Regionally-Grown barley is in demand for craft malting and brewing in Virginia. Barley lines suited to both Virginia's climate and craft brewing applications are currently under development. Free amino nitrogen (FAN) is a malt quality parameter that influences beer flavor directly and via yeast metabolism during fermentation. FAN and the individual amino acids making up FAN influence yeast health, beer color development, flavor, and flavor stability. Despite potential impacts on beer quality, individual amino acid concentrations in barley and malt are not generally measured or monitored. The objective of this project is to evaluate and assess the conversion of FAN and individual amino acid concentrations during the malting and mashing process of genetically distinct varieties. An additional objective is to evaluate their malt sensory characteristics, to understand genetic variability therein. Raw barley and malt samples were subject to low temperature aqueous extraction, and wort was produced using an isothermal hot water extract technique. FAN and amino acid composition were determined for each line for raw barley, malt and wort extracts, prepared as described. Statistical analysis revealed that these lines had significantly different changes in FAN and amino acid composition. Sensory characteristics of malt hot steep teas were evaluated by forty (N=40) panelists with brewing experience using a sorting task to group malts with similar sensory characteristics, and to assign descriptors to those groups. From the sixteen (N=16) breeding lines, five (5) distinct sensory groups were identified. Taken together, our findings will inform the selection process for barley lines for craft brewing, and add to the knowledge of the extent to which free amino acid composition varies among eastern barley lines and along the malting and mashing processes. / Master of Science in Life Sciences / Regionally-grown barley is in demand for craft malting and brewing in Virginia, and barley lines suited to both Virginia's climate and craft brewing applications are of interest. The production of beer requires high quality malt, which is defined by many parameters; one of which is free amino nitrogen (FAN), which plays a critical role in yeast nutrition and beer flavor through yeasts' metabolism and chemical reactions. FAN is a general measurement of nitrogen and its individual components (including amino acids) are rarely measured, despite their major role in beer flavor. The objective of this project was to assess the development and changes in FAN and amino acids that occur over the course of the barley supply chain – which will better inform breeders, growers, maltsters, and brewers of the raw materials they use and how they can affect their processes A second objective was to assess the flavor of newly developed barley lines and varieties, and compare them to established varieties currently being grown in Virginia through sensory evaluation methods designed to determine similarities between samples. Results from the analysis of FAN and the amino acid composition identified significant differences between varieties, and results from sensory evaluation identified 5 distinct groups with similar flavor characteristics. Taken together, these findings will help inform the selection process for barley lines for craft brewing, and add to the knowledge of the extent to which free amino acid composition varies among barley lines and along the brewing processes.

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Microfluidics is the most modern version of electrophoresis to be introduced into the analytical chemistry field. In this format, electrophoresis is performed in trough-like channels in a planar format. When combined with laser induced fluorescence detection, this remarkable technique has the potential to separate a simple mixture with high efficiency in a matter of seconds with better sensitivity than conventional methods. The research herein focuses on the development of microfluidic electrophoresis methods for the separation and identification of amino acids in consumable fish products. Preliminary studies utilized amino acid standards and yielded baseline resolution of mixtures of amino acid 1, 2 and 3 within 30 seconds at efficiencies of >1 x 106 plates per meter. In subsequent studies, free amino acids were analyzed in various species of fish obtained from commercial sources and local lakes. Finally, fish samples known to be contaminated with PCBs were also analyzed.

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<P> Fluorine-18 labeled 6-fluoro-3, 4-dihydroxy-phenyl-L-alanine (6-FDOPA) has been used in conjunction with Positron Emission Tomography (PET) to study the dopamine metabolism in the living human brain and also to monitor gastrointestinal carcinoid tumors. Elemental fluorination of L-DOPA in anhydrous HF (aHF) or aHF/BF3 has been shown to be an efficient method for the synthesis of 6-fluoro-L-DOPA. Utilization of aHF, however, is not desirable in a hospital environment owing to its hazardous nature. This work has consequently focused on the development of new methodologies for the direct electrophilic fluorination of aromatic amino acids, which circumvent the use of aHF. </p> <p> The present work has shown that the reactivity and selectivity of F2 towards L-DOPA in CF3S03H is comparable to that in aHF. The discovery and versatility of this new synthetic procedure has led to the production of 6-[18F]fluoro-L-DOPA, 6[18F]fluoro-D-DOPA, 4-[18F]fluoro-L-m-tyrosine (4-FMT) and 6-(8F]fluoro-L-m-tyrosine (6-FMT) in high radiochemical yields that are not only suitable for small animal imaging, but are also suitable for clinical use in human subjects. Because of the low volatility of CF3S03H, its removal from the reaction mixture was accomplished by use of an anion exchange resin in acetate form. The syntheses of2-, 4-and 6-FMT were also achieved by the direct fluorination of m-tyrosine (MT) in H20. The effect of temperature on the fluorination of MT was investigated and it was shown that, unlike CF3S03H, optimal conditions in H20 were attained at elevated reaction temperatures. </p> <p> There have been several reports relating to the formation of [18F]OF2 as a major byproduct (up to 20%) in the gas phase nuclear reaction, 180(p,n)18F. This reaction is used for the routine production of [18F]F2 which, in tum, is utilized for the syntheses of PET tracers such as radiofluorinated aromatic amino acids. Because the reactivity of OF2 has been reported to be similar to that of F2, its selectivity as a fluorinating agent towards aromatic amino acids was investigated. The effect of solvent acidity on the fluorination of MT using OF2 was studied and it was shown that, in contrast with the reactivity of F2 in superacids, OF2 is a more efficient fluorinating agent in less acidic solvent media. The use of H20 as the solvent medium for fluorination ofMT resulted in the formation of 19FFMT isomers in 4.35 ±0.04% yield. Consequently, the potential use of OF2 as a fluorinating agent for aromatic amino acids was also investigated for L-phenylalanine, 3nitro-L-tyrosine, 4-nitro-DL-phenylalanine, L-DOPA, 3-0-methyl-L-DOPA, 3,4dimethoxy-L-phenylalanine, m-, p-and a-tyrosine. In these studies, the only aromatic system fluorinated by OF2 was MT, indicating that the presence of [18F]OF2 as a byproduct resulting from the nuclear reaction, 180(p,n)18F, does not have a significant impact on the syntheses of radio fluorinated aromatic amino acids that have applications in PET imaging. </p> / Thesis / Master of Science (MSc)

Call number: LD2668 .T4 1961 R35

The penicillin tolerance of ammo acid-deprived re/A+ Escherichia coli is attributed to the stringent response. The β-lactam-induced lysis of amino acid-deprived bacteria resulting from relaxation of the stringent response was inhibited by cerulenin, or by glycerol deprivation in the case of a gpsA mutant (defective in the biosynthetic snglycerol 3-phosphate dehydrogenase). Therefore, β-lactam-induced lysis of amino acid-deprived cells was dependent on phospholipid synthesis. Both the priming and the lysis induction stages of β-lactam-induced lysis were shown to require phospholipid synthesis. It has been known for some time that phospholipid synthesis is inhibited by the stringent reponse. These results indicate that the inhibition of peptidoglycan synthesis and the induction of penicillin tolerance during the stringent response are both secondary consequences of the inhibition of phospholipid synthesis. Direct experimental evidence is presented for the first time indicating that the penicillin tolerance of amino acid-deprived E coli was directly attributable to action of guanosine 3',5'-bispyrophosphate (ppGpp) and not to some other effect of amino acid deprivation. The overproduction of ppGpp resulted in the inhibition of peptidoglycan and phospholipid synthesis and in penicillin tolerance. Penicillin tolerance and the inhibition of peptidoglycan synthesis were both suppressed when ppGpp accumulation was prevented by treatment of the bacteria with chloramphenicol, an inhibitor of ppGpp synthetase I activation. Glycerol-3-phosphate acyltransferase, the product of plsB gene, was recently identified as the main site of ppGpp inhibition in phospholipid synthesis. The overexpression of the cloned plsB gene reversed the penicillin tolerance conferred by ppGpp accumulation. This also indicates that the membrane-associated events in peptidoglycan metabolism were dependent on ongoing phospholipid synthesis. Interestingly , treatment with β-lactam antibiotics by itself induced re/A-dependent ppGpp accumulation, but the maximum levels attained were insufficient to confer penicillin tolerance. It was al so demonstrated that penicillin tolerance was induced when phospholipid synthesis was inhibited in normal growing E. coli. This penicillin tolerance was not the result a simple inhibition of growth or a decrease in the membrane levels of individual phospholipids (e.g., acidic phospholipids), but rather the direct result of the inhibition of net phospholipid synthesis. A number of factors that interfere with β-lactam-induced lysis were investigated. (i) It was demonstrated that de-energization of the E. coli cytoplasmic membrane resulted in penicillin tolerance due to the inhibition of both the priming and the lysis induction stages. (ii) Inhibition of protein synthesis in the absence of the stringent response promoted both the priming and the lysis induction stages resulting in a faster onset of β-lactam-induced lysis. (iii) The temperature sensitivity of β-lactam-induced lysis in amino acid-deprived E. coli was re-investigated. Penicillin tolerance resulting from a temperature up-shift was not due to the induction of the heat-shock response, as previously reported, but from a reversible inhibition of unidentified thermosensitive enzyme(s) involved in the lysis induction stage. / Graduate

Ribosomal incorporation of unnatural amino acids (AAs) into peptides or proteins has found broad applications in studying translation mechanism, discovering potential therapeutics, and probing protein structure and function. However, such applications are generally limited by the low incorporation efficiencies of the unnatural AAs. With in vitro kinetics studies using a purified E. coli translation system, we found that the natural N-alkyl AA carrier, tRNAPro, could hasten the incorporation of N-methyl AAs. Also, the incorporation rate increased remarkably with increasing pH in the range of 7 to 8.5, suggesting the rate was limited by peptidyl transfer, not accommodation. Competition experiments revealed that several futile cycles of delivery and rejection of the A site N-methyl AA-tRNA were required per peptide bond formation, and the incorporation yield could be increased by using a higher Mg2+ concentration. Kinetics of ribosomal polymerization, using AA-tRNA substrates prepared from the standard N-NVOC-AA-pdCpA chemoenzymatic ligation method, clarified that the inefficiency of incorporation was due to the penultimate dC. This dC prompted faster peptidyl-tRNA drop-off, leading to loss of processivities along consecutive incorporations. Circumventing the penultimate dC by using our N-NVOC-AA-pCpA chemoenzymatic ligation or the flexizyme charging method to prepare the AA-tRNA substrates was able to improve the efficiencies of ribosomal consecutive incorporations of unnatural AAs. By studying the translation steps after aminoacylation of tRNAPyl, the favored carrier for unnatural AAs in vivo, we demonstrated surprisingly slow biphasic kinetics of tRNAPyl-mediated amber suppression in vitro. The fast phase amplitude increased with increasing EF-Tu concentration, allowing measurement of Kd of EF-Tu binding. Results revealed ~25-fold weaker EF-Tu binding affinity of the tRNAPyl body than that of E. coli tRNAPhe. The fast phase rate was ~30-fold slower than that of native substrates, and this rate was limited by the ~10-fold less efficient AA-tRNAPyl:EF-Tu:GTP ternary complex binding to the ribosome. The incorporation was so slow that termination by RF2 mis-reading of the amber codon became a significant competing reaction. The processivity was unexpectedly impaired as ~40% of the dipeptidyl-tRNAPyl could not be elongated to tripeptide. This new overall understanding opens a window of improving unnatural AA incorporation both in vitro and in vivo.