This thesis involves the synthesis of two main classes of triaminocyclopropenium (tac) Ionic Liquids (ILs) (i) Amino Acid Ionic Liquids (AAILs) and (ii) reduced-symmetry cations. [C₃(NEt₂)₂(NRR’)]X (X = TFSA and MeSO₄) were prepared, whereby NHR is derived from amino acids. Optically pure AAILs, [E₄AminoAcid]X (X = TFSA and MeSO₄) were obtained as a mixture of the IL and its zwitterion. The ratios of these mixtures were determined by pH titration and microanalysis. The AAILs specific rotations and pKa values were determined. AAILs can be used for chiral discrimination and form diasterreomeric salts with the entioenriched sodium salt of Mosher’s acid. The AAILs were also successfully used as a solvent and/or catalyst in an aldol reaction and a Diels-Alder reaction. The low-molecular weight series, [C₃(NMe₂)₂(NRR’)]X and [C₃(NMe₂)₂(NR’2)]X was synthesized and characterized: protic ILs NRR’, where R = ethyl, propyl, allyl, butyl, - CH2CH2OCH₃ and pentyl, R’ = H and X = TFSA: and aprotic ILs NRR’, where R = Me, R’ = ethyl, allyl, propyl, butyl, -CH2CH2OCH₃ and hexyl and X = TFSA and DCA. ILs with C2v symmetry [C₃(NEt₂)₂(NH2)]X (X = TFSA and MeSO₄), [C₃(NEt₂)₂(NBu2)]I, [C₃(NEt₂)₂(NHex₂)]I and [C₃(NEt₂)₂(NHex₂)]OTf were also synthesized and characterized. The C₃h cations, [C₃(NMeR)₃]X (R = ethyl, allyl, -CH2CH2OCH₃ and phenyl, X = TFSA and DCA) were successfully prepared as well. The D₃h cation salts [C₃(NEt₂)₃]X (X = MeC6H4SO₃, OTf, I and F5C6O) and [C₃(NBu2)₃]X (X = B(CN)4 and FAP) were also prepared. The tac-based ILs [C₃(NEt₂)₃]+ and [C₃(NBu2)₃]+ were also complexed with metal halides - - 2- 2- forming salts with FeCl₄ , SnCl₃ , CuCl₄ and ZnCl₄ . Reaction of pentachlorocyclopropane (C₃Cl5H) with BuNH2 gave the open ring allylium product [H2C₃(NBuH)4]2+. This was characterized as Cl- and TFSA- salt. During the synthesis of [C₃(NMe₂)₃]Cl, the open ring cation [HC₃(NMe₂)4]+ was also isolated and was characterized as the TFSA- salt. XX Abstract The TGA, DSC, density, viscosity, conductivity, and molar conductivity properties for the ILs were measured where possible. The viscosity and conductivity data was fitted for the Arrhenius and Vogel-Fulcher Tamman equations. The entire tac-based ILs lie below the KCl ideal line in Walden plot. A fragility plot was obtained by fitting the viscosity data and all the tac-based ILs were fragile. The crystal structures of [C₃(NPhH)₃]TFSA, [C₃(NEt₂)₃]FeCl₄ and [HC₃(NMe₂)4]Cl.2CH₃Cl were determined.

Voltage-dependent anion-selective channels (VDAC), or mitochondrial porins,regulate the flow of metabolites across the mitochondrial outer membrane. They presumably span the membrane as β-barrels, but the residues forming the individual β-strands are unknown. This information is essential for understanding the structure and function of the protein. Using Neurospora VDAC as a template, published data were reassessed to delineate a unified model for porin structure Bay and Court 2002, which was subsequently refined in collaboration with Greg Runke Runke et al. 2006. The focus of this work was the development and analysis of systems for maintaining high levels of folded porin for the acquisition of high resolution data needed for model testing. The conformation of hexahistidinyl-tagged Neurospora porin in detergent was probed by fluorescence, near-UV circular dichroism and ultraviolet absorption spectroscopy. Derivatives of tryptophan and tyrosine were also examined by fluorescence spectroscopy and UV absorbance spectroscopy to model the interactions between the detergents and the amino acid side chains in the protein. Detergent-specific levels of β-strand and tyrosine exposure were observed. In all cases, the two tryptophan residues reside in weakly asymmetric, hydrophobic environments, suggesting transient tertiary interactions. Porin solubilized in these detergents forms functional channels in liposomes and membrane insertion is accompanied by increased levels of β-strand and loss of protease sensitivity. These data were used to develop mixed detergent folding systems. A mixture of SDS and dodecyl-β-D-maltopyranoside (DDM)supports a β-strand rich conformation at high protein concentrations. The tertiary contacts and protease resistance of the SDS/DDM solubilized porin are very similar to those of the protein following reconstitution into liposomes. Finally, the role of sterols in porin folding was examined, as the addition of sterols to detergent-solubilized VDAC is required for channel formation in artificial membranes. Sterols do not alter the secondary structure of VDAC, and subtle alterations to tertiary interactions were detected, suggesting that sterols do not promote an insertion-competent structure, but rather facilitate insertion into artificial bilayers. In summary, this analysis of the folded states of detergent-solubilized porin has revealed a system that maintains high concentrations of mitochondrial porin in a state that is very promising for structural studies.

Voltage-dependent anion-selective channels (VDAC), or mitochondrial porins,regulate the flow of metabolites across the mitochondrial outer membrane. They presumably span the membrane as β-barrels, but the residues forming the individual β-strands are unknown. This information is essential for understanding the structure and function of the protein. Using Neurospora VDAC as a template, published data were reassessed to delineate a unified model for porin structure Bay and Court 2002, which was subsequently refined in collaboration with Greg Runke Runke et al. 2006. The focus of this work was the development and analysis of systems for maintaining high levels of folded porin for the acquisition of high resolution data needed for model testing. The conformation of hexahistidinyl-tagged Neurospora porin in detergent was probed by fluorescence, near-UV circular dichroism and ultraviolet absorption spectroscopy. Derivatives of tryptophan and tyrosine were also examined by fluorescence spectroscopy and UV absorbance spectroscopy to model the interactions between the detergents and the amino acid side chains in the protein. Detergent-specific levels of β-strand and tyrosine exposure were observed. In all cases, the two tryptophan residues reside in weakly asymmetric, hydrophobic environments, suggesting transient tertiary interactions. Porin solubilized in these detergents forms functional channels in liposomes and membrane insertion is accompanied by increased levels of β-strand and loss of protease sensitivity. These data were used to develop mixed detergent folding systems. A mixture of SDS and dodecyl-β-D-maltopyranoside (DDM)supports a β-strand rich conformation at high protein concentrations. The tertiary contacts and protease resistance of the SDS/DDM solubilized porin are very similar to those of the protein following reconstitution into liposomes. Finally, the role of sterols in porin folding was examined, as the addition of sterols to detergent-solubilized VDAC is required for channel formation in artificial membranes. Sterols do not alter the secondary structure of VDAC, and subtle alterations to tertiary interactions were detected, suggesting that sterols do not promote an insertion-competent structure, but rather facilitate insertion into artificial bilayers. In summary, this analysis of the folded states of detergent-solubilized porin has revealed a system that maintains high concentrations of mitochondrial porin in a state that is very promising for structural studies.

The recognition of modified amino acids by reader proteins is governed by the competing interplay of weak, attractive, intermolecular forces and solvation effects. For the recognition of hydrophobic cations like methyl-lysines and methyl-arginines, native reader proteins utilize structural cages always containing multiple aromatic amino acids and sometimes an occasional acidic residue. Through the highly ordered arrangement of multiple aromatic surfaces, reader proteins can invoke the attractive forces of electrostatic, cation-pi, and in the case of arginine, pi-pi interactions. The hydrophobic effect can also significantly affect these binding events in aqueous environments. In this thesis, a number of small molecule, synthetic cages containing significant aromatic surface area have been synthesized. Variation in both total host hydrophobicity and degree of flexibility were explored to determine what effect they have on the overall binding of methylated amino acids in water. Significant flexibility in the first generation of highly aromatic hosts was shown to be detrimental to binding. However, strong binding was observed for guests with significant hydrophobic character despite this flexibility. The cause of the strong affinities in this family of synthetic cages was shown to be due to the hydrophobic effect, rather than any attraction due to cation-pi interactions. Synthetic efforts towards hosts with more rigid structures led to the use of Tröger’s base as a structural building block. Hosts incorporating Tröger’s bases into well-defined aromatic cavities were found to exhibit strong binding to both methyl-lysine and methyl-arginine derivatives in pure water. Differences in guest selectivity were due to the rigid altered host geometry introduced by the Tröger’s base cleft. / Graduate

Recent studies of the relationship between protein sequence and protein structure are reviewed. A detailed discussion of past attempts to predict the structure of a protein from its amino acid sequence, the protein folding problem, is presented and the strengths and weaknesses of these methods are examined. The root-mean-square deviation is studied and a benchmark for structural comparisons is established. A combinatorial approach to the protein folding problem is outlined and its advantages over existing methods is discussed. Specific algorithms based on the combinatorial approach are developed and applied to a variety of proteins. The success of this approach in terms of the root-mean-square deviation benchmark as well as the drawbacks of this method are presented.

Methylmalonic aciduria results from defects in the enzyme methylmalonyl-CoA mutase and from defects in the synthesis of the enzyme's cofactor adenosylcobalamin. Two patients who excrete methylmalonic acid have been shown to have a homozygous nonsense mutation in the methylmalonyl-CoA epimerase gene (MCEE). To further understand the causes of methylmalonic acid excretion, the MCEE gene was sequenced in 229 patients who excreted methylmalonic acid for which no cause was known. Mutations were detected in five patients. Fusion of fibroblast lines from two patients with a homozygous nonsense mutation in MCEE did not result in correction of [14C]propionate incorporation toward control values while the defect in these fibroblasts was complemented by mut, cblA, and cblB fibroblasts. Transfection with wild-type MCEE cDNA resulted in correction of the biochemical phenotype in cells from both patients. These experiments support the hypothesis that a defective epimerase enzyme can be a cause of elevated methylmalonic acid excretion.

Protein restriction during rodent pregnancy is a well-established model of developmental programming. Although the Southampton low-protein diet model of developmental programming has been accepted to produce hypertensive offspring, the mechanism of this programming remains unclear. Currently the effects of protein restriction in the Southampton low-protein diet are confounded by a relative elevation of the amino acid methionine. The aim of this project was thus to clarify the roles of protein reduction and methionine elevation within this model, especially within the context of amino acid and one-carbon metabolism. Pregnant Wistar rats were fed casein-based diets ad libitum varying in casein (18% or 9%) and methionine content (0.5% or 1.0%) from day 0 through 20 of pregnancy. Two diets exactly replicated the Southampton control and low-protein diets (Con: 18% casein, 0.5% methionine; LP: 9% casein, 0.5% methionine), while a third low-protein high-methionine diet (LP-MET: 9% casein, 1.0% methionine) was employed as a positive control for methionine stress. On day 20 of gestation dams were sacrificed and the feto-placental unit was excised; maternal and fetal blood was collected for HPLC analysis of free amino acids. Maternal plasma was also analyzed for homocysteine content using a spectrophotometric-based enzyme assay. Diet did not affect maternal weight gain, food consumption, litter size or fetal weight. In dams and fetuses, methionine was significantly elevated in both low-protein groups. Maternal homocysteine was significantly elevated only in dams fed the low-protein high-methionine diet. Reductions in maternal serine, proline and glycine levels also occurred in dams fed the low-protein high-methionine diet; fetuses of these dams had significantly reduced levels of all three branch chain amino acids (leucine, isoleucine and valine). Both low-protein diets resulted in drastic reductions in circulating threonine levels in dams and fetuses. Thus, ingestion of low-protein diets with a relative (0.5%) or overt excess (1.0%) of methionine appears to disrupt one-carbon metabolism at the level of homocysteine remethylation to methionine. This may place strain on the folate cycle, as may be indicated by the reduced levels of threonine, serine and glycine. Further testing is necessary to clarify the extent to which folate stores are being utilized for homocysteine remethylation. Increased competition for placental amino acid transport may explain the alternations in circulating free fetal amino acids. Further investigations into levels of other one-carbon metabolites in dams and fetuses are necessary to fully characterize the effect of low-protein high-methionine diets, particularly within the context of the Southampton model of developmental programming.

In this study, the effects of bioprocess operation parameters on aromatic amino acid synthesis performance of Bacillus acidocaldarius were investigated. Firstly, in laboratory scale shake-bioreactors, a defined medium was designed in terms of its carbon and nitrogen sources, to achieve the highest cell concentration. Thereafter, the effects of bioprocess operation parameters, i.e., pH and temperature were investigated / and the optimum medium contained (kg m-3): fructose, 8 / (NH4)2HPO4, 5 / CaCl2, 0.2 / KH2PO4, 2 / NaH2PO4.2H2O, 7.318 / Na2HPO4, 0.0438 / Mg(CH3COO)2.4H2O, 87&times / 10-3 / 1 , MgSO4.7H2O / 2&times / 10-3, FeSO4.7H2O / 2&times / 10-3, ZnSO4.7H2O / 15 &times / 10-5, MnSO4.H2O / 2&times / 10-5, CuSO4.5H2O with pH0 =5, T=55&amp / #61616 / C, N=175 min-1. In this medium, the bacteria produced L-tryptophan at the highest concentration of 0.204 kg m-3 and L-phenylalanine at a maximum concentration of 0.0106 kg m-3 with no L-tyrosine production. Finally the fermentation and oxygen transfer characteristics of the bioprocess were investigated in 3.0 dm3 pilot scale bioreactors. The effects of oxygen transfer were investigated at four different conditions at the parameters air inlet rates of QO/VR =0.2, and 0.5 vvm, and agitation rates of N= 250, 500, 750 min-1. The effect of pH was investigated at pH=5 uncontrolled and controlled operations. The variations in cell, fructose, amino acid and organic acid concentrations with the cultivation time / and using the dynamic method, the oxygen uptake rate and the liquid phase mass transfer coefficient values throughout the growth phase of the bioprocess / the yield and maintenance coefficients were determined. The aromatic amino acids produced at the highest and the least amount and frequency were L-tryptophan and L-tyrosine, respectively. The highest L-tryptophan production, 0.32 kg m-3 in 17 hour was at 0.2 vvm and 500 min-1. Among all operations, the highest L-tryptophan was produced at the lowest oxygen transfer condition. Controlled-pH conditions produced more L-tryptophan.

My dissertation encompasses five different studies that are linked by a common theme the investigation of transposable element (TE) contributions to eukaryotic gene sequences. A detailed analysis of exonization events of LTR elements in the human genome shows the preference towards the fixation of LTR elements in gene untranslated regions, which supports the existing concept of a major role of LTR elements as a natural source of regulatory sequences. The ability of different classes of sequence similarity search methods to detect TE-derived sequences was evaluated. In general, the different search methods are found to be complementary, and combined search approaches are needed to systematically check any data set for all potential TE-associated coding sequences. On average, TE-derived exon sequences have low protein coding potential. In particular, non-coding TEs, are frequently exonized but unlikely to encode protein sequences. Many of these non-coding exonized TEs may be actually involved in gene regulation via the formation of double stranded RNA complexes with complementary TE-derived exons. The investigation of the relationship between human miRNAs and TEs shows that 55 experimentally verified human miRNA genes (~12%) originated from TEs. Overall, TE-derived miRNA genes are less conserved than non TE-derived miRNAs. The potential regulatory and functional significance of TE-derived miRNAs was explored. An ab initio prediction algorithm I developed was used to discover putative cases of novel TE-derived miRNA genes. A miRNA gene family, hsa-mir-548, was found to be derived from Made1 family of MITEs. The palindromic structure of the Made1 elements, and MITEs in general, points to a specific mechanism by which these sequences can be recognized and processed by the miRNA biogenesis pathway. MITEs may also represent an evolutionary link between siRNAs and miRNAs. An original model for a siRNA-to-miRNA evolutionary transition mediated by DNA-type TEs is proposed. This model is supported by the presence of evolutionary intermediate TE sequences that encode both siRNAs and miRNAs in the Arabidopsis and rice genomes. The siRNA-to-miRNA evolutionary transition is representative of a number of other regulatory mechanisms that evolved to silence TEs and were later co-opted to serve as regulators of host gene expression.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 104-124). / Microfiche. / xii, 124 leaves, bound photos. 29 cm