ETD Search Results: instcode:osu

Skip navigation

ETD Center Search

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

[1] [2]

1. Boff, Jacqueline Christine. An Early-Life Infection with Escherichia coli in BALB/c Mice causes Long-Lasting Deficits in Behavior, Brain Development, and Microglial Reactivity.

Degree: PhD, Biochemistry Program, Ohio State, 2012, Ohio State University

► There is mounting evidence that inflammatory insults that occur early in life… (more)

▼ There is mounting evidence that inflammatory insults that occur early in life can significantly influence normal brain development and promote neurobehavioral deficits that manifest in young adulthood. The biochemical mechanisms underlying these complications, however, are poorly understood. We developed a model of early-life infection in BALB/c mice using a peripheral injection of Escherichia coli in neonatal mice. This transient infection had a profound influence on behavioral phenotype, brain development, and response to a secondary immune challenge later in young adulthood. For instance, early-life infected mice had impaired motor coordination and hyperactivity that was evident 1 and 2 months after the neonatal infection. In addition, these behavioral deficits were associated with hypomyelination in the subcortical white matter, cortex, and hippocampus. Hypomyelination was concomitant with a reduced number of oligodendrocytes in the subcortical white matter and a robust increase in L-ferritin in the brain of early-life infected mice that localized to the cytoplasm of neurons. Early-life infection also promoted microglial priming resulting in hyperactivation of these cells following a secondary immune challenge. Exaggerated inflammatory cytokine production corresponded to increased Iba1 immunoreactivity of microglia specifically in the amygdala and hippocampus of ELI mice 48 hours following LPS. Taken together, these data indicate that a peripheral infection during neonatal brain development promotes abnormal iron storage in neurons, reduces oligodendrocytes, markedly impairs myelination, and primes microglia. These biochemical changes corresponded to long lasting deficits in behavior and motor coordination. Understanding the biochemical and immune complications that result from an early-life insult will provide new therapeutic targets to attenuate neurobehavioral complications, such as attention deficit disorder, autism, and schizophrenia. Advisors/Committee Members: Godbout, Jonathan.

Subjects: Behaviorial Sciences; Cellular Biology; Immunology; Neurosciences

Keywords: neonatal infection; microglia; oligodendrocyte; myelination; L-Ferritin; BALB/c

2. Brown, Jessica Ann. Kinetic Mechanisms of DNA Polymerases.

Degree: PhD, Biochemistry Program, Ohio State, 2010, Ohio State University

► High-fidelity DNA polymerases accurately replicate an organism’s genomic DNA while low-fidelity DNA… (more)

▼ High-fidelity DNA polymerases accurately replicate an organism’s genomic DNA while low-fidelity DNA polymerases are specialized to function in DNA repair and DNA lesion bypass, two processes that are necessary to overcome the DNA damage induced by endogenous and exogenous sources. Therefore, understanding the molecular basis of polymerase nucleotide selectivity and fidelity is an important objective towards ascertaining the overall stability of an organism’s genome. Transient state kinetic techniques were used to elucidate the mechanisms of DNA polymerization catalyzed by high- and low-fidelity enzymes. Here, we established that the fidelity of Sulfolobus solfataricus DNA polymerase B1 synthesizing undamaged DNA to be in the range of 10-6 to 10-8 or one error per 1,000,000 to 100,000,000 nucleotide incorporations. PolB1 used an induced-fit mechanism to incorporate a correct nucleotide with a tight nucleotide binding affinity and fast rate of incorporation. In contrast, Saccharomyces cerevisiae DNA polymerase η and human Rev1, two enzymes that function in DNA lesion bypass, synthesized undamaged DNA with a fidelity of 10-2 to 10-4 and 100 to 10-5, respectively. The extremely low fidelity of hRev1 was due to the preferred misincorporation of dCTP with templating bases dA, dT, and dC. Human DNA polymerase λ (Pol λ), a low-fidelity enzyme involved in gap-filling DNA synthesis during DNA repair, utilizes unique mechanisms to select nucleotides and was shown to be potentially mutagenic in different situations. Pol λ prefered to insert deoxyribonucleotides over ribonucleotides by 3,000- to 50,000-fold due to a steric clash between the ribose 2′-hydroxyl group of a ribonucleotide and a backbone carbonyl group of Y505 in Pol λ’s active site. In addition, the unprecedented tight nucleotide binding affinity of both correct and incorrect nucleotides to the Pol λ•DNA complex was manifested in cooperative interactions with multiple active site residues. Furthermore, the fidelity of Pol λ was governed mostly by R517, a residue that interacts with the minor groove of the DNA template. During long gap-filling DNA synthesis, the fidelity of Pol λ dropped two orders of magnitude, and this downregulation of fidelity was controlled by Pol λ’s non-enzymatic N-terminal domains. Pol λ was error-prone when it encountered an 8-oxo-7,8-dihydro-2′-deoxyguanosine lesion in the DNA template, as dCTP and dATP incorporation proceeded with essentially equal efficiency and probability. A comprehensive mechanism for the bypass of cis-[Pt(NH3)2{d(GpG)-N7(1),-N7(2)}] intrastrand cross-links was established for Sulfolobus solfataricus DNA polymerase IV (Dpo4), an enzyme involved in DNA lesion bypass. Dpo4 was able to bypass this double-base lesion, although, the incorporation efficiency of dCTP opposite the first and second cross-linked guanine bases was reduced by 72- and 860-fold, respectively. Moreover, the fidelity of Dpo4 at the lesion decreased up to two orders of magnitude. Lastly, antiviral nucleotide analogs were determined to be substrates for six human DNA polymerases (Pols β, λ, η, ι, κ, and Rev1) involved in DNA repair and lesion bypass. The kinetic results suggested that nucleotide analog incorporation catalyzed by these six human enzymes may represent a potential mechanism of drug toxicity and also established a structure-function relationship for designing improved analogs. Advisors/Committee Members: Suo, Zucai.

Subjects: Biochemistry

Keywords: DNA polymerase, transient state kinetics

3. Das, Mom. Mechanistic Studies of Class II Bacterial Prolyl-tRNA Synthetase and YbaK Editing.

Degree: PhD, Biochemistry Program, Ohio State, 2012, Ohio State University

► Aminoacyl-tRNA synthetases (aaRSs) play an important role in translation by accurately pairing… (more)

▼ Aminoacyl-tRNA synthetases (aaRSs) play an important role in translation by accurately pairing amino acids with their cognate tRNAs, thereby providing the ribosome with building blocks for protein synthesis. To minimize errors in translation, synthetases achieve the amino acid substrate specificity either by preferentially binding the cognate amino acid and/or by selectively editing the near-cognate amino acids. Defects in aaRS editing can reduce the overall translational fidelity causing growth defects in bacteria, apoptosis and neurodegeneration in mammalian cells. In this work we have studied the roles of the editing domain of Escherichia coli prolyl-tRNA synthetase (ProRS) and its freestanding paralog, YbaK, in maintaining translation accuracy. The synthetic active site of E. coli ProRS acts as a coarse sieve by misactivating noncognate amino acids Ala and Cys along with cognate Pro. The cis-editing domain of bacterial ProRS (INS) is a fine sieve and can only edit smaller Ala-tRNAPro. The Cys paradox is solved by YbaK, which specifically deacylates Cys-tRNAPro in trans, employing a novel "triple-sieve" editing mechanism in bacteria. Using biochemical and computational approaches, we have probed the substrate specificities and mechanisms of these homologous editing domains. Our studies suggest that INS catalyzes hydrolysis of Ala-tRNA via a steric exclusion-based sieve, which is hydrophobic in nature and whose specificity can be re-engineered by mutagenesis implying that a general water-catalyzed mechanism is employed by INS. In contrast, substrate specificity of YbaK is not readily modulated as it employs a substrate-assisted sulfhydryl side chain chemistry, representing a novel chemical sieve. Our results support a critical role for the tRNA 2'-OH group in substrate binding and catalytic water activation and for the protein's main chain atoms of Gly261 in a proton relay that contributes substantially to catalysis in the INS domain. Further computational calculations have proposed a similar role for the backbone carbonyl of Phe29 in E. coli YbaK. YbaK has been shown to be a general Cys-tRNA deacylase in vitro. We have demonstrated that substrate specificity of YbaK is ensured, in part, through formation of a novel trans-editing ProRS/YbaK/tRNAPro ternary complex. The interactions of this complex have been probed in vivo and in vitro using tandem affinity purification and mass spectrometry-based protein footprinting, respectively. The latter approach has helped to map the sites on YbaK that likely play a role in interaction with ProRS/tRNAPro. Advisors/Committee Members: Musier-Forsyth, Karin.

Subjects: Biochemistry

Keywords: aminoacyl-tRNA synthetase, amino acid, tRNA, editing, QM/MM

4. Devaraj, Aishwarya. Molecular Studies of the Fidelity of Translation Elongation.

Degree: PhD, Biochemistry Program, Ohio State, 2011, Ohio State University

► Protein synthesis occurs in ribosomes, megadalton RNA-protein machines that use aminoacyl-tRNA (aa-tRNA)… (more)

▼ Protein synthesis occurs in ribosomes, megadalton RNA-protein machines that use aminoacyl-tRNA (aa-tRNA) molecules to translate messenger RNA (mRNA) with high fidelity. During translation elongation, the ribosome orchestrates 3 major events: decoding, peptidyl transfer and translocation. The process of proteins synthesis is also one of the major targets of antibiotics and hence understanding the basics of ribosome function should provide insight for the development of novel drugs. Genomes are maintained and expressed with remarkable fidelity and the accuracy of each process involved represents a compromise that optimizes the evolutionary fitness of the organism. The process of translation elongation is a complex one, and therefore there are potentially many ways the process can go awry. Chapter 1 introduces translation elongation errors and discusses the differences between missense, nonsense and frameshift errors. Mutations in the ribosome and other translation factors that affect the fidelity of translation elongation are also discussed. Chapter 2 is focused on the ribosomal exit (E) site and its role in maintaining the translational reading frame. It has been proposed that a critical role for the E site is in maintenance of translational reading frame, dependent on codon-anticodon pairing (191). Though several studies support the idea that codon-anticodon interaction in the E site contributes to frame maintenance (167), direct in vivo evidence for this hypothesis has been scant. In chapter 2, we investigated this fundamental question and found that the E site helps to maintain the reading frame, but does not contribute to the accuracy of decoding, as has been suggested (chapter 2, 204). We also showed that the mutation of the 30S E site does not inhibit EF-G-catalyzed translocation, in sharp contrast to the effects of mutations in 50S E site. These data provided evidence that the function of the E site in translocation is largely confined to the 50S subunit. One of the earliest identified examples of translational frameshifting occurs in the prfB gene of E. coli, encoding the peptide release factor 2 (RF2). While the genetic studies have identified the determinants of prfB programmed frameshifting and their relative importance, how these determinants act to promote frameshifting has remained unclear. In chapter 3, we compared ribosomal complexes with various spacer lengths between the SD sequence and P codon. We found that a close juxtaposition of the SD–ASD helix and P codon strongly destabilized P-site tRNA but had little or no effect on RF2-dependent termination or EF-Tu-dependent decoding. These data suggested that the intragenic SD of prfB destabilizes pairing of peptidyl-tRNALeu to the zero-frame CUU and promotes directional movement of the mRNA template with respect to the bound tRNA. In chapter 4, we have isolated 16S rRNA mutations that could suppress a +1 frameshift mutation in E. coli. In one of the screens (where the slippery sequence in the frameshift window had a stop codon), 31 independent mutations were identified and mapped to four different positions, of which C1054U was isolated 28 times. The C1054U mutation has also been isolated previously as a nonsense suppressor. Purine substitutions at this position also increased UGA readthrough and miscoding. While the C1054U mutation significantly increased nonsense readthrough and frameshift errors, the mutation had a hyperaccurate phenotype with respect to decoding (i.e., reduced misreading). Other substitutions at this position also had differential effects on the three reporters (missense, nonsense and frameshift). These interesting observations prompted us to characterize these A-site mutations as well as others in 16S rRNA (C1200U, G1491A and G299A) in vitro to get a better understanding of how the ribosome maintains its high fidelity (chapter 5). We investigated the effect of these mutations on RF2 function and found that all of the mutations tested had a defect in RF2-dependent termination. We directly tested the effect of these mutations on decoding by measuring the rate of GTP hydrolysis in both cognate and near-cognate mRNA. We found that all of the mutations tested (C1200U, G1491A, C1054U, C1054A, and G299A) had a substantial defect in initial selection, increasing the rate of GTP hydrolysis particularly on near-cognate mRNA. We also investigated the effect of these mutations on the stability of various tRNAs in the A site. Of the mutations analyzed, C1054U and G1491A seemed to differentially affect tRNA stability, suggesting that these mutations may stimulate GTP hydrolysis in a different way than the others. Advisors/Committee Members: Fredrick, Kurt.

Subjects: Biochemistry

Keywords: Ribosome, E site, Frameshifting, S7

5. Dewan, Varun. Lysyl-tRNA Synthetase-Capsid Interaction in Human Immunodeficiency Virus-1: Implications for the Priming of Reverse Transcription and Therapeutic Development.

Degree: PhD, Biochemistry Program, Ohio State, 2012, Ohio State University

► The primer for reverse transcription in human immunodeficiency virus type 1 (HIV-1),… (more)

▼ The primer for reverse transcription in human immunodeficiency virus type 1 (HIV-1), human tRNALys 3, is selectively packaged into the virion along with tRNALys 1, 2. Human lysyl-tRNA synthetase (LysRS), the only cellular factor known to interact specifically with all three tRNALys isoacceptors, is also selectively packaged into HIV-1. Previous work has defined a tRNALys packaging complex that includes the tRNALys isoacceptors, LysRS, Gag, GagPol, and viral RNA. Numerous studies support the hypothesis that during tRNALys packaging, a Gag/GagPol complex interacts with a tRNALys/LysRS complex, with the capsid (CA) domain of Gag interacting specifically with LysRS, and GagPol interacting with both Gag and tRNALys. In this work, we have identified critical residues along one face of the dimerization helix 7 (H7) of LysRS involved in packaging of LysRS into virions. Mutation of these residues affects binding to Gag in vitro, as well as the oligomerization state and aminoacylation activity of the synthetase. Taken together with previous work, these data support the conclusion that the LysRS H7–CA interaction interface represents a novel antiviral target. With this target in mind, a support-bound cyclic peptide (CP) library containing randomized amino acid sequences and different ring sizes was synthesized and screened against CA and the monomeric form of the CA C-terminal domain (WM-CA CTD). Out of 3 x 105 CPs screened, 21 hits were obtained and 6 CPs were chosen for detailed in vitro analysis. Two CPs, CP2 and CP4 showed strong binding (Kd ~ 500 nM) to both CA and WM-CA CTD in vitro. Scrambled variants of CP2 and CP4 and point mutants at each of the randomized positions eliminated binding, suggesting a sequence-specific mode of interaction. CP2 and CP4 also inhibited LysRS/CA interaction in vitro with an IC50 value of ~ 1 µM. Furthermore, nuclear magnetic resonance (NMR), mutational studies along with in silico analysis revealed that CP2 and CP4 bind to a site proximal to helix 4 (h4) of the CA-CTD, which is the known site of LysRS interaction. Preliminary studies to understand the packaging of host 7SL RNA in HIV-1 have also been carried out. Preliminary binding studies with N-terminally truncated LysRS showed that 7SL RNA bound with high affinity and specificity. The binding affinity obtained was comparable to that of cognate tRNALys, thereby suggesting structural homology between the packaged RNAs. These studies may provide insights into the mechanism/function of 7SL RNA in HIV-1. In summary, HIV-1 has evolved several intricate mechanisms for the packaging of host molecules. Targeting these interactions for anti-viral development may provide an opportunity to block the viral replication. Advisors/Committee Members: Musier-Forsyth, Karin.

Subjects: Biochemistry; Biophysics; Molecular Biology

Keywords: Host-viral interactions, Lysyl-tRNA synthetase, HIV-1 capsid

6. Henry, Christopher John. Role of Primed Microglia in the Aging Brain in Prolonged Sickness and Depressive Behavior Concomitant with Peripheral Immune Stimulation.

Degree: PhD, Biochemistry Program, Ohio State, 2011, Ohio State University

► In the elderly, systemic infection is associated with an increased frequency of… (more)

▼ In the elderly, systemic infection is associated with an increased frequency of behavioral and cognitive complications such as depression and delirium. These complications negatively affect quality of life and are associated with increased morbidity and mortality. An excessive neuroinflammatory response to peripheral infection may underlie the impairments in affect and cognition in the aged and this excessive response may be caused by microglia that have become primed in the course of normal aging to give a dysregulated immune response to peripheral immune activation. In support of this notion, we and others have shown an exaggerated neuroinflammatory response to peripheral immune stimulation in aged mice. Moreover, this exaggerated neuroinflammation is accompanied by prolonged sickness and depressive-like behavior. To clarify if the brain is the origin of the dysregulated immune response that leads to exaggerated neuroinflammation and prolonged sickness behavior in the aged, adult and aged mice were given an intracerebroventricular immune challenge and sickness behavior, inflammatory markers, and markers of glial activation were measured. Central immune challenge elicited a prolonged sickness response in aged mice which was accompanied by protracted expression of inflammatory cytokines in cerebellum and hippocampus. Plasma IL-6 concentrations were also elevated by central immune challenge and the magnitude and duration of these increases were greater in aged mice compared to adults. To determine if aging leads to an imbalance in LPS-induced microglial expression of pro- and anti-inflammatory mediators, microglia were isolated from adult and aged mice after intraperitoneal LPS injection and assayed for mRNA and protein expression of pro-inflammatory IL-1β and anti-inflammatory IL-10. Microglia isolated from aged mice showed higher mRNA and protein induction of both IL-1β and IL-10 compared to adults. Furthermore, primed (MHC II+) microglia from aged mice showed the most prominent induction of IL-1β. To determine if minocycline, a purported microglia inhibitor, could attenuate LPS-induced microglia activation, reduce neuroinflammation, and impact sickness behavior, mice were pretreated with minocycline then given an intraperitoneal injection of LPS. Minocycline pretreatment facilitated recovery from LPS-induced sickness; attenuated LPS-induced microglial expression of TLR2, a marker of microglial activation; and decreased LPS-induced markers of neuroinflammation (IL-1β, IL-6, and IDO) in the hippocampus and cortex. To determine if defects in IL-4 signaling could play a role in the exaggerated microglial response to peripheral immune stimulation in aged mice, adult and aged mice received an intraperitoneal injection of LPS and microglia were isolated and treated with IL-4 ex vivo. Lipopolysaccharide treatment led to an increase in expression of IL-4Rα in both adult and aged mice, but the microglial response to ex vivo IL-4 treatment was significantly lower in microglia from aged mice than adults. These findings support the hypothesis that aging primes microglia to give an exaggerated inflammatory response to peripheral immune stimulation, that this amplified neuroinflammation leads to behavioral deficits such as prolonged sickness and depressive-like behaviors, and that age-associated impairments in microglial anti-inflammatory signaling such as IL-4 signaling may play a significant role in the amplified and prolonged microglia activation in the aged brain following peripheral immune stimulation. Advisors/Committee Members: Godbout, Jonathan.

Subjects: Aging; Immunology; Neurosciences

Keywords: brain; lipopolysaccharide; microglia; aging; minocycline; mice; behavior; neuroinflammation; cytokines

7. Hessein Hassona, Mohamed Darwish. The Role of Profilin1 Gene in the Development of Cardiovascular Diseases: Insights From Profilin1 Transgenic Mouse Model.

Degree: PhD, Biochemistry Program, Ohio State, 2010, Ohio State University

► Hypertension represents a major risk factor for cardiovascular diseases. Increased mechanical stress/hypertension… (more)

▼ Hypertension represents a major risk factor for cardiovascular diseases. Increased mechanical stress/hypertension in the vessel wall triggers the hypertrophic signaling pathway, resulting in structural remodeling of vasculature. We have developed a novel transgenic mouse model by overexpressing the cDNA of human profilin1 in the blood vessels of transgenic mice. We showed that increased expression of profilin1 protein in the medial layer of the aorta induces stress fiber formation, triggering the hypertrophic signaling resulting in vascular hypertrophy and, ultimately, hypertension in older mice. Our results showed significant increases in the expression of alpha1integrin (280%), beta1 integrin (325%) and the activation of Rho/ROCKII pathway (260% and 350%, respectively) in profilin1 mesenteric arteries. The activation of Rho/ROCK led to the inhibition of eNOS expression (39%) and phosphorylation (35%), while an increase in myosin light chain phosphorylation (p-MLC20) (372%). There were also increases in hypertrophic signaling pathways i.e. phospho-ERK1/2 (p-ERK1/2) (312.15%) and phospho-JNK (p-JNK) (232.5%). Functional analyses of profilin1 mesenteric arteries showed significant increases in the vascular responses towards phenylephrine, while significant decreases in response towards ROCK inhibitor Y27632, acetylcholine (Ach), sodium nitrite NaNO2 and cytochalasin D. In addition, we assessed the effects of losartan, amlodipine or atenolol on vascular hypertrophy-associated hypertension, by treating the profilin1 transgenic mice for 4 weeks. Our myograph results showed improvement in the contraction response towards phenylephrine and in the relaxation response towards Ach and NaNO2 in losartan- and amlodipine-treated profilin1 mice. Western blot analyses using mesenteric arteries of lasortan and amlodipine-treated profilin1 mice showed significant decreases in their signaling respectively as follows: the expression of alpha1 integrin (104% and 93%) and beta1 integrin (116% and 109%); p-ERK1/2 (149% and 130% ) and p-JNK (171% and 137%); p-MLC20 (117% and 150%) and the ROCKII expression (125% and 180). Conversely, there were significant increases in the eNOS expression (82% and 80%) and activation (p-eNOS) (78 and 76%). On the other hand, atenolol-treated profilin1 mice showed no significant change in all measured parameters. Furthermore, we assessed the effects of elevated blood pressure on the cardiac structure and function of old hypertensive transgenic mice (OHP1). Our results showed significant increases in profilin1 expression (179%) and STAT3 activation (161.2%) in the hearts of OHP1. In addition, there was an increase in alpha1 integrin (215%), beta1 (305%) integrin and ROCKII (176%) in the hearts of OHP1 as well as p-ERK1/2 (185%) and p-JNK (265%). Echocardiography and magnetic resonance imaging demonstrated a significant decrease in left ventricle (LV) ejection fraction, fractional shortening while a significant increase in LV mass in OHP1. There was also an increase in heart/body weight ratio in OHP1. In conclusion, the changes in vascular responses in the mesenteric arteries of profilin1 mice are due to vascular hypertrophy and hypertension. In addition, the cardiac remodeling in OHP1 mice may enable us to understand the signaling mechanisms involved in the development of cardiac hypertrophy-associated hypertension in human and may reveal a new therapeutic target to inhibit the cardiac remodeling in chronic hypertensive patients. Advisors/Committee Members: Wani, Altaf.

Subjects: Biochemistry; Pharmacology; Pharmacy Sciences

Keywords: vascular remodelling; hypertension. cardiac hypertrophy; profilin1; transgenic mice

8. Huanyu, Wang. Characterization of N1/N2 Family Histone Chaperones: Hif1p and NASP.

Degree: PhD, Biochemistry Program, Ohio State, 2010, Ohio State University

► Recent studies revealed that a protein known as Hat1p Interacting Factor-1 forms… (more)

▼ Recent studies revealed that a protein known as Hat1p Interacting Factor-1 forms a complex with Hat1p/Hat2p in the nucleus and functions as a histone chaperone during chromatin assembly. This protein is a yeast homolog of the N1/N2 histone chaperone, which functions in both the storage and assembly of histone H3/H4 tetramers during the rapid rounds of DNA replication which occurs early in X. laevis embryogenesis. Hif1p functions as a chromatin assembly factor in vitro and associates with acetylated histone H4 in vivo in a Hat1p/Hat2p dependent manner. These findings demonstrated a physical connection between type B HATs and factors directly involved in the process of chromatin assembly. We have performed biochemical experiments to further characterize this protein. By using chromatographic techniques, we demonstrated that Hif1p forms complexes in both Hat1p/Hat2/-dependent and Hat1p/Hat2p-independent manners. We have developed a method combining both conventional and affinity chromatography to isolate and identify proteins associate with Hif1p. Our results also suggested a link between Hif1p and a H3-specific type B HAT. The human homolog of Hif1p, NASP, has been reported to be an H1-specific histone chaperone when all the other members of N1/N2 family are H3/H4-specific histone chaperones. To resolve this paradox, we have performed a detailed and quantitative analysis of the binding specificity of human NASP. Our results confirmed that NASP can interact with histone H1 and that this interaction occurs with high affinity. In addition, multiple in vitro and in vivo experiments, including native gel electrophoresis, traditional and affinity chromatography assays and surface plasmon resonance, all indicated that NASP also forms distinct, high specificity complexes with histones H3 and H4. The interaction between NASP and histones H3 and H4 is functional as NASP is active in in vitro chromatin assembly assays using histone substrates depleted of H1. We have also further characterized this protein in detail by directly mapping domains that are involved in interactions with each histone in vitro and developing a cell culture model to understand how the association with specific histones contributes to the cellular function of NASP in vivo. We identified two distinct domains that separately interact with linker histone or core histone through different mechanisms. We demonstrated that loss of native NASP increased the sensitivity of chromatin to digestion with micrococcal nuclease, therefore, identified NASP as a significant contributor to global chromatin structure. Advisors/Committee Members: Parthun, Mark.

Subjects: Biochemistry

Keywords: Histone Chaperone; chromatin assembly

9. Johnson, Eric K. A new model for the dystrophin associated protein complex in striated muscles.

Degree: PhD, Biochemistry Program, Ohio State, 2012, Ohio State University

► Dystrophin is a large, cytoskeletal protein localized to the intracellular side of… (more)

▼ Dystrophin is a large, cytoskeletal protein localized to the intracellular side of the muscle membrane, and is the central organizer of a large protein complex known as the dystrophin associated protein complex (DAPC). Through the DAPC, dystrophin functions not only to stabilize the membrane from the forces generated by muscle contraction, but also functions in intracellular signaling mediated through DAPC proteins. Absence of dystrophin destabilized the DAPC disrupting membrane integrity and muscle function leading to muscle damage. Clinically, mutations in the dystrophin gene give rise a group of muscular dystrophies, termed the dystrophinopathies. The functions of dystrophin are generally believed to be similar for all striated muscles. However, it has become clear that loss of dystrophin does not affect all muscles types equally, particularly the heart and diaphragm. Because the majority of dystrophin functions are facilitated though the DAPC, we have hypothesized that the tissue specific functions of dystrophin are mediated by unique protein interactions. In order to identify dystrophin associated proteins, we developed a high throughput proteomics approach that combines dystrophin immunoprecipitation with downstream shotgun mass spectrometry. Using this approach we identified major tissue-specific differences in the protein interactions of dystrophin in the heart and diaphragm. This included both differences in the composition of known DAPC proteins, in addition to novel cardiac and diaphragm-specific dystrophin associated proteins. Importantly, these proteins are important for muscle function, and have known roles in the regulation of calcium homeostasis and membrane repair. In addition to dystrophin, we chose to study β-dystroglycan, an essential component of the DAPC. Our current model of the DAPC is that in the absence of dystrophin, the DAPC is destabilized and lost from the sarcolemmal membrane. However, disruption of dystroglycan gives rise to a more severe pathology than dystrophin, and in the absence of dystrophin, β-dystroglycan and some other DAPC proteins are still expressed at the membrane. Together this suggests that dystroglycan must have additional functions not mediated directly by the DAPC and may be mediated by yet unidentified protein complexes. Using a similar approach for dystrophin we show that in skeletal muscle multiple complexes of β-dystroglycan exist and interact with a unique set of proteins compared to dystrophin. These findings suggest that distinct β-dystroglycan complexes exist in skeletal muscle, and we therefore propose a new model of β-dystroglycan function and organization in striated muscle. The studies presented here provide clear evidence that the DAPC is a dynamic protein complex with unique differences between individual muscle tissues. These novel differences suggest tissue specific functions of dystrophin in the heart and diaphragm. Furthermore, our studies on β-dystroglycan add additional complexity our understanding of components of the DAPC and highlight new possible functions of both β-dystroglycan and dystrophin. Importantly the technique described here overcomes a significant challenge in the field and offers a new method for studying dystrophin protein interactions. The generation of the method and the novel findings described here will likely prove vital to our understanding of the mechanisms leading to muscle disease for patients with dystrophinopathies. Dystrophin Muscular Dystrophy Proteomics Immunoprecipitation Advisors/Committee Members: Montanaro, Federica.

Subjects: Biochemistry

Keywords: Dystrophin Muscular Dystrophy Proteomics Immunoprecipitation

10. Jones, Christopher P. Primer tRNA annealing by human immunodeficiency virus type 1.

Degree: PhD, Biochemistry Program, Ohio State, 2012, Ohio State University

► Like all retroviruses, the human immunodeficiency virus type 1 (HIV-1) replicates in… (more)

Subjects: Biochemistry

Keywords: HIV; tRNA; RNA; primer; annealing; tRNA-like element; Gag; nucleocapsid; matrix; primer binding site; small-angle X-ray scattering; SAXS; retrovirus; assembly; packaging

11. Joyner, Jeff C. Synthesis and Evaluation of Catalytic Metallodrugs and Analysis of RNA Cleavage by Mass Spectrometry.

Degree: PhD, Biochemistry Program, Ohio State, 2012, Ohio State University

► Catalytic metallodrugs have been developed that include Rev-coupled transition metal chelates (M-chelate-Rev… (more)

Subjects: Biochemistry

Keywords: metallodrug, catalysis, MALDI-TOF MS, mass spectrometry, RNA, protein, oxidation, cleavage

12. Liu, Tao. Development Of Cyclic Peptidyl Ligands Through A Combinatorial Library Approach.

Degree: PhD, Biochemistry Program, Ohio State, 2011, Ohio State University

► Cyclic peptides are widely produced in nature and possess a broad range… (more)

▼ Cyclic peptides are widely produced in nature and possess a broad range of biological activities. Their enhanced proteolytic stability in vivo and improved receptor binding affinity/specificity makes them excellent drug candidates, molecular probes and targeting agents. In fact, many cyclic peptides are clinically used therapeutic agents. Combinatorial library approaches provide powerful tools for the rapid identification of compounds with desired properties from large pools in biological and biomedical studies. However, the synthesis and screening of cyclic peptide libraries in a combinatorial format has been challenging. To overcome the issue, we have successfully developed one-bead-two-compound (OBTC) libraries with a cyclic peptide displayed on the bead surface accessible for protein targets screening, while the bead interior contains the corresponding linear peptide served as an encoding tag for hit identification. The primary goal of my research is to identify novel biologically active cyclic peptides, beyond what nature has provided us. By applying cyclic peptide library approach, we have successfully identified high affinity ligands against various biological targets, including: extracellular protein receptors (human prolactin receptor), intracellular protein domains (the capsid domain of HIV-1 Gag polyprotein and calcineurin catalytic domain) and enzymes (Pin1 catalytic domains). In the meantime, we have continued to improve the methodologies associated with combinatorial chemistry. To facilitate the process and improve the screening results, such as avoiding false positives, we have developed many cyclic library approaches including libraries on different solid supports, reduced surface density libraries, high diversity libraries with different ring sizes and library compatible with rapid solution phase validation. These new approaches greatly facilitate the ligands discovery process. My final work focused on the intracellular delivery of cyclic peptides. Little is known about how cyclization would affect peptides membrane permeability and the results from existing studies are controversial. With a combination of biophysical approaches and cell based studies, we have found that cyclization has a dramatic effect on the cell permeation of peptides with certain residues. By applying the rules, we were able to design cell permeable cyclic peptide inhibitors against various intracellular protein targets. Our studies provide guiding principles for designing membrane penetrating cyclic peptidyl drugs. Advisors/Committee Members: Pei, Dehua.

Subjects: Biochemistry; Biomedical Research; Chemistry

Keywords: Cyclic Peptides; Combinatorial Chemistry; One-Bead-One-Compound (OBOC) Library; Prolactin Recepotr; Pin1; HIV Capsid; Calcineurin; Cell Penetrating Peptide (CPP)

13. Liu, Wei. Structural basis and functional impact of ligand-independent dimerization for human prolactin receptor.

Degree: PhD, Biochemistry Program, Ohio State, 2012, Ohio State University

► The human prolactin receptor (hPRLr) is a member of the class 1… (more)

▼ The human prolactin receptor (hPRLr) is a member of the class 1 cytokine receptor family, which also includes growth hormone receptor (GHr) and erythropoietin receptor (EPOr). The classic mechanism for class 1 cytokine receptor activation describes that ligand-induced receptor dimerization triggers downstream signaling and is supported by biochemical and biophysical evidence. However, the recent discoveries of ligand-independent dimerization of class 1 cytokine receptors in plasma membrane, including hPRLr, hGHr, and mouse EPOr, have challenged this classic mechanism. Several mechanistic models have been proposed for the activation of GHr and EPOr, including the scissor model, the piston model, and the rotation model. In contrast, it is unknown if hPRLr shares a similar mechanism. The specific amino acid residues that mediate ligand-independent hPRLr dimerization have not been determined, although the transmembrane (TM) domain has been suggested to be important. The role of ligand-independent hPRLr dimers in hPRLr activation is unclear. Furthermore, the presence of ligand-independent hPRLr dimers does not rule out the classic mechanism, because hPRLr may exist in equilibrium between monomers and preformed dimers in plasma membrane. This dissertation has focused upon the structural basis and the functional impact of ligand-independent dimerization of hPRLr. We aimed to systematically evaluate in hPRLr the proposed models for class 1 cytokine receptor activation. An extensive series of alanine or glycine insertions were introduced at the junctions between the TM domain and either the extracellular or intracellular domain to manipulate the relative orientations of different hPRLr domains. The basal and ligand-stimulated activities of these hPRLr insertion variants were examined in transiently transfected 293T cells. Our data demonstrate that altering the spatial relationships of hPRLr domains does not induce constitutive activity or impair ligand-induced activation. Such results do not support the rotation or piston model for hPRLr. We also identified a population of covalently linked ligand-independent hPRLr dimers that are redox-sensitive and investigated the involvement of intermolecular disulfides in ligand-independent hPRLr dimerization. Twelve cysteines in various domains of hPRLr were replaced with serines, and the dimerization status of these hPRLr mutants was examined under reducing and non-reducing conditions. Iodoacetamide, an alkylation reagent for cysteine, was employed to distinguish in vivo disulfides from ex vivo disulfides. Our data indicate that multiple cysteines from different domains of hPRLr, including but not limited to C184, C225, and C242, participate in forming intermolecular disulfides in ligand-independent hPRLr dimerization. We next examined the role of these disulfide-linked hPRLr dimers in hPRLr activation. Abolishing the formation of ligand-independent disulfide-linked hPRLr dimers by removing twelve C-terminal cysteines did not impair ligand-induced activation or affect the speed of activation, indicating that disulfide-linked hPRLr dimers are not required for hPRLr activation. Furthermore, wild-type disulfide-linked hPRLr dimers were not phosphorylated after ligand stimulation. Only hPRLrs that were not covalently linked became phosphorylated. We conclude that disulfide-linked hPRLr dimers are unlikely to participate in hPRLr activation. Collectively, the data in this dissertation suggest the validity of the classic mechanism of ligand-induced dimerization for hPRLr activation and illustrate the uniqueness of hPRLr in this receptor family. Advisors/Committee Members: Brooks, Charles.

Subjects: Biochemistry

Keywords: prolactin receptor; human; dimerization; disulfide; structure function

14. Liu, Yu-Yu. Modulation of Sodium/Iodide Symporter Expression and Function in Thyroid.

Degree: PhD, Biochemistry Program, Ohio State, 2011, Ohio State University

► Na+/I- Symporter (NIS) is a membrane glycoprotein that mediates active iodide uptake… (more)

▼ Na+/I- Symporter (NIS) is a membrane glycoprotein that mediates active iodide uptake into the thyroid gland for thyroid hormone synthesis. NIS-mediated iodide uptake and iodide organification is the basis for the post-operative use of radioiodide in detection and targeted ablation of differentiated thyroid cancer. However, about 20-30% patients with metastatic thyroid cancer do not benefit from radioiodine therapy due to reduced or absent NIS expression/function. Thus, it is of clinical importance to investigate the underlying mechanism of NIS modulation such that strategies to selectively upregulate NIS expression and/or functional activity can be devised. Micro-SPECT was used to examine and quantify temporal thyroidal and salivary radioiodine accumulation in both wild type and Tg-PTC1 mice treated with triiodothyronine (T3), bTSH and/or 17-AAG. The extent of thyroidal radioiodine accumulation stimulated by a single dose of exogenous bTSH in T3-supplemented endogenous TSH suppressed mice was much less than that in non-treated mice. Furthermore, the extent and duration of radioiodine accumulation stimulated by bTSH was reduced in thyroid tumor-bearing thyroid glands of Tg-PTC1 thyroid cancer mouse model compared to the thyroids in wild type mice. Lastly, the effect of 17-AAG on increasing thyroidal, but not salivary, radioiodine accumulation was validated in WT mice and in Tg-PTC1 thyroid cancer mouse model. We also aimed to understand the mechanism underlying regulation of NIS in vitro. In PC Cl3 rat thyroid cells, inhibition of PI3K by LY294002 increased NIS-mediated RAIU activity through upregulation of NIS expression. On the other hand, inhibition of mTORC1 by Rapamycin decreased NIS-mediated RAIU activity yet increased NIS protein levels. The discordance between NIS protein and NIS-mediated RAIU activity by Rapamycin is due to its effect on activating pERK and pAkt levels. The effect of Akt inhibition on NIS expression/function was further examined showing that Akti-1/2 markedly increased NIS-mediated RAIU activity by decreasing iodide efflux rate and increasing iodide transport rate and iodide affinity of NIS. The effect of Akti-1/2 on increasing NIS-mediated RAIU activity is restricted to thyroid cells encourages the use of Akt pharmacological inhibitors to selectively increase thyroidal radioiodine accumulation and decrease radioiodine accumulation in non-thyroid tissues. Advisors/Committee Members: Jhiang, Sissy.

Subjects: Endocrinology

Keywords: thyroidal iodide accumulation, NIS, SPECT, 17-AAG, Akt, PI3K, mTOR, MEK

15. Liu, Zhenzhen. The Roles of Interleukin-27 in Tumor Immunity.

Degree: PhD, Biochemistry Program, Ohio State, 2012, Ohio State University

► Interleukin-27 (IL-27) is a member of the IL-12 family of cytokines. IL-27… (more)

▼ Interleukin-27 (IL-27) is a member of the IL-12 family of cytokines. IL-27 is a heterodimer consisting of an IL-12 p40-related protein subunit, EBV-induced gene 3 (EBI3) and a p35-related subunit, p28. IL-27 is mainly produced by activated antigen presenting cells. It functions through engaging IL-27 receptor, which is expressed on a variety of immune cell types, including CD4+ and CD8+ T cells. Overexpression of IL-27 by tumor cells exerts potent anti-tumor activity through diverse mechanisms, in which CD8+ T cells were considered to be the main effector cells. However, the exact mechanisms by which IL-27 enhances anti-tumor CD8+ T cell response and leads to tumor rejection remain unclear. The impacts of IL-27 on the differentiation and activation of CD8+ T cells were studied by stimulating naïve tumor antigen-specific CD8+ T cells (P1CTL) with cognate P1A peptide in the presence and absence of IL-27. First, T cell proliferation and apoptosis were examined by thymidine incorporation and Annexin V/7-AAD staining, respectively. Second, expressions of activation and differentiation markers of T cells were analyzed by Real time PCR and Western Blotting. Third, cytokine production was evaluated by ELISA and flow cytometry. To investigate the in vivo roles of IL-27 in tumor immunity, mouse models involving tumor cells (J558 plasmacytoma and B16 melanoma) expressing IL-27 and IL-27-deficient (EBI3-/-) mice were used. To delineate the mechanisms by which IL-27 enhances antitumor CTL responses, tumor antigen specific CD8+ T cells were adoptively transferred into various genetically engineered mice with established tumors, and their in vivo proliferation and apoptosis were studied by CSFE staining and flow cytometry analysis. To examine the role of IL-27 in response and function of T regulatory cells, Anti-CD25 antibody was used to deplete Treg cells; IL-27 deficient or WT CD4+CD25+ Treg cells were adoptively transferred together with effector T cells into tumor-bearing mice; tumor establishment and metastases were evaluated by monitoring s.c. tumor growth and weighing lungs, respectively. Overall, we have uncovered four novel findings that can explain why IL-27 boosts antitumor CD8+ T cell responses: 1) IL-27 enhances the survival of activated tumor antigen specific CD8+ T cells both in vitro and in vivo. 2) IL-27 induces a unique memory precursor cell (MPC) phenotype in activated tumor antigen specific CD8+ T cells, which is characterized by up-regulation of SOCS3, Bcl-6, Sca-1 and IL-10. 3) IL-27 robustly induces IL-10 production by tumor antigen specific CD8+ T cells, which contributes to IL-27-mediated tumor rejection in vivo. 4) IL-27 inhibits the expansion and immunesuppressive ability of CD4+FoxP3+ T regulatory cells, resulting in more potent anti-tumor CTL responses. Our findings suggest that: 1) IL-27 has the potential to be used as an adjuvant to boost the efficacy of antitumor vaccines; 2) IL-27 can be used to culture tumor antigen-specific CTLs for adoptive transfer therapy of cancer patients. Advisors/Committee Members: Wu, Lai-Chu.

Subjects: Cellular Biology; Immunology; Pathology

Keywords: IL-27, IL-10, CTL response, Treg response, Tumor rejection

16. Mathur, Manjula. Molecular cloning, sequencing and characterization of a novel fatty acid synthase: mycocerosic acid synthase from mycobacterium tuberculosis var. bovis BCG.

Degree: PhD, Biochemistry Program, Ohio State, 1992, Ohio State University

Advisors/Committee Members: Kolattukudy, P. E.

17. Mehta, Payal. Molecular Analysis of Regulation of Macrophage Fcγ Receptor Function: Implications for Tumor Immunotherapy.

Degree: PhD, Biochemistry Program, Ohio State, 2011, Ohio State University

► Therapeutic monoclonal antibodies (MAB) are used as a part of induction as… (more)

▼ Therapeutic monoclonal antibodies (MAB) are used as a part of induction as well as salvage regimen for treatments of various malignancies. Despite a large number of MAB available, the efficacy of tumor immunotherapy remains suboptimal. Fcγ Receptor (FcγR), play a critical role in mediating cytotoxic effects of MAB. FcγR activation results in elimination of immune complexes (IC) and antibody-coated tumor targets via phagocytosis and antibody-dependent cell-mediated cytotoxicity (ADCC) respectively, generation of reactive oxygen and nitrogen species (ROS), and release of pro-inflammatory cytokines. These effector functions are tightly regulated by simultaneous engagement of activating and inhibitory FcγR as well as recruitment of downstream signaling kinases and phosphatases. A thorough understanding of the regulatory pathways that impede sustained and robust immune response is critical for designing MAB therapy with enhanced efficacy. For this study we have specifically examined the role of such regulatory pathways on FcγR-mediated functional responses and its influence on tumor immunotherapy. In the first part of the study, demonstrate that not all FcγR contribute equally to monocyte response against antibody-coated tumor targets. Further we show that FcγRI predominantly mediates ADCC and cytokine production but not FcγRIIa that preferentially associates with inositol phosphatase SHIP. In the second part of the study, we have identified a novel SHIP-interacting protein that negatively regulates FcγR function and in the last part of the study, we examined the regulation of FcγR expression and function by bacterial unmethylated CG containing DNA motif, CpG that activates TLR9 and further tested its role as an adjuvant for Rituximab therapy in B-CLL patients. Taken together, our finding have established novel ways by which FcγR-mediated signaling response are regulated in human monocytes. We have also demonstrated the importance of modulating these regulatory pathways specifically within the context of tumor immunotherapy. In future findings of this study may contribute to the design of better immunotherapeutic strategies. Advisors/Committee Members: Tridandapani, Susheela.

Subjects: Immunology

Keywords: FcγR, tumor immunotherapy, monoclonal antibody, SHIP

18. Mitchell, Sharnise Nicole. CHARACTERIZATION OF THE LYSINE-RESPONSIVE L BOX RIBOSWITCH.

Degree: PhD, Biochemistry Program, Ohio State, 2012, Ohio State University

► Regulation of gene expression is an essential process that organisms employ for… (more)

▼ Regulation of gene expression is an essential process that organisms employ for growth and survival. The activation or repression of genes is critical as it allows the cell to monitor environmental signals and adapt to changes in nutrient availability or environmental conditions. One method of regulation involves RNA elements termed riboswitches. Riboswitches are conserved RNA elements that regulate gene expression by modulation of the RNA structure. Typically, a structural change in the RNA occurs in response to environmental signals such as temperature, small RNAs, or small molecules. Most of the known riboswitches modulate gene expression in response to a small molecule effector. Recognition of the effector causes a structural rearrangement that prevents or promotes the formation of a regulatory structure such as an intrinsic transcriptional terminator. Riboswitches that regulate gene expression at the level of translation undergo a structural rearrangement that can occlude or expose the ribosomal binding site (RBS). In this work, we focused our analysis on the L box riboswitch that regulates gene expression during transcription in Bacillus subtilis and is predicted to regulate at the translational level in Escherichia coli. This conserved RNA regulates the expression of lysine biosynthetic genes in response to cellular lysine concentrations. Previous work revealed that the lysC leader RNA from B. subtilis promotes premature transcription termination in the presence of lysine and can discriminate against lysine analogs. Analysis of the leader region in vivo indicated that lysC expression is repressed when the RNA is transcribed in the presence of high lysine. Here we investigate the conserved sequence and structural features of the lysC leader riboswitch to elucidate the features that are required for lysine binding and the required structural transition. We have identified variants of the lysC leader RNA that stabilize the termination conformation in the absence of lysine. These results suggest that the energetic balance between the termination and antitermination conformations is sequence-dependent and the conserved nucleotides are critical to the stabilization of the ligand-free structure. In vitro evolution of a randomly mutated lysC leader indicated that regions with conserved structural motifs are also critical to the lysine sensitivity and the structural transition required for lysine-dependent repression. Mutation of non-conserved residues demonstrated that regions distal to the ligand-binding pocket affect ligand sensitivity. Finally, site-directed mutagenesis was used to identify the features of the lysine-binding pocket that are responsible for the lysine specificity. Our results indicate that the majority of the conserved nucleotides within the binding pocket are required for recognition of independent features of the lysine molecule. Mutation of the conserved residues yielded lysC leader variants with alternate ligand specificities. These mutations resulted in variants that are specific for lysine analogs and exhibit a loss of lysine recognition. These experiments provide a comprehensive view of the lysC leader RNA requirements for gene regulation and highlight the conserved sequence and structural features that are necessary for function of the L box riboswitch. Advisors/Committee Members: Henkin, Tina.

Subjects: Biochemistry; Molecular Biology

Keywords: riboswitch; gene regulation; regulatory RNA; L box

19. Nauerth, Michelle Jon. Role of the Discoidin Domain receptor proteins in atherosclerosis: Interaction with lipids and collagen.

Degree: PhD, Biochemistry Program, Ohio State, 2011, Ohio State University

► Discoidin domain receptors (DDR1 and DDR2) are unique tyrosine kinase receptors (RTKs)… (more)

▼ Discoidin domain receptors (DDR1 and DDR2) are unique tyrosine kinase receptors (RTKs) in that they bind to and become phosphorylated by collagens, particularly collagen type I; the most abundant protein in the extracellular matrix (ECM). It is currently not known if collagen is the only ligand for these proteins, since some members of the discoidin family also bind phospholipids via their discoidin domain. In either case, the mechanisms for binding any ligand by these two proteins are not completely understood. It is generally known that receptors from the RTK family bind a ligand and induce down-stream phosphorylation of the intracellular tyrosine kinase, thus controlling several cellular processes. The goal of this study is to demonstrate a possibility for alternative ligands, other than collagen, for the discoidin domain receptor proteins, and elucidate how a mechanism of binding may occur. The data presented here qualitatively addresses the possibility that phospholipids may be a ligand for the DDRs. Several proteins in the blood coagulation pathway bind phospholipids via their discoidin-like domains. In addition, the three-dimensional crystal structures of these proteins have been resolved. Utilizing their amino acid sequence, as well as their three-dimensional structure as a template, a series of experiments was designed to elucidate a possible DDRs:phospholipid interaction. There is a strong indication, as my results demonstrate that DDRs bind to phospholipids. Three-dimensional molecular models of the DDRs were proposed and used to understand how these proteins might bind to a plasma membrane, which is comprised of phospholipids. The finished structure of the DDRs mimics those proteins in the blood coagulation pathway which bind phospholipids and support my hypothesis that DDRs may favor phospholipids as a ligand. Based on the results of the molecular modeling study, the extracellular domain (ECD), which is the proposed ligand binding region of the DDRs, was employed in a series of experiments involving platelet aggregation. It is known that the preferred ligand for the DDRs is fibrillar collagen. The data presented in this study is novel, and shows that DDRs inhibit platelets from aggregating via an indirect interaction with collagen. These results may elucidate a possible role for the DDRs in arteriole plaque formation and rupture. The last aim of this research involves the role of DDR1 in the remodeling of the extracellular matrix (ECM). The ECM contains three morphological forms of collagen; monomeric (M), semi-polymeric (SP), and fibrillar (F). As cells secrete M collagen, it forms a polymeric intermediate SP form, and eventually composes the fibrillar F form. The results of the study indicate the preferred ligand for DDR1 is the intermediate SP form of collagen type I in addition to the known F form. These results provide a better understanding of morphological collagen usage-initially during collagen processing in the ECM in response to vascular damage, and their interactions with DDRs. Advisors/Committee Members: Brooks, Charles L.

Subjects: Biochemistry

Keywords: Discoidin Domain; protein engineering

20. Omar, Hany Ahmed Mostafa Mohamed. Molecular Pharmacology and Preclinical Studies of Novel Small-molecule Targeted Agents for The Treatment of Hepatocellular Carcinoma.

Degree: PhD, Biochemistry Program, Ohio State, 2010, Ohio State University

► Hepatocellular carcinoma (HCC) is one of the most common cancers in Asia… (more)

▼ Hepatocellular carcinoma (HCC) is one of the most common cancers in Asia and Africa, which is lethal in about 75% of cases. In the United States, there are 6,000-9,000 new cases of HCC per year. HCC is one of the highly chemo-resistant cancers, for which systemic treatments have been unsuccessful. The available therapies are surgical (10-20% of cases), locoregional or recently, chemical using Sorafenib, a multi-kinase inhibitor which has been shown in clinical trial to add two months to the lifespan of late stage HCC patients. To combat HCC with its assortment of genomic and cellular aberrations that occur during the progression of the disease, we have developed OSU-A9 and OSU-2S, novel small-molecule targeted agents for HCC therapy, using indole-3-carbinol (I3C) and fingolimod (FTY720) respectively, as scaffolds. In this study, we used pharmacological and molecular genetic approaches to investigate the mechanisms of action of the lead compounds of these classes (OSU-A9 and OSU-2S) and assess both the efficacy and safety in a series of preclinical studies carried out both in vitro and in vivo. OSU-A9 exhibits up to 100-fold greater in vitro efficacy relative to I3C in HCC cells and provides a considerable therapeutic advantage over I3C with respect to chemical stability. Mechanistic evidence indicates that OSU-A9 antitumor effect is mediated by blocking the Akt-NF-kB signaling network, leading to the inhibition of signaling pathways governing cell cycle progression, survival, and metastasis. Equally important , oral administration of OSU-A9 suppressed HCC xenograft tumor growth in mice without causing overt signs of toxicity. In addition, sub-toxic doses of OSU-A9 combined with the tumor necrosis factor-related apoptosis inducing ligand (TRAIL) effectively inhibited the resistance of HCC to TRAIL-induced apoptosis. The synergistic apoptotic effect is mediated by the ability of OSU-A9 to antagonize TRAIL-activated NF-κB cell survival pathway and to upregulate death receptor (DR) 5 expression in HCC cells. The ability of OSU-A9-TRAIL combination to selectively target HCC cells regardless of p53 status could shed the light on a promising combination therapy for HCC. Based on our finding that FTY720 mediates apoptosis in HCC cells by activating the reactive oxygen species (ROS)-protein kinase (PK)C delta signaling independent of S1P1 receptor, we developed OSU-2S, a novel PKCdelta-targeted non-immunosuppressive antitumor agent, by abrogating the S1P1 receptor activity of FTY720. Several lines of pharmacological evidence indicate that OSU-2S exhibits higher potency than FTY720 in suppressing HCC due to the metabolic inactivation of FTY720 through phosphorylation in the context of antitumor activity. As a single agent, OSU-2S exhibits high in vivo potency in suppressing xenograft tumor growth without overt toxicity, which supports its clinical promise as a component of therapeutic strategies for advanced HCC. Moreover, PKCdelta is a major downstream effector of DNA damage-induced apoptosis, so the activation of PKCdelta by OSU-2S is noteworthy, since it provides a rationale to combine OSU-2S with other genotoxic agents in HCC therapy. Advisors/Committee Members: Chen, Ching-Shih.

Subjects: Biochemistry; Molecular Biology; Pharmacology

Keywords: OSU-A9; OSU-2S

21. Ouseph, Madhu Micheal. Atypical E2F repressors E2F7 and E2F8: Balancing E2F activity in normal and variant cell cycles.

Degree: PhD, Biochemistry Program, Ohio State, 2012, Ohio State University

► Coordinated activation and repression of E2F-responsive genes is believed to be pivotal… (more)

▼ Coordinated activation and repression of E2F-responsive genes is believed to be pivotal for progression of normal cell cycle. Studies using lower organisms and mammalian cell culture systems support the idea that this balanced cumulative E2F activity in different phases of cell cycle is executed by canonical E2F activators (E2F1-E2F3) and repressors (E2F4-E2F6). But, recent studies in mouse models with ablation of these factors have failed to provide substantial molecular and phenotypic evidence to support this notion. The evolutionarily ancient arm of E2F family consisting of newly identified atypical E2F repressors, E2F7 and E2F8, is known to be critical for mammalian embryonic development. Germline ablation of E2f7 and E2f8 leads to severe placental defects culminating in embryonic lethality by embryonic age 11.5. Remarkably, the concomitant loss of E2f3a normalized placental and fetal gene expression programs, corrected placental defects and fostered the survival of E2f7/E2f8 deficient embryos to birth. Using expression profiling and biochemical approaches, we show that atypical E2F repressors and canonical E2F activator E2F3a form key antagonistic regulators of G1-S transcriptional program during placental development. We thus provide the first in vivo evidence to show that balanced repression by E2F7 and E2F8 and activation by canonical E2F activators coordinate expression of E2F-resonsive genes in mitotic cell cycle during mammalian development. Utilizing a combination of novel and established lineage-specific cre mice we also demonstrate that these two opposing arms of the E2F program, one driven by canonical transcription activation (E2F1, E2F2 and E2F3) and the other by atypical repression (E2F7 and E2F8), converge on the regulation physiological polyploidy in vivo. Ablation of atypical repressors diminished ploidy in the trophoblast giant cells in the placenta, hepatocytes in the liver and megakaryocytes in bone marrow, whereas ablation of canonical activators in the trophoblast giant cells and hepatocytes augmented genome ploidy. In addition, the severe reduction of ploidy caused by E2f7/E2f8 deficiency could be rescued significantly by added loss of E2f1 and E2f3a. Taken together, the results presented within provide the first in vivo evidence for a direct role of E2Fs in regulating non-traditional cell cycles in mammals. Though polyploidy has been demonstrated to be essential for metazoan development and is widely believed to have conserved physiological functions in mammals, to our surprise, reduction of ploidy caused by loss of E2f7 and E2f8 had no apparent adverse impact on placental, hepatic or megakaryocyte physiology. In summary, our studies reveal novel functions of mammalian atypical E2Fs and provide significant insight into mechanism of cell cycle phase dependent regulation of E2F-responsive genes in normal mitotic cell cycle and variant cell cycles in mammals. Advisors/Committee Members: Leone, Gustavo.

Subjects: Genetics; Molecular Biology

22. Qin, Daoming. Role of 16S Ribosomal RNA in Translation Initiation.

Degree: PhD, Biochemistry Program, Ohio State, 2011, Ohio State University

► In the cell, translation is the process by which polypeptides are synthesized… (more)

▼ In the cell, translation is the process by which polypeptides are synthesized on the ribosome based on the genetic information encoded in the messenger RNA (mRNA). The first step of translation, initiation involves recognition of the start codon by the initiator tRNA (fMet-tRNAfMet in bacteria) in the ribosomal peptidyl (P) site. The correct start codon is selected primarily based on the complementarity of codon-anticodon pairing. Initiation factors IF1 and IF3 have been implicated to negatively regulate translation initiation to promote its accuracy. 16S ribosomal RNA (rRNA) undoubtedly plays a role in initiation, but precisely how it participates in the process remains unclear. In this work, we use a combination of genetic and biochemistry approaches to investigate the role of 16S rRNA in start codon selection. We first screened a number of P-site mutations for their effects on start codon selection. Our results showed that most of these P-site mutations, while generally reducing the efficiency of translation, increase the stringency of start codon selection. These mutations confer a defect in fMet-tRNA binding, which likely makes initiation depend more heavily on the cognate codon-anticodon pairing. One exception, G1338A confers a higher affinity for tRNAfMet, which could partially compensate for the mismatches in the codon-anticodon helix and thereby allow initiation from non-canonical start codon. These data provide evidence that the affinity of the initiator tRNA for the 30S P site is tuned to balance efficiency and accuracy of initiation. Unlike G1338A, 30S subunits harboring A790G show defect in IF3 binding. We suspect that reduced IF3 binding explains the loss of fidelity in that case. Next, we employed random mutagenesis in a genetic screen and identified additional 16S rRNA mutations that increase initiation from non-canonical start codons. Most interestingly, a cluster maps to helix 44 (h44) just ‘down’ from the A site, a region known to be distorted by IF1. Several h44 mutations (e.g. A1413C) are predicted to change the non-canonical pair to a Watson-Crick pair, suggesting that they may decrease fidelity of translation initiation by altering the conformation of h44. Indeed, our subsequent work showed that these h44 mutations stimulate the second step of initiation (50S docking), which may explain for their defects in start codon recognition in vivo. To reveal possible structural changes of 16S rRNA upon start codon recognition, we used chemical probing methods to monitor 16S rRNA in canonical and non-canonical 30S initiation complex. Of ~110 nucleotides targeted, 6 showed altered reactivity in response to the nature of start codon. One of them is A1408, which lies in h44. In particular, the high reactivity of A1408 attributed to IF1 is specifically reduced in 30S initiation complexes containing AUG, even though the level of IF1 binding remains unchanged. Additionally, either mutation A1413C or streptomycin similarly reduces the reactivity of A1408. In light of the fact that both A1413C and streptomycin can stimulate premature 50S docking, we propose that the docking is controlled in part by a conformational switch in the 1408 region. IF1 stabilizes the A1408 region in a docking unfavorable conformation, which is reversed upon start codon recognition. Another group of the nucleotides lie in or near the P site. These nucleotides, which are protected by fMet-tRNA in the canonical 30S initiation complexes, are either unprotected or less protected in the non-canonical 30S initiation complexes. The loss of protections suggests that P site is largely unoccupied in the presence of non-canonical start codon. We propose that in this situation, fMet-tRNA associates to the 30S subunits in a liable manner. Finally, we also show that the selectivity of 30S.mRNA.fMet-tRNA ternary complex formation is high (K(AUG)/K(AUC)~100) in the absence of initiation factors – high enough to account for the accuracy in vivo. In the presence of all three factors, the selectivity is similar. Omission of IF3 in the latter case decreases selectivity to 4. These data support a simple model in which IF3 kinetically counteracts the stabilizing effects of the other factors to allow the inherent selectivity of the 30S P site to be utilized. Advisors/Committee Members: Fredrick, Kurt.

Subjects: Biochemistry

Keywords: Translation; initiation; IF1; IF2; IF3; h44; P site; start codon; ribosome; RNA

23. Qi, Wenbin. Studies of Iron-Sulfur Cluster Biogenesis and Trafficking.

Degree: PhD, Biochemistry Program, Ohio State, 2011, Ohio State University

► Fe/S cluster proteins are key players in diverse pathways, such as mitochondrial… (more)

▼ Fe/S cluster proteins are key players in diverse pathways, such as mitochondrial respiration, gene regulation, and DNA/RNA metabolism. It is generally believed that an Fe/S cluster is synthesized inside the mitochondria before it is exported out to the cytosol. But the details of this export pathway are still unclear; especially what is the substrate that is exported. This substance should be essentially small enough to go through the exporter Atm1p, stable enough to survive the transport process and labile enough to deliver the cluster to cytosolic proteins. It has been shown that glutathione (GSH) or its derivatives might be involved in this pathway. Several Glutaredoxin protein (Grx) structures revealed that Grx is able to coordinate a Fe/S cluster along with two molecules of GSH. The cluster transfer between Grx and scaffold protein ISU was found to be reversible, which indicates Grx may deliver the cluster to the export pathway and plays important role in Fe/S cluster biosynthesis. However, the role of this cluster is still not well understood. A complex of four GSH-coordinated Fe/S cluster was successfully synthesized and characterized and found to be stable under physiological conditions, but undergoes a reversible exchange with scaffold protein ISU. Considering the high cellular concentration of GSH and the stability of this complex under physiological conditions, this complex may contribute to a labile cellular Fe/S cluster pool. The GSH cluster complex is a very intriguing candidate for the substrate of mitochondrial Fe/S cluster exporter. A liposome system was constructed onto which transporter was successfully reconstituted. The stimulation of proteoliposome ATPase activity by GSH cluster complex indicates that the GSH cluster complex is very likely to be the exported substance. Evidence indicates frataxin to be the general iron donor protein in Fe/S cluster biosynthesis. However, a thorough search of the genomic database shows that based on sequence similarity, there is no homolog in some species. By utilizing a novel structural similarity search, a previously uncharacterized protein - Bacillus subtilis ydhG with very similar structure but no sequence similarity to frataxin was identified. B. subtilis yhdG shows the same function as human frataxin and is identified as the first structural homolog of this protein. This finding greatly expands the frataxin family and supports the role of frataxin being the general iron donor. Human ferredoxin-1 (hFd1) and human ferredoxin-2 (hFd2) share high sequence similarity but have distinct roles in different cellular pathways. HFd2 was found to undergo a unique conformational change upon heating to around 60 °C before cluster degradation is observed. Transition to the second conformation is entropy driven and enthalpy unfavorable. The transition is not reversible; hFd2 retains conformation when cooling from high temperature. This transition to a second stable conformation of hFd2 is a not observed for hFd1 and may help to explain their distinct cellular functions. Advisors/Committee Members: Cowan, James.

Subjects: Biochemistry; Biophysics; Cellular Biology; Chemistry

Keywords: Iron-sulfur cluster; iron; biogenesis; trafficking; Atm1p; glutathione; ISU; glutaredoxin; ferredoxin; proteoliposome; exporter

24. Rameez, Shahid. Engineering Cellular Hemoglobin-Based Oxygen Carriers For Use In Transfusion Medicine.

Degree: PhD, Biochemistry Program, Ohio State, 2011, Ohio State University

► The main goal of the research discussed in this dissertation is to… (more)

▼ The main goal of the research discussed in this dissertation is to create effective and safe cellular hemoglobin (Hb)-based oxygen (O2) carriers (HBOCs) for use in transfusion medicine. This dissertation focuses on the development and scale up production of cellular HBOCs composed of novel lipid formulations and polymers. The research concentrates on the development of cellular HBOCs over acellular HBOCs and abiotic O2 carriers, owing to their various advantages, such as, longer shelf-life, high Hb content, increased circulation half-life, biocompatibility, and absence of vasoactivity and hypertension in vivo. This dissertation presents various novel large scale methods for the production of cellular HBOCs. The methods discussed are very efficient as HBOCs are produced with very reproducible biophysical properties. All the methods are designed with cost effectiveness in mind. The methods presented are simple and easily scalable to an industrial scale. In addition, they eliminate the use of energy consuming equipment/methods used in the production of other cellular HBOCs. Moreover, all the scale up methods are designed to satisfy important design criteria required to develop an effective O2 carrier, such as, high Hb content, suitable biophysical properties to ensure proper O2 delivery to tissues and organs and absence of any free Hb in the dispersions. This dissertation also outlines detailed insights on the role of the intracellular diffusion barrier, as a result of Hb encapsulation, on binding/release of gaseous molecules to Hb. It emphasizes the importance of the cellular membrane and Hb encapsulation, as well as the pivotal role it plays in regulating O2 delivery or consumption of nitric oxide (NO) by Hb in cellular HBOCs. In addition, the ex vivo experiments in rat arterial segments discussed in this dissertation provide for the correlation between O2 delivery and NO consumption to Hb-induced vasoconstriction by using exogenous and endogenous sources of NO. Overall, this dissertation presents novel cellular HBOCs with a potential of being safe and effective O2 carriers for use in transfusion medicine. Advisors/Committee Members: Palmer, Andre.

Subjects: Alternative Medicine; Biochemistry; Chemical Engineering

Keywords: hemoglobin; red blood cell; liposomes; polymersomes; oxygen carriers; HBOC; poly(ethylene glycol)

25. Sherrer, Shanen Michelle. Mutagenic and Kinetic Effects of Various DNA Lesions on DNA Polymerization Catalyzed by Y-Family DNA Polymerases.

Degree: PhD, Biochemistry Program, Ohio State, 2011, Ohio State University

► Cell survival requires genomic stability through the conservation of DNA sequences. If… (more)

▼ Cell survival requires genomic stability through the conservation of DNA sequences. If DNA is damaged, most DNA polymerases will stall during DNA replication. The specialized Y-family DNA polymerases rescue stalled DNA replication sites and avoid cell death. Hence, gaining knowledge on the molecular basis of DNA polymerase’s nucleotide selectivity and fidelity during DNA lesion bypass improves our understanding of this process. However, the multiple bypass mechanisms that Y-family DNA polymerases employ are poorly understood. Sulfolobus solfataricus DNA Polymerase IV (Dpo4), a model Y-family member, has a fidelity range of 10-3-10-4 at 37 °C, which does not significantly change between 26 °C and 56 °C. However, the physiological temperature of the S. solfataricus is approximately 80 °C. To determine the kinetic and structural relevance of data collected below 80 °C, we employed a circular dichroism spectroscopic investigation to observe the secondary structural changes of Dpo4 over a large temperature range. We discovered that Dpo4 displayed a three-state cooperative unfolding trend with a hyperthermophilic melting temperature, and exhibited secondary structural stability until 87 °C. We also established that the Dpo4 unfolding intermediate originated from ionic interactions between the linker region and Palm domain. These interactions are considered important for DNA binding during binary complex formation and possibly nucleotide incorporations. Utilizing transient kinetics, we demonstrated that an active site mutation (Y12A) within Dpo4 caused an average 220-fold increase in matched ribonucleotide incorporation efficiency and an average 9-fold decrease in correct deoxyribonucleotide incorporation efficiency, leading to an average reduction of 2,000-fold in sugar selectivity. Therefore, the bulky side chain of Tyr12 is important for both ribonucleotide discrimination and efficient deoxyribonucleotide incorporation. To examine mutagenic outcomes of lesion bypass, we employed short oligonucleotide sequencing assay (SOSA) with all human Y-family members and DNA containing a non-informational abasic (AP) site. We observed complex mutagenic patterns of AP site bypass catalyzed by human Y-family enzymes including rare mutagenic events. This data suggested that human DNA Polymerase η (hPolη) is the likely enzyme to bypass AP sites in vivo. Furthermore, our other SOSA studies using DNA substrates containing a cis-syn cyclobutane pyrimidine dimer (a product of UV-exposure) or cisplatin-dGpG (a product of anticancer drug cisplatin) depicted each Y-family DNA polymerase performing lesion bypass uniquely for each DNA lesion and differently from other Y-family members. Both our pre-steady state kinetic and SOSA investigations on the bypass of N-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP), a product of incomplete fuel combustion, confirmed this hypothesis as Dpo4 was the most error-free while hPolη was the most efficient during lesion bypass. Our work also indicated that all Y-family enzymes utilized different dGAP bypass kinetic mechanisms, and that the dGAP presence decreased nucleotide incorporation efficiencies and accuracies upstream, downstream and opposite the lesion site. Beyond these findings, we elucidated the minimal dGAP bypass mechanism for Dpo4 and hPolη, as well as added more details to the kinetic mechanism employed by hPolη during DNA synthesis with undamaged DNA. Overall, our data contributes to the understanding of lesion bypass and potentially mutagenic outcomes of this vital biological process in vivo. Advisors/Committee Members: Suo, Zucai.

Subjects: Biochemistry

26. Shimko, John C. Synthetic Tools for the Preparation of Modified Histones.

Degree: PhD, Biochemistry Program, Ohio State, 2011, Ohio State University

► The eukaryotic genome is organized into nucleosomes consisting of 146 bp of… (more)

▼ The eukaryotic genome is organized into nucleosomes consisting of 146 bp of DNA wrapped around an octamer of histone proteins, two copies each of H2A, H2B, H3, and H4. Post-translational modification (PTM) of histones perturbs nucleosome structure and dynamics thereby regulating important biological processes including transcription, replication and DNA repair. To understand these processes, we have developed synthetic tools for the preparation of homogenous samples of modified histones. We established a novel ligation-desulfurization system for the preparation of modified histone H4 proteins enabling acetylation and phosphorylation adjacent to the C-terminus of the protein while retaining the native protein sequence. Modified H4 proteins were reconstituted into nucleosomes and nucleosome arrays. The effect of lysine 77 and 79 acetylation on nucleosome array stability was assessed. Furthermore, we demonstrated that the simultaneous incorporation of eight acetylated lysines within the LRS and dyad regions of the nucleosome does not significantly impact the structure or stability of the nucleosome. We introduced the total synthesis of histone H3 acetylated at lysine 56 (H3-K56ac) via the chemoselective condensation of three peptide segments prepared by manual solid phase peptide synthesis (SPPS) with Boc chemistry. Non-native cysteine residues at sites of ligation were converted to native alanine residues by free-radical-desulfurization. Reconstitution and characterization of H3-K56ac semi-synthetic nucleosomes revealed that the introduced modification increases DNA site-accessibility and protein invasion of the nucleosome. We developed a reversible protection strategy for 3,4-diaminobenzoic acid (Dbz), a Fmoc compatible thioester precursor, which allows for the use of highly activating conditions and acetyl capping during Fmoc peptide synthesis. Dbz protection maximizes product yield while minimizing the formation of deletion products. Further, we demonstrated the novel site-specific derivitization of the unsubstituted Dbz amine with biophysical probes including biotin and fluorophores. Finally, we adapted our initial total synthesis strategy for modified histone H3 to allow for the automated synthesis of a library of ~45 residue-modified peptide segments in high yield and purity by Fmoc chemistry. The combination of automated peptide synthesis protocols with sequential NCL allows for a widely accessible strategy for the combinatorial preparation of differentially-modified histone proteins suitable for biophysical characterization. Advisors/Committee Members: Ottesen, Jennifer J.

Subjects: Biochemistry

Keywords: histone; post-translational modification; peptide synthesis; native chemical ligation; total synthesis

27. Singh, Mamata. Insights into the Renal Protective Mechanisms of mRNA Binding Protein HuR.

Degree: PhD, Biochemistry Program, Ohio State, 2011, Ohio State University

► HuR is a ubiquitously expressed protein that resides primarily in nuclei under… (more)

▼ HuR is a ubiquitously expressed protein that resides primarily in nuclei under normal growth conditions but translocates to the cytosol during stress, where it binds and stabilizes a select subset of mRNAs bearing adenine and uridine-rich sequences in their 3’ untranslated regions. To clarify HuR’s role in renal stress, we knocked down or overexpressed it in proximal tubule cell lines that were subjected to ATP depletion, an in vitro procedure that mimics effects of ischemic injury to native kidneys. HuR was found to be protective against apoptosis that occurs when proximal tubule cells are ATP depleted. These results suggest that HuR’s function in native proximal tubules may be protective against ischemic stress. To understand what other cell survival signaling pathways might be induced by HuR, PCR array analysis was used to compare gene expression levels in ATP-depleted cells with normal or suppressed levels of HuR. Here, we show that in renal proximal tubule cells, HuR performs a central role in cell survival by amplifying Akt signaling in a positive feedback loop. Key to this feedback loop is HuR-mediated stabilization of mRNA encoding Grb10, an adaptor protein that enhances Akt activity by aiding in its transport to sites of activation. Under normal growth conditions, stimulation of Akt by HuR-Grb10 interactions then activates NFkappaB, which further enhances HuR expression at a transcriptional level. This study demonstrates a central role for HuR in cell survival mechanisms and reveals how only modest changes in HuR levels below or above normal can be amplified, resulting in cell death, or alternately, cellular transformation. The data presented here show that the expression of HuR during ATP depletion and recovery is upregulated by both transcriptional and translational mechanisms. It was previously demonstrated that HuR mRNA is expressed as two isoforms with vastly different 5’ untranslated regions and different translatabilities. Here we have demonstrated that one of these mRNAs is responsive to the BMP-7/Smad 1/5/8 signaling pathway, is more highly expressed during recovery from ATP depletion, and is the only form capable of being translated during ATP depletion. We also demonstrated that translation of the alternate form occurs during normal growth and recovery, and occurs through regulation by a post-transcriptional control element (PCE) present in its 5’ untranslated region. We further hypothesize that the differential translation of the HuR isoforms may be due to their alternate targeting to polyribosomes or stress granules. Stress granules are sites of aborted translation formed during various types of cell stress and recovery, including heat shock, arsenite poisoning, and UV-irradiation. We demonstrated that ATP-depleted epithelial cells form stress granules and activated their upstream kinases presumably for the purpose of regulating mRNA stability and translatability. During ATP depletion, some HuR mRNA is stored temporarily in stress granules. We hypothesize that this may be to avoid aberrant translation of HuR, which can lead to tumorigenicity. The presence of a PCE imposes another level of regulation on the translation of HuR and thus the translation of HuR is controlled during normal, stressed, and recovered conditions. Advisors/Committee Members: Lee, Beth S.

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

Keywords: apoptosis; HuR; acute kidney injury; stress granules; mRNA stability; Akt; Grb10

28. Smith, Brian A. Mechanistic insights into a reverse polymerase.

Degree: PhD, Biochemistry Program, Ohio State, 2012, Ohio State University

► tRNAHis guanylyltransferase (Thg1) is a highly conserved enzyme found in all domains… (more)

▼ tRNAHis guanylyltransferase (Thg1) is a highly conserved enzyme found in all domains of life including higher eukaryotes. Thg1 catalyzes the incorporation of a single guanosine residue to the -1 position (G-1) of tRNAHis, using an unusual 3'-5' nucleotidyl transfer reaction. G-1 serves as a necessary recognition element for histidyl-tRNA synthetase and is conserved among nearly all tRNAHis species. Thg1 is the only known enzyme that adds nucleotides in the 3'-5' direction and shares no identifiable sequence homology to any other known enzyme, thus its molecular mechanism is unknown. However, the high resolution crystal structure of Thg1 reveals remarkable structural similarities between canonical DNA/RNA polymerases and eukaryotic Thg1, suggesting that 3'-5' and 5'-3' nucleotide addition reactions share a common evolutionary origin. G-1 addition occurs via a complex mechanism involving three steps. In the first step, Thg1 activates the 5' end of tRNAHis by formation of an adenylylated intermediate via a 5' – 5' phosphoanhydride bond (App-tRNAHis). Subsequently, Thg1 utilizes the 3'-OH of GTP to attack the intermediate in a nucleotidyltransfer step yielding AMP and resulting in the addition of a single GTP to the 5' end of the tRNA. In the final step Thg1 removes pyrophosphate from the G-1 residue in a pyrophosphatase step yielding mature G-1-containing tRNAHis. A complete understanding of this complex reaction mechanism requires isolation and characterization of each catalytic step individually. I used transient kinetic assays to measure the pseudo-first order forward rate constants for each step of this reaction (kaden, kntrans and kppase). Using this kinetic framework in conjunction with data from the crystal structure of nucleotide-bound Thg1, we identified conserved residues involved in nucleotide binding and the adenylylation step of the G-1 addition reaction. A kinetic investigation of the third, pyrophosphate removal, step of the reaction reveals base pair-dependent effects on the rate of this step that affect the ability of the enzyme to add multiple nucleotides to some tRNA substrates. We also identified residues that when altered to alanine, cause severe defects in tRNA binding which are localized to a region distant from the putative active site, suggesting the possibility of structural rearrangements induced upon tRNA binding. Advisors/Committee Members: Jackman, Jane E.

Subjects: Biochemistry

29. Suh, Sung-Suk. A STUDY OF MICRORNAS ASSOCIATED WITH MULTIPLE MYELOMA PATHOGENESIS AND MICORRNAS/TP53 FEEDBACK CIRCUIT IN HUMAN CANCERS, MULTIPLE MYELOMA AND GLIOBLASTOMA MULTIFORME.

Degree: PhD, Biochemistry Program, Ohio State, 2012, Ohio State University

► MicroRNAs (miRNAs) are single-stranded RNAs of 19-25 nucleotides in length and play… (more)

▼ MicroRNAs (miRNAs) are single-stranded RNAs of 19-25 nucleotides in length and play a crucial role in regulating gene expression though post-transcriptional gene silencing which leads to mRNA degradation or translational repression. Recently, miRNAs are increasingly implicated in regulating cancer initiation and progression. Here, we first tried to understand the roles of miRNAs in the pathogenic progress of multiple myeloma (MM), a plasma cell malignancy. In profiling assay of miRNA expression in MM cell lines and CD138+ bone marrow plasma cells (PCs) from subjects with MM, monoclonal gammopathy of undetermined significance (MGUS), and normal donors, we identified overexpression of miR-21, miR-106b-25 cluster, miR- 181a and b in MM and MGUS samples with respect to healthy PCs. Furthermore, two miRNAs, miR-19a and 19b, that are part of the miR-17-92 cluster, were shown to down regulate expression of SOCS-1. We also identified p300-CBP-associated factor, a gene involved in p53 regulation, as a bona fide target of the miR-106b-25 cluster, miR-181a and b. Xenograft studies using human MM cell lines treated with miR-19a and b, and miR-181a and b antagonists resulted in significant suppression of tumor growth in nude mice. In summary, we have described a MM miRNA signature, which includes miRNAs that modulate the expression of proteins critical to myeloma pathogenesis. Next, we investigated the role of miRNAs in the p53 regulatory loop in human cancers, especially, Mutiple Myeloma (MM) and Glioblastoma Multiforme (GBM). In multiple myeloma (MM), we provide evidence that miR-192, 194, and 215, which are downregulated in a subset of newly diagnosed MMs, can be transcriptionally activated by p53 and then modulate MDM2 expression. Furthermore, ectopic re-expression of these miRNAs in MM cells increases the therapeutic action of MDM2 inhibitors in vitro and in vivo by enhancing their p53-activating effects. In addition, miR-192 and 215 target the IGF pathway, preventing enhanced migration of plasma cells into bone marrow. On the other hand, in human Glioblastom (GBM) two miRNAs, miR-25 and -32, are identified as p53-repressed miRNAs via p53-dependent, negative regulation of their transcriptional regulators, E2F1 and MYC. In addition, they result in p53 accumulation by directly targeting MDM2 and TSC1, negative regulators of p53 and the mTOR pathway, respectively, leading to inhibition of cellular proliferation through cell cycle arrest. Significantly, overexpression of transfected miR-25 and-32 in GBM cells inhibited growth of the GBM cells in mouse brain in vivo. Altogether, these results define microRNAs as positive regulators of p53 underscoring their role in tumorigenesis in MM and GBM. Advisors/Committee Members: Croce, Carlo M.

Subjects: Biology

Keywords: microRNA, TP53, Multiple Myeloma, Glioblastoma Multiforme

30. Sullivan, Brandon Joseph. Engineering Proteins from Sequence Statistics: Identifying and Understanding the Roles of Conservation and Correlation in Triosephosphate Isomerase.

Degree: PhD, Biochemistry Program, Ohio State, 2011, Ohio State University

► The structure, function and dynamics of proteins are determined by the physical… (more)

▼ The structure, function and dynamics of proteins are determined by the physical and chemical properties of their amino acids. Unfortunately, the information encapsulated within a position or between positions is poorly understood. Multiple sequence alignments of protein families allow us to interrogate these questions statistically. Here, we describe the characterization of bioinformatically-designed variants of triosephosphate isomerase (TIM). First, we review the state-of-the-art for engineering proteins with increased stability. We examine two methodologies that benefit from the availability of large numbers - high-throughput screening and sequence statistics of protein families. Second, we have deconvoluted what properties are encoded within a position (conservation) and between positions (correlations) by designing TIMs in which each position is the most common amino acid in the multiple sequence alignment. We found that a consensus TIM from a raw sequence database performs the complex isomerization reaction with weak activity as a dynamic molten globule. Furthermore, we have confirmed that the monomeric species is the catalytically active conformation despite being designed from 600+ dimeric proteins. A second consensus TIM from a curated dataset is well folded, has wild-type activity and is dimeric, but it only differs from the raw consensus TIM at 35 nonconserved positions. These two TIMs differ in the fraction of dataset sequences from eukaryotes and prokaryotes. These distribution differences have led to the breaking and altering of networks of statistical correlations at nonconserved positions which we demonstrate with mutual information and subset perturbation calculations. Additionally, we show that the curated consensus TIM is an extreme thermostable enzyme. The protein remains half folded at 95 °C and may be the only TIM to completely refold after thermal denaturation. Third, we wished to understand the determinants of protein stability – one of biochemistry's most difficult questions. It has been shown that consensus mutations improve the stability of native proteins approximately half the time, but there is no a priori technique to predict which consensus mutations will be stabilizing. We have developed a double-sieve filter that selects stabilizing mutations based on extent of conservation and statistical independence from other positions within the multiple sequence alignment. These two mathematical tests reliably predict stabilizing mutations with greater than 90% accuracy. The statistical algorithm was used to select 15 consensus mutations that together, significantly improved the melting temperature of wild-type TIM . Finally, we designed and characterized a model system for testing the effects of statistically correlated residues. The TIM-knockout from the Keio Collection was engineered for T7 expression and tested for TIM activity complementation. The single gene knockout exhibits differential growth that correlates well to in vitro specific activities. The design and characterization of two libraries are proposed to test the relationship between correlations and protein fitness. Advisors/Committee Members: Magliery, Thomas.

Subjects: Biochemistry; Bioinformatics; Biophysics; Molecular Biology

Keywords: Protein Engineering; Protein Sequence Statistics; Triosephosphate Isomerase

[1] [2]