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.

Thesis advisor: Abhishek Chatterjee / Abstract Unnatural amino acid (UAA) incorporation is a powerful tool used by biochemists to discover the nature of protein structure and function. The evolution of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs enables site-specific incorporation of UAAs proteins inside of living cells. The goal of this study was to further expand the repertoire of genetically encoded unnatural amino acids in E. coli as well as eukaryotes. We first attempted to engineer an aaRS, previously evolved for p-borono-phenylalanine (pBoF), to specifically charge 3-acetyl-p-borono-phenylalanine (AcpBoF). A randomized library of the pBoF-specific synthetases was generated and it was subjected to established selection schemes in a bacterial host. This report also describes the development of a yeast-based selection system to alter the substrate specificity of bacterial leucyl-tRNA synthetase, for genetic code expansion in eukaryotes. / Thesis (MS) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The purpose of the study is to fill the gap in the literature regarding sibling bereavement. This study explored how sibling bereavement from unnatural causes of death affects surviving siblings. Bereavement affects millions of Americans every year. Most grieve naturally, but some experience complicated grief or depression. Many studies address parental and spousal bereavement, but few focus on sibling bereavement. This study fills that gap in the literature so that mental health care professionals and the general public understand what bereaved siblings experience after the death of a sibling. The study was a phenomenological study using social constructivism as a theoretical lens to explore how sibling bereavement affects surviving siblings. Open-ended interviews were collected from 10 bereaved siblings. Those interviews were then transcribed and categorized using a 7 step process to review and organize all relevant statements. Results of this study shows that sibling bereavement can be a life changing event for surviving siblings affecting all aspects of life and leaving unanswered questions and feelings of guilt. Participants also state they felt overlooked after the death leading to delayed grief. Participants concluded that sibling grief is subjective, so any treatment plan should be catered to the individual based on their relationship to the deceased sibling and the role the sibling played. This study can bring about positive social change by helping mental health care workers understand sibling bereavement better so that they may help those suffering from complicated grief following the loss of a sibling.

Thesis advisor: Abhishek Chatterjee / Förster Resonance Energy Transfer (FRET) offers a powerful approach to study biomolecular dynamics in vitro as well as in vivo. The ability to apply FRET imaging to proteins in living cells provides an excellent tool to monitor important dynamic events such as protein conformational changes, protein-protein interactions, and proteolysis reactions. However, selectively incorporating two distinct fluorophores into the target protein(s) that are capable of FRET interaction within the complex cellular milieu is challenging. Consequently, terminal fusion to genetically encoded fluorescent proteins has emerged as the predominant labeling strategy for FRET studies in vivo. However, a major limitation of this strategy stems from the large size of the fluorescent proteins, which may perturb the native properties of the target, and restricted attachment only to the termini of the target. We reasoned that using genetically encoded fluorescent unnatural amino acids would overcome several of these challenges associated with currently available labeling strategies owing to their small size and the ability to introduce them site- specifically and co-translationally. Here, we report the use of the fluorescent unnatural amino acid “Anap” as a FRET donor with green and yellow fluorescent protein acceptors. We demonstrate the utility of this labeling strategy using proteolysis and conformational change models, and step towards in vivo studies by further developing a proteolysis system in cell lysates. / Thesis (MS) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Evan R. Kantrowitz / The understanding of how cells regulate and control all aspects of their function is vital for our ability to intervene when these control mechanisms break down. Almost all modes of cellular regulation can be related in some manner to protein conformational changes such as the quaternary conformational changes of allosteric enzymes that alter enzyme activity to regulate metabolism. The control of metabolic pathways by allosteric enzymes is analogous to a molecular valve with "on" and "off" positions. In the "off" position, flow through the pathway is severely hindered, while in the "on" position the flow is normal. For a comprehensive understanding of allosteric regulation we must elucidate in molecular detail how the allosteric signal is transmitted to the active site to alter enzyme activity. In this work we use unnatural amino acid mutagenesis to introduce a fluorescent amino acid into the allosteric binding site of aspartate transcarbamoylase (ATCase), the enzyme responsible for regulation of pyrimidine nucleotide biosynthesis. The fluorescence from the amino acid is exquisitely sensitive to the binding of the allosteric effectors ATP, CTP, UTP, and GTP. In particular we show how the asymmetric nature of the allosteric sites of the enzyme are used to achieve regulatory sensitivity over a broad range of mixed heterotropic effector concentrations as is observed in the cell. Furthermore, employing the method of random sampling - high dimensional model representation (RS-HDMR) we derived a model for how ATCase is regulated when all four nucleotides are present at fluctuating concentrations, consistent with physiological conditions. We've discovered the fundamental requirements to induce the allosteric transition to the R state by showing that although ATCase can accept L-asparagine as an unnatural substrate, the transition to the R allosteric state requires the correct positioning of the alpha-carboxylate of its natural substrate L-aspartate. However, linking the functionalities of L-asparagine and carbamoyl phosphate into a single molecule is sufficient to correctly position the bi-substrate analog in the active site to induce the allosteric transition to the R-state. The cooperative nature of ATCase was further investigated through the isolation of a unique quaternary structure of ATCase consisting of two catalytic trimers linked covalently by disulfide bonds. By relieving the quaternary constraints imposed by the bridging regulatory subunits of the native holoenzyme, the flexibility of the c6 subunit significantly enhanced enzyme activity over the native holoenzyme. Unlike the native c3 catalytic subunit, the c6 species displays homotropic cooperativity for L-aspartate demonstrating that, when two catalytic trimers are linked, a binding event at one or more active sites can be transmitted through the molecule to the other active sites in the absence of regulatory subunits. The catalytic reaction of ATCase follows an ordered sequential mechanism that is complicated by the transition from the T state to the R state upon the binding of the second substrate L-aspartate. Acquiring X-ray crystal structures at each step along the pathway has advanced our understanding of the catalytic mechanism, yet R-state structures are difficult to obtain. Using a mutant version of ATCase locked in the R-allosteric state by disulfide bonds we captured crystallographic images of ATCase in the R state bound to the true substrates (CP and Asp), products (CA and Pi), and in the process of releasing the final product (Pi) prior to reversion of the molecule to the T state. These structures depict the steps in the catalytic cycle immediately before the catalytic reaction occurs, immediately after the reaction, and after the first product has been released from the active site. This work also focuses on developing allosteric inhibitors of the enzyme fructose-1,6-bisphosphatase (FBPase), one of the enzymes responsible for regulation of the gluconeogenesis pathway. Inhibitors of FBPase could serve as potential therapeutic agents against type-2 diabetes. / Thesis (PhD) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Advances in molecular technologies have provided conservation biologist with the opportunity to quantify the genetic structure of a population and, in turn develop management guidelines and policies aimed at preserving the genetic diversity of fish stocks challenged by human activities. This thesis examines the status of genetics as applied to the management of freshwater fisheries by state natural resource agencies with a purpose of understanding the keys to a successful genetics program. An online survey was used to investigate the breadth of molecular marker application to freshwater fisheries management by state natural resource departments. Seven questions were posed to 50 state agencies addressing species of concern, type of genetic resources used, type of molecular marker used, and management concerns. Genetics was listed as a concern in the management of 18 freshwater fish families representing 70 distinct species, with Salmonid species the most frequently reported (20%#37;). A majority of agencies rely on outside resources to perform genetics testing (65%#37;). The most common analysis technique used by state agencies was microsatellite DNA analysis (35%#37;) and the most frequently reported management concerns were genetic stock identification and management boundaries (23%#37;). The application of a specific molecular technology to a conservation question was addressed by investigating the mechanisms of unnatural selection in the form of a study of trait heritability. Microsatellite parentage analysis was used to reconstruct familial relationships of juvenile Florida bass (Micropterus floridanus) displaying variable traits of growth and aggressiveness in a culture setting. Differences in the parentage of high growth and aggression (HGA) and baseline growth and aggression (BGA) offspring showed that certain parent-pairings contribute disproportionally to certain size classes and levels of aggression. These results suggest that the selective pressures of recreational harvest may negatively impact the fitness of wild fish stocks. Overall, this work provides natural resource managers with the basic information required to successfully develop and employ strategies aimed at preserving the genetic integrity of freshwater fisheries.

Unnatural amino acids (UAAs) have been used in bacteria and yeast to pinpoint protein binding sites, identify binding partners, PEGylate proteins site-specifically (vs. randomly), and attach small molecule fluorophores to proteins. The process of UAA incorporation involves the manipulation of the genetic code, which is established by the proper function of aminoacyl tRNA synthetases (RSs) and their cognate transfer RNAs (tRNAs). It has been discovered that certain regions of RS proteins can either block or enable cross-species reactivity of RSs. In essence, a bacterial RS can function with a human tRNA by transferring the human CP1 region to the bacterial RS, and vice versa. This knowledge has been used to engineer a tRNA capable of recognizing a stop codon (tRNA*), rather than an amino acid codon, and a cognate RS capable of recognizing only tRNA* and no endogenous tRNAs. We have previously described the use of this methodology to engineer a UAA incorporation system capable of amber stop codon suppression in HEK293T cells. Since UAAs are so useful, and their use has now been enabled in mammalian systems, we applied UAA incorporation to pluripotent cells. Stem and pluripotent cells have been the focus of cutting edge research for years, but much of the work done on these cell lines is done in the ignorance of basic biological processes underlying differentiation, dedifferentiation, and tumorigenesis. In order to facilitate the study of these basic biological processes and enable more adept manipulation of differentiation, dedifferentiation, and tumorigenesis, the development and use of two separate UAA incorporation systems is described herein. The overarching goal of this project is to facilitate the study of protein-protein interactions in stem and pluripotent cells. Since we have also previously described the development of a mammalian two-hybrid system, the use of that system in pluripotent cells is also described. / text

This study deals with the pastoral counselling of elderly persons after the loss of a mature child by unnatural death. Such a traumatic event leaves a significant void in a person's life, who is already experiencing other losses, and is in the eve of their lives. With the death of a mature child, the natural order of life is turned around: the child who is supposed to live and financially and emotionally support the elderly, is no more, while they who's own passing away is imminent, are alive. Elderly parents in this position experience this situation as an existential conflict, because it is to them as if they are burying the future with their child. Because the death is one of a mature child, the elderly parent does not necessarily receive the appropriate recognition from society for the trauma they experience. To further complicate the issue is the fact that they are not usually the primary care receivers being guided along the path of mourning and acceptance - the focus, on the contrary, is usually upon the spouse and children of the deceased. In the section dealing with basis-theoretical research, it is evident that the exceptional empathy and sympathy of God for the parent who lost a child, is repeatedly stressed by Scripture. Because of Jesus Christ's victory on the cross and also His victory over death, there is the assurance of hope for eventual healing for the traumatized parent finding himself/herself in such a situation. From the section dealing with meta-theoretical research, it is evident that the death of a mature child is one of the most alarming and traumatic experiences any parent can go through. The process of mourning is however a natural reaction and differs from parent to parent, according to circumstances. Specific phases have been identified through which parents will go under these circumstances and it is therefore very important that both the pastoral counselor, as well as the parents, are aware of the pertinent aspects. In the section dealing with empirical research, use has been made of both qualitative and quantitative research methods. In the quantitative section, the results obtained from questionnaires handed to a significant number of elderly parents who have lost mature children, were processed. Thereafter, in the qualitative section of the research, in depth interviews were conducted with two sets of parents from the original group. It became evident that this process of discussion had a significant healing and therapeutic effect on parents dealing with the loss of a mature child. In the section dealing with the practice theoretical research, a model has been formulated by which pastoral counsellors and ministers can generally assist elderly parents after the death of a mature child. / Thesis (Ph.D. (Pastoral))--North-West University, Potchefstroom Campus, 2009.

This study deals with the pastoral counselling of elderly persons after the loss of a mature child by unnatural death. Such a traumatic event leaves a significant void in a person's life, who is already experiencing other losses, and is in the eve of their lives. With the death of a mature child, the natural order of life is turned around: the child who is supposed to live and financially and emotionally support the elderly, is no more, while they who's own passing away is imminent, are alive. Elderly parents in this position experience this situation as an existential conflict, because it is to them as if they are burying the future with their child. Because the death is one of a mature child, the elderly parent does not necessarily receive the appropriate recognition from society for the trauma they experience. To further complicate the issue is the fact that they are not usually the primary care receivers being guided along the path of mourning and acceptance - the focus, on the contrary, is usually upon the spouse and children of the deceased. In the section dealing with basis-theoretical research, it is evident that the exceptional empathy and sympathy of God for the parent who lost a child, is repeatedly stressed by Scripture. Because of Jesus Christ's victory on the cross and also His victory over death, there is the assurance of hope for eventual healing for the traumatized parent finding himself/herself in such a situation. From the section dealing with meta-theoretical research, it is evident that the death of a mature child is one of the most alarming and traumatic experiences any parent can go through. The process of mourning is however a natural reaction and differs from parent to parent, according to circumstances. Specific phases have been identified through which parents will go under these circumstances and it is therefore very important that both the pastoral counselor, as well as the parents, are aware of the pertinent aspects. In the section dealing with empirical research, use has been made of both qualitative and quantitative research methods. In the quantitative section, the results obtained from questionnaires handed to a significant number of elderly parents who have lost mature children, were processed. Thereafter, in the qualitative section of the research, in depth interviews were conducted with two sets of parents from the original group. It became evident that this process of discussion had a significant healing and therapeutic effect on parents dealing with the loss of a mature child. In the section dealing with the practice theoretical research, a model has been formulated by which pastoral counsellors and ministers can generally assist elderly parents after the death of a mature child. / Thesis (Ph.D. (Pastoral))--North-West University, Potchefstroom Campus, 2009.

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