Traditional methods of pest control are threatened by the development of insecticide resistance, both to traditional insecticides and Bt toxins. Discovery of RNA interference (RNAi) has created opportunities to develop new insect control mechanisms. However, RNAi responses appear to be robust in coleopteran pests, but other orders, e.g. Lepidoptera and Hemiptera, present varied or ineffective RNAi responses. Current delivery strategies for double-stranded RNA (dsRNA) include microinjection, ingestion, and soaking. These approaches have benefits and problems. This study investigates the potential for porous silica nanoparticles (pSNPs) to improve the delivery of dsRNA and induce an RNAi response in Spodoptera frugiperda cells. Initially, the binding conditions of RNA onto porous and nonporous silica nanoparticles was examined, and the movement of RNA on and within pSNPs was observed. That information was then applied to in vitro studies for examining the capacity of silica nanoparticles to protect dsRNA from degradation by nucleases. This work culminated in an in vivo assay for measuring apoptosis when dsRNA is delivered to insect cells by pSNPs. Results of these studies show that silica nanoparticles bind nucleic acids and that dsRNA is mobile, pSNPs protect dsRNA from nuclease degradation, and pSNP/dsRNA complexes can induce apoptosis in lepidopteran insect cells.

The ZNF9 gene on chromosome 3 encodes the cellular nucleic acid binding protein (CNBP), a ubiquitously expressed, 177 amino acid (≈19.5kDa) protein that is highly conserved among vertebrates. The function of the protein is largely unknown, however an expansion in the first intron of the protein results in myotonic dystrophy type 2 (DM2), a multisystemic disease featuring cardiac arrhythmia, muscle wasting, cataracts, and a range of neuropathologies. Remarkably, we recently discovered that CNBP is involved in regulating the activity of β-secretase, the enzyme that produces the first cleavage event in the generation of the amyloid-β peptide (Aβ). The progressive fibrillization and deposition of Aβ is widely believed to be the primary causal factor in the development of Alzheimer’s disease (AD), and AD-like pathology in individuals with Down syndrome (DS). DS provides a unique model for evaluating how these factors change in the aged brain as compared to young brain, and how such changes affect the proportion of DS patients with AD. In the AD brain, both BACE1 and BACE2 increased from an early stage of disease; in DS brains, BACE1 significantly decreased (p<0.04) with age, whereas BACE2 was unchanged, even though the gene for BACE2 is located within the DS obligate region of chromosome 21. BACE1 and BACE2 activity levels were highly correlated in this series (r2 = 0.95), indicating that there may be a higher degree of shared regulation than previously believed. This implicates regulators of BACE as potentially critical for the development of AD, and our data suggests that CNBP may be one such regulator. In AD, CNBP increases early in the disease process, a change that does not occur in the normal aging process or in DS. CNBP and BACE protein levels were correlated in these cases (p<0.001), while there was no relationship between CNBP and age, or CNBP and Aβ, in either the human or mouse brain, indicating that CNBP does not increase as a consequence of normal aging. Thirty day overexpression of CNBP following adeno-associated viral delivery in murine gastrocnemius muscle resulted in an increase in BACE1 protein (p<0.01) and a consequential increase in Aβ production (p<0.01). Other experiments indicated that CNBP overexpression did not affect the half-life of BACE1 mRNA or protein, but resulted in an increase in BACE1 translation. These data indicate that CNBP is an important regulator of β-secretase, and may play an important role in the onset and progression of AD.

xii, 83 p. : ill. A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / My dissertation research has centered on understanding how nuclear encoded proteins affect chloroplast gene expression in higher plants. I investigated the functions of three proteins that belong to families whose members function solely or primarily in mitochondrial and chloroplast gene expression; the Whirly family (ZmWHY1) and the pentatricopeptide repeat (PPR) family (ZmPPR5 and ZmPPR10). The Whirly family is a plant specific protein family whose members have been described as nuclear DNA-binding proteins involved in transcription and telomere maintenance. I have shown that ZmWHY1 is localized to the chloroplast where it binds nonspecifically to DNA and also binds specifically to the atpF group II intron RNA. Why1 mutants show reduced atpF intron splicing suggesting that WHY1 is directly involved in atpF RNA maturation. Why1 mutants also have aberrant 23S rRNA metabolism resulting in a lack of plastid ribosomes. The PPR protein family is found in all eukaryotes but is greatly expanded in land plants. Most PPR proteins are predicted to localize to the mitochondria or chloroplasts where they are involved in many RNA-related processes including splicing, cleavage, editing, stabilization and translational control. Our results with PPR5 and PPR10 suggest that most of these activities may result directly from the unusually long RNA binding surface predicted for PPR proteins, which we have shown imparts two biochemical properties: site-specific protection of RNA from other proteins and site-specific RNA unfolding activity. I narrowed down the binding site for PPR5 and PPR10 to ∼45 nt and 19 nt, respectively. I showed that PPR5 contributes to the splicing of its group II intron ligand by restructuring sequences that are important for splicing. I used in vitro assays with purified PPR10 to confirm that PPR10 can block exonucleolytic RNA decay from both the 5' and 3' directions, as predicted by prior in vivo data. I also present evidence that PPR10 promotes translation by restructuring its RNA ligand to allow access to the ribosome. These findings illustrate how the unusually long RNA interaction surface predicted for PPR proteins can have diverse effects on RNA metabolism. This dissertation includes both previously published and unpublished co-authored material. / Committee in charge: Eric Selker, Chairperson, Biology; Alice Barkan, Advisor, Biology; Victoria Herman, Member, Biology; Karen Guillemin, Member, Biology; J. Andrew Berglund, Outside Member, Chemistry