Structural and Functional Characterization of a Novel Heterodimeric Kinesin in Candida albicans

Kinesins are molecular motors that transport intracellular cargos along microtubules (MTs) and influence the organization and dynamics of the MT cytoskeleton. Their force-generating functions arise from conformational changes in their motor domain as ATP is bound and hydrolyzed, and products are released. In the budding yeast Saccharomyces cerevisiae, the Kar3 kinesin forms heterodimers with one of two non-catalytic kinesin-like proteins, Cik1 and Vik1, which lack the ability to bind ATP, and yet they retain the capacity to bind MTs. Cik1 and Vik1 also influence and respond to the MT-binding and nucleotide states of Kar3, and differentially regulate the functions of Kar3 during yeast mating and mitosis. The mechanism by which Kar3/Cik1 and Kar3/Vik1 dimers operate remains unknown, but has important implications for understanding mechanical coordination between subunits of motor complexes that traverse cytoskeletal tracks. In this study, we show that the opportunistic human fungal pathogen Candida albicans (Ca) harbors a single version of this unique form of heterodimeric kinesin and we present the first in vitro characterization of this motor. Like its budding yeast counterpart, the Vik1-like subunit binds directly to MTs and strengthens the MT-binding affinity of the heterodimer. However, in contrast to ScKar3/Cik1 and ScKar3/Vik1, CaKar3/Vik1 exhibits weaker overall MT-binding affinity and lower ATPase activity. Preliminary investigations using a multiple motor motility assay indicate CaKar3/Vik1 may not be motile. Using a maltose binding protein tagging system, we determined the X-ray crystal structure of the CaKar3 motor domain and observed notable differences in its nucleotide-binding pocket relative to ScKar3 that appear to represent a previously unobserved state of the active site. Together, these studies broaden our knowledge of novel kinesin motor assemblies and shed new light on structurally dynamic regions of Kar3/Vik1-like motor complexes that help mediate mechanical coordination of its subunits.

Experimental and RANS-CFD Study of Nacelle Drag

The drag on a nacelle model was investigated experimentally and computationally to provide guidance and insight into the capabilities of RANS-based CFD. The research goal was to determine whether industry constrained CFD could participate in the aerodynamic design of nacelle bodies. Grid refinement level, turbulence model and near wall treatment settings, to predict drag to the highest accuracy, were key deliverables. Cold flow low-speed wind tunnel experiments were conducted at a Reynolds number of 6∙〖10〗^5, 293 K and a Mach number of 0.1. Total drag force was measured by a six-component force balance. Detailed wake analysis, using a seven-hole pressure probe traverse, allowed for drag decomposition via the far-field method. Drag decomposition was performed through a range of angles of attack between 0o and 45o. Both methods agreed on total drag within their respective uncertainties. Reversed flow at the measurement plane and saturation of the load cell caused discrepancies at high angles of attack. A parallel CFD study was conducted using commercial software, ICEM 15.0 and FLUENT 15.0. Simulating a similar nacelle geometry operating under inlet boundary conditions obtained through wind tunnel characterization allowed for direct comparisons with experiment. It was determined that the Realizable k-ϵ was best suited for drag prediction of this geometry. This model predicted the axial momentum loss and secondary flow in the wake, as well as the integrated surface forces, within experimental error up to 20o angle of attack. SST k-ω required additional surface grid resolution on the nacelle suction side, resulting in 15% more elements, due to separation point prediction sensitivity. It was further recommended to apply enhanced wall treatment to more accurately capture the viscous drag and separated flow structures. Overall, total drag was predicted within 5% at 0o angle of attack and 10% at 20o, each within experimental uncertainty. What is more, the form and induced drag predicted by CFD and measured by the wake traverse shared good agreement. Which indicated CFD captured the key flow features accurately despite simplification of the nacelle interior geometry.

Surface wave and storm surge prediction during Hurricane Sandy

Hurricane Sandy was the largest storm on historical record in the Atlantic Ocean basin with extensive coastal damage caused by large waves and high storm surge. The primary objectives of this thesis are to compare and evaluate three different spatially-varying surface wind fields of Hurricane Sandy to investigate the impact of the differences between the complex wind fields on predictions of the sea surface evolution, and to evaluate the impact of the storm on the hydrodynamics in Great South Bay (GSB) and the discharge of ocean water into the back-barrier bay from overwash over Fire Island. Three different spatially-varying surface wind fields were evaluated and compared to wind observations, including the parametric Holland (1980) model (H80), the parametric Generalized Asymmetric Holland Model (GAHM), and results from the WeatherFlow Regional Atmospheric Modelling System (WRAMS). The winds were used to drive the coupled Delft3D-SWAN hydrodynamic and ocean wave models on a regional grid. The results indicate that the WRAMS wind field produces wave model predictions in the best agreement with significant wave height observations, followed by the GAHM and H80 wind fields and that a regional atmospheric wind model is best for hindcasting hurricane waves and water levels when detailed observations are available, while a parametric vortex model is best for forecasting hurricane sea surface conditions. Using a series of four connected Delft3D-SWAN grids to achieve finer resolution over Fire Island and GSB, a higher resolution WRAMS was used to predict waves and storm surge. The results indicate that strong local winds have the largest influence on water level fluctuations in GSB. Three numerical solutions were conducted with varying extents of barrier island overwash. The simulations allowing for minor and major overwash indicated good agreement with observations in the east end of GSB and suggest that island overwash provided a significant contribution of ocean water to GSB during the storm. Limiting the overwash in the numerical model directly impacts the total discharge into GSB from the ocean through existing inlets. The results of this study indicate that barrier island overwash had a significant impact on the water levels in eastern GSB.