STRUCTURAL ANALYSIS OF RIBOSOME BINDING ELEMENTS OF THE 3´ UTR IN TWO POSITIVE-STRAND PLANT RNA VIRUSES

Turnip crinkle virus (TCV) and Pea enation mosaic virus (PEMV) are two positive (+)-strand RNA viruses that are used to investigate the regulation of translation and replication due to their small size and simple genomes. Both viruses contain cap-independent translation elements (CITEs) within their 3´ untranslated regions (UTRs) that fold into tRNA-shaped structures (TSS) according to nuclear magnetic resonance and small angle x-ray scattering analysis (TCV) and computational prediction (PEMV). Specifically, the TCV TSS can directly associate with ribosomes and participates in RNA-dependent RNA polymerase (RdRp) binding. The PEMV kissing-loop TSS (kl-TSS) can simultaneously bind to ribosomes and associate with the 5´ UTR of the viral genome. Mutational analysis and chemical structure probing methods provide great insight into the function and secondary structure of the two 3´ CITEs. However, lack of 3-D structural information has limited our understanding of their functional dynamics. Here, I report the folding dynamics for the TCV TSS using optical tweezers (OT), a single molecule technique. My study of the unfolding/folding pathways for the TCV TSS has provided an unexpected unfolding pathway, confirmed the presence of Ψ3 and hairpin elements, and suggested an interconnection between the hairpins and pseudoknots. In addition, this study has demonstrated the importance of the adjacent upstream adenylate-rich sequence for the formation of H4a/Ψ3 along with the contribution of magnesium to the stability of the TCV TSS. In my second project, I report on the structural analysis of the PEMV kl-TSS using NMR and SAXS. This study has re-confirmed the base-pair pattern for the PEMV kl-TSS and the proposed interaction of the PEMV kl-TSS with its interacting partner, hairpin 5H2. The molecular envelope of the kl-TSS built from SAXS analysis suggests the kl-TSS has two functional conformations, one of which has a different shape from the previously predicted tRNA-shaped form. Along with applying biophysical methods to study the structural folding dynamics of RNAs, I have also developed a technique that improves the production of large quantities of recombinant RNAs in vivo for NMR study. In this project, I report using the wild-type and mutant E.coli strains to produce cost-effective, site-specific labeled, recombinant RNAs. This technique was validated with four representative RNAs of different sizes and complexity to produce milligram amounts of RNAs. The benefit of using site-specific labeled RNAs made from E.coli was demonstrated with several NMR techniques.

Regenerative Stormwater Conveyance: Design Implications of an Urban Case Demonstration in Baltimore, Maryland

This research-design thesis explores the implementation of Regenerative Stormwater Conveyance (RSC) as a retrofit of an existing impervious drainage system in a small catchment in the degraded Jones Falls watershed in Baltimore City. An introduction to RSC is provided, placing its development within a theoretical context of novel ecosystems, biomimicry and Nassauer and Opdam’s (2008) model of landscape innovation. The case site is in Baltimore’s Hampden neighborhood on City-owned land adjacent to rowhomes, open space and an access point to a popular wooded trail along a local stream. The design proposal employs RSC to retrofit an ill-performing stormwater system, simultaneously providing a range of ecological, social and economic services; water quantity, water quality and economic performance of the proposed RSC are quantified. While the proposed design is site-specific the model is adaptable for retrofitting other small-scale impervious drainage systems, providing a strategic tool in addressing Baltimore City’s stormwater challenges.

MAXIMIZING LANDSCAPE PERFORMANCE AT ADVENTIST HOSPITAL: HEALING THE PEOPLE, HEALING SLIGO CREEK

This paper answers the question of whether a design intervention on Washington Adventist Hospital’s Takoma Park campus can combine stormwater Best Management Practices with outdoor healing spaces, to improve the health of the local creek (Sligo Creek) while creating a restorative environment for the hospital community. To improve the health of Sligo Creek, a campus-wide stormwater analysis was undertaken, in addition to an intervention-site-specific stormwater analysis, and a literature review of stormwater best management practices. To create a restorative environment, a literature review of healing gardens was undertaken, in addition to a campus-wide site analysis, to uncover the most ideally suited site to create a restorative environment.

Dual Quorum Quenching Capsules: Disrupting two bacterial communication pathways that lead to virulence

Healthcare Associated Infections (HAIs) in the United States, are estimated to cost nearly $10 billion annually. And, while device-related infections have decreased, the 60% attributed to pneumonia, gastrointestinal pathogens and surgical site infections (SSIs) remain prevalent. Furthermore, these are often complicated by antibacterial resistance that ultimately cause 2 million illnesses and 23,000 deaths in the US annually. Antibacterial resistance is an issue increasing in severity as existing antibiotics are losing effectiveness, and fewer new antibiotics are being developed. As a result, new methods of combating bacterial virulence are required. Modulating communications of bacteria can alter phenotype, such as biofilm formation and toxin production. Disrupting these communications provides a means of controlling virulence without directly interacting with the bacteria of interest, a strategy contrary to traditional antibiotics. Inter- and intra-species bacterial communication is commonly called quorum sensing because the communication molecules have been linked to phenotypic changes based on bacterial population dynamics. By disrupting the communication, a method called ‘quorum quenching’, bacterial phenotype can be altered. Virulence of bacteria is both population and species dependent; each species will secrete different toxic molecules, and total population will affect bacterial phenotype9. Here, the kinase LsrK and lactonase SsoPox were combined to simultaneously disrupt two different communication pathways with direct ties to virulence leading to SSIs, gastrointestinal infection and pneumonia. To deliver these enzymes for site-specific virulence prevention, two naturally occurring polymers were used, chitosan and alginate. Chitosan, from crustacean shells, and alginate, from seaweed, are frequently studied due to their biocompatibility, availability, self-assembly and biodegrading properties and have already been verified in vivo for wound-dressing. In this work, a novel functionalized capsule of quorum quenching enzymes and biocompatible polymers was constructed and demonstrated to have dual-quenching capability. This combination of immobilized enzymes has the potential for preventing biofilm formation and reducing bacterial toxicity in a wide variety of medical and non-medical applications.