PhD, University of Cincinnati, 2016, Arts and Sciences: Physics

Calbindin D9k (CAB) is a single domain calcium-binding protein and is the smallest members of the calmodulin superfamily, possessing a pair of calcium-binding EF-hands, and structures for all four states have been determined and extensively characterized experimentally. Because of the tremendous advancement in hardware and software computer technologies in recent years, longer and more realistic molecular dynamics (MD) simulations of a protein are possible now in reasonable periods of time. These advances were exploited to generate multiple, all-atom MD simulations of CAB via the AMBER software package, and the resulting trajectories were employed to calculate backbone order parameters of the apo, the singly and the doubly loaded states of calcium in CAB. The results are in very good agreement with corresponding experimental NMR-based (Nuclear Magnetic Resonance spectroscopy) results, and are improved in comparison to those calculated over a decade ago; use of modified force fields played a key role in the observed improvements. The apo state is the most flexible, and the singly loaded and the doubly loaded states are similar, thus supporting positive cooperativity in line with the experimental results. Further, B-factor calculations of backbone atoms for these calcium-binding states of calbindin D9k also support such cooperativity. Although changes in side-chain motions are not necessarily correlated to changes in protein backbone mobility, past studies on the comparison of experimental and simulated methyl side-chain NMR relaxation parameters of CAB for the doubly-loaded state reported significant improvements in the quantitative representation of side-chain motion by MD simulation. In this project, the order parameters for various side chains in apo, singly loaded and doubly loaded states of CAB were calculated. The primary goal of this work was to determine whether or not the allosteric effect of calcium binding, as observed via the backbone order parameters, (open full item for complete abstract)

Committee: Mark Rance Ph.D. (Committee Chair); Eric Johnson Ph.D. (Committee Member); Thomas Beck Ph.D. (Committee Member); David Mast Ph.D. (Committee Member); Rostislav Serota Ph.D. (Committee Member) Subjects: Physics

Bachelor of Sciences, Ohio University, 2016, Biological Sciences

We describe the distribution of calbindin (CAL), cholecystokinin (CCK), dynorphin (DYN), galanin (GAL), neurotensin (NT), somatostatin (SOM), and substance P (SP) immunoreactive elements in the primate parabrachial nucleus (PbN), a pontine structure with a crucial role in autonomic control. This study follows earlier work using calcitonin gene-related peptide (CGRP) as a marker for ascending visceral pathways in the human brain (de Lacalle & Saper, 2000). Our observations were made on horizontal sections through the brainstem from four neurologically normal human individuals and five male Cebus monkeys. Tissue was processed for immunocytochemistry using commercially available antibodies. Which revealed areas of dense peptide immunoreactivity in fibers, as well as in cell bodies. The extreme expansion of the superior cerebellar peduncle has altered the topographical organization of these pathways in the primate. However, using the peduncle as a marker, we found that similarities in the distribution of cell bodies and fibers remain conserved in some areas across species. The PbN of the Cebus monkey contained well defined external lateral and external medial subnuclei. The distribution of CAL-ir , CCK-ir, NT-ir, SOM-ir, and SP-ir elements were very similar in the human and monkey for the external lateral and external medial subnuclei, and DYN-ir elements were found in the external lateral PbN only in both species. GAL-ir elements were present in the central lateral PbN and dorsal medial PbN areas in both species. Our results contribute to define a neurochemical identity for different regions in the primate PbN, thus providing support to the delineation of physiological roles for the distinct subnuclei of this crucial visceral regulatory region.

Committee: Christine Griffin Dr. (Advisor) Subjects: Neurosciences

PHD, Kent State University, 2016, College of Arts and Sciences / School of Biomedical Sciences

Alzheimer's disease (AD) is considered a uniquely human disorder, characterized by amyloid plaques (AB), neurofibrillary tangles (NFTs), neuroinflammation, and calcium dyshomeostasis. Prior limited research on aged great apes demonstrated widespread AB pathology in absence of significant tauopathy. The objective of the current research was to evaluate a large group of elderly chimpanzees for AD pathology (n = 20, ages 37-62 years), and determine whether these lesions were accompanied by glial reactivity and calcium changes typically observed in humans with AD. We used immunohistochemical and stereological techniques to evaluate AB plaque and vessel volume, pretangle, NFT, and tau neuritic plaque (NP) densities, activated microglia and morphologic densities, and calbindin-immunoreactive (ir) and calbindin/tau-ir neuron density in the neocortex and hippocampus of aged chimpanzees. Moreover, an imbalance in the normal equimolar ratio of three-repeat (3R) and four-repeat (4R) tau isoforms has been identified in several neurodegenerative diseases, including AD. To determine normal levels of tau isoforms in adult chimpanzees and baboons, two species known to develop NFTs, sandwich ELISA was performed for 3R and 4R tau in the cortex and cerebellum. Volume occupied by AB-ir vessels was higher than plaques and corresponded with increases in all tau densities, particularly in the hippocampus. Chronological age correlated with greater volumes of AB-ir plaques and vessels but not with tau-related pathologies. Tangle pathology was found in five individuals, four of which exhibited moderate or severe cerebral amyloid angiopathy. Additionally, AB deposition was more predominant in blood vessels, suggesting amyloid buildup in the brain's vasculature may precede plaque formation in aged chimpanzees. Activated microglia densities were higher in CA3 compared to CA1 in the hippocampus. AB-ir plaque volume was positively correlated with greater microglial activation and intermediate mor (open full item for complete abstract)

Committee: Mary Ann Raghanti Ph.D. (Advisor); Heather Caldwell Ph.D. (Committee Member); Stephen Fountain Ph.D. (Committee Member); Ernest Freeman Ph.D. (Committee Member); David Riccio Ph.D. (Committee Member) Subjects: Biomedical Research; Neurobiology; Neurosciences; Physical Anthropology

MA, Kent State University, 2013, College of Arts and Sciences / Department of Anthropology

Schizophrenia reduces the fitness of an individual but continues to remain prevalent in 1% of the human population around the world and is thought to be a consequence of human brain evolution. Recent research has indicated that many symptoms of schizophrenia are associated with cortical disinhibition. Inhibitory processes are responsible for focusing on specific tasks and blocking out excess signals. This study focused on calbindin-immunoreactive (CB-ir) interneurons, a subset of the GABAergic neurons, in Brodmann's areas 9 and 46. These areas have been implicated in auditory hallucinations as revealed by fMRI. Interneurons that colocalize with calbindin are decreased in schizophrenia, and it is thought that this decrease contributes to deficits in language and auditory processing. The density of these interneurons was compared to those of the surrounding areas (Areas 44 and 41) to determine if there was a selective disinhibition within areas 9 and 46. Tissue samples were analyzed using immunohistochemistry in order to visualize CB-ir interneurons. Results showed a decrease in CB-ir interneurons in layers V-VI of areas 46 and 41 in schizophrenic subjects compared to normal controls. Alternately, there was an increase in schizophrenic subjects of CB-ir interneurons in layers II-III of area 41 compared to normal controls. These results indicate abnormal CB-ir interneuron densities in both the frontal and temporal lobes of schizophrenic patients that may result in deficits in audition, attention, and audition.

Committee: Mary Ann Raghanti (Advisor); Richard Meindl (Committee Member); Linda Spurlock (Committee Member) Subjects: Neurobiology; Physical Anthropology; Psychology

Doctor of Philosophy (PhD), Wright State University, 2011, Biomedical Sciences PhD

Locomotor development is dependent on the maturation of spinal cord circuits controlling motor output, but little is known about the development of the spinal interneurons that control motoneuron activity. This study focused on the development of Renshaw cells (RCs) and Ia inhibitory interneurons (IaINs), which mediate recurrent and reciprocal inhibition, respectively, two basic inhibitory circuits for motorneuron control. Both interneurons originate from the same progenitor pool (p1) giving rise to ventral spinal embryonic interneurons denominated V1. V1-derived interneurons (V1-INs) establish local inhibitory connections with ipsilateral motoneurons and express the transcription factor engrailed-1. This characteristic permitted the generation of transgenic mice that were used in this study to genetically label V1 interneuron lineages from embryo to adult. Adult V1-derived Renshaw cells and IaINs share some similar properties, both being inhibitory and establishing ipsilateral connections; but differ in morphology, location in relation to motor pools, expression of calcium binding proteins (calbindin vs. parvabumin), synaptic connectivity and function. These differences are already present in neonates, therefore the purpose of this study was to determine possible embryonic differentiation mechanisms. Using 5‟-bromodeoxyuridine birth-dating we demonstrated that V1-INs can be divided into early and late born groups. The early group quickly upregulates calbindin iv expression and includes the Renshaw cells, which maintain calbindin expression through life. The second group includes many cells that postnatally upregulate parvalbumin, including IaINs. This later born group is characterized by upregulation of the transcription factor FoxP2 as they start to differentiate and is retained up to the first postnatal week in many V1-derived IaINs. In contrast, Renshaw cells express the transcription factor MafB that seems relatively specific to them within the V1-INs. Furtherm (open full item for complete abstract)

Committee: Francisco Alvarez PhD (Advisor); Paula Bubulya PhD (Committee Member); Timothy Cope PhD (Committee Member); David Ladle PhD (Committee Member); James Olson PhD (Committee Member) Subjects: Neurology

Master of Science (MS), Wright State University, 2010, Anatomy

These experiments analyze differences in synaptic development in central auditory pathways between normal hearing (CBA/J) and congenitally deaf (dn/dn) mice, which provide valuable insight into central synaptic plasticity corresponding to human congenital deafness. Immunofluorescent analysis of the developmental expression of the calcium buffering proteins calretinin, calbindin d-28k, and parvalbumin at various postnatal time points was performed to assess the effects of altered neural activity on the level and/or pattern of protein expression within these nuclei. Results indicate that the pattern of calbindin and parvalbumin is unaffected by congenital deafness in dn/dn mice. However, the pattern of calretinin expression in the MNTB during development of dn/dn mice differed significantly from that of CBA/J mice, indicating that calcium buffering may be impaired in these synapses without appropriate afferent stimulation.

Committee: Robert E.W. Fyffe Ph.D. (Advisor); Larry J. Ream Ph.D. (Committee Member); John C. Pearson Ph.D. (Committee Member) Subjects: Biomedical Research; Neurology

Master of Science (MS), Wright State University, 2006, Anatomy

This experiment analyzes synaptic differences in the central auditory pathway between normal hearing and congenitally deaf (dn/dn) mice, and provides valuable insight into central changes that correspond with human congenital deafness. Specifically, this experiment analyzes developmental expression of the Calcium (Ca2+)-binding proteins Calretinin (CR), Calbindin D-28k (CB) and Parvalbumin (PV) in large excitatory synapses in the anteroventral cochlear nucleus (AVCN) and the medial nucleus of the trapezoid body (MNTB) of normal and dn/dn mice. Immunofluorescence imaging with primary antibodies detecting CR, CB or PV was used to analyze the expression of each at 9 days, 13 days, 20 days, 30 days and 49 days postnatal in normal and dn/dn mice. Results indicated that Ca2+-binding expression was similar at each location in normal and dn/dn mice at 9 days postnatal, prior to opening of the ear canal and the onset of hearing (which occurs around 11 days postnatal) . In normal mice, patterns of Ca2+-binding protein expression changed progressively after the onset of hearing. In dn/dn mice (which completely lack auditory nerve activity), however, patterns of expression did not change after the onset of hearing, suggesting that patterns of Ca2+-binding protein expression change during development in normal mice in response to evoked auditory nerve activity, and that patterns of Ca2+-binding protein expression do not change during development in dn/dn mice due to lack of evoked auditory nerve activity. As a result, Ca2+ buffering is impaired in synapses located in the AVCN and MNTB of dn/dn mice.

Committee: Robert Fyffe (Advisor) Subjects:

MS, University of Cincinnati, 2008, Arts and Sciences : Chemistry

The potential energy surfaces of both calcium binding sites in calbindin D9k were determined in DFT calculations using the Gaussian031 program. The potential energy surfaces were also determined using the CHARMM2 program; the default force field and a charge modified force field were used in the classical calculations. The potential energy surface was found for the interaction energy between the Ca2+ ion and the ligand oxygens plus limited neighbors. The binding sites are in the form of a pentagonal bipyramid around the calcium where 6 of the ligands are contributed by the protein and one is an oxygen atom from a water molecule. A large and significant difference is seen between the classical and quantum results; this dictates that any classical calculations involving the calcium ion in this and other EF-hand structures need to take this into account. A potential has been calculated using a Metropolis Monte Carlo simulated annealing procedure. Future work will implement this potential into a classical molecular dynamics program.

Committee: Thomas L. Beck PhD (Committee Chair); Bruce S. Ault PhD (Committee Member); William B. Connick PhD (Committee Member) Subjects: Biochemistry; Biophysics; Chemistry