DNA microarrays are chip-based, analysis tools which can perform hundreds of thousands of parallel assays to determine the identity of genes or gene expression levels present in a sample. They have been identified as a key technology in genomic sciences and emergent medical techniques; however, despite their abundant use in research laboratories, microarrays have not been used in the clinical setting to the fullest potential due to the difficulty of obtaining reproducible results. Microarrays work on the principle of DNA hybridization, and can only be as accurate as this process is robust. Fundamental, molecular-level understanding of hybridization on surfaces is needed to further refine these devices.This work shows how orientation of DNA probes with respect to the surface affects the thermodynamics and stability of hybridization. Ideal surface hybridization (a DNA duplex bound to the surface on one end) is compared to more realistic conditions such as interaction between DNA and the surface in multiple locations. This research also describes the effect of mismatch location and number of mismatches on a single target strand. The results clarify key details of the biophysics involved in microarray performance and this knowledge can be used to improve next-generation devices. The disparity between surface and bulk hybridization behavior is examined here in molecular level detail that is not currently possible with experimental techniques.

Discovering, screening, and associating changes in DNA sequence are important to a broad range of disciplines and play a central role in Forensic Science. The typical types of changes include sequence variations [single nucleotide polymorphisms (SNP)] and length variations [short tandem repeats (STR)]. The steps for forensic DNA sample processing are similar for both types of changes but diverge at the point of detection. A number of approaches are being explored for SNP genotyping while STR analysis primarily consists of size-based analysis by capillary electrophoresis. Limitations exist for all current detection methods that pose significant impacts to forensic analysis. Bi-allelic SNPs result in three possible genotypes with a minimal amount of information generated per marker. Limitations for SNP analysis are due to the inability to amplify a suitable number of SNP markers from low DNA content samples to provide an appropriate level of discrimination. Multi-allelic STR markers are currently the marker of choice for forensic typing but a variety of experimental artifacts are possible that consist of either biology or technology related causes. Molecular genotyping methods developed across other disciplines have potential to alleviate some of these shortcomings but no current approach is capable of genotyping both SNP and STR loci with a single chemistry. The need for a more effective, efficient, and generalized approach led to development of a unique method called Dye Probe Fluorescence Resonance Energy Transfer (dpFRET) and determination of its suitability for forensic analysis. The development phase of the research consisted of synthetic testing to establish proof of concept for the chemistry followed by polymerase chain reaction (PCR) based assays to demonstrate real world applications. Following successful development, the boundaries and limitations for the technology were established (sensitivity, allelic dropout, mixed samples) and efforts were made to improve the approach. In the process, parallel testing for other fields including molecular pathology and conservation biology were incorporated to explore potential widespread application of this new approach. The overall goal of this project was to develop and explore the limitations for a unique approach to genotyping both SNPs and STRs. A majority of the work involved development of the method itself with the ultimate objective of application for forensic science. The focus of this project was to address and alleviate some of the shortcomings of current approaches that result in potential limitations for forensic analysis. It is expected that future applications of this technology might impact a wide range of disciplines to aid in discovery, screening and association of changes in DNA sequence.

Cryptococcus neoformans is among the most common human pathogenic fungi and the leading cause of fungal meningitis. It consists of two divergent haploid serotypes A and D, as well as their hybrid serotypes AD. Hybridization between the two serotypes A and D is a widespread phenomenon in natural populations of this species. Studies have shown that serotype AD strains possess unique properties in several medically important traits: they are more virulent in animal models, more tolerant to antifungal drugs, more tolerant to UV radiation and other stresses than one or both parental serotypes. Despite the potential medical and evolutionary importance, relatively little is known about the phenotypic and genotypic consequences of the hybridization in C. neoformans. In my thesis, I present a series of studies that show: (1) recombination occurs at a very low frequency during hybridization between serotypes A and D; (2) serotypes A and D in C. neoformans have diverged from each other not only at the DNA sequence level, but also at the chromosomal structure level, both of which contributed to suppressed recombination and limited genetic introgression between the two serotypes; (3) though there is widespread hybridization, the hybrids seemed largely distinct and the genomes of the parental serotypes A and D populations are relatively unaffected by the hybrids in natural populations; and (4) the fixation of these serotype specific chromosomal rearrangements in the two serotypes, together with evidences of selective sweeps observed at both inversion junctions and genes located close to chromosomal inversions suggest that these rearranged regions (or genes surrounding these regions) were likely involved in the diversification between the two serotypes. My studies provide a genetic framework for future quantitative analyses of medically important traits of C. neoformans. / Thesis / Doctor of Philosophy (PhD)

<p> An improved method for the synthesis of oligoribonucleotides of defined sequence was developed. The general phosphotriester synthesis of Neilson and co-workers was modified by the introduction of a new condensing agent, mesitylenesulfonyl-1,2,4-triazole, and by the replacement of the bis(cyclohexylammonium) salt of mono-2,2,2-trichloroethyl phosphate with its acid salt. These modifications provided significant increases in the yields for the condensation of protected nucleosides - especially in the case of purine residues. Finally, modification of the three-step procedure for the deprotection of protected oligoribonucleotides resulted in the isolation of oligomers of exceptional purity and biological activity.</p> <p> Oligomers corresponding to natural sequences in transfer RNA molecules were obtained by this improved method of synthesis. These oligomers were then used to study: 1. The formation of short double-stranded RNA helices and 2. The interactions of aminoacyl-tRNA ligases with tRNA fragments.</p> / Thesis / Doctor of Philosophy (PhD)

A series of neutron scattering studies of unconventional superconductors is presented. These measurements are split into two parts. The first part considers the purely magnetic scattering in low-doped $La_{2−x}Ba_{x}CuO_{4}$. This study is comprehensive and elucidates much of the doping and temperature dependence of the low energy magnetic scattering in this system. It also clearly demonstrates that two dimensional incommensurate magnetic order in this system forms at the expense of three dimensional commensurate magnetic order. The remainder of the thesis is concerned with characterizing and determining the physics underlying pronounced enhancements of the inelastic scattering found to exist at 20 meV at equivalent two dimensional magnetic zone centers in both $La_{2−x}Ba_{x}CuO_{4}$ and $La_{2−x}Sr_{x}CuO_{4}$. Arguments are presented to interpret these features as a result spin-phonon hybridization in 214 cuprate superconductors. The measurements also explore the temperature and doping dependence of these features, determining that the enhancements are largely insensitive to doping and only present parametric response at temperatures relevant for three dimensional magnetic order in this system. In addition, the first evidence for a superconducting spin gap in $La_{2−x}Ba_{x}CuO_{4}$ is presented. The implications of these findings are discussed. / Thesis / Doctor of Philosophy (PhD) / This thesis discusses a series of measurements using brand new state of the art facilities to re-examine the family of high temperature superconductors with the simplest structure. By taking advantage of these new facilities and applying their capabilities to a, relatively speaking, simpler material family, the goal is to try and learn more about what may be the cause of high temperature superconductivity - a magnetism based phenomenon with profound implications for energy efficiency, energy storage, diagnostic healthcare, quantum computing and much more. While many interesting effects were observed, two of the more important findings were observations that unify the physical descriptions of $La_{2−x}Ba_{x}CuO_{4}$ and $La_{2−x}Sr_{x}CuO_{4}$, as is necessary to have a truly universal understanding of high temperature superconductivity, and a detailed characterization we interpret as hybridization. It is clear from this work that any complete solution of superconductivity must also explain this part magnetic and part crystalline structural hybridization phenomenon.

Water stress affects vine productivity, disease tolerance, and enological characteristics of grape. Florida Hybrid Bunch grape are developed through hybridization of local grape spp with Vitis vinifera. These cultivars are mostly grown in southeast region of United States. Water deficit conditions resulted due to failure of rains in the region has developed concern among Florida grape growers to increase water use efficiency of grape. The goal of this research is to identify genes and proteins differentially expressed in response to water stress and to correlate these changes with enological characteristics. Investigating transcripts and proteins will allow us to correlate them and confirm the involvement of specific genes responding to stress. Florida hybrid bunch ‘Suwannee’ grape plants were maintained under green house conditions. Water stress was induced by withholding irrigation. The leaf samples were collected from both irrigated and stressed plants at 5, 10, 15 and 20 day interval. We generated over 200 Subtractive Hybridization PCR products from control and water stressed leaf tissues. Cloning, sequencing and transcript analysis revealed that, 54 genes related to drought and defense regulated pathways out of 125 characterized transcripts. Proteins were extracted from leaf tissue with trichloroacetic acid /acetone and separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The proteins were sequenced in LC/Mass Spectrophotometer. The most important differentially expressed genes include sucrose synthase, actin, isoprene synthase, ABF3, SNF1 related protein kinase, WRKY type transcription factors, AP2, ASR2, glyoxalase I and, cytochrome b which play significant role in cell permeability, transportation, photosynthesis and, maintenance in osmotic stress. We have found that ribulose bisphosphate carboxylase and phosphoribulokinase, which play major role in photosynthesis, were suppressed in response to water stress in Florida hybrid bunch. The results suggested that water stress affects expression of cDNAs associated with defense and drought regulated functions. Such profiling studies will be used to explicate specific pathways disconcerted by water deficit treatments, and in the identification of varietal differences.

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