Senior Project Abstracts 2011

All Projects for Academic Year 2010-2011

Theodore G. Bean II Rebecca J. Beichner
Bridget F. Donnelly Shivani Dudhia
Shawn E. Dumbroski Leanne E. Ianniello Kyle J. Knupp
Ana M. Kuoman Erin Resetar Emma C. Wise

Synthesis of Dicamba Derivatives

Theodore G. Bean II, 2011 (Advisor: Dr. Shaun Murphree)

The auxins are an important class of plant growth hormones with dicamba being a common auxinic herbicide. The underlying mechanisms behind their function in plants is finally being uncovered after over 100 years of auxin research. These mechanisms allow for increased specificity of binding and phenotypic results of synthetic auxins. Herein, a total synthesis of two dicamba derivatives is described. Dicamba 5 was first reduced to the alcohol 9, which was subsequently brominated via PBr3 to the bromide 10. This was converted to two different final products 1) the acetic acid 11 through a Grignard with carbon dioxide and 2) the benzyl sulfonate 13 through an SN2 reaction with sodium sulfite.

FbxA-dependent ubiquitination and degradation of FbiA in Dictyostelium discoideum

Rebecca J. Beichner, 2011 (Advisor: Dr. Margaret Nelson)

FbiA (FbxA interacting protein) is a protein found in Dictyostelium discoideum cells that promotes the formation of prespore cells and inhibits prestalk cell formation. This protein is found in the early stages of cellular development (mound to pre-culminant) and may be regulated by interaction with FbxA, an F-box protein that functions in the SCF complex of the ubiquitin proteasome system (UPS). I hypothesize that FbxA regulates FbiA by ubiquitinating this protein and marking it for degradation by a proteasome in the UPS. Previous research suggests that the FbiA-GFP fusion protein can be immunopurified. Ubiquitination of FbiA-GFP, however, could not be confirmed with Western blotting techniques. The immunoprecipitation techniques were enhanced by increasing the time of incubation of the soluble lysate with GFP-resin, increasing the volume of GFP beads during incubation, changing the buffer used to elute the protein from the GFP-resin, modifying the lysis procedure, and adding NEM (a deubiquitinating enzyme inhibitor) to the lysis buffer. These optimized techniques were used to immunopurify co-transformations of cells containing both HA-ubiquitin and GFP or GFP-FbiA (GFP-741) plasmids. Because the level of GFP-FbiA protein immunopurified from these experimental cells was too low to be detected, the optimized immunoprecipitation techniques were used to analyze single transformations of cells transformed with GFP or GFP-741 plasmids. The level of GFP-FbiA protein immunopurified from cells in this experimental design was below detectable levels such that the ubiquitination of FbiA by interaction with FbxA remains inconclusive.

The effects of innexin RNA interference on immune function in Drosophila melanogaster

Bridget F. Donnelly, 2011 (Advisor: Dr. Brad Hersh)

Gap junction proteins allow communication between cells in an organism. In Drosophila melanogaster, gap junction proteins are encoded by the innexin family of genes. Previous work in caterpillars and flies suggests a role for innexin gap junctions in the insect innate immune response, an organism’s initial non-specific defense against pathogens. We hypothesized that by knocking down the expression of innexin genes in the hemocytes using Hml-Gal4 and UAS-Inx-RNAi, we would see reduced innexin expression in the hemocytes and we would reduce the immune response of Drosophila. To test this hypothesis, we infected flies orally with the gram-negative bacterium Serratia marcescens to study their survival. Additionally, we proposed to examine the relative levels of innexin expression in the hemolymph using Real Time-PCR. We found that S. marcescens is effective in decreasing the survival of Hml-Gal4 control flies but it is not effective in decreasing the survival of innexin 6 knockdown flies, indicating that the bacteria did not affect the innexin 6 knockdown flies. We were not able to determine the relative levels of innexin expression in the hemocytes because we were not able to perform Real Time-PCR due to methodological issues. We successfully isolated RNA from the hemolymph, however it contained genomic DNA. We then performed a DNase digestion in order to remove the DNA from the RNA samples but the DNase digested the RNA as well.

Role of Innexins and Vinnexins in Hemocyte Gap Junction Formation in Drosophila melanogaster

Shivani Dudhia, 2011 (Advisor: Dr. Brad Hersh)

Gap junctions are intracellular channels that span the plasma membrane at adhesion sites allowing for cell-to-cell communication through ion and small signaling molecule exchange. Innexins and vinnexins assemble gap junction channels in insects like Drosophila melanogaster and mutualistic ichnoviruses, respectively. Drosophila innexin genes are expressed individually or in combination during many essential processes; however, their role in immune function is poorly described even though it is clear that insect blood cells (hemocytes) form gap junctions in vitro during encapsulation of foreign invaders. Campoletis sonorensis ichnovirus (CsIV) vinnexins also form gap junctions in vitro. This ichnovirus is transmitted by parasitoid wasps during egg deposition, and disrupts cellular immunity in infected insects. It has, therefore, been hypothesized that CsIV vinnexins disrupt cellular immunity by interfering with gap junction-mediated intercellular communication. This experiment aimed to identify protein-protein interactions between Drosophila melanogaster innexins (2 and 4) and CsIV vinnexins (D, Q1, and Q2) using the yeast two-hybrid screen, and observe the effects of hemocyte-specific vinnexin expression on Drosophila melanogaster immunity after wasp infection. No interaction was observed between innexin 2 and vinnexin D and Q1. Low expression level of bait protein in the yeast Y2HGold strain and/or use of full-length genes as the bait may have led to these findings. Vinnexins D and Q1 were cloned and attempts were made to insert into the pUAST-attB vector to use for the hemocyte-specific vinnexin expression via the GAL4-UAS system.

Structural and Kinetic Consequences of an Arginine-Cysteine Substitution in Pea Ascorbate Peroxidase

Shawn E. Dumbroski, 2011 (Advisor: Dr. Ann Kleinschmidt)

As plants carry out normal metabolic activity, reactive oxygen species are formed. Enzymes are capable of reacting with reactive oxygen species, but the end product of hydrogen peroxide is still harmful to the plant, in high concentrations. Peroxidases are enzymes that are capable of reducing hydrogen peroxide, producing water. Ascorbate peroxidase (APX) is named after its reducing substrate, ascorbate. Comparison of APX in different species of plants reveals several interesting details. APX enzymatic activity varies between different species even though there is high sequence similarity. Sequence comparison of APX between the legume family and the viridiplantae family reveals at positions 167 and 168 legumes have an alanine-alanine motif where other viridiplantae have an arginine-cysteine motif. In addition to the arginine-cysteine motif, viridiplantae also have four more cysteines than legumes for five total. This study will examine an arginine-cysteine substitution at positions 167 and 168 in pea APX. The substitution is hypothesized to cause a structural change in the enzyme, changing the kinetic ability of the enzyme. Examination of electronic absorption data shows that the substituted forms of APX have a heme that is 6 coordinate low spin, where wild type APX has 5 coordinate high spin heme. Formation of Compound I was observed via transient state kinetics. Activity was observed for wild type and A168C A167R APX, but no activity was observed for A168C APX. The activity observed was minimal and this is believed to be from a minimal shift in Soret peak absorbance from resting state enzyme to the Soret peak of Compound I. FT-Raman was also utilized and confirmed the electronic absorption spectra in terms of heme coordination and heme spin. FT-Raman also showed that in A168C that one vinyl group was conjugated to the porphyrin ring, where wild type and A168C A167R APX have both groups conjugated to the porphyrin ring. Also, the substituted forms have only one vinyl group capable of bending, suggesting that the vinyl group is in a different confirmation compared to the wild type. The data suggests that the substituted forms of APX have different conformations, which cause the lack of or diminished ability to react with hydrogen peroxide.

Determination of efflux pump characteristics in Haemophilus ducreyi using efflux pump inhibitors CCCP and PAβN

Leanne E. Ianniello, 2011 (Advisor: Dr. Tricia Humphreys)

Haemophilus ducreyi is the causative agent of the sexually transmitted disease chancroid, which facilitates HIV infection. Although currently treatable, antibiotic resistance is a potential future problem for treating chancroid. One of the potential mechanisms for antibiotic resistance is antibiotic efflux pumps which prevent the antimicrobial drug from affecting the cell by extruding the chemical. A strategy for identifying strains of bacteria that have efflux pumps involves using efflux pump inhibitors and determining antibiotic susceptibility in the presence and absence of such chemicals. This study looked to determine if H. ducreyi strains HMC112 and 35000HP have efflux pumps using the efflux pump inhibitors PAβN and CCCP. Disc diffusion assays were performed in the presence and absence of either PAβN or CCCP and the zones of inhibition were measured for the antibiotics ampicillin, erythromycin, tetracycline, and amoxicillin/clavulanic acid. The EPI CCCP did not have a significant impact on the zones of inhibition for any of the antibiotics in either strain. However, PAβN had a significant effect on the zones of inhibition for both HMC112 (p = 0.0112) and 35000HP (p=0.0328). From these results, it is possible that H. ducreyi does possess efflux pumps that could contribute to antibiotic resistance. Therefore, it would be beneficial to investigate further into H. ducreyi efflux pump prevalence on a wider scale.

Identification of a sialometabolism gene cluster in Haemophilus ducreyi

Kyle J. Knupp, 2011 (Advisor: Dr. Tricia Humphreys)

Haemophilus ducreyi is a gram-negative bacterium that is able to sialylate its LOS in class I strains, but not in class II strains, because of differences in Lipooligosaccharide (LOS) structure. Other organism with the same ability to sialylate their LOS are able to catabolize sialic acid. Comparison of the genes from Haemophilus influenzae, a close relative, to H. ducreyi makes it plausible to think H. ducreyi may be capable of catabolizing sialic acid. Here I show how homologous the gene nanA (HI0142) from H. influenzae are to the gene sequences obtained for this gene in H. ducreyi. Other gene were shown to be able to be amplified such as nagB (HI0141) and nanE (HI0145), but no SiaR (HI0143) was able to amplified for either class. The gene nanA, encodes an N-acetylneuriminate lyase, which is essential for sialic acid degradation, was present in both class I and class II, with homologies of 64%, 67% and 65% to strains HD85-023233 (I), 33921 (II), and DMC64 (II), respectively. The DNA sequences were then translated to protein sequences and appear to encode a functional nanA enzyme. The above strains had homologies of 84%, 89%, and 89% without mutations to the presumptive active site of the N-acetylneuriminate lyase (NAL) subfamily. Other genes were unable to be fully analyzed, due to poor sequence quality, but could be amplified. nagB is present in both classes and nanE is present in class I strains. The findings show that all strains have the possibility of catabolizing sialic acid, because nanA is present in all classes, protein sequences of nanA analyzed conform to NAL subfamily, and the genes nagB and nanE are present which strengthens this hypothesis.

The Effects of Deterin Overexpression in the Central Nervous System of Drosophila melanogaster

Ana M. Kuoman, 2011 (Advisor: Dr. Brad Hersh)

Organisms eliminate unwanted cells through apoptosis. Homeostasis is maintained during development via apoptosis, making it a crucial process. Although regulation of apoptosis is vital, dysfunction of programmed cell death can occur giving rise to various neurodegenerative diseases or cancer. An important aspect of apoptosis is its conservation of genetic pathway throughout many species. The model organism Drosophila melanogaster contains a gene, named deterin, homologous to the mammalian inhibitor of apoptosis protein, survivin. Both genes contain first introns that are inserted in identical positions, despite their divergence in primary structures. The goal of this study was to examine the effects of deterin overexpression in the central nervous system in the fruit fly using transgenic flies expressing deterin. Our results revealed deterin overexpression to affect wall-climbing activity in two out of the four strains, DF2-4 and DF2-7, including changes in behavior compared to the control. This suggested that deterin inhibits apoptosis in the central nervous system.

The Effects of High Light Stress on Ascorbate Peroxidase 1 and Peroxidase 33 T-insertion Mutants in Arabidopsis thaliana: a Comparative Analysis

Emma C. Wise, 2011 (Advisor: Dr. Ann Kleinschmidt)

Excess light leading to oxidative stress results in the accumulation of hydrogen peroxide beyond homeostatic levels. If unchecked, the excess hydrogen peroxide has detrimental effects; thus, plants have evolved protective mechanisms including increased expression of scavenger enzymes. Primary among these is Ascorbate Peroxidase I (APX1). In knockout APX1 plants, a number of genes were found to be up-regulated in response to high light. While the response of APX1 to light stress is fairly well known, the function, if any, of class III POXs in responding to excess light has not been extensively characterized. The goal of this study was to compare the effects of light stress on T-DNA insertion mutants of the class III peroxidase 33 (T-per33) and APX1 (T-apx1). Whereas the T-apx1 plants grew more slowly than wild type, T-per33 plants were unimpaired. Analysis of hydrogen peroxide accumulation using amplex red staining of leaf tissue suggested that Per33 helps to prevent hydrogen peroxide from diffusing throughout the leaf, where it would cause oxidative damage. Hydrogen peroxide accumulation was much more apparent in T-apx1 plants, which implicates APX1 as a primary respondent to hydrogen peroxide that accumulates as a result of high light stress. Finally, quantitative RT-PCR was used to examine mRNA levels for multiple genes implicated in the high light response. Although most of the results were not statistically significant, a consistent elevation of HSP70 mRNA was observed. Overall, these results suggest that further studies of Class III peroxidases in the response to high light would be informative.