figures, bibliography, 427 titles. Retinal ganglion cell death has been determined to be the final common pathway in glaucoma. Continuous loss of retinal ganglion cells results in irreversible progressive visual field deterioration that culminates in blindness. No effective therapy is currently available to reverse retinal ganglion cell loss. Therefore, preventing the loss of retinal ganglion cells is a logical approach to maintaining vision in effected individuals. Of the methods of investigation, in vivo models of ganglion cell death provide a physiological system in which to study neuroprotective drugs and their effects, but these systems are inefficient for initial screening studies. We have addressed this by utilizing the RGC-5 clonal rat retinal ganglion cell line. Glutamate treatment of RGC-5 cells induces apoptotic death which can be attenuated by pretreatment with the anti-oxidants N-acetyl cysteine and thiourea, implicating oxidative stress as a major component of glutamate’s cytotoxicity. Also antioxidants, estrogens have been demonstrated to be potent neuroprotectants in a variety of in vitro and in vivo models of neurodegeneration. Estrogens’ antioxidant capacity has been attributed to the ability of the phenolic A ring to quench and resonance stabilize oxidative free radicals. It is also known that the estrogen A ring is responsible for binding of these hormones to estrogen receptors, producing feminizing phenotypes. The feminizing effects of estrogens narrow or preclude their use as neuroprotectants in males, and in females that may be predisposed to their deleterious effects. To address these shortcomings we screened 13, non-feminizing, non-receptor binding estrogen analogues in our glutamate-induced RGC-5 model of oxidative stress-induced cell death. The most effective of these drugs was ZYC-3. ZYC-3 was synthesized by the addition of an adamantly group to the C2 position on the A ring of estrone. This modification produced a neuroprotective compound with potency and efficacy at least equal to the prototypical estrogen, 17β-estradiol, but with no appreciable binding affinity for estrogens receptors α or β. Our preliminary findings suggest that ZYC-3 enhances glutathione synthesis and blocks mitochondrial apoptotic pathways. However, as a novel drug we are naïve to its effects on cellular physiology and as to how it affords neuroprotection. Understanding how this drug regulates cellular destructive and protective mechanisms could lead to further innovations in drug design and in methods to prevent retinal ganglion cell degeneration. In vivo studies of this drug may then form the bridge to a better clinical approach to managing ocular disorders in which ganglion cell loss is the culprit for vision loss. Although promising, evidence supporting the application of estrogen analogues in models of ocular neurodegenerative diseases are nearly non-existent. It is our objective to study the neuroprotective effects of ZYC-3 in glaucomatous models with the goal of maintaining retinal ganglion cell viability and preventing vision loss.Item Serum-Deprivation: A Model for Lens Cell Differentaition(2001-12-01) Ong, Marcia; Garner, Margaret; Agarwal, Neeraj; Roque, RouelOng, Marcia., Serum-Deprivation: A Model for Lens Cell Differentiation. Master of Science (Biomedical Sciences), December, 2001, 154 pp., 2 tables, 36 illustrations, bibliography, 91 titles. The purpose of thisi project was to develop an in vitro cell culture system in which mammalian lens epithelial cells differentiate into lens fiber cells. METHODS. Primary cultures were grown from lens epithelium explants obtained from bovine lenses and propagated in Minimum Essential Medium containing 10% calf serum. Subsequently, cell cultures were maintained in MEM supplemented with either 4%, 3% or 1% calf serum and left undisturbed for 21 days. Immunofluorescence experiments and Western Blot analysis were performed in order to determine expression of lens fiber-specific protein expression within these cells. RESULTS. The following lens fiber-cell markers, MP-26, beta and gamma crystalline and filensin were expressed in immunofluorescence micrographs and Western Blots, and cells propagated in 10% serum (high) did not express the fiber cell markers. CONCLUSIONS. Cultured lens epithelial cells maintained in reduced serum conditions differentiate into fiber cells.Item The Role of Calcium in Light-Induced Photoreceptor Apoptosis(2002-05-01) Krueger, Darrel Scott; Collier, Robert; Agarwal, Neeraj; Wordinger, Robert J.Krueger, Darrel Scott, The Role of Calcium in Light-Induced Photoreceptor Cell Apoptosis. Doctor of Philosophy (Biomedical Sciences), May, 2002; 305 pp., 15 tables, 31 illustrations, bibliography, 421 titles. Retinal degenerative diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD) result in loss of photoreceptors by apoptosis. Photo-oxidative stress accelerates the rate of photoreceptor apoptosis in models of retinal dystrophies. Thus, a better understanding of light-induced apoptosis is important for developing preventative and treatment options for persons with these blinding diseases. The goals of this dissertation were to investigate the effect of photo-oxidative stress on [Ca2+]I in rat photoreceptor cells and determine whether cell death could be prevented by altering cells’ abilities to manage [Ca2+]i. Thirty minutes of light exposure resulted in significant elevation of [Ca2+]i, determined using Fura-2 ratiometric imaging, which increased with additional light exposure. At 120 minutes, the F340/380 ratio was 3-times the beginning baseline ratio. Using multiple techniques indicative of early and late phases of apoptosis, changes consistent with apoptosis were observed, including early labeling (30 Min) with Annexin-V, activation of caspase-3 (2IIr), TUNEL labeling and Propidium Iodidc staining. TUNEL and Propidium labeling were more intense at 3Hrs light exposure, reflecting late phase changes. Apoptosis was confirmed using electron microscopy (TEM). TEM showed mitochondrial swelling, indicative of permeabilization, prior to chromatin condensation. Pharmacological agents were utilized to mediate participation of cellular calcium sources or storage sites in the maintenance of intracellular calcium homeostasis during photo-oxidative stress. Agents were separately added to the media prior to light exposure and their effects on cell viability were assessed using the Formazan assay. Mitochondria were confirmed to be the site of action affected by elevated [Ca2+]I, since prevention of calcium uptake by ruthenium red (1-100-μM) provided significant protection of cell viability in a dose-related manner. The 100- μM concentration resulted in complete maintenance of viability. BAPTA-AM also demonstrated some protection (50%) indicating that reduction of [Ca2+]I independent of source is beneficial is maintaining cell viability. Identification of mitochondrial uptake and cytosolic buffering of calcium as key viability determinants in light-induced apoptosis is a significant discovery for targeting future research for preventing or inhibiting photoreceptor cell apoptosis associated with retinal dystrophies such as RP and AMD.