Department: Molecular, Cellular and Developmental Biology ![[clear]](close-x.png "Remove this limiter")
51 matches in the database.
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1.
Abdulrahman, Basant Ahmed.
The Role of Autophagy in Burkholderia cenocepacia Infection.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2012, Ohio State University
► Cystic fibrosis (CF) is the most common inherited lethal disease of Caucasians…
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▼ Cystic fibrosis (CF) is the most common inherited lethal disease of Caucasians which results in multi organ dysfunction. However, 85% of the deaths are due to pulmonary infections. Infection by Burkholderia cenocepacia (B. cenocepacia) is a particularly lethal threat to CF patients because it causes severe and persistent lung inflammation and is resistant to nearly all available antibiotics. In this dissertation, we aimed to elucidate a strategy for controlling B. cenocepacia infection in CFTR delF508 (delF508) mouse model. We found that, in delF508 macrophages, B. cenocepacia persists in vacuoles that do not fuse with the lysosomes and mediates increased production of the inflammatory cytokine (IL-1β). It is believed that intracellular bacterial survival contributes to the persistence of the bacterium. Autophagy is a basic fundamental survival mechanism. An autophagosome is a compartment which engulfs non-functional organelles and parts of the cytoplasm then delivers them to the lysosome for degradation to produce nutrients during periods of starvation or stress. Here we showed for the first time that in wild-type macrophages (WT), many B. cenocepacia reside in autophagosomes that fuse with lysosomes at later stages of infection. Accordingly, association and intracellular survival of B. cenocepacia are higher in delF508 macrophages than in WT macrophages. Interestingly, B. cenocepacia down-regulates autophagy genes in WT and delF508 macrophages. Here, we demonstrate that the autophagy-stimulating agent, rapamycin markedly decreases B. cenocepacia infection in vitro by enhancing the clearance of B. cenocepacia via induced autophagy. In vivo, rapamycin decreases bacterial burden in the lungs of CF mice and drastically reduces signs of lung inflammation. As a second strategy to stimulate autophagy in delF508 macrophages, was to target SQSTM1/p62. p62 is a ubiquitin binding adaptor protein which is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 downregulation and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, downregulation of p62 in delF508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cenocepacia intracellular growth. Interestingly, the depletion of p62 from delF508 macrophages results in the release of the autophagy molecule beclin1 (BECN1), from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cenocepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cenocepacia infection in WT and delF508 macrophages. Therefore, targeting p62 inside the lung of CF patients could be a novel approach to control B. cenocepacia infection. Together, these studies reveal that if efficiently activated, autophagy is capable of controlling B. cenocepacia infection and the associated inflammation.
Advisors/Committee Members: Amer, Amal.
Subjects: Biochemistry; Biology; Biomedical Research; Immunology; Microbiology; Molecular Biology
Keywords: Autophagy, Burkholderia cenocepacia, p62
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2.
Ball, Sarah Lynnette.
Small molecule inhibitors, LLL12 and celecoxib, effectively inhibit STAT3 phosphorylation, decrease cellular viability and induce apoptosis in medulloblastoma and glioblastoma cell lines.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2011, Ohio State University
► Tumors of the central nervous system represent a major source of cancer-related…
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▼ Tumors of the central nervous system represent a major source of cancer-related deaths, with medulloblastoma and glioblastoma being the most common malignant brain tumors in children and adults respectively. While significant advances in treatment have been made, with the 5-year survival rate for medulloblastoma at 70-80%, treating patients less than 3 years of age still poses a problem due to the deleterious effects of radiation on the developing brain and the median survival for patients with glioblastoma is only 15 months. The transcription factor, STAT3, has been found constitutively activated in a wide variety of cancers and in recent years it has become an attractive therapeutic target. Previously, our collaborators designed a non-peptide small molecule STAT3 inhibitor, LLL12, using structure-based design. Here we show that LLL12 can inhibit STAT3 phosphorylation, decrease cell viability and induce apoptosis in medulloblastoma and glioblastoma cell lines with elevated levels of p-STAT3 (Y705). IC50 values for LLL12 were found to be between 1.07μM and 5.98μM in the five cell lines expressing phosphorylated STAT3. STAT3 target genes were found to be downregulated and a decrease in STAT3 DNA binding was observed following LLL12 treatment, indicating that LLL12 is an effective STAT3 inhibitor. LLL12 was also able to inhibit colony formation and wound healing and decreased IL-6 and LIF secretion. Our results suggest that LLL12 is a potent STAT3 inhibitor and that it may be a potential therapeutic treatment for medulloblastoma and glioblastoma. The process of developing a successful drug, getting it to clinical trials and gaining FDA approval takes years with the majority of drugs never making it past the first stages of development. This long and costly process has spurred scientists to reexamine some of the FDA approved drugs all ready on the market for possible chemotherapeutic potential. The nonsteroidal anti-inflammatory drug celecoxib is one such drug. Several studies have reported that celecoxib is able to treat and even prevent colon cancer, as well as reduce the risk of breast, prostate and lung cancer. Using computer modeling, we demonstrated that celecoxib has the ability to bind to STAT3 and we examined the affects of celecoxib on medulloblastoma and glioblastoma cells in vitro. Celecoxib was able to decrease cellular viability, inhibit STAT3 phosphorylation and induce apoptosis with IC50 values ranging from 35.97μM to 45.58μM. Celecoxib treatment also resulted in a decrease in the expression of STAT3 target genes and inhibited wound healing and colony formation. These results suggest that celecoxib is able to inhibit STAT3 and merits further investigation as a possible treatment for medulloblastoma and glioblastoma.
Advisors/Committee Members: Lin, Jiayuh.
Subjects: Biology; Molecular Biology; Oncology
Keywords: STAT3; LLL12; celecoxib; medulloblastoma; glioblastoma
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3.
Bebee, Thomas Wayne.
Temporally inducible SMN expression and splicing modulation of the SMN2 gene in SMA mouse models.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2012, Ohio State University
► Proximal spinal muscular atrophy (SMA) is a progressive neurodegenerative disease that is…
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▼ Proximal spinal muscular atrophy (SMA) is a progressive neurodegenerative disease that is caused by low levels of Survival Motor Neuron (SMN) protein. SMN protein is encoded by two genes in humans, SMN1 and SMN2. SMA patients have lost SMN1 expression by mutation or deletion, and SMN2 only partially compensates for SMN1 due to inefficient splicing of exon 7. The inefficiency of SMN2 exon 7 splicing is due to a C>T mutation in exon 7 that reduces exon definition by the splicing machinery. Increase survival in severe SMA mouse models by splicing correction of SMN2 and SMN replacement therapies has proven successful, however, the timing of SMN reintroduction will be crucial in therapeutic efficacy and may vary between severities of SMA. To address the temporal requirement of SMN replacement we generated an inducible SMN mouse model under the control of Cre-recombinase. Induction of SMN expression at varying time-points during development was verified using tamoxifen inducible Cre expression. We further evaluated the ability of our inducible SMN mouse to modulate survival of a severe SMA mouse model. Our lab previously developed a mild mouse model of SMA by introducing the SMN2 C>T mutation into the murine Smn gene, the homologue of SMN1. This mouse model exhibits motor function and behavioral deficits that recapitulate mild SMA. We further characterized these mice for neuropathological defects that may serve as markers for therapeutic benefit when SMN is reintroduced to determine if the same therapeutic window is required for mild SMA mouse models. Disease progression in severe SMA mice is associated with global alterations in splicing and induction of stress related genes. One such gene that is altered by splicing and increased expression is the hypoxia inducible hif3alpha gene. We observed induced skipping of SMN2 exon 7 under hypoxic stress and reduction of SMN protein. SMA mice treated with hyperoxia showed an improvement in motor function and inclusion of SMN2 exon 7. Respiratory assistance is a primary treatment in SMA and is capable of extending survival and quality of life of SMA patients. The role of hypoxic stress induced skipping of SMN2 exon 7 may shed light on additional therapeutic benefits of respiratory assistance in SMA patients, beyond compensation of progressively weakening respiratory muscles.
Advisors/Committee Members: Chandler, Dawn.
Subjects: Biology; Biomedical Research
Keywords: Spinal Muscular Atrophy; SMA; Survival Motor Neuron; RNA splicing
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4.
Biswas, Sayantanee.
Role of Protocadherins in Zebrafish Neural Development.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2012, Ohio State University
► Dynamically regulated cell-cell adhesion is an important component of morphogenesis. Protocadherins comprise…
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▼ Dynamically regulated cell-cell adhesion is an important component of morphogenesis. Protocadherins comprise a newly discovered and highly conserved group of surface glycoproteins within the cadherin superfamily. Although, the protocadherins belong to the same superfamily as the classical cadherins, which are bona fide cell adhesion proteins, the role of protocadherins in cell-cell adhesion is not well understood. Due to the distinct expression patterns of protocadherins members in the nervous system, their isoform diversity, and frequent presence at synaptic junctions, it has been postulated that differential adhesive interactions among protocadherin members may provide a mechanism for selective cell-cell interaction during brain circuit formation. Although very little is known about the cellular roles of protocadherins, an increasing number of clinical studies show that protocadherins are involved in a wide range of neurodevelopmental disorders: Protocadherin-19 in Epilepsy and Mental Retardation limited to Females (EFMR), Protocadherin-17 in schizophrenia, and Protocadherin-18 in intellectual impairment. In this thesis using the zebrafish model organism, we have investigated the mRNA expression patterns of non-clustered protocadherin family members: Protocadherin-17 (Pcdh17), Protocadherin-19 (Pcdh19) and Protocadherin-18b (Pcdh18b). Each of the above mentioned non-clustered protocadherins exhibit dynamic expression in nervous system. In addition, we have characterized the detailed neuronal expression pattern of clustered protocadherins using antibodies generated against zebrafish Pcdh-α and Pcdh-γ. To understand the biological function of Pcdh19 and Pcdh18b, we performed loss-of-function studies using morpholino-mediated knockdown. In vivo two-photon imaging of embryos injected with Pcdh19-specific morpholinos revealed convergent cell movement defects in the anterior neural plate, which ultimately led to malformed brains. Further investigation indicated that Pcdh19 functions synergistically with Neural-cadherin (N-cadherin), a member of the classical cadherin family, during these convergent cell movements. Functional studies show a role of Pcdh18b in filopodial dynamics and arborization of caudal primary motor axons. In an attempt to characterize the adhesive property Pcdh19, we have discovered a novel mechanism of adhesion mediated by purified complexes of Protocadherins and Classical cadherin ectodomains, which is non-compatible with the strand-swap mechanism of adhesion seen in classical cadherins. Although, Pcdh19 and Pcdh17 did not show intrinsic adhesive activity, they formed cis-complexes with the classical cadherin member, N-cadherin, to mediate trans-homophilic interactions. Interestingly, in the complex-mediated bead aggregation, the protocadherins partner determined the adhesive interface and adhesive specificity of the complex. We thus propose a mechanism by which different combinations of Pcdh-Ncad molecules could provide cellular recognition and specify neuronal connections by mediating differential adhesiveness between neurons expressing different repertoires of protocadherins and classical cadherins.
Advisors/Committee Members: Jontes, James.
Subjects: Neurosciences
Keywords: Protocadherin, zebrafish, neural development
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5.
Biyik, Rumeysa.
Understanding the Role of Stromal PTEN Regulated miR-101 and miR-130b in Tumor Microenvironment.
Degree: MS, Molecular, Cellular and Developmental Biology, 2012, Ohio State University
► Stromal cells from the tumor microenvironment significantly contribute tumor progression. However, the…
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▼ Stromal cells from the tumor microenvironment significantly contribute tumor progression. However, the signaling between tumor cells and stromal cells is not well-understood. Cancer associated fibroblasts (CAFs), a major component of tumor stroma, promote tumorigenesis by secreting growth factors, chemokines and pro-angiogenic factors. Previous study in our lab has shown that genetic inactivation of the tumor suppressor PTEN (Phosphatase and Tensin homologue) in stromal fibroblasts accelerates initiation and progression of mammary tumors. Transcriptome and proteome profiling have shown that deletion of PTEN in mammary fibroblasts triggers the oncogenic secretome which contributes to the transcriptional reprogramming of all cell types in the microenvironment. The role of miRNAs in cancer cells has been extensively studied but the role of miRNA in tumor-associated fibroblast cells remains to be elucidated. miRNA expression profiling shows that mir-101 and mir-130b are down-regulated in PTEN deleted mammary fibroblasts. In this study, we examined the role of mir-101 and mir-130b in PTEN deleted fibroblasts. We hypothesized that mir-101 and mir-130b would affect the fibroblasts secretome, thus influencing the other cells in tumor microenvironment. As a first step towards this goal, the conditioned media was collected from miR-101 and miR-130b re-expressing PTEN-deleted stromal fibroblasts in order to examine the affect on the other cells in tumor microenvironment, such as tumor cells and endothelial cells. Then, in silico analysis was performed to find putative targets of miR-101 and miR-130b. PDGFRA (platelet-derived growth factor receptor alpha) was found as a direct target of mir-130b. Paracrine PDGF signaling contributes tumor progression through recruitment of stromal cells such as fibroblasts, endothelial cells and immune cells to the tumor mass so we examined how PTEN deletion in mammary fibroblast influenced PDGF expression in other cells in tumor microenvironment.
Advisors/Committee Members: Ostrowski, Michael.
Subjects: Molecular Biology
Keywords: PTEN, Fibroblast, PDGFRA, Tumor Microenvironment, miR-101, miR-103b
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6.
Bosse, Kevin M.
A Framework Gene Regulatory Network Controlling Neural Crest Cell Diversification.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2010, Ohio State University
► The neural crest is a transient population of embryonic cells that diversifes…
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▼ The neural crest is a transient population of embryonic cells that diversifes into a wide variety of cell types including peripheral neurons, pigment cells and elements of the craniofacial skeleton. The mechanisms regulating neural crest cell diversification, however, remain incompletely understood. Previous studies using mutant zebrafish have indicated that the transcription factors foxd3 and tfap2a function early and differentially in the development of neural crest sublineages. We show that zebrafish foxd3zdf10;tfap2alow double mutant embryos completely lack all neural crest derivatives. While the initial induction of the neural crest is normal in these embryos, distinct neural crest sublineages fail to be specified. The failure of neural crest cell diversification is accompanied by a loss of neural crest SoxE family gene expression. Restoration of sox9a/b or sox10 function via misexpression differentially rescues sublineage specification and derivative differentiation. These results indicate the functional necessity of foxd3 and tfap2a for neural crest cell diversification and that this requirement is mediated in large part through the regulation of the expression of SoxE family genes. Our results represent a minimal genetic framework controlling neural crest cell diversification. Furthermore, we demonstrate the potential for other genes and regulatory elements to be involved in neural crest sublineage specification and have developed novel approaches to further investigate and identify these additional regulators.
Advisors/Committee Members: Henion, Paul.
Subjects: Biology; Genetics
Keywords: neural crest; cell diversification; zebrafish; cell fate specification
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7.
Cataldi, Marcela Patricia.
Diverse Effects of DNA Repair Pathways on the Outcome of Recombinant Adeno-Associated Virus (rAAV) Vector Gene Delivery.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2011, Ohio State University
► Because of Adeno-Associated Virus (AAV) nonpathogenicity and ability to promote sustained, long-term…
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▼ Because of Adeno-Associated Virus (AAV) nonpathogenicity and ability to promote sustained, long-term transgene expression in a wide variety of tissues including the brain, liver, muscle, retina, and vasculature, several recombinant AAV (rAAV) serotypes are emerging as attractive vectors for gene therapy. The linear DNA genomes of AAV are acted upon by multiple DNA repair and recombination pathways once released into the host nucleus. The palindromic terminal repeats (TR) on both ends of the AAV genome, which form DNA hairpin (HP) structures, serve as substrates for intramolecular and intermolecular recombination, leading to circularization and concatemer formation, respectively. Infrequently, AAV TRs can also recombine with the host genome, leading to chromosomal integration. However, the mechanism of recombination between AAV TRs is still unclear, and with respect to integration, recombination raises concern for genotoxicity. I have compared the fates of single-stranded rAAV (ssAAV) and self-complementary (scAAV) genomes in cell lines deficient in each of three signaling factors, ATM, ATR, and DNA-PKcs, which orchestrate major DNA double-strand break (DSB) repair pathways. The results suggest that there are qualitative and quantitative differences between both vectors in terms of their interactions with different host DNA repair and recombination pathways. A striking and unexpected observation from these experiments was that in cells deficient in ATM, transduction scored by GFP expression was increased relative to wild-type (WT) cells for both rAAV vectors. The augmented transduction was not reflected in Southern blots of nuclear vector DNA, suggesting that interactions with ATM lead to silencing in normal cells. The additional functional genomes in ATM-/- cells remained linear, and the number of circularized genomes was not affected by the mutation, consistent with compartmentalization of genomes into different DNA repair pathways. To investigate whether the TR HP has any specific effect on recombination, I compared DNA substrates containing the TR sequences in an open DNA duplex conformation or in a covalently closed hairpin conformation, or containing blunt DNA ends with no TR sequence. These DNA substrates were transfected into either WT or DNA-repair deficient cells. DNA molecules with the TR sequences constrained in the hairpin conformation at one or both ends were subject to a loss of gene expression, which was partially relieved in ATM-/- cells. This ATM-mediated effect was not due to a loss of DNA, suggesting that a single AAV TR in the hairpin conformation in cis is necessary and sufficient to mediate the ATM-dependent silencing of gene expression from rAAV genomes. The results also indicate that DNA substrates with AAV TR in a hairpin conformation circularize and integrate with the same efficiency as other forms of DNA ends in WT cells. Therefore, the specificity of the recognition of the HP structure of rAAV vector genomes by DNA repair/recombination pathways leads to loss of functional genomes rather than an enhancement of recombination. I further investigated rAAV recombination by comparing circularization and integration in dividing and non-dividing cultured cells. The results indicated that neither circularization nor integration requires active cell division, and that integration occurs rapidly after infection, over the same time period as circularization.
Advisors/Committee Members: McCarty, Douglas.
Subjects: Molecular Biology; Virology
Keywords: adeno-associated virus, gene therapy, DNA recombination, rAAV vector, DNA viral vectors
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8.
Chen, Wen-Yi.
Division of Labor Among Protein Subunits That Aid RNA Catalysis in Archaeal RNase P.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2010, Ohio State University
► Ribonuclease P (RNase P), a Mg2+-dependent catalytic ribonucleoprotein (RNP) complex, cleaves the…
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▼ Ribonuclease P (RNase P), a Mg2+-dependent catalytic ribonucleoprotein (RNP) complex, cleaves the 5'-leader from precursor tRNAs (pre-tRNAs). It is made up of a catalytic RNase P RNA (RPR) and a varying number of RNase P proteins (RPPs) depending on the source: one in bacteria, at least four and nine in archaea and eukarya, respectively. Since the four archaeal RPPs (POP5, RPP30, RPP21 and RPP29) have eukaryotic homologs, archaeal RNase P can be used as an experimental surrogate for the biochemically intractable eukaryotic variant. This dissertation documents a division of labor among archaeal RPPs that aid the cognate RPR's catalysis, and highlights their roles in chemical cleavage, substrate binding, cleavage-site selection and Mg2+ coordination. Previous studies established that archaeal POP5 and RPP30 function as a heterodimer (POP5•RPP30) as do RPP21 and RPP29 (RPP21•RPP29). I assessed the effect of these RPP pairs, either individually or together, on the cleavage step by using single-turnover kinetic measurements. The kinetic parameters obtained from these experiments showed that while POP5•RPP30 is solely responsible for enhancing the RPR’s rate of pre-tRNA cleavage by 60-fold, RPP21•RPP29 increases substrate affinity by 15-fold. We found that the protein-rich archaeal and eukaryal RNase P exhibit better fidelity than the bacterial counterpart while cleaving pre-tRNAGln and its mutant derivatives, which lack consensus structures/sequences. To dissect which archaeal RPPs contribute to this increased fidelity, I examined cis cleavage of pre-tRNAGln by Methanocaldococcus jannaschii (Mja) RPR, a strategy that allowed me to calculate the rates of correct and aberrant cleavage. I showed that RPP21•RPP29 rescued the RPR’s mis-cleavage without affecting the RPR’s rate of cleavage; in addition to promoting the correct cleavage, POP5•RPP30 enhanced cleavage rate by 600-fold for both correct and aberrant cleavage. Interestingly, RPP21•RPP29 specifically increases the rate of correct cleavage by 3-fold in the presence of POP5•RPP30 indicating a synergistic effect between the two RPP pairs. Collectively, these results suggest that protein-rich RNase P display better fidelity while allowing flexibility in substrate-recognition.Finally, I determined that deletion of a universally conserved uridine (-U) in a bulge-helix structure in archaeal RPR weakens Mg2+ coordination and lowers activity, a defect completely rescued by their cognate RPPs under multiple-turnover conditions. Cis cleavage of pre-tRNATyr by Mja -U RPR revealed an 80-fold decrease in the cleavage rate compared to the wildtype; both Mja RPP pairs partially ameliorate this defect and narrow the difference to ~12-fold, indicating their ability to coordinate catalytically relevant Mg2+ ions. In addition to archaeal RPPs rescuing an RPR mutation, we (in collaboration with the laboratory of Dr. Mark Foster, OSU) found that an N-terminal deletion mutant of RPP29, which fails to bind RPP21, is functional upon addition of RPR. Together, these findings illustrate the cooperative subunit interactions critical for generating the functional structure of archaeal RNase P. Overall, my results demonstrate the functional coordination among the RPR and different RPPs, and help rationalize why distinct protein cofactors were recruited by an ancient RNA enzyme during the transition from a primordial RNA to the extant RNP world.
Advisors/Committee Members: Gopalan, Venkat.
Subjects: Biochemistry
Keywords: Archaeal RNase P; RNase P protein; RPP; RNase P RNA; RPR
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9.
Chu, Hui-Yi.
Genome-wide Investigation of Cellular Functions for tRNA Nucleus-Cytoplasm Trafficking in the Yeast Saccharomyces cerevisiae.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2012, Ohio State University
► In eukaryotic cells tRNAs are transcribed in the nucleus and exported to…
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▼ In eukaryotic cells tRNAs are transcribed in the nucleus and exported to the cytoplasm for their essential role in protein synthesis. This export event was thought to be unidirectional. Surprisingly, several lines of evidence showed that mature cytoplasmic tRNAs shuttle between nucleus and cytoplasm and their distribution is nutrient-dependent. This newly discovered tRNA retrograde process is conserved from yeast to vertebrates. Although how exactly the tRNA nuclear-cytoplasmic trafficking is regulated is still under investigation, previous studies identified several transporters involved in tRNA subcellular dynamics. At least three members of the β-importin family function in tRNA nuclear-cytoplasmic intracellular movement: (1) Los1 functions in both the tRNA primary export and re-export processes; (2) Mtr10, directly or indirectly, is responsible for the constitutive retrograde import of cytoplasmic tRNA to the nucleus; (3) Msn5 functions solely in the re-export process. In this thesis I focus on the physiological role(s) of the tRNA nuclear retrograde pathway. One possibility is that nuclear accumulation of cytoplasmic tRNA serves to modulate translation of particular transcripts. To test this hypothesis, I compared expression profiles from non-translating mRNAs and polyribosome-bound translating mRNAs collected from msn5∆ and mtr10∆ mutants and wild-type cells, in fed or acute amino acid starvation conditions. Microarray data revealed that several amino acid biosynthetic pathways, including the sulfur assimilation, arginine biosynthesis, and leucine biosynthesis pathways, are primary targets of the tRNA trafficking processes. I confirmed the microarray data by both Northern and Western blot analyses. Levels of all tested target proteins involved in such amino acid biogenesis pathways are down-regulated when the tRNA nuclear import or re-export is disrupted. The steady state levels of target total RNAs are similar between wild-type cells and tRNA trafficking defective mutants. The data suggest that the reduction of target proteins most likely results from translation defects of the target mRNAs. This study provides information that tRNA nuclear-cytoplasmic dynamics is connected to amino acid biosynthesis via control at the level of translation.
Advisors/Committee Members: Hopper, Anita K.
Subjects: Cellular Biology; Genetics; Molecular Biology
Keywords: tRNA; export; import; Los1; Msn5; Mtr10; translation; polysome; microarray; sulfur; arginine; leucine
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10.
Cremer, Thomas John IV.
Mechanisms of Host-Defense Against Intracellular Bacterial Pathogens Through The PI3K/Akt Host Signaling Pathway.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2010, Ohio State University
► Intracellular bacterial pathogens have developed mechanisms to exploit host cells as a…
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▼ Intracellular bacterial pathogens have developed mechanisms to exploit host cells as a part of their lifecycle and these pathogens manipulate or suppress signaling events by the host to accomplish this. Elucidating host signaling and defense mechanisms is essential for advancing our understanding of the pathogenesis caused by many different microbes and for discovering novel therapeutic targets. Two different Gram-negative intracellular bacterial pathogens will be examined in the following chapters, Francisella tularensis and Burkholderia cenocepacia. Both organisms are capable of replicating within mononuclear phagocytes and thus subvert the immune clearance mechanisms of these cells. These bacterial pathogens also activate the PI3K/Akt signaling pathway, which regulates numerous cellular functions including immune responses. The focus of the following studies is to understand how the PI3K/Akt pathway is regulated during microbial infection and to investigate the role of this pathway in host-defense. The first part of this dissertation investigates the role of microRNAs as regulators of the immune response against Francisella. Previously it has been shown that activation of the PI3K/Akt pathway is beneficial to the host by enhancing NF-kappaB activation, the production of pro-inflammatory cytokines, promoting macrophage killing of bacteria, and overall host resistance to Francisella. These processes are all inhibited by the phosphatase SHIP, which limits Akt activation. Here it is shown that a microRNA, miR-155, negatively regulates SHIP to promote pro-inflammatory cytokine production following infection. The most interesting finding was that this microRNA is differentially induced between low virulence and high virulence Francisella subspecies. This study uncovers a key mechanism by which PI3K/Akt signaling is altered during the microbe-host interaction. In the second part of this dissertation the regulation of miR-155 during Francisella infection is explored in detail. This microRNA is essential for many immune functions and is also oncogenic. Therefore it is important to understand the regulation of this miR in a variety of contexts. Here it is shown that miR-155 is induced through an indirect response to infection and can be transferred through soluble bacterial factors. The induction requires NF-kappaB activation to up-regulate Fos and Jun transcription factors, which mediate transcription of the precursor to this microRNA. The third and final data section to this dissertation explores the role of PI3K/Akt signaling in the host-response to Burkholderia. This is a highly antibiotic resistant organism with limited therapeutic options. It causes an opportunistic respiratory infection in susceptible individuals who have a dramatic inflammatory response leading to progressive morbidity and mortality. Here it is shown that Akt promotes NF-kappaB activation and the production of pro-inflammatory cytokines from Burkholderia infected phagocytes. This occurs through a unique IKK-independent, GSK3beta-depenedent mechanism. Thus this provides a potential novel therapeutic target for the treatment of Burkholderia infections. The overall goal of these studies was to determine novel mechanisms of PI3K/Akt regulation during bacterial infection and to broaden the overall understanding of this pathway as a key regulator of host-defense.
Advisors/Committee Members: Tridandapani, Susheela.
Subjects: Immunology
Keywords: Francisella, Burkholderia, PI3K/Akt, SHIP, GSK3beta, miR-155
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11.
Dakhlallah, Duaa.
Cross-Talk Between Epigenetic Regulation And Mir-17~92 Cluster Expression In Idiopathic Pulmonary Fibrosis (IPF).
Degree: PhD, Molecular, Cellular and Developmental Biology, 2011, Ohio State University
► Interstitial lung disease/idiopathic pulmonary fibrosis (ILD/IPF) is the most progressive form of…
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▼ Interstitial lung disease/idiopathic pulmonary fibrosis (ILD/IPF) is the most progressive form of pulmonary fibrosis disorder and leads to death in patients afflicted. The etiology and pathogenesis of ILD/IPF in poorly understood with no known prevention or cure available. ILD/IPF is associated with increased expression of certain fibrogenic genes such as CTGF, VEGF, GM-CSF, and TSP-1; however, the precise mechanism responsible for the increased gene and protein expression is not known. Since there are a large number of genes differentially expressed in the lungs of patients with ILD/IPF compared to controls, we speculated that elements like microRNAs (miRNAs) may be involved. miRNAs, also known as miRs, are small regulatory RNAs that alter gene expression by causing degradation, translational repression, or epigenetic regulation resulting in a change in protein expression. Changes in miRNA expression are associated with up to 30% of cancers (Volinia et. al., 2010) and a variety of other diseases (Nana-Sinkam et. al., 2009). Using miRNA profiling, we identified a specific miRNA cluster that was reduced in expression in ILD/IPF lung tissue compared to normal lung tissue and lung tissue from patients with chronic obstructive pulmonary disease (COPD), the miR-17~92 cluster. The miR-17~92 polycistron cluster is ~ 1Kb located within the third intron of the open reading frame13q31.3 (C13orf25) which encodes six microRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92a-1). This locus is overexpressed in B-cell lymphomas and lung cancer. In ILD/IPF, miR-17~92 miRNA cluster targets genes, such as metalloproteinases, collagen, and transforming growth factor that are highly expressed. Our preliminary data indicated decreased expression of the miR-17~92 miRNA cluster family members in the lungs from patients with ILD/IPF. Notably, miR-19b and miR-20a decreased proportionally to disease severity (and pulmonary function) of ILD/IPF. In ILD/IPF lung tissue, miR-19b and miR-20a expression was reduced and its predicted targets, CTGF, VEGF, TGF-β, TSP-1 and Ets-1 were up-regulated. Importantly, we noticed that the 5’-region of the promoter is heavily occupied with CpG Islands (>90%) and sought to define if this area was affected by methylation in ILD/IPF, as the mechanism causing downregulation of the cluster expression in ILD/IPF. Our preliminary data suggested that this epigenetic silencing of miR-17~92 clusters was due to methylation of the promoter, and was increased with the severity of ILD/IPF. Indeed, we found this CpG island methylated in ILD/IPF and by relieving this methylation with 5’-aza-2'-deoxycytidine, enhanced cluster expression and decreased expression of the target genes. In addition, we found that the miR-17~92 cluster targeted DNA methyltransferases (DNMTs), which we believe are responsible for the methylation events in ILD/IPF. Interestingly, our data showed direct regulation of miR-17, miR-19b, miR-20a and miR-92 on the 3’UTR of DNMT-1 in lung fibroblasts isolated from patients with IPF, indicating a negative feedback mechanism of the cluster to its own promoter though an epigenetic modifier. In fact, inhibition of DNA methylation by 5’-aza-2'-deoxycytidine in our in vivo murine bleomycin-induced pulmonary fibrosis model recovered miR-17~92 cluster expression to that of normal lung tissue, rescued the animals’ lungs from fibrosis, and decreased fibrotic gene expression in their lungs. Thus, our data indicate that dysregulation of miRNA expression in IPF may contribute to the development and/or progression of the disease. 5’-aza-2'-deoxycytidine treatment, or miR-17~92 overexpression, could be a therapeutic intervention in ILD/IPF.
Advisors/Committee Members: Clay, Marsh.
Subjects: Biology; Biomedical Research; Molecular Biology
Keywords: IPF, miRNA, fibrosis, Lung desease, miR-17~92 cluster
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12.
Deatherage, Daniel E.
TGFΒ/SMAD4 Signaling and Altered Epigenetics Contribute to Increased Ovarian Cancer Severity.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2011, Ohio State University
► Ovarian cancer is the eighth most common cancer and is the fifth…
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▼ Ovarian cancer is the eighth most common cancer and is the fifth most common cause of cancer related death among women. Early stage ovarian cancer is very responsive to treatments and more than 93% of patients diagnosed with early stage disease achieve a five year survival rate. By contrast less than 30% of patients who are diagnosed with late stage disease achieve a five year survival rate, yet more than 60% of all cases present as late stage. Treatment is typically surgery followed by a chemotherapy regiment of a platinum-based chemotherapeutic and a taxane derivative. While this treatment plan works well for early stage disease, recurrent and late stage cancers are less responsive. Here we present work investigating the altered epigenetics and TGFΒ/SMAD4 signaling pathway in ovarian cancer in an effort to better understand the difference in disease severity at a molecular level. We have identified a microRNA hsa-mir-9-3 which is epigenetically repressed by DNA methylation in primary ovarian cancer patients. Quantitative analysis of DNA methylation in the CpG island which contains the hsa-mir-9-3 microRNA revealed significant hypermethylation in both patient samples and cell lines compared to normal tissue. Functional studies reveal that the repression of this microRNA leads to increased proliferation rates and a decrease in apoptosis. We believe that the hypermethylation of the hsa-mir-9-3 locus serves as a novel biomarker for ovarian cancer. TGFΒ/SMAD4 signaling is commonly dysregulated in ovarian cancers while being a key growth inhibition signal for the ovarian surface epithelium during menstruation. Here we present a genome-wide profile of SMAD4 binding by ChIP-sequencing following TGFΒ stimulation in the ovarian cancer cell line A2780. We believe this to be the first truly genome-wide profiling of SMAD4 binding using next generation sequencing approaches in an ovarian cancer model. Comparison of ChIP-seq results with previously reported ChIP-chip studies show dramatic biological and technical differences including more than 70% of all SMAD4 binding loci being more than 10kb away from the nearest transcription start site. Gene expression analysis following TGFΒ stimulation revealed a group of 318 genes whose expression changed following SMAD4 binding to the distal promoter of the gene. Of those 318 genes, a subset of them was used to predict patient survival in two independent patient cohorts. These results suggest that the loss of long distance SMAD4 gene regulation following TGFΒ stimulation may play a key role in ovarian carcinogenesis. Additionally, we identified a novel biomarker, CLDN11, whose epigenetic repression is associated increased cisplatin resistance in a tissue culture model system. Examination of CLDN11 expression levels in a previously reported patient cohort revealed lower expression levels associated with increased tumor grade. Finally, loss of CLDN11 expression is associated with increased cellular motility. In conclusion we have investigated and correlated several different epigenetic and signaling abnormalities associated with an increase in the severity of ovarian cancer while demonstrating the importance of recent technological advances in genome-wide methodologies. Together these results are likely to aid in both future discovery methods and patient prognosis and treatment.
Advisors/Committee Members: Huang, Tim.
Subjects: Bioinformatics; Biology; Biomedical Research; Cellular Biology; Molecular Biology
Keywords: Ovarian Cancer; Epigenetics; DNA hypermethylation; hsa-mir-9-3; Cisplatin resistance; CLDN11; TGFΒ; SMAD4; ChIP-sequencing; Survival data
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13.
Deol, Yadwinder S.
ROLE OF PSORIASIN (S100A7) IN ESTROGEN RECEPTOR POSITIVE BREAST CANCERS.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2012, Ohio State University
► Psoriasin (S100A7) is expressed in several epithelial malignancies including breast cancer. Although…
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▼ Psoriasin (S100A7) is expressed in several epithelial malignancies including breast cancer. Although S100A7 is associated with the worst prognosis in estrogen receptor α-negative (ERα-) invasive breast cancers, its role in ERα-positive (ERα+) breast cancers is relatively unknown. We investigated the significance of S100A7 in ERα+ breast cancer cells and observed that S100A7-overexpression in ERα+ breast cancer cells, MCF7 and T47D, exhibited decreased migration, proliferation, and wound healing. These results were confirmed in vivo in a nude mouse model system. Mice injected with S100A7-overexpressing MCF7 cells showed significant reduction in tumor size compared to mice injected with vector control cells. Mechanistic studies revealed that S100A7 mediates the tumor-suppressive effects by regulating β-catenin/TCF4 and p53 pathways. We observed down-regulation of β-catenin, p-GSK3β, TCF4, cyclin D1, and c-myc in S100A7-overexpressing ERα+ breast cancer cells. In addition, we observed increased expression of GSK3β. Treatment with GSK3β inhibitor CHIR 99021 increased the expression of β-catenin and its downstream target c-myc in S100A7-overexpressing cells. Tumors derived from mice injected with S100A7-overexpressing MCF7 cells also showed reduced activation of the β-catenin/TCF4 pathway. Our results also demonstrated that S100A7-overexpression in MCF7 cells increased the stability of p53 compared to vector control cells. Co-immunoprecipitation studies revealed that S100A7 binds to and co-localize with p53 in the nucleus of S100A7-overexpressing cells. In addition, we found that phosphorylation of p53 at Serine-15 residue was enhanced in S100A7-overexpressing MCF7 cells. Serine-15 is known to stabilize and activate p53 during cellular stresses. Further, real time PCR p53 array and immunoblotting experiments showed that S100A7-overexpression in MCF7 cells up-regulated ATR and its downstream molecules p-Chk1 and p-Chk2 which are known to phosphorylate serine-15 of p53. We further evaluated the role of p53 and S100A7 in vivo by generating a mouse model which was deficient in p53 expression but expressed murine S100A7 (mS100A7A10) in the mammary glands under doxycycline treatment. After 10 weeks of doxycycline treatment, we observed the development of spontaneous tumors in the fourth inguinal mammary gland of the mouse. S100A7-overexpression in MCF7 cells also exhibited decreased actin polymerization as evident from decreased formation of migratory structures. Actin staining revealed F-actin expression on the plasma membrane in vector control cells whereas in S100A7-overexpressing cells, actin staining was intracellular. Both serum and EGF-induced migration were reduced in S100A7-overexpressing cells and upon analysis of the mechanisms that regulate actin polymerization; we observed reduced activation of EGFR, Rac1 and cofilin. In conclusion, our studies reveal for the first time that S100A7-overexpressing ERα+ breast cancer cells exhibit tumor suppressor capabilities by down-modulation of the β-catenin/TCF4 and p53 pathways both in vitro and in vivo. Our results show that S100A7 stabilizes p53 in the nucleus which then activates the stress induced pathway. Moreover, we also show that S100A7 modulates the cytoskeleton by regulating actin polymerization. Since S100A7 has been shown to enhance tumorigenicity in ERα- cells, our studies suggest that S100A7 may possess differential activities in ERα+ compared with ERα- cells.
Advisors/Committee Members: Ganju, Ramesh.
Subjects: Oncology
Keywords: Psoriasin; S100A7; Breast Cancer; Beta-catenin; GSK3beta; TCF4; Actin Polymerization; p53; Transgenic Mice
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14.
Diaz, Greetchen M.
Targeting of Peripherally Associated Proteins to the Inner Nuclear Membrane in Saccharomyces cerevisiae: The Role of Essential Proteins.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2012, Ohio State University
► The nuclear envelope (NE) is composed of the inner nuclear membrane (INM)…
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▼ The nuclear envelope (NE) is composed of the inner nuclear membrane (INM) and the outer nuclear membrane (ONM) which is contiguous with the endoplasmic reticulum (ER). The appropriate location of NE proteins is important in cells. Integral INM proteins are proposed to be synthesized at the ER and then translocated through the nuclear pore complex (NPC). In contrast, peripherally associated INM proteins are proposed to follow a targeting mechanism to the nucleus that is similar to nucleoplasmic proteins. Our research aims to understand the mechanism of targeting of peripherally associated proteins to the INM. We employed yeast as a genetic model and the tRNA modification enzyme, Trm1-II, as a reporter. We screened a collection of temperature sensitive (ts) mutants for defects in galactose-inducible Trm1-II-GFP (Gal-Trm1-II-GFP) INM localization. We found that the majority (46%) of the ts mutations affecting Gal-Trm1-II-GFP localization were in genes that encode proteins involved in ER-Golgi homeostasis. Interestingly, about 35% of the mutated essential genes encode components of the Spindle Pole Body (SPB). In the SPB ts mutants, at the non-permissive temperature, Gal-Trm1-II-GFP accumulates as a spot that localizes to the ER, rather than being evenly distributed around the entire INM as in wild-type cells. Following the dynamics of Gal-Trm1-II-GFP we learned that its inappropriate distribution results from a failure to move from the initial contact with the NE (ONM) throughout the INM. Gal-Trm1-GFP accumulates to the ER with time, suggesting that this might be the initial Trm1-II tethering site. Surprisingly, SPB defects also affect targeting of an integral INM protein, but not a soluble nucleoplasmic protein which indicates that there is no defect in import and that appropriate SPBs are required for INM targeting of both integral and peripheral INM proteins, but not nucleoplasmic proteins. Our evidence suggests Gal-Trm1-II-GFP is alternatively transported via soluble mechanism when unable to tether to the ER. We propose a novel mechanism for peripherally associated INM proteins that combines targeting mechanisms for both integral and soluble proteins. We also learned that INM maintenance of Gal-Trm1-II-GFP was altered in SPB defective cells, which suggests that a general defect at the membrane that forms the ER and the NE occurs in SPB defective cells. The possible role of the SPB based in INM targeting and maintenance is discussed.
Advisors/Committee Members: Hopper, Anita K.
Subjects: Genetics
Keywords: yeast, inner nuclear membrane, protein targeting, spindle pole body, peripheral protein, essential genes
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15.
Doueiri, Rami.
CHARACTERIZATION OF THE HUMAN T-CELL LEUKEMIA VIRUS TYPE-2 P28 ACCESSORY PROTEIN.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2012, Ohio State University
► Human T-cell leukemia virus type 1 (HTLV-1) causes malignancy, several neurodegenerative and…
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▼ Human T-cell leukemia virus type 1 (HTLV-1) causes malignancy, several neurodegenerative and inflammatory diseases. On the other hand, HTLV-2 is less pathogenic with few cases of neurodegenerative diseases. Comparative studies of the regulatory genes of HTLV elucidated the role of these proteins in the viral life cycle and in part explained the different pathology of both viruses. However, increasing amount of evidence describes an essential role for the accessory genes in viral pathogenesis. The accessory protein p30 of HTLV-1 and p28 of HTLV-2 are encoded from ORF-II and share some amino acid homology. They are post-transcriptional negative regulators of viral replication, and act by retaining tax/rex mRNA in the nucleus. However, unlike p30, p28 is devoid of transcriptional activity. Several binding proteins, functional domains and post-translational modification have been identified for p30; however, no such studies have been performed for p28. Identification and characterization of p28 structure/function would facilitate comparative studies between p28 and p30, allow us to better understand the role of accessory genes in the viral life cycle, and ultimately to better understand the different pathobiology of HTLV-1 and HTLV-2. In Chapter 2, we identified binding partners of p30 and p28 using mass spectrometry, and then verified our data using molecular methods. We identified NIP30 as a unique binding partner for p30, while hnRNP H1 solely interacted with p28, and PRMT5 interacted with both proteins. We then knocked down PRMT5 in vivo to assess its role in the viral life cycle. Our data suggest that PRMT5 is involved in post-transcriptional control of HTLV-2 replication, while its effect on HTLV-1 might be at the level of DNA damage and cell cycle control. In Chapter 3, we identified and characterized the effect of phosphorylation on p28 in vivo. We conducted phosphoryl mapping of mammalian-expressed p28 protein using mass spectrometry and substitution mutational analysis. We identified seven phosphorylation events on p28 at Ser-33, Ser-160, Ser-170, Ser-172, Thr-199, Ser-200 and Ser-203. We evaluated the functional significance of these phosphorylation sites and found that phosphorylation at Ser-160 and Thr-199 reduced the ability of p28 to dimerize, while phosphorylation did not affect the post-transcriptional activity of p28 or its ability to interact with hnRNP H1 or PRMT5. In Chapter 4, we identified functional domains of p28. We created six p28 deletion mutations p28d”N (deletion of amino acid (aa) (d”)1-50), p28d”M1(d”51-100), p28d”M2 (d”101-150), p28d”C (d”151-216), p28d”M2-C (d”101-216) and p28d”M1-M2-C(d”51-216), and evaluated the functional significance of these domains. We identified a tripartite nuclear localization sequence (NLS) in regions N-M2-C (aa 1-50 and 100-216), while p28 interaction with hnRNP H1 requires domains N (aa 1-50) and C (aa 151-216), and its dimerization requires domains M1 (aa 51-100) and C (aa 151-216). Finally, we identified that p28 C terminus (150-216) exerts an inhibitory effect on p28 post-transcriptional function. This work is the first to identify p28 binding partners, phosphorylation sites and functional domains, providing insight into p28 mechanism of action to better understand its role in HTLV-2 replication and pathogenesis
Advisors/Committee Members: Green, Patrick.
Subjects: Virology
Keywords: HTLV-1; HTLV-2; p28; p30; PRMT5; hnRNP H1; phosphorylation; functional domains
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16.
Feller, Antje Christin.
ROLE OF THE MAIZE TRANSCRIPTION FACTOR R IN THE REGULATION OF ANTHOCYANIN BIOSYNTHESIS.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2010, Ohio State University
► The maize flavonoid biosynthetic pathway is one of the best characterized plant…
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▼ The maize flavonoid biosynthetic pathway is one of the best characterized plant model systems to study the combinatorial regulation of gene expression. Flavonoids are important secondary metabolites and important for the plant, such as in the case for anthocyanins as attractants to pollinators as well as for humans due to a number of biological activities. Anthocyanins in maize are regulated by the cooperation of the R2R3-MYB domain protein C1 and the basic helix-loop-helix (bHLH) protein R. In contrast, phlobaphene pigments, derived from a separate flavonoid biosynthetic branch, are regulated by the R2R3-MYB domain protein P1, which can activate transcription in the absence of a (known) bHLH. Our laboratory has established that the bHLH transcription factor R plays a key role in determining the biological specificity of C1. I describe here how R might contribute to regulatory specificity, on one hand by forming a platform for protein-protein interactions, and on another, by binding to different sites in the regulatory regions of its target genes depending on the interacting partners. Using a variety of techniques I have investigated three protein-protein interacting regions of R. I demonstrate that the highly conserved bHLH domain of R is involved in transcriptional regulation and histone functions. I show that R interacts with the EMSY-like protein RIF1 specifically via the bHLH domain and that this interaction is required for the regulation of endogenous flavonoid genes. RIF1 is part of the C1/R regulatory complex and I discuss how RIF1 links transcriptional regulation of flavonoid biosynthetic genes with chromatin function. In addition, I show that the region adjacent to the bHLH domain has structural similarity to a leucine zipper and that the extended bHLH-LZ-like region is able to homodimerize. The bHLH-LZ-like mediated dimerization is required for activation of a synthetic pG-box::Luc promoter::reporter construct in transient expression studies and for binding to a synthetic G-box probe, as well as to the Bz1 and C2 promoter in vitro. Furthermore, I demonstrate that the ACT domain at the C-terminus of R homodimerizes. This domain is necessary for anthocyanin pigment formation, for transcriptional activation of at least four anthocyanin biosynthetic genes and important for DNA-binding to the A1 and Bz1 promoters. I show that interplay between the functional domains described here is necessary for transcriptional activation and DNA- binding. I am also characterizing R-interacting partners which possibly tether R to as yet unknown target genes and therefore might show the involvement of R in other cellular processes. Taken together, these studies emphasize the importance of the bHLH transcription factor R in combinatorial regulation of gene expression of anthocyanin biosynthetic genes and open new possibilities for R to function in other cellular processes. Moreover, these studies highlight the complexity of biochemical pathway regulation and show novel mechanisms of how one TF can participate in several regulatory complexes.
Advisors/Committee Members: Grotewold, Erich.
Subjects: Biochemistry; Biology; Cellular biology; Molecular biology
Keywords: bHLH; Transcription Factor; RED1; dimerization; maize; transcriptional regulation; DNA binding; combinatorial regulation
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17.
Fleming, Jessica L.
Utilizing Cancer Resistant and Susceptible Mice to Identify the Genetic Contributions to Cutaneous Squamous Cell Carcinoma Susceptibility.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2012, Ohio State University
► Non-melanoma skin cancer (NMSC), consisting of cutaneous basal cell carcinoma and squamous…
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▼ Non-melanoma skin cancer (NMSC), consisting of cutaneous basal cell carcinoma and squamous cell carcinoma (cSCC), is the most common form of cancer in the world. In 2006, the estimated number of new NMSC cases in the United States rose to 3.5 million; of those, 700,000 were cSCCs. The most important factor for the development of primary cSCCs is cumulative exposure to ultraviolet radiation. Other environmental risk factors include viral infection, immunosuppression, chronic inflammation, and exposure to industrial carcinogens. Cancer development is not solely dependent on environmental factors as a genetic contribution for several cancers is well described. Population and family-based studies suggest that there is a genetic component to the development of cSCC. These studies suggest heritability for cSCC risk; however, the specific genetic factors remain elusive. This dissertation is comprised of three related projects that explore the genetic contribution to cSCC. These studies utilize both skin cancer susceptible and resistant mice and human samples. Because human populations are heterogeneous, the use of mouse models to map genetic risk factors is ideal. Cancer susceptibility genes identified in the mouse have been confirmed in human studies suggesting this is an effective approach to identify true genetic risk factors. Previous studies using these mice identified skin tumor susceptibility loci, including the one of focus for this study, Skts5, as well as a small number of microRNAs (miRNAs) showing differential expression. The goal of the first project was to identify and characterize novel skin cancer susceptibility genes in the mouse. We hypothesized that genes mapping to Skts5 which showed expression differences and/or sequence variations between cancer susceptible and resistant mice may be playing a role in tumorigenesis. We identified aryl hydrocarbon receptor (Ahr) as a potential candidate susceptibility gene. We performed in vitro functional analyses and revealed potential roles that Ahr may be playing in tumor development and progression. The focus of the second project was to identify cancer susceptibility genes at the human SKTS5 locus using allele-specific imbalance and risk association studies. We hypothesized that human cSCC tumors would show allele-specific somatic genetic changes at SKTS5 and that these alterations would contribute to cSCC risk. Candidate genes identified in the mouse were analyzed in human samples. We identified single nucleotide polymorphisms and haplotypes in HDAC9 that conferred risk, supporting my hypothesis. The goal of the third project was to identify and characterize miRNAs that are playing a role in cSCC tumorigenesis. This project utilized a miRNA microarray expression data set to compare expression in cSCC cancer susceptible and resistant mice. We hypothesized that miRNAs showing differential expression between the strains are important in the development of cSCC. This study identified six miRNAs and we performed in vitro functional characterization of miR-1. Genetic discovery is important to not only understand the biology of disease, but to also facilitate better preventative measures and treatment options. The three studies of this dissertation give insight to genes and miRNAs contributing to cSCC development.
Advisors/Committee Members: Toland, Amanda.
Subjects: Genetics
Keywords: HDAC9, microRNA-1, cutaneous squamous cell carcinoma, cancer risk alleles, allele-specific imbalance, Ahr, Skts5, skin cancer, ultraviolet light
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18.
Funk, Kristen E.
Alzheimer’s Disease Pathology as a Clue to Pathogenesis.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2012, Ohio State University
► Alzheimer’s disease is characterized pathologically by two intracellular lesions, granulovacuolar degeneration and…
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▼ Alzheimer’s disease is characterized pathologically by two intracellular lesions, granulovacuolar degeneration and neurofibrillary tangles. Because Alzheimer’s disease is primarily sporadic, the traditional starting point for studying disease is pathology. It is still debated whether these hallmark lesions are markers or mediators of disease progression; however, the correlation between these lesions and disease progression warrants their use as clues to underlying disease processes. The molecular mechanisms of their development, their role in the disease process, and their connection to one another are not fully understood. Granulovacuolar degeneration involves the accumulation of large, double membrane-bound bodies within certain neurons during the course of disease. Because of their ultrastructure, it is hypothesized that the bodies are autophagic. To test this, colocalization of autophagic and endocytic markers with a marker for granulovacuolar degeneration was investigated in hippocampal sections prepared from post mortem late stage Alzheimer’s disease cases using double-label confocal fluorescence microscopy. The resultant immunohistochemical signature suggests that granulovacuolar degeneration bodies accumulate at the nexus of autophagic and endocytic pathways and that failure to complete autolysosome formation may correlate with their formation. Due to its far-reaching roles in cells, disruption of the endocytic pathways has the potential to act as a nidus central to disease onset and the development of granulovacuolar degeneration as well as neurofibrillary tangles and amyloid plaques. In sporadic Alzheimer’s disease, neurofibrillary tangle formation is preceded by extensive post-translational modification of tau. To identify the modification signature associated with tau lesion formation at single amino acid resolution, paired helical filaments immunopurified from Alzheimer’s disease brain were subjected to liquid chromatography, tandem mass spectrometry analysis. The resulting spectra identified Lys monomethylation as a new tau modification distributed among seven residues located in the projection and microtubule biding domains of tau protein. One site, K254, was found to be a substrate for a competing Lys modification, ubiquitylation. Double label confocal fluorescence microscopy demonstrated that methylation is wide-spread among neurofibrillary tangles in hippocampal sections prepared from post mortem late-stage Alzheimer’s disease cases. Together these data provide the first evidence that tau in neurofibrillary lesions is modified by Lys methylation. The tau methylation signature involves sites known to mediate disease-related modifications including ubiquitylation and phosphorylation, suggesting that methylation is a candidate modification for influencing tau aggregation, toxicity, and protein turnover. To extend the correlation between methylation occupancy and disease, the modification state of soluble tau protein isolated from cognitively normal human brain was investigated using proteomic methods. Results showed that normal soluble tau is hypermethylated relative to disease-derived tau in its microtubule binding domain. Furthermore, recombinant human tau subjected to reductive methylation in vitro retains the normal function of tau as a microtubule-binding and stabilizing protein but its aggregation propensity is greatly attenuated. These data establish Lys methylation as a normal tau post-translational modification in human brain that may protect against pathological tau aggregation during aging. Furthermore, data suggests that enzymes responsible for tau methylation, including methyltransferases and demethylases, may be tractable targets for disease-modifying therapies focused on halting neurofibrillary lesion formation in Alzheimer’s disease.
Advisors/Committee Members: Kuret, Jeff.
Subjects: Biochemistry; Biomedical Research; Neurosciences
Keywords: Alzheimer’s Disease; Autophagy; Confocal Microscopy; Endosome; Granulovacuolar Degeneration; Mass Spectrometry; Lysine Methylation; Lysosome; Multivesicular Body; Neurofibrillary Tangle; Pathology; Post-Translational Modification; Tau
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19.
Gabilly, Stephane T.
A Disulfide-Reducing Pathway Required For Plastid Cytochrome c Assembly.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2012, Ohio State University
► The formation and reduction of disulfide bonds is essential for the folding,…
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▼ The formation and reduction of disulfide bonds is essential for the folding, stability and function of a large number of proteins. While the Dsb pathway (“Dsb” for Disulfide bond) controls thiol/disulfide chemistry in the bacterial periplasm, the question of how this process is performed in the thylakoid lumen, the topologically equivalent compartment in plastids and in cyanobacteria, has yet to be explored. The occurrence of disulfide-bonded proteins and the requirement for reduced cysteines in the assembly of cytochromes c in this compartment suggests that thiol/disulfide chemistry is also enzymatically assisted. From our study, using the unicellular green alga, Chlamydomonas reinhardtii as model organism, we have identified three novel components CCS4, CCS5 and CCDA operating in a trans-thylakoid disulfide-reducing pathway, which is required for the assembly of cytochromes c, a class of metalloproteins with a covalently attached heme. The ccs4 and ccs5 mutants are photosynthetic deficient and exhibit a block in the assembly of cytochromes f and c6, two c-type cytochromes of the algal photosynthetic apparatus, involved in electron transfer. CCS5 is a membrane anchored lumen-facing thioredoxin-like protein that we show is the functional homolog of Arabidopsis thaliana HCF164, previously identified as being involved in photosynthesis. A disulfide-reducing activity for CCS5 is inferred from the findings that ccs5 is chemically rescued by exogenous thiols and that recombinant CCS5 reduces a disulfide in the CXXCH heme binding motif of apocytochrome c. CCS4 is a small protein with a hydrophobic amino-terminus, a hydrophilic carboxyl-terminal domain containing several charged residues and no noticeable motif suggestive of a biochemical activity. Its involvement in a disulfide-reducing pathway is based on the thiol-dependent photosynthetic rescue of the ccs4 mutant and also on the ectopic expression of plastid CCDA, an ortholog of the bacterial thiol-disulfide transporter. Because heme is covalently linked to the heme binding motif, we propose that CCS5 controls plastid cytochrome c assembly by maintaining the CXXCH cysteines under a reduced form prior to the heme attachment in the thylakoid lumen. We postulate that CCS5 is maintained in a reduced state by the thylakoid membrane transporter CCDA whose activity/stability is regulated by CCS4 via a yet-to-be defined mechanism.
Advisors/Committee Members: Hamel, Patrice.
Subjects: Biology
Keywords: Disulfide reducing pathway; Cytochrome c; Chloroplast; Photosynthesis; Chlamydomonas
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20.
Hickey, Christopher Jon.
The Role of MicroRNA-181a in Acute Leukemia: Biological, Clinical, and Therapeutic Implications.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2011, Ohio State University
► Recent cytogenetic and molecular characterization along with gene expression profiling of acute…
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▼ Recent cytogenetic and molecular characterization along with gene expression profiling of acute myeloid leukemia (AML) patients have revealed previously unknown features associated with distinct subsets of the disease. Among these newly discovered moleclar features were mutations within the CCAAT/Enhancer Binding Protein Alpha (CEBPA) gene found in approximately 15% of patients lacking chromosomal abnormalities, thus characterized as cytogenetically normal AML. These mutations were shown to favorably impact the outcome of AML patients through unknown mechanisms. Notably, patients harboring CEBPA mutations also present with increased expression of microRNA-181a (miR-181a). High levels of miR-181a expression have been suggested to have tumor suppressor qualities in accord with CEBPA mutations. Therefore, it is logical to hypothesize that a mechanistic correlation exists between miR-181a expression levels and CEBPA mutations which contribute to favorable outcomes associated to this subset of AML patients. In this body of work, the correlation between different mutant isoforms of C/EBPα and miR-181a was identified and the potential therapeutic implications described. Experimentally, miR-181a expression was found to be higher in those cells expressing the N-truncated C/EBPα isoform. As a result of elevated miR-181a expression, targeted transcripts coding for innate immune effector molecules, Toll-Like Receptor 4 (TLR4) and Interleukin-1 Beta (IL-1β), were down modulated due to direct interaction of miR-181a. The miR-181a repression of these effector molecules ultimately contributed to the repression of the characteristic tumorigenic activity associated to NF-κB. Furthermore, smaller tumors were observed in mouse models xenografted with leukemia cells supplemented with miR-181a. In an effort to recapitulate the therapeutic benefits involving miR-181a, an immunomodulatory compound, lenalidomide, was investigated as an agent to induce miR-181a expression in leukemia cells. Lenalidomide was found to induce the expression of the N-truncated C/EBPα isoform similar to the N- mutant isoforms observed in AML patients. The mechanism favoring the expression of the N-truncated C/EBPα isoform was identified and involved the activity of the translational subunit eIF4E. Following the lenalidomide treatment and subsequent induction of the N-truncated C/EBPα isoform, miR-181a expression was increased. The in vitro data implicating lenalidomide to increased miR-181a expression were validated in xenograft mouse models. These tumors were found to have higher miR-181a expression and the tumors were physically smaller in size and weight. Finally, the in vivo mouse data were validated following analyses of miR-181a expression of individual leukemia patients who participated in two separate clinical trials utilizing lenalidomide as an anti-leukemia therapy. The therapeutic benefits associated to lenalidomide were also found to be applicable to acute lymphoblastic leukemia (ALL) cells which harbor the tumorigenic chimera protein, TEL/AML1. In two different ALL cell lines, the lenalidomide induction of miR-181a expression was found to lower the expression of TEL/AML1, subsequently lowered the expression of the pro-survival protein SURVIVIN, and ultimately induced apoptosis in those cells treated with the therapeutic drug. Taken together, increasing the expression of miR-181a has therapeutic benefits in acute leukemias. Indeed, this work served as a paradigm for designing and initiating clinical trials with lenalidomide followed by chemotherapy in distinct cohorts of AML patients with previously untreated or relapsed/refractory disease.
Advisors/Committee Members: Marcucci, Guido.
Subjects: Molecular Biology
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21.
Hilario, Jona Dela Cruz.
Molecular cues in pathfinding of axial motoneurons in the developing zebrafish.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2010, Ohio State University
► Establishing neuromuscular specificity is an important step during development to ensure proper…
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▼ Establishing neuromuscular specificity is an important step during development to ensure proper motor function. Motor axons may travel over relatively long distances from their positions in the CNS to muscle targets in the periphery. The zebrafish model has been useful for investigating the process of motor axon pathfinding. The embryonic motor system of the zebrafish is relatively simple and has been well studied. Both forward and reverse genetics have been used to study axon pathfinding of zebrafish trunk motor axons. Our study of two motor axon mutants, stumpy and topped, has led to the identification of two molecules that guide trunk motor axons to their muscle targets. stumpy mutants exhibit a phenotype wherein motor axons stall for prolonged periods at intermediate targets prior to them reaching their final target. Positional cloning mapped the mutation to the zebrafish homolog of the collagenXIXa1 (colXIX). colXIX is expressed at known intermediate targets during the time of axon outgrowth. Knocking down ColXIX using morpholinos (MO) phenocopies stumpy. Also stumpy mutants were rescued by knocking down ColXIX and adding back mouse colXIX RNA. This suggests that ColXIX functions to enable growth cones to navigate intermediate target during development. Semaphorin 5A (Sema5A) was initially identified as a candidate gene for the topped mutant but this does not appear to be a case. However, Sema5A appears to play a role in motor axon pathfinding. Sema5A is expressed in the myotome during the time of axon outgrowth. Knocking down Sema5A using MOs specifically affects the Caudal Primary (CaP) motor axon, inducing a delay in its extension to its muscle target as well as axon branching. This MO phenotype can be rescued by adding back rat sema5A RNA. Sema5A has been previously shown to act as a bifunctional cue in the rat habenula. In zebrafish, we saw that adding back RNA encoding the sema domain alone rescued the branching phenotype in sema5A morphants. Conversely, adding back RNA encoding the thrombospondin repeat (TSR) domain alone into sema5A morphants exclusively rescued delay in ventral motor axon extension. These data show that Sema5A is a bifunctional axon guidance cue for vertebrate motor axons in vivo. The addition of ColXIX and Sema5A to the list of molecules that are involved in this seemingly simple pathfinding process demonstrates that numerous factors and pathways may be involved in establishing precise neuromuscular connections. Both these molecules have been shown to function in other contexts in the nervous system thus, understanding the roles these molecules play in axonal pathfinding can reveal novel mechanisms involved in wiring the nervous system.
Advisors/Committee Members: Beattie, Christine.
Subjects: Biology
Keywords: zebrafish, axon guidance, semaphorin 5A, collagen xix
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22.
Hsu, Shu-hao.
The Biological Functions of miR-122 and its Therapeutic Application in Liver Cancer.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2012, Ohio State University
► miR-122, the most abundant liver-specific microRNA (miRNA), is involved in many different…
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▼ miR-122, the most abundant liver-specific microRNA (miRNA), is involved in many different biological functions, including cholesterol metabolism, hepatitis C virus replication, and hepatocarcinogenesis. Previous studies have shown that downregulation of miR-122 in hepatocellular carcinoma (HCC) correlates with metastasis and poor prognosis. Among the deregulated miRNAs in HCC, miR-122 is drastically reduced in HCC. Based on these observations, our hypothesis is that miR-122 is a liver-specific tumor suppressor and its loss may promote hepatocarcinogenesis. To test this hypothesis, we generated germ-line (KO) and liver-specific (LKO) miR-122 knockout mice. Both KO and LKO mice exhibited hepatic microsteatosis and hepatic inflammation at early adult stage. Lipid analysis showed accumulated hepatic triglyceride, which correlated with increased de novo triglyceride synthesis and reduced triglyceride secretion. By 6 month both KO and LKO mice develop hepatic steatosis, inflammation, and fibrosis. After twelve months, these mice produce spontaneous liver tumors resembling HCC. The HCC incidences were ~30% and ~50% in LKO and KO mice, respectively. Microarray and realtime RT-PCR analysis attributed these pathological phenotypes to dysregulated expression of signaling pathways involved in triglyceride synthesis, cytokine expression, and oncogenesis. Among the deregulated genes, Agpat1, Cidec and Mapre1 were identified for the first time as the direct targets of miR-122. Exploration of the mechanism leading to hepatic inflammation in KO and LKO mice led to the identification of CD11bhighGr-1+ subtype of inflammatory cells increased in the liver of KO mice. These cells were determined as the major source of high levels of IL-6 and TNF-α that accumulated in the livers of KO and LKO mice. Ccl2, a reported myeloid chemo-attractant, was induced in hepatocytes of KO and LKO mice and was inversely regulated by miR-122 in vitro. To establish further the tumor suppressor role of miR-122, we tested the feasibility of the therapeutic delivery of miR-122 to the liver and tumor cells by lipid-based nanoparticles, which is recognized as a safe delivery method due to its biocompatibility. We developed a novel LNP, designated LNP-DP1, consisting of a conditionally ionizable cationic lipid, 2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), egg PC, cholesterol (Chol) and Chol-polyethylene glycol (Chol-PEG). Ectopic expression of miR-122 in HCC cell lines by LNP-DP1 reduced expression of miR-122 targets, such as Adam10, by >95%. The biodistribution analysis revealed that hepatocytes and tumor epithelial cells preferentially took up systemic circulated LNP-DP1 conjugated fluorescence labeled siRNA. Serum profile of treated mice showed that LNP-DP1 did not induce significant liver or kidney damage in mice. Several target genes of miR-122 were downregulated after systemic delivery of LNP-DP1 conjugated miR-122. This result suggested that miR-122 was successfully released from LNP-DP1 and was functional in hepatocytes and tumor cells. Furthermore, intratumoral injection of LNP-DP1 conjugated miR-122 into HCC xenograft developed in nude mice resulted in suppression of tumor growth within 30 days of miR-122 delivery. In conclusion, these findings revealed critical functions for miR-122 in liver metabolism, hepatic immune response, and in hepatocarcinogenesis. The application of miR-122 delivery by LNP-DP1 in animal HCC model suggested potential utility of miR-122 therapy for selected HCC patients negative for HCV.
Advisors/Committee Members: Jacob, Samson.
Subjects: Biology; Biomedical Engineering; Biomedical Research; Oncology
Keywords: miR-122, cationic lipid based nanoparticle, liver cancer, knockout mice
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23.
Huss, David J.
Effector Th1 cells demonstrate self-regulation in a mouse model of Multiple Sclerosis.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2011, Ohio State University
► Multiple sclerosis (MS) is an immune-mediated demyelinating disorder of the central nervous…
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▼ Multiple sclerosis (MS) is an immune-mediated demyelinating disorder of the central nervous system (CNS) that is perpetuated by myelin-specific autoreactive CD4+ T cells. Interestingly, both healthy individuals and MS patients have myelin-specific T cells; however, these cells are of an effector-memory phenotype in MS patients, indicating previous exposure to antigen (Ag). Additional phenotypic analysis has demonstrated these cells are primarily of an effector-memory T helper (Th) 1 and Th17 lineage in MS patients. After activation in the periphery, myelin-specific T cells traffic to the CNS and enter a unique cytokine microenvironment that has the potential to influence the effector and/or regulatory mechanisms of these infiltrating cells. This project was initiated to determine how cytokines in the CNS microenvironment influence the pathogenicity of myelin-specific Th1 effector cells. Transforming growth factor (TGF)-β is a cytokine expressed in both the healthy and inflamed CNS with demonstrated suppressive effects on naïve CD4+ T cell differentiation; however, the effects of this cytokine on Ag-experienced Th1 effector cells are not well defined. Using myelin-specific T cell receptor (TCR) transgenic (Tg) mice in the experimental autoimmune encephalomyelitis (EAE) model of MS, we demonstrate that TGF-β elicits differential effects on proliferation, cellular activation, and cytokine secretion based on the differentiation state of a CD4+ T cell (i.e. naïve versus effector-memory). Additionally, Th1 effector cells begin to produce IL-10 as a self-regulatory mechanism that is enhanced with both activation in an environment rich in TGF-β, and repetitive Ag stimulation. TGF-β signaling caused direct binding of smad4 to the IL-10 promoter, providing molecular evidence for TGF-β-mediated regulation of IL-10 production in Th1 effector cells. Furthermore, the IL-10 produced by IFN-γ+IL-10+ Th1 effector cells decreased EAE severity by reducing the expression of encephalitogenic markers on IL-10R+ cells and preventing host-cell recruitment to the inflamed CNS. These data establish a regulatory mechanism by which highly activated Th1 effector cells modulate their pathogenicity through induction of IL-10. This work is a demonstration of the complex signaling networks that direct the functions of immune cells. Furthermore, the data presented highlight the importance of regulatory mechanisms that modulate the immune response, whether during normal inflammation or autoimmunity. In the context of MS, increasing the inherent regulatory potential of pathogenic effector-memory T cells may lead to novel therapies for the treatment of this debilitating disease.
Advisors/Committee Members: Lovett-Racke, Amy.
Subjects: Immunology
Keywords: Immune regulation; effector-memory T cell; EAE; MS; TGF-beta; IL-10
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24.
Hutzen, Brian John.
Development and Use of Recombinant Oncolytic Measles Viruses for the Treatment of Medulloblastoma.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2012, Ohio State University
► Medulloblastoma is the most common malignant brain tumor of early childhood. Our…
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▼ Medulloblastoma is the most common malignant brain tumor of early childhood. Our understanding of this disease, its etiology, and treatment has improved considerably over the past several years and is reflected in 5-year survival rates that now exceed 70%. Despite these advancements, numerous challenges in the effective treatment of medulloblastoma remain. Conventional therapy, which consists of surgical resection and craniospinal irradiation with or without chemotherapy, is associated with significant neurocognitive morbidity. In addition, a sizable subset of medulloblastoma patients will effectively remain incurable because of medulloblastoma’s propensity to disseminate along the spinal canal within the cerebrospinal fluid. Alternative treatment modalities for medulloblastoma are thus clearly needed. One promising approach is the development and use of recombinant oncolytic measles viruses. These viruses, which are based on the attenuated Edmonston vaccine strain virus, display a natural tropism for malignant cells and induce their fusion and subsequent death via apoptosis following infection. We have demonstrated that medulloblastoma tumor cells are highly susceptible to measles virus-induced oncolysis, and measles virus treatment can significantly prolong survival in mouse xenograft models of localized and disseminated disease. We have also utilized recombinant viruses expressing transgenes for the thyroidal sodium iodide symporter and the angiogenesis inhibitors endostatin and angiostatin to further augment the efficacy of measles virotherapy. Our initial results are encouraging and suggest that measles virus-based therapies may be of clinical utility in the treatment of medulloblastoma.
Advisors/Committee Members: Raffel, Corey.
Subjects: Biology; Molecular Biology; Oncology; Virology
Keywords: Medulloblastoma; measles virus
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25.
Ismail, Noura A.
CHARACTERIZATION OF PLASMA AND MACROPHAGE DERIVED MICROVESICLES.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2010, Ohio State University
► Microvesicles are small membrane-bound vesicles released under normal homeostatic and stimulatory conditions…
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▼ Microvesicles are small membrane-bound vesicles released under normal homeostatic and stimulatory conditions by a wide variety of cell types. Microvesicles are collectively referred to as exosomes and microparticles, which vary in size due to different cellular mechanisms responsible for their production. These microvesicles have a wide range of functions from facilitating communication to regulating cellular growth and differentiation. During their production; microvesicles become enriched in various molecules including proteins and nucleic acids. Previously, we have shown that plasma-microvesicles derived from many cell lineages contain microRNAs (miRNAs). We also found that the majority of the peripheral blood microvesicles are derived from platelets while those originating from monocytic cells including macrophages represent the second largest population. Since microvesicles derived from mononuclear phagocytes are a large subpopulation in the plasma; we were interested in understanding their content and function. We hypothesized that the content and/or quantity of macrophage-derived microvesicles could induce the maturation of monocytes. To address our hypothesis, peripheral blood monocytes were treated in vitro for 4hr with GM-CSF; washed and cultured in media devoid of cytokines for 24 h then microvesicles were collected. Flow cytometry and electron/ confocal microscropy were used to quantify and visualize microvesicles production. To examine the function of the microvesicles on macrophage maturation, the purified microvesicles were then cultured with freshly isolated monocytes. Macrophage differentiation was determined by cellular adherence using a crystal violet uptake assay and changes in surface antigen expression by flow cytometry. We also examined the genetic changes induced in monocytes incubated with the microvesicles compared to GM-CSF-treated cells. We found that freshly isolated monocytes treated with GM-CSF-derived microvesicles acquired phenotypic characteristics of a macrophage such as cellular adherence and surface antigen expression. We also found that treatment of naïve monocytes with the microvesicles induced molecular changes similar to GM-CSF treated monocytes. To begin dissecting the molecules contained in the microvesicles responsible for these changes, we performed mass-spectrometry and miRNA profiling. Many of the proteins found in the vesicles are important in facilitating protein: protein interactions and nucleic acid binding. Based on our observations we postulate that microvesicles in areas of inflammation may contribute to the inflammatory response through the maturation of immune cells and activation of cells responsible for tissue repair. Thus, defining key components of this response may identify targets to regulate inflammation.
Advisors/Committee Members: Marsh, Clay.
Subjects: Biology
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26.
Kabbout, Mohamed Nazih.
ETS1 AND ETS2 ROLE IN RAS ONCOGENIC TRANSFORMATION IN MOUSE EMBRYONIC FIBROBLASTS.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2010, Ohio State University
► Human soft-tissue sarcoma is a malignant tumor that is usually found in…
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▼ Human soft-tissue sarcoma is a malignant tumor that is usually found in children and young adults. In the United States more than 12000 new cases are diagnosed each year with soft tissue sarcomas. Oncogenic Ras has been shown to be involved in soft-tissue sarcoma development and K-Ras, H-Ras and N-Ras missense mutations at codons 12, 13 and 61 have been reported in 23 to 30% of soft tissue sarcoma tumors. The MAPK pathway is one of the main mediators of the Ras response, and it has been shown to be deregulated in cancer in general and soft tissue sarcoma in particular. Ets1 and Ets2 are the downstream effectors of the Ras/ERK pathway and have been shown to be over expressed in cancer and activated by Ras/ERK through the phosphorylation of their conserved MAPK phosphorylation domain next to the PNT domain. This phosphorylation is essential for Ets1 and Ets2 to regulate many genes that are important for diverse biological processes. Over expression of constitutively active Ras in mouse embryonic fibroblasts leads to cellular transformation, and injection of Ras transformed fibroblasts into nude mice leads to formation of tumors similar to fibroblastic soft tissue tumor, which makes the Ras transformation in vitro model a suitable system for studying soft-tissue sarcoma. The Ets families of transcription factors have been shown to play an essential role in Ras transformation, but the identity of the Ets family members that are essential for Ras transformation is still unkown. Using the Cre-lox system to delete Ets2 from Mouse Embryonic Fibroblasts (MEFs) that are Ets1-/- and have the Ets1 null allele, we showed that the deletion of both Ets1 and Ets2 is necessary to inhibit Ras oncogenic transformation and tumorigenesis in MEFs. Here we show that Ets1 and Ets2 play a post transcriptional repression role in Ras transformation through a C-myc dependent up regulation of miR17-92 microRNA. Ets1 and Ets2 bind to the C-myc promoter and regulate C-myc RNA and protein expression. We also show that Ets2 similar to C-myc binds to the promoter of miR17-92, and Ets1 and Ets2 through transient transfection are able to upregulate the miR17-92 cluster expression in C-myc -/- MEFs. C-myc and miR17-92 Cluster over expression in Ets1/Ets2 double knockout MEFs rescued Ras tumorigenesis in vivo upon injection in nude mice. Also we show that Ets1 and Ets2 play a transcriptional repression role through a c-myc dependent repression of Sfrp1, Fas and Lox tumor suppressor genes during Ras transformation. Fas, Lox and Sfrp1 are not repressed in Ets1/Ets2 double knockout MEFs even after Ras retroviral infection, but over expression of C-myc repressed Fas, Lox and Sfrp1 again in the Ets1/Ets2 double knockout MEFs. We found that Ets1, Ets2 and C-myc bind to the promoter of Fas, Sfrp1 and Lox genes during Ras transformation, and we show an enrichment of these promoters with H3K9 and H3K27 trymethylation markers which are usually present on silent promoters.
Advisors/Committee Members: Ostrowski, Michael.
Subjects: Biomedical research
Keywords: soft-tissue sarcoma; Ets1; Ets2; Ras; transformation; C-myc; MEFs; miR17-92
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27.
Kim, Dhohyung.
Mechanism of Maternal Antibody Inhibition and Vaccination Strategies in the presence of Maternal Antibodies.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2012, Ohio State University
► A crucial unsolved problem for all vaccines is the inhibition by maternal…
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▼ A crucial unsolved problem for all vaccines is the inhibition by maternal antibodies. Maternal antibodies confer protection against infections early in life. Over time, titers of maternal antibodies decline to non-protective titers that still inhibit vaccination. Generally, the generation of neutralizing antibodies is most significantly inhibited while T cell responses are not affected by maternal antibodies. Although this problem arises for many pathogens, no effective immunization strategy for vaccination in the presence of maternal antibodies has been defined because the underlying mechanism of interference was not known. The cotton rat is the only rodent in which measles virus replicates in the respiratory tract and lymphoid organs after intranasal inoculation. Using the cotton rat model of measles virus (MV) vaccination, we have been able to study the mechanism of maternal antibody inhibition and vaccination strategies in the presence of maternal antibodies. In the absence of experimental data and based on antibody feedback study, it has been suggested that epitope masking is one of the mechanism explaining the inhibition by maternal antibodies. However, we found that maternal antibodies inhibit B cell responses through cross-linking of the B cell receptor (BCR) with Fc gamma IIB receptor (FcgRIIB). This inhibition can be partially overcome by injection of MV-specific monoclonal IgM antibody. IgM stimulates the B cell through cross-linking the BCR via complement protein and antigen to the complement receptor 2 (CR2) signaling complex. To fully stimulate B cells in the presence of maternal antibodies, B cells require an extra stimulus to differentiate into antibody-secreting plasma cells. In order to provide such a stimulus, we generated a recombinant Newcastle disease virus (NDV) expressing the MV hemagglutinin (NDV-H). In contrast to MV, NDV-H induced high levels of type I interferon in plasmacytoid dendritic cells (pDCs) and in lung tissue. In cotton rats immunized with NDV-H, the induction of neutralizing antibodies was partially restored in the presence of maternal antibodies. These data indicated that type I interferon is an additional stimulus for B cell activation in the presence of maternal antibodies. Complete B cell activation resulting in secretion of neutralizing antibodies is the ultimate goal of vaccination in the presence of maternal antibodies. Co-stimulation of Toll-like receptor (TLR)-3 and TLR-9 with their respective agonists, Poly I:C (Polyinosinic polycytidylic acid, TLR-3 agonist) and ODN 2216 (CpG oligodeoxynucleotides 2216, TLR-9 agonist), induced synergistically higher type I interferon than that of NDV-H induced. In cotton rats, co-immunization of ODN 2216 and Poly I:C with MV vaccine induced normal B cell proliferation and neutralizing antibody response in the presence of maternal antibodies. These data support that type I interferon is crucial and sufficient to stimulate normal B cell responses in the presence of maternal antibodies. In addition, we have shown that CD21 functions as an interferon alpha receptor. In aggregate our data demonstrate that the shift in the balance of negative and positive signals by interferon alpha towards B cell stimulation is crucial in overcoming maternal antibody inhibition.
Advisors/Committee Members: Niewiesk, Stefan.
Subjects: Biology; Biomedical Research; Immunology; Medicine; Microbiology; Molecular Biology; Virology
Keywords: Maternal Antibody, Measles, Cotton Rat, Vaccination, Neonatal Immune Response, Neutralizing Antibody
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28.
Kim, Taewan.
The function of microRNAs in p53-regulated epithelial-mesenchymal transition.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2011, Ohio State University
► p53 suppresses tumor progression and metastasis. Epithelial-mesenchymal transition (EMT) is a key…
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▼ p53 suppresses tumor progression and metastasis. Epithelial-mesenchymal transition (EMT) is a key process in tumor progression and metastasis. The transcription factors ZEB1 and ZEB2 promote EMT. Here we show that p53 suppresses EMT by repressing expression of ZEB1 and ZEB2. By profiling 92 primary hepatocellular carcinomas (HCCs) and 9 HCC cell lines, we found that p53 upregulates microRNAs including miR-200 and miR-192 family members. The miR-200 family members transactivated by p53 then repress ZEB1/2 expression. p53-regulated miR-192 family members also repress ZEB2 expression. Inhibition or over-expression of the microRNAs affects p53-regulated EMT by altering ZEB1 and ZEB2 expression. Our findings indicate that p53 can regulate EMT, and that p53-regulated microRNAs are critical mediators of p53-regulated EMT.
Advisors/Committee Members: Croce, Carlo.
Subjects: Biomedical Research
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29.
Li, Qiming.
IL-1 Receptor Biology: Promoter Complex and Cell Type Specific Function.
Degree: PhD, Molecular, Cellular and Developmental Biology, 2010, Ohio State University
► Interleukin-1 (IL-1) is a pleiotropic factor involved in numerous aspects of multiple…
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▼ Interleukin-1 (IL-1) is a pleiotropic factor involved in numerous aspects of multiple systems. Considering the vast array of distinct IL-1 activities, it is surprising that IL-1 exerts its function mostly through a single receptor, the type 1 IL-1 receptor (IL-1R1). In this thesis study, we investigated how distinct promoters of IL-1R1 control its expression in different cell types and in different tissues, allowing differential regulation of IL-1R1 expression. By characterizing both human and mouse promoter complexes of IL-1R1, we discovered novel transcriptional regulation of the IL-1R1 by multiple promoters. We also investigated the cell type specific functions of IL-1R1, particularly in neuronal and endothelial cells. We identified a novel IL-1 receptor which appears to be neuronal specific in the brain. The importance of endothelial IL-1R1 was studied in the experimental autoimmune encephalomyelitis (EAE), a widely used animal model of human c¬entral nervous system (CNS) demyelinating diseases. We showed that endothelial IL-1R1-mediated leukocyte recruitment is critical in the induction and progression of EAE. This thesis contains four major parts. In the first part, seven human promoters were identified and characterized. These promoters exhibit cell type specific expression patterns and one of them was sensitive to the stimulation by glucocorticoids. In the second part, three mouse IL-1R1 (mIL-1R1) promoters were identified and characterized. Full length cDNAs produced from these promoters were cloned; IL-1R1 proteins expressed from these cDNAs can mediate IL-1-induced activation of NF-kappaB. The three mIL-1R1 promoters exhibit cell type specific expression patterns and their corresponding mRNAs exhibit tissue specific distribution in vivo. Further, glucocorticoids differentially regulate these promoters in a cell type specific manner. In the third part, we identified a novel mouse IL-1 receptor, IL-1R3, which appears to be neuronal specific in the brain. The mRNA and protein of IL-1R3 are expressed in normal and IL-1R1 KO animals. In IL-1R1 KO mice, IL-1R3 expression is significantly reduced by the introduction of an antisense RNA against IL-1R3. IL-1beta binds specifically to IL-1R3, but IL-1 stimulation of IL-1R3 does not activate NF-kappaB. In the last part, we show that selective knockdown of endothelial IL-1R1 (eIL-1R1 KD) expression by antisense RNA results in decreased incidence, severity, and delayed onset of EAE. In addition, eIL-1R1 KD mice show significant decrease of leukocyte recruitment in the CNS of EAE animals. These findings indicate a critical role for endothelial IL-1R1 in mediating the pathogenesis of EAE. In conclusion, we show that the different IL-1R1 promoters and the cell type-specific expression of IL-1R1 proteins contribute to the discrete and diverse actions of IL-1.
Advisors/Committee Members: Quan, Ning.
Subjects: Biomedical research; Cellular biology; Molecular biology
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30.
Madabusi, Narasimhan Kandaye.
Characterization of three SMN missense mutations using mouse models of Spinal Muscular Atrophy.
Degree: MS, Molecular, Cellular and Developmental Biology, 2012, Ohio State University
► Spinal Muscular Atrophy (SMA) is an autosomal recessive neurodegenerative disease characterized by…
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▼ Spinal Muscular Atrophy (SMA) is an autosomal recessive neurodegenerative disease characterized by loss of lower motor neurons and muscle atrophy. It is caused due to reduced levels of the ubiquitously expressed Survival Motor Neuron (SMN) protein. Humans have two SMN genes – SMN1 which produces full length SMN and SMN2 which mainly produces a truncated unstable protein and a small amount of full length SMN (~10%). SMN is a multi-functional protein with a known role in snRNP assembly. To date, it is still not known why the lower motor neurons are specifically affected in SMA although SMN is expressed in all tissues. Several missense mutations of SMN1 have been studied to understand the mechanism of SMA. One such synthetic mutation which affects the first glutamine (Q) of the QNQKE motif present in exon 7 is Q282A. A previous study has shown that this mutant construct cannot rescue the axonal defects in a zebrafish model of SMA. Since in vitro assays have suggested that SMN-Q282A can function in snRNP assembly, the authors proposed that restoring snRNP assembly function of SMN was not sufficient to rescue SMA. To analyze this mutant further, we generated transgenic mice carrying SMN-Q282A and crossed them to generate SMA mice with the following genotype: SMN-Q282A;SMN2;mSmn-/-. Currently, we have 25 such rescue mice and they show no physical or behavioral abnormality when compared to normal mice. This indicates that SMN-Q282A acts to complement the full length SMN produced by SMN2. However, as in previous studies with SMN missense mutations, the SMN-Q282A could not rescue a mSmn-/- mice. This is to be expected since no single mutant form of SMN has been able to rescue a mSmn-/- mouse by itself. These results suggest that snRNP assembly plays an important role in SMA and that the axonal defects in fish do not correlate with the critical function of SMN in SMA. We are currently performing snRNP assembly assays on brain and spinal cord lysates of these rescue mice. This will confirm our hypothesis that restoring snRNP assembly is sufficient to rescue SMA mice. Similar experiments were done with SMN-I116F, a mutation seen in SMA Type I patients. Initial results show that this mutant construct can provide only minimal complementation to SMN2. SMA mice of genotype SMN-I116F;SMN2+/+;mSmn-/- could survive only for 16 days, similar to the SMND7 SMA mice. The effect of another mutation SMN-D44V, seen in a Type III patient, was studied using scAAV9 vector. The viral vector carrying the SMN-D44V mutant was injected via the facial vein into SMA neonate pups at day 1. Partial recovery was seen with survival of the mice extended to 35±2 days. Transgenic mice having the SMN-D44V mutant have been generated and we expect them to fully complement SMN2, being a milder allele. Further experiments are being carried out to characterize these two transgenes, which will give us more input as to how different mutations affect the functioning of the SMN complex and its composition.
Advisors/Committee Members: Burghes, Arthur.
Subjects: Biochemistry; Genetics; Molecular Biology
Keywords: Spinal Muscular Atrophy, SMN, missense mutations, mouse models
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