Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: Joseph M. Fox, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

Thesis (M.S.)--Oregon State University, 1977. / Typescript (photocopy). Includes bibliographical references. Also available on the World Wide Web.

With the objective to functionalize proteins for the understanding of their biological roles and developing protein-based biosensors, I have been developing methods to synthesize proteins with defined modifications and applying them to study protein functional roles and generate proteins with new properties. These methods rely on the read-through of an in-frame stop codon in mRNA by a nonsense suppressor tRNA specifically acylated with a noncanoncial amino acid (NAA) by a unique aminoacyl-tRNA synthetase and the genetic incorporation of this NAA at the stop codon site. NAAs either provide chemical handles for site-specific manipulation or mimic the posttranslational modifications, which are critical for understanding cellular regulations and signal transduction. The pyrrolysine synthetase (PylRS) has been wildly used to incorporate NAAs into proteins in E. coli. Taking advantage of PylRS, I have developed method to genetically incorporate ketone-containing N--acetyl-L-lysine analog, 2-amino-8-oxononanoic acid (KetoK), into proteins for their site-specific modifications and used it to mimic the protein lysine acetylation process. I have also modified the ribosome in order to improve the amber suppression efficiency and therefore to achieve incorporation of multiple copies of NAA into one protein. By overexpressing a truncated ribosomal protein, L11C, I have demonstrated 5-fold increase of amber suppression level in E. coli, leading to higher expression levels for proteins incorporated with NAAs. I have also demonstrated this method can be applied successfully to incorporate at least 3 NAAs into one protein in E. coli. With the success of incorporating multiple NAAs into one protein, I have further introduced two distinct NAAs into one protein simultaneously. This is done by using a wild type or evolved PylRS-pylTUUA pair and an evolved M. jannaschii tyrosyl-tRNA synthetase (MjTyrRS)-tRNACUA pair. By suppressing both UAG and UAA stop codons in one mRNA, a protein incorporated with two NAAs is synthesized with a decent yield. There is of great interest to incorporate new NAAs into proteins, which is done by library selection. By introducing both positive and negative selective markers into one plasmid, I have developed a one-plasmid selection method. In this method, the positive and negative selections are accomplished by in a single type of cells hosting a single selection plasmid.

The effects of pH on the torsional flexibility of DNA bound to a nucleosome core particle were investigated by studying the time-resolved fluorescence anisotropy decays of ethidium bromide intercalated into the DNA of the core particle. As the torsional flexibility of DNA is affected by the presence of an intercalating dye, the decays were studied at different ethidium bromide to core particle binding ratios. The anisotropy decays were collected using the method of time-resolved single-photon counting and were fit to a model developed by J. M. Schurr (Schurr, 1984) using a non-linear least squares fitting algorithm developed by the author for this purpose. It was shown that below a binding ratio of 0.1 there was no demonstrable change in the anisotropy as a function of binding ratio. Our results show, that the apparent torsional flexibility of DNA of to a nucleosome core particle is dependent on the number of base pairs of the DNA between points of attachment to the histone core. If this number is as high as 30 base pairs, then the torsional flexibility of DNA on a nucleosome core particle is as high or higher than DNA free in solution. Also, for reasonable values of N, the friction felt by the DNA on a core particle is much higher than that felt by free DNA. This indicates that the DNA on a core particle is highly constrained in its motions. The hydrogen ion concentration was shown to have a substantial effect on the fluorescent anisotropy decays, particularly in the early regions of the decay. These analyses indicated that the observed change could be attributed to either a loosening of the contacts between the DNA and the histone core, or a relaxing of the torsional flexibility of the DNA. / Graduation date: 1991

Monounsaturated fatty acids (MUFA) have been viewed as either beneficial or neutral with respect to health; however, recent evidence suggests that MUFA may be associated with obesity and cardiovascular disease. Sex differences in MUFA composition have been reported in both rats and humans that may be mediated by sex hormones. Therefore, differences in fatty acid and enzyme expression were examined in male and female rats. HepG2 cells treated with 17β-estradiol, progesterone and testosterone (0, 10, 30 and 100 nM), and ovariectomized female rats with hormone implants (sham, no treatment, estradiol, progesterone, and estradiol plus progesterone) examined the role of sex hormones in MUFA metabolism. MUFA concentrations were determined by gas chromatography. The mRNA and protein expression of stearoyl-CoA desaturase and elongase 6, key enzymes involved in MUFA biosynthesis, were measured by real-time PCR and immunoblotting, respectively. Elongase 6 protein expression was higher in females as compared with males, increased with estradiol and progesterone treatment of HepG2 cells, and was higher in ovariectomized rats treated with estradiol. Elongase 6 expression was also decreased in HepG2 cells treated with testosterone. In contrast, the expression of stearoyl-CoA desaturase did not appear to be associated with sex or sex hormones in HepG2 cells, although ovariectomized rats treated with estradiol plus progesterone had increased stearoyl-CoA desaturase protein levels compared with sham controls. Sex differences and differences after hormonal treatments were observed in the fatty acid concentrations of MUFA and immediate MUFA substrates including 16:0, 16:1n-7, 18:0, 18:1n-7 and 18:1n-9. These differences in MUFA are consistent with the changes in elongase 6 expression. The effect of sex and hormone status on elongase 6 has not been previously examined, but these results suggest elongase 6 is an important factor in determining MUFA composition. These sex and hormonal differences in MUFA composition may contribute to sex differences in obesity and cardiovascular disease. Further work examining other factors involved in MUFA composition including oxidation, synthesis of complex lipids and the effect of diet is required.

Atmospheric particulates have been known to act as cloud condensation nuclei (CCN) and therefore their presence can indirectly affect important processes such as global radiation balance through cloud formation. Soot particles are well known to be atmospheric constituents, but the hydrophobic nature of fresh soot likely prohibits them from encouraging cloud development. Soot aged through contact with oxygenated organic compounds may become hydrophilic enough to promote water uptake. In this study I have observed the interaction between a number of carboxylic acids and soot from different fuel sources and formation mechanisms. A low pressure fast flow reactor was used to control the contact between the solid phase soot and gas phase organics, while chemical ionization-mass spectrometry was utilized to monitor concentrations of gas phase organics. Most acids irreversibly deposited on the soot surfaces, and the uptake coefficient was measured in the wide range of 9.0 x 10-4 to 1.0 x 10-1. The Brunauer, Emmett, and Teller (BET) surface areas of the soots were measured and the soot bulk and surface chemical compositions were investigated with Fourier transform infrared (FTIR) spectroscopy and attenuated total reflection (ATR) spectroscopy to help explain differences in uptake. By comparing the mono and dicarboxylic acids and the information gathered from soot physiochemical properties I have discussed possible uptake mechanisms.

This project focused on scaling up the laboratory fermentation of biomass to carboxylic acids. Four 1050-gallon tanks were used to simulate four-stage countercurrent fermentation. Most laboratory fermentations have been performed with 1-L fermentors. The purpose of the pilot plant was to show that the process is scalable. The inocula were marine and terrestrial microorganisms. Office paper was used as an energy source, and chicken manure provided the necessary nutrients. The substrate was 80 wt% office paper and 20 wt% chicken manure. Calcium carbonate was used as a neutralizing agent and iodoform served as a methane inhibitor. The fermentor temperature was 40 oC and the pH was 6.0. The highest total acid concentration obtained was 32.4 g/L, operating with a volatile solids loading rate (VSLR) of 1 g/(L liq ·d) and a liquid residence time (LRT) of 80 days. Typical laboratory VSLRs and LRTs are 3 to 10 g/(L liq ·d) and 10 to 30 days, respectively. Similar VSLRs and LRTs were not achieved at the pilot scale because the design was limited by the ability to effectively separate large amounts of solids and liquids. The bulk of the effort was concentrated on overcoming temperature control and solids-handling issues. Design modifications included a redesigned temperature control system and a new material transfer method.

Six Quarter Horse yearling fillies were used in a duplicated 3 x 3 Latin square designed experiment to evaluate the technique of nitrogen retention as a response criterion for amino acid studies in the horse. The yearlings were paired by age and randomly assigned to one of three concentrates fed with a medium quality Coastal Bermudagrass hay throughout the study. Diets were fed at approximately 1.9% of horse body weight per day, divided into twice daily feedings with a 60:40 concentrate: hay ratio. With the exception of lysine and threonine, proposed amino acid requirements for yearling horses were calculated using nutrient to calorie ratios of gilts weighing 80-120 kg and gaining 325 g/d. Diet A was amino acid sufficient, as provided by a soybean meal-based concentrate. Diet B was amino acid deficient, with a cottonseed hull-based concentrate. Diet A and Diet B were isonitrogenous, containing approximately 12% crude protein. Diet C used the identical concentrate as Diet B, with synthetic essential amino acids and cysteine orally dosed to match the amino acid levels in Diet A. Nitrogen retention was not different between Diet A and Diet B. Diet C resulted in differences from Diets A and B in nitrogen retention; however, differences were a consequence of nitrogen intake. Nitrogen retained as a percent of nitrogen absorbed was lower (P < 0.05) for Diet B than for Diet A, for data not accounting for endogenous fecal and urinary losses. There were no differences in nitrogen retained as a percent of nitrogen absorbed for horses fed Diet C, when compared to either Diet A or Diet B, for data not accounting for endogenous losses. It was concluded that differences in nitrogen retained as a percent of nitrogen absorbed were observed between amino acid sufficient diets and amino acid deficient diets. However, horses fed amino acid deficient diets and orally dosed with synthetic amino acids, likely require some modified dosage level to achieve the same or higher values in nitrogen retained as a percent of nitrogen absorbed as those values for amino acid sufficient diets.

The MixAlco process, a proprietary technology owned by Texas A&M University, converts biomass (e.g., municipal solid waste, sewage sludge, paper, agricultural residues, and energy crops) into usable chemicals (e.g., acetic acid) and fuels (e.g., ethanol). Historically, calcium carbonate has been used as the buffer. Recently, it was found that using ammonium bicarbonate as the buffering agent enhances the fermentation conversion. In this case, fermentation broth contains ammonium salts (e.g., ammonium acetate, propionate, butyrate, pentanoate). Therefore, the downstream processing steps (including extraction, purification, esterification, and product separation) must be compatible with the ammonium carboxylate salts formed in the fermentation. This research focuses on converting fermentation broth carboxylate salts into their corresponding acids via "acid springing." Reactive extraction and thermal conversion (distillation) are crucial parts of the acid springing process. Because the components of the fermentation broth are over 80% ammonium acetate and 20% other ammonium carboxylate salts (ammonium propionate, butyrate, pentanoate, etc.), all the initial experiments in this study were performed using reagentgrade ammonium acetate to simplify the reaction. Later, actual fermentation broth was employed. The primary objective of this study was to provide the optimal operating conditions to make the downstream processing steps of the MixAlco process compatible with ammonium carboxylate salts formed in the fermentation. The optimal initial concentration for reactive extraction should be 150-200 g/L and the volume ratio of aqueous phase and extractant should be 1:1. The distribution coefficient reaches the maximum value when the concentration of TOA is 20% (vol %) in n-octanol. The batch distillation study shows that there are two reaction stages: (1) water leaves the system at 100-106 °C and (2) the acid-amine complex decomposes at 160-180 °C.

Thesis--Faculté de médecine de Paris, 1871. / Title continuation and author statement transposed from head of title. "Faculté de médecine de Paris, no. 33." Dédicace (p. 3) followed by a manuscript revision of the dedicatory text in ink, apparently in the author's hand. Number 4 in a bound vol. of 6 dissertations. DNLM DNLM Also available online.