Department: Biochemistry and Molecular Biology ![[clear]](close-x.png "Remove this limiter")
21 matches in the database.
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1.
Balanarasimha, Madhumitha.
STRUCTURAL AND FUNCTIONAL ALTERATION OF FULL LENGTH PPARα AND LXRα BY FATTY ACIDS AND THEIR THIOESTERS.
Degree: MS, Biochemistry and Molecular Biology, 2011, Wright State University
► Peroxisome proliferator-activated receptors (PPAR) and liver X receptors (LXR) are known to…
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▼ Peroxisome proliferator-activated receptors (PPAR) and liver X receptors (LXR) are known to play important roles in fatty acid metabolism, interact with each other, and function as heterodimeric partners. Although previous studies indicate that PPARα is activated by long chain fatty acyl-CoA thioesters (LCFA-CoA) and polyunsaturated fatty acids, little is known about the effects of these ligands on the function or interaction of PPARα and LXRα. In this study, hPPARα and hLXRα were shown to directly interact by circular dichroism, fluorescent binding assays, and co-immunoprecipitation. Further experiments suggested that although fatty acids resulted in small structural changes, they significantly altered binding affinities; while LCFA-CoAs decreased the binding affinities, no observable trend was seen with respect to the number of carbon atoms or bonds. In addition, transactivation assays in the presence of certain fatty acids suggested that the combination of PPARα and LXRα increased the activity of the PPARα regulated gene - ACOX, while downregulating the LXRα regulated gene SREBP. As high levels of fatty acids are associated with certain metabolic disorders and also serve as natural ligands for PPARα, changes in structure and/or interaction between PPARα and LXRα may have significant effects on the normal functioning of a cell.
Advisors/Committee Members: Hostetler, Heather.
Subjects: Biochemistry; Molecular Biology
Keywords: PPARα, LXRα, fatty acids, circular dichroism, binding assay, transactivation, coimmunoprecipitation
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2.
Farkaly, Terry C.
Inhibition of Cell Invasion by Targeting PLD.
Degree: MS, Biochemistry and Molecular Biology, 2010, Wright State University
► Phospholipase D (PLD) is a crucial signaling enzyme involved in many cellular…
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▼ Phospholipase D (PLD) is a crucial signaling enzyme involved in many cellular processes. The catalytic activity of PLD is essential for the production of Phosphatidic Acid (PA), a critical second messenger in cell signaling cascades downstream. Using the highly invasive rat mammary adenocarcinoma cell line mTLn3 as a metastatic model, we investigated the proficiency of these cells to invade using matrigels that mimic the basement membrane of the extracellular matrix (ECM), their activity through PLD enzymatic assays, as well as the potency of our potential inhibitors to inhibit PLD-mediated cell invasion and lipase activity. This study reveals that PLD-mediated cell invasion is dependent on protein-protein interactions with other cell signaling molecules such as Grb2 and Rac2 and their effect on lipase activity. Regulation of PLD2 activity by phosphorylation on tyrosine residue sites within its Phox domain are examined; in which we elucidate the effects of specific kinases EGFR, Jak3 and Src on cell invasion and enzymatic activity. Based off this approach, a therapeutic resolution will be proposed to diminish cell invasion in metastasis.
Advisors/Committee Members: Gomez-Cambronero, Julian.
Subjects: Molecular Biology
Keywords: PLD; cell invasion; inhibition; lipase activity; mtln3
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3.
Forquer, Isaac P.
Characterization of Photosynthetic Reaction Centers from Bradyrhizobium strain BTAi 1.
Degree: MS, Biochemistry and Molecular Biology, 2005, Wright State University
► Forquer, Isaac Paul. M.S. Department of Biochemistry and Molecular Biology,WrightState University,…
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▼ Forquer, Isaac Paul. M.S. Department of Biochemistry and Molecular Biology,WrightState University, 2005. Characterization of Photosynthetic Reaction Centers from Bradyrhizobium strain BTAi 1 Photosynthetic rhizobia have been studied for about 15 years now. They are now considered to be metabolically aligned with a relatively recently discovered group of bacteria, the anoxygenic aerobic phototrophs (AAP’s).Rhizobia form symbiotic relationships with plants from the Fabaceae family. Photosynthetic rhizobia not only nodulate the roots, as most other rhizobia do, but they also form nodules on the stems of certain leguminous plants. The plant provides carbon to the bacteria and the bacteria provides the plant with soluble nitrogen fixed from the biologically inert but abundant atmospheric N2. A key question regarding photosynthetic rhizobia and other AAP’s derives from the observation that photosynthesis in these organisms shuts down under anaerobic conditions. It has been proposed, and is the hypothesis of this thesis that the primary electron acceptor (QA) in the photosynthetic reaction center has a higher midpoint potential than in reaction centers found in the AAP’s counterparts, the anaerobic purple bacteria. If QA had a higher midpoint potential, it would be more labile to overreduction under anoxic conditions, and if QA is reduced, then photosynthetic electron transport is blocked. A redox titration was done to measure the midpoint potential of Q in the reaction centers of BTAi 1. This was done by observing the level of P (primary electron donor) bleaching upon excitation with bright light at different ambient redox potentials. The level of P bleaching is proportional to the fraction of QA that is not reduced, since P cannot bleach and donate an electron if QA is already reduced. Reaction centers from BTAi 1 were purified using two techniques, both involving ion exchange chromatography and one involving ammonium sulfate precipitation. Reaction centers were characterized by spectrophotometric studies, mass spectroscopy studies (MALDI TOF) and the cofactor composition was determined.Themidpoint potential of QA in BTAi 1 is –44 mV vs. SHE. The molecular weights of the subunits are very comparable to other photosynthetic reaction centers, from both aerobic and anaerobic bacteria. The pigment stoichiometry of reaction centers from BTAi1 is 2:1 bacteriochlorophyll:bacteriopheophytin. Both absorbance and light minus dark absorbance spectra are nearly identical to that found in anaerobic photosynthetic bacteria.Photosynthetic reaction centers in BTAi 1 are very similar to reaction centers of anaerobic photosynthetic bacteria. The midpoint potential of QA cannot account for its overreduction under anaerobic conditions. It is likely that AAP’s lack a key enzyme that would participate in redox homeostasis of the photosynthetic electron transport chain.
Advisors/Committee Members: Fleischman, Darrell E.
Subjects: Chemistry, Biochemistry
Keywords: Photosynthetic Reaction Centers; Photosynthetic Rhizobia; Bacteriochlorophyll
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4.
HOLLYFIELD, JENNIFER LYNNE.
DOSE-DEPENDENT EFFECTS OF OXYGEN ON METABOLISM IN RAT CORTICO-HIPPOCAMPAL BRAIN TISSUE SLICES.
Degree: MS, Biochemistry and Molecular Biology, 2009, Wright State University
► Studies have shown that 95% oxygen increases neuronal excitability and ROS production.…
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▼ Studies have shown that 95% oxygen increases neuronal excitability and ROS production. We wanted to investigate the dose-dependent effects of oxygen on brain slice metabolism. We exposed rat brain cortico-hippocampal tissue slices to 0.40, 0.95, and 4.50 ATA O2 for 60 minutes, made dual-phase tissue extracts, and used multi-nuclear NMR experiments to elucidate the slice metabolism. We found that low doses of oxygen may shift metabolism toward anaerobic glycolysis. Elevated lactate suggests this shift, along with elevated ratios of NAD+/NADH which may drive the reactions toward the production of lactate. The results also suggest that high doses of oxygen may cause aerobic glycolysis and oxidative phosphorylation to accelerate. Lower lactate amounts along with a low NAD+/NADH ratio may be driving these reactions towards pyruvate and the TCA cycle and suggest a highly oxidized environment. The 0.40 ATA O2 results suggest a hypoxic environment while the 0.95 and 4.50 ATA O2 results suggest hyperoxic environments that lead to changes in metabolism on both ends of the oxygen spectrum.
Advisors/Committee Members: Reo, Nicholas.
Subjects: Biochemistry; Biology; Biomedical research
Keywords: NMR; nuclear magnetic resonance; brain slices; hippocampus; oxygen
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5.
Jani, Meghna.
Transcriptional regulation of LAMB3 by p53.
Degree: MS, Biochemistry and Molecular Biology, 2008, Wright State University
► The p53 tumor-suppressor plays a very important role in the prevention of…
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▼ The p53 tumor-suppressor plays a very important role in the prevention of cancer and it is known that about 50% of all human tumors possess p53 mutations. Although mutations in p53 are most prevalent in human cancers, inactivation of wild-type p53 occurs through many different mechanisms that are independent of p53 mutation or deletion. In an effort to determine novel p53 target genes, our lab employed a microarray method in which p53 was re-activated by RNAi mediated knockdown of Hdm2 and HdmX in MCF7 human breast cancer cell line, harboring wild-type p53 and elevated levels of Hdm2 and HdmX. Gene expression profiling of the RNAi treated MCF7 breast cancer cells led to the identification of Laminin-beta 3 (LAMB3) as a potential transcriptional target of p53. To test this hypothesis, I carried out validation experiments which confirmed the earlier microarray experimental results.Interestingly, I also found four p53 half binding sites downstream of the LAMB3 promoter. DNA-damage and overexpression studies established that p53 activation unexpectedly did not lead to any transcriptional increase in LAMB3 expression. In contrast, serum deprivation and senescence experiments demonstrated that LAMB3 expression paralleled p21 (a well-known p53 target involved in cell cycle arrest) expression. Taken together, the experimental results suggest that LAMB3 may be a p53 regulated gene, but is not a classical p53 target.
Advisors/Committee Members: Berberich, Steven.
Subjects: Molecular biology
Keywords: p53; LAMB3; Hdm2; HdmX; transcriptional; Laminin-5
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6.
KATRANGI, NADIA.
DUE-B IN CHROMATIN AND NUCLEAR SPECKLES.
Degree: MS, Biochemistry and Molecular Biology, 2007, Wright State University
► The DNA unwinding element binding protein (DUE-B) was first identified by using…
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▼ The DNA unwinding element binding protein (DUE-B) was first identified by using a yeast one hybrid screen with the DNA unwinding element (DUE) from the c-myc origin as bait. DUE-B’s orthologue in the yeast Saccharomyces cerevisiae lacks the last 60 C-terminal amino acids and has been identified as a D-tyrosyl-tRNA deacylase. A substantial group of evidence suggests a role for DUE-B in the regulation of replication initiation. Here we show that DUE-B is focused in nuclear speckles and colocalizes with spliceosome associated protein 145 (SAP145), an mRNA splicing factor 3B subunit. Mass spectrometry results show that SAP145 co-purifies with the 6xHis-tagged DUE-B. Surprisingly, ÄCT- DUE-B, the DUE-B mutant lacking the 60 C-terminal amino acids, appeared almost exclusively in nuclear speckles whereas its yeast orthologue was found to be cytoplasmic. DUE-B’s distribution pattern did not change in cells arrested in G1/S and it did not appear in replication foci despite the strong evidence of its involvement in replication initiation. Finally, DUE-B’s proposed interaction with DNA methyltransferase 1 (Dnmt1) is further investigated by immunofluorescence. Interestingly, results show that this interaction with Dnmt1 seems to occur in nuclear speckles. Based on DUE-B’s interacting proteins and its concentration in nuclear speckles, a possible role in the DNA damage response is discussed.
Advisors/Committee Members: LEFFAK, MICHAEL.
Subjects: Biology, Molecular
Keywords: DUE-B; Nuclear Speckles; Replication initiation; DNA repair
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7.
Kenche, Harshavardhan.
Validation Of A Custom-made Microarray To Study Human Intestinal Microflora.
Degree: MS, Biochemistry and Molecular Biology, 2008, Wright State University
► Intestinal microflora refers to all the different species of bacteria that reside…
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▼ Intestinal microflora refers to all the different species of bacteria that reside in the human gut and is an important organ of the human body because almost all the digestive reactions of the host occur in the intestine. The bacteria of the intestine play a key role in this process by supplementing the intestine with various enzymes and proteins that are required for the digestive process. At the same time, these bacteria were shown to be implicated in a variety of gastrointestinal disorders like Irritable Bowel Syndrome, Inflammatory Bowel Disorder and Gastrointestinal Cancer, but with the current knowledge about the microflora it is difficult to determine which exact species is responsible for a particular disease caused. The knowledge about the composition of the typical intestinal microflora is very limited, the cause at large being the lack of properculture techniques to isolate and study the microfloral species in artificial media. Majority of the species of the microflora are obligate anaerobes and selective culturing techniques provide very limited knowledge about the composition of such complex microflora. Phylogenetic microarrays are one such approach to study various members of the microflora because they contain probes for numerous species of bacteria on a single glass slide and are also known to provide robust and high throughput analysis. ENTREZ nucleotide database was used to compile a list of 16S ribosomal DNA (rDNA) sequences of bacterial species isolated from the human intestine and they were grouped into various phylo-species. Representative sequences for each phylo-species were extracted and the probes on the microarray were designed based on these representative sequences. 16 different bacterial species were used for validation experiments, which represented bacteria from various groups. The results showed that the microarray correctly identified 15 of a total 16 bacterial species. The detection sensitivity of the microarray was at least 1pg. As a test, fecal samples from adults and children were analyzed by the microarray. Clostridia were the dominant group of the microflora followed by Bacteroidetes in both adults and children. The analysis of the fecal samples showed clear differences between the microflora composition of adults and children.
Advisors/Committee Members: Paliy, Oleg.
Subjects: Biochemistry; Microbiology; Molecular biology
Keywords: Microflora, Microarrays
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8.
Khokhar, Shama Khan.
Differential effects of mutant TAp63γ on transactivation of p53 and/or p63 responsive genes and their effects on global gene expression.
Degree: MS, Biochemistry and Molecular Biology, 2007, Wright State University
► p63, a member of the p53 gene family, known to play a…
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▼ p63, a member of the p53 gene family, known to play a role in development, has more recently also been implicated in cancer progression. Mice lacking p63 exhibit severe developmental defects such as limb truncations, abnormal skin, and absence of hair follicles, teeth, and mammary glands. Germline missense mutations of p63 have been shown to be responsible for several human developmental syndromes including SHFM, EEC and ADULT syndromes and are associated with anomalies in the development of organs of epithelial origin. The contrasting phenotypes associated with the different classes of p63 mutations might be in part due to the differential regulation of target genes. A previous report has demonstrated that heterozygous p63 mutations display high predisposition to tumor formation. Moreover, it has been shown that both p63 and p73, another member of the p53 family, are required for p53 mediated DNA damage induced apoptosis. Finally, differential splicing of p63 gene gives rise to p63 isoforms which can either act as tumor suppressors or oncogenes. The goal of this study is to determine the effects of naturally occurring TAp63γ mutants on regulation of p53/p63 and p63 specific target genes and their effects on global gene expression. Our results indicate that both TAp63γ(R227Q) and TAp63γ(R298Q) mutants mimic wildtype TAp63γ effects on its target genes. TAp63γ(K194E) and TAp63γ(R280C) significantly induced genes regulated by p63 and p53, but not those specific for p63. TAp63γ(R279H) andTAp63γ(R204W) were unable to induce any of the targets tested in this study. Co-transfection of p63 mutants along with wildtype p63 was performed to assess the effects of p63 mutants on ability of wildtype p63 to induce its target genes, while co-transfection of TAp63γ(R279H) and TAp63γ(R204W) led to a complete inhibition of the wildtype TAp63γ mediated induction of p63 specific target genes, they had no effect on p53/p63 target genes. We demonstrated that the ability of these mutants to regulate wildtype activity was independent of their ability to either interact with wildtype TAp63γ or affect its localization. In addition, we demonstrated that the effects of these mutants on cell growth and survival were consistent with their ability to regulate the downstream targets when compared to wildtype TAp63γ. Furthermore, our analysis of the GeneChip data using GeneSpring led to the identification of several common and unique genes regulated by specific p63 mutants when compared to cells transfected with wildtype p63. Additionally, the specific genes regulated by the p63 mutants observed in EEC, SHFM and ADULT syndrome might offer unique insights in understanding the involvement of p63 in development, ectodermal-mesenchymal interactions and differentiation. In summary, we show that p63 mutants exhibit a differential effect on p63 specific and p53/p63 specific target genes and on induction of apoptosis. This, in turn might have a significant impact on p63 mutation associated abnormalities of human developmental syndromes. Taken together, our data shows that p63 mutants differentially regulate gene expression and provide an insight into the molecular biology of p63. Further, these results will aid in better understanding of role of p63 mutants in development and cancer.
Advisors/Committee Members: Kadakia, Madhavi P.
Subjects: Biology, Molecular
Keywords: TAp63gamma
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9.
Litteral, Vaughn.
mdm2 Amplification in NIH3T3L1 Preadipocytes Leads to Mdm2 Elevation in Terminal Adipogenesis.
Degree: MS, Biochemistry and Molecular Biology, 2008, Wright State University
► The p53 protein is a tumor suppressor protein that is mutated or…
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▼ The p53 protein is a tumor suppressor protein that is mutated or non-functional in nearly all cancers. The Mdm2 protein has the ability to functionally inactivate p53 and these two proteins have been studied extensively in the context of cellular proliferation. In this study, expression of the murine double minute 2 (mdm2) gene was examined in the mouse NIH3T3L1 cell line. Under the proper conditions, the immortalized NIH3T3L1 cells have the ability to differentiate from fibroblasts to adipocytes (Green et al., 1975). This well characterized cell line provides an excellent model to study mdm2 in differentiation. While evaluating the regulation of the mdm2 gene during adipogenesis, it was discovered that NIH3T3L1 preadipocytes possess a 36-fold elevation of mdm2 mRNA relative to A31 cells, another Balb/c 3T3 fibroblast cell line that lacks the capacity to differentiate (Berberich et al., 1999). Based on Southern blot analysis, this increase in mdm2 mRNA is the result of a 60 fold gene amplification (Berberich et al., 1999). This study evaluated how mdm2, p53 and other associated gene products change as NIH3T3L1 cells differentiate from preadipocytes to adipocytes. Most surprising, the mdm2 mRNA and protein levels remained high throughout differentiation and Mdm2 changes localization and interaction with key proteins. This work further proves adipogenesis can occur in the presence of high levels of Mdm2 expression. Herein, Mdm2 is shown to preferentially interact with the retinoblastoma protein, pRb, in terminal adipocytes versus preadipocytes. The p53 mRNA, protein levels and DNA binding (data not shown) decreased and the results suggest that Mdm2 could have a more p53 independent role in adipogenesis. Finally, this work leads to future experiments of determining the importance of high level Mdm2 expression in terminal adipogenesis.
Advisors/Committee Members: Berberich, Steve.
Subjects: Biochemistry; Biology; Biomedical research; Cellular biology; Molecular biology; Oncology
Keywords: mdm2; mdm-2; Rb; retinoblastoma; p53; cancer; adipogenesis; liposarcoma; adipose; oncogene; C/EBP; tumor suppressor; NIH3T3L1; NIH3T3-L1; NIH3T3; amplification; chromosomal amplification; Southern; Northern; Western; immunoprecipitation; mdm4; mdmX
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10.
Makley, Meghan Katherine.
NMR analyses show TCDD elicits differences in hepatic metabolism in female C57BL/6 mice and Sprague-Dawley rats.
Degree: MS, Biochemistry and Molecular Biology, 2008, Wright State University
► TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) elicits tissue-, sex-, and species-specific effects. This study compares the…
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▼ TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) elicits tissue-, sex-, and species-specific effects. This study compares the hepatic response to an oral dose of TCDD in immature ovariectomized (i.o.) C57BL/6 mice (30 µg/kg) and i.o. Sprague-Dawley rats (10 µg/kg), at 72, 120, and 168 h post-dose. Hepatic lipid extracts were analyzed by 13C and 31P NMR, and aqueous extracts by 1H and 31P NMR.Consistent with increased lipid content in mice (p≤0.05), TCDD induced increases in hepatic triacylglycerides (TAG), cholesterol, and fatty acids. Principal component analysis of 13C spectra show treatment groups separate in mice, but not rats. Mice showed decreases in the lactate/pyruvate ratio and dihydroxyacetone phosphate, consistent with decreased cytosolic NADH/NAD+ ratio and upregulated TAG synthesis. TCDD-treated rats exhibited decreased levels of sphingomyelin, and three-fold increase in phosphocholine, suggesting TCDD activates sphingomyelinase. Both species showed similar decreases in cardiolipin, indicating oxidative stress. These observations are compared with hepatic histopathology and gene expression findings.
Advisors/Committee Members: Reo, Nicholas.
Subjects: Biochemistry; Toxicology
Keywords: liver; NMR; metabolism; TCDD; dioxin
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11.
Mandke, Pooja P.
Study of MicroRNA-34a mediated post transcriptional regulation of MDM4.
Degree: MS, Biochemistry and Molecular Biology, 2012, Wright State University
► MDM4 is an important negative regulator of the tumor suppressor p53. In…
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▼ MDM4 is an important negative regulator of the tumor suppressor p53. In normal unstressed cells the activity of p53 is kept under control by MDM4 and its homologue MDM2. MDM4 is said to possess oncogenic potential based on the evidence of its overexpression in many cancers. Until recently it was believed MDM4 is constitutively transcribed; however a decrease in full length MDM4 in response to genotoxic stress was observed paving way for exploring the mechanism responsible for this. It was observed miR-34a a member of the miR34 family which is a direct transcriptional targets of p53 could have a potential role in regulation of MDM4 expression. The 3'untranslated region of MDM4 was also seen to contain several miR-34a binding sites. However reporter assays with select regions of the 3'UTR revealed that the 3'UTR was unresponsive to miR-34a mediated regulation. Reassessment of the MDM4 gene revealed presence of a potential miR-34a regulatory site in the protein coding exon eleven of MDM4. This site was further considered to check for functionality in response to miR-34a modulation. A reporter with the miR-34a site from the coding region was constructed. This reporter was responsive to overexpression or inhibition of endogenous miR-34a in H1299 and MCF7 cells respectively ascertaining the functionality of this site. A SNP leading to an A to C transversion in the seed region of this miR-34a site in the exon 11 was predicted to disrupt responsiveness to miR-34a. We confirmed this by creating point mutants and performing reporter assays. This study was designed to understand the regulation of MDM4 in absence of DNA damage conditions. Understanding the role of miR-34a in regulation of MDM4 will pave way for designing specific therapeutic strategy for reactivation of p53 via inhibition of MDM4 in cancer that overexpress MDM4 and retain wild type p53.
Advisors/Committee Members: Markey, Michael.
Subjects: Molecular Biology
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12.
Mpagi, Meldrick Daniel.
In Search For New p53 Regulated Genes.
Degree: MS, Biochemistry and Molecular Biology, 2008, Wright State University
► Mpagi, Meldrick. M.S., Department of Biochemistry and Molecular Biology, Wright State…
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▼ Mpagi, Meldrick. M.S., Department of Biochemistry and Molecular Biology, Wright State University, 2008. In Search For New p53 Regulated Genes.The p53 tumor suppressor protein has the ability to transactivate its target genes whose gene products are involved in carrying out cell cycle arrest, apoptosis, DNA repair, and senescence. Here, I report that two genes may be p53 regulated. Utilizing a microarray method to search for novel p53 target genes, I was able to identify a possible transcriptional target of p53 being solute carrier family 1a1 (SLC1a1). Along with that finding I also identified an E2F-target gene, minichromosome maintenance 10 (MCM10), as being p53 regulated. Gene expression profiling of MCF7 breast cancer cells treated with RNAi targeting Hdm2 and HdmX in order to reactivate p53 led to increased SLC1a1 transcript levels. DNA damage experiments in several cell lines and along with a p53 overexpression experiments established that p53 activation does not directly result in a transcriptional increase in SLC1a1 expression. Thus the results suggest that SLC1a1 is not a transcriptional target of p53 and may have been a false positive result from the microarray experiment. Gene expression profiling of MCF7 breast cancer cells treated with RNAi targeting Hdm2 and HdmX in order to reactivate p53 led to a transcriptional decrease in MCM10 expression. DNA damage experiments along with siRNA targeting Hdm2 and HdmX established that p53 activation leads to a reduction in MCM10 transcript levels. Furthermore, I established that the p53-mediated reduction of MCM10 mRNA levels is due to p53-mediated transactivation of p21, a well-known p53 target involved in cell cycle arrest. These results suggest that p53 activation leads to a reduction in gene expression of E2F target genes involved in cell cycle progression through transactivation of p21.
Advisors/Committee Members: Berberich, Steven.
Subjects: Molecular biology
Keywords: p53, Hdm2, HdmX, siRNA
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13.
Omolewu, Rachel.
Characterization of Three Mutations in Conserved Domain of Subunit III of Cytochrome c Oxidase from Rhodobacter sphaeroides.
Degree: MS, Biochemistry and Molecular Biology, 2010, Wright State University
► Cytochrome c oxidase (COX) is the final electron acceptor in mitochondrial respiratory…
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▼ Cytochrome c oxidase (COX) is the final electron acceptor in mitochondrial respiratory chain and in many bacterial species including Rhodobacter sphaeroides. Electron transfer is coupled with the pumping of protons across the membrane. Previous work has shown that reaction of beef COX with dicyclohexylcarbodiimide (DCCD) resulted in an inhibition of proton translocation by covalently binding to the conserved amino acid residue E90 located in a nonpolar region of subunit III (SIII). E90 is involved in a bonding pair with another conserved residue H212, possibly connected by a salt bridge or a hydrogen bond in the three dimensional structure of SIII. Our goal was to test whether the retention of the E90-H212 linkage and the spatial arrangements of these amino acid residues were critical for electron transfer and proton pumping activities of the enzyme. This work analyzes the functional role of these amino acids through the creation of three mutants in SIII-H212E, E90H, and E90H/H212E. SDS-PAGE verified all mutants contained SI-III. The first mutant, H212E, bacteria cultures grew significantly faster as compared to wild-type; while the other two mutant culture grew at comparable rates to wild-type. Additionally, the visible absorbance spectrum of H212E mutation in bacterial membranes showed little or no heme aa3 oxidase expression while the other two mutants exhibited properties similar to wild-type. Conversely, the spectrum of isolated and purified COX-SIII E90H mutant protein displayed a red shift while the double mutation COX-III E90H/H212E resembled that of wild-type. Electron transfer activity of two purified mutant proteins E90H and E90H/H212E revealed decreases in steady-state activities approximately 40% and 15% respectively. Lastly, the two mutants displayed a slight alkaline shift in pKa value for electron transfer activity. In summary, these results imply that the absolute positions of E90 and H212 are essential to COX activity. The single mutations resulting in two like charges caused changes in COX activity whereas the double mutation that retained the native salt bridge did not. Additionally, the increase in pKa may suggest the environment around the active site is perturbed, which is reflected by the decrease in electron transfer activity.
Advisors/Committee Members: Prochaska, Lawrence.
Subjects: Biochemistry; Biophysics
Keywords: cytochrome c oxidase; mitochondria, DCCD; rhodobacter sphaeroides; subunit III; site-directed mutagenesis
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14.
Rigsbee, Laura J.
Interrogation of the Distal Gut Microbiota of Healthy Adolescents and those with Irritable Bowel Syndrome.
Degree: MS, Biochemistry and Molecular Biology, 2011, Wright State University
► The human-associated microbiota has been the focus of much current research, with…
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▼ The human-associated microbiota has been the focus of much current research, with the microbiota inhabiting the gastrointestinal tract of particular interest. These organisms play many roles in human health and well-being. However, shifts in the composition of the intestinal microbiota have been associated with diseases such as irritable bowel syndrome, inflammatory bowel disease, and colon cancer. Several recent studies have reported on the distal gut microbiota composition of healthy adults and those with IBS, while there is a lack of studies devoted to adolescents. This study utilized a custom-designed Affymetrix Microbiota Array capable of detecting 775 phylo-species of intestinal bacteria to determine the composition of the distal gut microbiota of 22 adolescents suffering from IBS-D (diarrhea-predominant) and 22 healthy adolescents. High sample-to-sample variation was observed in both groups at genus level. While some differences were observed in mean relative abundance of several bacterial genera between IBS-D and healthy adolescents, including Bifidobacterium, Lactobacillus, Veillonella, and Prevotella, these differences were not significant. Sample groups also failed to separate in PCA space. Therefore, we cannot conclude that the distal gut microbiota of adolescents with IBS-D is significantly different than that of healthy adolescents.
Advisors/Committee Members: Paliy, Oleg.
Subjects: Microbiology; Molecular Biology
Keywords: microbiota; intestine; intestinal bacteria; distal colon; irritable bowel syndrome; IBS; microarray
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15.
Rotsinger, Joseph E.
EXPLORATION OF YPEL3 RESPONSE TO HORMONES AND ABILITY TO INDUCE SENESCENCE.
Degree: MS, Biochemistry and Molecular Biology, 2012, Wright State University
► p53 activation through different cellular senescence pathways can trigger cell cycle arrest…
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▼ p53 activation through different cellular senescence pathways can trigger cell cycle arrest via regulation of p53 target genes. One such target gene is YPEL3 which is expressed upon binding of tumor suppressor protein p53 at its p53 binding sites (Kelley, 2010). The ability of p53 to induce YPEL3 gene expression led to the discovery that YPEL3 is one of several p53 target genes which induce cellular senescence (Kelley, 2010). Additionally YPEL3 can be regulated independently of p53 by estrogen signaling through estrogen receptor α (Tuttle, 2011). The loss of estrogen receptor α or removal of estrogen induces YPEL3 gene expression and leads to cellular senescence, indicating that estrogen bound to estrogen receptor α represses YPEL3 gene expression (Tuttle, 2011). Although YPEL3 induction results in cellular senescence the mechanism by which YPEL3 elicits cellular senescence is not well understood. It is also unknown if other steroid hormones, such as testosterone play a role in regulating YPEL3 gene expression To further understand hormone regulation of YPEL3 the first part of this thesis tested if testosterone regulates YPEL3 gene expression in MCF7 breast cancer cells and LnCAP prostate cancer cells. Like MCF7 breast cancer cells, LnCAPs cultured in the absence of steroid hormones induced YPEL3 expression indicating that YPEL3 gene expression in LnCAPs is repressed by steroid hormones. This induction of YPEL3 expression was blocked by the addition of testosterone to LnCAP cells. In contrast the addition of testosterone to steroid deprived MCF7 cells resulted in YPEL3 induction. Based on the results in LnCAP prostate cancer cells and MCF7 breast cancer cells it appears that testosterones effect on YPEL3 gene expression is tissue specific. In part two of this thesis MCF7 and IMR90 cells were employed to determine if over expression of YPEL3 leads to increased reactive oxygen species (ROS) levels. First an optimized method for detecting reactive oxygen species levels in breast cancer cells using DCFDA was developed. Utilizing this method, MCF7 human breast cancer cells harboring a Tet-On system expressing YPEL3 induced with tetracycline did not show increased levels of reactive oxygen species over LacZ expressing MCF7 cells. Additionally Infecting MCF7 cells with lentivirus expressing YPEL3 and probing with DCFDA showed no increase of ROS levels. Alternatively IMR90 primary diploid human fibroblasts containing a normal repertoire of genes and fully functional pathways were infected with lentivirus expressing YPEL3 and also did not show an increase in ROS levels. These results suggest that YPEL3 activates senescence in a ROS independent manner. The third part of this thesis was to identify YPEL3 interacting proteins. Epitope tagged YPEL3 proteins obtained from MCF7 tetracycline responsive cells expressing YPEL3 were captured from cell extracts by co-immunoprecipitation, followed by elution and denaturing. Denatured proteins were separated by SDS-Page gel electrophoresis and potential protein bands excised for composition analysis by LC/MS/MS. LC/MS/MS analysis identified potential proteins that interact with YPEL3. The cumulative findings of this thesis were designed to aid in the understanding of YPEL3 regulation by testosterone and to assist in locating potential downstream targets of YPEL3 that may lead to senescence.
Advisors/Committee Members: Berberich, Steven.
Subjects: Biochemistry
Keywords: YPEL3; Testosterone; Reactive Oxygen Species; Co-Immunoprecipitation
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16.
Sibomana, Isaie.
‘Functional Metabolomics’ Enhances Assessment of Tissue Dysfunction as Demonstrated in a Rat Model of Sub-Acute D-serine Exposure.
Degree: MS, Biochemistry and Molecular Biology, 2011, Wright State University
► We describe a methodology that combines urinary metabolomics with a tissue-specific stressor…
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▼ We describe a methodology that combines urinary metabolomics with a tissue-specific stressor administration to enhance assessment of tissue function. Kidney function in rats was mildly compromised with a sub-acute dose of D-serine and stressed with furosemide. NMR-based metabolomics analyses showed no detectable effects due to D-serine alone; but furosemide or D-serine + furosemide groups, classified separately from each other, and from control. Furosemide alone caused a ca. 2-fold increase in glucose, lactate, choline, and a 30% decrease in TCA intermediates (p≤0.05). D-serine suppressed these effects and produced a 1.7-fold increase in a p-phenolic acid-derivative of tyrosine (PAdY) relative to control (p≤0.05). The PAdY/tyrosine ratio increased 2-fold relative to rats given furosemide alone. D-serine effects were only detectable in furosemide-challenged rats, suggesting that minor disruption in kidney function, induced by low-level D-serine, is manifested by this functional metabolomics methodology. This technique may improve sensitivity for assessment of tissue function and disease
Advisors/Committee Members: Reo, Nicholas V.
Subjects: Biochemistry
Keywords: NMR; metabolomics; D-serine toxicity; kidney toxicity
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17.
Vaiana, Christopher Anthony.
Bio-Functionalized Clay Nanoparticles for Wound Healing Applications.
Degree: MS, Biochemistry and Molecular Biology, 2011, Wright State University
► Wound healing is a complex, multi-step process that can be summarized into…
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▼ Wound healing is a complex, multi-step process that can be summarized into three stages, namely hemostasis and inflammation, proliferation, and finally tissue remodeling. Battlefield wound healing demands rapid hemostasis using clotting or cauterizing agents to immediately limit blood loss, but this occurs at the expense of proper tissue repair beyond hemostasis. Layered silicate clays such as kaolin and montmorillonite (MMT) have been previously shown to induce blood clotting due to their ability to form charged interactions with clotting factors. The charge characteristics of sodium MMT (Na-MMT) also enable functionalization with active biomolecules. Herein we first functionalize three types of alumoinosilicate clays, namely Na-MMT, kaolin, and halloysite with horseradish peroxidase (HRP) as a model system with which to study the binding and biological activity of biomolecules bound to MMT. We then functionalized Na-MMT with epidermal growth factor (EGF) via ion exchange reaction to create a nanocomposite (MMT-EGF) with EGF occupying approximately 0.12 % of the Na+ exchange sites and conduct biochemical analysis of keratinocytes after treatment with MMT-EGF. Our results demonstrate that EGF immobilized on MMT retains the ability to activate the epidermal growth factor receptor (EGRF), causing phosphorylation of the AKT and MEK1 pathways, as well as upregulation of its downstream target gene expression involved in cell growth and migration. This study also shows that like EGF, MMT-EGF treatment can stimulate cell migration in vitro, which is dependent on ERK1/2 phosphorylation.
Advisors/Committee Members: Kadakia, Madhavi.
Subjects: Biochemistry
Keywords: wound healing; layered silicates; EGF; Montmorillonite
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18.
Van Nostrand, Joseph E.
Detection and Destruction of Escherichia Coli Bacteria and Bacteriophage Using Biofunctionalized Nanoshells.
Degree: MS, Biochemistry and Molecular Biology, 2007, Wright State University
► The ability to detect chemical and biological agents is arguably one of…
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▼ The ability to detect chemical and biological agents is arguably one of the highest priority technical challenges today. The capability to obtain specific information at and near single-molecule resolution is the ultimate goal in chemical and biological agent detection. Metallic nanostructures, nanoshells and nanorods in particular, are attractive substrates because of their plasmonic properties. Combining the specificity of biomolecular recognition with these nanostructures might lead to increased sensitivity and selectivity. Localization of biological recognition motifs to the surface of these nanostructures could provide a mechanism for highly specific and directed energy transfer when bound to its target. This study utilizes nanoshells functionalized with antibodies specific for Escherichia coli, and investigates at both the microscopic and macroscopic scales the ability of these biofunctionalized nanoshells to bind and destroy their target micro-organism when excited using 808 nm near infrared laser radiation. Extension of the technique to Bacillus subtilis spores as well as bacteriophage specific to Escherichia coli are also explored. The bacteriophage is a viral surrogate, and provides a means to explore proof of principle of the interactions between nanoshells and viruses. It is demonstrated that appropriately biofunctionalized nanoshells recognize and bind to their target species, and that the nanoshell successfully couples the energy transfer from an IR laser to the target species. A ratio of nanoshells to Escherichia coli of ~104 for a 50% bacterial cell survival rate is determined, and a possible mechanism for this is discussed. Finally, this ratio is found to decrease by 4-5 orders of magnitude for the case of two Escherichia coli bacteriophage considered, and the significance of this is described.
Advisors/Committee Members: Kadakia, Madhavi P.
Subjects: Biology, Molecular
Keywords: NANOSHELLS; BIOFUNCTIONALIZED; BIOFUNCTIONALIZED NANOSHELLS; ESCHERICHIA; ESCHERICHIA COLI; COLI; BACTERIOPHAGE
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19.
Wang, Zhuo.
Genetic studies of genes involved in the initiation of DNA replication in the fission yeast Schizosaccharomyces pombe.
Degree: MS, Biochemistry and Molecular Biology, 2010, Wright State University
► The initiation of DNA replication is a highly conserved process in all…
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▼ The initiation of DNA replication is a highly conserved process in all eukaryotes. However, the underlying mechanism is not well understood. Genetic studies in the fission yeast S. pombe have contributed greatly to and will continue to provide insights to our understanding of this important biological process. In the first chapter, we have used a complementary method to test three recently identified human replication proteins DUE-B, Ticrr/Treslin, and GEMC1 as the candidate functional homologue of Sld3 in S. pombe. Sld3 is an essential replication initiation protein discovered in yeasts. Since no apparent sequence similarity can be found, its homologue in higher eukaryotes remains to be uncovered. In fact, among all yeast replication proteins, Sld3 is the only protein whose functional homologue has not been found in metazoan. Our preliminary results showed that all three human replication proteins failed to complement the function of Sld3 in fission yeast. Unlike DUE-B, whose expression does not perturb the cell cycle progression in fission yeast, overexpression of Ticrr and GEMC1 in S. pombe can suppress the cell growth. In the second chapter, we have developed a screening strategy in S. pombe to discover new gene(s) that may function in the initiation of DNA replication. A yeast strain has been made in which the promoter of Chk1, the effector kinase of the DNA damage checkpoint in fission yeast, was replaced with a thiamine-repressive promoter. The Chk1 expression in this strain can be completely shut off by adding thiamine in the culture medium. It is known that defects in the replication initiation require the checkpoint function for cell survival. A mutation in the gene involved in the initiation of DNA replication is expected to be sensitive to the depletion of the Chk1 function. In a small-scale screening, we obtained 15 so-called Kds (Chk1 dependent survival) mutants. Characterization of one the mutants Kds15 in the preliminary study identified a structure-destructive mutation in Psf2, a subunit of the essential GINS complex required for the initiation of DNA replication. This result validates the strategy and shows that the screening can be productive.
Advisors/Committee Members: Xu, Yong-jie.
Subjects: Biochemistry; Biology; Genetics
Keywords: Initiation of DNA replication; Homologue; Sld3; DUE-B; Ticrr; Treslin; GEMC1; Kds; Psf2; Schizosaccharomyces pombe
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20.
Whitlatch, Andrew J.
p63 and VDR are regulated by Vitamin D (VD3) and UV signaling.
Degree: MS, Biochemistry and Molecular Biology, 2010, Wright State University
► Skin cancers, such as squamous cell carcinoma (SCC), develop from accumulated mutations…
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▼ Skin cancers, such as squamous cell carcinoma (SCC), develop from accumulated mutations as a result of excessive exposure to Ultraviolet B (UVB) radiation. Intriguingly, UVB also catalyzes the synthesis of 1alpha, 25-dihydroxy Vitamin D3 (VD3), the hormonally active form of Vitamin D. Downstream VD3 signaling has been associated with promoting the inhibition of cell cycle progression, regulating calcium homeostasis, and inducing differentiation and apoptosis. VD3 mediates these processes via genomic mechanisms through interaction with its cognate receptor, the Vitamin D Receptor, (VDR). In addition, it was recently discovered that VD3 reduces UVB-mediated phosphorylation of the SAPK/c-Jun N-terminal kinase (JNK), which correlated with a reduction in apoptosis and an increase in cell survival. Furthermore, VD3 treatment of keratinocytes also promoted up-regulation of the pro-survival p63 isoform, deltaNp63alpha. Expression of deltaNp63alpha in the basal progenitor layer of the epidermis is required for maintaining epidermal integrity by promoting continual proliferation and early commitment to stratification. VDR has also been shown to be essential for maintaining the integrity of the epidermis as exhibited by VDR null mice, which display skin defects. Although the downstream effects of VDR and deltaNp63alpha are well documented, their upstream regulation has been underexplored. In this study, we hypothesized that deltaNp63alpha and VDR are regulated by VD3 and UV signaling. To address this, mouse embryonic fibroblasts (MEFs) and transformed keratinocyte cell lines were treated with VD3 in the presence or absence of UV radiation. We confirmed that UV treatment resulted in the phosphorylation and activation of the JNK pathway, and that this effect is reduced in keratinocytes pre-treated with VD3. Moreover, we observed that UV exposure resulted in a reduction in VDR protein levels, and led to an electrophoretic mobility shift in deltaNp63alpha. Although, a mobility shift in deltaNp63alpha upon UV treatment was previously attributed to phosphorylation by p38, it did not rule out JNK as an alternate kinase and upstream regulator. While VD3 treatment inhibited UV induced p-JNK and VDR degradation, it did not reverse the mobility shift in deltaNp63alpha. Furthermore, VD3 treatment did not result in a significant elevation of p63 or VDR RNA, but increased the protein levels of VDR and p63. We attributed the increased levels of deltaNp63alpha and VDR protein to increased stability since VD3 significantly increased the half-life of both proteins. Finally, we observed that siRNA knockdown of VDR did not affect the ability of VD3 to inhibit the formation of p-JNK, but instead resulted in reduced total JNK levels independent of VD3 treatment. Our data shows that UV and VD3 signaling merge to regulate both deltaNp63alpha and VDR, and the balance between the two signaling pathways could determine whether cells survive or undergo apoptosis following UV-mediated DNA damage.
Advisors/Committee Members: Kadakia, Madhavi.
Subjects: Biochemistry; Molecular biology
Keywords: p63, VDR UV, Vitamin D, VD3
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21.
Yao, Jianhong.
DUE-B, A NEW HUMAN DNA REPLICATION PROTEIN, IS THE FUNCTIONAL HOMOLOG OF S. CEREVISIAE SLD3.
Degree: MS, Biochemistry and Molecular Biology, 2009, Wright State University
► DNA unwinding elements (DUEs) are commonly found at DNA replication origins. The…
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▼ DNA unwinding elements (DUEs) are commonly found at DNA replication origins. The DUE binding protein (DUE-B) is crucial for the initiation of DNA replication in eukaryotes. The unique 59 amino acid C-terminal part of DUE-B shares nearly 50% similarity with yeast the C-terminus of Sld3. DUE-B plays a key role in eukaryotic DNA replication because it is required for the loading of Cdc45, the MCM helicase activator, on chromatin. Here we show that DUE-B, just like yeast Sld3, binds to Cdc45 and TopBP1 through its C-terminus in Sf9 cells and in vitro. We also show that DUE-B, Cdc45 and TopBP1 form a heterotrimeric complex in vitro. The mass spectrometric data show that dominant negative Sf9 DUE-B is not phosphorylated but functional HeLa DUE-B is phosphorylated. All these data suggest that human DUE-B is a functional homolog of yeast Sld3.
Advisors/Committee Members: Leffak, Michael.
Subjects: Biomedical research
Keywords: Cdc45; TopBP1; Cdc45 and TopBP1; SLD3; DNA; DNA REPLICATION; REPLICATION
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