The purpose of this study was to investigate the effect of four direct restorative materials that can be used in the proximal box elevation (PBE) technique. Materials and Methods: Seventy-five molar teeth were randomly assigned to one of five groups (n=15): Type II glass-ionomer (GI), Type II resin-modified glass-ionomer (RMGI), resin-based composite (RBC), bulk fill (BF) resin-based composite, and a control with no box elevation procedure. Specimens were prepared for a standard CAD-CAM ceramic onlay preparation with mesial cervical margins located 1 mm above CEJ (cemento-enamel junction) and distal cervical margins located 2 mm below the CEJ. PBE was used to elevate the distal margins to 1 mm above the CEJ in all groups except the control group. For the control group the onlay margin was placed directly on the prepared distal tooth without PBE. A Lava UltimateTM, CAD/CAM Resin, nano-Ceramic onlay Restorative (LAVU) was milled and bonded on all specimens with RelyX UltimateTMAdhesive Resin Cement. The margin quality of the tooth-PBE material and PBE material-onlay interface was evaluated with scanning electron microscopy (SEM) using epoxy replicas before and after mechanical loading (100,000 cycles, 1.2 Hz at 65N). In addition to margin quality, the fracture resistance of each group was measured using a universal testing machine. Fracture pattern was recorded by visual examination. One-way ANOVA was performed followed by Least Square Means. Results: For dentin margins, a statistically significant difference was detected between RMGI and control group at baseline (p=0.0442). All other groups GI, RBC, and BF showed no difference to control at baseline (p>0.05). No statistical significance was observed among groups for post-mechanical fatigue (p=0.8735). For onlay margins, no statistical significance was observed among groups for pre-mechanical fatigue, post-mechanical fatigue, or change (p=0.9713, p=0.528, p= 0.4385 respectively). No significant difference was observed for the fracture resistance among groups or for the type of break by material used (p=0.1593, p=0.77 respectively). Conclusion: Within the parameters of this study, following mechanical fatigue, the materials used for PBE: resin-modified glass-ionomer and glass-ionomer, resin-based composite and bulk-fill composite, did not influence results in terms of margin quality and fracture resistance. Therefore, collective findings suggest that these materials might be suitable for proximal box elevation procedures. Nevertheless, clinical caution is recommended with any PBE procedure and further testing of GI materials is needed.

Ribonuclease III is a highly-conserved bacterial enzyme that cleaves double-stranded (ds) RNA structures, and participates in diverse RNA maturation and decay pathways. Essential insight on the RNase III mechanism of dsRNA cleavage has been provided by crystallographic studies of the enzyme from the hyperthermophilic bacterium, Aquifex aeolicus. However, those crystals involved complexes containing either cleaved RNA, or a mutant RNase III that is catalytically inactive. In addition, neither the biochemical properties of A. aeolicus (Aa)-RNase III, nor the reactivity epitopes of its cognate substrates are known. The goal of this project is to use Aa-RNase III, for which there is atomic-level structural information, to determine how RNase III recognizes its substrates and selects the target site. I first purified recombinant Aa-RNase III and defined the conditions that support its optimal in vitro catalytic activity. The catalytic activity of purified recombinant Aa-RNase III exhibits a temperature optimum of 70-85°C, a pH optimum of 8.0, and with either Mg2+ or Mn2+ supports efficient catalysis. Cognate substrates for Aa-RNase III were identified and their reactivity epitopes were characterized, including the specific bp sequence elements that determine processing reactivity and selectivity. Small RNA hairpins, based on the double-stranded structures associated with the Aquifex 16S and 23S rRNA precursors, are cleaved in vitro at sites that are consistent with production of the immediate precursors to the mature rRNAs. Third, the role of the dsRBD in scissile bond selection was examined by a mutational analysis of the conserved interactions of RNA binding motif 1 (RBM1) with the substrate proximal box (pb). The individual contributions towards substrate recognition were determined for conserved amino acid side chains in the RBM1. It also was shown that the dsRBD plays key dual roles in both binding energy and selectivity, through RBM1 responsiveness to proximal box bp sequence. The dsRBD is specifically responsive to an antideterminant (AD) bp in pb position 2. The relative structural rigidity of both dsRNA and dsRBD rationalizes the strong effect of an inhibitory bp at pb position 2: disruption of one RBM1 side chain interaction can effectively disrupt the other RBM1 side chain interactions. Finally, a cis-acting model was developed for subunit involvement in substrate recognition by RNase III. Structurally asymmetric mutant heterodimers of Escherichia coli (Ec)-RNase III were constructed, and asymmetric substrates were employed to reveal how RNase III can bind and deliver hairpin substrates to the active site cleft in a pathway that requires specific binding configurations of both enzyme and substrate. / Chemistry