ETD Search Results: instcode:kent

1. Acharya, Kalpana D. Ms. ROLE OF MEMBRANE BOUND G-PROTEIN COUPLED ESTROGEN RECEPTOR GPR30 AND Z-LINKED RIBOSOMAL GENE S6 (RPS6) IN SEXUALLY DIMORPHIC DEVELOPMENT OF THE ZEBRA FINCH BRAIN.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2012, Kent State University

► Neural dimorphisms in the songbird represent one of the most extreme examples… (more)

Subjects: Neurology

Keywords: songbird, sex differences, zebra finch, brain dimorphisms, membrane estrogen receptor

More Like This

2. Agarwal, Udit. Factors Affecting Ventricular Remodeling Post Myocardial Infarction.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2010, Kent State University

► The term ventricular remodeling refers to the series of events that lead… (more)

▼ The term ventricular remodeling refers to the series of events that lead to the orchestration of the biological processes that determine left ventricular size, shape, and function following acute myocardial infarction (AMI). The present study focuses on defining the mechanisms of two distinct phenomena that are known to regulate this process; namely, extracellular matrix (ECM) degradation via protease inactivation and cardiac myocyte death. AMI is followed by degradation of ECM of the heart leading to thinning and rupture of the left ventricular wall. A critical regulator of ECM degradation is plasmin, a protease. The generation of plasmin in turn is regulated by the oxidation-sensitive enzyme, Plasminogen Activator Inhibitor -1 (PAI-1). PAI-1 is an inhibitor of urokinase-like plasminogen activator (uPA), which converts plasminogen into plasmin. PAI-1 deficient (Pai-1-/-) mice die of ventricular rupture within 7 days following infarction. It has been previously reported that the activity of PAI-1 is sensitive to oxidation and hence inhibited by leukocyte-derived oxidant-generating systems, such as Myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS). Furthermore, the expression of ceruloplasmin (CP), another oxidant- generating enzyme, is increased in the blood post-AMI. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity regulates the oxidant-generating capacity of MPO, iNOS, and CP. However, the relative contributions of each of these leukocyte-derived oxidant producing enzymes in PAI-1 oxidation is not known. We hypothesized that leukocyte-derived NADPH oxidase oxidizes PAI-1 and hence participates in ventricular rupture post-AMI. To address this issue, we transplanted Pai-1-/- mice with Pai-1-/-, wild type (WT), p47phox-/- (a subunit of NADPH oxidase complex, also known as neutrophil cytosolic factor-1), Mpo-/-, iNOS-/- and Cp-/- bone marrow, performed Left Anterior Descending (LAD) artery ligation in all the groups, and measured their survival for 21 days. Data demonstrate Pai-1-/- mice were dead by day 7 due to ventricular rupture and WT marrow partially rescued Pai-1-/- mice survival. Interestingly, p47phox-/ – /- marrow-transplanted mice had a significantly better survival compared to WT marrow-transplanted Pai-1-/- mice suggesting that, NADPH oxidase is critical in inhibiting PAI-1-induced protease inactivation and ventricular rupture. PAI-1 activity was higher in p47phox-/- marrow-transplanted Pai-1-/- animals compared to WT marrow-transplanted Pai-1-/- animals, indicating that leukocyte-derived NADPH oxidase inhibit PAI-1 activity in this model of ventricular remodeling. Another determinant of ventricular remodeling is cardiac myocyte death. Several studies have identified the stromal cell-derived factor-1 α (SDF-1α)/CXC chemokine receptor 4 (CXCR4) axis to be important for the homing and survival of stem cells at the site of injury post-AMI. It has also been reported that Sdf-1-/- and Cxcr4-/- mice exhibit defects in hematopoesis, neurogenesis, vacsulogenesis, and ventricular septum formation and die within a few days of birth. Multiple reports claim that overexpression of SDF-1 at the infarcted region improves ventricular function by preservation of cardiac myocytes and increased vasculogenesis. Since the role of cardiac myocyte-derived CXCR4 was not well-defined in this context, we hypothesized that normal cardiac development is dependent on cardiac myocyte-derived CXCR4 expression. In the adult heart, SDF-1α and its receptor, CXCR4 do not get expressed at the same time. Post-AMI, there is a short and immediate period of SDF-1α expression, which is followed by CXCR4 expression in cardiac myocytes. Therefore, we postulated that cardiac myocyte-derived CXCR4 expression is not critical in adulthood post-AMI. To address these issues, we developed congenital and conditional cardiac myocyte-specific Cxcr4-/- mouse models. Our results demonstrate that congenital deletion of CXCR4 has no effect on cardiac function and septal defect. Furthermore, the ventricular function of conditionally deleted cardiac myocyte-specific CXCR4 was not significantly different from their WT littermates. Collectively, our observations suggest that leukocyte-generated NADPH oxidase-derived free radicals participate in ventricular rupture post-AMI by oxidizing PAI-1 and cardiac myocyte-derived CXCR4 has no major role in cardiogenesis during development and ventricular remodeling post-AMI due to mismatch in the timing of expression of SDF-1 and CXCR4 in the heart. Advisors/Committee Members: Penn, Marc S.

Subjects: Biomedical research; Cellular biology

Keywords: Plasminogen Activator Inhibitor-1 oxidation; Cardiac myocyte specific CXCR4; Post Myocardial Infarction; Ventricular remodeling

More Like This

3. Armfield, Brooke Autumn. The Evolution and Development of Mammalian Tooth Class.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2010, Kent State University

► For centuries, the dentition has been important for morphological studies, while in… (more)

Subjects: Molecular biology

Keywords: Tooth development

More Like This

4. Awale, Prabha Sumant. Microglial alterations in valproic acid models of autism.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2012, Kent State University

► Autism spectrum disorder (ASD) is a neurodevelopmental disorder affecting approximately 1 in… (more)

▼ Autism spectrum disorder (ASD) is a neurodevelopmental disorder affecting approximately 1 in 110 children in North America. Valproic acid (VPA) is a multi-target drug widely used to treat epilepsy. It is also a histone deacetylase inhibitor and fetal exposure to it increases the risk of ASD. One important hallmark of ASD is an unusually large brain size around the age of 2 years. The cause of this enlargement is not known, but it is speculated that a lack of pruning of dendrites may be associated with the large brains. The effect of prenatal exposure to VPA on microglial survival during postnatal brain development has not been studied and may provide some potential hints regarding the etiology of this disorder. In this study, we have tried to delineate the effect of VPA on macrophage/microglia activation states/survival both in in vitro and in vivo to better understand VPA’s underlying mechanism in this disorder. Based on the experiments conducted, it was observed that VPA potentiated the response to endotoxin stimuli by increasing the production of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) from two separate macrophage cell lines. VPA was further evaluated using in vivo experiments and was shown that exposure during embryogenesis causes a reduction in the number of microglia in the primary motor cortex (PMC) at postnatal day 6 (P6) and postnatal day 10 (P10) in male mice. Similar reduction in microglial number was observed in female mice at P10. However, the effects of VPA were more pronounced in male mice than female mice. These data suggests that VPA alters the activation states of macrophage/microglia as well as affects the survival in early postnatal brain development. The effect of VPA on microglial survival/activation state may be partly responsible in mediating the unusual brain size observed in the pathology of ASD. In the future, pharmacological intervention aimed at protecting the macrophage/microglia population during crucial period of brain development may be beneficial in ASD. Advisors/Committee Members: Carroll, Richard T.

Subjects: Biomedical Research

Keywords: autism; valproic acid; microglia; macrophage; cytokines; primary motor cortex; mice; prenatal exposure; postnatal neurodevleopment

More Like This

5. Barton, Maria. Evaluating XMRV As An Indicator Of Prostate Cancer Risk.

Degree: MS, College of Arts and Sciences / School of Biomedical Sciences, 2011, Kent State University

► Xenotropic Murine Leukemia Virus-Related Virus (XMRV) is a retrovirus originally identified in… (more)

Subjects: Biomedical Research

Keywords: Prostate cancer; XMRV; envelope protein; amplicon

More Like This

6. Beard, LaMonta L. Regulation of Distal Lung Fluid Absorption by Membrane Components.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2011, Kent State University

► Near the end of gestation, the direction of ion and fluid flow… (more)

Subjects: Biomedical Research

Keywords: Cl transport, distal lung fluid absorption, fetal lung development, IL-1beta, Na transport

More Like This

7. Brett, Adina R. The effect of the Wilms' tumor gene 1 (WT1) on E-cadherin regulation and migration of prostate cancer cells.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2012, Kent State University

► Prostate adenocarcinoma is a complex disease and the molecular mechanisms that control… (more)

Subjects: Biomedical Research

Keywords: WT1, E-cadherin, prostate cancer

More Like This

8. Cardona, Sandra Milena. Understanding temporal and cellular constraints involved in the development of MLV-induced spongiform neurodegeneration.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2012, Kent State University

► Certain murine leukemia viruses (MLVs) are capable of inducing a progressive motor… (more)

▼ Certain murine leukemia viruses (MLVs) are capable of inducing a progressive motor neuron disease that clinically resembles amyotrophic lateral sclerosis (ALS) and results in vacuolar degeneration characteristic of prion diseases. Development of spongiosis requires neurotoxic Env protein expression within cells of the brain parenchyma, however the nature of the cell type that mediates pathology remains unresolved. Additionally, spongiosis requires developmental maturation of susceptible neural elements beyond postnatal day 10 (P10). In the first part of this study we investigated whether the P10 developmental maturation was the only requirement for neuropathology. Neural stem cell-mediated virus challenge of P10 “mature” brains revealed that neuronal susceptibility acquired at P9-10 is not sufficient to facilitate the appearance of acute spongiform changes; instead a 7-day delay in neuropathology appearance implicates the requirement for additional events beyond viral entry, spread or neurotoxic Env expression. In the second part of this document we investigated the cell type involved in the development of spongiform neurodegeneration. Here we provide evidence for a critical role of infection of astrocyte progenitors and their maturation and interaction with susceptible neurons in MLV-induced neuropathogenesis. Identification of astrocytes as novel targets of MLVs was accomplished by the utilization of transgenic mice as a readout for MLV infection, because in a scenario of sub-detectable viral protein expression, standard immunological probes in combination with cell type specific markers showed no differences in the CNS cell types infected with neurovirulent or non-neurovirulent viruses that could account for neuropathogenicity. Implication of astrocytes as critical players in MLV-induced neurodegeneration is supported by the utilization of an amphotropic virus (4070A) thought to be non-neurovirulent, but which was discovered by our laboratory to have neurotoxic effects only when pseudotyped by ecotropic Envs. This pseudotyping results in the dissemination of amphotropic neurotoxic sequences into astrocytes at a higher frequency in comparison to the restricted 4070A virus alone that infects a very low number of astrocytes and induces minimal spongiosis. Revealing the precise nature of the interactions between infected astrocytes and neuronal populations susceptible to undergoing vacuolar degeneration will provide more insight into the molecular mechanisms underlying neurodegenerative diseases. Advisors/Committee Members: Lynch, William.

Subjects: Neurosciences

More Like This

9. Chatterjee, Somik. STRUCTURE-FUNCTION ANALYSIS OF THE VIRULENCE PROTEIN ICP34.5 FROM HERPES SIMPLEX VIRUS TYPE 2.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2009, Kent State University

► The ICP34.5 gene of herpes simplex virus (HSV) is diploid, has unusually… (more)

▼ The ICP34.5 gene of herpes simplex virus (HSV) is diploid, has unusually high GC content and is expressed as a late protein in the viral cycle. The ICP34.5 protein is not essential for virus replication, but is essential for replication in neuronal and non-growing cells. The ICP34.5 protein from HSV-1 has an N-terminal arginine-rich region, a C-terminal mammalian GADD34-like region, and a central PAT (Proline-Alanine-Threonine) repeat region. The GADD34-like region of the HSV-2 ICP34.5 is almost identical to that of HSV-1, and the N-terminal region is also arginine-rich, although the protein sequence is considerably different elsewhere. The PAT repeat sequence is absent from HSV-2 ICP34.5, instead there is a 19 nucleotide repeat sequence in the ICP34.5 gene from HSV-2, which is excised as an intron. ICP34.5 from clinical sub-isolates of HSV-2 show extensive sequence homology with HSV-2 strain 333, but with a variable number of repeats of the 19 nucleotide sequence in the intron region. Differences in plaque morphology, efficiency of glycoprotein processing, and neuroinvasive disease potential of the virus are due to differences in the structure of ICP34.5 for HSV-1. My studies compared HSV type 1 and 2 variants of ICP34.5 with respect to structure and function. The HSV-2 strain 333 resembles the attenuated non-neuroinvasive HSV-1 strain KOS321 in that it forms large plaques in tissue culture cells, and glycoprotein processing is efficient. Cells expressing the ICP34.5 from HSV-2 strain 333 (by transfection) promoted full and efficient viral glycoprotein processing in cells infected with HSV-1 strain SP7. This shows that the ICP34.5 protein from HSV-2 strain 333, like the protein from HSV-1 KOS321, is sufficient to control and promote glycoprotein processing in HSV infected cells. However, unlike the KOS321 variant of ICP34.5, the HSV-2 strain 333 ICP34.5 protein localized in the cytoplasm rather than in the nucleus. Further studies show that the ICP34.5 from HSV-2 strain 333 binds to the mammalian autophagy protein beclin 1, as does the HSV-1 variants, despite considerable sequence differences. ICP34.5 proteins from HSV-2 and HSV-1, despite sequence differences, maintain the ability to bind to cellular proteins to enhance viral virulence. Advisors/Committee Members: Rosenthal, Kenneth.

Subjects: Biomedical research

Keywords: HSV-2, ICP34.5, structure-function, virulence

More Like This

10. Cheng, Lina. Regulation of Protein Phosphatase 1, PP1γ2, in Testis/Spermatozoa by PPP1r11, PPP1r7 and PPP1r2.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2008, Kent State University

► In mouse testis, PP1γ1 is restricted mainly to somatic cells, while PP1γ2… (more)

▼ In mouse testis, PP1γ1 is restricted mainly to somatic cells, while PP1γ2 is primarily located in secondary spermatocytes and germ cells, and the only isoform detectable in mammalian spermatozoa. Targeted deletion of the PP1γ gene results in sterile male mice. Because the most prominent marker of this phenotype is aberrant spermatid morphogenesis, its primary cause may be the absence of PP1γ2 since PP1γ1 is mainly in somatic cells. To better understand the role of PP1γ2 in mammalian testis/sperm, this dissertation was undertaken to explore PP1γ2 interactions with its regulatory proteins in testis/sperm. Wild-type and mutant isoforms of PPP1R11/inhibitor 3 (I3), PPP1R2/inhibitor 2 (I2) and a novel protein similar to I2 were studied.Testicular/sperm PP1γ2 was found to form a complex with I3, a potent protein phosphatase 1 inhibitor, PPP1R7/Sds22, the mammalian homologue of a yeast protein phosphatase 1 binding protein, and actin by binding assay, chromatography and native gel electrophoresis, and that in the spermatoozoon equivalent of this complex, PP1γ2 is catalytically inactive by phosphatase assay. In PP1γ-null testis, I3 levels drop significantly while increased dramatically when a transgenic source of testicular PP1γ2 is provided. Together with studies showing that PP1γ2 and I3 jointly reach their highest steady state levels during wild-type spermiogenesis, these data make a hypothesis that the formation of a complex containing PP1γ2 and I3 in testis and sperm, while inhibiting PP1γ2 catalytic activity, promotes sperm morphogenesis and subsequent function by stabilizing I3. To understand the role of a naturally occurring mutant isoform of I3 (t/t-I3) in mice involved in an abnormal sperm motility, curlicue, the properties of t/t-I3 and wt-I3 were compared, and found that they differed only in their phosphorylation in vitro. Finally, the presence of PPP1R2/inhibitor 2 (I2) in testis and sperm were demonstrated, and evidence was provided that a novel protein similar to I2 is transcribed in vivo and translated in vitro by Northern blot, RT-PCR and in vitro expression. These studies provide important insights into the regulation of PP1γ2 in testis and sperm. Advisors/Committee Members: Vijayaraghavan, Srinivasan.

Subjects: Biomedical research

Keywords: PP1γ2; PPP1R11; I3; PPP1R7; Sds22

More Like This

11. Cooper, Lisa Noelle. EVOLUTION AND DEVELOPMENT OF CETACEAN APPENDAGES.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2009, Kent State University

► The origin of cetaceans (whales, dolphins, and porpoises) has become a popular… (more)

▼ The origin of cetaceans (whales, dolphins, and porpoises) has become a popular case study in mammalian evolution. This dissertation utilizes a variety of methods to address the evolution and development of cetacean appendages. Comparative anatomical methods are used to investigate locomotor abilities of a fossil relative to cetaceans. Long bone cross-sectional areas were quantified and modern comparative phylogenetic methods were used to reconstruct character evolution in the limbs of cetaceans and artiodactyls. Experimental molecular techniques are also utilized to answer questions regarding the development of the dolphin forelimb. Fossil findings near the Jammu-Kashmir region along the India-Pakistan border show that a fossil artiodactyl family, Raoellidae, is closely related to cetaceans, lived in an aquatic environment, and never lost its ability to bear weight on land. Using comparative anatomical methods, the postcranial skeleton of the middle Eocene raoellid Indohyus was found to be a digitigrade, cursorial artiodactyl. Indohyus also had a thickened skeleton that may have afforded it a greater ability to swim compared to terrestrial artiodactyls. Vertebrates that occupy an aquatic environment (e.g., Hippopotamus, penguins) have an increased long bone cortical thickness compared to taxa that occupy a terrestrial niche. Little is known of cortical bone evolution in artiodactyls and cetaceans. This study reconstructed cortical bone evolution based on quantitative measures of cortical bone thickness of artiodactyls and cetaceans. Thickened bone evolved only once in artiodactyls, in the common ancestor of fossil anthracotheres, fossil raoellids, cetaceans and hippopotamids. Thickened bone cortices probably acted as skeletal ballast that allowed for the successful invasion of the marine environment. Most mammals have a generalized phalangeal formula with two phalanges in the thumb and three in the remaining digits. Cetaceans are the only mammals that have more than three phalanges per digit (hyperphalangy). Developmental mechanisms generating hyperphalangy are unknown. This study tests the hypothesis that dolphins exhibit greater duration of expression of the genes controlling development of limb growth from the body wall, and joint formation. Studies of protein signals during dolphin ontogeny indicate that the Fibroblast Growth Factors (Fgfs) are active while the forelimb is projecting from the body wall until 30 days gestation, experiences a brief hiatus, and Fgf-8 expression is recapitulated later in development after approximately six phalanges are formed. This recapitulation of Fgf-8 is unlike the generalized mammalian pattern, suggesting that heterochronic changes of expression took place. Cetaceans evolved a flipper consisting of soft tissue encasing bony digits that functions as a hydrofoil. While most mammals develop separated digits, cetaceans retain interdigital tissues during development. The genetic mechanisms that allow for the retention of interdigital tissues in cetaceans are unknown. This study found that a key activator of interdigital cell death, bone morphogenic proteins (Bmps) was active in the developing dolphin limbs, but action of Bmp was inhibited by overlapping signals of two Bmp-antagonists, Gremlin and Fibroblast Growth Factors (Fgfs). Gremlin directly inhibits the apoptotic effects of Bmp, while Fgfs promote the survival and proliferation of interdigital tissues. Advisors/Committee Members: Thewissen, J.G.M. "Hans".

Subjects: Biology

Keywords: Cetacea; Artiodactyla; Raoellidae; Indohyus; Hyperphalangy; Cortical Bone Thickness; Apical Ectodermal Ridge; Webbing

More Like This

12. Deshpande, Abhishek. Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) and blood vessel density changes in an experimental model of Chronic Hydrocephalus.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2010, Kent State University

► Chronic hydrocephalus (CH) is characterized by the presence of ventricular enlargement, decreased… (more)

▼ Chronic hydrocephalus (CH) is characterized by the presence of ventricular enlargement, decreased cerebral blood flow (CBF), and brain tissue oxygen delivery. Although the underlying pathophysiological role of vascular endothelial growth factor (VEGF) is not clear, ischemic-hypoxic events in CH are known to trigger its release. We investigated changes in neuronal and glial VEGFR-2 density and blood vessel density (BVd) in two clinically relevant areas of the brain, the hippocampus and caudate nucleus in an experimental model of CH. We also investigated changes in neuronal and glial VEGF receptor -2 density and BVd in the caudate nucleus after cerebrospinal fluid (CSF) shunting. Animals with CH were divided into short term (ST, 2-4 weeks) and long term (LT, 12-16 weeks) and were compared with surgical controls (SCs, 12-16 weeks) and CH-shunted animals (CH-S, 12-16 weeks). The cellular and BVds were estimated using immunohistochemical and stereological counting methods. Overall, percentage (%) VEGFR-2 neurons were approximately two–six times greater in CH (ST, LT) than in SC. There were differences in glial VEGFR-2 and BVd expression in the hippocampus and caudate nucleus. There was a six fold increase in VEGFR-2 glia in all regions of the hippocampus. By comparison, glial cell %VEGFR-2 was greater by 4-17% in CH compared with that in SC. Blood vessel density was six times greater in the hippocampus after CH but significantly lower in CH caudate compared to SC. There was a decrease in VEGFR-2 neuronal expression after shunting in the caudate regions compared to CH. After CSF shunting, the BVd initially decreased followed by an increase but only in the superficial caudate. Changes in CSF ventricular volume correlated with VEGFR-2 or BVd in the hippocampus but only partially in the superficial caudate region. VEGFR-2 may play an adaptive role in angiogenesis after CH-induced hypoxia. The dissociation of BVd and VEGF expression observed in the caudate differs from the hippocampus where uniform increases in BVd may have been facilitated by greater VEGFR-2 activation and better collateral blood supply. Modulation of the VEGF ligand and/or VEGFR-2 receptor may be important in developing treatments for hypoxic conditions including hydrocephalus and other forms of ischemia. Advisors/Committee Members: Luciano, Mark.

Subjects: Cellular biology; Neurology; Surgery

Keywords: angiogenesis, ischemia, canine, stereology, cerebrospinal fluid, shunting

More Like This

13. Dhakar, Monica B. The role of the vasopressin 1b receptor in the regulation of sensorimotor gating.

Degree: MS, College of Arts and Sciences / School of Biomedical Sciences, 2011, Kent State University

► The neuropeptide arginine vasopressin (Avp) plays an important role in the regulation… (more)

▼ The neuropeptide arginine vasopressin (Avp) plays an important role in the regulation of variety of social behaviors such as aggression, paternal care, social memory, and social motivation. Two types of receptors mediate these central effects: the vasopressin 1a receptor (Avpr1a) and the vasopressin 1b receptor (Avpr1b). Through the use of Avpr1b knockout mice (-/-) a critical role of the Avpr1b in the regulation of social behavior has been identified. Mice that lack the Avpr1b have impaired aggression, altered social recognition and reduced social motivation. In humans, Avp has also been implicated in several neuropsychiatric disorders such as schizophrenia and attention deficit hyperactivity disorder. One of the features of some neuropsychiatric disorders, such as schizophrenia, are deficits in sensorimotor gating, i.e., the ability to filter incoming sensory, cognitive and motor information. Deficits in sensorimotor gating can be modeled in animals by administering psychotomimetics and measuring prepulse inhibition (PPI) of the acoustic startle reflex. The objective of my thesis work was to examine the role of the Avpr1b in sensorimotor gating. PPI was assessed in male and female Avpr1b -/- and Avpr1b +/+ mice after injection of the following psychotomimetics: amphetamine, apomorphine and MK-801. We found that male Avpr1b -/- mice have significantly more disrupted PPI when treated with MK-801, which is a N-methyl-D-aspartate (NMDA) receptor antagonist, as compared to Avpr1b +/+ mice; no genotypic differences were found in females. These data suggest that Avpr1b’s modulation of the PPI neurocircuitry is likely via the glutamatergic system. We also found that Avpr1b -/- male mice have increased expression of NMDAR2A subunit expression in the hippocampus as compared to Avpr1b +/+ mice. It is likely that this alteration in the composition of NMDA receptors might be contributing to the PPI deficits seen in Avpr1b -/- mice following administration of a NMDA receptor antagonist. Advisors/Committee Members: Caldwell, Heather.

Subjects: Neurosciences

Keywords: sensorimotor gating; vasopressin 1b, knockout, schizophrenia

More Like This

14. Dike, Obianuju E. UNDERSTANDING THE ROLE OF OXYTOCIN IN SENSORIMOTOR GATING DEFICITS.

Degree: MS, College of Arts and Sciences / School of Biomedical Sciences, 2009, Kent State University

► Oxytocin (Oxt) is a nonapeptide synthesized in the magnocellular neurons of the… (more)

Subjects: Biomedical research

Keywords: Oxytocin, knockout mice, schizophrenia, glutamate, startle, PPI, MK-801, amphetamine

More Like This

15. Dunphy, Jaclyn Marie. Infection of Neural Stem Cells with Murine Leukemia Viruses Inhibits Oligodendroglial Differentiation: Implications for Spongiform Neurodegeneration.

Degree: MS, College of Arts and Sciences / School of Biomedical Sciences, 2012, Kent State University

► Certain murine leukemia viruses (MLVs) are capable of inducing progressive spongiform neurodegeneration… (more)

▼ Certain murine leukemia viruses (MLVs) are capable of inducing progressive spongiform neurodegeneration upon infection of the central nervous system (CNS). Clinical disease is characterized by hindlimb paralysis and loss of motor function that is ultimately fatal. The vacuolar changes associated with CNS infection arise at post-synaptic processes within the neuropil and also includes cells with intact nuclei that have lost cytoplasmic structure. These cytoplasmically effaced cells (CECs) have been suggested to result from direct neurovirulent virus infection of oligodendrocytes (OLs), however OL lineage cells are infected by a neurovirulent amphotropic MLV without inducing CECs or other vacuolar changes. Herein, we revisit this question. Neuropathology, infection status, and OL lineage differentiation state of uninfected mice was compared with mice neonatally exposed to neurovirulent (FrCasE; NV) or non-neurovirulent (Fr57E; NN) ecotropic viruses. CECs were detectable in the brainstems of FrCasE infected mice by 12 dpi, but not observed in Fr57E or control animals. Olig2 immunostaining showed half of the CECs in the FrCasE brainstems were OL lineage cells, but CECs showing a mature OL phenotype indicated by proteolipid protein promoter (PLP) driven enhanced green fluorescent protein (EGFP) expression were not detected. Double immunolabeling assessment of MLV Env+ cells for early (NG2), intermediate (Olig2), and late (CAII) OL lineage markers showed one third of infected CNS cells expressed the oligodendrocyte progenitor cell (OPC) markers NG2 and Olig2, but not mature OL markers CAII or PLP-EGFP, regardless of whether the mice were infected with FrCasE or Fr57E. In addition, no overt MLV-induced cytotoxicity was observed over 3 weeks in neurosphere (NPH) cultures, despite significant ex vivo infection by FrCasE (87.8%) and Fr57E (91.5%). Transplantation of ex vivo NN and NV-infected NPHs showed these cells could engraft and assume mature glial morphologies as well as uninfected NPH (tracked by mTomato expression). In contrast, transplantation of NPHs derived from PLP-EGFP mice showed that, unlike controls, cells exposed to FrCasE or Fr57E failed to differentiate into mature OLs by 2 weeks post-transplantation. This suggested that MLV infection of NSCs/OPCs was not directly cytotoxic, but rather interfered with differentiation into OLs in vivo. The implications for spongiogenesis are two-fold. First, inhibition of NG2+ cell differentiation would result in their persistence as excitable cells which may potentiate excitotoxicity initiated through the infection of astrocytes. Second, the differentiation-arrested OPCs are susceptible to excitotoxic damage due to their expression of Ca+2-permeable glutamate receptors, which would be down-regulated upon differentiation to OLs. Advisors/Committee Members: Lynch, William.

Subjects: Biomedical Research; Neurosciences

Keywords: neurodegeneration; retrovirus; oligodendrocyte progenitor cell

More Like This

16. Eisermann, Kurtis. Androgen Mediated Regulation of VEGF in Prostate Cancer.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2011, Kent State University

► An important goal for prostate cancer therapy is to identify novel mechanisms… (more)

▼ An important goal for prostate cancer therapy is to identify novel mechanisms of androgen signaling that may provide new targets for androgen blockade therapy. Androgen regulated target genes continue to be identified, and include genes with regulatory regions containing 1) classical dimeric androgen receptor elements (AREs), 2) sites for other transcription factors (TFs) that tether AR to a regulatory region lacking ARE sites, or 3) non-canonical ARE half-sites. Vascular endothelial growth factor (VEGF) is the most important signaling protein involved in angiogenesis and has been shown to be up-regulated by hormone, but the mechanisms of this regulation are unknown. Thus, this dissertation examined potential mechanisms of androgen-mediated regulation of VEGF in prostate cancer cells. Androgens were demonstrated to increase both VEGF mRNA and protein and the anti-androgen casodex attenuated this activation. Three non-canonical ARE half-sites were identified in the promoter of the VEGF gene. ChIP assays demonstrated binding of AR specifically to these sequences in vivo. Mutation of these monomeric ARE binding sites decreased activation of the VEGF promoter, but did not completely eliminate hormone response. Additional zinc finger transcription factor binding sites were discovered adjacent to these ARE sites, so these TFs were hypothesized to be playing a role in the androgen regulation of VEGF. Both Sp1 and WT1 bound the VEGF promoter at sequences adjacent to an ARE and formed a complex with AR as shown by co-immunoprecipitation assays. Although the VEGF core promoter lacks ARE sites, it also was upregulated by androgen. A specific Sp1 binding site in the core promoter was found to be responsible for the androgen regulation of this region, as mutation of this site specifically eliminated the hormone response. These results suggest a mechanism whereby AR is tethered to Sp1 bound to the VEGF promoter. It is important to delineate TFs that cooperate with AR because androgen signaling is vital to malignant prostate growth. Therapy targeting disruption of AR-ZFTF complexes may maintain the indolent form of prostate cancer and extend lives. Advisors/Committee Members: Fraizer, Gail.

Subjects: Genetics; Molecular Biology; Oncology

Keywords: Prostate cancer, androgen signaling, VEGF, AR, WT1, Sp1, transcriptional regulation

More Like This

17. Feng, Hailin. Srebp2 and Reverb-alpha Regulation of Human CYP8B1.

Degree: MS, College of Arts and Sciences / School of Biomedical Sciences, 2009, Kent State University

► Human CYP8B1 is a liver specific enzyme that catalyzes the synthesis of… (more)

▼ Human CYP8B1 is a liver specific enzyme that catalyzes the synthesis of cholic acid (CA) from cholesterol and determines the ratio of CA and chenodeoxycholic acid (CDCA) in the bile. Because CA is more hydrophilic than CDCA, CYP8B1 may regulate the hydrophobicity of the bile acid pool, which may determine the bile acid synthesis. In addition, CA plays an important role in enhancing the absorption of cholesterol in the intestine. Therefore, regulation of CYP8B1 may be crucial for both the synthesis of bile acids and the cholesterol homeostasis. Numerous regulators, such as sterol response element binding protein 2 (SREBP2), Reverb-α, have been shown to be involved in the transcriptional regulation of the synthesis of bile acids in rodents. We hypothesize that: 1) SREBP2 has inhibitory effects on human CYP8B1 gene expression by competing with PGC-1α recruitment to the CYP8B1 promoter. 2) Reverb-α inhibits human CYP8B1 promoter activity by binding at the RevRE on human CYP8B1 promoter. In the present study, using transient transfection assay, we revealed that both SREBP2 and Reverb-α exhibited significant inhibition on the transcriptional activity of human CYP8B1. Deletion analyses of the human CYP8B1promoter localized a region that mediated the inhibitory effects of SREBP2. Based on mammalian one-hybridization assay, our data indicated that SREBP2inhibited human CYP8B1 gene transcription by blocking HNF4α recruitment of PGC-1α. Using human CYP8B1 luciferase deletion constructs and mutagenesis analyses, we also identified a Reverb-α response element on human CYP8B1. An enhancement of the heme level may lead to a further decrease in CYP8B1 luciferase reporter activity. Indeed, our data from Real-time PCR assay demonstrated that an increase of heme levels inhibited CYP8B1 mRNA expression in human primary hepatocytes. In contrast, the expression level of human CYP8B1 mRNA was significantly increased in the presence of succinylactone, a specific inhibitor of de novo heme synthesis. Together, SREBP2 and Reverb-α may inhibit human CYP8B1 activity through different mechanisms; whereas SREBP2 blocks the HNF4α recruitment of PGC-1α, Reverb-α binds on the human CYP8B1 promoter and inhibits its transcriptional activity. Furthermore, heme, the ligand of Reverb-α, may be an important regulator of human CYP8B1 gene expression. Thus, our findings provided evidence for the regulation of human CYP8B1 by SREBP2 and Reverb-α. Advisors/Committee Members: Chiang, John.

Subjects: Biomedical research; Molecular biology

Keywords: srebp2; reverb-alpha; CYP8B1

More Like This

18. Flate, Elizabeth L. THE EFFECT OF EXTRACELLULAR MATRIX COMPONENTS ON MOTILITY AND CHEMOSENSITIVITY OF SELECT OVARIAN CANCER CELL LINES.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2012, Kent State University

► Ovarian cancer is the fifth leading cause of cancer-related death among women… (more)

▼ Ovarian cancer is the fifth leading cause of cancer-related death among women and is the deadliest of gynecologic cancers. During metastasis, cancer cells preferentially adhere to the peritoneal submesothelial extracellular matrix (ECM). In an effort to better understand the process of metastasis, the effect of ECM on migration and cisplatin (CDDP) resistance was tested in two ovarian cancer cell lines: OV2008 and C13. Both transwell migration and in vitro wound healing assays were used to show that OV2008 cells have a greater capacity for movement compared to the C13 cell line on uncoated surfaces. Total wound healing and directionality of individually migrating cells was assessed for all experiments. Results showed that collagen type I (COLL) increases the migratory ability of OV2008 and C13 cells by increasing the directional migration of cells. In contrast, it appears that fibronectin (FN) decreases the migratory ability of OV2008 cells by reducing their ability to directionally migrate. In order to determine if differences in gene expression were correlated with the different cell movement phenotypes observed when the cells were cultured on glass, COLL or FN, GeneChip microarrays were performed for each of the cells lines on all three surfaces. Of the genes that displayed differences between migratory phenotypes, PAK2 was chosen for further investigation. When both cell lines are cultured on COLL and treated with increasing concentrations of the PAK inhibitor (IPA-3), there is a dose-dependent response such that there is a decrease in migration with an increase in inhibitor concentration. Further experiments utilizing PAK2 knockdown via siRNA transfection demonstrated significantly reduced migration of OV2008 cells on COLL as compared to those receiving Control siRNA. FN appears to increase the sensitivity of C13 cells to 24 hours of 30 μM CDDP treatment. Further, PAK2 knockdown in OV2008 cells decreased survival following CDDP treatment. In conclusion, our results indicate that FN and COLL affect the motility of the selected ovarian cancer cells lines and this effect is likely mediated, at least in part, by PAK2. As PAK2 is involved in both motility and chemosensitivity, it should be further explored as a pro-metastatic gene in ovarian cancer. Advisors/Committee Members: Stalvey, John R.D.

Subjects: Biomedical Research

Keywords: Ovarian Cancer; Metastasis; Extracellular Matrix

More Like This

19. Gilbert, Erin V. Characterization of the Circadian Clock in Pet-1 Knockout Mice.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2010, Kent State University

► The suprachiasmatic nucleus (SCN) drives circadian rhythms of physiology and behavior and… (more)

▼ The suprachiasmatic nucleus (SCN) drives circadian rhythms of physiology and behavior and synchronizes the timing of circadian clocks in other tissues via unknown signals. One of the major input signals to the SCN is serotonin (5-HT), which is thought to communicate information about behavioral activity to the circadian clock. In mice lacking the transcription factor Pet-1 the 5-HT system fails to develop normally, resulting in mice with only a small fraction of their normal complement of 5-HT neurons. Pet-1 knockout mice show longer endogenous circadian periods indicating that 5-HT may function as an overall phase advancing signal to the clock. The loss of the advancing signal also causes a shift in peak wheel running activity from early to late night and a phase response curve to light which is reduced in amplitude when compared to wildtype leading to smaller phase delays in Pet-1 knockout animals. No alterations in response to pharmacological nonphotic stimuli (8-OH-DPAT) were seen and responses to non-pharmacological, nonphotic stimuli suggest that cage changes are a more significant stimulus for female animals. Within the cerebellum, a site of an extra-SCN oscillator, Pet-1 knockout animals show attenuation of Per1 expression 4 hours before lights off and generally attenuated levels of Per2 expression. The lack of 5-HT did not affect expression of the core clock genes Clock, Npas2, Bmal1, or the immediate early gene Fos indicating that 5-HT may play a role in modulating levels of Per gene expression. However, Fos expression in response to injection of 8-OH-DPAT was increased in wildtype but not Pet-1 knockout animals which suggests the possibility that 5-HT receptors in knockout mice are unable to respond to drugs which affect the 5-HT system. Together, these data suggest that a developmental deficit of the serotonergic system may alter the circadian clock in a way that differs from current understanding of the system and add to our knowledge of the role of 5-HT in circadian rhythm generation. Advisors/Committee Members: Mintz, Eric.

Subjects: Neurobiology; Neurosciences

Keywords: Pet-1; serotonin; circadian; suprachiasmatic; clock genes

More Like This

20. Gregg, Jennifer L. EGF-Mediated Regulation of EGR1 in Prostate Cancer Cells.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2010, Kent State University

► Prostate tumorigenesis is a complex and multi-faceted process. To examine differential expression… (more)

▼ Prostate tumorigenesis is a complex and multi-faceted process. To examine differential expression between tumor and normal prostate samples, paired tumor and non-neoplastic prostate tissues were assayed for various expression levels of growth factors, growth factor receptors, transcription factors, and cytokines. Notably, WT1 mRNA and protein expression was elevated in prostate tumor tissues and cell lines relative to non-neoplastic tissues and cells. The epidermal growth factor, EGF, was examined as a potential regulator of WT1 expression. While EGF did not induce WT1 expression, EGF was a potent and specific activator of EGR1 expression in prostate cancer cells. EGF induced EGR1 expression levels in LNCaP cells in a dose and time-dependent manner. In order to elucidate the mechanisms regulating the EGF-mediated EGR1 response, the specific inhibitors PD98059 and wortmannin were used. Both inhibitors attenuated EGR1 activation by EGF, suggesting that ERK and PI3K pathways regulate EGR1 expression. However, subsequent analysis of downstream phosphorylation events determined that the ERK pathway is the most likely mediator of EGR1 activation. Analysis of the EGR1 promoter using deletion constructs identified an EGF-responsive region in the proximal promoter (-771 to -245 bp) containing three potential SRE sites. In vivo chromatin immunoprecipitation assays demonstrated that Elk-1 binding at the SRE sites of the EGR1 promoter was enhanced by EGF treatment of LNCaP cells, although a significant amount of Elk-1 was bound in the absence of stimulus. Overexpression of the Elk-1 transcription factor enhanced activation of the EGF-responsive region indicating that Elk-1 is sufficient to activate EGR1 in PC3 cells. Similarly, a dominant-negative Elk-1 suppressed EGR1 promoter activity indicating that Elk-1 is necessary for EGF induced EGR1 transcription in both PC3 and LNCaP cells. Taken together, these results demonstrate for the first time that EGR1 expression in prostate cancer cells is mediated through an EGF-ERK-Elk-1 signaling cascade. Advisors/Committee Members: Fraizer, Gail.

Subjects: Genetics; Molecular biology; Oncology

Keywords: prostate cancer; gene expression; EGR1; EGF; Elk-1

More Like This

21. Gregory, L Tremaine. Socioecology of the Guianan bearded saki, Chiropotes sagulatus.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2011, Kent State University

► Bearded sakis (genus Chiropotes) are among the least studied primates in the… (more)

▼ Bearded sakis (genus Chiropotes) are among the least studied primates in the world. This study documented social behavior, ecology, habitat use, and responses to seasonal changes in the Guianan bearded saki (Chiropotes sagulatus) in continuous forest in Brownsberg Nature Park, Suriname. The results of this study provide further support for existing information about this genus and make new contributions to our knowledge of these elusive animals. Social behavioral data demonstrated highly affiliative relationships between males within groups. There was a strong bias towards male-male partnerships during social interactions, and interactions were affiliative in 100% of cases. There was also initial documentation of all-male (sub)groups, and higher rates of affiliative behaviors between males when in all-male groups suggest that these times are potential opportunities for bond reinforcement. Results of the ecological data set demonstrate a response to seasonal changes in resource availability, and during the long dry season group size decreased, and activity patterns and diet composition changed. Group size also interacted with travel distance during the dry season resulting in smaller groups and shorter travel distances. While other primate species use some of these strategies to withstand periods of lower resource production in the forest, this study demonstrates that, similar to fission-fusion in Ateles and Pan, flexibility in group size is also used frequently by Chiropotes. In this study, innovative spatial ecological analyses using GIS technology showed preference patterns in habitat use. Sakis tended to use the forests on the slopes of the mountain, and they demonstrated patterns of slope use that showed initial indications of strategies to increase navigation efficiency. This study not only provides insight into bearded saki behavior, but it also expands our knowledge of patterns of male-male affiliativeness and potential ecological responses to seasonal resources changes in a genus characterized by seed predation and large group size. Advisors/Committee Members: Noconk, Marilyn.

Subjects: Ecology; Geographic Information Science; Physical Anthropology; Zoology

Keywords: Bearded saki; Suriname; primate socioecology; platyrrhine; spatial ecology; Chiropotes sagulatus

More Like This

22. Guo, Jiami. The requirement of Smad4 in Mouse Early Embryonic Development.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2012, Kent State University

► This dissertation is divided into two Chapters: Chapter I focuses on the… (more)

▼ This dissertation is divided into two Chapters: Chapter I focuses on the requirement of maternal Smad4 in mouse preimplantation development; Chapter II focuses on the function of zygotic Smad4 in trophoblast lineage development. Chapter I: Maternally contributed transcripts and proteins are crucial for shaping the earliest developmental programs. Smad4 is a central mediator of the Transforming Growth Factor beta (TGF-β) superfamily, molecules of which are important regulators of development and tissue homeostasis. We found that Smad4 gene products are abundant in unfertilized eggs as well as cleaving blastomeres of mouse preimplantation embryos. To investigate the role of maternal Smad4 during mouse preimplantation development, we have conditionally inactivated Smad4 in mouse female germline. Here we report that Smad4 is a new member of mouse maternal effect genes that lacking of maternal Smad4 results in peri-implantation lethality severe preimplantation development defects in mitosis, polarization, and first lineage segregation between trophectoderm (TE) and inner cell mass (ICM). Not only have our studies uncovered novel functions of Smad4-dependent signaling in preimplantation development, but also have provided important insights into how maternal effect genes influence the earliest development programs in mammals. Chapter II: Trophoblast lineage gives rise to embryonic portion of the placenta, the function of which is essential for the establishment and maintenance of pregnancy. Mouse trophoblast stem (TS) cells established from the outgrowth of the polar trophectoderm of blastocysts or the extraembryonic ectoderm of post-implantation embryos are the precursors of trophoblasts and can contribute to all trophoblast lineage derivatives in vivo, providing a powerful in vitro system for the study of trophoblast stem cell self-renewal and differentiation. Although it is known that TGF-β/Nodal-related signaling together with FGF4 signaling is critical for TS self-renewal, the function of Smad4, the central mediator of the TGF-β related signaling pathways, in TS cell has not been fully investigated, partially due to the early lethality of Smad4 null embryos around peri-gastrulation stage. By studying Smad4-deficient TS cells, we have uncovered a critical requirement for Smad4 mediated TGF-β related signaling in trophoblast lineage development. We show that Smad4 deficiency alters the differentiation kinetics and trophoblast lineage distribution of the mutant TS cells. Moreover, Smad4 null TS cells display enhanced mobility and invasiveness and undergo epithelial-mesenchymal transition (EMT). Consistent with the changes in cellular properties, transcriptome profiling reveals misregulation of genes associated with EMT, tumorigenesis and cancer progression in Smad4 null TS cells. Our data provides evidence linking the differentiation defects and EMT properties seen in the Smad4 null TS cells to alterations in several signaling pathways, including the WNT pathway, whose role in TS cell biology has not been well appreciated previously. Taken together, our studies revealed a novel requirement for Smad4 in maintaining the differentiation potential, epithelial integrity and homeostasis of TS cells through Smad4-depedent TGF-β related signaling and its crosstalks with other developmentally important signaling pathways. Our analysis will provide insights into the molecular mechanisms that regulate trophoblast lineage development in vivo. Advisors/Committee Members: Chen, Yijing.

Subjects: Developmental Biology

Keywords: Smad4,TGF-β, mouse preimplantation, maternal effect genes, mouse early post-implantation, mouse trophoblast lineage, trophoblast stem cell, differentiation, epithelial-mesenchymal transition (EMT), tumorigenesis, cancer, WNT signaling, MAPK, ERK1/2

More Like This

23. Hammer, Steven Berlin. Interactions between Exercise, Aging and Ethanol and the Mammalian Circadian Timing System.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2009, Kent State University

► All organisms encounter uncontrollable factors such as disease, gene expression and general… (more)

▼ All organisms encounter uncontrollable factors such as disease, gene expression and general environmental conditions. Other factors such as exercise, ethanol consumption, and some aspects of aging can to some extent be controlled or altered. Encompassing all these factors is the circadian timing system, which controls many of the bodies’ physiological processes. These experiments were designed to examine the interaction of these factors in conjunction with the circadian timing system. We found that exercise in the Syrian hamster, forced and voluntary, modulates their ability to respond to photic or non-photic phase shifting treatments. Age plays a significant role in these modulations, presumably through alteration of serotonin pathways. With respect to age, there appears to be a peak in physiological responses within the circadian system around 10 to 14 months of age. Animals younger and older show attenuated physiological responses in general. We also found that the type of sensor used to measure activity is not equivalent between infrared or wheel sensors. Infrared sensors detect activity significantly earlier than wheel sensors and these results vary with age and activity levels. We also found that activity onset changes in response to ethanol consumption, with infrared activity becoming more delayed while wheel activity is unaffected. Ethanol consumption was also shown to be affected by exercise, attenuating voluntary ethanol consumption in young and old animals. Lastly we found that lighting condition can have an effect on ethanol consumption. In constant light Syrian hamsters have a propensity to display two activity onsets each day, “splitting.” Animals that displayed a split rhythm had a lower ethanol consumption compared to the animals that did not display splitting. This effect is possibly linked to a heightened state of glutamate within the circadian pathways. Overall we found that exercise and age do have significant effects upon the circadian timing system and that these effects extend into the realm of substance abuse, extending the overlapping potential for research in these fields. Advisors/Committee Members: Glass, J. David.

Subjects: Animals; Biology; Biomedical research; Experiments; Gerontology; Health; Mental health; Neurology; Physical education; Psychology; Sports medicine; Therapy

Keywords: Circadian, Aging, Exercise, Ethanol, Syrian hamster

More Like This

24. Hannibal, Luciana. Intracellular Processing of Cobalamins in Mammalian Cells.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2009, Kent State University

► In mammalian cells the sequence of events and the players involved in… (more)

▼ In mammalian cells the sequence of events and the players involved in the biosynthesis of adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl) from vitamin B12 (cyanocobalamin, CNCbl) are only partially understood. A central objective of this work was to gain a mechanistic understanding of how mammalian cells process incoming cobalamins for coenzyme biosynthesis. In Specific Aim 1 cobalamins with potential biological activity were synthesized and characterized. Nitrosylcobalamin, NOCbl, the elusive complex formed between nitric oxide and cobalamin, was synthesized via a novel reaction between aquacobalamin and the nitric oxide donor DEA-NONOate. NOCbl was obtained in high yield (85%) and purity (≥ 95% by 1H NMR spectroscopy) under alkaline and strictly anaerobic conditions. In addition to NOCbl, the synthesis and characterization of a number of other cobalamin forms were also carried out, some of which were utilized to assess the mechanisms of intracellular cobalamin processing in mammalian cells. In Specific Aim 2, a method for the accurate assessment of intracellular cobalamins, hereafter referred to as “cold-trapping”, was developed. The procedure, which was tested in cultured cells, facilitated the identification and quantification of intracellular cobalamin forms that present exchangeable beta-axial ligands. A series of in vivo and in vitro experiments describing a new role for the MMACHC gene product (cblC protein) is also presented. Our in vivo studies strongly suggested that the cblC protein is responsible for early processing of both CNCbl (decyanation) and alkylcobalamins (dealkylation). Our in vitro studies confirmed that the cblC protein catalyzed the dealkylation of Co-C bonded cobalamins by a reaction involving the nucleophilic attack of the Co-C bond by the thiolate anion of glutathione. In Specific Aim 3, I investigated the protein changes that accompany functional cobalamin deficiency in humans. The proteome of normal and cblC mutant fibroblasts was quantitatively examined by two-dimensional difference in-gel electrophoresis and mass spectrometry. Major changes were observed in the expression levels of proteins involved in cytoskeleton organization and assembly, the neurological system and cell signaling. The effect of hydroxycobalamin (HOCbl) supplementation on the proteome of normal and cblC fibroblasts was also examined. A bioinformatics analysis of the differentially expressed proteins was conducted using the Ingenuity Pathway Analysis® software. The in silico analysis established strong associations with neurological disorders, muscular and skeletal disorders, and cardiovascular diseases. Supplementation of the cell cultures with HOCbl did not restore the cblC proteome to the patterns of expression observed in the normal cell line. Our findings concur with the clinical manifestations of the cblC disorder, and the poor response of severely-ill patients to therapeutic doses of HOCbl. Advisors/Committee Members: Jacobsen, Donald.

Subjects: Biochemistry; Biology; Cellular biology; Chemistry; Pathology

Keywords: Cobalamin; vitamin B12; methylcobalamin; adenosylcobalamin; nitrosylcobalamin; transcobalamin; alkylcobalamin; synchrotron diffraction; processing; MMACHC; cblC; chaperone; crystal structure; inborn error; deficiency; proteomics; pathway analysis

More Like This

25. Han, Shuxin. VITAMIN D RECEPTOR REGULATION OF CHOLESTEROL 7α-HYDROXYLASE GENE TRANSCRIPTION AND BILE ACID SYNTHESIS IN HUMAN HEPATOCYTES.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2009, Kent State University

► Vitamin D receptor (VDR) plays an important role in the regulation of… (more)

▼ Vitamin D receptor (VDR) plays an important role in the regulation of calcium and phosphate homeostasis and bone formation. Lithocholic acid (LCA) is a potent endogenous ligand of VDR. In cholestasis, LCA levels increase in the liver and intestine. The objective of this study is to test the hypothesis that VDR plays a role in inhibiting the gene expression of cholesterol 7α-hydroxylase (CYP7A1) and bile acid synthesis in human hepatocytes. Immunoblot analysis has detected VDR proteins in the human hepatoma cell line HepG2 and primary hepatocytes. 1α, 25-dihydroxy-vitamin D3 (1α,25(OH)2-VD3) or LCA acetate-activated VDR inhibited CYP7A1 mRNA expression and bile acid synthesis, whereas small interfering RNA (siRNA) to VDR completely abrogated VDR inhibition of CYP7A1 in HepG2 cells. LCA or 1α, 25(OH)2-VD3 induced VDR translocation from the cytosol to the nucleus and plasma membrane in human primary hepatocytes. LCA or 1α, 25(OH)2-VD3 activated a tyrosine kinase c-Src and the VDR signaling pathway, which activated c-Raf, an upstream kinase of the MAPK pathway. Kinase inhibition and in vitro kinase assays showed that VDR specifically activated extracellular signal-regulated kinase 1/2 (ERK1/2), which phosphorylated VDR, retinoid X receptor α (RXRα) and hepatocyte nuclear factor 4α (HNF4α). Mammalian two-hybrid, coimmunoprecipitation, glutathione S-transferase pull-down, and chromatin immunoprecipitation assays show that ligand activated VDR specifically interacts with HNF4α to block HNF4α interaction with coactivators. ERK1/2 inhibitors suppressed VDR interaction with HNF4α. Electrophoretic mobility shift assay and mutagenesis analyses have identified the negative VDR response elements that bind VDR/RXRα in the human CYP7A1 promoter. Chromatin immunoprecipitation assays show that LCA or 1α, 25(OH)2-VD3 activated ERK1/2 stimulated the recruitment of VDR, RXRα and co-repressors, but decreased the occupancy of HNF4α and coactivators to human CYP7A1 chromatin, resulting in the inhibition of CYP7A1 gene transcription. In conclusion, LCA and 1α, 25(OH)2-VD3 caused intracellular translocation of VDR to the plasma membrane and nucleus in human hepatocytes. LCA and VDR activated the c-Raf/MEK/ERK1/2 pathway to regulate VDR inhibition of CYP7A1 in hepatocytes. This study identified a novel bile acid-activated VDR signaling pathway in human hepatocytes. LCA activation of VDR may inhibit bile acid synthesis and protect hepatocytes from cholestatic liver injury. Advisors/Committee Members: Chiang, John.

Subjects: Biomedical research

Keywords: VDR; BILE; BILE ACID; CYP7A1; 1Î±; 2-VD3; ERK1/2

More Like This

26. Herrman, Erin Rae. Estrous Cyclicity Modulates Circadian Rhythms In Female Syrian Hamsters.

Degree: MS, College of Arts and Sciences / School of Biomedical Sciences, 2008, Kent State University

► The suprachiasmatic nucleus of the hypothalamus (SCN) contains a circadian pacemaker that… (more)

Subjects: Behaviorial sciences; Biomedical research; Cellular biology; Gender; Molecular biology; Neurology; Technology

Keywords: Estradiol; Syrian hamster; Circadian rhythm; Estrous Cycle; Suprachiasmatic Nuclei (SCN); Laser Capture; Estrogen Receptor

More Like This

27. Ji, Jing. Sex Differences of Neurotransporters Within the Nigrostriatal Dopaminergic System.

Degree: MS, College of Arts and Sciences / School of Biomedical Sciences, 2008, Kent State University

► The purpose was to examine the sex differences of VMAT-2 and DAT… (more)

▼ The purpose was to examine the sex differences of VMAT-2 and DAT of the NSDA system in female and male mice. Two series experiments were performed: the first consisted of an in vivo experiment using reserpine-treated CD-1 mice; the second was in vitro experiment using DAT wild type and heterozygous mice. For the in vivo experiments, after treatment with reserpine, male mice showed significantly greater DA concentrations and K+-evoked DA output from the striatum. By contrast, no statistically significant sex differences were present in the response to MA-evoked DA output in reserpine-treated mice. As for the in vitro experiments, I measured DA and DOPAC contents in the corpus striatum and olfactory tubercle, and DA output induced by K+ and MA, and we compared effects of the VMAT-2 inhibiting drug reserpine upon the striatal dopaminergic function among male and female DAT wild type (+/+) and heterozygous (+/-), intact and gonadectomized mice. Striatal DA and DOPAC concentrations of female +/- DAT mice were significantly decreased as compared with wild type (+/+) controls and male +/- DAT mice. MA-evoked DA was increased in male and female +/- mice. While MA-evoked DA was significantly increased in +/+ males versus +/+ females, the +/- females showed the highest DA responses; thereby showing a reversal in the results seen in wild-type conditions. Potassium-stimulated DA was increased in male and female +/- DAT mice and maximally stimulated DA was obtained from +/- DAT females, although these mice showed the lowest DA concentrations. Infusion of reserpine-evoked DA output was significantly greater from intact +/+ and +/- female compared to male mice. The DA output profile differed markedly between +/+ and +/- females, but no such qualitative differences were present in males. Significantly increased reserpine-evoked DA responses were also obtained in +/+ and +/- gonadectomized female mice treated or not with estrogen as compared with respective responses from males. I can conclude that: 1) a deficiency in the DAT interacts with the sex of subject, 2) +/- DAT females show more extreme changes in dopaminergic responses, 3) females possess a more active VMAT-2/DA storage capacity, 4) the two transporter systems, DAT and VMAT-2, may be linked with each other and be affected differentially by the sex, 5) estrogen might contribute to this interaction between the DAT and VMAT-2, 6) the importance for understanding the sex differences present in the NSDA system in both healthy and diseased conditions. Advisors/Committee Members: Dluzen, Dean.

Subjects: Biomedical research

Keywords: DAT; DA; VMAT-2; mice; DA output; DOPAC; Reserpine

More Like This

28. Kallingal, George J. The Role of Gastrin-releasing Peptide in Photic Entrainment.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2008, Kent State University

► Physiological and behavioral rhythms are generated by the suprachiasmatic nucleus (SCN). The… (more)

▼ Physiological and behavioral rhythms are generated by the suprachiasmatic nucleus (SCN). The SCN can be roughly divided into two regions, the core and the shell. Within the core and the shell, various cellular markers and neuropeptides integrate afferent photic and non-photic signals that are essential to synchronize clock function. In Syrian hamsters (Mesocricetus auratus), gastrin releasing peptide (GRP) appears to be an important relay signal between these two SCN regions. GRP also serves as an output signal in other brain regions including the supraoptic nucleus (SON). In this dissertation, the role of GRP in the SCN will be examined in the context of regulating both afferent and efferent signals to and from the SCN in an effort to synchronize the clock. Administration of GRP to the SCN induces phase shifts in a manner similar to light. The behavioral experiments described seek to explore the effects of glutamate, serotonin, and neuropeptide Y (NPY) on GRP-induced phase shifts. AP5, an NMDA receptor antagonist, can attenuate GRP-induced phase shifts in the early subjective night and can additionally block the effects of light, but not GRP, when administered after the phase shifting stimulus. Furthermore, serotonin and NPY, carriers of non-photic signals, can oppose the action of GRP in the SCN. These results suggest that GRP signaling in the SCN is dependent on the regulation provided by glutamatergic, serotonergic, and NPYergic stimulation. GRP administered to the SCN additionally affects the cellular response of cells in the SON. GRP action in this SCN output appears not to be regulated by glutamate or serotonin. Administration of GRP to the SON additionally alters the cellular response of SCN cells and microinjection of GRP or bicuculline, a GABAA receptor antagonist, to the SON region can elicit phase shifts. These data suggest that the SCN and SON have reciprocal neuronal projections that may mediate photic entrainment through action of GRP. These experiments greatly increase our knowledge of the role of GRP in circadian rhythms by examining the interaction of other neurotransmitter systems on GRP signaling. These experiments additionally implicate the SON as a potential mediator of photic entrainment. Advisors/Committee Members: Mintz, Eric.

Subjects: Biomedical research

Keywords: suprachiasmatic, gastrin-releasing peptide, glutamate, serotonin, NPY

More Like This

29. Kirksey, Susan Lee. Effects of Physical Activity on the Stress-induced Rise in C-Reactive Protein in Female Rats.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2009, Kent State University

► Objective – Determine if acute psychosocial stress increases CRP; if increased voluntary… (more)

▼ Objective – Determine if acute psychosocial stress increases CRP; if increased voluntary physical activity decreases the rise in CRP caused by the stressor; if plasma norepinephrine (NE), corticosterone (CORT), interleukin-6 (IL-6) are physiological mediators underlying the stress-induced rise in CRP; and if blocking the estrogen receptor with tamoxifen (TAM) increases the rise in CRP caused by the stressor. Methods – Experiment 1: 36 WKY female rats, age 3.5-4 months, randomly divided into Control (C) = no treatment; Low Activity (LA) received Colony Stress (CS) and receptor antagonist treatments; and High Activity (HA)received 24/7 access to wheel, CS and receptor antagonist treatments. After HA animals had been running for 5 weeks, LA and HA were subjected to CS, then CS along with the following receptor antagonists, at two week intervals: alpha-adrenergic receptor antagonist, beta-adrenergic receptor antagonist, and CORT receptor antagonist. Six hours following each CS/Receptor antagonist treatment, blood samples were taken and plasma NE, IL-6, CORT, and CRP were measured. Experiment 2: 24 WKY female rats were randomly divided into C (CS only), LA (CS and TAM implant), and HA (24/7 access to wheel, CS and TAM implant). LA and HA were implanted with TAM capsules after HA had been running for 5 weeks. Blood was obtained and assayed as in Exp1. Results – CS caused significant increase in CRP in both experiments. Experiment 1: blocking adrenergic receptors significantly increased NE above Stress Baseline; blocking CORT receptors significantly increased CORT and IL-6 above Stress Baseline; however, stress-induced rise in NE and CORT were significantly lower for HA than LA. Experiment 2: NE for HA did not increase from Baseline to the final CS, while NE did increase from Baseline to the final CS for C and LA. TAM prevented the stress-induced rise in CRP for LA and HA but not for C. Conclusion - Increasing physical activity helped to minimize the NE and CORT response to stress. Blocking the estrogen receptor with TAM helped to prevent the CRP response to stress, potentially minimizing the harmful effects of the stress and inflammatory responses. Advisors/Committee Members: Ely, Daniel.

Subjects: Biology

Keywords: physical activity, C-reactive protein, psychosocial stress, interleukin-6, corticosterone, norepinephrine

More Like This

30. Koshy, Shyny. Low tidal volume ventilation as a strategy for inducing lung fluid absorption in the preterm guinea pig.

Degree: PhD, College of Arts and Sciences / School of Biomedical Sciences, 2009, Kent State University

► Respiratory distress syndrome (RDS) is one of many ailments that affect preterm… (more)

▼ Respiratory distress syndrome (RDS) is one of many ailments that affect preterm infants. This disease is characterized by impaired breathing caused by insufficient surfactant in the lungs and ion transport deficiencies. The main hypothesis for this project is that maternal stress hormone release is important for the regulation of fetal lung fluid absorption near term and that supportive low tidal volume ventilation is beneficial in the outcome of preterm guinea pigs at risk of developing RDS. The primary experimental focus of this project was to determine if stress hormones i.e., cortisol and epinephrine, release lead to fetal lung fluid absorption via epithelial sodium channel (ENaC) induction by the serum and glucocorticoid-regulated kinase (SGK)/neural precursor cell expressed, developmentally down-regulated protein 4-2 (Nedd4-2) pathway. The second experimental focus was to determine if different tidal volume-ventilation strategies affected distal lung fluid absorption via this pathway. My thesis was thus divided into principal sections, referred to as the lung development study and the ventilation study. In the lung development study, my principal findings demonstrated that the lungs of fetal guinea pigs at a gestation of 61 days (GD61; term=GD69), were still at the fluid-secretory stage, and lungs of GD68 fetuses were at the fluid absorptive stage. This developmental increase in distal lung fluid absorption corresponded to a cortisol-dependent regulation of SGK, Nedd4-2, and αENaC expression. In the ventilation study, my principal finding was that ventilation with low tidal volume (6 ml/kg body wt) improved distal lung fluid absorption in GD65-68 guinea pig fetuses. This observed induction in distal lung fluid clearance corresponded to a MAP kinase-dependent regulation of distal lung fluid absorption, as well as an induction of SGK/Nedd4-2 regulation of αENaC expression. Thus, the principal observation in this thesis suggests that low tidal volume-ventilation of preterm infants may be useful as a therapy to induce lung maturation and absorption of fetal lung fluid. Advisors/Committee Members: Folkesson, Hans.

Subjects: Molecular biology

Keywords: Respiratory distress syndrome; lung fluid absorption; αENaC; ventilation

More Like This

ETD Center Search