CHARACTERIZATION OF TOBACCO MOSAIC VIRUS DIRECTED REPROGRAMMING OF PHLOEM GENE EXPRESSION TO PROMOTE VIRAL PHLOEM LOADING AND SYSTMEIC MOVEMENT

Vascular phloem loading has long been recognized as an essential step in the establishment of a systemic virus infection. Yet little is known about this process and the mechanisms that control it. In this study, an interaction between the replication protein of Tobacco mosaic virus (TMV) and phloem specific auxin/indole acetic acid (Aux/IAA) transcriptional regulators was found to modulate virus phloem loading. Promoter expression studies show TMV 126/183 kDa interacting Aux/IAAs predominantly express and accumulate within the nuclei of phloem companion cells (CC). Furthermore, CC Aux/IAA nuclear localization is disrupted upon infection with an interacting virus but not during infection with a non-interacting virus. In situ analysis of virus spread shows the inability of TMV variants to disrupt Aux/IAA CC nuclear localization correlates with a reduced ability to load into the vascular tissue. Subsequent systemic movement assays also demonstrate that a virus capable of disrupting Aux/IAA localization is significantly more competitive at systemic movement than a non-interacting virus. Similarly, CC expression and over-accumulation of a degradation-resistant-interacting Aux/IAA protein was found to selectively inhibit TMV accumulation and phloem loading. Transcriptional expression studies demonstrate a role for interacting Aux/IAA proteins in the regulation of salicylic acid and jasmonic acid dependent host defense responses as well as virus specific movement factors including pectin methylesterase that are involved in regulating plasmodesmata size exclusion limits and promoting virus cell-to-cell movement. Further characterization of the phloem environment was done using two phloem specific promoters (pSUC2 and pSULTR2;2) to generate epitope-tagged polysomal-RNA complexes. Immuno-purification using the epitope tag allowed us to obtain mRNAs bound to polysomes (the translatome) specifically in phloem tissue. We found the phloem translatome is uniquely altered during TMV infection with 90% and 88% of genes down regulated in the pSUC2 and pSULTR2;2 phloem translatomes, compared to 31% of genes down regulated in the whole plant p35S translatome. Transcripts down regulated in phloem include genes involved in callose deposition at plasmodesmata, host defense responses, and RNA silencing. Combined, these findings indicate TMV reprograms gene expression within the vascular phloem as a means to enhance phloem loading and systemic spread.

Geography and Genetics of Ecological Speciation in Pea Aphids

During ecological speciation, divergent natural selection drives evolution of ecological specialization and genetic differentiation of populations on alternate environments. Populations diverging onto the same alternate environments may be geographically widespread, so that divergence may occur at an array of locations simultaneously. Spatial variation in the process of divergence may produce a pattern of differences in divergence among locations called the Geographic Mosaic of Divergence. Diverging populations may vary in their degree of genetic differentiation and ecological specialization among locations. My dissertation examines the pattern and evolutionary processes of divergence in pea aphids (Acyrthosiphon pisum) on alfalfa (Medicago sativa) and clover (Trifolium pretense). In Chapter One, I examined differences among North American aphid populations in genetic differentiation at nuclear, sequence-based markers and in ecological specialization, measured as aphid fecundity on each host plant. In the East, aphids showed high host-plant associated ecological specialization and high genetic differentiation. In the West, aphids from clover were genetically indistinguishable from aphids on alfalfa, and aphids from clover were less specialized. Thus, the pattern of divergence differed among locations, suggesting a Geographic Mosaic of Divergence. In Chapter Two, I examined genomic heterogeneity in divergence in aphids on alfalfa and clover across North America using amplified fragment length polymorphisms (AFLPs). The degree of genetic differentiation varied greatly among markers, suggesting that divergent natural selection drives aphid divergence in all geographic locations. Three of the same genetic markers were identified as evolving under divergent selection in the eastern and western regions, and additional divergent markers were identified in the East. In Chapter Three, I investigated population structure of aphids in North America, France, and Sweden using AFLPs. Aphids on the same host plant were genetically similar across many parts of their range, so the evolution of host plant specialization does not appear to have occurred independently in every location. While aphids on alfalfa and clover were genetically differentiated in most locations, aphids from alfalfa and clover were genetically similar in both western North America and Sweden. High gene flow from alfalfa onto clover may constrain divergence in these locations.

Sikh Sabad Kirtan as a Musical Construction of Memory

The performance of devotional music in India has been an active, sonic conduit where spiritual identities are shaped and forged, and both history and mythology lived out and remembered daily. For the followers of Sikhism, congregational hymn singing has been the vehicle through which text, melody and ritual act as repositories of memory, elevating memory to a place where historical and social events can be reenacted and memorialized on levels of spiritual significance. This dissertation investigates the musical process of Shabad Kirtan, Sikh hymn singing, in a Sikh musical service as a powerful vehicle to forge a sense of identification between individual and the group. As an intimate part of Sikh life from birth to death, the repertoire of Shabad Kirtan draws from a rich mosaic of classical and folk genres as well as performance styles, acting as a musical and cognitive archive. Through a detailed analysis of the Asa Di Var service, Shabad Kirtan is explored as a phenomenological experience where time, place and occasion interact as a meaningful unit through which the congregation creates and recreates themselves, invoking deep memories and emotional experiences. Supported by explanatory tables, diagrams and musical transcriptions, the sonic movements of the service show how the Divine Word as Shabad is not only embodied through the Guru Granth Sahib, but also encountered through the human enactment of the service, aurally, viscerally and phenomenologically.

VINTAGE MODERNISM: EARLY EXPERIMENTALIST MUSIC FOR VIOLIN IN THE UNITED STATES

The historical narrative of concert music in the early twentieth century has focused a great deal on the influence of European composers as well as the American composers who went to Europe to study. Often overlooked, however, is the influence of an entire generation of composers working in the United States during that time period. These artists experimented with polyrhythm, polytonality, dissonant counterpoint, and a whole host of other musical techniques in order to express their perceptions of a changing world. Over the years, the new techniques became associated with various movements including futurism, experimentalism and ultramodernism. Regardless of label, these composers were some of the first to introduce the new musical styles to the listening public.The recitals that make up this dissertation explore the sound world of experimentalist composers working in the United States during the early twentieth century. Serving as the foundation of these recitals are all four of the violin sonatas by Charles Ives, the “grandfather” of modernist music whose financial support helped to foster a whole generation of American composers. Also prominently featured is the music of Henry Cowell. His Suite for Violin and Piano, Mosaic Quartet (String Quartet No. 3), and Quartet Euphometric demonstrate the composer's use of cluster tones, dissonant counterpoint, polymeter, and indeterminate form. Additional works by George Antheil, Leo Ornstein, Wallingford Riegger, Dane Rudhyar, Carl Ruggles, and Ruth Crawford (Seeger) highlight other approaches taken by members of the ultra-modernist movement. Rounding out the repertoire for these recitals are works by Johanna Beyer and Conlon Nancarrow, both of whom either worked with or were influenced by Cowell in some way. All of the pieces selected date roughly from 1900 to the mid 1930's. Thus, the purpose of these recitals is not to provide a comprehensive overview of each composer's development, but rather to examine the influence and interconnections of a specific cross-section of the musical landscape.

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.

Integration of virus-like particle macromolecular bioreceptors in electrochemical biosensors

Rapid, sensitive and selective detection of chemical hazards and biological pathogens has shown growing importance in the fields of homeland security, public safety and personal health. In the past two decades, efforts have been focusing on performing point-of-care chemical and biological detections using miniaturized biosensors. These sensors convert target molecule binding events into measurable electrical signals for quantifying target molecule concentration. However, the low receptor density and the use of complex surface chemistry in receptors immobilization on transducers are common bottlenecks in the current biosensor development, adding to the cost, complexity and time. This dissertation presents the development of selective macromolecular Tobacco mosaic virus-like particle (TMV VLP) biosensing receptor, and the microsystem integration of VLPs in microfabricated electrochemical biosensors for rapid and performance-enhanced chemical and biological sensing. Two constructs of VLPs carrying different receptor peptides targeting at 2,4,6-trinitrotoluene (TNT) explosive or anti-FLAG antibody are successfully bioengineered. The VLP-based TNT electrochemical sensor utilizes unique diffusion modulation method enabled by biological binding between target TNT and receptor VLP. The method avoids the influence from any interfering species and environmental background signals, making it extremely suitable for directly quantifying the TNT level in a sample. It is also a rapid method that does not need any sensor surface functionalization process. For antibody sensing, the VLPs carrying both antibody binding peptides and cysteine residues are assembled onto the gold electrodes of an impedance microsensor. With two-phase immunoassays, the VLP-based impedance sensor is able to quantify antibody concentrations down to 9.1 ng/mL. A capillary microfluidics and impedance sensor integrated microsystem is developed to further accelerate the process of VLP assembly on sensors and improve the sensitivity. Open channel capillary micropumps and stop-valves facilitate localized and evaporation-assisted VLP assembly on sensor electrodes within 6 minutes. The VLP-functionalized impedance sensor is capable of label-free sensing of antibodies with the detection limit of 8.8 ng/mL within 5 minutes after sensor functionalization, demonstrating great potential of VLP-based sensors for rapid and on-demand chemical and biological sensing.