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Bleomycin, from start to finish: total synthesis of novel analogues to in vitro fluorescence microscopy imaging
The bleomycins are a family of glycopeptide-derived antibiotics isolated from various Streptomyces species and have been the subject of much attention from the scientific community as a consequence of their antitumor activity. Bleomycin clinically and is an integral part of a number of combination chemotherapy regimens. It has previously been shown that bleomycin has the ability to selectively target tumor cells over their non-malignant counterparts. Pyrimidoblamic acid, the N-terminal metal ion binding domain of bleomycin is known to be the moiety that is responsible for O2 activation and the subsequent chemistry leading to DNA strand scission and overall antitumor activity. Chapter 1 describes bleomycin and related DNA targeting antitumor agents as well as the specific structural domains of bleomycin. Various structural analogues of pyrimidoblamic acid were synthesized and subsequently incorporated into their corresponding full deglycoBLM A6 derivatives by utilizing a solid support. Their activity was measured using a pSP64 DNA plasmid relaxation assay and is summarized in Chapter 2. The specifics of bleomycin—DNA interaction and kinetics were studied via surface plasmon resonance and are presented in Chapter 3. By utilizing carefully selected 64-nucleotide DNA hairpins with variable 16-mer regions whose sequences showed strong binding in past selection studies, a kinetic profile was obtained for several BLMs for the first time since bleomycin was discovered in 1966. The disaccharide moiety of bleomycin has been previously shown to be a specific tumor cell targeting element comprised of L-gulose-D-mannose, especially between MCF-7 (breast cancer cells) and MCF-10A ("normal" breast cells). This phenomenon was further investigated via fluorescence microscopy using multiple cancerous cell lines with matched "normal" counterparts and is fully described in Chapter 4.
Due to its difficult nature, organic chemistry is receiving much research attention across the nation to develop more efficient and effective means to teach it. As part of that, Dr. Ian Gould at ASU is developing an online organic chemistry educational website that provides help to students, adapts to their responses, and collects data about their performance. This thesis creative project addresses the design and implementation of an input parser for organic chemistry reagent questions, to appear on his website. After students used the form to submit questions throughout the Spring 2013 semester in Dr. Gould's organic chemistry class, the data gathered from their usage was analyzed, and feedback was collected. The feedback obtained from students was positive, and suggested that the input parser accomplished the educational goals that it sought to meet.
The main objective of this project is to create a hydrogel based material system to capture and release CCRF-CEM Leukemia cancer cells via chemo-mechanical modulation. This system is composed of an aptamer-functionalized hydrogel thin film at the bottom of a microfluidic channel, which changes its film thickness as the temperature of the fluid in the system changes. The functionalized hydrogel film has been created as the primary steps to creating the microfluidic device that could capture and release leukemia cells by turning the temperature of the fluid and length of exposure. Circulating tumor cells have recently become a highly studied area since they have become associated with the likelihood of patient survival. Further, circulating tumor cells can be used to determine changes in the genome of the cancer leading to targeted treatment. First, the aptamers were attached onto the hydrogel through an EDC/NHS reaction. The aptamers were verified to be attached onto the hydrogel through FTIR spectroscopy. The cell capture experiments were completed by exposing the hydrogel to a solution of leukemia cells for 10 minutes at room temperature. The cell release experiments were completed by exposing the hydrogel to a 40°C solution. Several capture and release experiments were completed to measure how many cells could be captured, how quickly, and how many cells captured were released. The aptamers were chemically attached to the hydrogel. 300 cells per square millimeter could be captured at a time in a 10 minute time period and released in a 5 minute period. Of the cells captured, 96% of them were alive once caught. 99% of cells caught were released once exposed to elevated temperature. The project opens the possibility to quickly and efficiently capture and release tumor cells using only changes in temperature. Further, most of the cells that were captured were alive and nearly all of those were released leading to high survival and capture efficiency.