Biosynthesis and mode of action of ribosomally synthesized and post-translationally modified antimicrobial peptides

One of the greatest sources of biologically active compounds is natural products. Often these compounds serve as platforms for the design and development of novel drugs and therapeutics. The overwhelming amount of genomic information acquired in recent years has revealed that ribosomally synthesized and post-translationally modified natural products are much more widespread than originally anticipated. Identified in nearly all forms of life, these natural products display incredible structural diversity and possess a wide range of biological functions that include antimicrobial, antiviral, anti-inflammatory, antitumor, and antiallodynic activities. The unique pathways taken to biosynthesize these compounds offer exciting opportunities for the bioengineering of these complex molecules. The studies described herein focus on both the mode of action and biosynthesis of antimicrobial peptides. In Chapter 2, it is demonstrated that haloduracin, a recently discovered two-peptide lantibiotic, possesses nanomolar antimicrobial activity against a panel of bacteria strains. The potency of haloduracin rivals that of nisin, an economically and therapeutically relevant lantibiotic, which can be attributed to a similar dual mode of action. Moreover, it was demonstrated that this lantibiotic of alkaliphile origin has better stability at physiological pH than nisin. The molecular target of haloduracin was identified as the cell wall peptidoglycan precursor lipid II. Through the in vitro biosynthesis of haloduracin, several analogues of Halα were prepared and evaluated for their ability to inhibit peptidoglycan biosynthesis as well as bacterial cell growth. In an effort to overcome the limitations of in vitro biosynthesis strategies, a novel strategy was developed resulting in a constitutively active lantibiotic synthetase enzyme. This methodology, described in Chapter 3, enabled the production of fully-modified lacticin 481 products with proteinogenic and non-proteinogenic amino acid substitutions. A number of lacticin 481 analogues were prepared and their antimicrobial activity and ability to bind lipid II was assessed. Moreover, site-directed mutagenesis of the constitutively active synthetase resulted in a kinase-like enzyme with the ability to phosphorylate a number of peptide substrates. The hunt for a lantibiotic synthetase enzyme responsible for installing the presumed dehydro amino acids and a thioether ring in the natural product sublancin, led to the identification and characterization of a unique post-translational modification. The studies described in Chapter 4, demonstrate that sublancin is not a lantibiotic, but rather an unusual S-linked glycopeptide. Its structure was revised based on extensive chemical, biochemical, and spectroscopic characterization. In addition to structural investigation, bioinformatic analysis of the sublancin gene cluster led to the identification of an S-glycosyltransferase predicted to be responsible for the post-translational modification of the sublancin precursor peptide. The unprecedented glycosyltransferase was reconstituted in vitro and demonstrated remarkable substrate promiscuity for both the NDP-sugar co-substrate as well as the precursor peptide itself. An in vitro method was developed for the production of sublancin and analogues which were subsequently evaluated in bioactivity assays. Finally, a number of putative biosynthetic gene clusters were identified that appear to harbor the necessary genes for production of an S-glycopeptide. An additional S-glycosyltransferase with more favorable intrinsic properties including better expression, stability, and solubility was reconstituted in vitro and demonstrated robust catalytic abilities.

A Transformative Olympic Village: The Washington 2024 Post-Games Legacy

This thesis explores the Modern Olympic Games to strategically design an Olympic Village for Washington D.C. that plans not just to house athletes, but to provide a vision for the post-Games city. Through discovery of the spirit and meaning behind one of the world’s biggest events and analysis of various post-Games Villages, the proposed Olympic Village will innovate the future of Washington D.C.’s Southeast region. Study of existing mixed-use architecture, urban planning, and adaptation will help formulate an Olympic Village design. It is the intention that the Olympic Village, much like its athletes, will emulate the Olympic motto “Citius, Altius, Fortius,” meaning “Faster, Higher, Stronger.” The objective is to establish a village that allows for a faster turnaround in post-Olympic design, utilizes higher standards, and uses stronger applications to building a more sustainable city.

Pediatric Coronary Artery Revascularization Surgery: Development and Effects on Survival, Cardiac Events and Graft Patency for Children With Kawasaki Disease Coronary Involvements

Pediatric coronary artery bypass surgery gained wide acceptance with the introduction of internal thoracic arteries (ITAs) for bypass operations for post Kawasaki disease (KD) lesions. The technique is now established as the standard surgical choice, and its safety even in infancy, graft patency, growth potential, graft longevity and clinical efficacy have been well documented. In this article the author reviews the development of pediatric coronary bypass as the main indication for the treatment of coronary lesions due to KD. I believe that coronary revascularization surgery in pediatric population utilizing uni- or bilateral ITAs is the current gold-standard as the most reliable treatment, although percutaneous coronary intervention with or without a stent has been tried with vague long-term results in children.

The Japanese History Textbook Controversy Amid Post-War Sino-Japanese Relations

The relations between China and Japan are strained and continue to foster negative emotions partly because of China’s grievances about Japan’s actions during World War II and the allegedly false historiographical accounts found in Japanese history textbooks. This study will utilize historical analysis of the events leading up to the Nanjing Massacre in December of 1937, examine the Japanese Ministry of Education’s (MEXT) critical and contentious role in the selection of textbooks, used for primary and secondary schools, and will also juxtapose the controversial 2001 Atarashii rekishi kyōkasho with current Japanese history textbooks. The study will also include a syntactical analysis of key terms through my own original translations of multiple Japanese history textbooks, which are currently used in the Japanese school curriculum, to reveal that the textbook publishers, MEXT, and regulation councils are involved in adjusting the content causing the information to reveal various degrees of whitewashing.

Post-Revolutionary Post-Modernism: Central American Detective Fiction by the Turn of the 21st Century

Contemporary Central American fiction has become a vital project of revision of the tragic events and the social conditions in the recent history of the countries from which they emerge. The literary projects of Sergio Ramirez (Nicaragua), Dante Liano (Guatemala), Horacio Castellanos Moya (El Salvador), and Ramon Fonseca Mora (Panama), are representative of the latest trends in Central American narrative. These trends conform to a new literary paradigm that consists of an amalgam of styles and discourses, which combine the testimonial, the historical, and the political with the mystery and suspense of noir thrillers. Contemporary Central American noir narrative depicts the persistent war against social injustice, violence, criminal activities, as well as the new technological advances and economic challenges of the post-war neo-liberal order that still prevails throughout the region. Drawing on postmodernism theory proposed by Ihab Hassan, Linda Hutcheon and Brian MacHale, I argued that the new Central American literary paradigm exemplified by Sergio Ramirez’s El cielo llora por mí, Dante Liano’s El hombre de Montserrat, Horacio Castellanos Moya’s El arma en el hombre and La diabla en el espejo, and Ramon Fonseca Mora’s El desenterrador, are highly structured novels that display the characteristic marks of postmodern cultural expression through their ambivalence, which results from the coexistence of multiple styles and conflicting ideologies and narrative trends. The novels analyzed in this dissertation make use of a noir sensitivity in which corruption, decay and disillusionment are at their core to portray the events that shaped the modern history of the countries from which they emerge. The revolutionary armed struggle, the state of terror imposed by military regimes and the fight against drug trafficking and organized crime, are among the major themes of these contemporary works of fiction, which I have categorized as perfect examples of the post-revolutionary post-modernism Central American detective fiction at the turn of the 21st century.

The Coming of Post-reflexive Society: Commodification and Language in Digital Capitalism

Language is a unique aspect of human communication because it can be used to discuss itself in its own terms. For this reason, human societies potentially have superior capacities of co-ordination, reflexive self-correction, and innovation than other animal, physical or cybernetic systems. However, this analysis also reveals that language is interconnected with the economically and technologically mediated social sphere and hence is vulnerable to abstraction, objectification, reification, and therefore ideology – all of which are antithetical to its reflexive function, whilst paradoxically being a fundamental part of it. In particular, in capitalism, language is increasingly commodified within the social domains created and affected by ubiquitous communication technologies. The advent of the so-called ‘knowledge economy’ implicates exchangeable forms of thought (language) as the fundamental commodities of this emerging system. The historical point at which a ‘knowledge economy’ emerges, then, is the critical point at which thought itself becomes a commodified ‘thing’, and language becomes its “objective” means of exchange. However, the processes by which such commodification and objectification occurs obscures the unique social relations within which these language commodities are produced. The latest economic phase of capitalism – the knowledge economy – and the obfuscating trajectory which accompanies it, we argue, is destroying the reflexive capacity of language particularly through the process of commodification. This can be seen in that the language practices that have emerged in conjunction with digital technologies are increasingly non-reflexive and therefore less capable of self-critical, conscious change.

An investigation of nucleation events in a coastal urban environment in the southern hemisphere

The occurrence of and conditions favourable to nucleation were investigated at an industrial and commercial coastal location in Brisbane, Australia during five different campaigns covering a total period of 13 months. To identify potential nucleation events, the difference in number concentration in the size range 14-30 nm (N14-30) between consecutive observations was calculated using first-order differencing. The data showed that nucleation events were a rare occurrence, and that in the absence of nucleation the particle number was dominated by particles in the range 30-300 nm. In many instances, total particle concentration declined during nucleation. There was no clear pattern in change in NO and NO2 concentrations during the events. SO2 concentration, in the majority of cases, declined during nucleation but there were exceptions. Most events took place in summer, followed by winter and then spring, and no events were observed for the autumn campaigns. The events were associated with sea breeze and long-range transport. Roadside emissions, in contrast, did not contribute to nucleation, probably due to the predominance of particles in the range 50-100 nm associated with these emissions.