Browsing by Subject "Department of Microbiology"

Now showing 1 - 9 of 9
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    Analysis of a Genetic Adaptation for Glycerol Utilization: Implications for Microbial Fuel Cells
    (2010-04-21) Torchia, Mike
    Biodiesel promises a renewable source of energy yet is unable to be an economically viable alternative to petroleum. One way to solve this is to convert glycerol, a by-product of the biodiesel production process, to higher value commodities. Shewanella oneidensis can respire insoluble extracellular substrates such as electrodes. Furthermore, when the pGUT2PET plasmid is transformed into wild type S. oneidensis, the non-redox balanced conversion of glycerol to ethanol is permitted. This engineered bacterium permits the generation of two higher value products (ethanol and electricity) from the original glycerol feedstock. Since any future industrial application of this microbe will necessitate optimization of all its parameters, we were interested in studying how S. oneidensis grows faster on glycerol.
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    Candida albicans Mutagenesis: Response to Stress
    (2009-04-08) Bruck, David Joachim
    Candida albicans is a model eukaryotic yeast and an opportunist human pathogen. It generates novel drug resistance through mutation and mitotic recombination. The rate loss of heterozygosity, or rate LoH, is a measure of this mutation and recombination. Previous research has shown that some stresses like the anti-fungal drug fluconazole cause the yeast cells to increase their rate LoH while other stresses do not. On the assumption that fluconazole does not directly affect DNA replication, it is possible that the increased rate LoH is due to a stress response pathway. My UROP project aimed to create a list of computationally curated gene targets using microarray expression data analysis which would then be tested for mutant phenotype rate LoH. The majority of the time allocated to the project was spent doing ortholog searches of gene lists curated from another yeast, Saccharomyces cerevisiae, as well as studying any previous papers where any of the orthologs in question were directly examined.
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    Comparative Analysis of Hs6st and Sulf1-mcherry Expression Patterns in Drosophila
    (2011-04-13) Uk, Samantha; Dang, An
    Heparan sulfate proteoglycans (HSPGs) are molecules that are comprised of a core protein modified with heparan sulfate (HS), a negatively charged linear polysaccharide, consisting of uronic acid and N-acetyl-glucosamine (GlcNAc) disaccharide repeats. Generally, these molecules are located on the cell surface and the extracellular matrix. HSPGs have been known to be associated with various biological processes such as growth factor signaling, neuronal development, and cell adhesion. HS chains possess heterogeneous structures, and their diverse patterns of sulfation can determine the binding specificity for certain proteins. 6-O-sulfation of GlcNAc (or GlcNSO3) is the key modification of HS, since it can be dynamically regulated; heparan sulfate 6-sulfotransferase (Hs6st) catalyzes the transfer of sulfate groups of GlcNAc (or GlcNSO3), while heparan sulfate 6-O endosulfatase (Sulf1) removes it. However, how 6-O-sulfation is regulated during animal development remains largely unknown. In this poster, we will present expression analysis of these enzymes in the fruit fly Drosophila melanogaster. We generated a transgenic reporter fly for Sulf1 gene, and its expression pattern was compared with that of Hs6st gene using Hs6st-lacZ enhancer trapline. Our study will provide regulatory mechanisms of HS during the development of Drosophila as well as other multi-cellular organisms.
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    Comparative Efficacy Study of IL-15 Transfections in Various Mouse Cancer Cells (RM-1 Prostate Cancer, B16 Melanoma, and 4T1 Breast Cancer)
    (2009-04-08) Schwab, Molly
    Genes for IL-15 were transfected into RM-1 (prostate cancer), B16 (melonoma), and 4T1 (breast cancer) cancer cells. The transfected genes consisted of two types, one for the isoform of IL-15 which has a full length leader sequence and is secreted from the cell, and the second for the isoform of IL-15 which has a truncated leader sequence and accumulates in the cytoplasm of the cell. Both kinds of each cancer cell line were cloned, showing that there was little translation of the IL-15 genes in RM-1 and 4T1 cells, and good translation of the IL-15 genes in B16. The vaccine efficacy of the original three cancer cell lines reflected these levels of IL-15. A genetic modification of the IL-15 gene was made to remove the signal peptide of the truncated isoform to make a very short sequence peptide (VSSP-1). The translation of the new genetically modified IL-15 gene was determined by cloning it into all of the cell lines, where it was found to give greater translation of IL-15. The vaccine efficacy for the various IL-15 transfectants of B16 melanoma was determined in mouse tumor studies. The VSSP-1 transfected B16 melanoma vaccine was the most efficacious.
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    Effects of Rapamycin on Dia2 in S. cerevisiae
    (2012-04-18) Newhouse IX, Peter
    The SCF ubiquitin ligase is a protein complex which selectively ubiquitinates various proteins in order to mark them for degradation. Dia2 is an example of an F-box protein, one of the components of the SCF complex. Dia2 is known to operate at the rDNA locus within the cell’s nucleus; this localization may be related to Dia2’s role in regulating the cell’s progress through the S-phase checkpoint. In this way, it has some similarities to Tor1, which has been shown to associate with the rDNA promoter except when the cell is under stress from starvation or the chemical rapamycin. We therefore hypothesized that treatment with rapamycin would result in a loss of Dia2’s nuclear localization. We demonstrate via indirect immunofluorescence that this nuclear localization is partially lost after exposure to rapamycin. Current efforts focus on quantifying the amount of Dia2 present in rapamycin-exposed and rapamycin-free cells, both within the cell as a whole and within the nuclear and cytoplasmic cell fractions.
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    Expression and purification of GxcA, a c-type cytochrome involved with metal respiration by the bacterium, Geothrix fermentans
    (2011-04-13) Liu, Joanne
    The study of bacteria capable of respiring oxidized metals offers insights into the geochemical cycling of metals, including toxic heavy metal contaminants. These dissimilatory metal-reducing organisms couple oxidation of organic compounds with the reduction of substrates to gain energy. Improving understanding of the various bacterial metal-reducing strategies will increase our ability to produce renewable energy using microbial fuel cell technologies. The bacterium Geothrix fermentanshas long been ignored in the study of metal respiration, but it has recently been shown to employ a unique strategy involving more than one biochemical pathway that appears tuned to use of high potential metals, such as uranium and manganese. Membranes isolated from Fe(III)-respiring G. fermentans contain high levels of a decahemecytochrome, known as GxcA, which is suspected in electron transfer by G. fermentans. As a genetic system for G. fermentans is not yet available, GxcAwas targeted for expression and purification. The DNA sequence for GxcA, containing an in-frame hexahistidinesequence, was first cloned into E. coli, using the inducible expression vector pETlite. Then, the recombinant plasmid was co-transformed into E. coli with pEC86, a plasmid that contains genes for the ccmc-type cytochrome maturation system. Colonies were screened for c-type cytochromes by redox difference absorption analysis and hemestain analysis. Future work will involve GxcApurification for redox and localization experiments to determine GxcA’spotential role in G. fermentans.
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    Fishing for Function: Identifying Functional Binding Partners of CUG-Binding Protein 1
    (2009-04-08) Jeschke, Jonathan C.
    One of the most remarkable distinguishing features of living organisms is their ability to fluidly adapt to changing environmental conditions. The advances in molecular biology over the past 50 years have resolved the general outlines this capacity for adaptation on the scale of molecules. We now view an organism’s ability to adapt as the result of many complex programs of gene expression. As a basic science, the improved resolution of these expression programs has proved invaluable in understanding many clinical pathologies, the most dramatic being cancer. My laboratory work has focused on resolving a process cells use, at the level of messenger RNA (mRNA), to turn off genes before functional proteins are made. This enzymatic process involves the break down of mRNA polymers where the degrading enzymes are recruited by specifi c adaptor proteins to specifi c sets of mRNA. One of these adaptor proteins is CUG-binding protein 1 (CUGBP1) and identifying the mRNA set CUGBP1 targets has been a focus of our lab. The degradation proteins which CUGBP1 targets to the mRNA are only poorly resolved. I am using a yeast-two hybrid screen to identify CUGBP1 binding candidates. These candidates will then be con firmed by co-immunoprecipitation and mRNA a ffinity chromatography. This will allow me to characterize the specifi c mechanism of decay elicited by the function of CUGBP1.
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    Impact of a Genetic Deletion on the Capacity of Group B Strepococcus to Invade Human Epithelial Cells
    (2009-04-08) Lemnitzer, Liz
    Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is one of the most common causes of invasive infections including sepsis, meningitis, and pneumonia in neonates and immunocompromised patients. A major virulence factor encoded by all nine serotypes of GBS is the surface protein, C5a peptidase r (SCPB). This enzyme specifically degrades the human C5a chemotaxin and promotes intracellular invasion of epithelial cells. Previous experiments revealed that some clinical serotype V strains contain a partial deletion within the scpB gene. This study investigates whether these strains are a unique lineage and are functionally similar to each other but different from strains that lack the scpB deletion. I postulated that the strains containing the deletion are clonally related and that the deletion in scpB reduces the ability of strains to invade HEp2 cells. A western blot using antibodies against the SCPB protein was performed on both mutant and wild type strains in order to confirm the absence of SCPB in the mutant strains. Invasion assays were conducted on various serotype V strains and showed that the strains with the scpB deletion invaded at a similar rate in comparison to each other suggesting that these strains belong to the same lineage. The comparison of DNA fragments produced by restriction fragment length polymorphism (RFLP) on these mutant strains also supports this theory. However, contrary to my hypothesis, strains containing the deletion demonstrated a higher percent invasion versus the wild type strain. To further investigate this finding and the relatedness of these strains, an adherence assay will be performed to determine if there is a difference in the ability to adhere to epithelial cells between mutant and wild type strains.
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    Investigating Calcium Intake and Osteoporosis Risk Factors in Vietnamese-Americans
    (2010-04-21) Ton, Angie
    Asian Americans have a naturally higher risk for developing osteoporosis due to their genetics, lifestyle choices, and higher incidence of lactose intolerance. While data is available on osteoporosis status, risk factors, and knowledge about the disease groups in general Asian populations, little is known about osteoporosis in specific Asian sub-groups. The purpose of this investigation was to survey Vietnamese-Americans in the local community to assess daily calcium intake, general knowledge about osteoporosis and prevention, as well as other lifestyle choices that promote the development of osteoporosis. A cross-sectional survey of 80 Vietnamese-Americans between the ages of 18-70 who attended a local Vietnamese New Year event or Vietnamese Student Association gathering was used to collect data. It is predicted that Vietnamese Americans adults will not meet the recommended daily calcium intake and will not be knowledgeable about other lifestyle choices that increase their risk for developing osteoporosis. Through the information collected, we hope to gain insight into the Vietnamese community’s knowledge about osteoporosis and educate local communities on osteoporosis prevention.