ETD Search Results: instcode:wright

Skip navigation

ETD Center Search

45 matches in the database. These are records: 1 - 30.

[1] [2]

1. Bartoszek, Joseph E. Genetic Differentiation in Ambystomatid Salamanders Across a Fragmented Landscape.

Degree: PhD, Biomedical Sciences PhD, 2009, Wright State University

► It is generally recognized that amphibian populations declining across the globe. Habitat… (more)

▼ It is generally recognized that amphibian populations declining across the globe. Habitat loss is the primary cause of decline. The salamander family Ambystomatidae has experienced significantly more rapid decline than the average for amphibians. Coefficients of Conservatism (CoC) have been developed for amphibians in Ohio. These are based primarily on habitat requirements with high CoCs for amphibians with stringent habitat requirements (e.g. vernal pools associated with large, intact, undisturbed forest), and low CoCs for amphibians with less stringent habitat requirements. Genetic diversity in five species and one hybrid complex of ambystomatid salamander in southwest Ohio were examined and compared to their respective CoC. Less gene flow across inhospitable habitat and consequently lower genetic diversity in that species would be expected compared with a species with less restrictive habitat requirements, i.e. a lower CoC. Microsatellites were used to determine conformation to Hardy Weinberg Equilibrium expectations, allelic and genotypic differences between populations, and inbreeding coefficients Fis and Fst. Genetic differences between woodlots and between ponds within a woodlot were examined. It was found that, for different species in the same habitat, species with higher CoCs have lower genetic diversity than species with lower CoCs. It was also found that habitat quality influences genetic diversity in all species. In some habitats, the genetic diversity of all species in that habitat will be lower than that of the same species in a more suitable habitat. In some cases presence of the hybrid complex (kleptogens) appears to have negatively impacted species that act as a sperm donor to the hybrid complex. In some species (e.g. smallmouth salamanders) genetic structure can be seen in different ponds within a woodlot. In other species (e.g. tiger salamanders) no such structure appears. It was also found that a railroad track acts as a barrier to marbled salamanders creating two genetically distinct populations. It was found that maternal ancestor of the kleptogens, A. barbouri, also acts as a sperm donor but does not replace the A. laterale genome in the nucleus. In addition to species specific differences in genetic diversity, habitat quality, and presence of kleptogens also influence genetic diversity in ambystomatid salamanders. Advisors/Committee Members: Krane, Dan.

Subjects: Animals; Biology; Ecology; Environmental science; Genetics; Molecular biology

Keywords: Ambystoma, habitat fragmentation, genetic diversity, landscape genetics, amphibian

2. Benito González, Ana. V1-DERIVED RENSHAW CELLS AND IA INHIBITORY INTERNEURONS DIFFERENTIATE EARLY DURING DEVELOPMENT.

Degree: PhD, Biomedical Sciences PhD, 2011, Wright State University

► Locomotor development is dependent on the maturation of spinal cord circuits controlling… (more)

▼ 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. Furthermore, Renshaw cells appear attracted to the ventral root exit region and follow a unique migratory route to become specifically placed at this location. In contrast, other V1 interneurons settle more medially and far from the ventral root exit region. MafB expression is upregulated in Renshaw cells only after they have reached their final position among motor axons. Therefore, the specific migration of Renshaw cells might be responsible for their final differentiation and unique relationship with motor axons in adult. Advisors/Committee Members: Alvarez, Francisco.

Subjects: Neurology

Keywords: spinal cord, development, neurogenesis, locomotor circuits, V1-interneurons, engrailed-1, calbindin, parvalbumin, ventral horn, recurrent inhibition, reciprocal inhibition, motoneuron

3. Bullinger, Katie Leigh. Cellular Function of the Ia-motoneuron Circuit Following Peripheral Nerve Regeneration.

Degree: PhD, Biomedical Sciences PhD, 2009, Wright State University

► Successful regeneration of a severed peripheral nerve is insufficient to restore the… (more)

▼ Successful regeneration of a severed peripheral nerve is insufficient to restore the stretch reflex. This deficit occurs despite successful muscle reinnervation and functional restoration of the circuit components. For example, Ia afferents encode muscle stretch, Ia-motoneuron synapses reform to the extent of restoring low frequency transmission, and activation of motoneurons results in muscle force output. However, when recording intracellularly from reinnervated rat motoneurons during ramp hold and release muscle stretch, no excitatory synaptic potentials are detected in 2/3 motoneurons (Haftel et al., 2005), a clear sign that the synapses connecting Ia afferents with motoneurons are physiologically disrupted. This thesis examines extensively the cellular properties of the presynaptic Ia afferent, postsynaptic motoneuron, and the function of their central synapse to identify where along this circuit deficits are occurring. Using intraaxonal recordings from regenerated muscle afferents, we found that not only were regenerated afferents capable of encoding stretch information, but they were, by many parameters, indistinguishable from normal. In addition, intracellular recordings from regenerated motoneurons indicated that intrinsic motoneuron properties (rheobase, input resistance, and afterhyperpolarization potential) returned to control levels with reinnervation. To study synaptic function between regenerated afferents and motoneurons, muscle stretch and electrical stimulation at group I strength were used to activate regenerated afferents. Whereas electrical stimulation at both low (1 pps) and physiologic frequencies was capable of producing synaptic responses, muscle stretch was entirely ineffective in some motoneurons. Examination of individual synapses using spike triggered averaging suggested that many regenerated afferents responding to muscle stretch in patterns typical of Ia do not make physiologic monosynaptic connections with motoneurons. In total, these data demonstrate a disconnect between peripheral reinnervation by regenerated afferents and central connectivity with motoneurons: many afferents regaining normal stretch responses are not centrally connected to motoneurons, however, many group I afferents that do not reinnervate muscle spindles maintain central synaptic connections with motoneurons. This finding may result from factors including nonspecific reinnervation, synaptic stripping of afferent input at proximal/distal sites, and electrotonic decay of afferent input at distal synapses and may explain why the stretch reflex is not restored even after successful muscle reinnervation. Advisors/Committee Members: Cope, Timothy.

Subjects: Biomedical research

Keywords: afferents; motoneurons; Ia afferents; EPSPs; regenerated; synaptic; muscle

4. Chen, Xiaomi. Aberrant DNA Replication at an Ectopic Chromosomal Site in Human Cells.

Degree: PhD, Biomedical Sciences PhD, 2011, Wright State University

► Aberrant DNA replication, including over-replication or under-replication may lead to life-threatening mutation… (more)

▼ Aberrant DNA replication, including over-replication or under-replication may lead to life-threatening mutation or even cause human diseases. This thesis focused on three issues related to abnormal DNA replication in human chromosomes including: I) to define the function of DNA unwinding element (DUE) and DNA unwinding element-binding protein (DUE-B) to maintain an active c-myc replicator; II) to determine the role of trans-acting factors in defining a replication origin on human chromosomes; III) to investigate the mechanism by which hairpins affect DNA replication and instability of (CTG)n•(CAG)n trinucleotide repeat tracts in human cells. Our laboratory previously demonstrated that both DUE and DUE-B, are essential in c-myc DNA replication initiation. In part I, I have shown that the increased binding of DUE-B and Cdc45 correlated with the decrease of the DUE helical stability and increased origin activity for the chimeric c-myc/SCA10 replicators. However, tethered binding of DUE-B on a mutant c-myc replicator with DUE deletion could not confer the DNA replication activity. In part II, I explored the induction of DNA replication origin via trans-acting factors. My data suggested that tethered binding of transcription factor, E2F1, could induce replication activity likely by changing the chromatin structure. Tethered binding of pre-RC component, Orc2 and Cdt1 also can induce replication origin activity while Mcm7 and Cdc45 could not. Unlike episomal systems, our system revealed that the induction of replication origin activity on human chromosome also required the essential cis-acting elements including the DUE. Although it is widely accepted that unstable triplet nucleotide repeat (TNR) caused instability, it still remains elusive how and when the hairpins form during DNA replication. In part III, engineered zinc finger nucleases (ZFNs) and small pool PCR (spPCR) were applied to probe the hairpin formation in vivo in human cells. In our system, it was demonstrated that (CTG)n•(CAG)n repeat tracts could form hairpins on either lagging strand or leading strand template, and the formation of hairpins is DNA replication associated. Advisors/Committee Members: Leffak, Michael.

Subjects: Biomedical Research

Keywords: Replication, c-myc replicator GAL4 fusion protein, induction of replication origin activity, pre-replication complex, isogenic cell lines, FLP recombinase system, DM1, triplet repeat instability, zinc finger nuclease, hairpin formation.

5. Chimote, Ameet Ajit. Ion transport mechanisms during hyposmotic regulatory and isosmotic apoptotic volume decreases in a human lens epithelial cells line.

Degree: PhD, Biomedical Sciences PhD, 2009, Wright State University

► Living cells maintain constant volume in response to physiological stresses by altering… (more)

▼ Living cells maintain constant volume in response to physiological stresses by altering trans-membrane ion, solute and water flow. In the present study, early and late membrane transport changes in human lens epithelial (HLE-B3) cells under hyposmotic and apoptotic stress were compared. Cell potassium (Ki), rubidium (Rbi)-uptake and water content were measured by atomic absorption spectrophotometry and gravimetry, respectively. Intracellular chloride concentration [Cl]i was determined with the fluorescence dye N- (ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE), during regulatory volume decrease (RVD) after hyposmotic stress and apoptotic volume decrease (AVD) induced by staurosporine (STP), a protein-kinase inhibitor. Cell water increased in hyposmotic balanced salt solution (BSS) as compared to isosmotic BSS by > 2-fold at 5 min and decreased within 30 min to baseline values, accompanied by K efflux commensurate with cell swelling and RVD. Clotrimazole (CTZ) reduced Ki and water loss in hyposmotic BSS, and Rbi uptake, suggesting a role for intermediate conductance K (IK) channels during RVD. This was verified by reverse transcriptase polymerase chain reaction (RT-PCR), Western blot and immunochemistry of HLE-B3 cells. Apoptosis measured 2 h after 2 μM STP treatment was significant and accompanied by 20% and 41% water loss after 30 min and 2 h, respectively. During apoptosis, Ki fell at a rate of 0.007/min until 30 min and decreased to 42% by 2 h. [Cl]i decreased by 25% and 19% below baseline values at 30 min and 60 min, respectively. High extracellular K, and 2 mM 4-aminopyridine (4-AP) significantly reduced apoptosis whereas the presence of CTZ did not. Annexin labeling appeared at 15 min suggesting lipid changes preceded the water and K loss initiating AVD. In contrast, loss of K, water, and most likely Cl differed by an order of magnitude between RVD and AVD at early time points. Quantitative PCR revealed decreased expression of IK channels during prolonged hyposmotic and apoptotic stress. These studies suggest that in HLE-B3 cells, IK channels partake in RVD, but cannot yet be discounted in AVD, which in part commences through 4-AP-sensitive voltage-gated K channels Advisors/Committee Members: Lauf, Peter.

Subjects: Cellular biology

Keywords: Cell volume Regulation; Apoptosis; Hyposmotic Stress; Annexin; IK channels; MQAE

6. Cvetkov, Teresa L. Cytochrome c Oxidase from Rhodobacter sphaeroides: Oligomeric Structure in the Phospholipid Bilayer and the Structural and Functional Effects of a C-Terminal Truncation in Subunit III.

Degree: PhD, Biomedical Sciences PhD, 2010, Wright State University

► Cytochrome c oxidase (COX) of the mitochondrial electron transport chain catalyzes the… (more)

▼ Cytochrome c oxidase (COX) of the mitochondrial electron transport chain catalyzes the reduction of oxygen to water while concomitantly translocating protons across the inner mitochondrial membrane. This two part dissertation is a structural and functional investigation of COX using the bacterial model system from Rhodobacter sphaeroides (R.sph.). First, the oligomeric structure of R.sph. COX within the lipid bilayer was investigated using discontinuous sucrose gradient ultracentrifugation. Utilizing this technique, liposomes containing R.sph. COX (pCOV) were separated from liposomes lacking enzyme (COV). The net buffering capacity and degree of light scattering per COX molecule were reduced in pCOVs, making them well suited for low buffer spectroscopic studies. Also, pCOVs maintained high oxygen reduction and proton pumping activities relative to COVs, indicating minimal damage induced by the centrifugation process. Quantitative lipid and protein concentrations were used to estimate the number of COX molecules per vesicle in the pCOVs. There was only one R.sph. COX molecule per vesicle, indicating that within the lipid bilayer, R.sph. COX exists in the monomeric state in contrast to the bovine enzyme which is dimeric. As a monomer, therefore, R.sph. COX is capable of maximal electron transfer and proton pumping efficiency. Second, the structural and functional effects of a c-terminal subunit III truncation were characterized in R.sph. in order to gain insight into the critical role played by this subunit in proper COX functioning. The mutation was modeled after a human mitochondrial disease mutation which genetically truncates subunit III after the third of its seven helices (Delta114 COX). In R.sph. cells, Delta114 COX had lower expression levels and impeded rates of COX assembly. Altered levels of native in vivo processing of subunits II and IV were observed in Delta114 COX and in COX which had subunit III genetically removed (I-II COX). The truncated subunit III was incorporated into the COX complex with at least 70% stoichiometry and was subject to proteolytic processing at a specific cleavage site. Prior to enzymatic turnover induced inactivation, the proton pumping and oxygen reduction activities of Delta114 COX were half that of wildtype and equivalent to I-II COX at physiological pH. Delta114 and I-II COX had similar catalytic lifetimes in detergent micelle, but when supplemented with phospholipids from soybean, the catalytic lifetime of Delta114 COX was increased compared to I-II COX. Taken together, these results indicate that the c-terminal bundle of subunit III plays a role in the assembly of COX in R.sph. and in the native processing of subunits II and IV. They also highlight the role of the structural lipids within the v-shaped cleft of subunit III as being important for providing protection against turnover induced inactivation. Advisors/Committee Members: Prochaska, Lawrence J.

Subjects: Biochemistry; Biophysics

Keywords: cytochrome c oxidase; membrane protein; oligomeric structure; cytochrome c oxidase subunit III; mitochondrial disease; phospholipids in protein structure

7. Deng, Zhihui. SMALL CONDUCTANCE CALCIUM-ACTIVATED POTASSIUM (SK) CHANNELS IN MAMMALIAN SPINAL MOTONEURONS.

Degree: PhD, Biomedical Sciences PhD, 2009, Wright State University

► Three homologous small conductance calcium-activated potassium (SK) channel subunits (SK1, SK2, and… (more)

▼ Three homologous small conductance calcium-activated potassium (SK) channel subunits (SK1, SK2, and SK3) are expressed in distinct and overlapping patterns in mammalian central nervous system. SK channels likely mediate the medium afterhyperpolarization (mAHP), which plays an essential role in regulating neuron repetitive firing frequency. In spinal motoneurons (MNs) the mAHP duration is shorter on average in fast (F-type) MNs than that in slow (S-type) MNs. To better understand the molecular basis for mAHP, we determined the expression and sub-cellular distribution of SK channels in normal, axonally-injured, and developing spinal MNs in vivo using immunohistochemistry and quantitative confocal imaging techniques.SK2 and SK3 channels are clustered on the surface membrane of MN soma and proximal dendrites. SK clusters are post-synaptically localized at synapses associated with cholinergic C-terminals. In complementary pattern, SK2-immunoreactive (-IR) and SK3-IR clusters are expressed in different subpopulations of rat and mouse spinal α-MNs; on average, SK3-IR MNs are smaller than SK2-IR MNs. Comparison of SK3 expression in rat soleus versus gastrocnemius MNs, together with intracellular electrophysiological data suggests that SK3-IR MNs are S-type whereas SK2-IR MNs are F-type. Moreover a subpopulation of motor axon terminals innervating slow muscle fibers expresses SK3 channels. In postnatal developing mouse MNs, differential expression of SK2 and SK3 channels becomes apparent around the same time that muscle fiber differentiation occurs (around P9). The SK channel clustering develops in concert with the establishment and maturation of pre-synaptic cholinergic C-terminals, corresponding to the maturation of motor function. Injury to the motor axon results in a decreased AHP duration in S-type MNs but an increased AHP duration or no change in F-type MNs. Here, we characterized the effects on SK3 channel clustering in rat spinal MNs following nerve crush. SK3 clusters appear unaltered until the 3rd day after axotomy. By the 8th day post-injury, the average sizes of SK3 clusters are much smaller than in the normal control MNs. In contrast, co-localized Kv2.1 clusters start to fragment and become reduced in size within hours following injury, suggesting differential regulation and dynamics of discrete channel populations at these synapses. Advisors/Committee Members: Fyffe, Robert.

Subjects: Biomedical research

Keywords: MOTONEURONS; SK3; SK channels; SPINAL; Kv2.1; SPINAL MOTONEURONS; SK3-IR

8. Frey, Kenneth Gene. Induction of SOCS-1 in HSV-1-Infected Murine Keratinocytes: A Mechanism of Inhibition of Interferon Gamma.

Degree: PhD, Biomedical Sciences PhD, 2009, Wright State University

► Epidermal cells such as keratinocytes are the major site of Herpes Simplex… (more)

Subjects: Biomedical research; Cellular biology; Immunology; Microbiology; Virology

Keywords: SOCS-1, Interferon, HSV-1, keratinocytes

9. Gao, Yanzhe. Regulation of The DNA Unwinding Element Binding Protein DUE-B in The Cell.

Degree: PhD, Biomedical Sciences PhD, 2012, Wright State University

► Eukaryotic cells have evolved tightly controlled processes that make sure the entire… (more)

Subjects: Molecular Biology

Keywords: DNA replication; DUE-B; CDK; DDK; CKII; PP2A; phosphorylation; checkpoint response; Cdc45; TopBP1; MCM

10. Geyer, R. Ryan. Investigating the Role of Subunit III in the Structure and Function of Rhodobacter Sphaeroides Cytochrome C Oxidase.

Degree: PhD, Biomedical Sciences PhD, 2007, Wright State University

► The role of subunit III (SIII) in cytochrome c oxidase structure and… (more)

▼ The role of subunit III (SIII) in cytochrome c oxidase structure and function was investigated using enzyme isolated from the bacterium Rhodobacter sphaeroides. Energy minimization calculations suggested that in the absence of SIII, subunit I (SI) adopted a more open conformation. This observation was tested through the use of limited proteolysis using, á-chymotrypsin. The results showed that in the absence of SIII the solution structures of wild-type and I/II oxidase were not significantly different, and that proteolysis occurred exclusively at the N and C-termini of SI. Upon inactivation of I/II oxidase by catalytic turnover, and subsequent digestion with the protease á-chymotrypsin it was concluded that SI underwent significant conformational changes. This change in conformation was probably due to CuB, located in the active site, dissociating from SI, upon turnover induced inactivation. A similar digestion pattern was observed in a mutant lacking CuB, which indicated that the large scale change in conformation was due to the loss of CuB from the active site. The functional role of SIII was also investigated by predicting and mutating interhelical hydrogen bonds within SIII. A cluster of amino acids consisting of Glu90, His212, and Tyr246 form a highly conserved triad of interhelical connectivity, perhaps linked by hydrogen bonds. Mutation of Glu90 to an alanine (E90A) resulted in a significant loss of SIII content, accompanied by a phenotype of turnover induced inactivation and reduced proton pumping efficiency, when reconstituted in phospholipid vesicles. Mutation of His212 to either an alanine or phenyalanine (H212A and H212F) resulted in wild-type expression of SIII and when reconstituted into a phospholipid vesicle, these mutants exhibited turnover induced inactivation as well as reduced proton pumping efficiency. Mutation of Tyr246 to a phenyalanine (Y246F) resulted in a mutant that was indistinguishable from wild-type enzyme. Therefore, it was concluded that Glu90 and His212 were involved in an interhelical hydrogen bond, and Y246 did not participate in this interaction. The functional data also suggests that the Glu90-His212 interaction may function to regulate electron transfer activity under certain conditions. In a second study the C-terminal helix of SIII, helix 7 was deleted from SIII via mutagenesis. Advisors/Committee Members: Prochaska, Lawrence.

Subjects: Chemistry, Biochemistry

Keywords: membrane protein; limited proteolysis; site-directed mutagenesis; mass spectrometry; heme protein; cytochrome c oxidase; stopped flow spectroscopy; absorbance spectroscopy

11. Greenberg, Marc Samuel. Defining Benthic Organism Exposure: Bioavailability and Effects of Non-Polar Organics.

Degree: PhD, Biomedical Sciences PhD, 2002, Wright State University

► Laboratory and field tests were conducted to evaluate the hypothesis that factors… (more)

▼ Laboratory and field tests were conducted to evaluate the hypothesis that factors such as the total organic carbon (TOC) contents and groundwater-surface water interactions (GSI) in the sediments can affect chemical desorption, bioavailability and benthic organism exposure. Laboratory studies were conducted with the polycyclic aromatic hydrocarbon fluoranthene (FLU) and the herbicide trifluralin (TF). Toxicokinetic parameters were determined for Lumbriculus variegatus and Hyalella azteca in water-only exposures to 0, 5, 20 and 50 µg/L of the compounds and bioaccumulation was measured during exposures to 0, 100 and 200 mg/kg of FLU and TF spiked onto sediments from Lakes Erie and Huron. Mean uptake clearance rates ranged from 150-180 mL/g wet animal/h for FLU and 84-120 mL/g/h for TF, and elimination rates were 0.12-0.18 and 0.067-0.10/h for FLU and TF, respectively. The uptake clearances in sediments (ks) ranged from 0.021 to 0.070 g dry sed/g wet animal/h for FLU and 0.013 to 0.041 g/g/h for TF. The desorption kinetics of FLU and TF from spiked sediments were measured over 34 d by extraction with Tenax®. The rapidly desorbing fraction for FLU and TF ranged from 31.3 to 54.9% of the initial concentrations and rates of the rapidly (krap), slowly (kslow) and very slowly (kvs) desorbing fractions were on the order of 10-1/h, 10-2–3/h and 10-4/h, respectively. The influence of GSI on contaminant bioavailability was demonstrated with in situ exposures of benthic invertebrates to river sediments that were contaminated primarily with chlorobenzenes (CBs). Hydrologic and chemistry data from nested mini-piezometers explained the exposure-effects relationships. Overall, downwelling conditions reduced the in situ exposure of organisms in surficial sediments, and hence, the toxicity and bioaccumulation of CBs. Data from these field and laboratory investigations were combined with literature values of contaminant partitioning (i.e., Koc values), and L. variegatus feeding rates and chemical assimilation efficiencies to parameterize a bioaccumulation model. Simulated tissue concentrations at sites containing contaminated sediments were compared to the body burdens measured in the field. The model predicted field observations within an order of magnitude and indicated that TOC, GSI and organism feeding behavior were sensitive parameters. The bioaccumulation model represents a useful tool that can reduce resource expenditures associated with site assessments and provide more accurate risk characterizations. Advisors/Committee Members: Burton, G. Allen.

Subjects: Environmental Sciences

Keywords: Sediments, Desorption, Toxicokinetics, Bioavailability, Benthic, Model, Groundwater-Surface Water Interaction.

12. Heizer, Esley Marvin Jr. Extent and Effects of Selection to Reduce Synthetic Cost of Highly Expressed Proteins.

Degree: PhD, Biomedical Sciences PhD, 2010, Wright State University

► Organisms that preferentially utilize less biosynthetically expensive amino acids in highly expressed… (more)

▼ Organisms that preferentially utilize less biosynthetically expensive amino acids in highly expressed genes exhibit metabolic efficiency. Exploration of this phenomenon has been limited to six prokaryotes. I present a large scale analysis of metabolic efficiency in prokaryotic organisms and analysis of two eukaryotes (Saccharomyces cerevisiae and humans). Examination of 73 bacteria reveals the presence of metabolic efficiency in 66 organisms. The average correlation between amino acid biosynthetic cost and CAI scores in these organisms is -0.21. The seven organisms that did not exhibit metabolic efficiency are all Lactobacilli and highly auxotrophic. Saccharomyces cerevisiae exhibits significant trends between average amino acid biosynthetic cost and CAI both when aerobic and anaerobic costs are considered. The treatment of amino acids in the seven organisms and the dual amino acid costs of Saccharomyces cerevisiae led to the examination of perceived cost of amino acid acquisition. Linear regression was utilized to calculate amino acid perceived cost in 11 organisms auxotrophic for a single amino acid and 13 prototrophic for all amino acids. The resulting costs for the auxotrophic amino acids are, in general, highly negative implying that auxotrophic organisms receive an energy dividend for utilizing amino acids they cannot produce. The results make it unlikely that linear regression is a suitable method for determining perceived cost. In order to determine the effect of metabolic efficiency I examined the cost of conserved versus non-conserved positions within homologous proteins. Positions that were variable in human, mouse, dog and cow utilized less expensive amino acids while conserved positions utilized more expensive amino acids. These results indicate that natural selection generally results in proteins with lower average amino acid biosynthetic costs. Human genes with CpG islands have been shown to be highly expressed in all tissues. Comparing the cost of these genes with genes not associated with CpG islands shows a lower average cost for the highly expressed genes demonstrating that metabolic efficiency is a significant evolutionary force in humans. Metabolic efficiency appears to drive amino acid substitutions in both simple one celled organisms and complex multicellular organisms and is most strongly manifested in variable regions of a protein. Advisors/Committee Members: Krane, Dan.

Subjects: Bioinformatics; Biology

Keywords: codon usage bias; metabolic efficiency; protein conservation; amino acid biosynthesis

13. Heminger, Katherine Ann. LOSS OF HDMX LEADS TO ALTERATIONS IN GENE EXPRESSION AND INHIBITION OF CELL GROWTH IN TUMOR CELLS WITH WILD-TYPE p53.

Degree: PhD, Biomedical Sciences PhD, 2007, Wright State University

► Mutations in the p53 tumor suppressor gene are among the most prevalent… (more)

▼ Mutations in the p53 tumor suppressor gene are among the most prevalent molecular abnormalities in human cancer. While half of all human tumors possess p53 mutations, inactivation of wild-type p53 can also occur through a variety of mechanisms that do not involve p53 gene mutation or deletion. This dissertation focuses on human tumor cell lines harboring wild-type p53 protein and elevated levels of HdmX and/or Hdm2, two critical negative regulators of p53 function. My hypothesis is that loss of HdmX in tumor cells with wild-type p53 and over-expressed HdmX, will activate p53 and induce p53 target genes leading to growth inhibition. To test this hypothesis, RNA interference (RNAi) was utilized to knockdown HdmX protein from HdmX over-expressing breast, colon, and bone tumor cell lines. Alterations in gene expression and effects on cell proliferation following the removal of HdmX were examined. Two RNAi approaches were assessed in this study; transient small interfering RNA (siRNA) transfection and lentivirus-delivered short-hairpin RNA (shRNA) vectors. Multiple siRNA transfections were selected as the method of choice over the shRNA vectors due to the induction of off-target genes in cells expressing the shRNA vectors. Affymetrix GeneChips and subsequent quantitative real time-PCR validations were used to uncover a subset of p53 target genes encoding proteins associated with cell cycle arrest and growth inhibition that were induced upon HdmX knockdown. In contrast, only one p53 dependent pro-apoptotic gene (i.e. Fas) was increased. The induction of these p53 target genes following loss of HdmX was p53-dependent, as no increase in these p53 target genes were observed after HdmX knockdown in two different p53-null tumor cell lines. Cell cycle analysis and cell proliferation assays confirmed that the loss of HdmX led to a significant G1 cell cycle arrest. Similar findings were observed upon Hdm2 knockdown, and removing both HdmX and Hdm2 resulted in even greater p53 activation and a synergistic or additive induction of p53 target genes associated with cell cycle arrest. The increase in p53 transactivation following loss of HdmX was not due to p53 phosphorylation, suggesting a nongenotoxic or genotoxic stress independent p53 activation. Furthermore, the loss of HdmX did not appear to alter p53 localization or stabilization. Although the removal of over-expressed HdmX appears limited to an anti-proliferative effect in tumor cells harboring wild-type p53, loss of HdmX enhanced the cytotoxicity of several chemotherapeutic agents. Cell viability assays showed an increase in chemosensitivity in tumor cells following knockdown of HdmX and/or Hdm2. Taken together, these results suggest that removal of HdmX may be an important therapeutic target that would complement chemotherapy drugs currently used to treat tumors possessing wild-type p53. Advisors/Committee Members: Berberich, Steven J.

Keywords: HdmX; Hdm2; p53; RNAi; human tumor cells

14. Horstman, Gabrielle Marie. Limitations of Functional Recovery of Stretch Reflex Circuitry After Peripheral Nerve Regeneration.

Degree: PhD, Biomedical Sciences PhD, 2012, Wright State University

► Peripheral nerve regeneration fails to restore complete normal function after surgical repair… (more)

▼ Peripheral nerve regeneration fails to restore complete normal function after surgical repair of severed nerves, and this failure has primarily been attributed to errors in connecting with peripheral targets. However, recent evidence suggests that central deficits remain even after peripheral target reinnervation is largely successful. It has long been established that regeneration fails to restore the stretch reflex despite observation that many of the neural components are intact. Regenerated Ia afferents are largely successful in reinnervating muscle spindles, are capable of encoding stretch, and elicit EPSPs in homonymous motoneurons, while regenerated motor pools are capable of responding to uninjured sources of excitatory input. We now know this areflexia is due in part to a retraction of Ia afferent collaterals from motor pools in lamina IX (Alvarez et al., 2011). However, Ia afferents project not only to homonymous motoneurons but also to heteronymous synergist motor pools, even ones that are injury-spared. Ia afferents also project to antagonist motor pools through an interposed inhibitory interneuron. Therefore stretch of a muscle is capable of producing reflex contraction of both itself and synergist muscles while producing reflex inhibition of antagonist muscles. The function of these heteronymous projections after regeneration, however, remains unknown. The goal of this thesis is to determine the limitations of recovery of spinal circuit function after peripheral nerve regeneration by direct examination of stretch-evoked reflexes among synergists and antagonists. Direct examination of the force response to stretch in vivo is extremely valuable as changes in behavior, the muscle response to stretch, after peripheral nerve regeneration must necessarily reflect changes in the underlying circuitry. We found that heteronymous stretch reflexes initiated by reinnervated muscle were dramatically decreased in both regenerated and injury-spared synergist motoneuronal pools. Additionally, both homonymous and heteronymous stretch reflexes were reduced in an injury-spared synergist after regeneration. These results give physiological evidence for retraction of regenerated Ia afferents from all synergist motor pools, and this retraction may extend to afferents that are injury-spared. Dysfunction also extends to antagonist stretch-evoked reflexes as we found a shift from net inhibition to net excitation of the injury-spared muscle due to reinnervated antagonist stretch. This shift is readily explained by the differential preservation of synapses located in lamina where interneurons mediating these responses are presumably located (Alvarez et al., 2011). Therefore functional deficits after peripheral nerve regeneration extend to heteronymous connections of Ia afferents with synergists and antagonists, both reinnervated and injury-spared. Taken together, these findings suggest that there is a profound discoordination of spinal reflexes and reorganization of spinal circuits after peripheral nerve regeneration. Advisors/Committee Members: Cope, Timothy.

Subjects: Neurosciences

Keywords: stretch reflex; spinal circuits; reinnervation; areflexia; plasticity; heteronymous stretch reflex; reciprocal inhibition

15. Imber, Ann Nicole. The Role of Ca2+ in Central Respiratory Control Neurons of the Locus Coeruleus: Development of the Chemosensitive Brake.

Degree: PhD, Biomedical Sciences PhD, 2012, Wright State University

► Chemosensitive LC neurons increase their firing rate in response to increased CO2… (more)

▼ Chemosensitive LC neurons increase their firing rate in response to increased CO2 (hypercapnia) in part via inhibition of K+ channels. This increase gets smaller during the first two postnatal weeks (neonatal rats aged P3-P16). Alterations of this “accelerating” pathway may account for the developmental changes in the magnitude of the chemosensitive response in LC neurons. Alternatively, Ca2+ and Ca2+ channels may play a role in the response to hypercapnia, but little is known about the role of Ca2+ in central chemosensitivity. Whole cell patch clamp and fluorescence imaging microscopy were used to study a different basis for the developmental changes in the chemosensitive response of LC neurons, a “braking” pathway. In the presence of tetrodotoxin (TTX-inhibitor of Na+ channels), currents composed of both a rhythmic cycling (SRO-subthreshold rhythmic oscillation) and voltage-sensitive spikes were observed. These currents developed over the first 10 postnatal days. Since both currents were abolished by the L-type Ca2+ channel inhibitor nifedipine, both were assumed to be due to the activity of L-type Ca2+ channels. Hypercapnia increased the frequency of oscillations and the accompanying spikes in a HCO3 – dependent but pH-independent fashion. Voltage clamp studies supported the presence of L-type Ca2+ currents in LC neurons that increased in postnatal rats aged P3 to P12 and were enhanced by hypercapnia, resulting in increased intracellular Ca2+ (Ca2+i). Hypercapnia activated Ca2+ channels in LC neurons via a HCO3 – dependent pathway involving soluble adenylate cyclase-cAMP-channel phosphorylation. The hypercapnic increase in Ca2+i activated hyperpolarizing Ca2+-activated K+ currents (KCa). The BK (a large conductance KCa) channel inhibitor paxilline and voltage clamp were used to study this KCa current. Inhibition of the BK current removed the “brake” and increased the firing rate response to hypercapnia in LC neurons. Notably, the “braking” pathway increased during neonatal development in a fashion that paralleled the decrease in the chemosensitive firing rate response to hypercapnia. These findings suggest a unique role for Ca2+ in controlling the magnitude of the firing rate response to hypercapnia. Abnormalities in this pathway could be associated with disorders involving elevated central sensitivity to changes in CO2, such as sleep apnea and panic disorders. Advisors/Committee Members: Putnam, Robert W.

Subjects: Biomedical Research; Biophysics; Cellular Biology; Neurobiology; Neurosciences; Physiology

Keywords: control of breathing; brainstem; calcium; electrophysiology; potassium currents; calcium currents; neuron

16. Jordan, Heidi Lynn. The Role of AMP-Activated Protein Kinase (AMPK) in Hypoxic Chemotransduction by the Carotid Body.

Degree: PhD, Biomedical Sciences PhD, 2012, Wright State University

► The carotid bodies are small sensory organs located along the bifurcation of… (more)

▼ The carotid bodies are small sensory organs located along the bifurcation of the carotid arteries. They detect changes in blood gases and relay this information to the brain to allow for initiation of appropriate respiratory and cardiovascular responses. A decrease in oxygen (hypoxia) sensed by the carotid body results in an increase in firing of the carotid sinus nerve and ultimately a change in one's breathing pattern. An inability to respond to an acute hypoxic (low oxygen) episode via increased ventilation may result in death or lead to pathological or chronic conditions such as stroke and hypertension (Prabhakar et.al., 2005). The carotid bodies are therefore responsible for initiating the acute hypoxic ventilatory response (HVR) and blunting or attenuation of the HVR has been implicated in sudden infant death syndrome (SIDS) in ‘at risk' infant groups (Calder et al., 1994; Horne et al., 2005; Gauda et. al., 2007). The exact mechanism(s) responsible for hypoxic chemotransduction by the carotid body remains controversial and are the subject of intense investigation. There is evidence indicating the energy-sensing enzyme AMP-activated protein kinase (AMPK) may play a critical role in the transduction of an acute hypoxic stimulus by the carotid bodies (Evans et. al., 2005; Wyatt et.al., 2007). Global AMPK α1 and AMPK α2 subunit knockout mice were used to gain further insight into the role AMPK has in acute oxygen-sensing in whole animal and in isolated oxygen-sensing cells of mice carotid bodies. A two-chamber plethysmography system was used to measure baseline breathing during normoxia (21% O2) and the hypoxic ventilatory response to 8% oxygen. AMPK α1 subunit knockout mice had a significant attenuation in percent change in breathing frequency during hypoxic exposure and AMPK α2 knockouts also showed a significant decrease in percent change in minute ventilation. The plethysmography data shows AMPKα subunits may be involved in baseline breathing and generation of acute hypoxic ventilatory responses. Isolated type I cells from global knockout mice were also used in calcium imaging experiments. Hypoxia induced Ca2+ signaling was inhibited by 85% in cells from AMPK α2 knockout mice whereas AMPK α1 KO mice showed a mixed response to hypoxia. The cellular data seem to show that a decrease in response to hypoxia may be due to an effect at the level of the carotid body. These findings provide further support for the hypothesis that AMPK regulates acute hypoxic chemotransduction and thereby energy supply at the whole body level. Advisors/Committee Members: Wyatt, Christopher.

Subjects: Biomedical Research

Keywords: Hypoxic chemotransduction; Carotid body; AMP-activated protein kinase; Plethysmography

17. Kelley, Kevin Daniel. Understanding the Molecular Dynamics of YPEL3 and FHIT Gene Expression.

Degree: PhD, Biomedical Sciences PhD, 2010, Wright State University

► A comprehensive understanding of the molecular signaling pathways that regulate cell growth… (more)

▼ A comprehensive understanding of the molecular signaling pathways that regulate cell growth and proliferation is essential in the realization of new therapeutic options to facilitate early detection and eradication of malignancy. Understanding the transcriptional regulation of the YPEL3 and FHIT genes forms the basis of this dissertation. YPEL3, or Yippee-like 3, is a newly identified p53 target gene that inhibits tumor cell growth and is thus itself, a novel tumor suppressor gene. FHIT, or Fragile histidine triad, is a well known tumor suppressor gene and is regulated at the transcriptional level by another growth inhibitory protein, FOXO3a, a Forkhead box transcription factor. Our laboratory has determined that YPEL3 is a direct transcriptional target of the tumor suppressor gene p53. The first section of this dissertation provides significant experimental evidence to validate this observation. Briefly, YPEL3 was shown to be upregulated downstream of p53 protein stabilization in a microarray screen that explored global gene expression modulation after RNAi-mediated reduction of p53's negative regulators, Hdm2 and HdmX. Genotoxic stress induced by treatment with DNA-damaging agents resulted in stabilization of p53 protein along with elevation of YPEL3 transcript and protein levels. Moreover, there exists a cis-acting p53 response element within the YPEL3 promoter that is bound by p53 in response to this stress. YPEL3 also elicits growth inhibition and decreases in colony formation when expressed in tumor cells. It is apparent that cells which express exogenous YPEL3 are forced into a permanent cell cycle arrest, termed “premature senescence.” Similar growth suppressive phenotypes are typical of many other known p53 target genes. However, most of these genes are associated with the regulation of transient inhibition of cell division or the induction of apoptosis. YPEL3 is unique in its ability to trigger premature cellular senescence. Indeed, YPEL3 stands out among many other p53 targets because it is among the first to play a role in this process. To further understand the mechanism of YPEL3-induced senescence, the second portion of this dissertation focuses on the generation of a three-dimensional model of YPEL3's protein structure. It was hypothesized that predicting YPEL3's protein structure may also aid in understanding the molecular events involved in the induction and maintenance of premature senescence. Identifying structural homology between a predicted model of YPEL3 and other known structures may provide new insights into this process. This would especially be the case if homology existed between the predicted structure of YPEL3 and other proteins that have established functions in stress-responsive or senescence-associated molecular pathways. By using two independent structural prediction algorithms (Rosetta ab initio and I-TASSER), I have been able to estimate a three-dimensional model of the YPEL3 protein which has significant structural homology to a family of Methionine oxidoreductases. These enzymes catalyze redox-mediated antioxidant reactions which dissipate intracellular reactive oxygen species (ROS) through the repair of proteins damaged by ROX-mediated methionine to methionine sulfoxide oxidation. It is even plausible that YPEL3 could be involved in the mediation of cellular senescence in response to severe oxidative stress. If this is the case, the implications of this speculation, linking the oxidative stress response to YPEL3-dependent cellular senescence, are indeed profound. YPEL3 may not only play a role in the molecular biology of cancer but also may be involved in the cellular oxidative stress response as it relates to senescence and aging. In the third part of this dissertation, FHIT, a tumor suppressor gene often deleted in epithelial-derived tumors, was also initially postulated to be regulated by p53 (just as in the case of YPEL3). However, FHIT gene expression was instead found to be modulated by FOXO3a. This gene’s expression was induced coincidentally under similar conditions as p53 such as cell cycle arrest. FHIT is suppressed by the AKT/PI3K pathway upon growth factor-dependent stimulation. These protooncogenic kinases phosphorylate and, thereby prevent FOXO3a from entering the nucleus and activating FHIT gene expression. Thus, FOXO3a is only able to enter the nucleus in the absence of mitogenic stimulation. FHIT transcript levels are elevated in response to the removal of mitogen-containing serum from breast, colon carcinoma, and immortalized mammary epithelial cells. Induction of FHIT gene expression is dependent on decreased PI3K kinase activity and subsequent reduced AKT phosphorylation which occur in the absence of growth factor stimulation. As such, quiescence-dependent elevation of FHIT mRNA can be elicited independently of mitogen removal, by treatment with PI3K inhibitors, or attenuated by ectopic expression of constitutively active AKT during serum deprivation. Furthermore, ectopic expression of FOXO transcription factors result in the upregulation of FHIT mRNA and protein levels. Additionally, nuclear shuttling experiments with a FOXO3a-estrogen receptor fusion protein have demonstrated that FOXO must enter the nucleus to modulate expression of FHIT. Finally, experiments using shRNA targeting FOXO3a have revealed that the modulation of FHIT gene expression, downstream of PI3K inactivation, is dependent on endogenous FOXO3a. In summary, this project began with investigating the regulation of two genes derived from a microarray screen to search for new p53 targets. Validation of YPEL3 as a novel p53 target gene was very straightforward. All assays employed in characterizing YPEL3 revealed that it behaved exactly as a classic p53 transcriptional target. The latter of the two genes, FHIT, however was determined not to be a p53 target but rather a transcriptional target of FOXO3a – another tumor suppressive transcription factor. It is the hope of the author that this information will serve to enhance our basic understanding of the paradoxically both, overwhelmingly complex and elegantly simple molecular dynamics of gene regulation, as it pertains to the biology of cancer. Advisors/Committee Members: Berberich, Steven J.

Subjects: Molecular biology

Keywords: FHIT; fragile histidine triad; YPEL3; yippee; yippee-like 3; yippie; gene expression; gene; transcription; regulation; molecular biology; DNA damage; tumor suppressor; p53; oncogene; oncology; cancer; transformation; p53 target; FOXO; FOXO3a; cell cycle

18. Kemp, Michael George. Regulation of DNA Replication Initiation by Histone Acetylation and the DNA Unwinding Element Binding Protein DUE-B.

Degree: PhD, Biomedical Sciences PhD, 2006, Wright State University

► Duplication of the genome during S phase of the mitotic cell cycle… (more)

▼ Duplication of the genome during S phase of the mitotic cell cycle begins at thousands of sites along chromosomes termed origins of replication. Although many of the essential protein components catalyzing events at these sites are known and are conserved throughout eukaryotes, the likelihood or efficiency of initiation of DNA synthesis at any given genomic site is expected to be influenced by other novel factors, including aspects of chromatin and DNA structure. Here I show that increased histone H4 acetylation at replication origin loci occurs after treatment with the histone deacetylase inhibitor TSA and coincides with a loss of specific initiation site selection both within origin loci and throughout the genome. Furthermore, new replication initiation sites become activated or used with greater frequency after treatment with TSA, and TSA promotes the activation of replication origins earlier during the S phase of the cell cycle. These data suggest a physiological role for histone acetylation in controlling the initiation of DNA synthesis at specific chromosomal sites. Regions of helically unstable DNA termed DNA unwinding elements (DUEs) are commonly found at replication origins, and our laboratory identified a DUE-binding protein (DUE-B) using the c-myc DUE in a yeast one-hybrid screen. Here I demonstrate that DUE-B is required for efficient entry into S phase in human cells and for efficient replication in the Xenopus egg extract replication system. Structural analyses show the N-terminal portion of the protein to be identical to that of bacterial D-aminoacyl-tRNA deacylases. Human DUE-B possesses this function in vitro and the ability to hydrolyze ATP, suggesting that DUE-B may be a multifunctional esterase. Unique to vertebrate homologs of DUE-B is a C-terminal extension of 62 amino acids that binds DNA and is targeted for phosphorylation by CK2. The addition of the C-terminal domain to DUE-B in higher eukaryotes may have coincided during evolution with the development of a novel function for this protein in the initiation of DNA replication. Together these two sets of data argue that previously uncharacterized factors regulate the initiation of DNA replication in higher eukaryotes, possibly to deal with the complex chromosomal architecture found in these organisms. Advisors/Committee Members: Leffak, Ira Michael.

Keywords: DNA replication; histone acetylation; DNA unwinding element; replication origin; tRNA metabolism

19. Kolawole, Abimbola Olayinka. The Molecular Basis of the Interaction Between the Coxsackievirus and Adenovirus Receptor (CAR) and MAGI-1.

Degree: PhD, Biomedical Sciences PhD, 2011, Wright State University

► A major factor in virus entry into cells is localization and abundance… (more)

▼ A major factor in virus entry into cells is localization and abundance of the primary receptor. The Coxsackievirus and adenovirus receptor (CAR) is the primary receptor for group B coxsackievirus and many serotypes of adenovirus. In most epithelia, a seven exon isoform of CAR (CAREx7) is exclusively localized at the basolateral surface where it behaves as a homophilic adhesion protein and is inaccessible for viral infection. However, in well-differentiated human airway epithelia, we recently discovered an alternatively spliced, low abundance isoform of CAR (CAREx8) that is apically localized where it may initiate apical viral infection. The two isoforms differ only in the last 26 (CAREx7) or 13 (CAREx8) amino acids of the cytoplasmic domain, which suggests that some intracellular interactions may differ. One such differential interaction involves MAGI-1, an essential PDZ-domain containing protein known to be involved in cell polarization and cancer. In non-polarized COS7 cells, the CAREx8 protein level is regulated by MAGI-1b. CAR-MAGI-1 interactions were investigated by MAGI-1 siRNA knockdown, in vitro translation, immunocytochemistry, co-immunoprecipitation-Western blot analysis, fluorescence resonance energy transfer, direct binding assays, and adenovirus infection. Data showed that both CAR isoforms were expressed in several cell lines with CAREx7 RNA consistently more highly expressed than CAREx8 and MAGI-1 siRNA knockdown improved adenovirus infection in polarized cells. Both isoforms strongly interacted with PDZ3. CAREx8 also interacted with PDZ1. Whereas co-expression of PDZ1 with CAREx8 did not affect adenovirus infection, it could compete with full length MAGI-1 to protect CAREx8 from loss. Co-expression of CAREx8 with PDZ3 significantly reduced CAREx8 cell surface expression and adenovirus infection but did not reduce total CAREx8 protein levels suggesting that this may be the MAGI-1 PDZ domain responsible for holding CAREx8 within the cell but it is likely that other MAGI-1 interacting proteins/domains are required for loss of CAREx8. This dissertation provides the molecular basis for MAGI-1-mediated regulation of CAREx8 which will allow further investigation into the mechanisms of CAREx8 cell surface expression and hence viral infection of polarized epithelia. Advisors/Committee Members: Excoffon, Katherine.

Subjects: Biomedical Research

Keywords: coxsackievirus and adenovirus receptor, CAR, MAGI-1, adenovirus, epithelia

20. Kommagani, Ramakrishna. DIFFERENTIAL REGULATION OF VITAMIN D RECEPTOR (VDR) BY p53, p63 AND p73.

Degree: PhD, Biomedical Sciences PhD, 2009, Wright State University

► The tumor suppressor p53 is the single most altered gene in human… (more)

▼ The tumor suppressor p53 is the single most altered gene in human cancers. p53 homologues, p63 and p73 play a major role in development and in human cancer. Both p63and p73-null mice exhibit profound developmental abnormalities, suggesting a vital role for p63 and p73 in development. Although the role of p73 in human cancers is well established, the role of p63 still remains to be understood. While p63 plays a major role in development, p73 plays a major role in tumor suppression as well as in development. Although a functional co-operation is evident between each member of p53 family, additional studies are required to understand the functional cross-talk between each member of the p53 family, and their ability to govern multiple biological functions. Identifying common and specific transcriptional networks of p53 family is essential for understanding the existing functional co-operation between each member.This dissertation focuses on defining the functional relevance of the regulation of vitamin D receptor (VDR) by p53 family members. Secosteroid hormone vitamin D, through its cognate receptor VDR, regulates genes involved in mineral homeostasis, bone formation and in epidermal differentiation. Vitamin D and its analogues also exhibit anti- proliferative activities and are widely used as cancer chemotherapeutic agents. Findings from this study demonstrated VDR as a direct target of p63 and p73; however p53 does not appear to regulate VDR directly or indirectly. One part of this dissertation underpinned the role of VDR in p63 mediated biological functions. Down regulation of endogenous p63 in human epidermoid cancer cells resulted in complete loss of endogenous VDR expression. In addition, up-regulation of VDR by p63 appears to inhibit the migration and invasion of human epidermoid cancer cells. the second part of this dissertation is involved in understanding the p73 mediated regulation of VDR in vitamin D-mediated differentiation. Findings from this section of study demonstrated that DNA damage-induced expression of VDR is dependent on p73. In addition, p73 was proven to be essential for vitamin D mediated osteoblastic differentiation. Furthermore, we demonstrated that DNA damage sensitized the cells to vitamin D mediated differentiation through p73. Taken together, while understanding the regulation of VDR by p63 will provide new insights on the molecular mechanisms of p63 biology, determining the role of p73 in vitamin D-mediated differentiation may aid in vitamin D based cancer chemotherapeutics. Advisors/Committee Members: Kadakia, Madhavi.

Subjects: Biochemistry; Biomedical research; Cellular biology; Molecular biology; Oncology

Keywords: p53, p63, p73, VDR and Vitamin D

21. Kulkarni, Kashmira. HIF-1 alpha: a master regulator of trophoblast differentiation and placental development.

Degree: PhD, Biomedical Sciences PhD, 2009, Wright State University

► Early embryonic development occurs under low oxygen levels. The placenta is an… (more)

Subjects: Cellular biology; Molecular biology

Keywords: Hypoxia, Hypoxia inducible factor-1 alpha, Trophoblast, Differentiation, Placenta.

22. Lehman, Jason Alexander. Novel Redox and DNA-Dependent Conformational Changes in Human Ku, a DNA-Double Strand Break Repair Protein.

Degree: PhD, Biomedical Sciences PhD, 2008, Wright State University

► Ionizing radiation (IR) and radiomimetic drugs used in cancer chemotherapy cause DNA… (more)

▼ Ionizing radiation (IR) and radiomimetic drugs used in cancer chemotherapy cause DNA double-strand breaks which are repaired by the nonhomologous end joining (NHEJ) pathway. Ku is a heterodimeric protein comprised of 70 and 80 kDa subunits and recognizes free DNA ends. Once Ku is bound to DNA, it binds to the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and forms a heterotrimeric DNA-PK complex for repair and damage signaling. We have analyzed the Ku protein using multiple biochemical techniques and uncovered a novel reversible redox change (Andrews, Lehman and Turchi, (2006) JBC 281(19):13596-603). From this data, we hypothesized that a redox-dependent conformational change was responsible for the unexplained differences in our DNA binding assays. This was confirmed by limited proteolysis and mass spectrometry analysis of oxidized and reduced Ku. The conformational change was manifested by alterations in the Ku70 subunit in proximity to the DNA binding site. Our results suggest a model for a Ku-DNA interaction that is altered by redox status upon DNA damage in the cell. In order to understand the complex structure of DNA-PK, we have initially examined Ku-DNA interactions using advanced biochemical techniques. Structural studies on Ku by other groups have utilized truncated versions missing disordered C-terminal domains (CTDs) from both subunits. Chemical modification with NHS-biotin and mass spectrometry were used to identify biotinylated reactive lysines. Biotinylation of free Ku revealed several lysines on Ku which were reduced or eliminated upon DNA binding. Interestingly, in the predicted C-terminal SAP domain of Ku70, biotinylation patterns suggest a structural change at this site induced by DNA binding. Limited proteolytic digests of free and DNA-bound Ku revealed a series of unique peptides correlating to a change in the accessibility of the Ku70 and Ku80 CTDs. A 10 kDa peptide was also identified which was preferentially generated under non-DNA-bound conditions and mapped to the Ku70 CTD. These results indicate a DNA-dependent movement or structural change in the CTDs of Ku70 and Ku80 that may contribute to DNA-PKcs binding and activation. This is the first demonstration of DNA-dependent conformational changes in Ku and will aid in discerning the mechanism of DNA-PK activation. Advisors/Committee Members: Turchi, John.

Subjects: Biochemistry

Keywords: DNA repair; Ku; DNA-PK; non-homologous end joining; mass spectrometry; conformational changes; redox

23. Leonard, Mary Kathryn. Regulation of the Transcription and Subcellular Localization of the Tumor Suppressor PTEN by ΔNp63α.

Degree: PhD, Biomedical Sciences PhD, 2012, Wright State University

► Non-melanoma skin cancers (NMSCs) are the most common form of cancer in… (more)

▼ Non-melanoma skin cancers (NMSCs) are the most common form of cancer in the United States with an estimated 3.5 million new cases each year. Surgical excision is the main treatment for NMSC, but leaves the potential for disfiguring scars and does not fully reduce the risk of recurrence since the surrounding tissue is also sun damaged and may contain tumor-promoting mutations. By understanding the molecular etiology of NMSC we may be able to expand treatment options to more than just resection. Normal epidermal development is dependent upon the expression of the transcription factor p63. Amplification of the ΔNp63α isoform is also frequently observed in NMSC, making it an excellent candidate for understanding the etiology of NMSC. This dissertation focuses on how ΔNp63α influences keratinocyte proliferation by regulating phosphatase and tensin homologue deleted on chromosome ten (PTEN). The tumor suppressor PTEN has been called “the second guardian of the genome” for its widespread role in preventing tumor formation and is second only to p53 in the frequency of observed mutations or loss in human cancers. The studies presented in this dissertation demonstrate that ΔNp63α is able to maintain the proliferative potential of keratinocytes by activating Akt through transcriptional repression of PTEN. The inhibition of PTEN by ΔNp63α was independent of any repressive effects of ΔNp63α towards other p53 family members. Since the discovery of nuclear PTEN the tumor suppressive functions of PTEN have expanded to include induction of cell cycle arrest, accentuation of apoptotic signaling and maintenance of chromosome stability. ΔNp63α was also shown to inhibit the nuclear localization of PTEN, which may further the increase the proliferative potential of basal layer keratinocytes. Inhibition of NEDD4-1 by ΔNp63α was implicated in reducing the ubiquitination of PTEN, thereby preventing its import into the nucleus. The studies presented here also identify a novel pool of PTEN that is localized to centrosomes only during mitosis. Coordinated control of both Akt and PTEN regulate centrosome composition and integrity during mitosis and provides insight into how PTEN functions as a multifaceted tumor suppressor. The importance of the ΔNp63α/PTEN/Akt signaling loop in epidermal biology was highlighted by the significant disruption of ΔNp63α and PTEN levels in NMSC. Altogether, these studies provide important molecular insight into the control of keratinocyte proliferation by the ΔNp63α-PTEN-Akt signaling loop. Advisors/Committee Members: Kadakia, Madhavi.

Subjects: Biomedical Research; Cellular Biology; Molecular Biology; Oncology

Keywords: p63; PTEN; keratinocyte; Akt; NEDD4-1; non-melanoma skin cancer; centrosome

24. Mahle, Deirdre A. 'Omic' Evaluation of the Region Specific Changes Induced by Non-Cholinergic Diisopropylfluorophosphate (DFP) Exposure in Fischer 344 Rat Brain.

Degree: PhD, Biomedical Sciences PhD, 2012, Wright State University

► Organophosphorous compounds (OPs) are a class of serine esterase inhibitors that have… (more)

▼ Organophosphorous compounds (OPs) are a class of serine esterase inhibitors that have widespread application as pesticides, veterinary pharmaceuticals and chemical warfare agents. Environmental contamination is ubiquitous. The threat of exposure is a concern for both military and civilian populations. Acute inhibition of acetylcholinesterase by OPs triggers a cholinergic crisis that results in muscle flaccidity, paralysis, convulsions and death. At low doses OPs can alter neuronal differentiation, cell signaling, behavior and cognition through unknown mechanisms. An imbalance of reactive oxygen species may be implicated in the adverse effects of OPs. An integrated approach using both metabolomic and transcriptomic techniques was used to reveal some of the non-cholinergic effects of diisopropylfluorophosphate (DFP), a model OP, in rat brain. Adult male Fischer 344 rats were administered 1 mg/kg DFP or saline via subcutaneous injection at 10 mL/kg. Cortex, brainstem, cerebellum and hippocampus were collected at multiple time points ranging from 0.5 - 48 hr. Total RNA was isolated from each region for differential gene expression analysis using the Affymetrix 1.0 ST gene array at 1 hr post dose. Lipid and aqueous extracts were prepared from each brain region at 2 hr post dose, and profiles of small molecule metabolites, lipids and phospholipids were measured using multinuclear NMR spectroscopy. Because the dose was below the threshold for cholinergic toxicity, it was hypothesized that DFP exposure would up-regulate inflammatory pathways and down-regulate processes that result in cellular degradation, such as apoptosis, and that these changes would correlate with perturbations in the small molecule and lipid profiles as well as gene expression. All brain regions reached minimum acetylcholinesterase activity (40-55%) at 1-2 hr post dose with the exception of cortex, which had minimum activity at 12 hr post dose. No brain region showed significant increases in lipid peroxidation. After 1 hr, pathways associated with prostaglandin D2 synthesis were up-regulated in cortex. Brainstem showed increased expression of genes associated with an inflammatory response and ascorbate transport. Cortex showed the most changes in the lipid profile with significant decreases in phosphatidylcholine, phosphatidylethanolamine, cholesterol, n3 and n6 fatty acids. The mitochondrial phospholipid cardiolipin was significantly decreased after 2 hr in brainstem. By evaluating the impact of low level OP exposure on the neuronal phenotype of specific brain regions, we hope to gain a greater understanding of the non-cholinergic mechanisms of action and sensitive target areas in order to improve the development of therapeutic targets for individuals exposed to OPs. Advisors/Committee Members: Reo, Nicholas.

Subjects: Biomedical Research

Keywords: organophosphate; DFP; non-cholinergic toxicity; brain; metabolomic; transcriptomic

25. Maken, Deborah Suzanne. Central Mechanisms Regulating Pituitary-Adrenal Activity in Infant Guinea Pigs (Cavia porcellus) during Exposure to Psychological Stressors: Independent and Combined Effects of Maternal Separation and Novelty.

Degree: PhD, Biomedical Sciences PhD, 2009, Wright State University

► Separation from the maternal attachment figure, particularly when it occurs in a… (more)

Subjects: Biomedical research; Developmental psychology; Psychobiology

Keywords: Maternal Separation; c-Fos protein; CRF mRNA; ACTH; Cortisol; Psychological Stressor; Novelty; MeA; BNST; PVN; Hypothalamus; Pituitary; Adrenal; Medial Amygdala; Bed Nucleus of the Stria Terminalus; Paraventricular Nucleus; Novel Environment; HPA axis

26. Mauck, Brena S. EFFECT OF PYRIDOSTIGMINE BROMIDE AND STRESS ON NEURONAL APOPTOSIS AND MUSCARINIC RECEPTOR DENSITY IN C57Bl MICE.

Degree: PhD, Biomedical Sciences PhD, 2003, Wright State University

► Mauck, Brena S. Ph.D., Biomedical Sciences Ph.D. Program, Wright State University,… (more)

▼ Mauck, Brena S. Ph.D., Biomedical Sciences Ph.D. Program, Wright State University, 2003. Effect of Pyridostigmine Bromide and Stress on Neuronal Apoptosis and Muscarinic Receptor Density in C57Bl Mice. Gulf War Syndrome is a name given to a group of neurological and neuro-muscular symptoms experienced by soldiers of the 1991 Persian Gulf War. These soldiers were prescribed pyridostigmine bromide (PB) as a prophylactic against nerve gases. PB inhibits cholinesterases, enzymes necessary for the termination of cholinergic transmission. One hypothesis is that extended exposure to PB, coupled with stress, allowed the drug to cross the blood brain barrier resulting in neuronal damage. The purpose of this study was to determine if a 7 day exposure to PB and stress was sufficient to alter muscarinic receptor density and to induce apoptosis in male C57Bl mice. Physostigmine, a cholinesterase inhibitor that crosses the blood brain barrier, was used as a positive control. Subcutaneously implanted osmotic mini-pumps delivered a constant dose of drug for 7 days. The stress paradigm involved random shaking of the mice for 2 minutes out of every 30 minutes. This was sufficient to raise blood corticosterone levels. Significant changes were seen in cholinergic receptor densities, primarily in cholinergic nuclei and their projection fields. Stress alone and physostigmine alone produced changes in the diagonal band nuclei and in the projection fields, the amygdala, the limbic cortex, and the hippocampus at the end of a 7 day exposure. PB produced changes in diagonal band nuclei at day 7 but changes in the amygdala and the limbic cortex were not seen until day 37. This delayed effect is important in that soldiers did not experience symptoms until months after their exposure. The combination of stress and drug did not increase the magnitude of density changes as expected. In fact, stress and physostigmine interacted in an antagonistic manner with the combination producing a change in receptor density of smaller magnitude than that produced by either component alone. This neuroprotective effect of stress is supported by studies found in the literature. The regions showing altered receptor densities in this study are known to be involved in memory and learning, areas where veterans experience problems. This study does not provide any evidence that shaker stress altered the leakiness of the blood brain barrier to PB as evidenced by the lack of significant differences between PB treated and PB + stress treated animals. The receptor density changes seen at day 7 due to physostigmine exposure do suggest that if PB is able to cross the blood brain barrier it might be capable of producing cellular changes in areas of the brain responsible for learning and memory. The changes seen at day 37 due to PB exposure suggest that the drug is capable of producing delayed effects, another characteristic of the symptoms experienced by the soldiers. Initial apoptosis studies showed high levels of apoptotic cells, which while consistent with levels found in the literature seemed unlikely to be real. Such high levels of neuronal apoptosis would be expected to lead to modified animal behavior or higher mortality. Modifications of the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) protocol designed to eliminate non-specific binding of the avidin and biotin conjugates eliminated any positive staining in the brains of treated animals. A compound known to produce low levels of apoptosis was used to confirm that the modified assay was capable of detecting low levels of apoptosis. This study shows that exposure to PB or stress leads to altered muscarinic receptor density but neither exposure leads to detectable levels of neuronal apoptosis. Advisors/Committee Members: Grubbs, Robert D.

Keywords: Pyridostigmine Bromide-adverse effects; Stress; Aptosis; Muscarinic Receptors; Persian Gulf Syndrome-etiology

27. Mercer, Carol A. Structural and Signaling Elements Important for the Efficient Degradation of BHMT through Macroautophagy.

Degree: PhD, Biomedical Sciences PhD, 2007, Wright State University

► Healthy cells maintain a dynamic and responsive intracellular environment that is marked… (more)

Keywords: Autophagy; BHMT; mTOR; S6 kinase

28. Merrill, Elaine Alice. A Mechanism-Based Model to Describe GABAA Receptor Trafficking and Benzodiazepine Pharmacoresistance during Status Epilepticus.

Degree: PhD, Biomedical Sciences PhD, 2012, Wright State University

► Status epilepticus (SE) is a perplexing pathology involving a sudden and long… (more)

▼ Status epilepticus (SE) is a perplexing pathology involving a sudden and long disruption of the brain's normal electrical activity. The study of relevant cellular processes has been useful in identifying therapeutic targets. As a result, many novel drugs are being studied which target receptor systems involved in neuronal membrane excitability. Yet, the standard treatment for SE remains benzodiazepines (BZs), a class of GABAA agonist drugs. Unfortunately, the targeted receptors undergo a desensitization mechanism via enhanced endocytosis (receptor trafficking), leading to rapidly reduced BZ efficacy (pharmacoresistance) within minutes of seizure onset. A comprehensive understanding of the complex interplay between the anticonvulsant's pharmacokinetics and its effect during SE is still lacking. Quantitative information regarding how the trafficking mechanisms of the targeted receptor contribute to the drug's overall pharmacodynamic profile is especially important for the development and assessment of SE countermeasures. This is because the testing of seizure therapeutics can only be performed in animals. Therefore computational modeling of pharmacodynamics provides a useful approach for extrapolations to humans. This dissertation links a physiologically-based pharmacokinetic (PBPK) model for the therapeutic agent, with a cellular level pharmacodynamic (PD) model of the targeted receptors. The latter explicitly takes into accounts the targeted receptor's surface expression and disrupted trafficking during seizures and the binding of the therapeutic drug. This approach is demonstrated for the interaction of diazepam and its major active metabolite, with the GABAA receptor, the major therapeutic target. The GABAA receptor is known to be rapidly modulated during seizure activity. Hypothetically, by accounting for diazepam's pharmacokinetics and occupancy of BZ-sensitive GABAA receptors, as well as the cellular trafficking of those receptors during SE, one should be able to mathematically describe the rapid pharmacoresistance. The model developed suggests that approximately 55% occupancy of the original receptor number is required to reverse ongoing seizures. This is up from a reported 37% occupancy required to prevent seizures in the rat, when diazepam is administered just before seizure onset. The physiological basis of the model allows for extrapolation to humans and dose optimization. In addition, the modeling approach used may serve to explain why some drugs may be more or less effective than BZs in treating SE and to offer suggestions for alternative therapeutics. Advisors/Committee Members: Gearhart, Jeffrey.

Subjects: Pharmacology

Keywords: GABAA receptor; PBPK/PD modeling; receptor trafficking; status epilepticus; pharmacoresistance; benzodiazepine

29. Miller, Kelly Lynn Robbins. Induction of p53 Dependent Cellular Senescence Through HdmX Inhibition or YPEL3 Expression.

Degree: PhD, Biomedical Sciences PhD, 2010, Wright State University

► Mutations in p53 that compromise its function have been reported in approximately… (more)

▼ Mutations in p53 that compromise its function have been reported in approximately half of all human cancers (Vousden et al., 2002). The other half of human tumors that retain wild-type p53 have a dysfunctional p53 pathway through other mechanisms (Wade et al., 2009). Activation of p53 leads to cell cycle arrest, DNA repair, apoptosis and senescence, however, the pathway leading to cellular senescence is the focus of this study. Cellular senescence is a process leading to irreversible arrest of cell division. Under normal physiologic conditions, the activity of p53 is kept in check by its negative regulators, Mdm2 and MdmX. While MdmX overexpression has been linked to human tumor formation, the mechanism has yet to be elucidated. The first part of this thesis describes the ability of HdmX to block oncogenic Ras mediated senescence in human diploid fibroblasts (HDFs) through interactions with Hdm2 and p53, elucidating a potential mechanism for HdmX's contribution to tumor formation. Furthermore, senescence induction was demonstrated when HdmX was reduced in prostate adenocarcinoma cells (mutant Ras, wild-type p53, high HdmX), suggesting that targeting HdmX in tumor cells with this genotype may be a useful anti-cancer therapeutic approach. In part two of this thesis, a novel-p53 target, YPEL3, was described as a senescence inducer in non-transformed and human tumor cell lines. YPEL3, acting downstream of p53, was shown to be required for oncogenic Ras mediated senescence but functions as a senescence inducer independent of p16 or p21 expression in HDFs, suggesting its critical nature to senescence signaling. In accordance with its potential activity as a tumor suppressor protein, YPEL3 expression was found to be differentially regulated in breast tumor versus normal tissue. In the third part of this thesis, I discovered that YPEL3 is down-regulated by ERα signaling in a p53 independent manner. Estrogen depletion of MCF7 cells led to activation of YPEL3 and induction of cellular senescence of this breast carcinoma cell line, suggesting that YPEL3 may be a valid therapeutic target for treating ER+ breast tumors. This study describes novel mechanisms that are involved in the activation of p53 and subsequent downstream targets leading to cellular senescence. Advisors/Committee Members: Berberich, Steven.

Subjects: Biomedical research

Keywords: senescence, p53, HdmX, YPEL3, ERα

30. Myers, Shere Lynne. Cellular Effects of Replicating a Polypurine-Polypyrimidine Sequence and the Interactions of DUE-B with Replication Proteins.

Degree: PhD, Biomedical Sciences PhD, 2010, Wright State University

► This work investigates two questions regarding DNA replication. The first aim examines… (more)

▼ This work investigates two questions regarding DNA replication. The first aim examines the interactions of DUE-B with replication proteins and the second explores the cellular effects of replicating a polypurine polypyrimidine sequence in human cells. DUE-B siRNA decreases the chromatin binding of essential replication proteins Cdc45, PCNA and RPA. DUE-B also co-immunoprecipitates with Cdc45 and TopBP1. In vitro kinase assays suggest that the checkpoint protein ATR may phosphorylate DUE-B. These experiments lend further evidence that DUE-B plays an important role in the initiation of eukaryotic DNA replication. To investigate the effects of replicating a polypurine-polypyrimidine sequence prone to secondary structure (PKD1), our laboratory constructed four cell lines. In TTR and TTF cells, the PKD1 sequence is inserted in opposite orientations in place of the triplex-prone region of the c-myc replicator (which is stably integrated into the same genomic locus in all four cell lines). In DTR and DTF cells, the PKD1 sequence is inserted in opposite orientations in place of the DNA Unwinding Element of the c-myc replicator. The probability of secondary structure formation by the PKD1 sequence varies based on the orientation due to changes in direction of replication. In these cells, the orientation of the insert affects the cells' growth rate, overall health, dependence on a functional checkpoint, and ability to replicate DNA after synchronization with aphidicolin or mimosine. A nearby origin is not required for these effects. When the PKD1 insert was removed from TTR cell lines, the normal phenotype was partially restored. These cells showed an intermediate response to aphidicolin and mimosine synchronization and to checkpoint inhibition. These conclusions argue for the efficiency of replication origins and suggest that the differences between the two cellular phenotypes are caused by the direction of replication fork movement through the polypurine-polypyrimidine sequence. Importantly, these data indicate that a single genomic lesion may have the ability to alter cell growth and recovery from DNA damage. Advisors/Committee Members: Leffak, Michael.

Subjects: Molecular Biology

Keywords: polypurine; polypyrimidine; triplex; quadruplex; replication; mirror repeat; polycystic kidney disease; checkpoint

[1] [2]