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Formins are potent regulators of actin dynamics that can remodel the actin cytoskeleton to control cell shape, cell cytokinesis, and cell morphogenesis. The defining feature of formins is the formin homology 2 (FH2) domain (Paul and Pollard, 2008), which promotes actin filament assembly while processively moving along the polymerizing filament barbed end. INFT-1 is one of six formin family members present in Caenorhabditis elegans (Hunt-Newbury et al., 2007) and is most closely related to vertebrate INF2, an inverted formin with regulatory domains in the C- rather than N-terminus. Nematode INFT-1 contains both formin homology 1 (FH1) and formin homology 2 (FH2) domains. However, it does not share the regulatory N-terminal Diaphanous Inhibitory Domain (DID) domain and C-terminal Diaphanous Autoregulatory Domain (DAD) domain found in mammalian INF2. In contrast to mammalian INF2, the sequence of INFT-1 starts immediately at FH1 domain and C-terminal region of INFT-1 shares little homology with INF2, suggesting that elegans INFT-1 is regulated by other mechanisms. We used fluorescence spectroscopy to determine the effect of INFT-1 FH1FH2 on actin assembly and total internal reflection fluorescence microscopy to investigate how INFT-1 formin homology 1 and formin homology 2 domains (FH1FH2) mediate filament nucleation and elongation. INFT-1 FH1FH2 nucleates actin filament and promote actin assembly. However, INFT-1 FH1FH2 reduces filament barbed-end elongation rates in the absence or presence of profilin. Evidences demonstrated that INFT-1 is non-processive, indicating a unique mechanism of nucleation. INFT-1 nucleation efficiency is similar to the efficiency of Arabidopsis FORMIN1 (AFH1), another non-processive formin. High phosphate affected the assembly activity of INFT-1 FH1FH2 in the absence or presence of profilin. INFT is thus the second example of a non-processive formin member and will allow a more detailed understanding of the mechanistic difference between processive and non-processive formins. / Master of Science

Water utilities can experience problems from bubble formation during conventional treatment, including impaired particle settling, filter air binding, and measurement as false turbidity in filter effluent. Coagulation processes can cause supersaturation and bubble formation by converting bicarbonate alkalinity to carbon dioxide by acidification. A model was developed to predict the extent of bubble formation during coagulation which proved accurate, using an apparatus designed to physically measure the actual volume of bubble formation. Alum acted similar to hydrochloric acid for initializing bubble formation, and higher initial alkalinity, lower final solution pH, and increased mixing rate tended to increase bubble formation. Lastly, the protocol outlined in Standard Methods for predicting the degree of supersaturation was examined, and when compared to this work, the Standard Methods approach produces an error up to 16% for conditions found in water treatment. Air entrainment and ozonation are the key causes of dissolved gas supersaturation and eventual bubble formation in water treatment plants. Total dissolved gas probes (TDGP) are now available to directly measure supersaturation and have many advantages compared to conventional techniques. Bubble formation during coagulation-flocculation hindered particle sedimentation, producing settled turbidities double that of solutions without dissolved gases. In a filtration study, run time to one half of initial flow was decreased by 54% when the source water was increased from 0.1 to 0.2 atm supersaturation. Indeed, even at 0.05 atm supersaturation, run length was only 21 hours in solutions without added particulate matter. A case study confirmed that bubble formation can interfere with coagulation and filtration processes at conventional treatment plants. / Master of Science

A new paradigm for the formation of calcified skeletons suggests mineralization proceeds through amorphous calcium carbonate (ACC) precursors. The implications of this strategy in carbonate crystallization are widespread, particularly for understanding factors controlling impurity and isotopic signatures in calcium carbonates. The first chapter is a literature review of the biomineralization processes used by two important model organisms: the sea urchin larva and the foraminifera. Sea urchin larvae provide a thoroughly studied example of mineralization by an ACC pathway that is under biological control through regulation of protein chemistry and the local mineralization environment. A review of how foraminifera produce their test structures is also examined to explore the question of how organisms regulate the Mg content in proportion to the temperature their environments of formation. The second chapter demonstrates that acidic biomolecules regulate the composition of ACC for a suite of model carboxylated molecules. The physical basis for the systematic trend in Mg content is related to the ability of the affinity of the biomolecule for binding Ca versus Mg. The third chapter builds on these findings to explore the transformation of Mg-rich ACC precursors to calcites of exceptionally high Mg-contents that could not be produced by classical step-dominated growth processes. The data indicate that these materials are likely a result of a nucleation-dominated pathway. The final, fourth chapter develops Raman spectroscopy-based calibrations for determining Mg contents in ACC. The calibrations are based upon peak position or peak width of the carbonate υ₁ stretch. / Ph. D.

Since the 1970s, a growing body of research has suggested that bacteria play an active role in precipitation. These bacteria are capable of catalyzing the formation of ice at relatively warm temperatures utilizing a specific protein family which aids in the binding of water molecules. However, the overall biodiversity, concentration, and relationship of ice nucleation active (ice+) bacteria with air mass trajectories and precipitation chemistry is not well studied. Precipitation events were collected over 15 months in Blacksburg, VA and ice+ bacteria were isolated from these samples. From these samples, 33,134 total isolates were screened for ice nucleation activity (INA) at -8 °C. A total of 593 of these isolated positively confirmed for INA at the same temperature in subsequent tests. The precipitation events had a mean concentration of 384±147 colony forming units per liter. While the majority of confirmed ice+ bacteria belonged to the gammaproteobacteria, a well-studied class of bacteria, including ice+ species of Pseudomonas, Pantoea, and Xanthomonas, two isolates were identified as Lysinibacillus, a Gram-positive member of the Firmicute phylum. These two isolates represent the first confirmed non-gammaproteobacteria with INA. After further characterization, the two isolates of Lysinibacillus did not appear to use a protein to freeze water. Instead, the Lysinibacillus isolates used a secreted, nanometer-sized molecule that is heat, lysozyme, and proteinase resistant. In an attempt to identify the mechanism responsible for this activity, species type strains were tested for INA and UV mutants were generated to knock out the ice+ phenotype. Based on these results, only members of the species L. parviboronicapiens exhibit INA and the genes responsible for the activity may lie within a type-1 polyketide synthase/non-ribosomal peptide synthase gene cluster. This gene cluster is absent from the genomes of all non-ice+ strains of Lysinibacillus, and contains mutations in five of the nine ice nucleation inactive mutants generated from the rain isolated strain. To better understand the phylogenetic relationship among ice+ Lysinibacillus, a comprehensive reference guide was compiled to provide the most up-to-date information regarding the genus and each of its species. This reference will be available to other researchers investigating Lysinibacillus species or other closely related genera. / PHD / It is a common misconception that water freezes at 0°C (32°F). In clouds, water may remain liquid until -37 to -40°C (-35 to -40°F). At temperatures warmer than this, water molecules must collect around small particles that can help form ice, called ice nuclei. Numerous ice nuclei have been identified, ranging from dirt and dust, to volcanic ash, and even to pollen, fungi, and bacteria. One of these bacteria, Pseudomonas syringae, was identified as an ice nucleus in the 1970’s when it was discovered that it was increasing susceptibility of corn to frost damage. Since then, other Pseudomonas species as well as other bacteria within the same family of bacteria have been shown to have the ability to freeze water at relatively warm temperatures utilizing a specialized protein. Despite numerous studies on how these bacteria can exist in the atmosphere and how they can freeze water, the extent of this freezing ability, the concentration of bacteria in precipitation, and how cloud chemistry affects these bacteria has not been widely studied. In this study, precipitation was collected over the course of 15 months and the bacteria found within the collected precipitation were checked to see if they could act as ice nuclei. We found many of the previously described bacterial ice nuclei in the precipitation samples, but also identified a previously unidentified bacterium capable of freezing water. This bacterium, Lysinibacillus parviboronicapiens, does not use the same method of freezing as the other described bacterial ice nuclei. As such, we set out to determine the method it uses. We have determined that this bacterium utilizes a heat-stable, nanometer-sized particle that is not a protein. To better understand this molecule, representative strains of each species of this genus of bacteria were tested for their ability to freeze water, however, only this species has the ability. To further identify the molecule, UV radiation was used to disrupt the bacteria’s ability to freeze water, and the genes responsible were identified. Based on these results, we have tentatively identified the responsible genes as part of a polyketide synthase gene cluster. This gene cluster is responsible for producing small molecules that provide some survival advantage for the bacteria, in our case, possibly the ability to freeze water. As a final step, and to help serve other researchers, a comprehensive analysis of the entire Lysinibacillus genus has been performed and a reference guide has been generated to help describe and distinguish individual species.