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The kinesin-14 Kar3 from Saccharomyces cerevisiae (Sc) is a C-terminal motor that forms a heterodimer with the kinesin-accessory protein Vik1. Although Vik1 possesses a typical kinesin motor domain (MD) fold, it lacks a nucleotide-binding site. However, it binds microtubules with affinities that can be regulated Kar3’s nucleotide state. This implies intermolecular communication between its subunits. This thesis aimed to understand this communication by studying the structures and functions of Kar3Vik1 orthologs. First, we biochemically characterized Kar3 from Ashbya gossypii (Ag) and determined the crystal structure of its MD. It was shown that the active site features of the AgKar3MD are similar to that of the ScKar3 R598A mutant, and that the β1 lobe at the edge of the MD was unique in structure and amino acid content. These results may provide a rationale for the unique enzymatic properties of this motor that could be relevant to its interaction with AgVik1 and function in Ashbya gossypii. We also determined the crystal structures of Kar3 and Vik1 orthologs from Candida glabrata (Cg). While the CgKar3MD structure was very similar to that of ScKar3MD, crystals of CgVik1 captured three novel conformations of the Vik1 motor homology domain (MHD). We observed that when the N-terminal neck helix docks against the MHD core in two unique positions, the C-terminus resembling neck mimics of kinesin-14 motors also docks against the neck-core junction. However, when the neck is non-helical and disengaged from the MHD, the C-terminus is undocked and disordered. To assess the functional importance of these N- and C-terminal segments of Vik1 MHD, we created CgKar3Vik1 constructs whose Vik1 subunit contained either a point mutation or complete truncation of the C-terminus (neck mimic), and analyzed their biophysical properties. All mutants showed defective ATPase activity and microtubule-gliding ability. Characterization of the mutations in CgVik1MHD by molecular dynamics simulations showed that residues Ile578 and Asn580 are not only involved in stabilizing interactions between the neck and neck mimic but they also influence and respond to conformational changes of the neck. These observations implicate the N- and C-termini of Vik1 as a key element of Kar3Vik1 function and communication. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2013-07-23 10:31:52.885

Anacampserotaceae pertence à ordem Caryophyllales, subordem Portulacineae, atualmente constituída por três gêneros (Anacampseros L., Grahamia Gill ex Hook e Talinopsis A. Gray) antes pertencentes à família Portulacaceae. Os representantes de Anacampserotaceae apresentam estruturas axilares lignificadas muito características, como tricomas, escamas (scales) e cerdas (bristles) e também apresentam células do tipo WBT que ocorre somente em mais outras duas famílias de Caryophyllales, Aizoaceae e Cactaceae. Os caracteres axilares são muito informativos para o clado ACPT (Anacampserotaceae, Cactaceae, Portulacaceae e Talinaceae) e vêm revelando importantes dados aos estudos filogenéticos da subordem Portulacineae, no entanto, há uma controvérsia quanto a origem ontogenética das estruturas axilares de Anacampserotaceae, o que pode levar a interpretações equivocadas das relações filogenéticas do grupo. Diante disso, este trabalho realizou um estudo ontogenético das estruturas presentes na região da axila foliar de oito espécies de Anacampserotaceae e analisou as WBTs do caule e de folhas quanto seu tipo de espessamento e medidas quantitativas de seis espécies. Os resultados apontam a 1) origem estipular das escamas e não protodérmica, como mencionado na literatura; 2) primeiro relato de profilos na família e 3) WBTs ocorrem em Anacampseros e estão ausente em Talinopsis, grupo considerado basal na família. / Anacampserotaceae belongs to the order Caryophyllales, suborder Portulacineae, currently consists of three genus (Anacampseros L., Grahamia Gill ex Hook and Talinopsis A. Gray) formerly circumscribed to the family Portulacaceae. Anacampserotaceae\'s representative presents lignified axillary structures very distinctive, like trichomes, scales and bristles and also WBT cells that is presented only by two other families of Caryophyllales, Aizoaceae e Cactaceae. The axillary characters are very informative for the ACPT clade (Anacampserotaceae, Cactaceae, Portulacaceae and Talinaceae) and has been revealing important data for the phylogenetic studies on the Portulacineae suborder, nevertheless, there is a controversy regarding the Anacampserotaceae axillary structures ontogenetic origin, what may lead to misguided interpretations for the ACPT phylogenetic relationships. Before it, this research have accomplished the ontogenetic study of the structures present on the leaf axil of eight Anacampserotaceae species and examined stem and leaf WBTs, as for its secondary thickening type and quantitative measurements of six species. The results points 1) that at least one structure (scale) has leaf origin instead of protodermic origin, as stated by some authors; 2) first report of prophylls for the family and 3) WBTs occur in Anacampseros but they are absent in Talinopsis, a basal group of Anacampserotaceae.

The use of remote sensing technology has made it possible for the non-contact measurement of soil moisture content (SMC). Many remote sensing techniques can be used such as microwave sensors, electromagnetic waves sensors, capacitance, and thermal infrared sensors. Some of those techniques are constrained by their high fabrication cost, operation cost, size, or complexity. In this study, a thermal infrared technique was used to predict soil moisture content with the aid of using weather meteorological variables. <p>The measured variables in the experiment were soil moisture content (%SMC), soil surface temperature (Ts) measured using thermocouples, air temperature (Ta), relative humidity (RH), solar radiation (SR), and wind speed (WS). The experiment was carried out for a total of 12 soil samples of two soil types (clay/sand) and two compaction levels (compacted/non-compacted). After data analysis, calibration models relating soil moisture content (SMC) to differential temperature (Td), relative humidity (RH), solar radiation (SR), and wind speed (WS) were generated using stepwise multiple linear regression of the calibration data set. The performance of the models was evaluated using validation data. Four mathematical models of predicting soil moisture content were generated for each soil type and configuration using the calibration data set. Among the four models, the best model for each soil type and configuration was determined by comparing root mean of squared errors of calibration (RMSEC) and root mean of squared errors of validation (RMSEV) values. Furthermore, a calibration model for the thermal infrared sensor was developed to determine the corrected soil surface temperature as measured by the sensor (Tir) instead of using the thermocouples. The performance of the thermal infrared sensor to predict soil moisture content was then tested for sand compacted and sand non-compacted soils and compared to the predictive performance of the thermocouples. This was achieved by using the measured soil surface temperature by the sensor (Tir), instead of the measured soil surface temperature using the thermocouples to determine the soil-minus-air temperature (Td). The sensor showed comparable prediction performance, relative to thermocouples. <p>Overall, the models developed in this study showed high prediction performance when tested with the validation data set. The best models to predict SMC for compacted clay soil, non-compacted clay soil, and compacted sandy soil were three-variable models containing three predictive variables; Td, RH, and SR. On the other hand, the best model to predict SMC for compacted sandy soil was a two-variable model containing Td, and RH. The results showed that the prediction performance of models for predicting SMC for the sandy soils was superior to those of clay soils.

The use of remote sensing technology has made it possible for the non-contact measurement of soil moisture content (SMC). Many remote sensing techniques can be used such as microwave sensors, electromagnetic waves sensors, capacitance, and thermal infrared sensors. Some of those techniques are constrained by their high fabrication cost, operation cost, size, or complexity. In this study, a thermal infrared technique was used to predict soil moisture content with the aid of using weather meteorological variables. <p>The measured variables in the experiment were soil moisture content (%SMC), soil surface temperature (Ts) measured using thermocouples, air temperature (Ta), relative humidity (RH), solar radiation (SR), and wind speed (WS). The experiment was carried out for a total of 12 soil samples of two soil types (clay/sand) and two compaction levels (compacted/non-compacted). After data analysis, calibration models relating soil moisture content (SMC) to differential temperature (Td), relative humidity (RH), solar radiation (SR), and wind speed (WS) were generated using stepwise multiple linear regression of the calibration data set. The performance of the models was evaluated using validation data. Four mathematical models of predicting soil moisture content were generated for each soil type and configuration using the calibration data set. Among the four models, the best model for each soil type and configuration was determined by comparing root mean of squared errors of calibration (RMSEC) and root mean of squared errors of validation (RMSEV) values. Furthermore, a calibration model for the thermal infrared sensor was developed to determine the corrected soil surface temperature as measured by the sensor (Tir) instead of using the thermocouples. The performance of the thermal infrared sensor to predict soil moisture content was then tested for sand compacted and sand non-compacted soils and compared to the predictive performance of the thermocouples. This was achieved by using the measured soil surface temperature by the sensor (Tir), instead of the measured soil surface temperature using the thermocouples to determine the soil-minus-air temperature (Td). The sensor showed comparable prediction performance, relative to thermocouples. <p>Overall, the models developed in this study showed high prediction performance when tested with the validation data set. The best models to predict SMC for compacted clay soil, non-compacted clay soil, and compacted sandy soil were three-variable models containing three predictive variables; Td, RH, and SR. On the other hand, the best model to predict SMC for compacted sandy soil was a two-variable model containing Td, and RH. The results showed that the prediction performance of models for predicting SMC for the sandy soils was superior to those of clay soils.

A Penning-type surface-plasma negative hydrogen (H^-) ion source has been delivering beam at the ISIS pulsed spallation neutron and muon facility for over thirty years. It is one of the most powerful and well-renowned H^- sources in the world. Although long-term experience has allowed the source to be operated reliably and set up in a repeatable way, it is treated as something of a 'black box': the detailed plasma physics of why it works has always been unclear. A vacuum Vessel for Extraction and Source Plasma Analyses (VESPA) has been developed to understand the ISIS ion source plasma and improve the beam extracted from it. The VESPA ion source is operated in a completely new regime whereby the analysing sector dipole magnet housed inside a refrigerated 'cold box', presently used on ISIS, is replaced by an on-axis extraction system. The new extraction system incorporates a novel einzel lens with an elliptical aperture. This is the first demonstration of an elliptical einzel being used to focus an asymmetric H^- ion beam. With the dipole magnet removed, the ion source has been shown to produce 85 mA of H^- beam current at normal settings; of which 80 mA is transported through the new einzel lens system, with a normalised RMS emittance of 0.2 &pi; mm mrad. Optical emission spectroscopy measurements have shown a plasma density of 10¹⁹ m^–3, an H₂ dissociation rate of 70%, an almost constant electron temperature of 3.5 eV and an atomic temperature which linearly increases above the electron temperature. In support of these principal measurements, rigorous particle tracking, electrostatic and thermal simulations were performed. In addition, a suite of new equipment was manufactured by the author. This includes a fast pressure gauge, a temperature controller, a high voltage einzel lens circuit, a fast beam chopper and a caesium detection system.

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