William Eimer, Department of Biological Sciences, Carnegie Mellon University
Mentor: Dr. Jonathan Minden
Improvement of Difference Gel Electrophoresis for Potential Applications in Medicine
The focus of my project is to improve the clarity and resolution of blood serum protein spots observed on DIGE gels (difference gel electrophoresis), a method developed in the Minden lab. DIGE works by dying two different samples of proteins with different fluorescent dyes and then mixing them together. The proteins in the mixed sample are first separated by their charge using isoelectric focusing, and secondly by their molecular mass using a SDS-PAGE gel. When analyzed in an imager, this creates a gel where each dot observed represents a specific protein. The two samples can be compared against each other to identify protein differences between the samples. Currently, gels prepared in this manner exhibit some large streaking and blurring areas rather than proteins appearing as single dots. As a partial solution to this problem, a method extracting Albumin and IGG from the samples was designed and applied to the process. Since these two proteins compose a large part of blood serum, when dyed they overshadowed many of the other proteins present. This extraction process involved running the mixed sample out on a preparatory gel and physically cutting out the undesired proteins. The remaining proteins were then eluted back into a concentrated sample. The next modification involved removal of sugars which were altering the results of isoelectric focusing. The charges on the sugars were creating variability in the proteins resulting in streaks on the DIGE gel. The removal of sugars is accomplished by treating the sample with PNGase F, a deglycosilation enzyme. Tests have shown that treatment with the enzyme is effective in increasing clarity of some proteins on the 2-D gel.While the spot clarity of the proteins on 2-D gels have increased, the resolution still needs addition processes before the desired clarity is achieved. Future tests which are planed are to use a revolutionary column which uses a protein hook to hold the protein while removing all other contaminants. This procedure can help purify and concentrate the sample to the level needed for effective 2-D gel analysis. Once completed, the complete method can be used to analyze and compare blood serum of healthy and diseased human patients, aiding in the identification of proteins associated with specific diseases. This will enable researches to diagnose and treat diseases at a much earlier phase in their development.
Ryan Malecky, Department of Chemistry, Carnegie Mellon University
Mentor: Dr. Terry Collins
Catalytic Degradation of the Environmental Pollutant Bisphenol A using Hydrogen Peroxide and an Fe III -TAML Catalyst
The bisphenol class of compounds is a growing environmental concern as recent studies have suggested links between these compounds and adverse health effects such as early onset of puberty, reduced sperm count and increased embryo mortality. Billions of pounds of bisphenols, in particular bisphenol A (BPA), are used every year in plastics, coatings, epoxies and other applications. They have been detected in industrial waste streams, residential sewage, and rivers and estuaries. Because current wastewater treatment techniques do not completely remove bisphenols, we are exposed to them on a daily basis. Hydrogen peroxide, activated by Fe III -TAML (Tetra Amido Macrocyclic Ligand) catalysts, has shown the potential to safely and effectively remove bisphenols from waste streams. Degradation of BPA by the Fe III -TAML/ H 2 O 2 system is achieved in minutes at room temperature at pH 8 and 12. The treatment is effective for BPA concentrations -as high as 5 mM. The Fe III -TAML catalyst concentrations are minute, a 0.1% molar ratio was sufficient and H 2 O 2 in the range of 0.15 - 250 mM (complete BPA mineralization requires 36 equivalents of H 2 O 2 , in this study upto 50 equivalents were used) were effective. The removal of bisphenol A occurs rapidly. Under the conditions examined the reaction was completed in a matter of minutes. The degradation process was monitored by UV-Visable spectroscopy and HPLC. These techniques indicate that the removal of BPA is occurring via different mechanisms at pH 8 and 12. Proton NMR spectroscopy was used to analyze reaction products. The Microtox® test for acute toxicity indicated that treatment dramatically reduced the toxicity BPA. Thus the Fe III -TAML/ H 2 O 2 system has the potential to provide an effective means for remediating bisphenols in the environment.
Samantha Spath, Department of Biological Sciences, Carnegie Mellon University
Mentor: Dr. Chuck Ettensohn
Localization of Maternal Components of the Wnt Signaling Pathway in the Sea Urchin Embryo
Signaling pathways are highly conserved in development and have been found to regulate various processes, including axis formation and cell differentiation. One such pathway that has been studied extensively is the canonical Wnt pathway. It involves a Wnt ligand binding to Frizzled and LRP receptors and initiating a cascade that stabilizes the transcriptional activator ß-catenin. In the sea urchin, it is already known that ß-catenin is localized to the nuclei of vegetal blastomeres in early cleavage, but not in animal blastomeres. This has been shown to be necessary for vegetal cell specification. It is not known, however, what causes this differential localization of ß-catenin. A similar asymmetry of ß-catenin is found in Xenopus embryos along the dorsal/ventral axis and it has been determined that maternal Wnt11 is responsible for ß-catenin stabilization on the dorsal side. While there is no Wnt11 in sea urchins, a similar mechanism may be in effect for the animal/vegetal axis of the urchin embryo. Thus, the purpose of this project is to look at the upstream regulators of the Wnt pathway and determine if any are involved in axis formation. Specifically, I am looking at the localization of maternal Wnt and Frizzled mRNAs in the urchin egg and early embryo using in situ hybridization and RT-PCR techniques. In situ hybridization on unfertilized eggs reveals an asymmetric localization of mRNA in approximately 30% of eggs for SpFz4, SpFz5/8, SpWnt1, SpWnt6, SpWnt7 and SpWnt16 genes. RT-PCR using isolated animal and vegetal blastomeres demonstrates that SpFz mRNA is located throughout the early embryo, while SpFz5/8 mRNA is restricted to the vegetal blastomeres. SpFz9/10 mRNA appears to be absent from the early embryo. Given these results, I have identified potential candidates in the pathway for specification of the animal/vegetal axis. This and future work will allow for comparison among organisms, thus increasing our understanding of axis formation and patterning as a whole.
Stefanie Sydlik , Department of Chemistry, Carnegie Mellon University
Mentor: Dr. Richard McCullough
Regioregular Random Copolymers of Poly-3-alkylthiophene and Polythiophene
The goal of this project was to synthesize new conductive polymers for use as plastic transistors. The polymers I synthesized are regioregular random copolymers of poly-3-substituted thiophenes and unsubstituted polythiophene. The McCullough group discovered regioregular polythiophenes in 1990 and regioregular polythiophenes are an important class of conjugated polymers that are excellent sensors, are the best plastic transistors known and have the highest efficiencies as solar cells. Regioregular materials have superior electronic and photonic properties when compared to regioirregular analogues. Thus far, most research has been focused on enhancing the physical properties of the polymer by synthesizing homopolymers, block copolymers or alternating copolymers, but little research has been done with random copolymers. This summer, I focused on these copolymers because random copolymers provide the most synthetically convenient way to combine the desirable properties of unsubstituted polythiophene with soluble poly-3-alkylthiophenes. In my research, I found that random copolymerization reactions do give rise to a reproducible polymer. In addition, the polymers I synthesized have some of the highest conductivities ever reported for organic polymers and On-Off ratios an order of magnitude higher than homopolymers of the corresponding poly-3-alkylthiophene. Although further work must be done, test thus far indicate that random copolymers are a simple and reliable method of enhancing the electronic properties of regioregular polythiophenes.