Tryptophan, 5-methyl tryptophan, glucosamine, and galactosamine can be separated from each other and hydrolysis products including lysinoalanine by chromatography on a 6 × 260-mm column of W-3H resin. The column is developed at 70°C for 20 min with pH 3.95 (0.4 m Na+) buffer, followed by pH 6.4 (1 m Na+) buffer for 55 min using a Beckman 119 CL amino acid analyzer. The recovery of the internal standards, 5-methyl tryptophan and galactosamine, can then be used to correct for tryptophan and glucosamine losses, respectively. The procedure uses the column and buffers normally employed for protein hydrolysate analysis and does not require additional resin columns, special buffers, or flow rate changes.

Recent epidemiological studies have suggested that the development and progression of several chronic diseases may be initiated or augmented by oxidative stress. Reactive oxygen species and reactive nitrogen species react readily with and can damage nucleic acids, proteins, and lipids. While biological systems are equipped antioxidant defenses to cope with oxidative stress, oxidative damage may still occur when oxidative stress overwhelms antioxidant defenses. This damage, if left unchecked, may lead to a variety of degenerative diseases, including heart disease, Alzheimer's Disease, Parkinson's Disease and cancer. Several assays have been designed to describe the antioxidant activity of various phytochemicals, vitamins, and other compounds. The ORAC and TOSC assays have emerged as industry standards for measuring antioxidant activity due to their high reliability and sensitivity. Until recently, however, little has been done to assess the relative correlation between these two assays. Furthermore, no assay has been developed to measure changes in antioxidant activities of cells in response to oxidative stress. The current work investigates the correlation between measured antioxidant activities of samples in the both the ORAC and TOSC assays. Recent antioxidant research also focuses on relating chemical structure to antioxidant activity. Previous research in this area has included a broad range of chemical groups, but no study has attempted to formulate a structure-function framework that has applicability to compounds of any group. The current work uses amino acids as a simplest-case model for studying the relationships between chemical structure and antioxidant activity. One particular area of emerging research has centered around comparing organic and conventionally grown food products. The impetus of these investigations lies in claims made by organic supporting groups that these food products are generally more beneficial than their conventional counterparts. Despite the rapid rise in popularity of organic foods, there remains a dearth of research investigating these claims. The current work compares the antioxidant activities of organic and conventionally grown blueberries and apples.

A protein's properties are influenced by both its amino-acid sequence and its three-dimensional conformation. Ascertaining a protein's sequence is relatively easy using modern techniques, but determining its conformation requires much more expensive and time-consuming techniques. Consequently, it would be useful to identify a method that can accurately predict a protein's secondary-structure conformation using only the protein's sequence data. This problem is not trivial, however, because identical amino-acid subsequences in different contexts sometimes have disparate secondary structures, while highly dissimilar amino-acid subsequences sometimes have identical secondary structures. We propose (1) to develop a set of metrics that facilitates better comparisons between dissimilar subsequences and (2) to design a custom set of inputs for machine-learning models that can harness contextual dependence information between the secondary structures of successive amino acids in order to achieve better secondary-structure prediction accuracy.

In eukaryotes, sequences that code for the amino acid structure of proteins represent a small fraction of the total sequence space in the genome. These are referred to as coding sequences, whereas the remaining majority of the genome is designated as noncoding. Studies of translation, the process in which a ribosome decodes a coding sequence to synthesize proteins, have primarily focused on coding sequences, mainly due to the belief that translation outside of canonical coding sequences occurs rarely and with little impact on a cell. However, recently developed techniques such as ribosome profiling have revealed pervasive translation in a diverse set of noncoding sequences, including long noncoding RNAs (lncRNAs), introns, and both the 5’ and 3’ UTRs of mRNAs. Although proteins with amino acid sequences derived partially or entirely from noncoding regions may be functional, they will often be nonfunctional or toxic to the cell and therefore need to be removed. Translation outside of canonical coding regions may further expose the noncoding genome to selective pressure at the protein level, leading to the generation of novel functional proteins over evolutionary timescales. Despite the potentially significant impact of these processes on the cell, the cellular mechanisms that function to detect and triage translation in diverse noncoding regions, as well as how peptides that escape triage may evolve into novel functional proteins, remain poorly understood.This thesis will describe novel findings that offer new insight into the process of noncoding translation mitigation revealed by a combination of high-throughput systems-based approaches and validated by biochemical and genetic approaches. Chapter 1 will discuss general concepts in the translation of noncoding sequences and the relevant cellular systems and impacts on human health. Chapter 2 will discuss the results of a high-throughput reporter assay investigating translation in thousands of noncoding sequences from diverse sources. The results discussed in this chapter revealed two factors involved in the mitigation of proteins derived from noncoding sequences: C-terminal hydrophobicity and proteasomal degradation. Chapter 3 will build on Chapter 2 and discuss the results of a genome-wide CRISPR/Cas9 knockout screen that identified the BAG6/TRC35/RNF126 membrane protein chaperone complex as a key cellular pathway in the detection and degradation of proteins with translated noncoding sequences. Having identified the BAG6 complex as targeting a specific reporter of translation of the 3’ UTR in the AMD1 gene, a series of knockout cell lines validated these results and demonstrated the participation of two additional genes, SGTA and UBL4A. Through coimmunoprecipitation western blots and rescue assays with flow cytometry as a readout, we confirmed physical interaction between BAG6 and the 3’ UTR of AMD1, and a similar experiment confirmed interaction between BAG6 and a readthrough mutant of the SMAD4 tumor suppressor gene. Finally, by combining our high-throughput reporter library with our BAG6 knockout cell line, we demonstrated that BAG6 targets hydrophobic C-terminal tails in many noncoding sequences of diverse origin. Finally, Chapter 4 will discuss the evolutionary perspective of noncoding translation through analyses of the sequence content of human and mouse genomes. The findings of this chapter demonstrate a significant trend for increased uracil content in noncoding regions of the genome, which frequently results in the translation of hydrophobic amino acids. We also find that many functional translated noncoding peptides localize to membranes, providing a theoretical link between the shuttling of translated noncoding sequences to a protein complex involved in membrane protein quality control and the emergence of newly evolving proteins from the noncoding genome.

Genetic modification of plastids in the model plant tobacco (Nicotiana tabacum) has demonstrated that numerous foreign gene products can accumulate to high levels in this setting. Plastid biotechnology is maturing to encompass the improvement of food and feed species and the production of biopharmaceutical proteins for oral delivery necessitating development of stable transplastomic edible plants. In the interest of establishing an edible platform we have investigated the use of native and foreign regulatory elements in relation to foreign gene expression in plastids. Multiple sequence alignments of intergenic regions for 20 species of angiosperm showed that despite 95% identity in the coding region, identity in the psbA upstream region is 59% across all taxa examined, other gene coding regions displayed sequence identity of 80-97%, whereas the non-coding regions were 45-79% suggesting that our physical data can be extrapolated beyond the model presented. We found that by exchanging psbA untranslated regions (UTRs) between N. tabacum and lettuce (Lactuca sativa), the expression of the CTB-proinsulin (CTB-Pins) monocistronic transcript declined by 84% and foreign protein accumulation was reduced by as much as 97% in mature leaves. Polyribosome association assays suggest that ribosome-free transgenic transcripts are stabilized where the native UTR is employed. RNA EMSA revealed that binding proteins interacted with psbA 5' UTRs in a species specific manner and the half life of the L. sativa 5'UTR-CTB-Pins mRNA was reduced by 3.7 fold in N. tabacum stromal extracts. Our data indicate that the use of species-specific regulatory elements could lead to establishment of reproducible plastid transformation in desirable target species such as L. sativa. Using transplastomic L. sativa for oral delivery of bioencapsulated CTB-Pins we delayed the onset of diabetes in NOD mice when retinyl acetate supplement was provided compared to untouched mice. In this 30 week study we monitored blood glucose levels and evaluated the in vitro suppressive capacity of regulatory T cells isolated from diabetic mice. Whether delay or prevention was achieved appeared to be a function of antigen dose as high dose resulted in a nine week delay of onset while low dose reduced the incidence of diabetes by 36%. In addition we have evaluated metabolic engineering in the N. tabacum model where we generated cis-genic lines expressing nucleus-encoded methionine pathway enzymes in plastids. Transplastomic expression of Cystathionine gamma-Synthase led to a three-fold increase in enzyme activity and a doubling of methionine content in leaves without a deleterious phenotype. In exploring molecular mechanisms supporting gene expression in plastids and applying transplastomic technology to real human problems this work seeks address the potential of plastid biotechnology for improvement of commodity crops and production of biopharmaceuticals.

Arginine transport is primarily mediated by the cationic amino acid transporters (CATs) in mammalian cells, but in aves the y+, b0,+ and B0,+ transport systems have also been observed. Arginine is the limiting catabolic substrate required for the production of nitric oxide (NO), a highly reactive compound that acts as a signaling molecule or killing compound. NO is synthesized by inducible nitric oxide synthase (iNOS) by macrophages for pathogen clearance. In mammals, CAT-2B is responsible for ARG import in the macrophage for NO synthesis, but the chicken CAT-2B isoform does not transport ARG. Therefore the objective of these studies was to identify the CAT(s) involved in mediating ARG uptake during a NO response in the chicken macrophage. Experiments were performed to measure: 1) ARG transporter mRNA and NO production from three sources of macrophages (HD11 cell line, n=6; primary 32d Cobb 500, n=8; Hyline W36, n=7) in response to Escherichia coli lipopolysaccharide (LPS); 2) the effect of CAT over-expression on NO production in response to LPS (HD11 cell line; n=8). In response to LPS iNOS mRNA abundance increased (P<0.05) 8.5-fold in the HD11 macrophages, 3.22-fold in broiler macrophages and 2.79-fold in layer macrophages. In all cells, CAT-1 was induced and CAT-2A increased (P<0.05) between 1.28 and 1.68-fold. CAT-2B was not detected at any time point or treatment condition. In the virally transformed chicken macrophage cell line (HD11) CAT-3 mRNA was induced, but in primary cells CAT-3 increased (P<0.05) 1.27-fold in broilers and 1.23-fold in layers. Transiently transfected chicken macrophages produce NO independent of LPS treatment by 6h, mock transfected controls did not respond by 6h. In the presence of LPS, CAT-1 transfected macrophages produced 50.0% more NO than mock transfected cells (P<0.05). CAT-2A and CAT-3 transfected macrophages produced only 17.6% and 72.1% of the total NO produced by controls (P<0.05). These results indicate that CAT-1 and CAT-3 are both sufficient to sustain ARG import for NO production in the chicken macrophage, but that CAT-1 produces a maximal response. These results also show that iNOS, despite its name, is constitutively present and can be activated by induction of CATs to import ARG.

Novel terpolymers of leucine (L), aspartic acid (D), and valine (V) were developed as targeting drug delivery carriers that specifically interact with the α 4 β 1 integrin, which is over-expressed in human malignant melanoma cells. As such, copolymerization and terpolymerization of leucine-N-carboxyanhydride (NCA), β-benzyl-aspartate-NCA, and valine-NCA, in dioxane, initiated with triethylamine, were investigated to determine the random nature of the terpolymer composition, for potential application as a targeting drug delivery carrier. The reactivity ratio of each monomer was determined by using Fineman-Ross, Kelen-Tüdös, and nonlinear least-squares curve fitting methods. The product of the estimated reactivity ratios from the monomers in the binary copolymerizations indicated that random polymers were predominantly formed. Based on the reactivity ratios determined from the binary copolymers, Alfrey-Goldfinger equations were used to estimate the composition of the terpolymers. There was no statistical difference between the actual monomer compositions and the calculated compositions of the terpolymers, which validate the randomness of the terpolymers. Therefore, the poly (Leucine-Aspartate-Valine) synthesized in this study is primarily a random terpolymer. The probability of the appearance of LDV sequences occurring within the random polymers were assessed by analyzing the influence of tacticity on the 13 C NMR signals of LDV terpolymers, a statistical method based on the terminal terpolymerization model, and the Poisson distribution, with the highest probability (∼13%) of LDV occurring in a random terpolymer of LDV to be approximately 8 to 9 triad units. Poly (LDV) polymers were shown to exhibit strong binding affinity for A-375 human malignant melanoma cells. The effectiveness of poly (LDV) to target malignant melanoma was evaluated and it showed that 21.3 ± 2.10% of melanoma cells adhered to poly (LDV) films compared to 39.0 ± 3.90% with the positive control fibronectin, whereas binding to HTB-129 human breast carcinoma cells and NHEK normal human keratinocytes were not significant. Poly (LDV)-doxorubicin conjugates displayed excellent selectivity and cytotoxicity in the delivery of doxorubicin. It was shown that the poly (LDV)doxorubicin conjugates exhibited cytotoxicity toward human malignant melanoma cells, but were less toxic than free doxorubicin. In addition, poly (LDV)-doxorubicin conjugates displayed a substantial reduction in toxicity per mole of doxorubicin against normal human keratinocyte cells when compared to free doxorubicin. Fluorescence microscopy showed poly (LDV)-FITC conjugates bound to the A-375 cells and were internalized within 30 minutes. Scatchard plots of poly (LDV)-doxorubicin conjugates were generated, which determined the association constants and established that there is one class of binding sites. It was shown that poly (LDV) could be internalized in a target specific manner by human malignant melanoma cells, which is dependent on the number of LDV targeting moieties in the polymer. These results established that poly (LDV) could be used as a drug delivery carrier that specifically targets the α 4 β 1 integrin.

Thesis advisor: Abhisheck Chatterjee / The genetic codon expansion (GCE) is a technique that uses an orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pair to incorporate noncanonical amino acids (ncAA) into proteins, to enable more protein-based chemistry. In the past two decades, more than 200 ncAAs have been site-specifically introduced into proteins in E. coli, and facilitated studies of protein structures, functions and interaction with other molecules. Although a large variety of ncAAs are available for incorporation in the bacterial systems, significantly fewer ncAAs are accessible for incorporation in eukaryotic cells. An expanded GCE toolbox will be beneficial for numerous applications in eukaryotic systems. Currently, introducing ncAAs in eukaryotes predominantly relies on the archaeal pyrrolysyl tRNA/aaRS pair. Such a strong dependence on a single platform has precluded genetic encoding of many desirable ncAAs, including structural mimics of many important post-translational modifications. The work presented in this thesis first developed an engineered E. coli leucyl tRNA/aaRS pair to enable site-specific incorporation of citrulline, an important PTM, into proteins expressed in mammalian cells. This technology was used to reveal the role of citrullination on site R372 and R374 of PAD4. Additionally, aiming at genetically encoding more diverse ncAAs, all 20 E. coli derived tRNA/aaRS pairs were screened for their ability to suppress TAG and TGA in mammalian cells. This study revealed several tRNA/aaRS pairs that are suitable for ncAA incorporation in mammalian cells, including those selective for phenylalanine, lysine, arginine, serine and glutamine. Efforts are currently under way to engineer these pairs to genetically encode new structural classes of ncAAs. / Thesis (PhD) — Boston College, 2022. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

<p dir="ltr">The objective of this thesis was to evaluate the nutrient digestibility in alternative animal and plant sources of feed ingredients for chickens and pigs. Therefore, 5 studies were carried out to determine the nutrient utilization in poultry meal (<b>PM</b>), faba beans (<b>FB</b>), and 3 cultivars field peas (<b>FP</b>).</p><p dir="ltr">In the first study, 2 experiments investigated the energy and phosphorus utilization of PM for broiler chickens. Poultry meal was used to substitute corn and soybean meal in the reference diet at 0, 80, or 160 g/kg in Experiment 1. Whereas PM was included in the diet at 0, 50, or 100 g/kg in Experiment 2. A total of 192 birds were allotted to 3 experimental diets in both experiments. The estimated ileal digestible energy (<b>IDE</b>), metabolizable energy (<b>ME</b>), and nitrogen-corrected metabolizable energy (<b>MEn</b>) for PM were 4,002, 3,756, and 3,430 kcal/kg DM, respectively. In Experiment 2, the true ileal digestibility (<b>TID</b>) and true total tract utilization (<b>TTTU</b>) of P in PM were 77.5 and 79.0%, respectively.</p><p dir="ltr">The second study consisted of 3 experiments. In the first experiment, 240 birds were assigned to 5 diets to determine the energy values of FB and DS admiral FP (<b>FPD</b>). In Experiment 1, the test ingredients were incorporated into a corn-soybean meal-based diet at 0, 150 or 300 g/kg. The IDE, ME, and MEn for FB were 2,541, 2,628, and 2,394 kcal/kg, respectively. The respective values for FPD were 2,254, 2,540, and 2,331 kcal/kg DM. In each of Experiments 2 and 3, 162 birds were assigned to 3 diets. Faba beans was included at 21, 42, or 63% and FPD at 16, 32, or 48% in Experiments 2 and 3, respectively. The TID and TTTU of P in FB were 66.5 and 66.7%, respectively. The corresponding values for FPD were 73.4 and 73.8%.</p><p dir="ltr">The third study consisted of 3 experiments. In Experiment 1, the energy values for Hampton FP (<b>FPH</b>) and 4010 FP (<b>FP4</b>) fed to broiler chickens were estimated. Two hundred and forty birds were assigned to 5 diets. The test ingredients were included at 0, 150 or 300 g/kg into a corn-soybean meal-based reference diet. With regression analysis, the determined IDE, ME, and MEn were 3,274, 3,033, and 2,850 kcal/kg DM in FPH, respectively, in FP4 the energy values were 3,019, 3,155, and 2,991 kcal/kg DM, respectively. The P utilization in FPH and FP4 were determined in Experiments 2 and 3, respectively. The corresponding TID and TTTU of P in FPH were 74.6% and 68.3%, and 74.3 and 61.7% in FP4.</p><p dir="ltr">Two experiments were conducted in the fourth study to estimate the digestible energy (<b>DE</b>) and ME in FB and FP fed to pigs. Twenty-four barrows were assigned to 3 dietary treatments in each of the experiments. Faba beans or FPD in Experiment 1 and FPH or FP4 in Experiment 2 were included in the diet at 0 or 300 g/kg. The determined DE and ME values for FB using the total collection method were 3,772 and 3,606 kcal/kg DM and in FPD were 3,683 and 3,589 kcal/kg DM, respectively. In Exp. 2, the respective DE and ME for FPH were 4,164 and 4,014 kcal/kg DM and for FP4 were 3,574 and 3,467 kcal/kg DM.</p><p dir="ltr">In the last study, standardized ileal digestibility (<b>SID</b>) of amino acids (<b>AA</b>) in faba beans and three cultivars of FP between broiler chickens and pigs were compared. The test ingredients were the only source of protein providing 160 g/kg crude protein and a nitrogen-free diet was prepared to estimate the basal endogenous losses of AA. The same set of five diets was used across both species. The SID of Lys in FB, FPD, and FPH exceeded 90% but in FP4 it was 85.1% for broiler chickens. Whereas for pigs the SID of Lys in FB, FPD, and FPH exceeded 80% but for FP4 it was 89.8%. The SID of Met in the test ingredients ranged from 72.1 to 89.8% and 68.1 to 81.8% for broiler chickens and pigs, respectively. In general, the SID of AA in the test ingredients were greater compared with chickens. The energy, P, and AA digestibility of the test ingredients determined in the five studies could be used in diet formulation for chickens and pigs.</p>

Silicone stands as an indispensable material for numerous applications; however, its high energy-cost synthesis poses significant environmental challenges. To address these concerns, bio-based silicone has gained considerable attention, showcasing its potential to dilute energy density while offering inherent functional benefits. Despite promising prospects, existing incorporation methods often involve protecting groups, rare metal catalysts, and multistep synthesis, which contradict green chemistry principles. The aza- Michael reaction emerges as a superior choice due to its high atom economy and mild reaction conditions. However, it still suffers from prolonged reaction times, hindering its overall efficiency and sustainability. This thesis utilizes self-activated beta-hydroxy acrylates to greatly enhance aza-Michael kinetics, achieving a 3-fold rate enhancement in solvent-free silicone synthesis. This fast aza-Michael reaction acts as the platform for the incorporation of Vitamin C and amino acids into silicone materials. Vitamin C-modified silicone demonstrates the potential for controlled antioxidant activity release, while amino acid-functionalized silicones are synthesized using choline amino acid ionic liquids, presenting a protecting-group-free and solvent-free synthesis method. Moreover, the synthesized choline amino acid-functional polymers and elastomers are investigated for their dielectric properties revealing promising potential for dielectric elastomer actuator applications. These innovative methods offer green alternatives for incorporating hydrophilic biomolecules into hydrophobic silicone systems, providing new functionalities that address both environmental and functional requirements. / Thesis / Doctor of Science (PhD)