Further characterization of Dictyostelium protein FbiA: ubiquitination via FbxA
Jonathan Buggey, 2008 (Advisor: Dr. Margaret Nelson)
The FbiA protein is a novel protein within Dictyostelium discoideum. It is highly conserved among many higher organisms, but its function remains unknown. Recent studies suggest FbiA may interact with the F-box protein FbxA. Based on FbxA’s known role as part of an SCF complex that targets RegA for ubiquitin-mediated degradation, FbiA may also be a target of FbxA. Using immunopurification and Western blot analysis, an FbiA-GFP fusion protein was pulled down from cell extracts prepared from cells in the vegetative, stream, mound, and first finger stages, and then checked for ubiquitination. In each stage the protein was successfully recovered; however, it was not found to be ubiquitinated. Cells were created that over-expressed the FbiA-GFP and FbxA proteins, in an attempt to saturate the proteasome with ubiquitinated FbiA-GFP. Again, the FbiA-GFP protein was recovered from the vegetative stage but it was not ubiquitinated. Taken together, these results suggest the FbiA-GFP fusion protein used cannot be ubiquitinated in vivo and future research still needs to be done to accurately characterize the possible FbiA ubiquitination process.
This research was supported in part by the Class of 1939 Senior Research Fund and the Dextra Baldwin McGonagle Fund.
Attempts towards (E)-3,7-dimethylnona-6,8-dien-1-ol: A Key Intermediate in the Formation of a Furoic Acid Anti-Fouling Agent
Dustin Butch, 2008 (Advisor: Dr. Shaun Murphree)
A synthetic strategy towards (E)-3,7-dimethylnona-6,8-dien-1-ol, an intermediate in the formation of a natural product antifouling agent, was investigated. The alcohol group of citronellol was successfully protected by a tert-butyldimethylsilane group in good yields. However, olefinic oxidative cleavage via ruthenium teraoxide only resulted in moderate yields over a variety of reaction conditions.
Shear Bond Strength and Debonding Characteristics of Radiopaque Light CUred Glass Ionomer Lining Cement
Brent M. Chaney, 2008 (Advisor: Dr. David Statman)
The objective of this study was to determine the effectiveness of an argon laser to polymerize a light-cured orthodontic adhesive. Orthodontic brackets were bonded to 50 teeth, divided into two groups with 25 teeth each as follows: laser at 488 nm for five seconds and laser at 514 nm for 5 seconds. All of the bonded teeth were placed in water for two weeks. The brackets were removed using shear force of specialized debonding pliers. The sites of bond failure and bond strength were observed. The difference in average bond strength between the two groups was .33 MPa while the adhesive remnant index scores were essentially equal. There were no enamel fractures in either group.
A Mechanistic Investigation Between the Reaction of Horseradish Peroxidase and Ascorbic Acid in the Presence of Hydrogen Peroxide Using Stopped-Flow Spectroscopy
Bryan F. Davis, 2008 (Advisor: Dr. Alice Deckert)
The overall three step mechanism between horseradish peroxidase and ascorbic acid was studied using stopped-flow spectroscopy. 0.05 mM, 0.085 mM, and 0.10 mM concentrations of horseradish peroxidase were combined with hydrogen peroxide in a 10:1 or 6:1 molar ratio, depending on the enzyme concentration, in order to produce compound I. Upon the determination of the spectrum that correlated to each HRP compound, the enzyme solution was reacted with ascorbic acid concentrations of 0.0056 mM, 0.011 mM, 0.017 mM, 0.022 mM, 0.50 mM, 0.75 mM, 1.0 mM, 2.0 mM, and 5.0 mM, again depending on the enzyme concentration. Data produced from the investigation with these varying concentrations of enzyme solution, oxidizing agent, and reducing substrate, at 410 nm, 416 nm, and 443 nm and at 25º C, was used to provide evidence for a suitable mechanism to the reaction. From the evidence that was gained, the proposed mechanism was supported as being an acceptable mechanism for the chemical reaction between horseradish peroxidase and ascorbic acid, in the presence of an oxidizing agent, H2O2.
Salicylic Acid’s Effect on Peroxidase Expression in Brassica rapa
John E. Dudzinski, 2008 (Advisor: Dr. Ann Kleinschmidt)
Since a wide variety of pathogens and insect herbivores pose a threat, plants have evolved several pathways of defense. The hypersensitive response (HR) is characterized by rapid and localized tissue collapse, which seals off the invading pathogen from the rest of the plant. Closely related to the HR is the production of reactive oxygen species (ROS), salicylic acid (SA), and peroxidases. Plant peroxidases have a variety of functions, including the final step of lignin synthesis in a structural defense response. The ultimate goal of this research was to provide insight into the role of peroxidases in defense response, and in the long-term aid in broadening the understanding of specific components of defense mechanisms. Experiments were done where both time and concentration were varied to determine the effect of salicylic acid treatment on peroxidase activity. The peroxidase activity of an acidic protein in Brassica rapa stem tissue was induced when 1.0 mM SA was applied 24 hours prior to tissue isolation. The protein was purified using a preparative agarose gel system and a size exclusion column. Followed by separation by SDS-PAGE two separate protein bands were submitted for peptide analysis. Surprisingly, neither the 28.6 kDa nor the 45.1 kDa band contained a peroxidase protein. In another attempt to identify a peroxidase that is up-regulated when plants are treated with salicylic acid, reverse transcriptase-polymerase chain reactions were conducted. Of all the peroxidases evaluated, the only one to be up-regulated was peroxidase 32 by 1.6 fold. If peroxidase 32 was found in one of SDS-PAGE gel bands, one may postulate that this peroxidase is influenced by treatment with salicylic acid, however it is not known whether it is directly within the SA pathway, or part of another pathway influenced by SA.
This research was supported in part by the Class of 1939 Senior Research Fund.
A Study of the Binding Affinity of Bacteriophage Felix O1 to Salmonella Serotypes based upon Modifications to the O Antigen
Ryan M. Farmer, 2008 (Advisor: Dr. Brandi Baros)
Detecting food-borne pathogens is paramount to halting their spread, and a quick, inexpensive, and easy method currently does not exist for the detection of Salmonella. Therefore, progress has been made towards the ability to detect Salmonella by the reporter bacteriophage Felix O1::mCherry. Felix O1 binds to Salmonella using the N-acetyl glucosamine sugar that is situated on the end of the core of the lipopolysaccharide, and to increase Felix’s binding affinity (and therefore its ability to detect Salmonella) the O antigen must be shortened to relieve steric hindrance. After cold temperature incubations, down to 17°C, an increase in the binding affinity was demonstrated after just 1 hour.
Comparisons of sequences and mRNA levels of two class III peroxidases in Arabidopsis thaliana, Brassica rapa, and Alliaria petiolata
Megan L. Fekete, 2008 (Advisor: Dr. Ann Kleinschmidt)
The importance of class III peroxidases in higher land plant viability has been shown in a variety of studies. With implications in auxin metabolism, cell-wall cross linking, cell elongation, and stress responses, the classification and understanding of these proteins is important to our overall understanding of plant physiology. Although fairly large numbers of peroxidases have been expressed and classified in Arabidopsis thaliana, their specific functions remain elusive. In this study, a comparison of peroxidase sequence and mRNA activity was completed for two peroxidases across three species in the Brassicaceae family: Arabidopsis thaliana, Brassica rapa, and Alliaria petiolata. Using cloning techniques, two peroxidase sequences from A. petiolata were determined. Comparison to sequences in A. thaliana revealed an 87% gene homology to peroxidase 23 and an 89% homology to putative peroxidase 23 in B. rapa. The other A. petiolata peroxidase revealed an 86% gene sequence similarity to A. thaliana peroxidase 37 and 89% homology to putative peroxidase 37 in B. rapa. The mRNA level of peroxidase 23 and 37 was investigated in old and young leaves in all three plant species by semi-quantitatve PCR. There was a marked increase in mRNA level of both peroxidases between “old” and “new” leaf in A. petiolta with relative level values of 22.1 to 921 for peroxidase 23 and 1 to 626 for peroxidase 37, indicating a “developmental switch” that may be influencing their expression. Peroxidase 23 and 37 may have roles in formation and growth of these new leaves. Relative level in A. thaliana remained constant, and shifted from 157 to 152 for peroxidase 22 and 11.2 and 13.2 for peroxidase 37 between rosette and caluine leaves, respectively. B.rapa peroxidase 23 levels changed from 4.31 to 17.9 between first and seconds leaves, while changing from 17.9 to 28.4 between first and second leaves for peroxidase 37. Differences in the mRNA levels across species can be attributed to the unique shape and size of the leaves in each species.
This research was supported in part by the Senior Research Fund.
Synthetic Approaches Towards Generalization of the 5-Position of 2-Methyl-3-(phenylsulfonyl)methyl-2-cyclopentenones from 2,3-Dibromo-1-phenylsulfonyl-1-propene
Sean Garrity, 2008 (Advisor: Dr. Shaun Murphree)
2-Cyclopentenones with various substitution patterns are highly desired as both natural products as well as versatile synthetic intermediates used in the construction of other molecules. By modifying a methodology for the synthesis of 2-methyl-3-(phenylsulfonyl)methyl-2-cyclopentenones through the reaction of ß-substituted diketones and 2,3-dibromo-1-phenylsulfonyl-1-propene, attempts were made to generalize the 5-position of the product 2-cyclopentenones. The target ß-substituted diketones were successfully synthesized, and their reaction with DBP yielded intermediate adducts, precursors to the target 2-cyclopentenones. The results of this work suggest the viability of this approach towards the ultimate generalization of the 5-position of 2-methyl-3-(phenylsulfonyl) methyl-2-cyclopentenones.
Progress Toward 2,3-dibromo-1-phenylsulfonyl-1-propene(DBP) mediated synthesis of (5’E)-5-(2′,6′-dimethylocta-5′,7′-dienyl)furanyl-3-carboxylic acid
Evan T. Geller, 2008 (Advisor: Dr. Shaun Murphree)
Research conducted by the Murphree lab group has elicited a cyclization methodology that yields 2,4-disubstituted furans. This synthetic approach utilizes 2,3-dibromo-1-phenylsulfonyl-1-propene (DBP) to convert 1,3-diketones into the elusive 2,4-disubstituted furan moiety. Selectivity of the DBP mechanism favors the most sterically hindered substituent on the 1,3-diketone, which places this substituent at the 2-position of the newly formed furan ring. A practical compound to showcase the feasibility of this methodology is the natural antifouling agent (5’E)-5-(2′-6′-dimethylocta-5′-7′-dienyl)furanyl-3-carboxylic acid 4. Viable synthetic strategies towards natural antifouling products have become increasing valuable in recent years as metal-based alternatives have been banned due to harmful environmental effects. Investigation into a methodology that utilizes commercially available starting materials has laid the groundwork for synthesis of the key intermediate 24 in the strategy toward natural antifouling compound 4.
Molecular Analysis of the Interaction between FbxA and FbiA in Dictyostelium discoideum
Karen A. Giovannitti, 2008 (Advisor: Dr. Margaret Nelson)
Protein-protein interactions are vital to understanding an abundance of biological processes. In Dicytostelium discoideum, the FbxA protein is a key component in the SCF complex of the Ubiquitin Proteasome System and controls cellular differentiation and development. It is hypothesized that the FbxA protein causes the degradation of a substrate protein, FbiA, through its recruitment to the SCF complex. It was shown in a previous study that a physical interaction takes place between the two proteins. In this study, negative controls were run to verify previous results and a smaller fragment of the FbiA protein was tested through co-immunoprecipitation experiments and analyzed via Western blotting. FbxA and FbiA were tagged with a myc epitote tag and fused with GFP respectively in order to probe for the proteins on a nitrocellulose blot. Results suggest an interaction between the FbxA protein and the GFP tag. There also was an inability to reproduce a physical interaction between FbxA and FbiA in Dictyostelium. This refutes previous evidence that suggested an interaction, but these results are somewhat inconclusive and therefore optimizing western conditions and repeating the experiments are necessary. A continuation of this analysis is important to understanding the interaction of these two proteins, and ultimately cell fate in Dictyostelium.
This research was supported in part by the Class of 1939 Senior Research Fund.
fbiA Promoter Identification, Isolation, and Characterization in Dictyostelium discoideum
Alison M. Helfrich, 2008 (Advisor: Dr. Margaret Nelson)
Dictyostelium discoideum is a cellular slime mold that begins its life cycle as an amoebae and eventually forms a full-culminant when starved. Its specific development phases allow it to be an ideal organism to use when studying cellular signaling and differentiation. Its FbiA protein, F-box interacting protein, displays evidence of interacting with the F-box region of FbxA. FbxA is an F-box protein that interacts in the SCF complex found in the ubiquitin-proteasome degradation pathway. By learning more about the FbiA protein, we can understand its relation to the ubiquitin-proteasome degradation pathway. It may also affect cell-type differentiation, giving another reason to study the FbiA protein. Our goal was to isolate the fbiA gene promoter and insert it upstream of lacZ then transform it back into D. discoideum to study the expression pattern of the FbiA gene via ß-galactosidase levels. Use of stable and unstable ß-galactosidase proteins would better approximate timing as to how long the gene is turned on or off. Our goal was to isolate a full-length and half-length promoter DNA fragments. With some difficulty, the half-length promoter, along with pBluescriptTM has successfully subcloned into upstream of lacZ and transformed into E. coli for use in plasmid amplification.
Effects of over expression of Apoptotic Inhibitor Deterin on Drosophila melanogaster Development
Katie Hinzman, 2008 (Advisor: Dr. Glen Wurst)
Apoptosis or programmed cell death is universally important process. It is involved in development and eliminates potentially dangerous cells. Misregulated apoptosis can result in heterotrophy and underdeveloped structures or unchecked growth and cancer. Survivin is a human inhibitor of apoptosis protein (IAP) whose over expression has been associated with many of the more lethal cancers. Like many other genes involved in the apoptosis pathway, survivin is highly conserved and is a homolog of the Drosophila IAP, deterin. The goal of this study was to study the over expression of deterin and the occurrence of phenotypic rescue by co-expressing the pro-apoptotic gene reaper. Although, studies were not completed on the phenotypic rescue by deterin and reaper co-expression, control studies on embryonic apoptosis by reaper over expression were conducted. The Gal-4-reaper embryos showed increased apoptosis as expected. Results suggest that reaper is responsible for apoptosis during the formation of the procephalic lobe and supraosophageal ganglia, as well as structures along the dorsal ridge and ventral cord. A presence of apoptosis normally which is amplified in these areas points to reaper’s involvement. Further testing will be needed to see if reaper is the sole pro-apoptotic protein in these regions, or if grim is also involved.
The effect of Deterin overexpression in Drosophila melanogaster S2 cells exposed to UV-B radiation
Rachel E. Keaton, 2008 (Advisor: Dr. Glen Wurst)
Apoptosis is an essential process in which multicellular organisms coordinate proper development and homeostasis. It is an important process, and as such, involves many regulatory pathways. Positive regulation is carried out by pro-apoptotic genes and caspases, while negative control is performed by inhibitor of apoptosis proteins (IAPs). One such IAP in the human system is Survivin, noted to be highly prevalent in some of the more debilitating forms of cancer, a result of faulty regulation. Survivin’s homologue within Drosophila melanogaster, Deterin, has been the subject of many recent experiments trying to characterize the nature of the negative regulatory pathway within the fruit fly. This study was interested in the relationship between an overexpression of the gene deterin within Drosophila S2 cells and exposure to UV-B radiation, which has been shown to normally increase apoptotic rates. S2 cells were to be transfected with plasmid vectors containing deterin and Green Fluorescent Protein (GFP). Unfortunately, the cells were contaminated before any conclusions could be made. Even so, successful bacterial transformations indicate that the transfection process would have yielded conclusive results, allowing radiation to take place.
Surface Immobilization of an Ascorbate Peroxidase Utilizing Organosulfur Self Assembled Monolayers on Gold
Mark Kline, 2008 (Advisor: Dr. Deckert)
The surface immobilization of Ascorbate peroxidase (APX) from Pisum sativum (Ps) and Brassica rapa (Br) was completed on a gold surface. Three separate molecules with mono-, di-, and tricarboxylic acid functional groups were used to immobilize the proteins. The molecules 5,5′-Dithiobis(2-nitro-benzoic acid) (DNBA), 5,5′-Dithiobis(2-nitrobenzamide-N-aspartic acid) (DNAA), and 5,5′-Dithiobis[2-nitrobenzamide,N-[[4-carboxy-4-bis(carboxymethyl)amino]butyl]] (DNCB) adsorbed to a gold surface through cleavage of the disulfide bond. Both DNAA and DNCB were synthesized in the laboratory. The self assembled monolayers (SAM) formed on gold were chelated with a 1M NiCl(aq) solution and exposed to a protein solution mix of APX_Ps and APX_Br. Contact angles measured at various steps of the process using water demonstrated chelation of the proteins. Of the three SAM molecules tested, DNBA appears more desirable as a chelator due to its variations in contact angle measurements, ability to chelate protein, cost effectiveness, and ease of use.
Investigation of cost-effective production of different Aspergillus sp. cellulases and xylanase: Applications in deinking mixed office waste paper
Yuling Kristin Khor, 2008 (Advisor: Dr. Brandi Baros)
Biological deinking in paper recycling has become an efficient and effective alternative over conventional chemical processes in the removal of thermally fused ink used in laser and xerographic printing. However, commercially produced enzymes are expensive for large-scale industrial applications. This study investigated the production of cellulases and xylanase in Aspergillus niger, A. oryzae and A. flavus through solid state fermentation (SSF) using agro-waste substrates wheat bran, corncob, and sawdust as a possible alternative for efficient deinking. The effectiveness of these whole-cell culture biocatalysts in deinking paper was evaluated. A. niger and A. oryzae, using wheat bran during SSF, yielded the highest amounts of cellulase (5.350 ± 0.014 IU/ml and 5.937 ± 0.027 IU/ml), endoglucanase (3.450 ± 0.003 IU/ml and 3.025 ± 0.006 IU/ml), and xylanases (6.47 ± 0.001 IU/ml and 6.230 ± 0.001 IU/ml) activity, respectively. Significantly lower enzyme yields were obtained from all cultures containing A. flavus and those grown using corncob and sawdust substrates. Whole-enzyme cultures of A. niger and A. oryzae from wheat bran substrate-SSF were also highly effective in reducing residual ink count per area, in comparison to the other cultures studied. The average percentage of black spots per unit area of 0.0004 m2 was reported at 24.78 ± 9.65% and 30.55 ± 5.88% respectively. It was concluded that agro waste wheat bran substrate and A. niger and A. oryzae enzymes offer the prospect for a low cost alternative in production of the enzymes required for effective paper deinking.
The effects of mefloquine on gene expression of the NR2B subunit of the NMDAR
Ashley L. Laux, 2008 (Advisor: Dr. Rodney Clark)
Many treatments for human diseases are becoming expendable as new advancements in the causes of these diseases are found. Various diseases, especially autoimmune disorders, are now being treated with older drugs such as anti-malarials. Mefloquine has shown many behavioral similarities with PCP but no biological pathway connection has been found to date. Female Sprague-Dawley rats were exposed to mefloquine (1.0 mg/mL, 1.7 mg/mL, 3.0 mg/mL) with frontal cortex and cerebellum tissues harvested. RNA isolation yielded total RNA while RT-PCR provided no results. Further analysis of must be done in order to draw any conclusions as to the effects of mefloquine on the NR2B subunit gene expression.
Deterin Expression during Drosophila melanogaster Embryogenesis
Nicoletta C. Machin, 2008 (Advisor: Dr. Glen Wurst)
Programmed cell death (PCD) or apoptosis plays a critical role in developing organisms. Tight control of apoptotic machinery is crucial for proper development. Regulation of PCD falls to the Inhibitors of Apoptosis Proteins (IAPs). The IAPs repress caspases, the protein executors of apoptosis. Survivin, a human IAP, is expressed in a larger variety of cancers than any other IAP. Therefore survivin is an IAP of interest across many fields of cancer research. It is believed to associate with the mitotic spindle and aid with the proper division of chromosomes during mitosis. This has placed a particular interest on deterin, the Drosophila homolog of survivin. Deterin is a short protein encoded by approximately 611 genomic nucleotides and composed of 153 amino acids and is a known inhibitor of apoptosis. Deterin is expressed during embryogenesis, but the amount and duration of the expression is unknown. This experiment aimed to survey deterin expression at various stages of embryogenesis through RT-PCR and to relate expression levels with known developmental events. Embryos were collected at hourly intervals and RNA isolations were conducted. RNA samples were tested for concentration and purity then subjected to an RT reaction. Due to technical difficulties no results were collected from the PCR analysis. RNA collection and RT procedures were successful, but the PCR reaction failed in all samples but the control. The primers should be tested on a genomic template, to test primer success.
Using Photosensitizers as Antimicrobials for Food Pathogens
Timothy S. McClung, 2008 (Advisor: Dr. Brandi Baros)
Photodynamic antimicrobial chemotherapy (PACT) is a successful method for inhibiting bacteria. Because high levels of inhibition are achievable in a short period of time using low concentrations of photosensitizers, it has the potential to become a sterilization technique for food. Rose bengal, cyanocobalamin, 5-aminolevulinic acid (ALA), and riboflavin were tested as photosensitizers against common food pathogens; Escherichia coli DH5α, Staphylococcus aureus, Salmonella enterica serotype typhimurium, and Listeria monocytogenes; by exposing the samples to a light projector for 10 minutes. Rose bengal had the highest antimicrobial properties, completely inhibiting the Gram-positive bacteria at a concentration of 1 µM. Gram-negative bacteria were reduced 1,000 to 10,000 fold when using 10 mM rose bengal. ALA was found to have a 100-1,000 fold level of inhibition of bacteria at a concentration of 74.6 µM. Neither cyanocobalamin nor riboflavin had any antimicrobial effects. By increasing the power of the light used in the experiment it is expected that inhibition of bacteria will increase greatly.
This research was supported in part by the Class of 1939 Senior Research Fund.
The molecular role of microdiscs in the developing eye-antennal imaginal discs in Drosophila melanogaster
Brandon L. Panaro, 2008 (Advisor: Dr. Glen Wurst)
The fruit fly, Drosophila melanogaster, has long been studied as a model system for developmental genetics. In particular the retinal determination pathway, which governs the development of the fly’s compound eye, acts as an ideal network for study due to its ability to be manipulated and for its ease of visualizing key developmental events on the eye-antennal imaginal discs such as the progression of the morphogenetic furrow. A gene called microdiscs has been identified as a key player in the development of imaginal discs in Drosophila larvae, and is likely to play a role in the development of the eye-antennal imaginal disc as well. In order to study the role of microdiscs in the developing imaginal discs, it is necessary to procure custom polyclonal antibodies for use during Western Blotting and in situ protein detection experiments. The pET-14b bacterial expression vector was used to produce the protein product of the microdiscs gene with a his-tag attached to it for affinity purification purposes. Because the protein was insoluble initially, attempts at purification were unsuccessful. This project offers numerous modifications and additions to the purification process that may prove useful for the progression of this study.
This research was supported in part by the Senior Project Fund.
Comparison of Structure and Kinetics of Ascorbate Peroxidases from Pisum sativum and Brassica rapa
Anthony M. Russo, 2008 (Advisor: Dr. Ann Kleinschmidt)
Cytosolic ascorbate peroxidase is an essential enzyme in higher plants for its ability to scavenge H¬2¬O2. Among many different species of plants ascorbate peroxidase show 70-90% amino acid sequence homology. Despite being from different plant families, ascorbate peroxidase isozymes in Pisum sativum and Brassica rapa show over a 80% homology. However, there are nonconservative differences at amino acid locations near the cation binding site. The cation binding site, unique to ascorbate peroxidase, is located on the proximal side of the heme active site. This research attempts to determine if amino acid residues away from the active site can influence the structural environment of the active site and the enzyme kinetics. UV/Vis analysis displayed spectra consistent with a 6-coordinate iron for APX of B. rapa. In contrast, the same analysis displayed spectra consistent with a 5- coordinate iron for APX of P. sativum. Kinetic studies also showed higher relative rates for P. sativum APX using both ascorbate and o-dianisidine as a substrate. This lower rate further suggests that APX of B. rapa is more six-coordinate, because typical six-coordinate heme proteins have reduced kinetic activity. As a result, this research suggests that structure environments of the two APX active sites are different, and may be a result of the amino acid residues near the cation binding ring. Specifically, the data suggests that B. rapa APX contains a six-coordinate iron heme, which lowers its enzymatic activity for both substrates.
This research was supported in part by the Class of 1939 Senior Research Fund.
Analysis of Inter-Plant Signaling by Measuring Threonine Deaminase mRNA Levels in Brassica napus
Timothy W. Vollmer, 2008 (Advisor: Dr. Ann Kleinschmidt)
Plants are commonly stressed in their everyday environment. Many of these stresses activate defense genes directed toward healing and defense. Defense genes respond to the stresses by generating a defense response that includes creating a chemical signal that is emitted systemically through the plant, and emitted outside the plant. This chemical message is perceived by neighboring plants as a warning that a threat is near. This entire process is mediated by jasmonic acid (JA). A gene that is believed to be involved in the defense response pathway in Brassica napus is the threonine deaminase (TDA) gene. This gene was investigated to determine if plants subjected to exogenous application of JA would induce higher mRNA levels, which are then correlated to whether or not the treated plant created a defense response. TDA mRNA levels were also studied in unstressed neighbor plants to determine if signaling exists. No elevated levels of TDA mRNA in unwounded neighbor plants were found regularly. In every experiment, the addition of 0.125% Triton X-100 detergent solution increased TDA mRNA levels in B. napus. Also, exogenous application of JA showed gave no reproducible concrete evidence that the TDA gene is affected by JA.