| Carolyn H. Baloh | Jillian G. Bolton | Erin C. Buckley |
| Nicholas S. Duca | Meredith T. Hanlon | Alyssa B. Lypson |
| Megan M. McGuire | Maggie M. Mucha | Michael VanHeyst |
| Courtney Walker | David M. Zielinski |
Development of a bacterial expression system for the analysis of Brassica rapa ascorbate peroxidase in response to oxidative stress
Carolyn H. Baloh, 2009 (Advisor: Dr. Ann Kleinschmidt)
Cytosolic ascorbate peroxidase in Brassica rapa (BrAPX) protects the plant from damage due to oxidative stress. Both pea and soybean ascorbate peroxidase have been extensively studied; however, it is interesting to note that while each of these sequences contains one Cysteine (Cys) in the entire protein, all other homologues of this enzyme sequenced contain more than one Cys, including BrAPX which contains five. The goal of this project was to develop a bacterial expression system as a mechanism for studying whether modification of the Cys residues occurs in BrAPX due to oxidative stress. BrAPX was expressed in Escherichia coli that were treated with varying concentrations of H2O2. RT-PCR analysis revealed that oxidative stress was present in both the H2O2 treated and untreated cultures. BrAPX was isolated, and electronic absorption spectra and kinetic analyses were performed. Differences in the electronic absorption spectra suggested that there might be some alteration in BrAPX structure in the location of the heme group due to high H2O2 concentrations (1 mM). Comparison of the BrAPX spectra and kinetics with those published for pea and soybean ascorbate peroxidase suggest differences in structure in the area of the heme group and reaction kinetics.
Localization of Carbonic Anhydrase II in the Nasal Cavities of Mice: Immunocytochemical Approach
Jillian G. Bolton, 2009 (Advisor: Dr. Lee Coates)
Carbonic anhydrase (CA) is the active enzyme in the chemoreceptors in the nasal cavities of many vertebrates. Chemoreceptors have become a focus for studying Sudden Infant Death Syndrome (SIDS). It is hypothesized that the chemoreceptors in nasal cavities are under developed and not functional, which can cripple the function of the chemoreceptor which contributes to probability of suffocation in infants. There are seven different major isozymes (I-VII) of CA in the vertebrate system. The purpose of this study was to determine a protocol for immunocytochemistry and use immunocytochemistry to locate CAII-positive cells in the olfactory epithelium throughout the entire nasal cavities of mice. There was positive staining for CAII found in the cells of the olfactory epithelium. There were CAII-positive olfactory epithelium cells found in ectoturbinate 2, endoturbinate IV, periodically in the septum, in glandular cells, and possibly along the mucosal surface. These results help determine a proper protocol for immunocytochemistry and help characterize the location of CAII. As a result, this helps determine the role CA plays in chemoreceptors. The more understanding we have of chemoreception in vertebrates, the more knowledge we will have to prevent and understand the cause of SIDS.
Determining the Influence of Group II (G-U) Single-Nucleotide Bulges on the Thermodynamic Parameters of RNA Duplex Formation to Help Develop a Model to Predict Secondary Structure
Erin C. Buckley, 2009 (Advisor: Dr. Marty Serra)
Thermodynamic parameters (ΔG°37, ΔH°, ΔS°, and TM) of oligonucleotide sequences containing single nucleotide bulge loops were determined through optical meeting in 1M NaCl. Seven sequences containing Group II bulge loops adjacent to a wobble base pair (G-U) were analyzed to see if the non-nearest neighbor model currently used for Group I single nucleotide bulges is an accurate model for Group II bulges. The identity of the bulge did not show an effect on the stability of the duplex containing the bulge loop; therefore, all Group II (G-U) bulges were considered one set. The bulge sequence was studied at varying positions along the duplex: 5′, 3, and/or middle. There was a correlation between the least stable stem and the destabilization of the duplex. The free energy increments at 37°C for the introduction of a Group II (G-U) single nucleotide bulge loop ranged between 2.0 and 5.2 kcal/mol. The thermodynamic parameters ΔG°37 and ΔH°, were combined with previous data gathered for Group II (G-U) (Serra unpublished) and the free energies were compared to Group I (Blose et al. 2007). Based on this investigation the characteristics of Group II (G-U) bulges were found to show a relationship to Group I bulges; therefore, the non-nearest neighbor model would appear to be an accurate model for predicting the stability of duplexes containing a Group II (G-U) single nucleotide bulge.
Total Synthesis of a Potential Marine Natural Product Antifouling Agent
Nicholas S. Duca, 2009 (Advisor: Dr. Shaun Murphree)
Natural product antifouling agents have become important targets for organic synthesis due to their effective yet environmentally benign character. The total synthesis of (E)-5-(2,6-Dimethyl-5,7-octadienyl)-3-Furancarboxylic Acid 16, a coral metabolite with antifouling activity is described. Citronellyl acetate 28 is first converted to ß-diketone 22 which is reacted with 2,3-dibromo-1-(phenylsulfonyl)-1-propene under basic methanolic conditions to provide phenylsulfonylfuran 26. Using a novel oxidative cleavage of sulfones, phenylsulfonylfuran 26 can be converted to the desired furoic acid 16.
Roles of Auxin and Peroxidases in the Formation of the Arbuscular Mycorrhizal Symbiosis between Glomus intraradices and Tomato (Lycopersicon esculentum)
Meredith T. Hanlon, 2009 (Advisor: Dr. Catharina Coenen)
The mutually beneficial arbuscular mycorrhiza (AM) fungal symbiosis is known to occur between fungi of the order Glomales and nearly 80% of all land plants, but the mechanism behind the interaction is poorly understood. A slight induction of defense mechanisms has been shown, and a role for hormones has been speculated upon, neither, however, has been confirmed as a definite regulatory mechanism of AM colonization. Here, tomato (Lycopersicon esculentum) was used to observe the interactions between an auxin resistant mutant (diageotropica, dgt), an auxin-hyper polar transporting mutant (polycotyledon, poc) and the AM fungus Glomus intraradices. Fungal interactions with the mutants were compared to those seen in the parent cultivar, Ailsa Craig. Plants were grown either as whole seedlings in the greenhouse or as excised root organ cultures (ROC), inoculated with G. intraradices, and assessed for colonization and peroxidase isoenzyme activity at various time points. Peroxidase activity was highly variable, but changes in dgt and poc ROC peroxidase activity early in the symbiosis seem to be indicative of later colonization patterns. Measurements of root and shoot architecture of the plants does indicate differences in phytohormone levels may exert control over symbiosis formation. From these observations, a model is proposed that speculates the interacting roles between auxin and cytokinin during early symbiosis formation. The complicated nature of hormone interaction leaves much space for this model to be improved and speculated upon in further studies.
This research was supported in part by the Harold M. State Research Fund and by USDA seed grant 2004-35304-14995.
The thermodynamic investigation of Group III bulge loops adjacent to GU wobble base pairs in RNA duplexes
Alyssa B. Lypson, 2009 (Advisor: Dr. Martin Serra)
Eleven RNA duplexes containing single nucleotide purine bulge sequences were optically melted, and the thermodynamic parameters (ΔG°37, ΔH°, ΔS°, and TM) were determined for each sequence. Data from this study were combined with data from previous thermodynamic investigations (Serra unpublished; Hasson, unpublished 2005) to compare differences between Group III purine bulges to pyrimidine bulges, as well as differences between Group I sequences, those in which the bulge is not identical to its nearest neighbors, and Group III sequences, those in which the bulge is adjacent to a GU wobble base pair. The bulge identity, order of the possible bulge loops (G/A or A/G), nearest neighbors effects, and non-nearest-neighbor effects were also examined to determine the influence of Group III purine bulge sequences on RNA duplex stability. The stability of purine bulges in Group III sequences was best predicted when the sequence was modeled to contain a bulged guanine and Watson-Crick nearest neighbors. Neither the order of the bulge nor the nearest-neighbor base pairs had an effect on the influence of the bulge loop on duplex stability. The stability of the duplex was primarily affected by non-nearest neighbor interactions. There was a direct correlation between duplex destabilization and the less stable duplex stem. It is possible the non-nearest neighbor model may be applied to predict the stability of duplexes containing Group III single nucleotide bulge loops. However, Group III purine and pyrimidine bulge data appear to behave differently. Therefore, two separate models may be needed to categorize purine and pyrimidine sequences for Group III single nucleotide bulge loops. Plans: I am currently waiting to hear back from a job interview at Magee Women’s Research Institute doing HIV research. Also, I recently took the MCAT and have been accepted at Drexel Medical College. However, in order to attend the school in the fall I have to receive a certain score on the MCAT, and therefore I will not know if I am attending until I receive my scores. As a back up plan, I recently spoke with Carnegie Mellon University and the University of Pittsburgh regarding their graduate programs in biochemistry and chemistry. They agreed to accept my application late and will consider me for entry in the fall graduate program.
Effects of the anti-auxin, PCIB, and the general proteasome inhibitor, MG132, on auxin-inducible promoter activity
Megan M. McGuire, 2009 (Advisor: Dr. Catharina Coenen)
Auxins are one of five classes of phytohormones that play an essential role in the regulation of plant growth, development, and functioning. One of auxin’s functions is the transcriptional regulation of auxin-inducible genes through the ubiquitin-mediated protein degradation system. Auxin-inducible genes contain auxin response elements (AuxREs) in their promoter sequences which are bound by auxin response factors (ARFs) acting as either transcriptional repressors or activators depending on auxin concentration. High auxin concentrations promote homodimerization of ARF proteins and induce transcription, while low auxin concentrations lead to decreased transcription due to heterodimerization between ARFs and Aux/IAA repressor proteins. In the nucleus, auxin is bound by F-box protein TIR1, a subunit of the E3 ubiquitin ligase SCFTIR1 complex. Upon binding, auxin mediates interaction between SCFTIR1 and its substrate, Aux/IAA proteins, which are targeted for degradation by the 26S proteasome via ubiquitination, ultimately activating transcription. To more closely examine auxin binding and Aux/IAA degradation, an anti-auxin, PCIB, and general proteasome inhibitor, MG132 were used to disrupt the signal transduction pathway in this study. The auxin-responsive GH3::luc promoter::reporter construct was used to quantify transcriptional activity in hypocotyl segments of 6 to 7 day old Nicotiana plumbaginifolia seedlings. In the presence of 0.1 µM IAA, application of 500 µM PCIB resulted in complete inhibition of promoter activity, while partial inhibition was caused by 100 µM MG132. When both inhibitors were applied at the sub-optimal inhibitory concentration of 100 µM, an additive inhibitory response was achieved. Additive inhibitory effects of PCIB and MG132 on promoter activity support a model in which the inhibitors increase the competitive inhibition at TIR1 between the auxin, anti-auxin, and ubiquitinated/un-ubiquitinated Aux/IAA proteins. The results of these experiments offer further insight into auxin binding and repressor protein degradation, while confirming the complexities surround auxin-inducible transcription.
The effect of atrazine exposure on female gonadal development, reproductive success, clutch size, and hatchability in the ring-legged earwig, Euborellia annulipes
Maggie M. Mucha, 2009 (Advisor: Dr. Susan Rankin)
The effect of atrazine exposure on female gonadal development, reproductive success, clutch size, and hatchability was examined in the ring-legged earwig, Euborellia annulipes. During the first experiment, newly eclosed females were injected with 2 µl of atrazine (1.5, 0.15, or 0.015 µg/µl) every other day for one week for a total of four treatments. Atrazine was diluted in ethanol and milli-Q water. Following treatment, the ovaries of the resulting eight day adults were excised, basal follicle length measured, and gross morphology examined. Food intake was monitored. Exposure to 1.5 µg/µL atrazine significantly slowed basal follicular and ovariole development. In the second experiment females were subjected to the same injection regime. Following the injection series, eight day old females were mated in twenty minute trials. Time to and duration of copulation was noted. Reproductive success was subsequently monitored as a function of oviposition, clutch size, maternal care, and proportion hatched. Exposure to atrazine did not affect these reproductive parameters. In addition to the experimental parameters examined, the ability of this model system to scan for endocrine-disrupting effects in vertebrates including humans was considered.
Microwave Assisted Synthesis of Carborane Derivatives Within the Framework of Closo-12-alkyl-CB11H11–
Michael VanHeyst, 2009 (Advisor: Dr. Mark Juhasz)
The goals of this experimentation were to improve on techniques required to produce 12-alkyl-CB11H12– anion derivatives from the 12-iodo-CB11H11– anion by Pd-catalyzed Kumada cross-coupling reaction with alkyl Grignard reagents. Complete synthesis of 12-R-CB11H11– (R=Methyl, Ethyl, and Phenyl) by both thermal and microwave reactions are reported. The results of this experimentation show that the use of microwave irradiation to procure the desired 12-alkyl-CB11H12– anion derivatives proved to be successful in not only decreasing the preparatory steps and reaction time but also in increasing the percent yield of literature values. Microwave irradiation assisted synthesis was able to increase percent yield of the thermal Kumada cross-coupled reactions by 3%-18%.
Phagocytic Resistance of Class II Haemophilus ducreyi
Courtney Walker, 2009 (Advisor: Dr. Tricia Humphreys)
The LspA proteins are antiphagocytic proteins secreted by Haemophilus ducreyi which are required for pustule formation for the genital ulcer sexually transmitted disease, chancroid. This study investigated the lspA2 gene and LspA2 protein sequence differences between class I and II of H. ducreyi using a Western Blot and sequence alignment software. I expected to see a band shift (LspA2) and a missing band (LspA1) when examining protein expression in the Western Blot. The cellular proteins were separated successfully, however only the standard was detected using chemiluminesence after transfer. It was determined that the gene and protein sequences aligned well between the 2 classes of H. ducreyi, except for a large deletion in class I of 186 nucleotides. The amino acid changes found between the two classes mostly had the same side chain polarity, with 9 exceptions. The examination of these protein sequences could be helpful in developing a vaccine for chancroid.
This research was supported in part by the Class of 1939 Senior Research Fund.
The Characterization of Brassica rapa Annexin 1 and Development of a Bacterial Expression System
David M. Zielinski, 2009 (Advisor: Dr. Ann Kleinschmidt)
The cellular functions of annexin proteins in plants are poorly understood. Previous work has identified a protein complex from Brassica rapa that displays peroxidase activity, and a protein was identified within this complex as annexin 1. In this study, experiments were completed to determine the coding sequence of the annexin 1 protein from B. rapa and to investigate its properties using a bacterial expression system. An annexin 1 cDNA was synthesized by RT-PCR using RNA from flower buds and annexin 1 specific forward and reverse primers. Sequencing revealed an annexin 1 sequence with an open reading frame of 951bp and a predicted protein sequence of 317 amino acids. A comparative alignment of the predicted B. rapa protein sequence and annexin 1 homologs showed a high amount of conservation among all sequences. The characteristic residues that compose the S3 cluster observed in certain annexin sequences are seen in the predicted protein sequence of annexin 1 from B. rapa. The predicted annexin 1 protein sequence also showed non-conserved glycine residues, so a second annexin 1 cDNA sequence was cloned from B. rapa. Differences were observed between the two annexin 1 cDNA clones obtained, including non-conserved amino acid changes. Attempts were made towards the development of an expression system for B. rapa annexin 1 and initial results were promising. This study has provided a foundation for further investigations of the structure and function of annexin 1 in B. rapa.