UMD Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/3
New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a given thesis/dissertation in DRUM.
More information is available at Theses and Dissertations at University of Maryland Libraries.
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Item Nucleic Acid Extraction and Detection Across Two-Dimensional Tissue Samples(2010) Armani, Michael Daniel; Shapiro, Benjamin; Smela, Elisabeth; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Visualizing genetic changes throughout tissues can explain basic biological functions and molecular pathways in disease. However, over 90% of mammalian messenger RNA (mRNA) is in low abundance (<15 copies per cell) making them hard to see with existing techniques, such as in-situ hybridization (ISH). In the example of diagnosing cancer, a disease caused by genetic mutations, only a few cancer-associated mRNAs can be visualized in the clinic due to the poor sensitivity of ISH. To improve the detection of low-abundance mRNA, many researchers combine the cells across a tissue sample by taking a scrape. Mixing cells provides only one data point and masks the inherent heterogeneity of tissues. To address these challenges, we invented a sensitive method for mapping nucleic acids across tissues called 2D-PCR. 2D-PCR transfers a tissue section into an array of wells, confining and separating the tissue into subregions. Chemical steps are then used to free nucleic acids from the tissues subregions. If the freed genetic material is mRNA, a purification step is also performed. One or more nucleic acids are then amplified using PCR and detected across the tissue to produce a map. As an initial proof of concept, a DNA map was made from a frozen tissue section using 2D-PCR at the resolution of 1.6 mm per well. The technique was improved to perform the more challenging task of mapping three mRNA molecules from a frozen tissue section. Because the majority of clinical tissues are stored using formalin fixation and not freezing, 2D-PCR was improved once more to detect up to 24 mRNAs from formalin-fixed tissue microarrays. This last approach was used to validate genetic profiles in human normal and tumor prostate samples faster than with existing techniques. In conclusion, 2D-PCR is a robust method for detecting genetic changes across tissues or from many tissue samples. 2D-PCR can be used today for studying differences in nucleic acids between tumor and normal specimens or differences in subregions of the brain.Item EVALUATION OF MASS FILTERED, TIME DILATED, TIME-OF-FLIGHT MASS SPECTROMETRY(2010) Demoranville, Leonard Thomas; Mignerey, Alice C; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Naval Research Laboratory's Trace Element Accelerator Mass Spectrometer (NRL-TEAMS) system offers a unique opportunity to develop a new type of time-of-flight (TOF) SIMS. This opportunity derives from use of a Pretzel magnet as a recombinator and mass filter in the injector to the accelerator. Mass filtering prior to time-of-flight analysis removes extraneous species, shortening the analysis time for a single beam pulse, thereby improving the duty cycle. Using this approach, it is possible to obtain an expanded portion of a narrow segment of the entire time-of-flight spectrum created by a single beam pulse. A longer flight path for greater momenta in the Pretzel magnet introduces time dilation. Potential benefits derived from time dilation and mass filtering include improved duty cycle, shorter analysis time, increased precision, and better resolution. While the NRL-TEAMS system is not designed for TOF work, it has been used as a test bed to prove the theoretical benefit of such a design. Theoretical treatments of the spectrometer have shown improved resolution is possible under certain conditions, when compared to a traditional TOF spectrometer. SIMION 8.0 computer simulations were used to model the system and provide insight to the theoretical capabilities of the Pretzel magnet. As expected, models have shown that as field decreases, and therefore path length increases, mass resolution improves. Generally, the model matched well to experimental results provided by the NRL TEAMS system. These experimental results have predicted fundamental parameters of the system accurately and consistently, and confirmed the validity of the model. This research improved the current system's performance through improved electronics and pulsing and further uses the model to predict the theoretical benefits of a system designed for use with a Pretzel magnet.Item STRUCTURAL STUDIES OF THE PEANUT ALLERGEN PROTEIN ARA H 2(2010) Li, Jinxi; FENSELAU, CATHERINE; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Presented in this dissertation are comprehensive studies of the structures of the peanut allergen protein Ara h 2 and the effect of food processing (roasting) on it. A detailed elucidation of the primary structure and PTM of Ara h 2 from the raw peanuts has been described. Ara h 2 isoforms were purified and cleaved via microwave accelerated trypsin digestion. The peptide mixtures were analyzed by LC-MS/MS and targeted CID. De novo sequencing of the MS/MS spectra revealed the protein sequence of each Ara h 2 isoform. Several hydroxyproline sites have been discovered while disulfide bond structures have been partially determined. Using anti-Ara h 2 antibodies, Western blotting of 1-D gels of the raw and dark roasted peanuts was carried out in order to characterize the changes of Ara h 2 between these two samples. The result indicates that Ara h 2 may present in a much heavier form in the roasted peanuts, possibly due to crosslinking and aggregation with other proteins. Subsequent LC-MS/MS studies of trypsin digestion of five gel pieces (>100, 100-50, 50-25, 25-16 kDa) from 1-D gels of the raw and dark roasted peanuts suggests that roasting process causes the crosslinking of Ara h 2 with other proteins. This supports our results from the immunological studies.