Master of Science (MS), Wright State University, 2023, Biological Sciences

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that causes the degeneration of motor neurons which leads to loss of motor function and eventual death. Over 20 genes have been implicated in ALS's pathogenesis, one being TARDBP, which codes for TDP-43. TDP-43 mislocalizes from the nucleus, accumulates, and then aggregates in the cytoplasm and is linked to cellular toxicity. We have modeled this aggregation and toxicity of TDP-43 in budding yeast. From genetic screens of human genes and their ability to modulate TDP-43 toxicity, we found 50 human genes that were able to reduce TDP-43 toxicity. 12 of these genes expressed the strongest rescue phenotype. Interestingly, these 12 genes did not lower the protein level or aggregation of TDP-43, nor did they exhibit any protein-protein interactions with TDP-43. We then investigated a possible cellular pathway that could be associated with TDP-43 toxicity and examined how our suppressors were able to reverse its effects. We identified four suppressors that were implicated in the cAMP/PKA pathway, with two of them directly downregulating it. Further analysis revealed that out of these four genes, two led to significantly increased cAMP levels compared to TDP-43 alone. We then studied a downstream target of the cAMP/PKA pathway, stress granule formation. Our observation indicated that TDP-43 not only colocalized with stress granules but also stimulated their formation.

Committee: Shulin Ju Ph.D. (Advisor); Michael Markey Ph.D. (Committee Member); Andrew Voss Ph.D. (Committee Member) Subjects: Biology; Cellular Biology; Molecular Biology

Master of Science (MS), Wright State University, 2020, Chemistry

Acid dissociation constants, often expressed as pKa values, afford vital information with regards to molecular behavior in various environments and are of significance in fields of organic, inorganic, and medicinal chemistry. Several quantitative structure-activity relationships (QSARs) were developed that correlate experimental pKas for a given class of compounds with a descriptor(s) calculated using density functional theory at the B3LYP/6-31+G** level utilizing the CPCM solvent model. A set of carbon acids provided a good final QSAR model of experimental aqueous pKas versus ΔEH2O (R2 = 0.9647) upon removal of three aldehydes as outliers. A study of saturated alcohols offered a final QSAR model with R2 = 0.9594, which was employed to confirm the behavior of the three aldehydes as hydrated species in aqueous solution. Finally, a study restricted to ketones was conducted to estimate their pKas in dimethyl sulfoxide solution. QSAR models of experimental pKas versus ΔEDMSO for the keto and enol tautomers were modest at best (R2 = 0.8477 and 0.7694, respectively). A binary linear regression was employed to incorporate descriptors representing both the keto and enol tautomers, improving the final R2 to 0.9670 upon removal of one outlier. The QSAR models presented may be utilized to estimate pKas for related compounds not offered in the existing literature or that are challenging to measure experimentally.

Committee: Paul Seybold Ph.D. (Advisor); Eric Fossum Ph.D. (Committee Member); David Dolson Ph.D. (Committee Member) Subjects: Chemistry; Organic Chemistry; Physical Chemistry

PHD, Kent State University, 2018, College of Arts and Sciences / Department of Biological Sciences

Cyclic-AMP plays an important role in sperm motility and activation. The actions of cAMP in sperm involve Protein Kinase A mediated protein phosphorylation. The crucial roles of sperm cAMP and PKA have also been demonstrated using genetic approaches. Understanding how a cAMP act to sustain sperm motility and fertility requires identification of the protein substrates of PKA. Identification of substrates of specific protein kinases and determination of changes in phosphorylation of these substrates during sperm motility initiation and fertilization will be a major advance in understanding signaling mechanisms underlying male gamete function. In our study we are using a novel chemical-genetic approach to identify proteins which are phosphorylated by PKA during sperm motility. In this approach the structurally conserved ATP-binding pocket in Protein Kinase A is genetically modified (analog-sensitive-Protein Kinase A) to generate mutant allele, this mutant protein will in addition to ATP, can also utilize specific ATP analogs. The mutation replaces a conserved amino acid with a bulky side chain with a smaller residue (alanine or glycine) creates a “gap” or an enlarged ATP binding pocket. The engineered ``gap'', located in the active site of the enzyme where the N6 amino group on the purine moiety of ATP is positioned, allows binding of not only ATP but also structurally modified ATP analogs with substitutions at the N6 position, such as N6-(benzyl)-ATP. Only the as-mutant kinase, but not the wild type kinase, can use N6-substituted ATP analogs as phosphate donors. Thus, only substrates of the as-mutant kinase are labeled by the ATP analogs. Another recent advance in this approach was the development of an affinity tagging technique where the ATP-analog labeled proteins can be recognized by specific antibodies. In this approach following labeling of substrates with N6-(benzyl)-ATP-¿S, the thio-phosphate group on the polypeptides is alkylated by para-nitrobenzylmesylate (open full item for complete abstract)

Committee: Srinivasan Vijayaraghavan PHD (Committee Chair); Douglas Kline PHD (Committee Member); Gary Koski PHD (Committee Member); Sanjaya Abeysirigunawardena PHD (Committee Member); Aaron Jasnow PHD (Committee Member) Subjects: Biochemistry; Biology; Cellular Biology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2018, Chemistry

Sea spray aerosols (SSA) are known to influence the climate directly by affecting the absorption and scattering of solar radiation and, indirectly, by acting as cloud condensation nuclei and ice nuclei. The chemical composition of SSA is driven by organic species and ions present in the sea surface microlayer (SSML), the organic film that coats the ocean surface. These organics are taken up into SSA and affect aerosol climate properties such as hygroscopicity and albedo. In addition, metal ions have been shown to alter the surface organization, speciation, and solubility of organic surfactants. Thus understanding the surface behavior of organics in SSA in response to factors including but not limited to surface area, pH and interactions with cations is necessary to fully realize the impact of SSA on climate. Langmuir monolayers of dipalmitoylphosphatidic acid (DPPA), a simple surfactant phospholipid, are utilized as proxies for studying the organic coating at the ocean surface and on marine aerosols. There are two themes of study in this work. The first theme addresses selectivity of metal cations binding to organics at the ocean surface resulting in enrichment in the sea surface microlayer (SSML) and sea spray aerosol (SSA), and their impact on surface properties upon binding. The second theme addresses the phenomena that speciation of organics is different at interfaces such as the SSML and surface of SSA. The speciation of organic surfactants on SSA is considered to be an important factor controlling the interfacial and climate properties of SSA. However, correctly predicting the surface speciation requires the determination of the surface dissociation constants (surface-pKa) of the protic functional group(s) present. Phase behavior, stability, and surface morphology of DPPA films with and without metal cations were studied using surface pressure compression isotherms and imaged with Brewster angle Microscopy (BAM). The surface speciation of DPPA was studied (open full item for complete abstract)

Committee: Heather Allen (Advisor); Terry Gustafson (Committee Member); Abraham Badu-Tawiah (Committee Member) Subjects: Cellular Biology; Chemical Oceanography; Chemistry

Doctor of Philosophy, The Ohio State University, 2018, Chemistry

Upon wind action on the ocean surface, sea spray aerosols (SSA) are generated and released to the atmosphere. SSA are enriched in the organics and ions present in the sea surface microlayer (SSML) due to the selective transfer of these species to the aerosol phase, and can impact the climate through multiple mechanisms. Aerosols and clouds contribute the largest uncertainty in the prediction of climate change, so an understanding of SSA chemistry is of importance. The organic coatings identified on SSA contain a diverse array of surface-active molecules such as fatty acids. These coatings impact the reactivity, reflectivity, lifetime, and nucleating abilities of the aerosol particles. As these organic films impact the climate-relevant properties of SSA, an understanding of the fundamental physical chemistry phenomena of lipids and ions at these interfaces warrants investigation. In this dissertation, surface sensitive techniques were utilized to probe the structure and properties of fatty acid model systems under various pH and ion conditions. In the first part of this study, the surface-pKa values of medium-chain (C8-C10) fatty acids were quantified through the use of surface tension titration. Our simple surface tension titration technique quantified the surface-pKa of medium-chain octanoic (C8), nonanoic (C9), and decanoic (C10) fatty acids as 4.9, 5.8, and 6.4, respectively. The surface-pKa determined with surface tension differs from the bulk value obtained during a standard acid-base titration, and differences between surface- and bulk-pKa are observed starting at chain lengths of nine carbon atoms. In the titration curves of the C8 and C9 acids, surface tension minima are observed near the surface-pKa as a result of the formation of highly surface active acid-soap complexes. The direction of the titration was shown to affect the measured surface-pKa of the C9 system due to differences in Na+ concentration in the solution at pH values near the pKa. Palmi (open full item for complete abstract)

Committee: Heather Allen (Advisor) Subjects: Chemistry

Master of Science (MS), Wright State University, 2016, Chemistry

Pyrimidines and related heterocycles are an important class of compounds with a wide variety of applications. As a result, there is great interest in the chemical properties of these compounds, specifically their pKa's. Despite the importance of these compounds their reported pKa's are often only approximations or are completely absent from literature. Experimentally measuring pKa's can be challenging, especially if the pKa is below zero. Alternatively, pKa's can be estimated computationally using a Quantitative Structure-Activity Relationship (QSAR). However, most of the compounds included in this study exist as two or more forms that arise from tautomerism. Tautomerism complicates the estimation of pKa's because proton transfers result in a rearrangement of electron density that alters chemical behavior. As a result, pKa's are unique to each tautomeric species. This research identifies the most stable tautomer associated with each of the compounds included in this study. From here QSAR models have been constructed using a variety of molecular descriptors. The resulting QSAR models are then used to estimate the pKa's of compounds for which there are currently no literature values.

Committee: Paul Seybold Ph.D. (Advisor); Eric Fossum Ph.D. (Committee Member); David Dolson Ph.D. (Committee Member) Subjects: Physical Chemistry

MS, Kent State University, 2014, College of Arts and Sciences / Department of Biological Sciences

PPP1CC1 and PPP1CC2 are alternatively spliced transcripts of PPP1CC gene. They are mostly identical, with difference only at their extreme C-termini. PPP1CC1 is ubiquitously expressed in somatic cells, while PPP1CC2 is germ cell specific. Complete deletion of PPP1CC leads to sterility due to impaired sperm morphogenesis. It has been previously documented, that fertility can be restored by optimum expression of PPP1CC2, by incorporating PPP1CC2 transgene on a PPP1CC null (-/-) background. It has also been seen that higher activity levels of PP1, renders sperm immotile but low activity levels initiate motility, as seen in caudal sperms (23). So, we wanted to determine if overexpressing PPP1CC2 in wild type (+/+) testis affected¿spermatogenesis¿ and sperm function. Mouse line was generated, having two copies of PPP1CC2 transgene on a wild type background, and was compared to wild type controls. It was found that PPP1CC2 protein as well as activity levels were highly regulated both in testis and sperm, and remained unchanged and comparable to that of wild type. Sperm were phenotypically indistinguishable from their wild type controls in terms of sperm morphology, motility and number. PPP1CC2 mRNA levels however showed a doubling in their levels in comparison to the wild type controls. Meanwhile, PPP1CC2 protein levels were found out to be drastically reduced in the mouse line, having one copy of PPP1CC1 transgene on a wild type background. There was no evidence of Protein Kinase-A mediated serine-threonine phosphorylation in these mice. Mice having one copy of PPP1CC1 transgene on a wild type background was found to be sub-fertile. Our studies suggest, perhaps, the high level of regulation of PPP1CC2 and maintenance of optimal levels of PPP1CC2 expression is crucial for maintaining normal spermatogenesis and sperm function.

Committee: Srinivasan Vijayaraghavan Dr (Advisor); Wen-Hai Chou Dr (Committee Member); Douglas Kline Dr (Committee Member) Subjects: Cellular Biology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2014, Molecular, Cellular and Developmental Biology

The establishment and maintenance of the osteoblastic phenotype requires a complex network of transcription factors as well as signaling pathways. Some bone tumors seem to arise from genetic lesions that block differentiation of normal bone-forming osteoblasts. Patients with Carney Complex (CNC) are at risk for the development of osteochondromyxomas, caused by an immature expansion of osteoblasts precursors. Similarly, patients with McCune-Albright syndrome (MAS) develop fibrous dysplasia, a histologically similar bone lesion. Interestingly, it is known that uncontrolled cAMP-dependent protein kinase (PKA) signaling is involved in both MAS and CNC. In addition to this genetic evidence, proper regulation of PKA signaling has been demonstrated functionally to be essential for bone mineralization both in vitro and in vivo, although different effects have been observed. The work presented in this dissertation is focused on the involvement of PKA signaling itself as well as its crosstalk with the Wnt pathway in osteoblast biology. PRKAR1A is the gene encoding the type 1A regulatory subunit of PKA and it is the cause of CNC. Data from our lab has demonstrated that Prkar1a loss causes tumors in multiple cell lineages, including neural crest cells and osteoblasts. We have proposed that one mechanism by which tumorigenesis occurs is through failure of terminal differentiation. In chapter two we directly test the effects of Prkar1a reduction on osteogenic differentiation in mouse and human cells in vitro. We found that Prkar1a levels noticeably increased during osteoblastic differentiation, indicating a positive correlation between the expression of Prkar1a and osteogenic potential. To validate this hypothesis, we generated stable Prkar1a knockdown in both mouse and human cells. These cells displayed significantly suppressed bone nodule formation and decreased expression of osteoblast markers such as osteocalcin and osteopontin. Further, because Runx2 is a key mediator of (open full item for complete abstract)

Committee: Lawrence Kirschner (Advisor); Denis Guttridge (Committee Member); Virginia Sanders (Committee Member); Qianben Wang (Committee Member) Subjects: Cellular Biology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2013, Integrated Biomedical Science Graduate Program

The goal of this dissertation was to determine the mechanism by which beta-2 adrenergic receptor (ß2AR) stimulation on a CD40L/IL-4-primed B cell regulates the level of IgE produced. IgE is the mediator of allergic asthma, an inflammatory condition treated with ß2AR agonists to relieve bronchoconstriction. However, our laboratory and others reported previously that ß2AR stimulation in vivo or in vitro by norepinephrine or an agonist drug increase the amount of IgE produced by a B cell on a per cell basis, without affecting class switch recombination, potentially worsening allergic asthma symptoms due to IgE in the long term. The proximal mechanism responsible for the increase in IgE involves a ß2AR-dependent activation of the cAMP/PKA/HePTP/p38 MAPK signaling pathways. The distal mechanism involves an increase in the generation of soluble CD23 (sCD23), a positive mediator of IgE production, which results from the cleavage of membrane-bound CD23 by A Disintegrin And Metalloprotease 10 (ADAM10). What remained unknown was the mechanism responsible for the ß2AR-mediated increase in sCD23, and if sCD23 was the ultimate mediator of the ß2AR-mediated increase in IgE. Recently, CD23 and ADAM10 were described on B cell-derived vesicles known as exosomes, suggesting a novel pathway by which ß2AR stimulation could regulate CD23 cleavage. Thus, the hypothesis tested in this dissertation is that ß2AR stimulation on a B cell enhances IgE production by increasing CD23 and ADAM10 expression on B cell-derived exosomes that play a role in mediating the ß2AR-associated increase in IgE production. The present data using an in vitro cell culture system are the first to show that ß2AR stimulation on a CD40L/IL-4-primed B cell enhanced the expression of CD23 and ADAM10 in the B cell in a PKA- and p38 MAPK-dependent manner. Using ß2AR-deficient mice and pharmacological agents, we showed that the ß2AR-dependent enhancement of CD23 and ADAM10 expression was detectable on exosomes, rather t (open full item for complete abstract)

Committee: Virginia M Sanders Ph.D. (Advisor); Ian Davis DVM Ph.D. (Committee Member); Susheela Tridandapani Ph.D. (Committee Member); Joanne Turner Ph.D. (Committee Member) Subjects: Biology; Immunology; Neurobiology; Pharmacology

Doctor of Philosophy, The Ohio State University, 0, Molecular, Cellular and Developmental Biology

Thyroid cancer is the most commone endocrine malignancy in the population and incidence rates continue to rise. The two most common types of thyroid cancer, papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC) are generally treatable with good prognoses. However, in a small subset of patients, progression to invasive and distantly metastatic disease occurs. Once progression occurs, treatment options are limited and many patients ultimately die of metastatic disease. Study of the genetics of sporadic human PTCs and FTCs has found that the Ras/Raf/Mek/Erk signaling pathway is a key reglator of PTC growth, and mouse models harboring mutations in this pathway in the thyroid generally develop PTC in good analogy to the human disease. While some mutations have been identiifed in sporadic FTC, a central pathway has not been identified, and mouse models of many of the mutations found in humans fail to produce FTC when modeled in the mouse. In addition to studying the genetics of patients with sporadic FTC in order to identify important signaling pathways and druggable targets, another source of information about the genetic etiology of this disease is the study of inherited syndromes with FTC in the clinical spectra of disease. The work described here describes two new mouse models of FTC with genetic alterations that mimic those in both inherited and sporadic FTC cases. These mouse models point to dysregulation of Protein Kinase A (PKA) as a key signaling node in FTC and that the PKA pathway can synergize with the PI3K/Akt pathway, also implicated in sporadic and inherited FTC, to foment disease progression. Thus, these models, in agreement with previous data, further support the hypothesis that at least two genetic alterations are necessary for FTC to progress to aggressive and distantly metastatic disease. This work also identifies strong activation of PKA in a set of sporadic human FTC, confirming the relevance of these mouse models to human d (open full item for complete abstract)

Committee: Lawrence Kirschner (Advisor); Sissy Jhiang (Committee Member); Krista La Perle (Committee Member) Subjects: Genetics; Molecular Biology; Oncology

Master of Science (MS), Wright State University, 2009, Chemistry

Quantum chemical analysis was used to examine nucleophilic aromatic substitution reactions of fluorinated benzophenones, diphenyl sulfones, and triphenylphosphine oxides. Some experimental results for these compounds were contrary to conventional wisdom, which holds that calculated atomic charges for the aromatic sites and 13C-NMR and 19F-NMR chemical shifts should allow prediction of the preferred sites for aromatic substitution. Density functional theory (B3LYP/6-31+G*//RM1) and semi-empirical (RM1) quantum chemical calculations were employed to study the intermediates in the reaction pathways in order to identify the preferred paths for aromatic substitution. In most cases studied para substitution pathways had the lower energy intermediates and were favored. Experimental acid dissociation pKa's for a set of 41 aliphatic carboxylic acids were compared with quantum chemical indices for the compounds in an attempt to find correlations that might help explain how the electronic structures of the compounds influence their tendencies to dissociate. The quantum chemical indices included the charges on atoms and groups of atoms, calculated vibrational frequencies, calculated nuclear magnetic resonance (NMR) chemical shifts, and reaction energy differences both in vacuum and in an aqueous phase solvent model. Several of these calculated quantities yielded respectable correlations, with the vibrational frequency of the carboxylic acid proton (R2 = 0.874) and the vibrational frequency of the carbonyl stretch of the carboxylate anion (R2 = 0.852) giving the best results. As was observed in earlier work, the RM1 semi-empirical calculations yielded better correlations than the more sophisticated density functional theory approach.

Committee: Paul Seybold PhD (Advisor); David Dolson PhD (Committee Chair); Eric Fossum PhD (Committee Member) Subjects: Chemistry

PhD, University of Cincinnati, 2005, Medicine : Molecular Genetics, Biochemistry, and Microbiology

Checkpoint proteins block progression through the cell cycle in order to prevent cell division when a cell has damaged or incompletely replicated DNA. This dissertation will focus on the DNA damage checkpoint signal transduction pathways which prevent mitotic progression following DNA damage. Mutations in the genes that encode for checkpoint proteins can lead to genomic instability, uncontrolled cell growth and cancer. Checkpoint proteins are being considered as drug targets for treatment of cancers, based on the hypothesis that the inactivation of a checkpoint protein enhances the rate at which cells fail to stop division with damaged DNA, which leads to accumulation of damaged DNA, and increases the probability of death for the cancer cell. Therefore, identifying novel checkpoint proteins could lead to the identification of new drug targets. The checkpoint pathways are conserved in the genetically amenable Saccharomyces cerevisiae, making it a good model system in which to identify novel proteins involved in regulating mitosis. The conserved checkpoint kinases Chk1 and Rad53 prevent mitotic progression by blocking the degradation of the securin, Pds1, thus preventing separation of the sister chromatids, and by inhibiting activation of the mitotic exit network (MEN), respectively. Chk1 and Rad53 play supporting roles in preventing mitosis following DNA damage. Although Chk1 prevents the degradation of Pds1 following DNA damage, Pds1 is degraded faster in cells containing a mutation in an upstream checkpoint kinase than in a chk1 mutant, suggesting that another pathway can regulate the destruction of Pds1. We show that the cAMP dependent protein kinase (PKA) pathway has a supporting role to Chk1 in preventing the degradation of the mitotic inhibitors Pds1 and Clb2 via regulation of the mitotic inducer Cdc20. We also found that proteins that regulated the levels of cAMP, and the checkpoint dependent phosphorylation of the PKA regulatory subunit were required to suppo (open full item for complete abstract)

Committee: Yolanda Sanchez (Advisor) Subjects:

PhD, University of Cincinnati, 2001, Medicine : Pathobiology and Molecular Medicine

Cyclic AMP signaling has been shown to be essential for growth, morphology and virulence in fungal pathogens of plants and animals. However, the function of this pathway is unknown in the opportunistic pathogen, Aspergillus fumigatus. As a first step toward elucidating the function of this pathway in A. fumigatus the genes encoding both the regulatory and catalytic subunits were cloned and sequenced and the response to exogenous cAMP was characterized. The availability of pkaR and pkaC sequences was used to construct vectors, facilitating future studies into the function of these genes. The regulatory subunit gene, pkaR, is expressed from an intronless coding sequence and the predicted translation product shares 79% sequence identity with the regulatory subunit of Aspergillus nidulans, both of which are defined as a type II regulatory subunits by the presence of a conserved autoinhibition site and two cAMP-binding domains. The gene encoding the A. fumigatus catalytic subunit, pkaC, is interrupted by three introns. The predicted PkaC protein shares 83% sequence identify with A. nidulans PkaC, and contains domains that are characteristic of PkaC proteins. Both pkaR and pkaC mRNAs are expressed throughout the asexual lifecycle of A. fumigatus. In regard to the effects of exogenous cAMP, both cAMP and phosphodiesterase inhibitors markedly reduced radial growth rate of A. niger, which has previously been demonstrated to respond exogenous cAMP, after 48 hours on minimal media with glucose as the carbon source, whereas growth of A. fumigatus was not affected. However, when glycerol, which does not initiate carbon catabolite repression, was used as a carbon source, cAMP inhibited the radial growth rate of only A. fumigatus (p<0.05). The addition of cAMP to glycerol-minimal medium resulted in a two-fold increase in protein kinase A activity in A. fumigatus cell extracts when compared with negative controls. The PKA activity in A. fumigatus cell extracts from cultures grown i (open full item for complete abstract)

Committee: Dr. Judith Rhodes (Advisor) Subjects: Biology, Microbiology

Doctor of Philosophy, The Ohio State University, 2010, Molecular, Cellular and Developmental Biology

Eukaryotic cells integrate information from multiple sources in order to respond appropriately to changes in the environment. Our work is focused on a signaling pathway that is essential for the coordination of cell growth with nutrient availability, namely the Ras/cAMP-dependent protein kinase (PKA) pathway. This pathway has been proposed to respond to carbon levels in the environment and impacts diverse processes in the cell including, stress response, translation, autophagy and carbohydrate metabolism. To better understand how PKA signaling modulates growth in response to changes in the environment, we examined three different aspects of PKA biology: coordination of PKA signaling with Tor signaling, regulation of P-body formation by PKA and substrate recognition by PKA. In the study described in Chapter 2, we examined the relationship between Ras/PKA and Tor signaling pathways in Saccharomyces cerevisiae. Although these pathways control a similar set of processes important for growth, it was not clear how their activities were integrated in vivo. The experiments here examined this coordination and found that the inhibition of TORC1 was generally associated with elevated levels of PKA activity. Similarly, TORC1 activity appeared to increase in response to lower levels of PKA signaling. In all, these data suggested that the PKA and TORC1 pathways were functioning in parallel to promote cell growth but that each pathway might restrain, either directly or indirectly, the activity of the other. In addition to post-translational regulation, cells respond to changing nutrient levels by modulating mRNA turnover in the cell, to regulate gene expression as well as translation. In the recent years, the mRNP aggregates known as P-bodies have emerged as a major site and player in mRNA decapping and decay. Very little is known about the regulation of P-body formation in response to nutrient deprivation. To this end, our study in Chapter 3 describes a novel aspect of P-body bi (open full item for complete abstract)

Committee: Dr. Paul K. Herman (Committee Chair); Anita K. Hopper (Other); Stephen A. Osmani (Other); Mark R. Parthun (Other) Subjects: Molecular Biology

Doctor of Philosophy, The Ohio State University, 2009, Molecular, Cellular, and Developmental Biology

Proper regulation of cAMP-dependent protein kinase (PKA) is essential to normal cell proliferation, but PKA dysregulation leads to tumor formation in a range of endocrine tissues. Resulting from mutations in the main PKA regulatory subunit, PRKAR1A, Carney complex is characterized by tumors of various endocrine glands, as well as bone and cartilage tumors, Schwann cell tumors, and skin discoloration. Since PKA dysregulation affects several neural crest-derived tissues, we sought to examine the role of PKA in the neural crest.The cranial neural crest (CNC) undergoes complex molecular and morphological changes during embryogenesis in order to form the vertebrate skull, and nearly 75% of all birth defects result from defective craniofacial development. The molecular events leading to CNC differentiation have been extensively studied; however the role of PKA during craniofacial development has only been described in palate formation. Selective inactivation of Prkar1a in the CNC results in perinatal lethality caused by dysmorphic craniofacial bone and cartilage development and subsequent asphyxiation. Aberrant differentiation of Prkar1a-null CNC mesenchymal cells resulted in anomalous intramembranous ossification and cartilage dysplasia. These observations provide new evidence for the importance of PKA regulation in craniofacial development, which may be beneficial to understanding and treating craniofacial birth defects. In addition to the facial structures, proper PKA regulation is required in Schwann cells. Tissue-specific ablation of Prkar1a in a subset of neural crest precursor cells caused schwannomas with high frequency. Previously, signaling events leading to Schwann cell tumor initiation have been characterized in the context of Neurofibromatosis (NF). At the molecular level, Prkar1a-null tumors revealed loss of both NF proteins, despite the fact that transcript levels were increased, implying post-transcriptional regulation. Furthermore, the small G-proteins Ra (open full item for complete abstract)

Committee: Lawrence Kirschner MD, PhD (Advisor); Sissy Jhiang PhD (Committee Member); Gustavo Leone PhD (Committee Member); Denis Guttridge PhD (Committee Member); Matthew Ringel MD (Committee Member) Subjects: Biology; Cellular Biology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2005, Molecular Genetics

Eukaryotic cells utilize a network of signal transduction pathways to sense their environment and control their growth and proliferation. Protein kinases are a large group of enzymes that coordinate responses to extracellular and intracellular stimuli via phosphorylation of specific downstream targets. In S. cerevisiae , growth is controlled, in part, by the Ras signaling pathway via the cAMP-dependent protein kinase, PKA. PKA is a serine/threonine-specific protein kinase that has been shown to regulate any aspects of cell growth and metabolism in this budding yeast and other eukaryotes. Unfortunately, finding protein kinase substrates by conventional methods is a difficult and time-consuming task. As a result, few targets of any given protein kinase are known. To simplify this task, we developed an evolutionary proteomics strategy for the identification of PKA substrates in S. cerevisiae and related yeast species. This evolutionary proteomics approach is sequenced-based and takes advantage of the fact that most PKA substrates contain the consensus sequence, R-R-x-S/T-B. In this consensus, “x” refers to any amino acid, “B” to hydrophobic residues and “S” or “T” to the site of phosphorylation. The general approach consists of two basic steps. In the first, we identified all of the proteins in the S. cerevisiae proteome that contain this PKA target consensus sequence. In the second, we asked whether these potential target sites are conserved in the orthologous proteins present in other budding yeast species. For this latter step, we used the recently released genome sequences of six different yeast, including five Saccharomyces species and Candida albicans. The underlying premise of this approach is that PKA sites important for general aspects of cell biology are more likely to be conserved across these evolutionary distances. We are presently testing this basic premise with a small number of proteins predicted to be physiologically relevant PKA substrates. In this th (open full item for complete abstract)

Committee: Paul Herman (Advisor) Subjects: Biology, Molecular

Doctor of Philosophy, The Ohio State University, 2003, Biochemistry

The Cys-34 thiol of serum albumin has been suggested to be a carrier and stabilizer of nitric oxide in plasma. The pKa of the Cys-34 thiol of serum albumins from human, bovine, equine, and canine species were determined kinetically and spectroscopically by following the absorbance of the thiolate anion. All serum albumins gave pKa's of ~8.5 for their Cys-34 thiols. The reactions between serum albumin and its model compounds, glutathione and N-acetyltryptiophan, with fast acting vasodilating drugs, sodium nitroprusside and isoamylnitrite, and with sodium nitrite were studied. It was found that the reactions with serum albumin are too slow to account for the immediate physiological effects of these compounds; serum albumin probably participate in their hypotensive effects indirectly through nitrosation by S-nitrosothiols formed in plasma. The rate of oxidation and reduction reactions for Cys-34 reacting with various disulfides and thiols commonly found in plasma were measured. The thiol content of bovine and human serum albumin (BSA and HSA) obtained from various sources, and HSA from freshly isolated plasma were also measured. The thiol contents of commercial preparations of HSA were only a fraction compared to those of HSA in fresh plasma and of BSA. The rates of Michael addition reactions between acrylamide, a neurotoxin recently discovered to be present in many common foods, and GSH and Cys-34 of HSA, were measured under physiological conditions. The half-life of acrylamide in blood was calculated to be < 7 hours. The mechanism of fatty acid binding as well as the influence of that on the reactivity of Cys-34 was examined. The cooperative binding of 4 to 5 molecules of fatty acids to each serum albumin was concluded from results of kinetic and mass analyses. It was also found that Cys-34 became more reactive upon fatty acid binding. This is presumed to be due to a conformational change that causes the enlargment of the Cys-34 cavity.

Committee: Gary Means (Advisor) Subjects:

PHD, Kent State University, 2010, College of Arts and Sciences / Department of Chemistry

The transcription factor, liver receptor homolog-1 (LRH-1/NR5A2), has roles in several developmental and steroidogenic processes; as well as being important for the expression of the liver enzyme, cholesterol 7α-hydroxylase. hLRH-1 can interact with different cofactors of transcription, such as steroid receptor coactivator-1 (SRC1), prospero-related homeobox (ProxI), silencing mediator of retinoic acid and thyroid hormone receptors (SMRT), small heterodimer partner (SHP), also might interact with hepatocyte nuclear factor 4α (HNF4α) and chicken ovalbumin upstream promoter transcription factor II (COUP –TFII). To test if this nuclear hormone receptor could serve as a substrate for protein kinases, recombinant LRH-1 was reacted with commercially available protein kinases and [32P]ATP. Full-length and segments of LRH-1 were found to be robustly labeled in these in vitro assays. Active PKA and JNK1 could differentially regulate the interaction of the LRH-1 ligand binding domain (LBD) with the cofactors, SRC-1 and COUP-TFII in Mammalian Two-Hybrid assays conducted in the human hepatoma cell line, HepG2. The amino acid residues identified as involved in this effect were mapped on computer models based on published protein crystal structures. The amino acid residues phosphorylated by PKA, T341 and S510 were found to flank ligand binding cleft of hLRH-1 LBD. Phosphorylation of LRH-1 is a potential mechanism for rapid modulation of the activity of this transcription factor by differentially modulating interactions with cofactors.

Committee: Diane Stroup (Advisor); Arne Gericke (Committee Member); Srinivasan Vijayaraghavan (Committee Member); John Chiang (Committee Co-Chair); Chun-che Tsai (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, Case Western Reserve University, 2009, Biochemistry

The general acid/base mechanism proposed for HDV catalysis involves a cytosine, C75 (genomic) / C76 (antigenomic), in proton transfer. Biochemical studies suggest that C75/C76 has a pKa perturbed to near neutrality. In this thesis, the first objective is to directly measure the pKa of C75 in a genomic HDV ribozyme using Raman crystallography. The results presented here reveal pKa values for C75 that reflect anticooperative thermodynamic coupling with Mg2+ binding, with values of 6.15 and 6.40 in the presence of 20 and 2 mM Mg2+, respectively. These studies provide the first direct physical measurement of a pKa near neutrality for a catalytic residue in a ribozyme. The second objective is to investigate metal-RNA interactions in crystals of HDV ribozyme. Metal cation binding lies at the heart of much of RNA chemistry and is crucial for RNA folding and ribozyme catalysis. Here, we show Raman crystallography and Raman difference spectroscopy provide a unique means for probing metal-RNA interactions in HDV. The present studies focus on: (1) detecting the inner-sphere interactions between magnesium hydrate and the phosphate backbone of HDV by identifying and analyzing the vibrational signatures of inner-sphere coordinated magnesium hydrate (~322 cm-1) and the PO2- symmetric stretch (~ 1100 cm-1) perturbed by the formation of a PO2- —Mg2+ (H2O)x (x≤5) inner-sphere complex. About 5 inner-sphere Mg2+—-O2P contacts per HDV molecule are observed in the presence of 20 mM magnesium; (2) characterizing Mg2+—HDV and Co(NH3)63+—HDV interactions. Our results reveal that Mg2+ and Co(NH3)63+ binding induce modest changes in HDV molecular conformation and Co(NH3)63+ is able to displace some inner-sphere coordinated Mg2+ ions in HDV. Finally, using Raman technique combined with ribozyme kinetic studies (the latter contributions are from the research group of Dr. B.Golden (Purdue University)), we observe an inner-sphere interaction of the catalytic Mg2+ ion with the N7 of guanine in t (open full item for complete abstract)

Committee: Paul Carey (Advisor); Vernon Anderson (Committee Chair); Michael Harris (Committee Member); Focco van den Akker (Committee Member); Philip Bevilacqua (Committee Member) Subjects: Biochemistry