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Thesis (M.A.)--Boston University / In studies on the formation of 2,3-diphosphoglycerate (2,3-DPG) from inosine by erythrocyte preparations it was observed that the inosine interfered with the enzymatic assay for 2,3-DPG. Phosphoglyceromutase, for which 2,3-DPG is a coenzyme, and enolase were used in a coupled assay. The inosine appeared to interfere by inhibiting the enolase [TRUNCATED]

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This experiment compares the implications of two methods of measuring viral particles, specifically Influenza particles in human cell lines in vitro. These strategies include plaque assays and xCELLigence screening. Plaque assays, also known as reduction assays, are plates that are overlaid with semi-solid medium that limits the spread of the virus and shows where each particle is located based on the "plaque" or empty space on the plates where cells have died and been removed. xCELLigence screening is a newer program that checks for "impedance", an artificial number that will measure the cells killed by virus as well as cell to cell interaction on a 96 well plate that utilizes gold microelectrodes. Both methods have variables that can make them useful in certain situations, however, the focus is on how reliable the xCELLigence program is in comparison to more traditional methods of quantifying viral particles.

Optically pure aromatic D-amino acids, such as D-phenylalanine (D-Phe) and its derivatives, are high-value building blocks for the pharmaceutical industry. These compounds can be prepared using biocatalytic methods relying on various enzymes, including aminotransferases (ATs). ATs, also called transaminases (EC 2.6.1.X), are a subclass of pyridoxal 5′-phosphate-dependent enzymes that catalyze the transfer of the amino group from a donor substrate to a ketone acceptor. Synthesis of optically-pure amino acids using whole-cell biocatalytic cascades based on ATs possess several advantages compared to traditional chemical methods, including excellent enantioselectivity and increased process and step efficiency, which is achieved through the catalysis of multiple steps in one-pot reactions without requirement for intermediate work-ups, cofactor recycling, or toxic metals. However, enzyme biocatalysts typically need to be engineered to alter their substrate specificity or to increase their catalytic efficiency, which has limited their industrial application. Therefore, to facilitate the engineering process of ATs broadly and to produce aromatic D-amino acids, we developed a high-throughput assay for the testing of a broad range of ATs against libraries of potential substrates, and developed a biocatalytic cascade to produce optically pure aromatic D-amino acids.

Understanding the signaling mechanisms that determine juvenile-to-mature transition and bud fate is vital for controlling tree reproduction. FD-like proteins also appear to be important for initiating reproductive development. In this study, phylogenetic analysis showed that three FD-like genes (FDL1, FDL2, and FDL3) are present in the poplar genome. FDL1 and FDL2 are products of a recent whole genome duplication event while FDL3 escaped such duplication. Yeast two-hybrid assays demonstrated that FT1 and FT2 proteins interact with FDL3 protein, but not with FDL1 or FDL2 protein. Analysis of the expression levels of FD-like transcripts in Populus deltoides via quantitative real-time PCR showed that FDL3 abundantly expresses in the shoot apex where it probably interacts with FT1 in late winter and early spring. Following the duplication event, FDL1 and FDL2 appear to have diverged in function as they express in a number of tissues in the fall, winter, and spring.

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A variety of novel and effective inhibitors of NQO1 was synthesized. The inhibitors were classified as 'asymmetrical' and 'halfway stage' analogues of dicoumarol. The synthesis of these inhibitors was achieved through the application of different techniques such as 'borrowing hydrogen methodology', thermal and microwave irradiation and reductive C-C cleavage using NaBH3CN. One of the most potent analogues was toxic (IC50 = 9.2 ± 0.3 μM) towards the non-small cell lung cancer cell line, A549.A selection of the most potent inhibitors was re-modified as prodrugs in order to improve drug penetration through the barrier of the cell membrane. This was achieved by conjugation with a delivery agent related to the natural product antheminone A. The synthesis involved a multi-step reaction sequence involving the use of natural product (-)-quinic acid as a precursor. A range of prodrugs were synthesized which exhibited toxicity towards the A549 cancer cell line.

Carboxyl esterases, particularly arylesterases, were predicted from 16 microbial genomes, and then expressed in E. coli. Of the more than 175 cloned genes, 86 were expressed in soluble form. These were screened for activity using a range of both commercial and natural substrates. Forty-eight proteins were active on pNP-acetate at pH 8 whereas 38 proteins did not exhibit any activity towards any substrates. Among the 48 active proteins, 20 proteins showed arylesterase activity. To date, 8 bacterial esterases and 2 archaeal arylesterases were characterized in terms of pH stability and optima, thermal inactivation, solvent stability, and kinetics. To our knowledge there is only one other published report of arylesterases from archaea. The synthetic capability of arylesterases can transform phenolic acids to value-added chemicals. Accordingly, this project provides an arsenal of industrially significant activities that can extend the antioxidant properties of lignin-derived molecules in a broader range of renewable products.