Idealized modeling of the role of stability and shear on mesoscale gravity wave evolution

Mesoscale Gravity Waves (MGWs) are large pressure perturbations that form in the presence of a stable layer at the surface either behind Mesoscale Convective Systems (MCSs) in summer or over warm frontal surfaces behind elevated convection in winter. MGWs are associated with damaging winds, moderate to heavy precipitation, and occasional heat bursts at the surface. The forcing mechanism for MGWs in this study is hypothesized to be evaporative cooling occurring behind a convective line. This evaporatively-cooled air generates a downdraft that then depresses the surface-based stable layer and causes pressure decreases, strong wind speeds and MGW genesis. Using the Weather Research and Forecast Model (WRF) version 3.0, evaporative cooling is simulated using an imposed cold thermal. Sensitivity studies examine the response of MGW structure to different thermal and shear profiles where the strength and depth of the inversion are varied, as well as the amount of wind shear. MGWs are characterized in terms of response variables, such as wind speed perturbations (U'), temperature perturbations (T'), pressure perturbations (P'), potential temperature perturbations (Θ'), and the correlation coefficient (R) between U' and P'. Regime Diagrams portray the response of MGW to the above variables in order to better understand the formation, causes, and intensity of MGWs. The results of this study indicate that shallow, weak surface layers coupled with deep, neutral layers above favor the formation of waves of elevation. Conversely, deep strong surface layers coupled with deep, neutral layers above favor the formation of waves of depression. This is also the type of atmospheric setup that tends to produce substantial surface heating at the surface.

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.