Genetic engineering

Despite the many epidemiological studies which have shown some effects of EMF on biological systems, there has yet to be any data that indicates the molecular mechanisms by which this effect takes place. My goal was to genetically engineer a yeast strain that would have a clear biological effect to the EMF's. The strategy involved using a yeast strain which requires histone function from a plasmid, and the plasmid construct that has a Gal1 promoter controlling the histone function. The plasmid construct could then be engineered to contain a promoter sequence for a known EMF-repressed gene in yeast, which would control the histone production. Without a functional histone gene, the cells will die, and the effects will be easily visualized. Although the genetic screening for the desired transformants appeared to work, the molecular analysis of those transformants did not show the promoter insertion. There are a few possible reasons for why this happened, including possible reversions from one of the original mutations of the chromosomal histone H4 genes, or the mutation of the Gal1 promoter which would no longer repress the histone H4 gene and allow the cells to grow on glucose.

This research is concerned with analyzing a set of viral genomes to elucidate the underlying characteristics and determine the information-theoretic aspects of the genomic signatures. The goal of this study thereof, is tailored to address the following: (i) Reviewing various methods available to deduce the features and characteristics of genomic sequences of organisms in general, and particularly focusing on the genomes pertinent to viruses; (ii) applying the concepts of information-theoretics (entropy principles) to analyze genomic sequences; (iii) envisaging various aspects of biothermodynamic energetics so as to determine the framework and architecture that decide the stability and patterns of the subsequences in a genome; (iv) evaluating the genomic details using spectral-domain techniques; (v) studying fuzzy considerations to ascertain the overlapping details in genomic sequences; (vi) determining the common subsequences among various strains of a virus by logistically regressing the data obtained via entropic, energetics and spectral-domain exercises; (vii) differentiating informational profiles of coding and non-coding regions in a DNA sequence to locate aberrant (cryptic) attributes evolved as a result of mutational changes and (viii) finding the signatures of CDS of genomes of viral strains toward rationally conceiving plausible designs of vaccines. Commensurate with the topics indicated above, necessary simulations are proposed and computational exercises are performed (with MatLabTM R2009b and other software as needed). Extensive data gathered from open-literature are used thereof and, simulation results are verified. Lastly, results are discussed, inferences are made and open-questions are identified for future research.

With antimicrobial resistance to current drugs steadily rising, the development of new antibiotics with novel mechanisms of action has become an imperative. The majority of life-threatening infections worldwide are caused by "ESKAPE" pathogens which are encountered in more than 40% of hospital-acquired infections, and are resistant to the majority of commonly used antibiotics. Naturally occurring cyclic depsipeptides, microbial secondary metabolites that contain one or more ester bonds in addition to amide bonds, have emerged as an important source of pharmacologically active compounds or lead structures for the development of novel antibiotics. Some of those peptides are either already marketed (daptomycin) or in advanced stages of clinical development (ramoplanin). Structurally simple, yet potent, fusaricidin/LI-F and lysobactin families of naturally occurring antibiotics represent particularly attractive candidates for the development of new antibacterial agents capable of overco ming infections caused by multidrug-resistant bacteria. These natural products exhibit potent antimicrobial activity against a variety of clinically relevant fungi and Gram-positive bacteria. Therefore, access to these classes of natural products and their synthetic analogs, combined with elucidation of their mode of action represent important initial steps toward full exploitation of their antmicrobial potential. This dissertation describes a general approach toward the solid-phase synthesis of fusaricidin/LI-F and lysobactin analogs and an extensive structure-activity relationship (SAR) study. We have devised a simple and robust preparation strategy based on standard Fmoc solid-phase peptide synthesis protocols.

In my thesis work, I attempted to construct a plasmid that would allow stable integration of genes into the Saccharomyces cerevisiae yeast genome under the control of the repressible TetO promoter. The yeast ACO1 gene was cloned under the control of the TetO operator and the tTA transactivator. This construct was inserted into yeast cells in order to observe the effects of aconitase overexpression on aging. Unfortunately, the transformed cells appeared incapable of aconitase expression as determined by glutamic acid auxptrophy, a phenotype of aconitase mutants. We have sequenced the pIT1ACO1 plasmid and have found many abnormalities in the promoter region. If the plasmid can be made to function as intended, the resulting yeast strain can be used in the future to determine if aconitase plays an important role in cellular aging.