Jamie Conklin, Department of Biological Sciences, Carnegie Mellon University
Mentor: Dr. Javier López

Lariat Stabilization in vivo : Methods and Application to Study the Autoregulation Mechanism of Sxl
Alternative splicing generates distinct proteins from the same gene by producing mRNAs with different exon structures. It plays an important role in normal development and in disease. In this project I developed new methods for analysis of alternative splicing based on RNA lariats. Lariats are generated by the 5' splice site cleavage reaction, in which the 2' hydroxyl group of the branchpoint adenosine (located 15-40 nucleotides upstream of the 3' splice site) attacks the phosphodiester bond at the exon/intron boundary, forming a lasso-like structure. A free lariat is then released when the 3'OH of the upstream exon attacks the phosphodiester bond at the intron/exon boundary to ligate the two exons. The splice sites (and therefore the exons) joined by any splicing event can be identified by examining the sequences that span the branch point of the resulting lariat. An important advantage of lariat analysis is that it provides information about the mechanisms that generate differences in alternative mRNA levels in vivo , but it is difficult because lariats are degraded rapidly after linearization by debranching enzyme (DBR). I developed two approaches to stabilize lariats in Drosophila , using regulated RNAi constructs to induce degradation of DBR mRNA and controlled expression of a genetically engineered dominant negative allele of DBR to interfere with enzyme activity. To demonstrate the utility of lariat analysis, I used these methods to study the mechanism of alternative splicing of Sxl transcripts, which produces distinct mRNAs in males and females. In vitro experiments had led to the proposal that Sxl protein regulates alternative splicing of its own mRNA at the second step of the splicing reaction, but my in vivo experiments show that Sxl acts at the first step by regulating selection of competing branchpoints.

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 dyeing 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. Further tests were performed by reducing and alkalating the sample with TCEP and iodoacetamide respectively. This procedure also clarified the protein spots. While dialysis and filtration were two methods tested to separate Albumin and IGG from the other proteins, elution of a prep gel remained the reliable form of separation. 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.

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.

Ashley Krankowski, Department of Chemistry, Carnegie Mellon University
Mentor: Dr. Rick McCullough

New Conductive Elastomers based on Poly (3-hexylthiophene)
The goal of this project was to synthesize poly(3-hexylthiophene)- b -polydiene conductive elastomers. An elastomer is a polymer that recovers its shape after being stretched or deformed. Polydienes are well-known elastomers, which are extensively used in the production of tires. The novelty of poly(3-hexylthiophene)- b -polydiene copolymers consists in combining the conductive properties of regioregular poly(3-hexylthiophene) with the elastic behavior of polydienes. The work is significant because conducting elastomers would be ideally suited for applications such as flexible electronic circuits. I accomplished a multi-step method for the synthesis of poly(3-hexylthiophene)- b -polydiene copolymers. In step one, 2,5-dibromo-3-hexylthiophene is first reacted with t -butyl magnesium chloride followed by [1,3-bis(diphenylphosphino)propane]-dichloronickel (II) (Ni(dppp)Cl 2 ) generating regioregular poly(3-hexythiophene) (PHT) (>98% head-to-tail coupling). Separately, a block of polyisoprene with a small, living block of polystyrene was prepared. Finally, coupling of living polydienyl lithium with allyl-terminated PHT was performed. The block copolymer was analyzed by ¹ H-NMR and MALDI-TOF mass spectrometry.

Ryan Malecky, Carnegie Mellon University
Mentor: Dr. Terry Collins

The Degradation of Tartrazine by a Fe-TAML Catalyst and H₂O₂
Tartrazine is an extremely common dye: it and two other dyes in the azo family account for more than half of the dyes produced every year. It is used in food, paper, textiles and many other applications. Because the dye is used in such great quantities it makes up much of the color pollution produced by dye houses. The wastewater from these dye houses often ends up in rivers and streams, where it blocks light from reaching aquatic ecosystems. In the United States there is regulatory pressure to reduce this pollution in a safe and cost-effective way. Fe-TAML is a catalyst that allows hydrogen peroxide to oxidize some common pollutants. It is environmentally friendly since it does not introduce toxic elements such as chromium to the environment . Also, since the chemistry is done with hydrogen peroxide rather than chlorine, there is no potential for chlorinated organics to be generated during reaction. The Fe-TAML-catalyzed oxidation of tartrazine by hydrogen peroxide was studied by UV-Visible spectroscopy to find optimal reaction conditions. Tartrazine's reaction products were identified using NMR spectroscopy and mass spectrometry. 4-Phenolsulfonic acid, sulfanilic acid, and 3-carboxy-1-(4-sulfophenyl)-5-pyrazolone were used as model compounds to better understand how specific parts of the dye were oxidized. The Fe-TAML and hydrogen peroxide system oxidized tartrazine effectively as the yellow color of the starting material was completely removed. Analysis of the solution after treatment revealed the presence of small organic acids and other oxidized products. The process of oxidation of tartrazine and its model compounds will be presented.